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The human cytokine I-309 is a monocyte chemoattractant
Abstract
The human cytokine I-309 is a small glycoprotein secreted by activated T lymphocytes and structurally related to a number of inflammatory cytokines. To investigate the biological activities of I-309 protein, we produced a stable Chinese hamster ovary cell transfectant, CDI.10, which constitutively secretes I-309 protein into culture supernatant. Affinity chromatography on a heparin-Sepharose matrix followed by reverse-phase HPLC was used to purify to homogeneity a glycoprotein doublet of 15-16 kDa from culture supernatant. Biochemical analysis showed the purified recombinant I-309 glycoprotein to be indistinguishable from the natural I-309 glycoprotein constitutively secreted by the T-cell line IDP2. Purified recombinant I-309 stimulated migration of human monocytes but not neutrophils when tested by in vitro chemotaxis assay. Furthermore, the purified protein transiently increased cytoplasmic free calcium concentration in human peripheral blood monocytes but did not do so in lymphocytes or neutrophils. These results demonstrate that the I-309 gene encodes an inflammatory mediator that specifically stimulates human monocytes.
... Miller and Krangel (3,25) identified the human homologue of TCA3, termed 1309. Sequence homology indicated that TCA3 and I309 proteins were only 42% homologous, but these are the only chemokines that possess a third conserved pair of cysteines, express a lysine residue at the N-terminus, use a single N-glycosylation site, and share similar patterns of tissue expression (26). ...
... Sequence homology indicated that TCA3 and I309 proteins were only 42% homologous, but these are the only chemokines that possess a third conserved pair of cysteines, express a lysine residue at the N-terminus, use a single N-glycosylation site, and share similar patterns of tissue expression (26). Although I309 is chemotactic for human monocytes in vitro it has no affect on human neutrophils (3,25). The failure of I309 to affect human neutrophils may reflect technical differences or interspecies variation. ...
The murine beta-chemokine TCA3 was purified to homogeneity. The biologic activities of the purified glycoprotein were evaluated in vivo and in vitro. Mice injected i.p. with 1- to 100-ng purified rTCA3 exhibited a rapid influx of neutrophils and macrophages. Increased numbers of neutrophils and monocytes were observed in peripheral blood within 15 min and peak at 45 min. After 45 min neutrophil and macrophage levels were increased in the peritoneal exudate with peak levels occurring at 2 h, followed by a subsequent decline by 24 h. Inflammatory responses were induced in a dose-dependent fashion. The in vivo inflammatory responses were mirrored by the pattern of TCA3-induced chemotaxis in vitro. Neutrophils and macrophages responded to similar concentrations of TCA3 (3 x 10(-9) to 10(-8) M). Lymph node cells responded to other chemokines but did not migrate to TCA3. We also demonstrated that rTCA3 stimulates a transient increase in cytoplasmic free calcium in monocytic cells through a PTX-sensitive pathway. Cross-desensitization studies indicate that TCA3 acts independently of other beta-chemokines (MIP-1 alpha and RANTES) and the alpha-chemokine IL-8. Furthermore, TCA3 does not induce a Ca2 lux in cells transfected with cDNA for the C-C CKR-1 chemokine receptor, supporting the conclusion that there are distinct receptors for TCA3.
... One of the proteins that portrayed the greatest expression change following the chlorogenic acid treatment was the small inducible cytokine A1 (CCL1) or I-309. This is the first identified CCL chemokine secreted by activated T lymphocytes [23]. Although CCL1 is mainly expressed in monocytes, mast cells, and dendritic cells in the skin, the CCL1-CCR8 signaling is yet to be clearly understood, mainly because its expression by inflammatory signals is controversial. ...
... Another limitation of this study is that we could not draw a specific conclusion regarding the immunological responses of keratinocytes induced by chlorogenic acid treatment because it triggered pleiotropic responses. Some of the cytokines that were significantly upregulated with chlorogenic acid treatment are associated with hypersensitive inflammatory disorders, including atopic dermatitis [23,29]. Therefore, further studies are required to correlate the functions of chlorogenic acid with the development of these diseases. ...
Chlorogenic acid is one of the most abundant polyphenols found in human diet. It is well-documented that chlorogenic acid has a significant impact on human cells, especially in the regulation of inflammation and metabolic processes. However, its role in regulating skin functions, especially with respect to the dermal collagen network or epidermal skin barrier, has not yet been elucidated. Here, we report that chlorogenic acid treatment can induce production of procollagen type I in human dermal fibroblast, Hs68 cell lines. Moreover, this treatment can stimulate upregulation of skin barrier genes, including the ones encoding filaggrin (FLG), involucrin (IVL), and envoplakin (EVPL), in epidermal keratinocytes. Chlorogenic acid also triggered a multifaceted response in the cytokine profile of keratinocytes. Therefore, we suggest that chlorogenic acid can be used to restore the impaired dermal matrix network as well as the epidermal skin barrier.
... Most prominently, CCL1, CCL17, CD206, FAP, SNAI1 and VEGFA were strongly upregulated in M0-macrophages. CCL1 and CCL17 are cytokines functioning as a chemoattractants for immune cells, like monocytes, NK cells, as well as T-and B cells [78][79][80]. CD206 (MRC1) is a membrane receptor mediating endocytosis of glycoproteins by macrophages, and FAP (fibroblast-activation protein) is a membrane gelatinase / serine protease postulated to regulate FB growth, epithelial-mesenchymal interactions and ECM re-modelling during tissue repair and carcinogenesis [81]. SNAI1 is involved in epithelial-mesenchymal-transition (EMT) by promoting repression of E-Cadherin, as well as triggering tumor growth and metastasis in breast carcinoma cells, while VEGFA is a mitogen promoting angiogenesis, Fig. 6. ...
Germ cell tumors (GCT) are the most common solid tumors in young men of age 15 - 40. In previous studies, we profiled the interaction of GCT cells with cells of the tumor microenvironment (TM), which showed that especially the 3D interaction of fibroblasts (FB) or macrophages with GCT cells influenced the growth behavior and
cisplatin response as well as the transcriptome and secretome of the tumor cells, suggesting that the crosstalk of these cells with GCT cells is crucial for tumor progression and therapy outcome. In this study, we shed light on the mechanisms of activation of cancer-associated fibroblasts (CAF) in the GCT setting and their effects on GCT cells lines and the monocyte cell line THP-1. Ex vivo cultures of GCT-derived CAF were established and characterized molecularly and epigenetically by performing DNA methylation arrays, RNA
sequencing, and mass spectrometry-based secretome analysis.
We demonstrated that the activation state of CAF is influenced by their former prevailing tumor environment in which they have resided. Hereby, we postulate that seminoma (SE) and embryonal carcinoma (EC) activate CAF, while teratoma (TER) play only a minor role in CAF formation. In turn, CAF influence proliferation and the
expression of cisplatin sensitivity-related factors in GCT cells lines as well as polarization of in vitro-induced macrophages by the identified effector molecules IGFBP1, LGALS3BP, LYVE1, and PTX3.
Our data suggests that the vital interaction of CAF with GCT cells and with macrophages has a huge influence on shaping the extracellular matrix as well as on recruitment of immune cells to the TM. In conclusion, therapeutically interfering with CAF and / or macrophages in addition to the standard therapy might slow-down
progression of GCT and re-shaping of the TM to a tumor-promoting environment.
... Although we observed no significantly different levels of cytokines/chemokines in neonates from MAR+ mothers compared to neonates from AB− and AB+ mothers after adjusting for covariates, we observed several notable differences when assessing their effect sizes. Neonates from MAR+ mothers had lower levels of the chemokines I-309 (CCL1) and eotaxin-1, as well as IL-4 when compared to neonates from AB− mothers, all of which are considered to be involved in the Th2 immune response (Miller and Krangel 1992;Teran et al. 1999). Notably, when we compared the effect sizes of neonates from AB+ mothers to those of neonates from MAR+ mothers, we found that the MAR+ group had higher levels of the proinf lammatory cytokine IL-16, which preferentially activates Th1 cells (Wilson et al. 2004). ...
