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Cross-linking of Intercellular Adhesion Molecule-1 Induces Interleukin-8 and RANTES Production through the Activation of MAP Kinases in Human Vascular Endothelial Cells
Abstract
We investigated whether intercellular adhesion molecule-1 (ICAM-1) transduces outside-in signals for the production of chemokines IL-8 and RANTES in endothelial cells. Cross-linking of ICAM-1 induced IL-8 and RANTES mRNA expressions and increased their protein synthesis and secretions in endothelial cells. Furthermore, ICAM-1 cross-linking activated 44- and 42-kDa mitogen-activated protein (MAP) kinases (ERK1 and ERK2) in endothelial cells, as indicated by the electrophoretic mobility shift of MAP kinases on SDS-polyacrylamide gels. Finally, the specific MEK inhibitor PD98059 inhibited ICAM-1-induced IL-8 and RANTES production in endothelial cells. Taken together, these results indicate that stimulation of ICAM-1 induces IL-8 and RANTES production through the activation of 44- and 42-kDa MAP kinases in endothelial cells, suggesting that ICAM-1-induced chemokine production in endothelial cells would further attract and activate leukocytes to induce intense inflammation.
... Chelating calcium or inhibitors of PKC or Src block leukocyte migration across endothelial cells [12,24,25]. In other reports, ICAM-1 activates extracellular signal-regulated kinase 1/2 (ERK1/2) and/or c-Jun N terminal kinase (JNK) [20,26,27]. However, the mechanisms for ICAM-1 activation of XO, PKC and ERK1/2 are not known. ...
... It is reported that ICAM-1 binding induces activation of extracellular signal-regulated kinase 1/2 (ERK1/2) [20,26,27]. In addition, ICAM-1-dependent ROS production is reported to activate mitogen-activated protein (MAP) kinases [3,6789 . ...
... This suggests that since ERK1/2 activation was not affected by tocopherols but ERK1/2 activation was dependent on XO, the antioxidant properties of the tocopherols did not function to block the XO-induced ERK1/2 that leads to activation of PKCa. Separate reports indicate that ICAM-1 can activate PKC, XO, PLC or ERK1/2 [20,26,27], but the mechanisms for activation of these signals in endothelial cells was not known. In brain endothelial cell lines, ICAM-1 binding induces a calcium/ PLCc 1 /PKC pathway [24] . ...
ICAM-1-dependent leukocyte recruitment in vivo is inhibited by the vitamin E isoform d-α-tocopherol and elevated by d-γ-tocopherol. ICAM-1 is reported to activate endothelial cell signals including protein kinase C (PKC), but the PKC isoform and the mechanism for ICAM-1 activation of PKC are not known. It is also not known whether ICAM-1 signaling in endothelial cells is regulated by tocopherol isoforms. We hypothesized that d-α-tocopherol and d-γ-tocopherol differentially regulate ICAM-1 activation of endothelial cell PKC.
ICAM-1 crosslinking activated the PKC isoform PKCα but not PKCβ in TNFα-pretreated human microvascular endothelial cells. ICAM-1 activation of PKCα was blocked by the PLC inhibitor U73122, ERK1/2 inhibitor PD98059, and xanthine oxidase inhibitor allopurinol. ERK1/2 activation was blocked by inhibition of XO and PLC but not by inhibition of PKCα, indicating that ERK1/2 is downstream of XO and upstream of PKCα during ICAM-1 signaling. During ICAM-1 activation of PKCα, the XO-generated ROS did not oxidize PKCα. Interestingly, d-α-tocopherol inhibited ICAM-1 activation of PKCα but not the upstream signal ERK1/2. The d-α-tocopherol inhibition of PKCα was ablated by the addition of d-γ-tocopherol.
Crosslinking ICAM-1 stimulated XO/ROS which activated ERK1/2 that then activated PKCα. ICAM-1 activation of PKCα was inhibited by d-α-tocopherol and this inhibition was ablated by the addition of d-γ-tocopherol. These tocopherols regulated ICAM-1 activation of PKCα without altering the upstream signal ERK1/2. Thus, we identified a mechanism for ICAM-1 activation of PKC and determined that d-α-tocopherol and d-γ-tocopherol have opposing regulatory functions for ICAM-1-activated PKCα in endothelial cells.
... While the extracellular domains of ICAM-1 are sufficient for the attachment of leukocytes to the endothelium, in vitro experiments have shown signaling through the cytoplasmic tail to be critical for transendothelial migration of multiple subsets including neutrophils, monocytes, and T cells [84,85,89]. ICAM-1 intracellular signaling via MAPKs p38 and ERK has also been shown to up-regulate expression of multiple pro-inflammatory cytokines including CXCL8, CCL3, CCL4, and COX-2 in human brain microvascular endothelial cells and HUVECs [90,91]. ...
... While the extracellular domains of ICAM-1 are sufficient for the attachment of leukocytes to the endothelium, in vitro experiments have shown signaling through the cytoplasmic tail to be critical for transendothelial migration of multiple subsets including neutrophils, monocytes, and T cells [84,85,89]. ICAM-1 intracellular signaling via MAPKs p38 and ERK has also been shown to up-regulate expression of multiple pro-inflammatory cytokines including CXCL8, CCL3, CCL4, and COX-2 in human brain microvascular endothelial cells and HUVECs [90,91]. . ICAM-1 intracellular signaling after integrin binding. ...
Simple Summary
Intercellular adhesion molecule 1 (ICAM-1) is a protein produced by cells in blood vessel walls, as well as many other cell types. It localizes to the surface of these cells to allow them to communicate with adjacent cells. The production of ICAM-1 is induced by inflammation, such as occurs during infection, when one of its main functions is to direct white blood cells to the site of the infection. However, in autoimmune diseases and certain types of infection, ICAM-1 can be involved in directing cells to damage healthy tissue. ICAM-1 can also promote or protect against cancer, depending on the type of cancer and the cell populations that are producing it. In this review article, the ICAM1 gene and ICAM-1 protein are introduced, and their regulation is summarized. The roles of ICAM-1 in the immune system and a spectrum of diseases, as well as therapeutics that target ICAM-1, are discussed.
Abstract
Intercellular adhesion molecule 1 (ICAM-1) is a transmembrane protein in the immunoglobulin superfamily expressed on the surface of multiple cell populations and upregulated by inflammatory stimuli. It mediates cellular adhesive interactions by binding to the β2 integrins macrophage antigen 1 and leukocyte function-associated antigen 1, as well as other ligands. It has important roles in the immune system, including in leukocyte adhesion to the endothelium and transendothelial migration, and at the immunological synapse formed between lymphocytes and antigen-presenting cells. ICAM-1 has also been implicated in the pathophysiology of diverse diseases from cardiovascular diseases to autoimmune disorders, certain infections, and cancer. In this review, we summarize the current understanding of the structure and regulation of the ICAM1 gene and the ICAM-1 protein. We discuss the roles of ICAM-1 in the normal immune system and a selection of diseases to highlight the breadth and often double-edged nature of its functions. Finally, we discuss current therapeutics and opportunities for advancements.
... Additionally, in the present study with OLENDO cells, the effects of TLR1/2 and TLR4 receptors on LPS-induced ICAM 1 expression were assessed. ICAM 1 is an important adhesion molecule involved in the attachment and recruitment of leukocytes to the endothelium [30,43,44]. Its activation induces inflammation and vascular disruption, as shown e.g., in rat brain endothelial cells or human umbilical vein endothelial cells [30,43,44]. ...
... ICAM 1 is an important adhesion molecule involved in the attachment and recruitment of leukocytes to the endothelium [30,43,44]. Its activation induces inflammation and vascular disruption, as shown e.g., in rat brain endothelial cells or human umbilical vein endothelial cells [30,43,44]. Similarly, recent results from our group showed that LPS disrupts in vitro capillary morphogenesis and endothelial barrier function, in association with increased ICAM 1 expression and altered gap junctional intercellular communication, mediated particularly by Cx43 in OLENDO cells [31]. ...
Escherichia coli (E. coli) is the most common Gram-negative bacterium causing infection of the uterus or mammary gland and is one of the major causes of infertility in livestock. In those animals affected by E. coli driven LPS-mediated infections, fertility problems occur in part due to disrupted follicular and luteal functionality. However, the molecular mechanisms by which LPS induces inflammation, and specifically, the role of LPS in the disruption of capillary morphogenesis and endothelial barrier function remain unclear. Here, we hypothesized that LPS may lead to alterations in luteal angiogenesis and vascular function by inducing inflammatory reactions in endothelial cells. Accordingly, OLENDO cells were treated with LPS followed by evaluation of the expression of selected representative proinflammatory cytokines: NF-kB, IL6, IL8, TNFα, and ICAM 1. While TNFα was not affected by treatment with LPS, transcripts of NF-kB, IL6, and IL8 were affected in a dosage-dependent manner. Additionally, the activity of TLR2 and TLR4 was blocked, resulting in suppression of the LPS-induced expression of ICAM 1, NF-kB, IL6, and IL8. Inhibition of the PKA or MAPK/ERK pathways suppressed the LPS-stimulated expression of NF-kB, IL6, and IL8, whereas blocking the PKC pathway had the opposite effect. Furthermore, LPS-induced phosphorylation of Erk1 and Erk2 was inhibited when the TLR4 or MAPK/ERK pathways were blocked. Finally, LPS seems to induce inflammatory processes in OLENDO cells via TLR2 and TLR4, utilizing different signaling pathways.
... ICAM-1 allows attachment of leukocytes to the endothelium and recruits them to areas of inflammation (Wittchen, 2009). Its activation also increases vascular permeability and production of pro-inflammatory cytokines such as interleukins (IL)1β and IL8 in vascular endothelial cells, as shown in rat brain endothelial cells and human umbilical vein endothelial cells (Dragoni et al., 2017;Sano et al., 1998). This indicates the involvement of vascular endothelial cells in the initiation of inflammatory responses mediated through TLRs, ICAM-1 and other factors. ...
... This is also consistent with observation made in other studies, in which treatment of human brain microvascular endothelial cells (Wong and Dorovini-Zis, 1992), neonatal dermal lymphatic microvascular endothelial cells (Sawa et al., 2008) or umbilical vein endothelial cells (Yan et al., 2002) with LPS increased the expression of ICAM-1 as well as transendothelial migration of leukocytes. Additionally, by activating MAPKs in rat brain endothelial cell lines (Dragoni et al., 2017) and human umbilical vein endothelial cells (Sano et al., 1998), ICAM-1 increases vascular inflammation and barrier dysfunction. ...
Gram-negative bacteria, in particular Escherichia coli with its cell wall lipopolysaccharide (LPS), often cause metritis and mastitis in domestic animals. Ovarian LPS accumulation may initiate local inflammatory reactions mediated through cell surface Toll-like receptors (TLRs). This may disrupt ovarian functionality leading to infertility. Possible adverse effects of LPS on luteal activity are not yet well explored. We hypothesized that LPS could lead to alterations in luteal vascular functionality. Therefore, we established an in vitro cell line model (OLENDO) by immortalizing microvascular endothelial cells isolated from ovine corpus luteum (CL) with a potent Simian Virus 40 T-antigen (SV40-Tag). OLENDO exhibit endothelial cell characteristics, like low-density lipoprotein (LDL) uptake, express BSL-I, and VEGFR2, as well as TLR2 and TLR4 receptors. LPS-treatment of OLENDO altered in vitro tube formation, had no effects on cell viability and decreased gap junctional intercellular communication (GJIC). LPS did not impair GJA1/Cx43 protein expression, but altered its cellular localization showing signs of internalization. Taken together, we demonstrated the mechanisms underlying LPS induced impairment of luteal GJIC and immune processes in a novel and well-characterized OLENDO cell line.
... Proinflammatory cytokines increase ICAM-1 expression in endothelial cells in an NF-kB-dependent manner (20). Activation of ICAM-1 by integrin molecules on the leukocyte surface leads to a change in its distribution on the endothelial cell surface and initiates outside-in signaling (21). We have demonstrated that cytokines, such as TNF-a, can prime ICAM-1 in a phosphorylation-dependent manner, which leads to an increase in binding activity (22). ...
... We have demonstrated that cytokines, such as TNF-a, can prime ICAM-1 in a phosphorylation-dependent manner, which leads to an increase in binding activity (22). ICAM-1 ligation induces the activation of Src tyrosine kinases, eNOS (23,24), p38 MAPK, and Rho pathways (21,25,26). Recent studies have suggested that ICAM-1 activation-dependent signaling pathways also regulate the molecular events that are associated with diapedesis (23,24). ...
Intercellular adhesion molecule-1 (ICAM-1) mediates the firm adhesion of leukocytes to endothelial cells and initiates subsequent signaling that promotes their transendothelial migration (TEM). Vascular endothelial (VE)-cadherin plays a critical role in endothelial cell-cell adhesion, thereby controlling endothelial permeability and leukocyte transmigration. This study aimed to determine the molecular signaling events that originate from the ICAM-1-mediated firm adhesion of neutrophils that regulate VE-cadherin's role as a negative regulator of leukocyte transmigration. We observed that ICAM-1 interacts with Src homology domain 2-containing phosphatase-2 (SHP-2), and SHP-2 down-regulation via silencing of small interfering RNA in endothelial cells enhanced neutrophil adhesion to endothelial cells but inhibited neutrophil transmigration. We also found that VE-cadherin associated with the ICAM-1-SHP-2 complex. Moreover, whereas the activation of ICAM-1 leads to VE-cadherin dissociation from ICAM-1 and VE-cadherin association with actin, SHP-2 down-regulation prevented ICAM-1-VE-cadherin association and promoted VE-cadherin-actin association. Furthermore, SHP-2 down-regulation in vivo promoted LPS-induced neutrophil recruitment in mouse lung but delayed neutrophil extravasation. These results suggest that SHP-2-via association with ICAM-1-mediates ICAM-1-induced Src activation and modulates VE-cadherin switching association with ICAM-1 or actin, thereby negatively regulating neutrophil adhesion to endothelial cells and enhancing their TEM.-Yan, M., Zhang, X., Chen, A., Gu, W., Liu, J., Ren, X., Zhang, J., Wu, X., Place, A. T., Minshall, R. D., Liu, G. Endothelial cell SHP-2 negatively regulates neutrophil adhesion and promotes transmigration by enhancing ICAM-1-VE-cadherin interaction.
... ICAM-1 activation also increases inflammatory gene expression, e.g., that of IL-1B (15), CXCL8 (IL-8), CCL5 (RANTES) (16), and VCAM-1 (17), although it is unclear how, or if, this relates to leukocyte TEM. ...
... Endothelial ICAM-1 stimulation induces or enhances a variety of inflammatory responses including vascular permeability, inflammatory gene expression, and the licensing of leukocyte TEM (2,5,6,15,16). However, the underlying signal transduction networks responsible for these diverse effects are incompletely understood. ...
Lymphocyte transendothelial migration (TEM) is critically dependent on intraendothelial signaling triggered by adhesion to ICAM-1. Here we show that endothelial MAPKs ERK, p38, and JNK mediate diapedesis-related and diapedesis-unrelated functions of ICAM-1 in cerebral and dermal microvascular endothelial cells (MVECs). All three MAPKs were activated by ICAM-1 engagement, either through lymphocyte adhesion or Ab-mediated clustering. MAPKs were involved in ICAM-1-dependent expression of TNF-? in cerebral and dermal MVECs, and CXCL8, CCL3, CCL4, VCAM-1, and cyclooxygenase 2 (COX-2) in cerebral MVECs. Endothelial JNK and to a much lesser degree p38 were the principal MAPKs involved in facilitating diapedesis of CD4(+) lymphocytes across both types of MVECs, whereas ERK was additionally required for TEM across dermal MVECs. JNK activity was critical for ICAM-1-induced F-actin rearrangements. Furthermore, activation of endothelial ICAM-1/JNK led to phosphorylation of paxillin, its association with VE-cadherin, and internalization of the latter. Importantly ICAM-1-induced phosphorylation of paxillin was required for lymphocyte TEM and converged functionally with VE-cadherin phosphorylation. Taken together we conclude that during lymphocyte TEM, ICAM-1 signaling diverges into pathways regulating lymphocyte diapedesis, and other pathways modulating gene expression thereby contributing to the long-term inflammatory response of the endothelium.
... Cytokine-derived activation of all these pathways was described previously [4,33,35,36]. It was also confirmed that TNF-α and IFN-γ induced the phosphorylation of ERK [36,37], which was sensitive to OA-NO 2 inhibition. These results indicate that OA-NO 2 decreases the phosphorylation of signaling molecules that are strongly induced by pro-inflammatory mediators and thus modifies EC function during immune responses. ...
... These results are in accordance with the effect of OA-NO 2 on individual signaling pathway responses (e.g. STATs, MAPKs, and NF-κB) [2,4,33,34,36,37]. ...
Background:
Inflammatory-mediated pathological processes in the endothelium arise as a consequence of the dysregulation of vascular homeostasis. Of particular importance are mediators produced by stimulated monocytes/macrophages inducing activation of endothelial cells (ECs). This is manifested by excessive soluble pro-inflammatory mediator production and cell surface adhesion molecule expression. Nitro-fatty acids are endogenous products of metabolic and inflammatory reactions that display immuno-regulatory potential and may represent a novel therapeutic strategy to treat inflammatory diseases. The purpose of our study was to characterize the effects of nitro-oleic acid (OA-NO2) on inflammatory responses and the endothelial-mesenchymal transition (EndMT) in ECs that is a consequence of the altered healing phase of the immune response.
Methods:
The effect of OA-NO2 on inflammatory responses and EndMT was determined in murine macrophages and murine and human ECs using Western blotting, ELISA, immunostaining, and functional assays.
Results:
OA-NO2 limited the activation of macrophages and ECs by reducing pro-inflammatory cytokine production and adhesion molecule expression through its modulation of STAT, MAPK and NF-кB-regulated signaling. OA-NO2 also decreased transforming growth factor-β-stimulated EndMT and pro-fibrotic phenotype of ECs. These effects are related to the downregulation of Smad2/3.
Conclusions:
The study shows the pleiotropic effect of OA-NO2 on regulating EC-macrophage interactions during the immune response and suggests a role for OA-NO2 in the regulation of vascular endothelial immune and fibrotic responses arising during chronic inflammation.
General significance:
These findings propose the OA-NO2 may be useful as a novel therapeutic agent for treatment of cardiovascular disorders associated with dysregulation of the endothelial immune response.
... fMLP activation of PMNs also induced Src activation and Src-dependent phosphorylation of Cav-1 in RLMVEC monolayers, and these effects were In the rat lung preparation, we observed that administration of anti-ICAM-1 blocking mAb (1A29), but not the isotypematched control mAb, significantly reduced PMN activationdependent increase in pulmonary vascular 125 I-albumin PS product ( Figure 6F). Using the ICAM-1 crosslinking method in endothelial cells, 29,41 we observed activation of Src and Cav-1 phosphorylation ( Figure 7A and supplemental Figure IIC). Neither Src activation nor Cav-1 phosphorylation was seen following treatment with anti-ICAM-1 mAb, control mouse IgG plus antimouse IgG (FabЈ) 2 (secondary Ab), or secondary Ab IgG (FabЈ) 2 alone ( Figure 7A and supplemental Figure IIC). ...
... We also observed that anti-ICAM-1 blocking mAb prevented the PMN activation-induced increase in 125 I-albumin endocytosis and transcytosis in endothelial cells. To address whether ICAM-1 may be directly responsible for signaling, we crosslinked cell surface ICAM-1 29,41 to induce clustering and observed activation of Src and Cav-1 phosphorylation. Moreover, ICAM-1 crosslinking increased albumin endocytosis and transendothelial albumin permeability, thus mimicking the effects of PMN activation of endothelial cells. ...
... In addition to its role in actin cytoskeleton rearrangements, crosslinking of ICAM-1 with monoclonal antibodies has also been shown to activate pro-inflammatory cascades, via activation of MAPK kinases ERK-1/2 and/or JNK (Etienne et al., 1998;Lawson et al., 1999;Sano et al., 1998). Activation of ERK-1 leads to AP-1 activation (Lawson et al., 1999) and ERK-dependent production and secretion of IL-8 and RANTES (Sano et al., 1998), expression of VCAM-1 expression on the cell surface (Lawson et al., 1999;Lawson et al., 2001). ...