Associations between maternal immune dysregulation (including autoimmunity and skewed cytokine/chemokine profiles) and offspring neurodevelopmental disorders such as autism have been reported. In maternal autoantibody-related autism, specific maternally derived autoantibodies can access the fetal compartment to target eight proteins critical for neurodevelopment. We examined the relationship between maternal autoantibodies to the eight maternal autoantibody-related autism proteins and cytokine/chemokine profiles in the second trimester of pregnancy in mothers of children later diagnosed with autism and their neonates’ cytokine/chemokine profiles. Using banked maternal serum samples from 15 to 19 weeks of gestation from the Early Markers for Autism Study and corresponding banked newborn bloodspots, we identified three maternal/offspring groups based on maternal autoantibody status: (1) mothers with autoantibodies to one or more of the eight maternal autoantibody-related autismassociated proteins but not a maternal autoantibody-related autism-specific pattern, (2) mothers with a known maternal autoantibody-related autism pattern, and (3) mothers without autoantibodies to any of the eight maternal autoantibody-related autism proteins. Using a multiplex platform, we measured maternal second trimester and neonatal cytokine/chemokine levels. This combined analysis aimed to determine potential associations between maternal autoantibodies and the maternal and neonatal cytokine/chemokine profiles, each of which has been shown to have implications on offspring neurodevelopment independently.
... The effect of C. angustifolia leave extract on the modulation production of inflammatory cytokines as IL-10, IL-1 alpha and beta, and TNF-alpha, the modulation caused increasing or decreasing influence for promote inflammation and limiting immune response to pathogens and stimulating macrophage, give T cell development and differentiation (26)(27)(28). Allium cepa (onion) was consumed through the world, it has many phytochemical as sapoines, quercetin, falvonoids, cepanens, phenolic compounds, and organ sulfurs all the antioxidant and immunomodulatory effect by reduced Th cell cytokines but increasing CD4 cells cytokines (29). Chemokines I-TAC is a mediator to the T cells migration and activation and it is role influence by INF-gamma level (30). ...
Cassia angustifolia Vahl. is a medicinal plant known for its efficacy in treating various, including respiratory conditions and skin inflammation. It possesses antibacterial and anticancer properties. This work investigated the immunomodulatory and anti-inflammatory effects of C. angustifolia. The ethanol leaf extract of C. angustifolia was utilized to examine gene expression related to angiogenesis cytokines in RAW 264.7 macrophage cells. The results demonstrated a significant increase in the viability of treated macrophage RAW 264.7 cells, accompanied by an improvement in angiogenesis cytokines expression and a dose-dependent inhibition of nitric oxide production. GC-MS analysis identified 11 active components within the extract, each exhibiting distinct biological activities such as antioxidant, antitumor and anti-inflammatory effects. Notable compounds include hexadecanoic acid, 2-pentadecanone, phthalic acid, oxalic acid, carbonic acid, tricosane, undecanal and many others. In conclusion, ethanol leaf extract of C. angustifolia exhibits immunomodulatory and anti-inflammatory effects by inhibiting nitric oxide production and enhancing the expression of angiogenesis cytokines.
... The CCL1 ligand, glycosylated at N52 as expected 39 , is intricately engaged with CCR8 through an interface formed by contributions from the receptor N-terminus and all three ECLs (Fig. 3a). This extensive interaction interface, mediated by multiple key polar interactions, can be grouped into previously defined chemokine recognition sites (CRS) 1, 1.5 and 2 26,27,30,40 and a unique binding site within CRS2 that appears critical to CCL1-CCR8 binding, termed CRS2.5 ( Fig. 3b-d). ...
The C-C motif chemokine receptor 8 (CCR8) is a class A G-protein coupled receptor that has emerged as a promising therapeutic target in cancer. Targeting CCR8 with an antibody has appeared to be an attractive therapeutic approach, but the molecular basis for chemokine-mediated activation and antibody-mediated inhibition of CCR8 are not fully elucidated. Here, we obtain an antagonist antibody against human CCR8 and determine structures of CCR8 in complex with either the antibody or the endogenous agonist ligand CCL1. Our studies reveal characteristic antibody features allowing recognition of the CCR8 extracellular loops and CCL1-CCR8 interaction modes that are distinct from other chemokine receptor - ligand pairs. Informed by these structural insights, we demonstrate that CCL1 follows a two-step, two-site binding sequence to CCR8 and that antibody-mediated inhibition of CCL1 signaling can occur by preventing the second binding event. Together, our results provide a detailed structural and mechanistic framework of CCR8 activation and inhibition that expands our molecular understanding of chemokine - receptor interactions and offers insight into the development of therapeutic antibodies targeting chemokine GPCRs.
... CCR8 is activated by CCL1 and mediates the recruitment of T helper 2 cells to sites of inflammation [95]. Notably, CCL1 has no significant chemotactic effect on neutrophils [96]. Olsen et al. [95] have demonstrated that elevated circulating CCL1 is strongly associated with prognosis and mortality in colorectal cancer. ...
Background: Cardiovascular diseases, particularly acute myocardial infarction, are the leading cause of disability and death. Atherosclerosis, the pathological basis of AMI, can be accelerated by chronic inflammation. Ulcerative colitis (UC), a chronic inflammatory disease associated with immunity, contributes to the risk of AMI development. However, controversy continues to surround the relationship between these two diseases. The present study unravels the pathogenesis of AMI and UC, to provide a new perspective on the clinical management of patients with these comorbidities.
Methods: Microarray datasets GSE66360 and GSE87473 were downloaded from the Gene Expression Omnibus database. Common differentially expressed genes (co-DEGs) between AMI and UC were identified, and the following analyses were performed: enrichment analysis, protein-protein interaction network construction, hub gene identification and co-expression analysis.
Results: A total of 267 co-DEGs (233 upregulated and 34 downregulated) were screened for further analysis. GO enrichment analysis suggested important roles of chemokines and cytokines in AMI and UC. In addition, the lipopolysaccharide-mediated signaling pathway was found to be closely associated with both diseases. KEGG enrichment analysis revealed that lipid and atherosclerosis, NF-κB, TNF and IL-17 signaling pathways are the core mechanisms involved in the progression of both diseases. Finally, 11 hub genes were identified with cytoHubba: TNF, IL1B, TLR2, CXCL8, STAT3, MMP9, ITGAX, CCL4, CSF1R, ICAM1 and CXCL1.
Conclusion: This study reveals a co-pathogenesis mechanism of AMI and UC regulated by specific hub genes, thus providing ideas for further mechanistic studies, and new perspectives on the clinical management of patients with these comorbidities.
... Compared with the G1 testes, there were 23 (2 coding) and 31 (9 coding) commonly down-and up-regulated genes in the G2-G5 testes (Fig. S2). In the two down-regulated coding genes, LYG2 encodes the antibacterial proteins and associated with regulation of innate immunity [25], whereas CCL1 encodes an inflammatory cytokine that chemoattracts monocytes [26]. In the up-regulated nine genes, CLEC4C and PDCD1LG2 also encode proteins associated with immune system. ...
In human, endo- or exogeneous factors might alter the cellular composition, the endocrine and inflammatory micro-environments and the metabolic balance in testis. These factors will further impair the testicular spermatogenesis capacity and alter the transcriptome of testis. Conversely, it should be possible that the alteration of the transcriptomes in testes be used as an indicator to evaluate the testicular spermatogenesis capacity and to predict the causing factors. In this study, using the transcriptome data of human testes and whole blood which were collected by the genotype-tissue expression project (GTEx), we analyzed the transcriptome differences in human testes and explored those factors that affecting spermatogenesis. As a result, testes were clustered into five clusters according to their transcriptomic features, and each cluster of testes was evaluated as having different spermatogenesis capacity. High rank genes of each cluster and the differentially expressed genes in lower functional testes were analyzed. Transcripts in whole blood which may be associated with testis function were also analyzed by the correlation test. As a result, factors such as immune response, oxygen transport, thyrotropin, prostaglandin and tridecapeptide neurotensin were found associated with spermatogenesis. These results revealed multiple clues about the spermatogenesis regulation in testis and provided potential targets to improve the fertility of men in clinic.
... CXC chemokines containing glutamate-leucine-arginine (ELR) motifs such as C-X-C motif chemokine ligand 8 (CXCL8), also known as IL-8, are more specialized in polymorphonuclear leukocyte (PMN) recruitment [118,119]. Meanwhile, other CC cytokines, including C-C motif ligand 1 (CCL1) act as monocyte [120] and lymphocyte [121] attractants. The expression of these chemokines must be firmly regulated during tissue repair to avoid dysregulation of inflammatory responses. ...