... In addition to its role in actin cytoskeleton rearrangements, crosslinking of ICAM-1 with monoclonal antibodies has also been shown to activate pro-inflammatory cascades, via activation of MAPK kinases ERK-1/2 and/or JNK (Etienne et al., 1998;Lawson et al., 1999;Sano et al., 1998). Activation of ERK-1 leads to AP-1 activation (Lawson et al., 1999) and ERK-dependent production and secretion of IL-8 and RANTES (Sano et al., 1998), expression of VCAM-1 expression on the cell surface (Lawson et al., 1999;Lawson et al., 2001). ICAM-1 cross-linking also upregulates tissue factor production (Schmid et al., 1995) and expression of proinflammatory cytokines including IL-1β (Koyama et al., 1996). ...
Background:
Scleroderma (SSc) is a complex autoimmune disorder that can be characterised by the presence 2of circulating autoantibodies to nuclear, cytoplasmic and cell surface antigens. In particular antibodies directed against endothelial cell antigens (anti-endothelial cell antibodies; AECA) have been detected. ICAM-1 is an adhesion molecule expressed on the surface of human endothelial cells. We have previously shown that cross-linking ICAM-1 with monoclonal antibodies leads to pro-inflammatory activation of human endothelial and vascular smooth muscle cells and that cardiac transplant recipients with transplant associated vasculopathy make antibodies directed against ICAM-1.
Objectives:
To determine whether SSc patients make antibodies directed against ICAM-1 and whether these antibodies induce pro-inflammatory activation of human endothelial cells in vitro.
Methods:
Using recombinant ICAM-1 as capture antigen, an ELISA was developed to measure ICAM-1 antibodies in sera from SSc patients. Antibodies were purified using ICAM-1 micro-affinity columns. HUVEC were incubated with purified anti-ICAM-1 antibodies and generation of reactive oxygen species, and expression of VCAM-1 was measured.
Results:
Significantly elevated levels of anti-ICAM-1 antibodies were detected in patients with diffuse (dSSc; 10/31 32%) or limited (lSSc; 14/36 39%) scleroderma. Cross-linking of HUVEC with purified anti-ICAM-1 antibodies caused a significant increase in ROS production (2.471±0.408 fold increase above untreated after 150 min p<0.001), and significant increase in VCAM-1 expression (10.6±1.77% vs 4.12±1.33%, p<0.01).
Conclusion:
AECA from SSc patients target specific endothelial antigens including ICAM-1, and cause pro-inflammatory activation of human endothelial cells, suggesting that they are not only a marker of disease but that they contribute to its progression.
... ICAM-1 was also con�rmed to transduce signals �outside in" [63,64]. e cross-linking of ICAM-1 with monoclonal antibodies was reported to activate the mitogen-activated protein kinase (MAPK) kinases ERK-1/2 and/or JNK [65][66][67]. e activation of ERK-1 lead to AP-1 activation [66], the ERK-dependent production and secretion of IL-8 and RANTES [67], and upregulation of VCAM-1 on the cell surface [66,68]. ICAM-1 cross-linking can also upregulate tissue factor production [69] and proin�ammatory cytokines, including IL-1 [70]. ...
... e cross-linking of ICAM-1 with monoclonal antibodies was reported to activate the mitogen-activated protein kinase (MAPK) kinases ERK-1/2 and/or JNK [65][66][67]. e activation of ERK-1 lead to AP-1 activation [66], the ERK-dependent production and secretion of IL-8 and RANTES [67], and upregulation of VCAM-1 on the cell surface [66,68]. ICAM-1 cross-linking can also upregulate tissue factor production [69] and proin�ammatory cytokines, including IL-1 [70]. ...
Autoantibodies against integral membrane proteins are usually pathogenic. Although anti-endothelial cell antibodies (AECAs) are considered to be critical, especially for vascular lesions in collagen diseases, most molecules identified as autoantigens for AECAs are localized within the cell and not expressed on the cell surface. For identification of autoantigens, proteomics and expression library analyses have been performed for many years with some success. To specifically target cell-surface molecules in identification of autoantigens, we constructed a serological identification system for autoantigens using a retroviral vector and flow cytometry (SARF). Here, we present an overview of recent research in AECAs and their target molecules and discuss the principle and the application of SARF. Using SARF, we successfully identified three different membrane proteins: fibronectin leucine-rich transmembrane protein 2 (FLRT2) from patients with systemic lupus erythematosus (SLE), intercellular adhesion molecule 1 (ICAM-1) from a patient with rheumatoid arthritis, and Pk (Gb3/CD77) from an SLE patient with hemolytic anemia, as targets for AECAs. SARF is useful for specific identification of autoantigens expressed on the cell surface, and identification of such interactions of the cell-surface autoantigens and pathogenic autoantibodies may enable the development of more specific intervention strategies in autoimmune diseases.
... Further signalling components initiated by ICAM-1 crosslinking on brain microvascular EC are intracellular calcium release and activation of protein kinase C, which are thought to lead to phosphorylation of cortactin (Etienne- Manneville, Manneville et al. 2000). MAPK-kinase pathways ERK-1/2 and/or JNK have been shown to the triggered depending on experimental design and cell type (Etienne, Adamson et al. 1998; Sano, Nakagawa et al. 1998; Lawson, Ainsworth et al. 1999; Lawson, Ainsworth et al. 2001). We have demonstrated that AP-1 activation is the result of ERK-1 activation (Lawson, Ainsworth et al. 1999), together with the ERK-dependent production and secretion of IL-8 and RANTES (Sano, Nakagawa et al. 1998) as well as upregulation of VCAM-1 on the cell surface (Lawson, Ainsworth et al. 1999; Lawson, Ainsworth et al. 2001). ...
... MAPK-kinase pathways ERK-1/2 and/or JNK have been shown to the triggered depending on experimental design and cell type (Etienne, Adamson et al. 1998; Sano, Nakagawa et al. 1998; Lawson, Ainsworth et al. 1999; Lawson, Ainsworth et al. 2001). We have demonstrated that AP-1 activation is the result of ERK-1 activation (Lawson, Ainsworth et al. 1999), together with the ERK-dependent production and secretion of IL-8 and RANTES (Sano, Nakagawa et al. 1998) as well as upregulation of VCAM-1 on the cell surface (Lawson, Ainsworth et al. 1999; Lawson, Ainsworth et al. 2001). Recently we have also shown that patient auto-reactive antibodies against ICAM-1 can also cause endothelial cell signalling (Lawson, Holder et al. 2005). ...
... ECs play a fundamental role in leukocyte migration. ICAM-1 triggers a EC calcium flux and protein kinase C (PKC), while VCAM-1 activates release of EC intracellular calcium, calcium channels and Rac-1 which generate EC NADPH oxidase activity [73]. In turn, NADPH oxidase activity produces low amounts of H 2 O 2 which activate EC-associated matrix metalloproteinase (MMPs) which are required for VCAM-1-dependent lymphocyte migration [73]. ...
... ICAM-1 triggers a EC calcium flux and protein kinase C (PKC), while VCAM-1 activates release of EC intracellular calcium, calcium channels and Rac-1 which generate EC NADPH oxidase activity [73]. In turn, NADPH oxidase activity produces low amounts of H 2 O 2 which activate EC-associated matrix metalloproteinase (MMPs) which are required for VCAM-1-dependent lymphocyte migration [73]. ...
The continuous antigenic triggering has greatly contributed to the evolution of the immune system and, therefore, animals have developed cells able to cope with a broad variety of microbial antigens and or their toxins, e.g., endotoxins. At the same time, intestinal commensals have evolved along with human progress and introduction of new foods, thus empowering both regional and systemic immune response. In this review, some important steps in the evolution of the immune system will be analyzed such as organization of lymphoid organs, formation of germinal centers, leukocyte attraction to tissue function of Toll like receptors and role of antimicrobial peptides. In particular, the major phylogenetic acquisitions of living organisms in the assessment of the immune machinery will be emphasized. Finally, fish will be taken into consideration as animal models of human diseases also in view of drug design strategies. Special attention will be focused on vaccinated salmon and zebrafish models.
... Furthermore, it was suggested that SHP-2 recruitment to PECAM-1 might dominate over the recruitment of ligands to ICAM-1 due to the lack of a conserved protein binding site on ICAM-1's cytoplasmic domain, which results in a reduced binding affinity of SHP-2 to ICAM-1 [12]. Depending on the experimental design and cell type investigated, the cross-linking of ICAM-1 with monoclonal antibodies may activate the MAPK kinases ERK-1/2 and/or JNK [17, 41, 76]. The activation of ERK-1 leads to AP-1 activation [41] , and the ERKdependent production and secretion of IL-8 and RAN- TES [76], as well as upregulation of VCAM-1 on the cell surface [41, 42] (Fig. 3). ...
... Depending on the experimental design and cell type investigated, the cross-linking of ICAM-1 with monoclonal antibodies may activate the MAPK kinases ERK-1/2 and/or JNK [17, 41, 76]. The activation of ERK-1 leads to AP-1 activation [41] , and the ERKdependent production and secretion of IL-8 and RAN- TES [76], as well as upregulation of VCAM-1 on the cell surface [41, 42] (Fig. 3). ICAM-1 cross-linking can also upregulate tissue factor production [78] and proinflammatory cytokines including IL-1b [37]. ...
Intercellular adhesion molecule-1 (ICAM-1; CD54) is a 90 kDa member of the immunoglobulin (Ig) superfamily and is critical for the firm arrest and transmigration of leukocytes out of blood vessels and into tissues. ICAM-1 is constitutively present on endothelial cells, but its expression is increased by proinflammatory cytokines. The endothelial expression of ICAM-1 is increased in atherosclerotic and transplant-associated atherosclerotic tissue and in animal models of atherosclerosis. Additionally, ICAM-1 has been implicated in the progression of autoimmune diseases. We and others have shown that the ligation of ICAM-1 on the surface of endothelial or smooth muscle cells with monoclonal antibodies, via its main leukocyte ligand, lymphocyte function associated molecule (LFA)-1, or with antibodies derived from patient serum, leads to the activation of several proinflammatory signaling cascades, and to the rearrangement of the actin cytoskeleton. A circulating or soluble form of ICAM-1 (sICAM-1) has been measured in various body fluids, with elevated levels being observed in patients with atherosclerosis, heart failure, coronary artery disease and transplant vasculopathy. sICAM-1 has signaling properties in several cell types, including EC, and invokes a range of proinflammatory responses. Thus, we propose that in addition to acting as a leukocyte adhesion molecule, ICAM-1 directly contributes to inflammatory responses within the blood vessel wall by increasing endothelial cell activation and augmenting atherosclerotic plaque formation.
... Together, these studies unveil ROS/Src/MEKK(3/6)/ p38 MAPK/ HSP27 and ROS/Src/cytoskeleton-associated proteins (Fig. 4) as important signaling cascades that are activated by PMN adhesion to mediate the cytoskeletal changes and subsequent PMN TEM. Ligation of ICAM-1 can also cause activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and/or c-Jun N terminal kinase (JNK) depending on the experimental system (89,134,154). Additionally, ICAM-1 ligation promotes RhoA activation and the formation of actin stress fibers (2, 45) (Fig. 4). ...
... Pretreatment of cells with MEK inhibitor PD98059 inhibited expression of VCAM-1 in response to ICAM-1 cross-linking, suggesting that the ERK pathway is involved in ICAM-1-mediated VCAM-1 expression. Activation of ERK1/2 is also required for the production of IL-8 and RANTES (134) and expression of ICAM-1 (35) in response to ICAM-1 cross-linking. In addition, ICAM-1 ligation induces the transcription of c-fos and rhoA expression in endothelial cells (153) (Fig. 4). ...
Stable adhesion of leukocytes to endothelium is crucial for transendothelial migration (TEM) of leukocytes evoked during inflammatory responses, immune surveillance, and homing and mobilization of hematopoietic progenitor cells. The basis of stable adhesion involves expression of intercellular adhesion molecule-1 (ICAM-1), an inducible endothelial adhesive protein that serves as a counter-receptor for beta(2)-integrins on leukocytes. Interaction of ICAM-1 with beta(2)-integrins enables leukocytes to adhere firmly to the vascular endothelium and subsequently, to migrate across the endothelial barrier. The emerging paradigm is that ICAM-1, in addition to firmly capturing leukocytes, triggers intracellular signaling events that may contribute to active participation of the endothelium in facilitating the TEM of adherent leukocytes. The nature, duration, and intensity of ICAM-1-dependent signaling events may contribute to the determination of the route (paracellular vs. transcellular) of leukocyte passage; these aspects of ICAM-1 signaling may in turn be influenced by density and distribution of ICAM-1 on the endothelial cell surface, the source of endothelial cells it is present on, and the type of leukocytes with which it is engaged. This review summarizes our current understanding of the "ICAM-1 paradigm" of TEM with an emphasis on the signaling events mediating ICAM-1 expression and activated by ICAM-1 engagement in endothelial cells.
... In cultured cells (e.g., endothelial cells and leukocytes) [13,14], ligation of membrane ICAM-1 activates transcription factors (e.g., AP-1 and FAST-1) [30,31], membrane receptors (e.g., IGF-1R and HGFR) [31], nonreceptor kinases (e.g., Src family) [32], signaling molecules (e.g., MAPK, Akt, and Rho GTPases) [33][34][35][36], and cytoskeletal-associated proteins (e.g., focal adhesion kinase and paxillin) [34]. Ligation of membrane ICAM-1 also increases expression of early response genes (e.g., c-fos) [37], cytokines (e.g., IL-1ß and MIP-1α) [30,38,39], and adhesion molecules (e.g., VCAM-1 and ICAM-1) [40,41]. Soluble ICAM-1, which is generated through proteolytic cleavage of membrane ICAM-1 and alternative splicing of the ICAM-1 gene, has also been reported to enhance macrophage chemotaxis and cytokine production [42][43][44]. ...
The local inflammatory environment of injured skeletal muscle contributes to the resolution of the injury by promoting the proliferation of muscle precursor cells during the initial stage of muscle regeneration. However, little is known about the extent to which the inflammatory response influences the later stages of regeneration when newly formed (regenerating myofibers) are accumulating myonuclei and undergoing hypertrophy. Our prior work indicated that the inflammatory molecule ICAM-1 facilitates regenerating myofiber hypertrophy through a process involving myonuclear positioning and/or transcription. The present study tested the hypothesis that ICAM-1 enhances global transcription within regenerating myofibers by augmenting the transcriptional activity of myonuclei positioned in linear arrays (nuclear chains). We found that transcription in regenerating myofibers was ~2-fold higher in wild type compared with ICAM-1-/- mice at 14 and 28 days post-injury. This occurred because the transcriptional activity of individual myonuclei in nuclei chains, nuclear clusters, and a peripheral location were ~2-fold higher in wild type compared with ICAM-1-/- mice during regeneration. ICAM-1’s enhancement of transcription in nuclear chains appears to be an important driver of myofiber hypertrophy as it was statistically associated with an increase in myofiber size during regeneration. Taken together, our findings indicate that ICAM-1 facilitates myofiber hypertrophy after injury by enhancing myonuclear transcription.
... Intercellular adhesion molecule 1 (ICAM1; CD54) is a representative ligand of integrin that is key to mediating leukocyte adhesion to the endothelial cell surface 40 . ICAM1-mediated endothelial chemokines attract and activate leukocytes, leading to a severe inflammatory response 41 . Silvia Dragoni et al. proved that ICAM-1-mediated intra-endothelial signaling plays a critical role in regulating lymphocyte transendothelial migration and modulating vascular permeability, thereby propagating chronic endothelial inflammation 42 . ...
This investigation seeks to dissect coronary artery disease molecular target candidates along with its underlying molecular mechanisms. Data on patients with CAD across three separate array data sets, GSE66360, GSE19339 and GSE97320 were extracted. The gene expression profiles were obtained by normalizing and removing the differences between the three data sets, and important modules linked to coronary heart disease were identified using weighted gene co-expression network analysis (WGCNA). Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and genomes (KEGG) pathway enrichment analyses were applied in order to identify statistically significant genetic modules with the Database for Annotation, Visualization and Integrated Discovery (DAVID) online tool (version 6.8; http://david.abcc.ncifcrf.gov). The online STRING tool was used to construct a protein–protein interaction (PPI) network, followed by the use of Molecular Complex Detection (MCODE) plug-ins in Cytoscape software to identify hub genes. Two significant modules (green-yellow and magenta) were identified in the CAD samples. Genes in the magenta module were noted to be involved in inflammatory and immune-related pathways, based on GO and KEGG enrichment analyses. After the MCODE analysis, two different MCODE complexes were identified in the magenta module, and four hub genes (ITGAM, degree = 39; CAMP, degree = 37; TYROBP, degree = 28; ICAM1, degree = 18) were uncovered to be critical players in mediating CAD. Independent verification data as well as our RT-qPCR results were highly consistent with the above finding. ITGAM, CAMP, TYROBP and ICAM1 are potential targets in CAD. The underlying mechanism may be related to the transendothelial migration of leukocytes and the immune response.
... To our knowledge, this is the first report on upregulated RANTES expression in uremic conditions in vitro. RANTES is a well-established mediator of atherogenic processes [38]. Moreover, the importance of RANTES in renal disease was established in a study of renal transplants undergoing rejection [39], since rejecting grafts exhibited large amounts of RANTES bound to the vascular endothelium. ...
Deletion polymorphism of glutathione S-transferase M1 (GSTM1), a phase II detoxification and antioxidant enzyme, increases susceptibility to end-stage renal disease (ESRD) as well as the development of cardiovascular diseases (CVD) among ESRD patients and leads to their shorter cardiovascular survival. The mechanisms by which GSTM1 downregulation contributes to oxidative stress and inflammation in endothelial cells in uremic conditions have not been investigated so far. Therefore, the aim of the present study was to elucidate the effects of GSTM1 knockdown on oxidative stress and expression of a panel of inflammatory markers in human umbilical vein endothelial cells (HUVECs) exposed to uremic serum. Additionally, we aimed to discern whether GSTM1-null genotype is associated with serum levels of adhesion molecules in ESRD patients. HUVECs treated with uremic serum exhibited impaired redox balance characterized by enhanced lipid peroxidation and decreased antioxidant enzyme activities, independently of the GSTM1 knockdown. In response to uremic injury, HUVECs exhibited alteration in the expression of a series of inflammatory cytokines including retinol-binding protein 4 (RBP4), regulated on activation, normal T cell expressed and secreted (RANTES), C-reactive protein (CRP), angiogenin, dickkopf-1 (Dkk-1), and platelet factor 4 (PF4). GSTM1 knockdown in HUVECs showed upregulation of monocyte chemoattractant protein-1 (MCP-1), a cytokine involved in the regulation of monocyte migration and adhesion. These cells also have shown upregulated intracellular and vascular cell adhesion molecules (ICAM-1 and VCAM-1). In accordance with these findings, the levels of serum ICAM-1 and VCAM-1 (sICAM-1 and sVCAM-1) were increased in ESRD patients lacking GSTM1, in comparison with patients with the GSTM1-active genotype. Based on these results, it may be concluded that incubation of endothelial cells in uremic serum induces redox imbalance accompanied with altered expression of a series of cytokines involved in arteriosclerosis and atherosclerosis. The association of GSTM1 downregulation with the altered expression of adhesion molecules might be at least partly responsible for the increased susceptibility of ESRD patients to CVD.
1. Introduction
Endothelial dysfunction is an underlying mechanism of cardiovascular diseases (CVD) which are the leading cause of death among patients with end-stage renal disease (ESRD) [1–4]. The vascular endothelium is likely the primary target of uremic toxins generated in ESRD. In these conditions, the endothelium is continuously exposed to accumulated uremic toxins hence inducing oxidative stress and inflammation, which can lead to endothelial impairment [2, 5–7].