There are well-established links between acute inflammation and successful tissue repair across evolution. Innate immune reactions contribute significantly to pathogen clearance and activation of subsequent reparative events. A network of molecular and cellular regulators supports antimicrobial and tissue repair functions throughout the healing process. A delicate balance must be achieved between protection and the potential for collateral tissue damage associated with overt inflammation. In this review, we summarize the contributions of key cellular and molecular components to the acute inflammatory process and the effective and timely transition toward activation of tissue repair mechanisms. We further discuss how the disruption of inflammatory responses ultimately results in chronic non-healing injuries.
... Journal of Immunology Research correlated with CD68 + CD163 + M2-like macrophages of NSCLC patients after radiotherapy, suggesting that eotaxin could emerge as a potential marker for the prediction of radiotherapy efficacy and recruitment of CD68 + CD163 +-M2-like macrophages of NSCLC patients in radiotherapy. MIP-1β (CCL4), RANTES, and the human cytokine I-309 are all monocyte and lymphocyte chemoattractant [48]. Our results showed that MIP-1β was obviously elevated in NSCLC patients after radiotherapy, in comparison with these patients before radiotherapy, and there were more expressions of RANTES and I-309 of NSCLC patients in the post-RT group than in the HC group. ...
Background:
Recent studies show that myeloid-derived suppressor cells (MDSCs) and M2-like macrophages are involved in the treatment of tumors; however, their therapeutic response role is rarely known in non-small cell lung cancer (NSCLC) during radiotherapy. We aim to explore the dynamic alteration of the circulating MDSCs and M2-like macrophages, to examine their relationship, and to evaluate their therapeutic response value for NSCLC patients in radiotherapy.
Methods:
Peripheral blood mononuclear cells from healthy controls and NSCLC patients with different radiotherapy phases were isolated to examine the circulating MDSCs and M2-like macrophages by flow cytometry. 40 plasma inflammatory cytokines were measured by multiplex ELISA.
Results:
In comparison with healthy controls, the percentages of MDSCs and CD68+CD163+M2-like macrophages of NSCLC patients were significantly elevated and were distinctly higher in radiotherapy than in preradiotherapy. MDSCs were correlated positively with CD68+CD163+M2-like macrophages in NSCLC patients in radiotherapy and postradiotherapy. Especially, we found that in comparison with those in the poor group, the percentages of two cells in the good response group were markedly increased during radiotherapy and they had a significantly positive correlation. During radiotherapy, the proportions of MDSCs were clearly increased in adenocarcinoma patients and the percentages of CD68+CD163+M2-like macrophages were markedly elevated in squamous carcinoma patients. We found that after radiotherapy, the expressions of eotaxin, MIP-1β, MCP-1, and BLC were significantly increased in NSCLC patients. Further results showed that the low levels of eotaxin and TNF RII expression before radiotherapy could predict a good therapeutic response. IL-1ra and MIP-1β had a positive relation with MDSCs or CD68+CD163+M2-like macrophages in NSCLC patients during radiotherapy, and eotaxin was correlated with CD68+CD163+M2-like macrophages but not MDSCs in NSCLC patients after radiotherapy.
Conclusions:
MDSCs and CD68+CD163+M2-like macrophages serve as therapeutic response biomarkers and are associated with the expressions of plasma inflammatory cytokines for NSCLC patients during radiotherapy.
... CCR8 was absent on any other type of immune cell, except for a few γδ T cells and NK cells (see below). Interestingly, human blood monocytes were originally reported to respond to CCL1/I-309 [86] and to express CCR8 mRNA [60]; however, these early findings were not corroborated by subsequent CCR8 protein expression studies [77,84]. In fact, healthy human skin turned out to be the primary residence of CCR8 + cells [76][77][78]. ...
Simple Summary
Antibodies directed at so-called immune checkpoint molecules represent a substantial improvement in cancer therapy. These biological reagents highlight the exquisite interplay between cancer and our own immune system. Cancer progression is enabled by establishing a compartment of suppressive immune cells within the tumor. Therefore, elimination of these suppressor cells is an attractive strategy to augment beneficial anti-tumor immunity and to further improve the newly established immune checkpoint therapy. This review focuses on CCR8, a chemokine receptor highly expressed on suppressive immune cells, and its potential value as a novel target in cancer therapy.
Abstract
Immune checkpoint blockade inhibitors (CBIs) targeting cytotoxic T lymphocyte associated protein-4 (CTLA-4) and program death receptor-1 (PD-1) or its ligand-1 (PD-L1) have transformed the outlook of many patients with cancer. This remarkable progress has highlighted, from the translational point of view, the importance of immune cells in the control of tumor progression. There is still room for improvement, since current CBI therapies benefit a minority of patients. Moreover, interference with immune checkpoint receptors frequently causes immune related adverse events (irAEs) with life-threatening consequences in some of the patients. Immunosuppressive cells in the tumor microenvironment (TME), including intratumoral regulatory T (Treg) cells, tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), contribute to tumor progression and correlate with a negative disease outlook. Recent reports revealed the selective expression of the chemokine receptor CCR8 on tumor Treg cells, making CCR8 a promising target in translational research. In this review, I summarize our current knowledge about the cellular distribution and function of CCR8 in physiological and pathophysiological processes. The discussion includes an assessment of how the removal of CCR8-expressing cells might affect both anti-tumor immunity as well as immune homeostasis at remote sites. Based on these considerations, CCR8 appears to be a promising novel target to be considered in future translational research.
... Increased concentrations of CCL1, CRP, VEGF, and CXCL10 in ATB compared to LTBI are in line with previous reports (30,31,(38)(39)(40)(41)54) and may reflect the activity of infected macrophages, innate immune cells such as natural killer (NK) cells and innate lymphoid cells (ILCs), as well as activated T lymphocytes. CCL1, also known as I-309, is a glycoprotein secreted by activated T lymphocytes and stimulates chemotaxis of monocytes (55). VEGF and CXCL10 are produced by activated macrophages and antigen-presenting cells and regulate cell growth and chemotaxis and may be the driving forces for stimulated angiogenesis as observed in ATB lesions (56)(57)(58). ...
Introduction
There is an urgent medical need to differentiate active tuberculosis (ATB) from latent tuberculosis infection (LTBI) and prevent undertreatment and overtreatment. The aim of this study was to identify biomarker profiles that may support the differentiation between ATB and LTBI and to validate these signatures.
Materials and Methods
The discovery cohort included adult individuals classified in four groups: ATB (n = 20), LTBI without prophylaxis (untreated LTBI; n = 20), LTBI after completion of prophylaxis (treated LTBI; n = 20), and healthy controls (HC; n = 20). Their sera were analyzed for 40 cytokines/chemokines and activity of adenosine deaminase (ADA) isozymes. A prediction model was designed to differentiate ATB from untreated LTBI using sparse partial least squares (sPLS) and logistic regression analyses. Serum samples of two independent cohorts (national and international) were used for validation.
Results
sPLS regression analyses identified C-C motif chemokine ligand 1 (CCL1), C-reactive protein (CRP), C-X-C motif chemokine ligand 10 (CXCL10), and vascular endothelial growth factor (VEGF) as the most discriminating biomarkers. These markers and ADA(2) activity were significantly increased in ATB compared to untreated LTBI (p ≤ 0.007). Combining CCL1, CXCL10, VEGF, and ADA2 activity yielded a sensitivity and specificity of 95% and 90%, respectively, in differentiating ATB from untreated LTBI. These findings were confirmed in the validation cohort including remotely acquired untreated LTBI participants.
Conclusion
The biomarker signature of CCL1, CXCL10, VEGF, and ADA2 activity provides a promising tool for differentiating patients with ATB from non-treated LTBI individuals.
... G-CSF can induce the migration of hematopoietic stem cells CD34+ cells [53]. I-309 can promote the migration of human monocytes [54]. VEGF is well known to promote the migration of endothelial cells, and leptin and is reported to induce the migration of cancer cells [55,56]. ...
Ionizing radiation is known to cause cell apoptosis at high dose range, but little is known about the cellular response to low dose radiation. In this study, we found that conditioned medium harvested from WI-38 lung fibroblasts and H1299 lung adenocarcinoma cells exposed to 0.1Gy to 1Gy could enhance the migration and invasion of unirradiated H1299 cells in both 2D and 3D culturing circumstances. Low dose radiation did not induce apoptosis, but induced senescence in irradiated cells. We next examined the expression of immediately early genes including c-Myc and K-Ras. Although both genes could be up-regulated by low dose radiation, induction of c-Myc was more specific to low dose range (0.5Gy) at transcriptional and translational levels. Knockdown of c-Myc by shRNA could repress the senescence induced by low dose radiation. The conditioned medium of irradiated cells induced migration of unirradiated cells was also repressed by knockdown of c-Myc. The c-Myc inhibitor 10058-F4 could suppress low dose radiation induced cell senescence, and the conditioned medium harvested from irradiated cells pretreated with 10058-F4 also lost the ability to enhance the migration of unirradiated cells. The cytokine array analysis revealed that immunosuppressive monocyte chemoattractant protein-1 increased by low dose radiation could be repressed by 10058-F4. We also showed that 10058-F4 could suppress low dose radiation induced tumor progression in a xenograft tumor model. Taken together, current data suggest that -Myc is involved in low dose radiation induced cell senescence and potent bystander effect to increase the motility of unirradiated cells.