Homozygous deletion of glutathione S-transferase M1 (GSTM1), which is a phase II detoxification enzyme, leads to accumulation of oxidative stress byproducts which indicates its role in antioxidant protection as well [8]. Between 30 to 50 percent of different human population are homozygous for GSTM1 deletion (usually denoted as GSTM1-null genotype), thus completely lacking the GSTM1 enzyme [9]. The GSTM1 deficiency is linked to higher susceptibility to CVD as individuals with GSTM1-null genotype were shown to have significantly increased risk for developing resistant hypertension [10], coronary artery disease/atherosclerosis [11, 12], and stroke [13, 14]. In addition, the GSTM1-null genotype increases susceptibility to ESRD and leads to shorter overall and cardiovascular survival of the patients on haemodialysis [8, 15–17]. The association between GSTM1 deletion and oxidative stress in ESRD patients was supported by our previous results that demonstrated elevated levels of several byproducts of protein and lipid oxidative damage in ESRD patients with GSTM1-null genotype compared to those with GSTM1-active genotype [8]. It has been suggested that in inflammatory and prooxidant environment observed in patients with ESRD, the endothelium responds by expressing intracellular and vascular cell adhesion molecules (ICAM-1 and VCAM-1) that facilitate migration and adhesion of leukocytes to the endothelial cells [18]. We have recently shown that soluble ICAM-1 and VCAM-1 (sICAM-1 and sVCAM-1) levels have a strong predictive role in terms of overall and cardiovascular survival in ESRD patients on haemodialysis [17]. Nevertheless, the potential influence of GSTM1 deletion on the aforementioned inflammatory markers has not been elucidated before in dialyzed patients or in endothelial cells exposed to uremic toxins commonly present in ESRD.
Despite the convincing findings in human cohorts showing the importance of GSTM1 deletion on susceptibility and development of CVD among dialyzed patients [15, 16, 19, 20], its role in the vascular pathophysiology has not been established yet. A functional role of GSTM1 was investigated in vascular smooth muscle cells (VSMC), showing that the reduction in GSTM1 expression in these cells caused increased cell proliferation, oxidative stress, and migration [21]. However, the mechanisms associated with GSTM1 downregulation in endothelial cells in uremic conditions have not been investigated so far.
Therefore, the aim of the present study was to elucidate the effects of GSTM1 knockdown on oxidative stress and expression of a panel of inflammatory markers in human umbilical vein endothelial cells (HUVECs) exposed to uremic serum. Additionally, we aimed to discern whether the GSTM1-null genotype is associated with serum levels of adhesion molecules in ESRD patients.
2. Materials and Methods
2.1. Cell Cultures
HUVECs (ATCC Manassas, Virginia, USA) were seeded on 0.2% gelatine-coated culture plates and grown in a MV2 growth medium (Endothelial Cell Growth Medium MV2, PromoCell, Germany) under standard cell culture conditions (humidified atmosphere, 5% CO2, 37°C). Cells were seeded in gelatine-coated plates for viability assays, Western blot analyses, oxidative stress measurements, and assessment of cytokine expression. HUVECs were incubated in a pool of human serums obtained from either healthy volunteers (control serum, ) or ESRD patients on haemodialysis (uremic serum, ). All patients underwent haemodialysis 3 times a week for at least 3 months before the study onset using polysulfone dialysis membranes and conventional bicarbonate-buffered dialysate. Participants with any form of malignancy, autoimmune disease, or infectious comorbidities (HIV, HBV, or HCV infections) were excluded. The blood was taken from patients prior to a haemodialysis session at the Center for the Renal Diseases, Zvezdara University Medical Center, Belgrade. All patients signed informed consent agreeing to participate in the study. This study was approved by the Ethical Committee of the Faculty of Medicine, University of Belgrade (No. 1550/V-30), and conducted in accordance with the Helsinki Declaration from 2013.
2.2. Cell Viability Assay
Cell viability was assessed by a colorimetric method based on measuring mitochondrial dehydrogenase activity, using the MTS Cell Proliferation Assay Kit (Abcam, UK), according to the manufacturer’s protocol. 5000 cells/well were cultured in a 96-well plate. After 24 h, growth media were discarded and cells were treated with media, 10%, 20%, or 30% control or uremic serum for 4 h and 6 h. The formazan salt produced by viable cells was quantified by measuring the absorbance at 490 nm on the FLUOstar® Omega plate reader (BMG Labtech, Germany). The viability of cells incubated in pooled human sera for 6 h did not change significantly compared to the cells incubated in the normal growth medium (Figure 1S A&B, Supplement). Therefore, all further HUVEC treatments were performed using 30% control or uremic serum-containing media for 6 h, before cytokine expression and oxidative stress measurements were performed.
2.3. GSTM1 Knockdown Using siRNA in HUVECs
To silence GSTM1 protein expression, HUVECs were seeded at a density of cells per well in 6-well plates and grown in a MV2 growth medium. The following day, cells were transfected with 100 nM GSTM1 small interfering RNA (siRNA) (Thermo Fisher Scientific, UK) using the DharmaFECT transfection reagent (GE Healthcare, USA) or treated with DharmaFECT as a control. Ninety-six hours posttransfection, the silencing effect was confirmed by Western blot.
2.4. Western Blot Analysis
HUVECs were lysed in a RIPA buffer (50 mM Tris-HCL pH 8.0, 150 mM NaCl, 1% IGEPAL, 0.5% sodium deoxycholate, and 10% SDS) supplemented with protease inhibitor cocktail (Roche, UK). After extraction, protein concentrations were determined by the Bicinchoninic Acid Protein Assay kit (BCA, Thermo Fisher Scientific, UK). An equal amount of proteins was loaded on 10% polyacrylamide gel, and electrophoresis was performed at 80 V for 10 min, then 100 V for 90 min. After wet transfer, nitrocellulose membranes were blocked in 5% nonfat milk (Bio-Rad, UK) for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies: monoclonal mouse anti-GSTM1 (1 : 1000, R&D Systems, USA), monoclonal mouse anti-ICAM1 (1 : 200, Santa Cruz, USA), polyclonal goat anti-VCAM1 (1 : 200, Santa Cruz, USA), and monoclonal mouse anti-β actin (1 : 10 000, Thermo Fisher Scientific, UK). The following day, membranes were incubated with appropriate HRP-conjugated secondary antibodies: anti-mouse 1 : 5000 (Abcam, UK) and anti-goat 1 : 1000 (RayBiotech, USA) for 1 h at room temperature. Chemiluminescent bands were detected using the West Femto Maximum sensitivity substrate (Thermo Fisher Scientific, UK) on the G-box (Kodak, UK) or ChemiDoc (BioRad, USA). Densitometry analysis was performed using ImageJ software (National Institutes of Health, Bethesda, USA).
2.5. Measurement of the Activity of Antioxidant Enzymes in HUVECs
The activity of antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GPX), in cell lysates was determined by spectrophotometric methods. SOD activity was assessed as previously described by Misra and Fridovich [22]. This method is based on the ability of SOD to inhibit the autoxidation of adrenaline at pH 10.2. The production of colored adrenochrome in reaction mixtures with cell protein extracts (sample) or without them (control) was followed at 480 nm. Activity of SOD was expressed as the percentage of inhibition of adrenaline autoxidation. GPX activity was assessed as reported by Günzler et al. [23]. GPX activity was assayed by the subsequent oxidation of NADPH at 340 nm with t-butyl-hydroperoxide as the substrate. One unit of enzyme activity was expressed as nmol NADPH oxidized per minute, assuming /l/mol/cm to be the molar absorbance of NADPH at 340 nm.
2.6. Measurement of Malondialdehyde Levels in HUVECs
Malondialdehyde (MDA) levels in cell lysates were measured using the competitive ELISA kit (Elabscience, Wuhan, China) in accordance with the manufacturer’s instructions. In brief, 50 μl of standards, samples, and blanks was added to each well of the MDA precoated ELISA plate with consecutive addition of 50 μl biotinylated antibody. After the 45 min of incubation, wells were washed in order to eliminate excess conjugate and unbound sample or standard, and HRP-conjugated antibody was added. The color change was measured spectrophotometrically at a wavelength of 450 nm.
2.7. Measurement of Total Reactive Oxygen Species in HUVECs
The total reactive oxygen species (ROS) production was assessed by flow cytometry (FACS) using 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA; Invitrogen, California, USA) stain. HUVECs were seeded in 6-well plates ( cells/well) and transfected with GSTM1 siRNA as described above. After 90 h incubation, the transfection solution was discarded, and cells were incubated for the next 6 h with the 30% control or uremic serum-containing media. Treatments were removed, and cells were trypsinised. Cell pellets were resuspended in 5 ml flow cytometry buffer (1% FBS in PBS) and incubated with 5 μl H2DCFDA stain for 30 min at 37°C. FACS tubes were centrifuged at 400 g for 8 min, supernatants were removed, and cells were allowed to recover for 15 min at 37°C in 1 ml of MV2 growth media. In the final step, cells were resuspended in 500 μl FACS buffer, and 5 μl 7-AAD-viability staining solution (eBioscience, San Diego, USA) was added prior to performing measurements on the Attune NxT Acoustic Focusing Flow Cytometer (Invitrogen, California, USA). The results were analysed using FlowJo, ver. 10.4 (Stanford Jr. University, USA).
2.8. Analysis of Cytokine Expression in HUVECs
To explore the effect of uremic serum and GSTM1 silencing on endothelial cell inflammation, the relative expression of 105 cytokines was assessed simultaneously in cell protein extracts using the Proteome Profiler™ Human XL Cytokine Array Kit (R&D Systems, UK) according to the manufacturer’s instruction. HUVECs were seeded in 6-well Petri dishes at a density of cells per well. Following the transfection, GSTM1+/+ and GSTM1+/- cells were incubated in 30% control or uremic serum-containing media for 6 h. After the incubation time expired, treatments were removed and cells were rinsed with PBS. Cells were then scraped in a lysis buffer 17 (R&D Systems, UK), supplemented with 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 10 μg/ml pepstatin. Cell lysates were obtained after centrifugation at 14 000 g for 5 min. Pooled cell lysates (/group) were probed on four separate nitrocellulose membranes. Each membrane contained capture and control antibodies spotted in duplicate, which allowed simultaneous measuring of 105 cytokine expressions. Chemiluminescent spots were visualised on the G-box (Kodak, UK). Results were quantified using HLimage++ software and normalized to the reference spots positioned at three of the corners of each blot (Western Vision Software, US).
2.9. Analysis of Circulating Adhesion Molecules in Plasma of ESRD Patients
Circulating adhesion molecules were determined in plasma of ESRD patients by enzyme immunoassays, according to the manufacturer’s protocols. The description of a cohort of 199 ESRD patients involved in this study has been described in details previously [17]. sICAM-1 was assayed by commercial solid-phase sandwich ELISA (Thermo Fisher Scientific, Waltham, Massachusetts, USA). sVCAM-1 was determined by a solid-phase sandwich ELISA kit (Novex, Life Technologies). Absorbances were read at 450 nm on the LKB 5060-006 Micro Plate Reader (Vienna, Austria). sICAM concentrations were expressed as pg/ml, and sVCAM levels were expressed as ng/ml.
3. Statistical Analysis
Statistical analysis was performed using the SPSS version 17.0 statistical package (IBM SPSS Statistics). All analysed parameters were tested for normality of the data using the Shapiro-Wilk test. For normally distributed data, differences between the groups were evaluated by the independent sample -test or one-way ANOVA with Fisher’s least significant difference (LSD) post hoc. For non-normally distributed data, the Mann-Whitney or Kruskal-Wallis test was used. The results were considered statistically significant if .
4. Results
To determine the effects of GSTM1 expression on oxidative stress and expression of a panel of inflammatory markers, we used specific siRNA to silence GSTM1 gene in HUVECs. Following 96 h of transfection, diminished GSTM1 expression was confirmed by immunoblotting which showed around ~90% reduction in GSTM1 protein levels in HUVECs treated with GSTM1 siRNA (GSTM1+/-) compared to the control (GSTM1+/+) (; Figure 2S, Supplement).
4.1. The Influence of Uremic Serum and GSTM1 Knockdown on Biomarkers of Oxidative Stress (SOD, GPX, MDA, and ROS) in HUVECs
Antioxidant enzyme activity and byproducts of oxidative stress were assessed in GSTM1+/+ and GSTM1+/- HUVECs incubated in control or uremic serum-containing media. The incubation of HUVECs with uremic serum led to a significant decrease in the activity of SOD and GPX antioxidant enzymes in GSTM+/+ HUVECs compared to control serum conditions (; Figures 1(a) and 1(b)). To determine the extent of GSTM1 activity loss on redox status of HUVECs, we silenced the GSTM1 gene by corresponding siRNA. Silencing of the GSTM1 gene did not affect antioxidant enzyme activity in any of the observed settings (Figures 1(a) and 1(b)). With respect to oxidative stress byproducts, the exposure to the uremic serum led to the significantly higher MDA concentrations () in GSTM1+/+ HUVECs compared to their counterparts incubated in control serum (Figures 1(c) and 1(d)). However, the GSTM1 knockdown did not have statistically significant impact on total oxidative stress byproducts in HUVECs in either control or uremic serum (Figures 1(c) and 1(d)). Only a trend towards increased MDA levels was observed in GSTM1+/- HUVECs compared to GSTM1+/+ HUVECs in control serum ().
(a)
... These pathways correlate well with the above data demonstrating that SCF can induce chemokine production. Recent studies have associated production of specific chemokines with signal transduction pathways involving MAP kinases (70,71), thus closely associating the responses with one another. Preliminary data in our laboratory indicate that inhibitors of some of these pathways, such as protein tyrosine kinase and MAP kinase, block chemokine production from SCFstimulated mast cells. ...
... IVD2) (3,15,540,543,557). In addition, ICAM-1 engagement has a number of transcriptional effects through activation of MAPKs ERK, p38, and JNK (122,136,269,439). ...
A central function of the vascular endothelium is to serve as a barrier between the blood and the surrounding tissue of the body. At the same time, solutes and cells have to pass the endothelium to leave or to enter the bloodstream to maintain homeostasis. Under pathological conditions, for example, inflammation, permeability for fluid and cells is largely increased in the affected area, thereby facilitating host defense. To appropriately function as a regulated permeability filter, the endothelium uses various mechanisms to allow solutes and cells to pass the endothelial layer. These include transcellular and paracellular pathways of which the latter requires remodeling of intercellular junctions for its regulation. This review provides an overview on endothelial barrier regulation and focuses on the endothelial signaling mechanisms controlling the opening and closing of paracellular pathways for solutes and cells such as leukocytes and metastasizing tumor cells.
... A chelation of intracellular calcium or the inhibition of PKC in IFN- treated brain EC lines blocks ICAM-1 dependent lymphocyte migration without affecting lymphocyte adhesion (Etienne-Manneville et al., 2000). ICAM-1 stimulates ECs to produce chemokines through the activation of ERK1 and ERK2 (Sano et al., 1998) suggesting that ICAM-1 induces EC signals that are required for lymphocyte migration. ICAM-1 signals can also regulate the EC actin cytoskeletion. ...
Introduction
The vascular endothelium is a specific organ consisting of the endothelial cell (EC)
monolayer, weighting about 1 kg which makes a unique border between the circulating
blood and the underlying tissues. Because of its strategic location, the endothelium interacts
with cellular and neurohumoral mediators, thus controlling the vascular contractile state
and cellular composition. Endothelial cells (ECs) used to be considered a layer of ”nucleated
cellophane“, an inert lining to blood vessels, endowed with negative properties, and the
most important of which is its ability to act as a non-thrombogenic substrate for blood.
Further, the endothelium was thought to participate in tissue reactions as target for
injurious agents. Now the endothelium is recognized as a semipermeable barrier that
regulates the transfer of small and large molecules, a highly specialized, metabolically active
organ having vital metabolic, secretory, synthetic and immunologic functions. It performs
significant autocrine, paracrine and endocrine actions and exerts influences on smooth
muscle cells, platelets and peripheral leucocytes. Therefore, haemostasis, inflammatory
reactions and immunity involve close interactions between immunocompetent cells and
vascular endothelium.
ECs arise from hemangioblasts, blast-like bipotential cells (Choi et al., 1998). They can also
transdifferentiate into mesenchymal cells and intimal smooth muscle cells. It is known that
there is a marked phenotypic variation between ECs in different parts of the vascular
system. The cells from different location in the same person not only express different
surface antigens and receptors, but can generate different responses to the same stimulus.
Further, cells from the same part of the vasculature can have varied responses (Galley &
Webster, 2004).
The vascular endothelium senses mechanical stimuli, such as pressure and shear stress, and
hormonal stimuli, such as vasoactive substances. In respons, it releases agents that regulate
vasomotor function, trigger inflammatory processes and affect hemostasis. Among the
vasodilatory substances the endothelium produces are nitric oxide (NO), prostacyclin,
different endothelium derived hyperpolarizing factors and C-type natriuretic peptide.
Vasoconstricting molecules include endothelin-1 (ET-1), angiotensin II (Ang II),
thromboxane A2 and reactive oxygen species (ROS) (Endemann & Schiffrin, 2004).
Inflammatory modulators include NO, intercellular adhesion molecule-1 (ICAM-1), vascular
adhesion molecule-1 (VCAM-1), E-selectin, and NF-κB. The endothelium realizes the
modulation of hemostasis by releasing: plasminogen activator, tissue factor inhibitor, von
Advances in the Etiology, Pathogenesis and Pathology of Vasculitis
154
Willebrand factor, NO, prostacyclin, thromboxane A2, plasminogen/activator, inhibitor/1
and fibrinogen. The endothelium also contributes to mitogenesis, angiogenesis, vascular
permeability and fluid balance. The endothelium is indispensable for body homeostasis. An
controlled endothelium cell response is involved in many disease processes including
atherosclerosis, hypertension, pulmonary hypertension, sepsis and inflammatory
syndromes including vasculitis. These diseases are related to endothelial injury, dysfunction
and activation.
... Similarly, this cross-linking can lead to the induction of ICAM-1 and VCAM in endothelial cells and fibroblasts (Clayton et al., 1998). In addition, the ligation of ICAM-1 can lead to induction and secretion of RANTES and IL-8 from endothelial cells (Blaber et al., 2003;Clayton et al., 1998;Sano et al., 1998). ...
... The physiologic outcome of engagement of ICAM-1 by β2 integrins and the resultant cell activation depend, in part, upon the type of cells. Activation of ICAM-1 on endothelial cells might elicit increased production of cytokines, such as IL-8 and RANTES, or increased expression of adhesion molecules (including ICAM-1 and VCAM), leading to enhanced leukocyte trafficking [22]. Human AVICs express high levels of ICAM-1 in response to pro-inflammatory stimulation [7,23]. ...
Calcific aortic valve disease (CAVD) is a chronic inflammatory condition and affects a large number of elderly people. Aortic valve interstitial cells (AVICs) occupy an important role in valvular calcification and CAVD progression. While pro-inflammatory mechanisms are capable of inducing the osteogenic responses in AVICs, the molecular interaction between pro-inflammatory and pro-osteogenic mechanisms remains poorly understood. This study tested the hypothesis that intercellular adhesion molecule-1 (ICAM-1) plays a role in mediating pro-osteogenic factor expression in human AVICs. AVICs were isolated from normal human aortic valves and cultured in M199 medium. Treatment with leukocyte function-associated factor-1 (LFA-1, an ICAM-1 ligand) up-regulated the expression of bone morphogenetic protein-2 (BMP-2) and resulted in increased alkaline phosphatase activity and formation of calcification nodules. Pre-treatment with lipopolysaccharide (LPS, 0.05μg/ml) increased ICAM-1 levels on cell surfaces and exaggerated the pro-osteogenic response to LFA-1, and neutralization of ICAM-1 suppressed this response. Further, ligation of ICAM-1 by antibody cross-linking also up-regulated BMP-2 expression. Interestingly, LFA-1 elicited Notch1 cleavage and NF-κB activation. Inhibition of NF-κB markedly reduced LFA-1-induced BMP-2 expression, and inhibition of Notch1 cleavage with a γ-secretase inhibitor suppressed LFA-1-induced NF-κB activation and BMP-2 expression. Ligation of ICAM-1 on human AVICs activates the Notch1 pathway. Notch1 up-regulates BMP-2 expression in human AVICs through activation of NF-κB. The results demonstrate a novel role of ICAM-1 in translating a pro-inflammatory signal into a pro-osteogenic response in human AVICs and suggest that ICAM-1 on the surfaces of AVICs contributes to the mechanism of aortic valve calcification.
... Assembly of Crk, p130CAS and C3G [48] leads to the activation of JNK, which then translocates to the nucleus and induces the transcription of the c-jun gene. c-Fos and c-Jun proteins together form the activator protein complex-1 (AP-1), which controls expression of cytokines and genes encoding other proteins, and ICAM-1 and VCAM-1, in particular [22,49,50]. ICAM-1 molecules lacking the intracellular domain are not capable of activating Rho proteins upon cross-linking [51 ]. ...
IntroductionIntercellular Adhesion Molecule 1 (ICAM-1) Adhesion Molecules on the Surface of APC and Target CellsICAM-1 Structure and Topology on the Cell SurfaceICAM-1 as Co-stimulatory Ligand and ReceptorICAM-1-mediated SignalingRole of ICAM-1 in Endothelial Response to LeukocytesICAM-1 Association with Lipid RaftsMajor Histocompatability Complex (MHC) MHC MoleculesMolecular Associations of MHC-I MoleculesAssociation of MHC-I and ICAM-1Could APC and Target Cells Play an Active Role in Ag Presentation?Identical pMHCs are Clustered in the Same MicrodomainIdentical pMHC can be Recruited to the Same Microdomain During Target Cell-T Cell InteractionCo-clustering of MHC and Accessory MoleculesRole of CytoskeletonConclusion Adhesion Molecules on the Surface of APC and Target CellsICAM-1 Structure and Topology on the Cell SurfaceICAM-1 as Co-stimulatory Ligand and ReceptorICAM-1-mediated SignalingRole of ICAM-1 in Endothelial Response to LeukocytesICAM-1 Association with Lipid Rafts MHC MoleculesMolecular Associations of MHC-I MoleculesAssociation of MHC-I and ICAM-1Could APC and Target Cells Play an Active Role in Ag Presentation?Identical pMHCs are Clustered in the Same MicrodomainIdentical pMHC can be Recruited to the Same Microdomain During Target Cell-T Cell InteractionCo-clustering of MHC and Accessory MoleculesRole of Cytoskeleton
... We have previously demonstrated that following burn, aged animals have elevated circulating levels of pro-inflammatory cytokines and chemokines which likely promotes upregulation of ICAM-1. Moreover, human studies have revealed the cross-linking of ICAM-1 stimulates production of IL-8, a human neutrophil chemokine [71]. This upregulation in IL-8 may be to further enhance neutrophil chemoattraction, but may also aid in the neutrophil transmigratory response to apical chemokines. ...
We have previously demonstrated that aging is associated with prolonged pulmonary inflammation in a murine model of thermal injury. To further investigate these observations, we examined lung congestion, markers of neutrophil chemotaxis and adhesion, and lung endothelia responses in young and aged mice following burn injury. Analysis of lung tissue and bronchoalveolar lavage fluid 24 hours after injury revealed that more neutrophils accumulated in the lungs of aged mice (p<0.05), but did not migrate into the alveoli. We then sought to determine if accumulation of neutrophils in the lungs of aged mice was due to differences in the peripheral neutrophil pool or local changes within the lung. Following burn injury, aged mice developed a pronounced peripheral blood neutrophilia (p<0.05) in comparison to their younger counterparts. In aged animals, there was a reduced frequency and mean fluorescent intensity of neutrophil CXCR2 expression (p<0.05). Interestingly, in uninjured aged mice, peripheral blood neutrophils demonstrated elevated chemokinesis, or hyperchemokinesis, (p<0.05), but showed a minimal chemotatic response to KC. To determine if age impacts neutrophil adhesion molecules, we assessed CD62L and CD11b expression on peripheral blood neutrophils. No age-dependent difference in the frequency or mean fluorescent intensity of CD62L or CD11b was observed post-burn trauma. Examination of pulmonary vasculature adhesion molecules which interact with neutrophil selectins and integrins revealed that intracellular adhesion molecule-1 (ICAM-1) was elevated in aged mice at 24 hours after burn as compared to young mice (p<0.05). Overall, our data suggests that age-associated pulmonary congestion observed following burn injury may be due to differences in lung endothelial adhesion responses that are compounded by elevated numbers of hyperchemokinetic circulating neutrophils in aged mice.
... A chelation of intracellular calcium or the inhibition of PKC in IFN- treated brain EC lines blocks ICAM-1 dependent lymphocyte migration without affecting lymphocyte adhesion (Etienne-Manneville et al., 2000). ICAM-1 stimulates ECs to produce chemokines through the activation of ERK1 and ERK2 (Sano et al., 1998) suggesting that ICAM-1 induces EC signals that are required for lymphocyte migration. ICAM-1 signals can also regulate the EC actin cytoskeletion. ...
... It has been reported that there is increased activity of MAPKs with ligation of ICAM-1, VCAM-1 or E-selectin ligation in epithelial [39,40,41] and endothelial cells [12,42,43,44,45]. Adherence of neutrophils or ICAM-1 crosslinking activates ERK in endothelial cells [42,43,46,47,48], epithelial cells [39,40,49,50], and smooth muscle cells [13]. Moreover, ligation of E-selectin leads to shear-dependent ERK1/2 phosphorylation in HUVECs [12,44]. ...
During inflammation, adhesion molecules regulate recruitment of leukocytes to inflamed tissues. It is reported that vascular cell adhesion molecule-1 (VCAM-1) activates extracellular regulated kinases 1 and 2 (ERK1/2), but the mechanism for this activation is not known. Pharmacological inhibitors of ERK1/2 partially inhibit leukocyte transendothelial migration in a multi-receptor system but it is not known whether VCAM-1 activation of ERK1/2 is required for leukocyte transendothelial migration (TEM) on VCAM-1.
In this study, we identified a mechanism for VCAM-1 activation of ERK1/2 in human and mouse endothelial cells. VCAM-1 signaling, which occurs through endothelial cell NADPH oxidase, protein kinase Cα (PKCα), and protein tyrosine phosphatase 1B (PTP1B), activates endothelial cell ERK1/2. Inhibition of these signals blocked VCAM-1 activation of ERK1/2, indicating that ERK1/2 is activated downstream of PTP1B during VCAM-1 signaling. Furthermore, VCAM-1-specific leukocyte migration under physiological laminar flow of 2 dynes/cm(2) was blocked by pretreatment of endothelial cells with dominant-negative ERK2 K52R or the MEK/ERK inhibitors, PD98059 and U0126, indicating for the first time that ERK regulates VCAM-1-dependent leukocyte transendothelial migration.
VCAM-1 activation of endothelial cell NADPH oxidase/PKCα/PTP1B induces transient ERK1/2 activation that is necessary for VCAM-1-dependent leukocyte TEM.
... Es zeigte sich ferner im Tiermodel (Rattenkarotiden) für c-Jun eine Involvierung in die intimale Hyperplasie nach Angioplastie (Khachigian et al., 2002; Hu et al., 1997)., 1999) und durch TNFα (Ozes et al., 1999). NFκB ist ein Mediator der Proliferation von SMC (Hoshi et al., 2000) und scheint involviert zu sein in die proliferative Antwort in hSMC auf Thrombin (Nakajima et al., 1994) Prozesse (Kyriakis und Avruch, 2001), aber auch proliferative Prozesse in SMC (Zhao et al., 2002) beschrieben, wie z.B. die PDGF-induzierte Mitogenese von SMC (Ratte) (Yamaguchi et al., 2001).Lawson et al., 1999) und in hSMC (Lawson et al., 2001) und die Produktion von Chemokinen in EC (Sano et al., 1998). In Bezug auf die Interaktion von Fibrinogen mit ICAM-1 wurde über proliferative bzw. ...
Fibrinogen ist ein bedeutender, unabhängiger Risikofaktor für kardiovaskuläre Erkrankungen, welches zahlreiche prospektive epidemiologische Studien bestätigten. Fibrin(ogen) und seine Spaltprodukte bieten vielfache potentielle Pathomechanismen für das kardiovaskuläre Risiko, wie u.a. die Induktion der Mitogenese. Die Wirkung von intaktem Fibrinogen auf die Mitogenese humaner glatter Gefäßmuskelzellen (hSMC) wurde bisher nicht genau untersucht. In der vorliegenden Arbeit wurde der Effekt von löslichem Fibrinogen auf die Desoxyribonukleinsäure(DNA)-Synthese mittels (3H)-Thymidin-Inkorporation und die mitogenen Signaltransduktionswege mittels Western Blot in kultivierten hSMC der V. saphena untersucht. 10 µM Fibrinogen (physiologische Plasmakonzentration) induzierte die DNA-Synthese und die mitogene Signaltransduktion, wie die Phosphorylierung der Mitogen-aktivierten Proteinkinasen (MAPK) ERK(extrazelluär regulierte Kinasen)-1/2, Akt als Komponente des Phosphoinositol-3(PI3)-Kinase Signalweges, p38-MAPK und die Expression von c-Jun. Durch die Hemmung der Fibrinogen-induzierten DNA-Synthese nach Inhibition der MAP/ERK-Kinase-1 (MEK-1), der PI3-Kinase und der p70 S6-Kinase wurde eine wichtige Bedeutung des ERK-1/2 Signalweges und des PI3-Kinase Signalweges für die Fibrinogen-induzierte Mitogenese in hSMC bestätigt. Mittels blockierender Antikörper gegen das Intercellular adhesion molecule-1 (ICAM-1) und Blockade des AlphaVbeta3-Integrins durch Abciximab (Reopro) wurde nachgewiesen, daß diese Fibrinogenrezeptoren als Mediatoren der Fibrinogen-induzierten DNA-Synthese ausscheiden. Ferner zeigte sich eine Involvierung von ICAM-1 in die Fibrinogen-induzierte p38-MAPK- und Akt-Aktivierung, wodurch vermutlich andere zelluläre Reaktionen als die Mitogenese der hSMC vermittelt werden. Die nachgewiesenen promitogenen Effekte von Fibrinogen auf hSMC könnten zu einer gesteigerten Proliferation dieser Zellen im Rahmen atherosklerotischer Prozesse beitragen. Als weitere potentiell proatherogene Eigenschaft von Fibrinogen wurde die Induktion der Expression von Adhäsionsmolekülen beschrieben, die zur Rekrutierung von Leukozyten beitragen. In dieser Arbeit wurde die Wirkung von Fibrinogen auf die Expression der Rezeptoren ICAM-1 und AlphaVbeta3 an der Zelloberfläche der hSMC durchflußzytometrisch untersucht. Dabei wurde für beide Fibrinogenrezeptoren basal ein geringes Expressionsniveau beobachtet. Allerdings hatte Fibrinogen auf die Expression von ICAM-1 und AlphaVbeta3 keine Effekte, was möglicherweise methodisch bedingt ist, z.B. durch eine zu geringe Sensitivität der vorhandenen Meßmethoden.
... The expression of RANTES probably enhanced the expression of its receptors. Sano and colleagues demonstrated that ICAM-1 induced RANTES mRNA expression and also increased its protein synthesis and secretion by endothelial cells [31]. It is likely that the P. yoelii 17XL-induced RANTES production observed in the current study would attract and activate leukocytes towards inflammatory sites to mediate localized hyper-inflammatory responses that could exacerbate the disease pathology in the cerebellum. ...
Abstract
Background
Malaria afflicts 300–500 million people causing over 1 million deaths globally per year. The immunopathogenesis of malaria is mediated partly by co mplex cellular and immunomodulator interactions involving co-regulators such as cytokines and adhesion molecules. However, the role of chemokines and their receptors in malaria immunopathology remains unclear. RANTES (Regulated on Activation Normal T-Cell Expressed and Secreted) is a chemokine involved in the generation of inflammatory infiltrates. Recent studies indicate that the degradation of cell-cell junctions, blood-brain barrier dysfunction, recruitment of leukocytes and Plasmodium -infected erythrocytes into and occlusion of microvessels relevant to malaria pathogenesis are associated with RANTES expression. Additionally, activated lymphocytes, platelets and endothelial cells release large quantities of RANTES, thus suggesting a unique role for RANTES in the generation and maintenance of the malaria-induced inflammatory response. The hypothesis of this study is that RANTES and its corresponding receptors (CCR1, CCR3 and CCR5) modulate malaria immunopathogenesis. A murine malaria model was utilized to evaluate the role of this chemokine and its receptors in malaria.
Methods
The alterations in immunomodulator gene expression in brains of Plasmodium yoelii 17XL-infected mice was analysed using cDNA microarray screening, followed by a temporal comparison of mRNA and protein expression of RANTES and its corresponding receptors by qRT-PCR and Western blot analysis, respectively. Plasma RANTES levels was determined by ELISA and ultrastructural studies of brain sections from infected and uninfected mice was conducted.
Results
RANTES (p < 0.002), CCR1 (p < 0.036), CCR3 (p < 0.033), and CCR5 (p < 0.026) mRNA were significantly upregulated at peak parasitaemia and remained high thereafter in the experimental mouse model. RANTES protein in the brain of infected mice was upregulated (p < 0.034) compared with controls. RANTES plasma levels were significantly upregulated; two to three fold in infected mice compared with controls (p < 0.026). Some d istal microvascular endothelium in infected cerebellum appeared degraded, but remained intact in controls.
Conclusion
The upregulation of RANTES, CCR1, CCR3, and CCR5 mRNA, and RANTES protein mediate inflammation and cellular degradation in the cerebellum during P. yoelii 17XL malaria.
... It should be noted that ICAM-1 may function as a cell surface receptor capable of initiating intracellular signaling. In this regard, several studies on human endothelial cells showed that although the cytoplasmic tail of ICAM-1 lacks intrinsic enzymatic activity, engagement of ICAM-1 triggers several signaling pathways, including activation of Src family kinase leading to phosphorylation of actin-associated proteins (17), including p44/p42 mitogen-activated protein kinase (MAPK; ref. 18), and p38 MAPK (19). Several studies showed that ICAM-1 expression correlated with melanoma thickness and suggested that it may contribute to the metastatic potential of melanoma cells (20,21). ...
Human primary melanoma cells (T1) were found to be more susceptible to lysis by a Melan-A/MART-1-specific CTL clone (LT12) than their metastatic derivative (G1). We show that this differential susceptibility does not involve antigen presentation by target cells, synapse formation between the metastatic target and CTL clone, or subsequent granzyme B (GrB) polarization. Although PI-9, an inhibitor of GrB, was found to be overexpressed in metastatic G1 cells, knockdown of the PI-9 gene did not result in the attenuation of G1 resistance to CTL-induced killing. Interestingly, we show that whereas T1 cells express high levels of intercellular adhesion molecule-1 (ICAM-1), a dramatically reduced expression was noted on G1 cells. We also showed that sorted ICAM-1+ G1 cells were highly sensitive to CTL-induced lysis compared with ICAM-1- G1 cells. Furthermore, incubation of metastatic G1 cells with IFN-gamma resulted in the induction of ICAM-1 and the potentiation of their susceptibility to lysis by LT12. More importantly, we found that the level of ICAM-1 expression by melanoma cells correlated with decreased PTEN activity. ICAM-1 knockdown in T1 cells resulted in increased phosphorylation of PTEN and the subsequent activation of AKT. We have additionally shown that inhibition of the phosphatidylinositol (3,4,5)-triphosphate kinase (PI3K)/AKT pathway by the specific inhibitor wortmannin induced a significant potentiation of susceptibility of G1 and ICAM-1 small interfering RNA-treated T1 cells to CTL-induced lysis. The present study shows that a shift in ICAM-1 expression, which was associated with an activation of the PI3K/AKT pathway, can be used by metastatic melanoma cells to escape CTL-mediated killing.
During inflammation, leukocytes extravasate the vasculature to areas of inflammation in a process termed transendothelial migration. Previous research has shown that transendothelial migration hotspots exist, areas in the vasculature that are preferred by leukocytes to cross. Several factors that contribute to hotspot-mediated transmigration have been proposed already, but whether one leukocyte transmigration hotspot can be used subsequently by a second wave of leukocytes and thereby can increase the efficiency of leukocyte transmigration is not well understood. Here, we show that primary neutrophil adhesion to the endothelium triggers endothelial transmigration hotspots, allowing secondary neutrophils to cross the endothelium more efficiently. Mechanistically, we show that primary neutrophil adhesion increases the number of endothelial apical filopodia, resulting in an increase in the number of adherent secondary neutrophils. Using fluorescence resonance energy transfer (FRET)-based biosensors, we found that neutrophil adhesion did not trigger the activity of the small GTPase Cdc42. We used kinase translocation reporters to study the activity of mitogen-activated protein (MAP) kinases and Akt in endothelial cells on a single-cell level with a high temporal resolution during the process of leukocyte transmigration and found that c-Jun N-terminal kinase (JNK) is rapidly activated upon neutrophil adhesion, whereas extracellular regulated kinase (ERK), p38, and Akt are not. Additionally, we show that short-term chemical inhibition of endothelial JNK successfully prevents the adhesion of neutrophils to the endothelium. Furthermore, we show that neutrophil-induced endothelial JNK1 but not JNK2 increases the formation of filopodia and thereby the adhesion of secondary neutrophils. JNK1 needs its downstream substrate MARCKSL1 to trigger additional apical filopodia and consequently neutrophil adhesion. Overall, our data show that primary neutrophils can trigger the endothelial transmigration hotspot by activating JNK1 and MARCKSL1 to induce filopodia that trigger more neutrophils to transmigrate at the endothelial hotspot area.
Endothelial Rho proteins are essential signalling molecules that mediate endothelial responses to adherent leukocytes through ICAM-1, allowing them to migrate through brain endothelial monolayers. In order to be functionally active, Rho proteins are required to undergo post-translational prenylation. In vitro inhibition of brain endothelial protein isoprenylation with protein prenyl transferase inhibitors, resulted in a significant reduction in the ability of brain endothelial cells to support transendothelial migration of myelin basic protein (MBP)-specific T-lymphocytes. The effect of these inhibitors was specific for the process of migration and did not significantly affect the ability of T-lymphocytes to adhere to endothelial cells. Protein prenyl transferase inhibitors were also effective at inhibiting leukocyte migration into the CNS and attenuating clinical signs of disease in an in vivo model of neuroinflammation. Similar studies using the 3-hydroxy-3-methylglutaryl Coenzyme A reductase inhibitor, lovastatin to limit the availability of isoprenoid groups for protein prenylation showed a significant reduction in both the transendothelial migration of MBP-specific T-lymphocytes and the infiltration of leukocytes into the CNS. These studies demonstrate the potential of pharmacologically targeting CNS endothelial cell signalling responses, and particularly limiting endothelial protein prenylation, as a means of attenuating leukocyte recruitment to the CNS and subsequent neuroinflammatory disease. Studies with VCAM-1 suggest that it may transduce intracellular signals in brain endothelial cells which may be important in the transvascular migration of monocytes.