... Within the cytokinecytokine receptor interaction pathway, DP showed increased levels of 54/292 genes and specific evaluation of effector associated genes that were upregulated in the DP compared with the DN population identified 17 genes to further pursue ( Figure 3B and Table 2). Of note, one of the most upregulated genes in the data set was Ccl1, the chemokine associated with recruitment of innate myeloid derived cells through binding its cognate receptor CCR8 (45,46). DP cells also displayed increased transcript levels for genes associated with classical antiviral cytokines (Ifng, Tnf), T cell survival and activation (Il2), chemokines (Ccl3, Ccl4, Cxcl9), cell differentiation (Gmcsf) and cytolytic components (Prf1, Gzma, Gzmb,Gzmc, Fasl, Tnfsf10) ( Table 2). ...
CD8 T cell memory offers critical antiviral protection, even in the absence of neutralizing antibodies. The paradigm is that CD8 T cell memory within the lung tissue consists of a mix of circulating TEM cells and non-circulating TRM cells. However, based on our analysis, the heterogeneity within the tissue is much higher, identifying TCM, TEM, TRM, and a multitude of populations which do not perfectly fit these classifications. Further interrogation of the populations shows that TRM cells that express CD49a, both with and without CD103, have increased and diverse effector potential compared with CD49a negative populations. These populations function as a one-man band, displaying antiviral activity, chemokine production, release of GM-CSF, and the ability to kill specific targets in vitro with delayed kinetics compared with effector CD8 T cells. Together, this study establishes that CD49a defines multiple polyfunctional CD8 memory subsets after clearance of influenza infection, which act to eliminate virus in the absence of direct killing, recruit and mature innate immune cells, and destroy infected cells if the virus persists.
... Homing of CCR8 + cells is mainly driven by CCL1 (16,19), which could be detected in ex vivo Panc02 tumors at both the RNA (Fig. 1C) and protein level (Fig. 1, D and E), and was detected in the tumor microenvironment at considerably higher levels than other organs (Fig. 1D). Activated T cells have previously been reported to produce CCL1 (20). Consistent with this, we found that tumor-infiltrating T cells produced high amounts of CCL1 upon stimulation (Fig. 1F). ...
CAR T cell therapy remains ineffective in solid tumors, due largely to poor infiltration and T cell suppression at the tumor site. T regulatory (T reg ) cells suppress the immune response via inhibitory factors such as transforming growth factor–β (TGF-β). T reg cells expressing the C-C chemokine receptor 8 (CCR8) have been associated with poor prognosis in solid tumors. We postulated that CCR8 could be exploited to redirect effector T cells to the tumor site while a dominant-negative TGF-β receptor 2 (DNR) can simultaneously shield them from TGF-β. We identified that CCL1 from activated T cells potentiates a feedback loop for CCR8 ⁺ T cell recruitment to the tumor site. This sustained and improved infiltration of engineered T cells synergized with TGF-β shielding for improved therapeutic efficacy. Our results demonstrate that addition of CCR8 and DNR into CAR T cells can render them effective in solid tumors.
... CCL1 is a chemokine that attracts monocytes, helper T cells (Ths), and regulatory T cells (Tregs) 27,28 and selectively interacts with C-C motif chemokine receptor 8 (CCR8). 29 CCR8 is a member of the β chemokine receptor family and is a seven-transmembrane protein, similar to G protein-coupled receptors. ...
Tumor-associated macrophages (TAMs) promote tumor progression. The number of infiltrating TAMs is associated with poor prognosis in esophageal squamous cell carcinoma (ESCC) patients; however, the mechanism underlying this phenomenon is unclear. Our previous cDNA microarray analysis had revealed that the expression of C-C motif chemokine ligand 1 (CCL1) is upregulated in peripheral blood monocyte (PBMo)-derived macrophages stimulated using conditioned media from ESCC cells (TAM-like macrophages). In this study, we evaluated the role of CCL1 in ESCC progression. We confirmed that CCL1 is overexpressed in TAM-like macrophages, and that C-C motif chemokine receptor 8 (CCR8), a CCL1 receptor, is expressed on ESCC cell surface. TAM-like macrophages significantly enhanced the motility of ESCC cells, and neutralizing antibodies against CCL1 or CCR8 suppressed this increased motility. Recombinant human CCL1 promoted ESCC cell motility via the Akt/proline-rich Akt substrate of 40 kDa (PRAS40)/mammalian target of rapamycin (mTOR) pathway. PI3K or Akt inhibitors, CCR8 silencing, and neutralizing antibody against CCR8 could significantly suppress these effects. The overexpression of CCL1 in stromal cells or CCR8 in ESCC cells was significantly associated with poor overall survival (P = 0.002 or 0.009) and disease-free survival (P = 0.009 or 0.047) in ESCC patients. These results indicated that the interaction between stromal CCL1 and CCR8 on cancer cells promotes ESCC progression via the Akt/PRAS40/mTOR pathway, providing novel therapeutic targets.
... I-309 can activate endothelial cell functions, 76 and it is a product of activated T-lymphocytes, mast cells, and monocytes and is antiapotoic. 77,78 Angiogenin can promote endothelial cells invasion and neovascularization via regulating the immune response in corneal fibroblasts. 79,80 MIF, apart from its proinflammatory role in T cell activation, 81 it also has a tumorassociated angiogenic role. ...
Purpose
Keratoconus is considered a non-inflammatory condition. Recently however, increased proinflammatory cytokines have been detected in the tears of keratoconic patients and clinical and immunohistochemical observations reported infiltration of matured dendritic cells and leukocytes. Our laboratory utilized cytokine antibody arrays to elucidate the inflammatory aspects of keratoconus.
Methods
Protein was extracted from 42 corneal buttons (14 keratoconic and 28 non-keratoconic) and incubated with cytokine antibody arrays scanning 120 cytokines. Mann Whitney U test with a p-value of <0.05 was considered significant.
Results
Pathways for wound healing, neuroprotection, angiogenesis, and inflammation were activated in keratoconic samples with 23 cytokines showing significant elevation. Fifteen were expressed only in keratoconus with 8 cytokines elevated 1.7–42-fold.
Conclusion
This study identified elevated inflammatory pathways covering immune responses in keratoconus. Our results support the evidence for inflammatory pathway activation in keratoconus and a possible redefinition of keratoconus as a chronic inflammatory corneal disease.
... Our data indicated that Eotaxin was positively correlated with CD68 + CD163 + M2-like macrophages in NSCLC patients in post-RT, suggesting that the upregulated Eotaxin could emerge as a potential marker for the recruitment of CD68 + CD163 + M2-like macrophages in NSCLC patients after radiotherapy. MIP-1β (CCL4), RANTES and the human cytokine I-309 are all monocyte and lymphocyte chemoattractant [48]. Our results showed that MIP-1β was obviously elevated in NSCLC patients in post-RT compared with these patients in pre-RT, and there was more expression of RANTES and I-309 in NSCLC patients in post-RT than HC group. ...