Our recent finding that resistance to lymphoma cell metastasis in intercellular adhesion molecule-1-(ICAM-1)–deficient mice was manifested after homing suggested that the mechanism could involve the capacity of ICAM-1 to induce, via leukocyte function-associated antigen-1 (LFA-1) signaling, the expression of new genes necessary for migration and survival of lymphoma cells after homing. This hypothesis would imply that lymphoma cells, on repeated metastatic cycles, would acquire such a highly aggressive phenotype that they no longer require contact with ICAM-1 at later stages of metastasis. We addressed this question by generating highly aggressive lymphoma variants to determine if increased tumorigenicity would allow lymphoma cells to grow into tumors in ICAM-1–deficient mice. We found that on repeated in vivo passages, a selective pressure favored the lymphoma cells that constitutively express high levels of matrix metalloproteainse-9 (MMP-9), a gene associated with a poor clinical outcome in non-Hodgkins's lymphoma. We further found that although the parent lymphoma cells could not grow tumors in ICAM-1–deficient mice, the aggressive lymphoma variants could. This indicates that, at late stages of the disease, tumor cells with a high metastatic efficiency, encoded by the repertoire of selected genes, no longer require some of the signals normally delivered by cell adhesion molecules. In light of these findings, the possibility of inhibiting dissemination of lymphoma cells at the late stage of the disease by acting against cell adhesion molecules must be reconsidered. (Blood. 2000;95:314-319)
Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) in humans. Both diseases are considered to be immunologically mediated but the exact pathological mechanisms are still poorly understood. Neutrophils are considered the first line of defense against invading microbes but little is still known of their role in virus infections. We wanted to study the role of neutrophils in HFRS using blood and tissue samples obtained from Puumala hantavirus (PUUV)-infected patients. We found that neutrophil activation products myeloperoxidase and neutrophil elastase, together with interleukin-8 (the major neutrophil chemotactic factor in humans), are strongly elevated in blood of acute PUUV-HFRS and positively correlate with kidney dysfunction, the hallmark clinical finding of HFRS. These markers localized mainly in the tubulointerstitial space in the kidneys of PUUV-HFRS patients suggesting neutrophil activation to be a likely component of the general immune response toward hantaviruses. We also observed increased levels of circulating extracellular histones at the acute stage of the disease supporting previous findings of neutrophil extracellular trap formation in PUUV-HFRS. Mechanistically, we did not find evidence for direct PUUV-mediated activation of neutrophils but instead primary blood microvascular endothelial cells acquired a pro-inflammatory phenotype and promoted neutrophil degranulation in response to PUUV infection in vitro. These results suggest that neutrophils are activated by hantavirus-infected endothelial cells and may contribute to the kidney pathology which determines the severity of HFRS.
Historically, it was believed that the central nervous system (CNS) fails to mount an inflammatory response due to its protection from the systemic immune system mediated by the blood-brain barrier (BBB). However, in the last decades, participation of neuronal and glial cells in inflammatory events occurring in infectious, autoimmune and degenerative neuropathologies became increasingly evident (Morganti-Kossmann et al. 2000). With regard to traumatic brain injury (TBI), it has been reported in clinical (Bell et al. 1997; Morganti-Kossmann et al. 1997; Holmin et al. 1998) as well as experimental studies (Taupin et al. 1993; Shohami et al. 1994; Fan et al. 1995; Fan et al. 1996; Carlos et al. 1997; Hans et al. 1999; Csuka et al. 2000) that, despite the presence of the BBB, a profound inflammatory response occurs within the brain immediately following injury. It is nowadays accepted that 1) immune surveillance is present in the CNS, 2) an immune response can be induced within the brain, 3) most of the molecules and mechanisms modulating the inflammatory response are present and active within the brain and 4) bi-directional communication exists between the nervous and the immune systems.
Aim: To investigate whether homocysteic acid could induce cultured human umbilical vein endothelial cells to express regulated upon activation, normal T cell expressed and secreted (RANTES) and protein, and analyze the mechanism by which the expression of RANTES was induced. Methods: The experiment was conducted in Molecular Biology Laboratory of Xinxiang Medical University from November 2004 to November 2005. (1) The human umbilical vein endothelial cells were separated with pancreatin digestion. (2) When the human umbilical vein endothelial cells were confluent, they were divided into three, groups including control group, homocysteic acid group, and pyrrolidine dithiocarbamate pretreatment group at random. The human umbilical vein endothelial cells of the control group were cultured with the medium without serum, and the homocysteic acid group was exposed to homocysteic acid at a concentration of 0.1 mmol/L. The two groups mentioned above were incubated for 8 hours, respectively. The pyrrolidine dithiocarbamate pretreatment group was incubated with pyrrolidine dithiocarbamate at a concentration of 100 μmol/L for 20 minutes at first, and then exposed to homocysteic acid at a concentration of 0.1 mmol/L for 8 hours. 3 The expression of RANTES mRNA in human umbilical vein endothelial cells were detected with dot blot hybridization. 4 The expression of RANTES protein in human umbilical vein endothelial cells was determined with Western blot method. 5 The expressions of NF-ΚB P65 protein in the control and homocysteic acid groups were detected with immunocytochemical method. Results: (1) The expression of RANTES mRNA in human umbilical vein endothelial cells: The image analysis showed, in human umbilical vein endothelial cells treated with homocysteic acid or pyrrolidine dithiocarbamate, the integral absorbance values were 18 790 and 16 420, which were 1.48-fold and 1.29-fold as much as that of the control group (12 680), respectively. (2) The expression of RANTES protein in human, umbilical vein endothelial cells: The image analysis showed that the integral absorbance values of RANTES protein in homocysteic acid group was 0.794 2, and that in pyrrolidine dithiocarbamate pretreatment group was 0.501 3, which were 2.72-fold and 1.72-fold as much as that in the control group (0.293 0), respectively. (3) The expression of nuclear factor (NF)-ΚB P65 protein in human umbilical vein endothelial cells: NF-ΚB P65 protein was mainly located in the cytoplasma of the human umbilical vein endothelial cells in the control group. The percentage of nuclear positive cells of NF-ΚB P65 protein in the control group was 8.3%, after exposure of human umbilical vein endothelial cells to homocysteic acid, which increased to 43.5% (P < 0.01). Conclusion: Homocysteic acid can induce the expression of RANTES mRNA and protein in human umbilical vein endothelial cells. NF-ΚB may, play an important role in the process. Homocysteic acid may play a critical role in atherogenesis through inducing endothelial cells to express RANTES.
MUC1 has conventionally been studied as an epithelial cell surface molecule. Its glycosylation and expression change when those cells are transformed into adenocarcinomas. These changes have led to focus on MUC1 as a tumor antigen and also its role in adhesion to blood vessels and signaling within the tumor cell. The recent discovery that T cells also express MUC1 on their surface extends the physiological role of MUC1, with the possibility that functions observed in tumors may be reproduced on T cells. Expression of MUC1 on T cells was first characterized in terms of timing, location and structure. T cells activated both in vivo and in vitro express MUC1. Expression in vitro is maintained over long time periods as the T cell population acquires the memory phenotype. Activated T cells induced to polarize by inflammatory conditions focus MUC1 expression to their leading edge, the sensory compartment of polarized T cells. Reactivity with glycosylation-sensitive antibodies and induction of glycosyltransferases indicates that the glycosylation of MUC1 on T cells is similar to that on normal epithelial cells. A MUC1-negative T cell line was transfected with MUC1 cDNA and used as a model to investigate consequences of MUC1 expression on the T cell surface. Interaction of MUC1+ T cells with resting or activated endothelial cells revealed that MUC1 aids in adhesion under both normal and inflammatory conditions. Analysis of interactions with individual adhesion molecules demonstrated MUC1 specific enhancement of binding to ICAM-1 but inhibition of binding to E-selectin. Phosphorylation of the MUC1 intracytoplasmic tail is constitutive but decreases upon interaction with activated endothelium. MUC1 expression on T cells is also associated with differential phosphorylation of proteins in the molecular weight ranges of 39 kDa, ~80 kDa and 190 kDa, with the ~80 kDa band identified as beta-catenin. While human T cells express MUC1 on their surface upon activation, this does not appear to be a characteristic of mouse T cells from the human MUC1 transgenic mouse model. However, as recent work indicates that mouse T cells express mouse Muc-1 after activation, human and mouse T cells may similarly depend on MUC1 for normal functioning.
Vascular manifestations can be seen early in the pathogenesis of inflammatory rheumatic diseases. Animal experiments, laboratory and clinical findings indicated that acute or long-term vibration exposure can induce vascular abnormalities. Recent years, in addition to Raynaud's phenomenon (RP), vibration as a risk factor for other rheumatic diseases has also received corresponding considered. This review is concentrated upon the role of vibration in the disease of systemic sclerosis (SSc). In this review, we are going to discuss the main mechanisms which are thought to be important in pathophysiology of vascular injury under the three broad headings of “vascular”, “neural” and “intravascular”. Aspects on the vibration and vascular inflammation are briefly discussed. And the epidemiological studies related to vibration studies in SSc and other rheumatic diseases are taken into account.
Lymphocyte recruitment to the central nervous system (CNS) is a critical step in the pathogenesis of diseases such as multiple sclerosis (MS), meningitis and posterior uveitis. The principle sequential stages that control lymphocyte emigration from the blood have been widely reported, but only recently has attention been directed towards the role of the vascular endothelium in actively supporting transvascular migration. It has now been shown that adhesion molecules, particularly those of the immunoglobulin super family (eg ICAM-1, VCAM-1 and PECAM-1), not only act as ligands for leucocyte receptors but can also serve as signal transducers. Engagement of these receptors initiates endothelial signalling cascades that result in downstream effector mechanisms which in turn influence the progression of neuroinflammation. In particular, it has been shown that ICAM-1-mediated signalling in brain endothelial cells is a crucial regulatory step in the process of lymphocyte migration through the blood-brain barrier and as such represents an additional phase in the multistep paradigm of leucocyte recruitment. In this article we review current understanding of endothelial cell ICAM-1 signalling and discuss the importance of these findings in relation to leucocyte trafficking to the CNS.
The pathophysiology of traumatic axonal injury (TAI) is only partially understood. In this study, we investigated the inflammatory response as well as the extent of neurological deficit in a rat model of traumatic brain injury (TBI). Forty-two adult rats were subjected to moderate impact-acceleration brain injury and their brains were analyzed immunohistochemically for ICAM-1 expression and neutrophil infiltration from 1 hr up to 14 days after trauma. In addition, the chemotactic factors MIP-2 and MCP-1 were measured in brain homogenates by ELISA. For evaluating the neurological deficit, three sensorimotor tests were applied for the first time in this model. In the first 24 hr after trauma, the number of ICAM-1 positive vessels increased up to 4-fold in cortical and subcortical regions compared with sham operated controls (P < 0.05). Maximal ICAM-1 expression (up to 8-fold increase) was detected after 4 days (P < 0.001 vs. 24 hr), returning to control levels in all brain regions by 7 days after trauma. MCP-1 was elevated between 4 hr and 16 hr post-injury as compared with controls. In contrast, neither neutrophil infiltration nor elevation of MIP-2, both events relevant in focal brain injury, could be detected. In all neurological tests, a significant deficit was observed in traumatized rats as compared with sham operated animals from Day 1 post-injury (grasping reflex of the hindpaws: P < 0.001, vibrissae-evoked forelimb placing: P = 0.002, lateral stepping: P = 0.037). In conclusion, after moderate impact acceleration brain injury ICAM-1 upregulation has been demonstrated in the absence of neutrophil infiltration and is paralleled by a selective induction of chemokines, pointing out that individual and distinct inflammatory events occur after diffuse vs. focal TBI. J. Neurosci. Res. 63:438–446, 2001. © 2001 Wiley-Liss, Inc.
Interleukin-8 (IL-8) is an angiogenic CXC chemokine produced by a variety of cell types. Although initially described as a
chemokine for neutrophils, IL-8 has become known as a potent angiogenic factor, involved in normal physiological processes
such as wound healing, and abnormal processes such as cancer metastasis. IL-8 is secreted by numerous solid tumour types and
associated inflammatory cells, and has been shown to exert a potent angiogenic effect via paracrine and autocrine routes in
tumourigenesis, and as such represents an valid opportunity for intervention in cancer metastasis.
Cannabinoids inhibit cancer cell invasion via increasing tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). This study investigates the role of intercellular adhesion molecule-1 (ICAM-1) within this action. In the lung cancer cell lines A549, H358, and H460, cannabidiol (CBD; 0.001-3 μM) elicited concentration-dependent ICAM-1 up-regulation compared to vehicle via cannabinoid receptors, transient receptor potential vanilloid 1, and p42/44 mitogen-activated protein kinase. Up-regulation of ICAM-1 mRNA by CBD in A549 was 4-fold at 3 μM, with significant effects already evident at 0.01 μM. ICAM-1 induction became significant after 2 h, whereas significant TIMP-1 mRNA increases were observed only after 48 h. Inhibition of ICAM-1 by antibody or siRNA approaches reversed the anti-invasive and TIMP-1-upregulating action of CBD and the likewise ICAM-1-inducing cannabinoids Δ(9)-tetrahydrocannabinol and R(+)-methanandamide when compared to isotype or nonsilencing siRNA controls. ICAM-1-dependent anti-invasive cannabinoid effects were confirmed in primary tumor cells from a lung cancer patient. In athymic nude mice, CBD elicited a 2.6- and 3.0-fold increase of ICAM-1 and TIMP-1 protein in A549 xenografts, as compared to vehicle-treated animals, and an antimetastatic effect that was fully reversed by a neutralizing antibody against ICAM-1 [% metastatic lung nodules vs. isotype control (100%): 47.7% for CBD + isotype antibody and 106.6% for CBD + ICAM-1 antibody]. Overall, our data indicate that cannabinoids induce ICAM-1, thereby conferring TIMP-1 induction and subsequent decreased cancer cell invasiveness.
Our recent finding that resistance to lymphoma cell metastasis in intercellular adhesion molecule-1-(ICAM-1)-deficient mice was manifested after homing suggested that the mechanism could involve the capacity of ICAM-1 to induce, via leukocyte function-associated antigen-1 (LFA-1) signaling, the expression of new genes necessary for migration and survival of lymphoma cells after homing. This hypothesis would imply that lymphoma cells, on repeated metastatic cycles, would acquire such a highly aggressive phenotype that they no longer require contact with ICAM-1 at later stages of metastasis. We addressed this question by generating highly aggressive lymphoma variants to determine if increased tumorigenicity would allow lymphoma cells to grow into tumors in ICAM-1-deficient mice. We found that on repeated in vivo passages, a selective pressure favored the lymphoma cells that constitutively express high levels of matrix metalloproteainse-9 (MMP-9), a gene associated with a poor clinical outcome in non-Hodgkins's lymphoma. We further found that although the parent lymphoma cells could not grow tumors in ICAM-1-deficient mice, the aggressive lymphoma variants could. This indicates that, at late stages of the disease, tumor cells with a high metastatic efficiency, encoded by the repertoire of selected genes, no longer require some of the signals normally delivered by cell adhesion molecules. In light of these findings, the possibility of inhibiting dissemination of lymphoma cells at the late stage of the disease by acting against cell adhesion molecules must be reconsidered. (Blood. 2000;95:314-319)
Clinical and laboratory evidence suggests that alcohol consumption dysregulates immune function. Burn patients who consume alcohol before their injuries demonstrate higher rates of morbidity and mortality, including acute respiratory distress syndrome, than patients without alcohol at the time of injury. Our laboratory observed higher levels of proinflammatory cytokines and leukocyte infiltration in the lungs of mice after ethanol exposure and burn injury than with either insult alone. To understand the mechanism of the increased pulmonary inflammatory response in mice treated with ethanol and burn injury, we investigated the role of intercellular adhesion molecule (ICAM)-1. Wild-type and ICAM-1 knockout (KO) mice were treated with vehicle or ethanol and subsequently given a sham or burn injury. Twenty-four hours postinjury, lungs were harvested and analyzed for indices of inflammation. Higher numbers of neutrophils were observed in the lungs of wild-type mice after burn and burn with ethanol treatment. This increase in pulmonary inflammatory cell accumulation was significantly lower in the KO mice. In addition, levels of KC, interleukin-1beta, and interleukin-6 in the lung were decreased in the ICAM-1 KO mice after ethanol exposure and burn injury. Interestingly, no differences were observed in serum or lung tissue content of soluble ICAM-1 24 hours postinjury. These data suggest that upregulation of adhesion molecules such as ICAM-1 on the vascular endothelium may play a critical role in the excessive inflammation seen after ethanol exposure and burn injury.
Cellular adhesion molecules were initially defined as cell surface structures mediating cell-cell and cell-extracellular matrix (ECM) interactions. Adhesion molecules involved in immune responses have been classified into three families according to their structure: selectins, immunoglobulin (Ig) superfamily, and integrins. It has been well documented that adhesion molecules of these family members (E-selectin, ICAM-1, and VCAM-1) are expressed on brain microvessel endothelial cells in active lesions of multiple sclerosis (MS) brain. In addition, accumulating data show that glial cells can express some of these adhesion molecules upon activation: astrocytes can express ICAM-1, VCAM-1, and E-selectin, and microglia express ICAM-1 and VCAM-1. In vitro studies show that these adhesion molecules are actively regulated by several cytokines which have relevance to MS or experimental autoimmune encephalomyelitis (EAE). In addition, soluble forms of adhesion molecules have been found in the serum and cerebrospinal fluid (CSF) of MS patients, and may be useful diagnostically. Experimental therapy of EAE using antibodies against several adhesion molecules clearly shows that adhesion molecules are critical for the pathogenesis of EAE. Thus far, the function of adhesion molecule expression on brain endothelial and glial cells has not been clearly elucidated. Studies on the possible role of adhesion molecules on brain endothelial and glial cells will be helpful in understanding their involvement in immune responses in the central nervous system (CNS).
Introduction
Following an inflammatory or infectious stimulus, the body’s defense mechanism initiates recruitment of circulating leukocytes toward the inflammatory stimulus. The emigration of leukocytes into extravascular tissues occurs in a highly coordinated fashion in multiple steps, including rolling and tethering of blood cells along the vascular endothelium and their firm attachment and subsequent transmigration and invasion toward the inflammatory site.1 During these sequential steps, transcellular recognition of different adhesion receptor/counterligand pairs, such as selectins/sialyl LewisX-carbohydrates,2 integrins/ immunoglobulin superfamily cell adhesion molecules (ICAMs),3 or binding to (provisional) extracellular matrix components, such as fibrinogen/fibrin, vitronectin, or fibronectin, control the strength and duration of interactions between leukocytes (neutrophils [polymorphonucleocytes (PMN)], eosinophils, monocytes and macrophages, mast cells, lymphocytes) and the vessel wall.4 The importance of these cellular interactions is evident from patients with the rare congenital disorders of “leukocyte-adhesion-deficiency,” which are either caused by a lack or dysfunction of ß2-integrins (LAD I) or a deficiency in the expression of sialyl-LewisX carbohydrates (LAD II).5 The interdependent adhesion processes are regulated by vascular cell-derived chemokines and chemoattractants that may directly influence the expression profile and activation state of adhesion molecules, such as ß2- and ß1-integrins, the shedding of selectins, and the nonthrombogenic properties of endothelial cells.6 Prior to transmigration, leukocyte adhesion may induce the disruption of vascular endothelial (VE)-cadherin mediated endothelial cell-to-cell junctions7 involving the proteasome machinery.8
The spatio-temporal cellular expression of juxtacrine adhesion and signaling receptors–particularly on PMN, endothelial cells, and platelets–contribute to the coordination of adhesion and inflammatory mechanisms required for vascular homeostasis9 and prothrombotic outcome under imbalanced conditions. Not only do monocytes express tissue factor (a receptor for the protease factor VII/VIIa) on their surface after stimulation with endotoxin or cytokines, but PMN contain cell surface receptors, such as the factor X/Xa-binding ß2-integrin Mac-1 or effector cell protease receptor (EPR)-1, that link cellular activation and inflammation with the induction of the blood clotting cascade and serve as an alternate pathway for thrombin formation.10,11 Moreover, defects in natural anticoagulant mechanisms, such as the thrombomodulin/protein C pathway, are potential risk factors for vascular thrombotic complications, as in myocardial infarction.12 Pathophysiological stimuli, such as dysregulated direct (i.e., adhesive contact) or indirect (i.e., release of soluble factors) activation of leukocytes, serious infectious agonists, or autoantibodies, may result in endothelial cell dysfunction or injury with the amplification of inflammatory and prothrombotic responses. In the following, some of the principal juxtacrine interactions between leukocytes, platelets, and endothelium, together with their direct or indirect influence on hemostasis and consequences for vascular thrombotic disease, will be discussed. Further understanding of the bidirectional cross-talk of adhesion receptors and the contribution of connecting points, such as protease receptors, may lead to promising therapeutic strategies that aim to protect or regain the endothelial defense mechanisms.