Background
Recent studies showed that myeloid-derived suppressor cells (MDSCs) and M2-like macrophages are involved in the treatment of non-small cell lung cancer (NSCLC) as immunosuppressive cells; however, the changes of MDSCs and M2-like macrophages, and their prognostic value are rarely unknown in NSCLC patients during radiotherapy. In this article, we aim to explore dynamic alteration of the circulating MDSCs and M2-like macrophages, to examine their relationship, and to evaluate their prognostic value for NSCLC patients in radiotherapy.Method:Peripheral blood mononuclear cells from healthy controls and NSCLC patients in radiotherapy were isolated to examine the circulating MDSCs and M2-like macrophages. The peripheral MDSCs defined as CD11b ⁺ CD33 ⁺ HLA-DR ⁻ and M2-like Macrophages signified as CD68 ⁺ CD163 ⁺ were determined by flow cytometry. 40 plasma inflammatory cytokines were measured by multiplex ELISA.ResultsCompared with health controls, the percentages of MDSCs and CD68 ⁺ CD163 ⁺ M2-like macrophages of NSCLC patients were significantly elevated, and were distinctly higher in NSCLC patients in radiotherapy than in pre-radiotherapy. Moreover, MDSCs correlated positively with CD68 ⁺ CD163 ⁺ M2-like macrophages in NSCLC patients in radiotherapy and post-radiotherapy. Interestingly, the alterations of the percentages of MDSCs and CD68 ⁺ CD163 ⁺ M2-like macrophages in RT were irrelated with radiotherapy area. During radiotherapy, the proportions of MDSCs were clearly increased in adenocarcinoma patients but not in squamous carcinoma patients, while the proportions of CD68 ⁺ CD163 ⁺ M2-like macrophages were markedly elevated in squamous carcinoma patients but not in adenocarcinoma patients. In addition, the percentages of MDSCs and CD68 ⁺ CD163 ⁺ M2-like macrophages were also increased in NSCLC patients reached PR in radiotherapy compared to NSCLC patients reached SD and PD. IL-1ra and MIP-1β have a positive relation with MDSCs or M2 macrophages in NSCLC patients in radiotherapy, and Eotaxin was correlated with CD68 ⁺ CD163 ⁺ M2-like macrophages but not MDSCs in NSCLC patients after radiotherapy.Conclusions
The dynamic alterations of MDSCs and M2-like macrophages, as well as their connection with plasm inflammation cytokines, could provide potentially prognostic and sensitive markers for NSCLC patients in radiotherapy.Trial Registration: Jinshan Hospital of Fudan University, IEC-2020-S01. Registered 22 May 2020.
... CCL1 and CXCL9 were found to be upregulated in the lung tissues of pigs infected with A/swine/Korea/ s803/2018 (H1N2) and A/swine/Korea/s806/2018 (H1N2). Compared to the lung tissues of PBS-mock-infected pigs, the lungs of pigs infected with A/swine/Korea/s803/2018 (H1N2), exhibited a 65.7-fold increase in CCL1, which attracts monocytes, natural killer (NK) cells, immature B cells, and dendritic cells (P < 0.001), those of pigs infected with A/swine/Korea/s806/2018 (H1N2) shared a 48.2-fold increase [45]. The increase in CXCL9, which attracts cytotoxic lymphocytes (CTLs), natural killer (NK) cells, natural killer T (NKT) cells, and macrophages, in the lung tissue of pigs infected with A/swine/Korea/s803/2018 (H1N2) and A/swine/Korea/s806/2018 (H1N2) was 29.85-(P < 0.05) and 20.3-fold, respectively [46]. ...
Pigs are capable of harbouring influenza A viruses of human and avian origin in their respiratory tracts and thus act as an important intermediary host to generate novel influenza viruses with pandemic potential by genetic reassortment between the two viruses. Here, we show that two distinct H1N2 swine influenza viruses contain avian-like or classical swine-like hemagglutinins with polymerase acidic (PA) and nucleoprotein (NP) genes from 2009 pandemic H1N1 influenza viruses that were found to be circulating in Korean pigs in 2018. Swine H1N2 influenza virus containing an avian-like hemagglutinin gene had enhanced pathogenicity, causing severe interstitial pneumonia in infected pigs and mice. The mortality rate of mice infected with swine H1N2 influenza virus containing an avian-like hemagglutinin gene was higher by 100% when compared to that of mice infected with swine H1N2 influenza virus harbouring classical swine-like hemagglutinin. Further, chemokines attracting inflammatory cells were strongly induced in lung tissues of pigs and mice infected by swine H1N2 influenza virus containing an avian-like hemagglutinin gene. In conclusion, it is necessary for the well-being of humans and pigs to closely monitor swine influenza viruses containing avian-like hemagglutinin with PA and NP genes from 2009 pandemic H1N1 influenza viruses.
... Secreted by activated T cells, CCL1 binds to CCR8 33 and acts as a chemokine for monocytes, NK cells and dendritic cells. 34 The up-regulation of CCL1 could suggest an increased ability to attract monocytes into tissue for macrophage differentiation and ultimately improved clearing of cellular debris and wounds. However, it is important to note CCL1 is not the only cytokine with monocyte chemotactic properties and that it is the only monocyte chemotactic agent up-regulated in this study. ...
A greater demand for food animal production without antibiotics has created the common practice of feeding food animals dietary immunomodulatory feed additives (IFA) throughout their life cycle. However, little is known about the impact of IFA on cytokine and chemokine signaling in non-stressed, non-pathogen-challenged food animals during the early feeding period. We evaluated the expression of 82 genes related to cytokine and chemokine signaling in the whole blood of growing Angus heifers to determine the effect of IFA supplementation on cytokine and chemokine signaling during the first 28 d of feeding. One gene (CCL1) was significantly up-regulated and 14 genes (17%) were significantly down-regulated by IFA feeding during the entire early feeding period including 5 of 21 (24%) evaluated chemokine and IL receptors (CCR1, CCR2, IL1R1, IL10RA, IL10RB). These data when taken together suggest providing an IFA in the diet of growing beef cattle during the early feeding period may suppress the inflammatory response through cytokine–cytokine receptor signaling.
... This model is used in atherosclerosis studies due to its resemblance to microvascular inflammation [27][28][29][30]. We show that AZ6983 suppressed several pro-inflammatory cytokines, including: CCL1, CXCL10, MIP3α (CCL20) and TNFα [14,[31][32][33][34] after 6 h of inflammation. Taken together, the affected cytokines indicate a general reduction in myeloid cell response. ...
Background and aims:
Alpha 7 nicotinic acetylcholine receptor (α7nAChR) stimulation can regulate acute inflammation, and lack of α7nAChR accelerates atherosclerosis in mice. In this study, we aimed to investigate the effects of the novel α7nAChR agonist, AZ6983, on atherosclerosis and assess its possible immunomodulating effects.
Methods:
AZ6983 was tested in vitro in LPS-challenged mouse and human blood and in vivo using the acute inflammatory air pouch model. Thereafter, long-term effects of AZ6983 treatment on atherosclerosis and immune responses were assessed in apoE-/- mice after 8 and 12 weeks. Atherosclerosis was investigated in the aortic root and thoracic aorta, serum levels of cytokines were analysed and RNAseq was used to study aortic gene expression. Further, bone-marrow-derived macrophages were used to assess phagocytosis in vitro.
Results:
α7nAChR activation by AZ6983 decreased pro-inflammatory cytokines in acute stimulations of human and mouse blood in vitro, as well as in vivo using the air pouch model. Treating apoE-/- mice with AZ6983 decreased atherosclerosis by 37-49% and decreased serum cytokine levels. RNAseq analysis of aortae suggested the involvement of several specific myeloid cell functions, including phagocytosis. In line with this, AZ6983 significantly increased phagocytosis in bone marrow-derived macrophages.
Conclusions:
This study demonstrates that activation of α7nAChR with AZ6983 inhibits atherosclerosis in apoE-/-mice and that immunomodulating effects on myeloid cells, such as enhanced phagocytosis and suppression of inflammatory cytokines, could be part of the athero-protective mechanisms. The observed anti-inflammatory effect in human blood supports the idea that AZ6983 may decrease disease also in humans.
... Production of inflammatory chemokines, such as CCL1 and CCL21a were also reduced in Nox1 −/− mice relative to WT controls after DEN challenge ( Figure 3E). CCL1 is mainly secreted by monocytes and activated macrophages and attracts monocytes and subsets of T cells [50][51][52] , by signaling through its receptor CCR7 53 . In support of this, DEN-challenged Nox1 −/− mice had decreased numbers of macrophage (F4/80) and exhibited reduced hepatocyte compensatory proliferation at 8 days post DEN challenge ( Figure S2C and S2D). ...
Background & aims:
Although there are associations among oxidative stress, reduced nicotinamide adenine dinucleotide phosphate oxidase (NOX) activation, and hepatocellular carcinoma (HCC) development, it is not clear how NOX contributes to hepatocarcinogenesis. We studied the functions of different NOX proteins in mice after administration of a liver carcinogen.