Airway epithelial cells which are the initial site of influenza virus (IV) infection are suggested to participate in airway inflammatory response by expressing various cytokines including RANTES; however, the intracellular signal that regulates RANTES expression has not been determined. In the present study, we examined the role of p38 mitogen-activated protein (MAP) kinase, extracellular signal-regulated kinase (Erk), and c-Jun-NH2-terminal kinase (JNK) in RANTES production by IV-infected human bronchial epithelial cells. The results showed that IV infection induced increases in p38 MAP kinase, and Erk and JNK phosphorylation and activity. SB 203580, PD 98059, and CEP-1347 attenuated IV-infection induced p38 MAP kinase activity, Erk activity, and JNK activity, respectively. SB 203580 and CEP-1347 attenuated RANTES production by 45.3% and 45.2%, respectively, but a combination of these inhibitors additively attenuated by 69.1%. In contrast, PD 98059 did not attenuate. Anti-IL-1alpha mAb, anti-IL-1beta mAb, anti-TNF-alpha mAb, anti-IL-8 mAb, anti-IFN-beta mAb, anti-RANTES mAb, and a combination of these mAbs did not affect IV infection-induced increases in p38 MAP kinase, Erk, and JNK phosphorylation, indicating that each cytokine neutralized by corresponding Ab was not involved in IV infection-induced phosphorylation of MAP kinases. N-acetylcysteine (NAC) did not affect IV infection-induced increases in MAP kinase phosphorylation, whereas NAC attenuated RANTES production by 18.2%, indicating that reactive oxygen species may act as a second messenger leading to RANTES production via p38 MAP kinase- and JNK-independent pathway. These results indicate that p38 MAP kinase and JNK, at least in part, regulate RANTES production by bronchial epithelial cells.
The events that lead to an inflammatory response are characterized by recognition of the site of injury by inflammatory cells, specific recruitment of subpopulations of leukocytes into tissue, removal of the offending agent and "debridement" of the injured cells/tissue, and repair of the site of injury with attempts to reestablish normal parenchymal, stromal, and extracellular matrix relationship. The molecular regulation of this complex physiologic process involves the interaction between cell surface, extracellular matrix, and soluble mediators, such as chemokines. Chemokine activities are mediated through G-protein coupled receptors. This is the largest known family of cell-surface receptors, which mediate transmission of stimuli as diverse as hormones, peptides, glycopeptides, and chemokines. In this review, we will focus on the signaling pathways involved in the production and function of chemokines as they relate to the inflammatory response.
Cytokine mediators and leukocyte-endothelial cell adhesion molecules are critical and interdependent components of the acute inflammatory response in sepsis. We hypothesized that the administration of monoclonal antibodies to intercellular adhesion molecule-1 (CD54) or E- and L-selectin (CD62E/L) would decrease serum levels of the proinflammatory cytokines interleukin-1beta (IL-1), IL-6, and IL-8 and tumor necrosis factor receptor (TNFR-1) in baboons during sepsis. Adult male baboons received infusions of 1 x 10(9) colony forming units (CFU)/kg heat-killed Escherichia coli (E. coli) followed 12 h later by live E. coli (1 x 10(10) CFU/kg). At the time of live bacterial infusion, six septic animals were treated with a monoclonal antibody to CD54 and six with an antibody to CD62E and L (1 mg/kg). Eight untreated septic animals served as controls. Sequentially drawn serum samples were assayed for IL-1, IL-6, IL-8, and TNFR-1 using enzyme-linked immunoassay (ELISA). Data were compared using Mann-Whitney U tests and Chi-square analyses. Median survival was decreased in both treatment groups compared to controls (P < 0.05). Peak IL-1 level was higher than controls in septic animals treated with anti-CD54 but not anti-CD62E/L (P < 0.05, P = NS, respectively). Elevations in IL-6, IL-8, and TNFR-1 were increased and prolonged in both antibody treated groups compared to controls (P < 0.05). These results provide the first in vivo evidence that leukocyte-endothelial adhesion molecules CD54 and CD62E/L regulate cytokine production in sepsis.
Lung cancer is a leading cause of cancer-related death in the United States. For this reason we chose to study the specific cellular effects that one chemotherapeutic agent, paclitaxel, has on lung carcinoma. In addition to its known mechanism of action, which is to stabilize microtubules, paclitaxel has been shown to have other interesting and relevant cellular effects. In this report, we demonstrate that a subset of human lung carcinoma cell lines respond to paclitaxel treatment with an up to a fivefold increase in the production of interleukin-8 (IL-8). We demonstrate that this increased production is specific to IL-8 but not to other chemokines, and is both dose- and time-dependent. Increased IL-8 mRNA is seen as early as 45 min with a peak at 4 h after paclitaxel treatment. This increase in mRNA is due to transcriptional activation because actinomycin D treatment blocked the increase. Paclitaxel also activates the mitogen-activated protein kinase family member, JNK1, in dose-dependent fashion. IL-8 enhancement is completely abolished with the use of an inhibitor of NF-kappaB, the super-repressor IkappaB. Similar results were obtained upon the inhibition of AP-1 activation with the MEK1/2 inhibitor, U0126. By gaining a better understanding of the differences in cellular response to paclitaxel chemotherapy, these findings might lead to either improved patient selection or to the development of adjuvant therapy targeted at specific-cell signaling proteins.
The collective interaction between cells is, in part, mediated by different families of adhesion molecules. Intercellular adhesion molecules (ICAMs) are structurally related members of the immunoglobulin supergene family and are ligands for the beta2 integrin molecules present on leukocytes. Of the five ICAMs identified, ICAM-1 is the most extensively studied. Although ICAM-1 is expressed constitutively at low levels on endothelial cells and on some lymphocytes and monocytes, its expression can be significantly increased in the presence of cytokines (TNFalpha, IL-1, IFNgamma) and reactive oxygen species. Depending upon cell type, ICAM-1 participates in trafficking of inflammatory cells, in cell:cell interactions during antigen presentation, in microbial pathogenesis, and in signal transduction through outside-in signaling events. Again, depending upon cell type examined, ICAM-1 engagement has been documented to activate specific kinases through phosphorylation, resulting in transcription factor activation and increased cytokine production, increased cell membrane protein expression, reactive oxygen species production, and cell proliferation.
PD 098059 has been shown previously to inhibit the dephosphorylated form of mitogen-activated protein kinase kinase-1 (MAPKK1)
and a mutant MAPKK1(S217E,S221E), which has low levels of constitutive activity (Dudley, D. T., Pang, L., Decker, S. J., Bridges,
A. J., and Saltiel, A. R.(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7686-7689). Here we report that PD 098059 does not inhibit Raf-activated MAPKK1 but that it prevents the activation of
MAPKK1 by Raf or MEK kinase in vitro at concentrations (IC = 2-7 μM) similar to those concentrations that inhibit dephosphorylated MAPKK1 or MAPKK1(S217E,S221E). PD 098059 inhibited
the activation of MAPKK2 by Raf with a much higher IC value (50 μM) and did not inhibit the phosphorylation of other Raf or MEK kinase substrates, indicating that it exerts its
effect by binding to the inactive form of MAPKK1. PD 098059 also acts as a specific inhibitor of the activation of MAPKK in
Swiss 3T3 cells, suppressing by 80-90% its activation by a variety of agonists. The high degree of specificity of PD 098059
in vitro and in vivo is indicated by its failure to inhibit 18 protein Ser/Thr kinases (including two other MAPKK homologues) in vitro by its failure to inhibit the in vivo activation of MAPKK and MAP kinase homologues that participate in stress and interleukin-1-stimulated kinase cascades in
KB and PC12 cells, and by lack of inhibition of the activation of p70 S6 kinase by insulin or epidermal growth factor in Swiss
3T3 cells. PD 098059 (50 μM) inhibited the activation of p42 and isoforms of MAP kinase-activated protein kinase-1 in Swiss 3T3 cells, but the extent of inhibition depended on how potently
c-Raf and MAPKK were activated by any particular agonist and demonstrated the enormous amplification potential of this kinase
cascade. PD 098059 not only failed to inhibit the activation of Raf by platelet-derived growth factor, serum, insulin, and
phorbol esters in Swiss 3T3 cells but actually enhanced Raf activity. The rate of activation of Raf by platelet-derived growth
factor was increased 3-fold, and the subsequent inactivation that occurred after 10 min was prevented. These results indicate
that the activation of Raf is suppressed and that its inactivation is accelerated by a downstream component(s) of the MAP
kinase pathway.
The cellular infiltrates of certain inflammatory processes found in parasitic infection or in allergic diseases consist predominantly of eosinophilic granulocytes, often in association with activated T cells. This suggests the existence of chemotactic agonists specific for eosinophils and lymphocyte subsets devoid of neutrophil-activating properties. We therefore examined four members of the intercrine/chemokine superfamily of cytokines (monocyte chemotactic peptide 1 [MCP-1], RANTES, macrophage inflammatory protein 1 alpha [MIP-1 alpha], and MIP-1 beta), which do not activate neutrophils, for their ability to affect different eosinophil effector functions. RANTES strongly attracted normal human eosinophils by a chemotactic rather than a chemokinetic mechanism with a similar efficacy as the most potent chemotactic myeloid cell agonist, C5a. MIP-1 alpha also induced eosinophil migration, however, with lower efficacy. RANTES and MIP-1 alpha induced eosinophil cationic protein release in cytochalasin B-treated eosinophils, but did not promote leukotriene C4 formation by eosinophils, even after preincubation with interleukin 3 (IL-3), in contrast to other chemotactic agonists such as C5a and formyl-methionyl-leucyl-phenylalanine (FMLP). RANTES, but not MIP-1 alpha, induced a biphasic chemiluminescence response, however, of lower magnitude than C5a. RANTES and MIP-1 alpha both promoted identical transient changes in intracellular free calcium concentration ([Ca2+]i), with kinetics similar to those induced by chemotactic peptides known to interact with G protein-coupled receptors. No cross-desensitization towards other peptide agonists (e.g., C5a, IL-8, FMLP) was observed, suggesting the presence of specific receptors. Despite its weaker eosinophil-activating properties, MIP-1 alpha was at least 10 times more potent on a molar basis than RANTES at inducing [Ca2+]i changes. Interestingly, RANTES deactivated the MIP-1 alpha-induced [Ca2+]i changes, while the RANTES response was preserved after MIP-1 alpha stimulation. MCP-1, a potent monocyte chemoattractant and basophil agonist, as well as MIP-1 beta, a peptide with pronounced homology to MIP-1 alpha, did not activate the eosinophil functions tested. Our results indicate that RANTES and MIP-1 alpha are crucial mediators of inflammatory processes in which eosinophils predominate.
The cellular infiltrates of certain inflammatory processes found in parasitic infection or in allergic diseases consist predominantly of eosinophilic granulocytes, often in association with activated T cells. This suggests the existence of chemotactic agonists specific for eosinophils and lymphocyte subsets devoid of neutrophil-activating properties. We therefore examined four members of the intercrine/chemokine superfamily of cytokines (monocyte chemotactic peptide 1 [MCP-1], RANTES, macrophage inflammatory protein 1 alpha [MIP-1 alpha], and MIP-1 beta), which do not activate neutrophils, for their ability to affect different eosinophil effector functions. RANTES strongly attracted normal human eosinophils by a chemotactic rather than a chemokinetic mechanism with a similar efficacy as the most potent chemotactic myeloid cell agonist, C5a. MIP-1 alpha also induced eosinophil migration, however, with lower efficacy. RANTES and MIP-1 alpha induced eosinophil cationic protein release in cytochalasin B-treated eosinophils, but did not promote leukotriene C4 formation by eosinophils, even after preincubation with interleukin 3 (IL-3), in contrast to other chemotactic agonists such as C5a and formyl-methionyl-leucyl-phenylalanine (FMLP). RANTES, but not MIP-1 alpha, induced a biphasic chemiluminescence response, however, of lower magnitude than C5a. RANTES and MIP-1 alpha both promoted identical transient changes in intracellular free calcium concentration ([Ca2+]i), with kinetics similar to those induced by chemotactic peptides known to interact with G protein-coupled receptors. No cross-desensitization towards other peptide agonists (e.g., C5a, IL-8, FMLP) was observed, suggesting the presence of specific receptors. Despite its weaker eosinophil-activating properties, MIP-1 alpha was at least 10 times more potent on a molar basis than RANTES at inducing [Ca2+]i changes. Interestingly, RANTES deactivated the MIP-1 alpha-induced [Ca2+]i changes, while the RANTES response was preserved after MIP-1 alpha stimulation. MCP-1, a potent monocyte chemoattractant and basophil agonist, as well as MIP-1 beta, a peptide with pronounced homology to MIP-1 alpha, did not activate the eosinophil functions tested. Our results indicate that RANTES and MIP-1 alpha are crucial mediators of inflammatory processes in which eosinophils predominate.
Activation of human neutrophils by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) induces tyrosine phosphorylation of several polypeptides, including a prominent band of approximately 41 kDa. A polypeptide of identical electrophoretic mobility was recognized by a monoclonal antibody raised against a sequence corresponding to amino acids 325-345 of ERK-1, one of a family of mitogen-activated protein (MAP) kinases. To establish the possible identity of these polypeptides, extracts from control and fMLP-treated cells were immunoprecipitated with immobilized antiphosphotyrosine antibodies. Reactivity with anti-ERK-1 antibodies was observed only in the precipitate of chemoattractant-stimulated cells. These data imply that a MAP kinase constitutes at least part of the tyrosine-phosphorylated 41-kDa polypeptide. By using an in vitro renaturation assay, treatment of intact cells with fMLP was found to stimulate several protein kinases, including one of approximately 41 kDa. Renaturation of samples immunoprecipitated with antiphosphotyrosine antibodies revealed the presence of an active protein kinase in chemoattractant-stimulated, but not in control cells. The immunoprecipitated kinase comigrated with the 41-kDa tyrosine phosphorylated polypeptide and the anti-ERK-1 reactive band. We conclude that a MAP kinase closely related or identical to ERK-1 is tyrosine phosphorylated and activated when human neutrophils are stimulated by chemotactic peptides. The rapid phosphorylation of this kinase, which is apparent within seconds, is compatible with a role in the activation of the respiratory burst and/or other neutrophil responses.
MAP kinase is activated and phosphorylated during M phase of the Xenopus oocyte cell cycle, and induces the interphase-M phase transition of microtubule dynamics in vitro. We have carried out molecular cloning of Xenopus M phase MAP kinase and report its entire amino acid sequence. There is no marked change in the MAP kinase mRNA level during the cell cycle. Moreover, studies with an anti-MAP kinase antiserum indicate that MAP kinase activity may be regulated posttranslationally, most likely by phosphorylation. We show that MAP kinase can be activated by microinjection of MPF into immature oocytes or by adding MPF to cell-free extracts of interphase eggs. These results suggest that MAP kinase functions as an intermediate between MPF and the interphase-M phase transition of microtubule organization.
The leukocyte function-associated antigen 1 (LFA-1) molecule is well established as a surface protein involved in cellular adhesion and interaction, but there has been little information about whether engagement of this molecule can also directly modify cellular activation. These studies demonstrate that crosslinking the LFA-1 molecule on human T cell clones transmits a unique signal to the cell. Crosslinking LFA-1 alone did not increase intracellular calcium ([ CA2+]i), nor did crosslinking LFA-1 activate the cells as measured by IL-2 production or [3H]thymidine incorporation. However, when CD3 and LFA-1 were crosslinked, a more prolonged calcium signal was observed than when CD3 alone was crosslinked. Moreover, IL-2 production and DNA synthesis were greatly augmented. These responses could be demonstrated when LFA-1 was crosslinked via either the alpha or the beta chain, and required surface expression of the LFA-1 molecule as no enhancement was observed in T cell clones from a child with leukocyte adhesion deficiency. The enhancement of cellular activation by LFA-1 did not require that it be directly crosslinked to the CD3 complex. Thus, crosslinking LFA-1 alone with isotype-specific secondary antibodies on cells also pretreated with an anti-CD3 mAb of a different Ig isotype stimulated the cells as effectively as crosslinking both surface antigens with GaMIg. Similarly, a delayed, but sustained increase in [Ca2+]i was elicited. This increase in [Ca2+]i and the enhanced functional responses required engagement of CD3 with an intact bivalent anti-CD3 mAb, as crosslinking LFA-1 on cells also reacted with Fab fragments of an anti-CD3 mAb did not increase [Ca2+]i, nor activate the cells. These data indicate that LFA-1 can convey activation signals to T cells. Synergism in signaling can be observed upon crosslinking of LFA-1 and independently crosslinking CD3. In the physiologic interaction between T cells and accessory cells, the interaction of LFA-1 with its ligand, intercellular adhesion molecule 1, may therefore not only facilitate cellular adhesion, but also may amplify T cell activation by delivering costimulatory signals.
Endothelial cells were isolated from freshly obtained human umbilical cords by collagenase digestion of the interior of the umbilical vein. The cells were grown in tissue culture as a homogeneous population for periods up to 5 mo and some lines were subcultured for 10 serial passages. During the logarithmic phase of cell growth, cell-doubling time was 92 h. Light, phase contrast, and scanning electron microscopy demonstrated that cultured human endothelial cells grew as monolayers of closely opposed, polygonal large cells whereas both cultured human fibroblasts and human smooth muscle cells grew as overlapping layers of parallel arrays of slender, spindle-shaped cells. By transmission electron microscopy, cultured endothelial cells were seen to contain cytoplasmic inclusions (Weibel-Palade bodies) characteristic of in situ endothelial cells. These inclusions were also found in endothelial cells lining umbilical veins but were not seen in smooth muscle cells or fibroblasts in culture or in situ. Cultured endothelial cells contained abundant quantities of smooth muscle actomyosin. Cultured endothelial cells also contained ABH antigens appropriate to the tissue donor's blood type; these antigens were not detectable on cultured smooth muscle cells or fibroblasts. These studies demonstrate that it is possible to culture morphologically and immunologically identifiable human endothelial cells for periods up to 5 mo.
Treatment of cells with a variety of growth factors triggers a phosphorylation cascade that leads to activation of mitogen-activated protein kinases (MAPKs, also called extracellular signal-regulated kinases, or ERKs). We have identified a synthetic inhibitor of the MAPK pathway. PD 098059 [2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one] selectively inhibited the MAPK-activating enzyme, MAPK/ERK kinase (MEK), without significant inhibitory activity of MAPK itself. Inhibition of MEK by PD 098059 prevented activation of MAPK and subsequent phosphorylation of MAPK substrates both in vitro and in intact cells. Moreover, PD 098059 inhibited stimulation of cell growth and reversed the phenotype of ras-transformed BALB 3T3 mouse fibroblasts and rat kidney cells. These results indicate that the MAPK pathway is essential for growth and maintenance of the ras-transformed phenotype. Further, PD 098059 is an invaluable tool that will help elucidate the role of the MAPK cascade in a variety of biological settings.
Some agonists of G protein-coupled receptors, such as thrombin and lysophosphatidic acid (LPA), can promote cell proliferation via a pertussis toxin (PTX)-sensitive signaling pathway. While these agonists stimulate phospholipase C and inhibit adenylate cyclase, it appears that other, as-yet-unidentified, effector pathways are required for mitogenesis. Here we report that LPA and a thrombin receptor agonist peptide rapidly activate the protooncogene product p21ras in quiescent fibroblasts. This activation is inhibited by PTX and yet not attributable to known PTX-sensitive G protein pathways, including stimulation of phospholipases, inhibition of adenylate cyclase, or modulation of ion channels. LPA- and peptide-induced p21ras activation is inhibited by the tyrosine kinase inhibitor genistein, at doses that do not affect epidermal growth factor-induced p21ras activation. Thus, a heterotrimeric G protein of the Gi subfamily regulates activation of p21ras by LPA and thrombin, possibly through an intermediary tyrosine kinase. This pathway may critically participate in mitogenic signaling downstream from certain G protein-coupled receptors.