Methods:
Fourteen-day-old Nox1-/- mice, Nox4-/- mice, Nox1-/-Nox4-/- (double-knockout) mice, and wild-type (WT) C57BL/6 mice were given a single intraperitoneal injection of diethylnitrosamine (DEN) and liver tumors were examined at 9 months. We also studied the effects of DEN in mice with disruption of Nox1 specifically in hepatocytes (Nox1ΔHep), hepatic stellate cells (Nox1ΔHep), or macrophages (Nox1ΔMac). Some mice were also given injections of the NOX1-specific inhibitor ML171. To study the acute effects of DEN, 8-12-week-old mice were given a single intraperitoneal injection, and liver and serum were collected at 72 hours. Liver tissues were analyzed by histologic examination, quantitative polymerase chain reaction, and immunoblots. Hepatocytes and macrophages were isolated from WT and knockout mice and analyzed by immunoblots.
Results:
Nox4-/- mice and WT mice developed liver tumors within 9 months after administration of DEN, whereas Nox1-/- mice developed 80% fewer tumors, which were 50% smaller than those of WT mice. Nox1ΔHep and Nox1ΔHSC mice developed liver tumors of the same number and size as WT mice, whereas Nox1ΔMac developed fewer and smaller tumors, similar to Nox1-/- mice. After DEN injection, levels of tumor necrosis factor, interleukin 6 (IL6), and phosphorylated signal transducer and activator of transcription 3 were increased in livers from WT, but not Nox1-/- or Nox1ΔMac, mice. Conditioned medium from necrotic hepatocytes induced expression of NOX1 in cultured macrophages, followed by expression of tumor necrosis factor, IL6, and other inflammatory cytokines; this medium did not induce expression of IL6 or cytokines in Nox1ΔMac macrophages. WT mice given DEN followed by ML171 developed fewer and smaller liver tumors than mice given DEN followed by vehicle.
Conclusions:
In mice given injections of a liver carcinogen (DEN), expression of NOX1 by macrophages promotes hepatic tumorigenesis by inducing the production of inflammatory cytokines. We propose that upon liver injury, damage-associated molecular patterns released from dying hepatocytes activate liver macrophages to produce cytokines that promote tumor development. Strategies to block NOX1 or these cytokines might be developed to slow hepatocellular carcinoma progression.
... Consistent with RNA data from dLNs ( Figure 2B), Ccl1-mCherry expression was unaffected, whereas Ccl8-eGFP expression was induced by Th2 immunization ( Figure 5B). CCL1 has been described to be produced by CD3 + T cells as well as the lymphatic endothelium (Miller and Krangel, 1992;Qu et al., 2004). We were unable to detect Ccl1-mCherry expression in lymphatic endothelial cells, blood endothelial cells, or fibroblastic reticular cells of the dLN ( Figure S5C). ...
The migration of mature dendritic cells (DCs) into the draining lymph node (dLN) is thought to depend solely on the chemokine receptor CCR7. CD301b+ DCs migrate into the dLN after cutaneous allergen exposure and are required for T helper 2 (Th2) differentiation. We found that CD301b+ DCs poorly upregulated CCR7 expression after allergen exposure and required a second chemokine signal, mediated by CCR8 on CD301b+ DCs and its ligand CCL8, to exit the subcapsular sinus (SCS) and enter the lymph node (LN) parenchyma. After allergen exposure, CD169+SIGN-R1+ macrophages in interfollicular regions produced CCL8, which synergized with CCL21 in a Src-kinase-dependent manner to promote CD301b+ DC migration. In CCR8-deficient mice, CD301b+ DCs remained in the SCS and were unable to enter the LN parenchyma, resulting in defective Th2 differentiation. We have defined a CCR8-dependent stepwise mechanism of DC-subset-specific migration through which LN CD169+SIGN-R1+ macrophages control the polarization of the adaptive immune response.
... The receptor CCR8 has been shown to be associated with phagocytic macrophages and activated microglia in MS lesions and directly correlated with demyelinating activity (Trebst et al., 2003). Expressions of CCL1 and CCR8 mRNA in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) (Fischer et al., 2000;Godiska, Chantry, Dietsch, & Gray, 1995) (Devi, Laning, Luo, & Dorf, 1995;Le, Zhou, Iribarren, & Wang, 2004;Miller & Krangel, 1992;Murdoch & Finn, 2000;Ono et al., 2003;Roos et al., 1997;Stuart et al., 2015;Trebst et al., 2003) CCL2 (Le et al., 2004;Murdoch & Finn, 2000;Ono et al., 2003;Stuart et al., 2015) CCL6 (C10, MRP-2) ...
... Chemokine (C-C motif ) ligand 1 (CCL1) is a small glycoprotein secreted by monocytes, activated macrophages, and T-lymphocytes that belongs to a family of inflammatory cytokines known as chemokines [15,16]. It has been reported that CCL1 is overexpressed in ATL cells, mediating an autocrine anti-apoptotic loop, along with its receptor, CC chemokine receptor 8 (CCR8), for in vivo growth and survival of leukemic cells [17]. ...
Background
Chemokine (C-C motif) ligand 1 (CCL1) is produced by activated monocytes/ macrophages and T-lymphocytes, and acts as a potent attractant for Th2 cells and a subset of T-regulatory (Treg) cells. Previous reports have indicated that CCL1 is overexpressed in adult T-cell leukemia cells, mediating an autocrine anti-apoptotic loop. Because CCL1 is also known as a potent chemoattractant that plays a major role in inflammatory processes, we investigated the role of CCL1 in the pathogenesis of human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP).
Results
The results showed that: (1) CCL1 was preferentially expressed in HAM/TSP-derived HTLV-1-infected T-cell lines, (2) CCL1 expression was induced along with Tax expression in the Tax-inducible T-cell line JPX9, (3) transient Tax expression in an HTLV-1-negative T-cell line activated the CCL1 gene promoter, (4) plasma levels of CCL1 were significantly higher in patients with HAM/TSP than in HTLV-1-seronegative patients with multiple sclerosis and HTLV-1-infected asymptomatic healthy carriers, and (5) minocycline inhibited the production of CCL1 in HTLV-1-infected T-cell lines.
Conclusions
The present results suggest that elevated CCL1 levels may be associated with the pathogenesis of HAM/TSP. Although further studies are required to determine the in vivo significance, minocycline may be considered as a potential candidate for the long-term treatment of HAM/TSP via its anti-inflammatory effects, which includes the inhibition of CCL1 expression.
... Chemokine C-C motif ligand-1 (CCL1), interacting exclusively with the chemokine (C-C motif) receptor 8 (CCR8), is an inflammatory mediator [8]. Studies showed that CCL1 induced tumor cells metastasis into lymph nodes [9]. ...
Purpose:
Cancer-associated fibroblasts (CAFs) activated by cancer cells has a central role in development and malignant biological behavior in colorectal cancer (CRC). Adult fibroblasts do not express Snail, but Snail-positive fibroblasts are discovered in the stroma of malignant CRC and reported to be the key role to chemoresistance. However, the reciprocal effect of CAFs expressed Snail to chemoresistance on CRC cells and the underlying molecular mechanisms are not fully characterized.
Materials and methods:
Snail-overexpressed 3T3 stable cell lines were generated by lipidosome and CT26 mixed with 3T3-Snail subcutaneous transplanted CRC models were established by subcutaneous injection. Cell Counting Kit-8, flow cytometry and western blotting assays were performed, and immunohistochemistry staining was studied. The cytokines participated in chemoresistance was validated with reverse transcriptase-polymerase chain reaction and heatmap.
Results:
Snail-expression fibroblasts are discovered in human and mouse spontaneous CRCs. Overexpression of Snail induces 3T3 fibroblasts transdifferentiation to CAFs. CT26 co-cultured with 3T3-Snail resisted the impairment from 5-fluorouracil and paclitaxel in vitro. The subcutaneous transplanted tumor models included 3T3-Snail cells develop without restrictions even after treating with 5-fluorouracil or paclitaxel. Moreover, these chemoresistant processes may be mediated by CCL1 secreted by Snail-expression fibroblasts via transforming growth factor β/nuclear factor κB signaling pathways.
Conclusions:
Taken together, Snail-expressing 3T3 fibroblasts display CAFs properties that support 5-fluorouracil and paclitaxel chemoresistance in CRC via participation of CCL1 and suggest that inhibition of the Snail-expression fibroblasts in tumor may be a useful strategy to limit chemoresistance.
Introduction
Obesity is a multi-factorial disease frequently associated with poor nutritional habits and linked to many detrimental health outcomes. Individuals with obesity are more likely to have increased levels of persistent inflammatory and metabolic dysregulation. The goal of this study was to compare four dietary patterns differentiated by macronutrient content in a postmenopausal model. Dietary patterns were high carbohydrate (HC), high fat (HF), high carbohydrate plus high fat (HCHF), and high protein (HP) with higher fiber.
Methods
Changes in body weight and glucose levels were measured in female, ovariectomized C57BL/6 mice after 15 weeks of feeding. One group of five mice fed the HCHF diet was crossed over to the HP diet on day 84, modeling a 21-day intervention. In a follow-up study comparing the HCHF versus HP dietary patterns, systemic changes in inflammation, using an 80-cytokine array and metabolism, by untargeted liquid chromatography-mass spectrometry (LCMS)-based metabolomics were evaluated.
Results
Only the HF and HCHF diets resulted in obesity, shown by significant differences in body weights compared to the HP diet. Body weight gains during the two-diet follow-up study were consistent with the four-diet study. On Day 105 of the 4-diet study, glucose levels were significantly lower for mice fed the HP diet than for those fed the HC and HF diets. Mice switched from the HCHF to the HP diet lost an average of 3.7 grams by the end of the 21-day intervention, but this corresponded with decreased food consumption. The HCHF pattern resulted in dramatic inflammatory dysregulation, as all 80 cytokines were elevated significantly in the livers of these mice after 15 weeks of HCHF diet exposure. Comparatively, only 32 markers changed significantly on the HP diet (24 up, 8 down). Metabolic perturbations in several endogenous biological pathways were also observed based on macronutrient differences and revealed dysfunction in several nutritionally relevant biosynthetic pathways.
Conclusion
Overall, the HCHF diet promoted detrimental impacts and changes linked to several diseases, including arthritis or breast neoplasms. Identification of dietary pattern-specific impacts in this model provides a means to monitor the effects of disease risk and test interventions to prevent poor health outcomes through nutritional modification.
Study question: Are the molecular signatures of cumulus cells (CCs) and follicular fluid (FF) of adolescents undergoing fertility preservation differ from that of reproductively adult oocyte donors?
Summary answer: The microenvironment immediately surrounding the oocyte, including the CCs and FF, is altered in adolescents undergoing fertility preservation compared to oocyte donors.
What is known already: Adolescents experience a period of subfecundity following menarche. Recent evidence suggests that this may be at least partially due to increased oocyte aneuploidy. Reproductive juvenescence in mammals is associated with suboptimal oocyte quality.
Study design, size, duration: This was a prospective cohort study. Adolescents (10-19 years old, N=23) and oocyte donors (22-30 years old, N=31) undergoing ovarian stimulation and oocyte retrieval at the Northwestern Fertility and Reproductive Medicine Center between November 1, 2020 and May 1, 2023 were enrolled in this study.
Participants/materials, setting, methods: Patient demographics, ovarian stimulation, and oocyte retrieval outcomes were collected for all participants. The transcriptome of CCs associated with mature oocytes was compared between adolescents (10-19 years old, n=19), and oocyte donors (22-30 years old, n=19) using bulk RNA-sequencing. FF cytokine profiles (10-19 years old, n=18 vs. 25-30 years old, n=16) were compared using cytokine arrays.
Main results and the role of chance: RNA-seq analysis revealed 581 differentially expressed genes (DEGs) in cumulus cells of adolescents relative to oocyte donors, with 361 genes downregulated and 220 upregulated. Genes enriched in pathways involved in cell cycle and cell division (e.g., GO:1903047, p= 3.5 x 10-43; GO:0051983, p= 4.1 x 10-30; GO:0000281, p= 7.7 x 10-15; GO:0044839, p= 5.3 x 10-13) were significantly downregulated, while genes enriched in several pathways involved in cellular and vesicle organization (e.g., GO:0010256, p= 1.2 x 10-8; GO:0051129, p= 6.8 x 10-7; GO:0016050, p= 7.4 x 10-7; GO:0051640, p= 8.1 x 10-7) were upregulated in CCs of adolescents compared to oocyte donors. The levels of 9 cytokines were significantly increased in FF of adolescents compared to oocyte donors: IL-1 alpha (2-fold), IL-1 beta (1.7-fold), I-309 (2-fold), IL-15 (1.6-fold), TARC (1.9-fold), TPO (2.1-fold), IGFBP-4 (2-fold), IL-12-p40 (1.7-fold) and ENA-78 (1.4-fold). Interestingly, 7 of these cytokines have known pro-inflammatory roles. Importantly, neither the CC transcriptomes or FF cytokine profiles were different in adolescents with or without cancer.
Large scale data: Original high-throughput sequencing data will be deposited in Gene Expression Omnibus (GEO) before publication, and the GEO accession number will be provided here.
Limitations, reasons for caution: This study aims to gain insights into the associated gamete quality by studying the immediate oocyte microenvironment. The direct study of oocytes is more challenging due to sample scarcity, as they are cryopreserved for future use, but will provide a more accurate assessment of oocyte reproductive potential.
Wider implications of the findings: Understanding the underpinnings of altered immediate oocyte microenvironment of adolescent patients may provide insights into the reproductive potential of the associated gametes in the younger end of the age spectrum. This has implications for the fertility preservation cycles for very young patients.
Introduction:
Initial macrophage response to biostimulatory substances is key in determining the subsequent behavior of fibroblasts and the organization of newly synthesized collagen. Though histological studies suggest that calcium hydroxylapatite (CaHA) filler initiates a regenerative healing response with collagen and elastin deposition similar to natural, healthy tissue rather than an inflammatory response with fibrosis, the relative activity of macrophages stimulated by CaHA, as well as how this activity compares to that induced by other biostimulatory fillers, has not been explored. The aim of the study is to characterize the in vitro macrophage response to two biostimulory fillers, CaHA and PLLA (poly-L lactic acid), and to evaluate their inflammatory potential.
Methods:
Primary human macrophages were incubated with two dilutions (1:50 and 1:100) of commercially available CaHA or PLLA. After 24 h incubation, an inflammation array was used to screen for the expression of 40 cytokines, released by macrophages. ELISA was used to confirm array results.
Results:
Four cytokines were significantly upregulated in M1 macrophages incubated with PLLA compared to both unstimulated controls and CaHA: CCL1 (p < 0.001), TNFRII (p < 0.01), MIP-1α (p < 0.05), and IL-8 (p < 0.001). In M2 macrophages, MIP-1α (p < 0.01) and MIP-1β (p < 0.01) were significantly upregulated by PLLA compared to CaHA and unstimulated controls.
Conclusion:
Together, these findings indicate that the CaHA mode of action is a non-inflammatory response while PLLA initiates expression of several cytokines known to play a role in inflammation. Our study supports the concept that these two "biostimulatory" fillers follow distinct pathways and should be considered individually with regard to mechanism of action.
Macrophage M2 polarization has been identified in the pathogenesis of pulmonary fibrosis (PF), but the mediators that drive the macrophage M2 program in PF need to be clarified. We showed that the expression of AMFR and CCR8, two known receptors of CCL1, was increased in macrophages from lungs of mice with bleomycin (BLM)-induced PF. Deficiency in either AMFR or CCR8 in macrophages protected mice from BLM-induced PF. In vitro experiments revealed that CCL1 recruited macrophages by binding to its classical receptor CCR8 and drove the macrophage M2 phenotype via its interaction with the recently identified receptor AMFR. Mechanistic studies revealed that the CCL1-AMFR interaction enhanced CREB/C/EBPβ signaling to promote the macrophage M2 program. Together, our findings reveal that CCL1 acts as a mediator of macrophage M2 polarization and could be a therapeutic target in PF.
Acute inflammation is crucial to the immune responses of fish. The process protects the host from infection and is central to induction of subsequent tissue repair programs. Activation of proinflammatory signals reshapes the microenvironment within an injury/infection site, initiates leukocyte recruitment, promotes antimicrobial mechanisms and contributes to the resolution of inflammation. Inflammatory cytokines and lipid mediators are primary contributors to these processes. Uncontrolled or persistent induction results in delayed tissue healing. The kinetics by which inducers and regulators of acute inflammation exert their actions is essential for understanding the pathogenesis of fish diseases and identifying potential treatments. Although, a number of these are well-conserved across, others are not, reflecting the unique physiologies and life histories of members of this unique animal group.