The mitogen-activated protein kinase (MAP kinase) pathway is thought to play an important role in the actions of neurotrophins. A small molecule inhibitor of the upstream kinase activator of MAP kinase, MAP kinase kinase (MEK) was examined for its effect on the cellular action of nerve growth factor (NGF) in PC-12 pheochromocytoma cells. PD98059 selectively blocks the activity of MEK, inhibiting both the phosphorylation and activation of MAP kinases in vitro. Pretreatment of PC-12 cells with the compound completely blocked the 4-fold increase in MAP kinase activity produced by NGF. Half-maximal inhibition was observed at 2 microM PD98059, with maximal effects at 10-100 microM. The tyrosine phosphorylation of immunoprecipitated MAP kinase was also completely blocked by the compound. In contrast, the compound was without effect on NGF-dependent tyrosine phosphorylation of the pp140trk receptor or its substrate Shc and did not block NGF-dependent activation of phosphatidylinositol 3'-kinase. However, PD98059 completely blocked NGF-induced neurite formation in these cells without altering cell viability. These data indicate that the MAP kinase pathway is absolutely required for NGF-induced neuronal differentiation in PC-12 cells.
Stimulation of Gi-coupled receptors leads to the activation of mitogen-activated protein kinases (MAP kinases). In several cell types, this appears to be dependent on the activation of p21ras (Ras). Which G-protein subunit(s) (G alpha or the G beta gamma complex) primarily is responsible for triggering this signaling pathway, however, is unclear. We have demonstrated previously that the carboxyl terminus of the beta-adrenergic receptor kinase, containing its G beta gamma-binding domain, is a cellular G beta gamma antagonist capable of specifically distinguishing G alpha- and G beta gamma-mediated processes. Using this G beta gamma inhibitor, we studied Ras and MAP kinase activation through endogenous Gi-coupled receptors in Rat-1 fibroblasts and through receptors expressed by transiently transfected COS-7 cells. We report here that both Ras and MAP kinase activation in response to lysophosphatidic acid is markedly attenuated in Rat-1 cells stably transfected with a plasmid encoding this G beta gamma antagonist. Likewise in COS-7 cells transfected with plasmids encoding Gi-coupled receptors (alpha 2-adrenergic and M2 muscarinic), the activation of Ras and MAP kinase was significantly reduced in the presence of the coexpressed G beta gamma antagonist. Ras-MAP kinase activation mediated through a Gq-coupled receptor (alpha 1-adrenergic) or the tyrosine kinase epidermal growth factor receptor was unaltered by this G beta gamma antagonist. These results identify G beta gamma as the primary mediator of Ras activation and subsequent signaling via MAP kinase in response to stimulation of Gi-coupled receptors.
Insulin stimulates tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and She in Rat1 fibroblasts overexpressing wild type insulin receptors. We investigated the relative role of IRS-1 and She in insulin activation of guanine nucleotide releasing factor (GNRF) and p21ras-GTP formation. The time course of insulin-stimulated tyrosine phosphorylation of IRS-1 was rapid, whereas Shc phosphorylation was relatively slow. Growth factor receptor bound protein-2 (Grb2) associated with IRS-1 rapidly and gradually dissociated after 5 min, whereas Grb2 association with Shc was slower and reached a maximum at 10 min after insulin stimulation. Thus, the kinetics of Grb2 association with IRS-1 and She corresponded closely to the time course of tyrosine phosphorylation of IRS-1 and Shc, respectively. Importantly, 3-13-fold more Grb2 was associated with Shc than with IRS-1. In addition, the kinetics of insulin-stimulated GNRF activity and p21ras-GTP formation corresponded more closely to the time course of Shc phosphorylation than to the kinetics of IRS-1 phosphorylation. Furthermore, immunoprecipitation of Shc proteins from cell lysates of insulin-stimulated cells removed 67% of the GNRF activity, whereas precipitation of IRS-1 had a negligible effect on GNRF activity. Thus, although both IRS-1 and Shc associate with Grb2, the current results indicate that Shc plays a more important role than IRS-1 in insulin stimulation of GNRF activity and subsequent p21ras-GTP formation.
An explosion of new information linking activation of cell surface signal initiators to changes in gene expression has recently emerged. The focus of much of this information has centered around the agonist-dependent activation of the mitogen-activated protein (MAP) kinases. Although this intracellular signal transduction pathway is extremely complex, conservation of many of its components has been observed in yeast, nematodes, Drosophila, and mammals. Thus, these signaling proteins may participate in the regulation of a variety of cellular processes.
Immune complexes are thought to be the major cause of cutaneous necrotizing vasculitis, but the mechanism of immune complex targeting to specific vessels is largely unknown. In myelomonocytic cells, immune complex binding and receptor-mediated endocytosis are mediated by Fc gamma R. We asked whether dermal microvascular endothelial cells (DMEC) express Fc gamma Rs. In cryostat sections of normal human skin, mAb IV.3 or AT10, both recognizing CD32 (Fc gamma RII), localizes to the luminal surface of DMEC of the superficial but not of the deep vascular plexus. All DMEC do not express CD16 (Fc gamma RIII) or CD64 (Fc gamma RI) molecules. Adult skin-derived DMEC in culture express CD32 (Fc gamma RII) molecules, as measured by FACS, but are negative for CD16 or CD64. HUVEC, tested for comparison, do not express CD16, 32, or 64 proteins. By reverse-transcriptase PCR and subsequent Southern blot analysis, the isoform of the CD32 molecule expressed on DMEC is determined as Fc gamma RIIa. HUVEC do not contain Fc gamma RIIa or Fc gamma RIIb mRNA. In DMEC, Fc gamma RIIa cross-linking results in immediate intracellular free Ca2+ ([Ca2+]i) concentration fluxes and in rapid internalization of the occupied receptors. We conclude that DMEC are equipped with fully functional Fc gamma RIIa molecules.
Eosinophil accumulation is a distinctive feature of lung allergic inflammation. Here, we have used a mouse model of OVA (ovalbumin)-induced pulmonary eosinophilia to study the cellular and molecular mechanisms for this selective recruitment of eosinophils to the airways. In this model there was an early accumulation of infiltrating monocytes/macrophages in the lung during the OVA treatment, whereas the increase in infiltrating T-lymphocytes paralleled the accumulation of eosinophils. The kinetics of accumulation of these three leukocyte subtypes correlated with the levels of mRNA expression of the chemokines monocyte chemotactic peptide-1/JE, eotaxin, and RANTES (regulated upon activation in normal T cells expressed and secreted), suggesting their involvement in the recruitment of these leukocytes. Furthermore, blockade of eotaxin with specific antibodies in vivo reduced the accumulation of eosinophils in the lung in response to OVA by half. Mature CD4+ T-lymphocytes were absolutely required for OVA-induced eosinophil accumulation since lung eosinophilia was prevented in CD4+-deficient mice. However, these cells were neither the main producers of the major eosinophilic chemokines eotaxin, RANTES, or MIP-1alpha, nor did they regulate the expression of these chemokines. Rather, the presence of CD4+ T cells was necessary for enhancement of VCAM-1 (vascular cell adhesion molecule-1) expression in the lung during allergic inflammation induced by the OVA treatment. In support of this, mice genetically deficient for VCAM-1 and intercellular adhesion molecule-1 failed to develop pulmonary eosinophilia. Selective eosinophilic recruitment during lung allergic inflammation results from a sequential accumulation of certain leukocyte types, particularly T cells, and relies on the presence of both eosinophilic chemoattractants and adhesion receptors.
Cross-linking the receptors for the Fc domain of IgG (Fc gamma R) on leukocytes induces activation of protein tyrosine kinases. The intermediary molecules that transduce to the nucleus the signals leading to induction of the diverse biological responses mediated by these receptors are not clearly identified. We have investigated whether mitogen-activated protein kinases (MAPK) are involved in transmembrane signaling via the three Fc gamma R present on monocytic, polymorphonuclear, and natural killer (NK) cells. Our results indicate that occupancy of Fc gamma RI and Fc gamma RII on the monocytic cell line THP-I and on polymorphonuclear leukocytes (PMN) induces, transiently and with fast kinetics, MAPK phosphorylation, as indicated by decreased electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and increased amounts of the proteins in antiphosphotyrosine antibody immunoprecipitates. This, associated with increased enzymatic activity, also occurs upon stimulation of the transmembrane isoform of CD16 (Fc gamma RIIIA) in NK cells and in a T cell line expressing transfected Fc gamma RIIIA alpha ligand-binding chain in association with zeta, but not upon stimulation of the glycosil-phosphatidylinositol-anchored Fc gamma RIIIB on PMN. Using the specific MAP kinase kinase inhibitor-PD 098059, we show that activation of MAPK is necessary for the Fc gamma R-dependent induction of c-fos and tumor necrosis factor alpha mRNA expression in monocytes and NK cells. These results underscore the role of MAPK as signal-transducing molecules controlling the expression of different genes relevant to leukocyte biology upon Fc gamma R stimulation.
Intercellular adhesion molecule 1 (ICAM-1) (CD54) is an adhesion molecule of the immunoglobulin superfamily. The interaction between ICAM-1 on B lymphocytes and leukocyte function-associated antigen 1 on T cells plays a major role in several aspects of the immune response, including T-dependent B cell activation. While it was originally believed that ICAM-1 played a purely adhesive role, recent evidence suggests that it can itself transduce biochemical signals. We demonstrate that cross-linking of ICAM-1 results in the up-regulation of class II major histocompatibility complex, and we investigate the biochemical mechanism for the signaling role of ICAM-1. We show that cross-linking of ICAM-1 on the B lymphoma line A20 induces an increase in tyrosine phosphorylation of several cellular proteins, including the Src family kinase p53/p56(lyn). In vitro kinase assays showed that Lyn kinase was activated within 1 min after ICAM-1 cross-linking. In addition, ICAM-1 cross-linking resulted in activation of Raf-1 and mitogen-activated protein kinases, as determined by gel mobility shift. Activation of these kinases may represent important components in the cascade of signals that link ICAM-1 to various ICAM-1-elicited cellular responses. These data confirm the important role of ICAM-1 as a signaling molecule in B cell activation.
Eosinophil infiltration of tissue is a hallmark of nasal polyposis in both nonatopic and atopic patients. These cells are thought to play a key role in the nasal polyp inflammatory process.
The objective of this study was to investigate whether cultured nasal polyps derived from nonatopic and atopic patients release RANTES both spontaneously and after phytohemagglutinin (PHA) stimulation.
Nasal polyps were obtained from 12 subjects (6 nonatopic and 6 atopic), cut into 2 to 3 mm large specimens, and cultured for 48 hours with or without PHA. RANTES was measured in the culture supernatant by ELISA (R&D Systems, U.K.).
Immunoreactive RANTES was found to be present in the culture supernatant of nasal polyps derived from both nonatopic and atopic patients with no difference between the two groups (median: 3.8 vs 2.9 pg/mg/ml). On incubation with PHA, nasal polyps from both nonatopic and atopic patients released sevenfold and 11-fold greater amounts of RANTES than unstimulated samples. As determined by immunohistochemistry, RANTES was localized to the vascular endothelium in nasal polyps from both groups of patients.
This study demonstrates that cultured nasal polyps derived from both nonatopic and atopic patients release RANTES spontaneously and after PHA stimulation. This observation and the finding that RANTES is present in nasal polyp endothelial cells suggest that this chemokine may be an important mediator of eosinophil and lymphocyte recruitment in both nonatopic and atopic nasal polyposis.
Publisher Summary This chapter focuses on interleukin-8 (IL-8) and related chemotactic cytokines—namely, CXC and CC chemokines. IL-8 is the best known member of a new class of cytokines that are widely studied because of their ability to attract and activate leukocytes, and their potential role as mediators of inflammation. IL-8 was originally isolated from the culture supernatants of stimulated human blood monocytes and was identified as a protein of 72 amino acids with a molecular weight of 8383. The three-dimensional structure of IL-8 has been studied by nuclear magnetic resonance spectroscopy and X-ray crystallography. In concentrated solution, and on crystallization, IL-8 is present as a dimer. The first CC chemokine was identified after cloning by differential hybridization from human tonsillar lymphocytes and was termed LD78. The CC and CXC chemokines are similar in size and have an overall structure that is characterized by the two intrachain disulfide bonds, short N-terminal and long C-terminal sequences. It discusses the role of chemokines in pathology with skin inflammation because psoriasis was the first disease to be linked to overproduction of IL-8. Several independent studies document the occurrence of high levels of IL-8 in the synovial fluid of inflamed joints of patients with different forms of rheumatic diseases, osteoarthritis, and gout.
The leukocyte function-associated antigen 1 (LFA-1) molecule is well established as a surface protein involved in cellular adhesion and interaction, but there has been little information about whether engagement of this molecule can also directly modify cellular activation. These studies demonstrate that crosslinking the LFA-1 molecule on human T cell clones transmits a unique signal to the cell. Crosslinking LFA-1 alone did not increase intracellular calcium ([ CA2+]i), nor did crosslinking LFA-1 activate the cells as measured by IL-2 production or [3H]thymidine incorporation. However, when CD3 and LFA-1 were crosslinked, a more prolonged calcium signal was observed than when CD3 alone was crosslinked. Moreover, IL-2 production and DNA synthesis were greatly augmented. These responses could be demonstrated when LFA-1 was crosslinked via either the alpha or the beta chain, and required surface expression of the LFA-1 molecule as no enhancement was observed in T cell clones from a child with leukocyte adhesion deficiency. The enhancement of cellular activation by LFA-1 did not require that it be directly crosslinked to the CD3 complex. Thus, crosslinking LFA-1 alone with isotype-specific secondary antibodies on cells also pretreated with an anti-CD3 mAb of a different Ig isotype stimulated the cells as effectively as crosslinking both surface antigens with GaMIg. Similarly, a delayed, but sustained increase in [Ca2+]i was elicited. This increase in [Ca2+]i and the enhanced functional responses required engagement of CD3 with an intact bivalent anti-CD3 mAb, as crosslinking LFA-1 on cells also reacted with Fab fragments of an anti-CD3 mAb did not increase [Ca2+]i, nor activate the cells. These data indicate that LFA-1 can convey activation signals to T cells. Synergism in signaling can be observed upon crosslinking of LFA-1 and independently crosslinking CD3. In the physiologic interaction between T cells and accessory cells, the interaction of LFA-1 with its ligand, intercellular adhesion molecule 1, may therefore not only facilitate cellular adhesion, but also may amplify T cell activation by delivering costimulatory signals.
Treatment of PC12 cells with nerve growth factor (NGF) induces a rapid increase in tyrosine phosphorylation of multiple cellular proteins. Expression of a dominant inhibitory Ras mutant specifically blocked NGF- and TPA-induced tyrosine phosphorylation of two proteins of approximately 42 and 44 kd. Conversely, expression of an oncogenic variant of Ras induced tyrosine phosphorylation of the same 42 and 44 kd proteins. The 44 kd protein was immunoprecipitated with an antibody directed against extracellular signal-regulated kinase 1/mitogen-activated protein kinase (MAPK) and the 42 kd protein comigrated with a 42 kd MAPK, indicating that at least one and probably both Ras-regulated phosphoproteins are MAPKs. In addition, MAPK activation, as measured by in vitro phosphorylation of myelin basic protein, was also regulated by Ras. Ras was not required for NGF-induced activation of Trk or tyrosine phosphorylation of PLC-gamma 1. Thus, NGF-induced tyrosine phosphorylation occurs both prior to and following Ras action, and Ras plays a critical role in the NGF- and TPA-induced tyrosine phosphorylation of MAPKs.
A synthetic peptide modeled after the major threonine (T669) phosphorylation site of the epidermal growth factor (EGF) receptor was an efficient substrate (apparent Km approximately 0.45 mM) for phosphorylation by purified p44mpk, a MAP kinase from sea star oocytes. The peptide was also phosphorylated by a related human MAP kinase, which was identified by immunological criteria as p42mapk. Within 5 min of treatment of human cervical carcinoma A431 cells with EGF or phorbol myristate acetate (PMA), a greater than 3-fold activation of p42mapk was measured. However, Mono Q chromatography of A431 cells extracts afforded the resolution of at least three additional T669 peptide kinases, some of which may be new members of the MAP kinase family. One of these (peak I), which weakly adsorbed to Mono Q, phosphorylated myelin basic protein (MBP) and other MAP kinase substrates, immunoreacted as a 42 kDa protein on Western blots with four different MAP kinase antibodies, and behaved as a approximately 45 kDa protein upon Superose 6 gel filtration. Another T669 peptide kinase (peak IV), which bound more tightly to Mono Q than p42mapk (peak II), exhibited a nearly identical substrate specificity profile to that of p42mapk, but it immunoreacted as a 40 kDa protein only with anti-p44mpk antibody on Western blots, and eluted from Superose 6 in a high molecular mass complex of greater than 400 kDa. By immunological criteria, the T669 peptide kinase in Mono Q peak III was tentatively identified as an active form of p34cdc2 associated with cyclin A. The Mono Q peaks III and IV kinases were modestly stimulated following either EGF or PMA treatments of A431 cells, and they exhibited a greater T669 peptide/MBP ratio than p42mapk. These findings indicated that multiple proline-directed kinases may mediate phosphorylation of the EGF receptor.
The mammalian shc gene encodes two overlapping, widely expressed proteins of 46 and 52K, with a carboxy-terminal SH2 domain that binds activated growth factor receptors, and a more amino-terminal glycine/proline-rich region. These shc gene products (Shc) are transforming when overexpressed in fibroblasts. Shc proteins become phosphorylated on tyrosine in cells stimulated with a variety of growth factors, and in cells transformed by v-src (ref. 2), suggesting that they are tyrosine kinase targets that control a mitogenic signalling pathway. Here we report that tyrosine-phosphorylated Shc proteins form a specific complex with a non-phosphorylated 23K polypeptide encoded by the grb2/sem-5 gene. The grb2/sem-5 gene product itself contains an SH2 domain, which mediates binding to Shc, and is implicated in activation of the Ras guanine nucleotide-binding protein by tyrosine kinases in both Caenorhabditis elegans and mammalian cells. Consistent with a role in signalling through Ras, shc overexpression induced Ras-dependent neurite outgrowth in PC12 cells. These results suggest that Shc tyrosine phosphorylation can couple tyrosine kinases to Grb2/Sem-5, through formation of a Shc-Grb2/Sem-5 complex, and thereby regulate the mammalian Ras signalling pathway.
An important process in the immune response is the migration of different populations of lymphocytes at the proper time to sites of antigenic challenge. Although several chemoattractants are known for broad classes of lymphocytes, such as T and B cells, the process by which lymphocytes of specific subsets, such as helper, cytotoxic or memory T cells, migrate to the appropriate sites remains obscure. Interleukin-8 is a chemoattractant for T cells and neutrophils and is a member of a superfamily of soluble molecules related by a conserved motif containing four cysteine residues. IL-8 and related molecules, including platelet factor 4, constitute the C-X-C class of the superfamily and a group of cytokines produced by haematopoietic cells constitute the RANTES/sis or C-C class. The roles of most of these molecules are not well known, although murine MIP-1 alpha of the C-C branch is a specific inhibitor of haematopoietic stem cell proliferation and some members of the C-X-C branch are neutrophil-targeted inflammatory agents. Here we report that the RANTES protein of the C-C class causes the selective migration of human blood monocytes and of T lymphocytes expressing the cell surface antigens CD4 and UCHL1. CD4+/UCHL1+T cells are thought to be prestimulated or primed helper T cells involved in memory T cell function. The preferential attraction of T-cell subsets by specific cytokines could in part explain how lymphocytes are targeted, and may provide insight into the workings of T cell memory.
Proliferative response of resting T cells generally requires not only cross-linking of the T cell receptor (TcR) but also co-stimulatory signals from accessory molecules. We here have used a "three-cell" model consisting of: (a) resting human CD4+ T cells as responders; (b) CD3 monoclonal antibody (mAb) OKT3 on latex beads as surrogate stimulators; (c) autologous monocytes as source of co-stimulation. As described by Kawakami et al. (J. Immunol. 1989, 142: 1818), T cell proliferation in this system is observed with paraformaldehyde-fixed monocytes if they have been activated and interleukin (IL) 1 beta/IL 6 is supplied. Since this three-cell system provides TcR cross-linking at a site spatially "remote" from co-stimulation, they help distinguish adhesion from signal transduction but the molecules that mediate co-stimulation in this system have not been identified. Our studies now demonstrate that co-stimulation by the monocytes is dependent on each of two receptor/ligand pathways CD2/LFA-3 and LFA-1/ICAM-1 since it is inhibited by each relevant mAb but not a variety of control mAb. The hypotheses that CD2 and LFA-1 could each mediate co-stimulation was tested in simplified model systems in which the monocyte was replaced with immobilized CD2 mAb or purified ICAM-1 presented on a separate surface from the CD3 mAb. The results in these simplified models demonstrate that on resting T cells either CD2 or LFA-1 molecules alone can mediate "remote" co-stimulation unlike most other T cell surface molecules. Co-stimulation requires IL 1 beta/IL6 both in the weaker LFA-1 ligand-mediated co-stimulation and at lower CD2 mAb concentrations in the stronger CD2 mAb-mediated co-stimulation. Thus: (a) the accessory cell function of stimulated fixed monocytes in T cell proliferation requires both the LFA-1/ICAM-1 and CD2/LFA-3 pathways; and (b) the T cell molecules CD2 and LFA-1 can give co-stimulatory signals that can act in a "remote" fashion.
Production of the neutrophil-activating peptide (NAP)-1/IL-8 by mononuclear phagocytes from patients with RA and from control subjects was studied under various conditions. Mononuclear cells from bone marrow (BMMC), PBMC, and synovial fluid (SFMC) were cultured for up to 48 h in the absence or presence of Escherichia coli LPS, different interleukins, interferon-gamma, zymosan, or immune complexes, and the neutrophil-stimulating activity released into the culture medium was determined. As shown by neutralization with an antiserum raised against human recombinant NAP-1/IL-8, over 90% of this activity could be attributed to NAP-1/IL-8. In unstimulated mononuclear cells from control individuals and BMMC from RA patients, the production of NAP-1/IL-8 was very low and was enhanced moderately by stimulation with LPS. By contrast, the spontaneous production of NAP-1/IL-8 was 3- to 10-fold higher in PBMC and even much higher in SFMC from RA patients. In all instances, the yield of NAP-1/IL-8 could be enhanced by stimulation in culture. In addition to LPS, rheumatoid factor-containing immune complexes, zymosan, and IL-1 were highly effective in inducing NAP-1/IL-8 production, while IL-3, GM-CSF, tumor necrosis factor (TNF), and IL-2 were somewhat less potent. An inhibitory effect was obtained with IFN-gamma, which significantly decreased the spontaneous NAP-1/IL-8 release from SFMC and the IL-1- and LPS-induced NAP-1/IL-8 from RA and control PBMC. Inhibition was also observed with glucocorticoids. The production of NAP-1/IL-8 was markedly reduced by dexamethasone in phagocytosis-stimulated PBMC, and almost totally inhibited in SFMC obtained from joints after intraarticular administration of betamethasone. By contrast, the cyclooxygenase inhibitor, indomethacin, tended to increase the NAP-1/IL-8 yield from PBMC in culture.
Cells of the synovial microenvironment may recruit neutrophils (PMN) and lymphocytes into synovial fluid, as well as lymphocytes into the synovial tissues, of arthritic patients. We have investigated the production of the chemotactic cytokine IL-8 by using sera, synovial fluid, synovial tissue, and macrophages and fibroblasts isolated from synovial tissues from 75 arthritic patients. IL-8 levels were higher in synovial fluid from rheumatoid (RA) patients (mean +/- SE, 14.37 +/- 5.8 ng/ml), compared with synovial fluid from osteoarthritis patients (0.135 +/- 17 ng/ml) (p less than 0.05) or from patients with other arthritides (5.52 +/- 5.11 ng/ml). IL-8 from RA sera was 8.44 +/- 2.33 ng/ml, compared with nondetectable levels found in normal sera. IL-8 levels from RA sera and synovial fluid were strongly positively correlated (r = 0.96, p less than 0.05). Moreover, RA synovial fluid chemotactic activity for PMN in these fluids was inhibited 40 +/- 5% upon incubation with neutralizing polyclonal antibody to IL-8. Synovial tissue fibroblasts released only small amounts of constitutive IL-8 but could be induced to produce IL-8 by stimulation with either IL-1 beta, TNF-alpha, or LPS. In contrast, unlike normal PBMC or alveolar macrophages, macrophages isolated from RA synovial tissue constitutively expressed both IL-8 mRNA and antigenic IL-8. RA synovial macrophage IL-8 expression was not augmented by incubation with either LPS, TNF-alpha, or IL-1 beta. Immunohistochemical analysis of synovial tissue showed that a greater percentage of RA macrophages than osteoarthritis macrophages reacted with anti-IL-8. Whereas macrophages were the predominant cell for immunolocalization of IL-8, less than 5% of synovial tissue fibroblasts were positive for immunolocalized IL-8. These results suggest that macrophage-derived IL-8 may play an important role in the recruitment of PMN in synovial inflammation associated with RA.
The adhesive interactions of cells with other cells and with the extracellular matrix are crucial to all developmental processes, but have a central role in the functions of the immune system throughout life. Three families of cell-surface molecules regulate the migration of lymphocytes and the interactions of activated cells during immune responses.
We recently described the purification and cloning of extracellular signal-regulated kinase 1 (ERK1), which appears to play a pivotal role in converting tyrosine phosphorylation into the serine/threonine phosphorylations that regulate downstream events. We now describe cloning and characterization of two ERK1-related kinases, ERK2 and ERK3, and provide evidence suggesting that there are additional ERK family members. At least two of the ERKs are activated in response to growth factors; their activations correlate with tyrosine phophorylation, but also depend on additional modifications. Transcripts corresponding to the three cloned ERKs are distinctly regulated both in vivo and in a differentiating cell line. Thus, this family of kinases may serve as intermediates that depend on tyrosine phosphorylation to activate serine/threonine phosphorylation cascades. Individual family members may mediate responses in different developmental stages, in different cell types, or following exposure to different extracellular signals.
Human neutrophils exposed to protein-coated polystyrene or cultured endothelial monolayers produce large quantities of H2O2 in response to soluble stimuli that elicit little or no secretion of reactive oxygen species from cells in suspension. To characterize the mechanisms involved in this adherence-dependent respiratory burst, we have investigated the possible role of one integrin known to participate in the adhesion of neutrophils to endothelial cells, CD11b/CD18 (Mac-1). H2O2 production was examined with chemotactic factor-stimulated human and canine neutrophils exposed to protein-coated surfaces and cultured human and canine endothelial cells. The two protein-coated surfaces used were type I collagen-coated glass or plastic, a surface to which neither human nor canine neutrophils adhered, and keyhole limpet hemocyanin (KLH)-coated glass or plastic, a surface to which human and canine neutrophils adhered only after chemotactic stimulation. FMLP-stimulated human neutrophils and platelet activating factor-stimulated canine neutrophils failed to produce detectable H2O2 when in contact with type I collagen, but secreted large amounts of H2O2 when adherent to KLH or endothelial cell monolayers. FMLP-stimulated neutrophils from patients with CD18-deficiency failed to adhere to any of these surfaces and failed to produce H2O2 under these conditions. mAb reactive with CD18 and CD11b were equally effective in markedly inhibiting the adhesion of normal human neutrophils to these surfaces and markedly inhibited the production of H2O2. A mAb reactive with CD18 blocked adhesion of stimulated canine neutrophils, and mAb directed against both CD18 and CD11b blocked H2O2 production by canine neutrophils on KLH and endothelium. A nonbinding mAb and a mAb reactive with CD11a did not inhibit H2O2 production of human cells on KLH or endothelial monolayers, and nonbinding and binding control mAb did not inhibit H2O2 production by canine neutrophils. These results indicate that Mac-1 (CD11b/CD18) can mediate adhesion-dependent H2O2 production by human and canine neutrophils exposed to chemotactic factors.
The presence of neutrophils in the synovial joint of patients with rheumatoid arthritis (RA) is thought to be due to the activity of chemotactic factors released by activated cells in the joint. We have shown in this report, for the first time, the abundance of one such factor, interleukin 8 (IL 8), in the synovial fluid of patients both with RA and other non-RA joint diseases, and the spontaneous production of IL 8 mRNA by RA synovial cells in culture. There was no correlation between the levels of chemotactic activity and IL 8 protein, suggesting that other factors with similar neutrophil chemotactic activity are also present in the synovial fluid exudate. In support of this concept neither the level of chemotactic activity nor IL 8 protein levels correlated with neutrophil or leukocyte infiltration, indicating that the mechanism of migration into the inflammatory environment of the joint is complex. Such migration is likely to be due to a number of chemotactic signals in addition to IL 8, which may either synergize with, or inhibit, the action of IL 8.
Human endothelial cells produced a neutrophil chemotactic factor (NCF) upon stimulation with tumor necrosis factor-alpha (TNF-alpha),
interleukin-1 beta (IL-1 beta), or lipopolysaccharide (LPS). The expression of endothelial cell-derived NCF messenger RNA
and biological activity was both time- and concentration-dependent. Maximal NCF mRNA expression occurred at 10 and at 2 nanograms
per milliliter for TNF and IL-1 beta, respectively; mRNA expression was first observed 1 hour after stimulation and was maintained
for at least 24 hours. In situ hybridization analysis showed that NCF mRNA peaked in treated cells by 24 hours, whereas unstimulated
cells were negative. These studies demonstrated that endothelial cells may participate in neutrophil-mediated inflammation
by synthesizing a chemotactic factor in response to specific monokines and LPS.
PD 098059 has been shown previously to inhibit the dephosphorylated form of mitogen-activated protein kinase kinase-1 (MAPKK1) and a mutant MAPKK1(S217E,S221E), which has low levels of constitutive activity (Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 7686-7689). Here we report that PD 098059 does not inhibit Raf-activated MAPKK1 but that it prevents the activation of MAPKK1 by Raf or MEK kinase in vitro at concentrations (IC50 = 2-7 microM) similar to those concentrations that inhibit dephosphorylated MAPKK1 or MAPKK1(S217E,S221E). PD 098059 inhibited the activation of MAPKK2 by Raf with a much higher IC50 value (50 microM) and did not inhibit the phosphorylation of other Raf or MEK kinase substrates, indicating that it exerts its effect by binding to the inactive form of MAPKK1. PD 098059 also acts as a specific inhibitor of the activation of MAPKK in Swiss 3T3 cells, suppressing by 80-90% its activation by a variety of agonists. The high degree of specificity of PD 098059 in vitro and in vivo is indicated by its failure to inhibit 18 protein Ser/Thr kinases (including two other MAPKK homologues) in vitro by its failure to inhibit the in vivo activation of MAPKK and MAP kinase homologues that participate in stress and interleukin-1-stimulated kinase cascades in KB and PC12 cells, and by lack of inhibition of the activation of p70 S6 kinase by insulin or epidermal growth factor in Swiss 3T3 cells. PD 098059 (50 microM) inhibited the activation of p42MAPK and isoforms of MAP kinase-activated protein kinase-1 in Swiss 3T3 cells, but the extent of inhibition depended on how potently c-Raf and MAPKK were activated by any particular agonist and demonstrated the enormous amplification potential of this kinase cascade. PD 098059 not only failed to inhibit the activation of Raf by platelet-derived growth factor, serum, insulin, and phorbol esters in Swiss 3T3 cells but actually enhanced Raf activity. The rate of activation of Raf by platelet-derived growth factor was increased 3-fold, and the subsequent inactivation that occurred after 10 min was prevented. These results indicate that the activation of Raf is suppressed and that its inactivation is accelerated by a downstream component(s) of the MAP kinase pathway.
Intercellular adhesion molecules (ICAM)-1 and -3 coexist on T lymphocytes and are counter-receptors for the integrin LFA-1. Signaling through ICAM-3 stimulates a number of T cell functions and involves phosphorylation of Fyn, Lck, CD45, and other proteins. In contrast, this type of specific signaling event has not been described for signaling through ICAM-1. Here, tyrosine phosphorylation of cellular proteins was examined after cross-linking of ICAM-1. Tyrosine phosphorylation of the 34-kDa cdc2 protein kinase was induced transiently after stimulation of the leukemic T cell line, Molt-3, or peripheral blood T cells. Stimulation through ICAM-1 had no effect on constitutive presence of cdc2 or phosphorylation of cdc2 on threonine. cdc2 kinase activity was constitutive in peripheral blood T cells, and transient inhibition of kinase activity after ICAM-1 stimulation correlated kinetically with phosphorylation of cdc2 on tyrosine.
Cell adhesion molecules were first described as accessory molecules simply to bridge one cell to another. More recently, it has been realized that these molecules also transmit signals from outside of the cell to inside. We show that cross-linking of the ICAM-1 on the cell membrane with anti-ICAM-1 mAb and F(ab')2 fragments of goat anti-MIgG in the presence of suboptimal levels of the bacterial peptide FMLP results in co-stimulation of an oxidative burst from CD14 expressing PBMCs. The amplitude of the oxidative response was less than the oxidative burst induced by CD18 cross-linking, whereas the response was more prolonged. On the other hand, cross-linking by anti-L-selectin mAb plus F(ab')2 fragments of goat anti-MIgG induced a minimal oxidative burst that was not significantly greater than the response generated by anti-L-selectin mAb alone. The addition of an excess of soluble ICAM-1 to compete for the anti-ICAM-1 mAb inhibits the oxidative burst in response to ICAM-1 cross-linking but not to CD18 cross-linking. These results suggest that ICAM-1 is capable of delivering a transmembrane signal into CD14-positive PBMC.
Chemokines are a family of low molecular mass proteins with chemotactic and cell activating activities. Reverse transcription-polymerase chain reaction and Northern hybridization were used to examine their expression during murine experimental allergic encephalomyelitis (EAE), an autoimmune disease used as a model of multiple sclerosis. The mRNAs encoding RANTES, MIP-1 alpha, MIP-1 beta, TCA3 (I-309), IP-10, JE (MCP-1), KC (MGSA/gro), and MARC (MCP-3) were induced in the spinal cord 1-2 days before clinical signs were apparent. SDF, a cDNA predicted to encode a chemokine-like product, was expressed in normal as well as diseased spinal cords. No expression of C10 or MIP-2 was detected. Activated encephalitogenic T cells expressed message for RANTES, MIP-1 alpha, MIP-1 beta, and TCA3. These results define a subset of chemokines that may play an important role in the inflammatory process during murine EAE.
Mitogen-activated protein (MAP) kinases mediate the phosphorylation and activation of nuclear transcription factors that regulate cell growth. MAP kinase activation may result from stimulation of either tyrosine-kinase (RTK) receptors, which possess intrinsic tyrosine kinase activity, or G-protein-coupled receptors (GPCR). RTK-mediated mitogenic signalling involves a series of SH2- and SH3-dependent protein-protein interactions between tyrosine-phosphorylated receptor, Shc, Grb2 and Sos, resulting in Ras-dependent MAP kinase activation. The beta gamma subunits of heterotrimeric G proteins (G beta gamma) also mediate Ras-dependent MAP kinase activation by an as-yet unknown mechanism. Here we demonstrate that activation of MAP kinase by Gi-coupled receptors is preceded by the G beta gamma-mediated tyrosine phosphorylation of Shc, leading to an increased functional association between Shc, Grb2 and Sos. Moreover, disruption of the Shc-Grb2-Sos complex blocks G beta gamma-mediated MAP kinase activation, indicating that G beta gamma does not mediate MAP kinase activation by a direct interaction with Sos. These results indicate that G beta gamma-mediated MAP kinase activation is initiated by a tyrosine phosphorylation event and proceeds by a pathway common to both GPCRs and RTKs.
Stimulation of human polymorphonuclear neutrophils with the chemotactic peptide FMLP induces an increase in the phosphorylation of several proteins on tyrosine residues. Immunoblotting of whole cell lysates with antiphosphotyrosine antibodies demonstrated this increase to be predominant in two proteins with apparent m.w. of 40,000 and 42,000, respectively. We identify these two proteins as members of the mitogen-activated-protein (MAP) kinase family. These results are based on comigration of the tyrosine phosphorylated proteins with two proteins of same apparent m.w., recognized by a series of antibodies raised against sequences contained within members of this family. This was further confirmed by immunoprecipitation with either an antiphosphotyrosine antibody or a MAP kinase-specific antibody. To assess whether neutrophil stimulation with FMLP increased MAP kinase activity, the cells were stimulated with various doses of FMLP and the phosphotransferase activity in cell lysates was measured against two identified substrates of MAP kinase: myelin basic protein and T-669, the epidermal growth factor receptor Thr 669 synthetic peptide modeled after the major site of phosphorylation of the receptor. A dose- and time-dependent increase in MAP kinase activity was detected in the lysates from FMLP-stimulated PMN, which closely correlated with the dose and time course of tyrosine phosphorylation of the 40- and 42-kDa proteins. Additionally, the MAP kinases in crude lysates were enriched by DEAE-cellulose chromatography and further resolved on a MonoQ column by fast performance liquid chromatography. The respective eluates contained the two proteins, each recognized by anti-MAP kinase antibodies and, after stimulation, each was phosphorylated on tyrosine residues and expressed T-669 phosphotransferase activity. Treatment with genistein, a tyrosine kinase inhibitor, reduced the tyrosine phosphorylation of the 40- and 42-kDa proteins in a dose-dependent manner, decreased the activation of the MAP kinases, and inhibited the production of superoxide anion by FMLP-stimulated neutrophils. Therefore, this study demonstrates that human polymorphonuclear neutrophils contain two distinct members of the MAP kinase family, antigenically related to sea star p44mpk and rat p43erk1 and that both MAP kinase isoforms are implicated in the cascade of protein phosphorylation induced by neutrophil stimulation with FMLP. Furthermore, these data suggest that tyrosine phosphorylation and activation of these two enzymes may play a regulatory role in the signal transduction pathway leading to the respiratory burst induced by FMLP.
Leukocytes adhere to target cells through their integrins and play a crucial role in self-defense, inflammation, and differentiation. Intercellular adhesion molecule-1 (ICAM-1; CD54) is a representative ligand for integrins and is expressed on many cell types, some of which are targets for leukocyte adhesion. Recent studies suggest that adhesion molecules function not only as a cellular glue, but also as a signal transducer. However, it remains to be clearly defined whether engagement of ICAM-1 is able to induce activation signals in target cells. In rheumatoid synovium, synovial cells are known to express abundant ICAM-1 and produce multiple inflammatory cytokines, such as IL-1beta. In this study, we provide the first evidence that ICAM-1 engagement induces activation of the transcription factor AP-1 and transcription of the IL-1beta gene using a specific Ab to cross-link ICAM-1 on a rheumatoid synovial cell line (E11 cells). This evidence includes ICAM-1 cross-linking-dependent induction of 1) in situ IL-1beta transcription and protein synthesis, 2) transiently transfected chloramphenicol acetyltransferase (CAT) reporter plasmids containing both the IL-1beta LPS-responsive enhancer (between -3134 and -2729) as well as multiple copies of an AP-1 site from this enhancer (between -3117 and -3111), and 3) the binding of a Jun/Fos family complex to this AP-1 site. Thus, ICAM-1 not only functions as a glue for integrin binding, but also as a transducer for AP-1 activation signals important for IL-1beta gene transcription.
Fc gamma R cross-linking on murine macrophages resulted in the activation of mitogen-activated protein kinase (MAPK) family members p42MAPK, p38, and c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase (SAPK). The temporal pattern of activation was distinct for each kinase. p42MAPK activation peaked at 5 min after receptor cross-linking, while peak p38 activity occurred 5 to 10 min later. Maximal JNK/SAPK activation occurred 20 min after Fc gamma R cross-linking. The selective MAPK/extracellular signal-regulated kinase-1 (MEK-1) inhibitor PD 098059 inhibited activation of p42MAPK induced by Fc gamma R cross-linking, but not p38 or JNK/SAPK activation. PD 098059 also inhibited the synthesis of TNF-alpha induced by Fc gamma R cross-linking (IC50 approximately 0.1 microM). Together, these results suggest that 1) the activation of MAPKs may play a role in Fc gammaR signal transduction, and 2) the activation of p42MAPK is necessary for Fc gamma R cross-linking-induced TNF-alpha synthesis.
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