Idiopathic pulmonary fibrosis (IPF), characterized by progressive worsening of dyspnea and irreversible decline in lung function, is a chronic and progressive respiratory disease with a poor prognosis. Chronic or repeated lung injury results in inflammation and an excessive injury-repairing response that drives the development of IPF. A number of studies have shown that the development and progression of IPF are associated with dysregulated expression of several chemokines and chemokine receptors, several of which have been used as predictors of IPF outcome. Chemokines of the CC family play significant roles in exacerbating IPF progression by immune cell attraction or fibroblast activation. Modulating levels of detrimental CC chemokines and interrupting the corresponding transduction axis by neutralizing antibodies or antagonists are potential treatment options for IPF. Here, we review the roles of different CC chemokines in the pathogenesis of IPF, and their potential use as biomarkers or therapeutic targets.
Increased understanding of molecular pathophysiology has led to the detection of clinically applicable biomarkers across medicine, which allow for minimally invasive detection, management, and monitoring of disease processes. Although biomarkers have traditionally played a more significant role in malignancy, these goals also pertain to benign disease. Herein, the authors review ongoing research into biomarker investigation and application in urethral stricture disease, benign prostatic hyperplasia, bladder outlet obstruction, and overactive bladder. No biomarkers for these entities are currently in clinical use; however, numerous physiologic pathways provide targets for current and future study.
Objective
Eosinophils are hallmarks in allergic type 2 inflammation and are known to release cytotoxic granule proteins that contribute to inflammation. Eosinophils develop in the bone marrow from hematopoietic stem cells and once mature, have a limited lifespan in culture, making them difficult to study ex vivo. IL-33 has increasingly been shown as a key regulator of type 2 inflammation via signaling through its receptor, ST2. The present study was conducted to detail a method of eosinophil differentiation from hematopoietic stem cells and determine the response to IL-33.
Methods
CD34+ and CD14+ cells were isolated from donor apheresis cones and differentiated into eosinophils or macrophage controls, respectively. Morphologic, transcriptional and protein analyses were performed to validate this method of eosinophil differentiation. The effect of IL-33 on differentiated eosinophils was assessed using qPCR, immunofluorescence, and multiplex cytokine array.
Results
CD34 differentiated eosinophils appear morphologically similar by H&E and express eosinophil peroxidase (EPX) protein as well as the conventional eosinophil transcripts EPX, CLC, and MBP. In addition, differentiated eosinophils expressed both isoforms of the IL-33 receptor, ST2L and sST2 throughout the differentiation process. Transcript levels of both IL-33 receptors were up-regulated by treatment with IL-33 at earlier timepoints in the differentiation. These cells also expressed IL-4 and IL-13 mRNA which were up-regulated by IL-33 as well. Notably, IL-13 expression was significantly higher with IL-33 treatment compared to media control at every timepoint measured. IL-33 significantly increased cellular secretion of IL-13 protein at most timepoints throughout differentiation. IL-8, LIF, CCL1, CCL5, CCL7, and CCL8 were also significantly secreted after IL-33 stimulation.
Conclusions
Our findings suggest that CD34 differentiated eosinophils are morphologically and phenotypically similar to peripheral eosinophils. The release of specific cytokines in direct response to IL-33 may contribute to the pathogenesis of type 2 inflammation and facilitates new avenues for studying eosinophils as effector cells in vitro.
Background:
Macrophages, with many different phenotypes play a major role during wound healing process, secreting the cytokines crucial to angiogenesis, cell recruitment and ECM remodeling. Therefore, macrophage-derived cytokines may be attractive therapeutic resource for wound healing.
Methods:
To obtain a conditioned media (CM) from macrophages, human monocyte THP-1 cells were seeded on TCP or human fibroblast-derived matrix (hFDM) and they were differentiated into M1 or M2 phenotype using distinct protocols. A combination of different substrates and macrophage phenotypes produced M1- and M2-CM or M1-hFDM- and M2-hFDM-CM, respectively. Proteome microarray determines the cytokine contents in those CMs. CMs-treated human dermal fibroblast (hDFB) was analyzed using collagen synthesis and wound scratch assay. Concentrated form of the CM (CCM), obtained by high-speed centrifugation, was administered to a murine full-thickness wound model using alginate patch, where alginate patch was incubated in the M2-CCM overnight at 4 °C before transplantation. On 14 day post-treatment, examination was carried out through H&E and Herovici staining. Keratinocyte and M2 macrophages were also evaluated via immunofluorescence staining.
Results:
Cytokine analysis of CMs found CCL1, CCL5, and G-CSF, where CCL5 is more dominant. We found increased collagen synthesis and faster wound closure in hDFB treated with M2-CM. Full-thickness wounds treated by M2-hFDM-CCM containing alginate patch showed early wound closure, larger blood vessels, increased mature collagen deposition, enhanced keratinocyte maturation and more M2-macrophage population.
Conclusion:
Our study demonstrated therapeutic potential of the CM derived from M2 macrophages, where the cytokines in the CM may have played an active role for enhanced wound healing.
CD8 T cell memory offers critical antiviral protection, even in the absence of neutralizing antibodies. The paradigm is that CD8 T cell memory within the lung tissue consists of a mix of circulating T EM cells and non-circulating T RM cells. However, based on our analysis, the heterogeneity within the tissue is much higher, identifying T CM , T EM , T RM , and a multitude of populations which do not perfectly fit these classifications. Further interrogation of the populations shows that T RM cells that express CD49a, both with and without CD103, have increased and diverse effector potential compared with CD49a negative populations. These populations function as a one-man band, displaying antiviral activity, chemokine production, release of GM-CSF, and the ability to kill specific targets in vitro with delayed kinetics compared with effector CD8 T cells. Together, this study establishes that CD49a defines multiple polyfunctional CD8 memory subsets after clearance of influenza infection, which act to eliminate virus in the absence of direct killing, recruit and mature innate immune cells, and destroy infected cells if the virus persists.
Contribution to the field
Protection from previously seen infections requires specialized immune memory cells properly positioned throughout the body to combat the newly invading pathogen. In the case of re-exposure to influenza virus, CD8 T cells resident within the respiratory tract (T RM ) are critical for eliminating the virus. Previously, T RM were viewed as mostly homogenous, with a limited range of immune functions. In this study, lung T RM were compared with circulating memory CD8 T cells transiently present within the lung, to define the breadth of their effector capabilities. Using T RM defining surface proteins CD49a and CD103 to identify different memory CD8 T cell subsets, gene and protein expression were evaluated. In addition to demonstrating higher levels of diversity than previously reported, multiple polyfunctional subsets were identified. This polyfunctionality was primarily associated with cell populations expressing CD49a, and these cells produced multiple antiviral factors, chemokines to recruit other immune cells, a growth factor associated with improved antigen presenting cell function, and cytolytic granules. Functional assays further demonstrated killing of target cells by T RM . This study paints a more holistic, complete picture of the phenotype and functions of lung CD8 T cells after viral infection, revealing CD49a as a marker of cells with high effector capacity.
We previously reported higher levels of C-C chemokine ligand (CCL) 1 in the bronchoalveolar lavage (BAL) fluid (BALF) of patients with sarcoidosis than in BALF of patients with immunoglobulin G4 (IgG4)-related disease (IgG4-RD), indicating that CCL1 might act as a marker of disease activity in sarcoidosis. Notably, less invasive sampling sources are desirable, as BAL cannot always be performed due to its inherent risk. In this study, we sought to decipher the correlation between serum levels of CCL1 and clinical characteristics of sarcoidosis. Serum samples were obtained from 44 patients with clinically confirmed sarcoidosis, 14 patients with IgG4-RD, and 14 healthy controls. The clinical and radiological findings were retrospectively evaluated. Serum levels of CCL1 were measured using a sandwich enzyme-linked immunosorbent assay. Serum levels of other 17 cytokines and chemokines were measured using a MILLIPLEX® MAP KIT and Luminex® magnetic beads. Serum levels of CCL1 were significantly higher in patients with sarcoidosis than in patients with IgG4-RD and healthy controls. Serum CCL1 was positively correlated with the degree of hilar lymph node swelling on chest computed tomography and serum levels of soluble interleukin 2 receptor. Positive correlations were also observed between serum CCL1 and total cell counts, lymphocyte counts in BALF, and serum T helper 1 mediators such as IP-10 and TNF-α in patients with sarcoidosis. Serum CCL1 levels were significantly elevated in sarcoidosis and correlated with clinical parameters of the disease. In addition, serum and BALF levels of CCL1 were positively correlated in a statistically significant manner. Although further research in this field is necessary, CCL1 might have the potential to be a reliable serological marker of disease activity in sarcoidosis.
Human T cell leukemia virus type 1 (HTLV-1) is a human retrovirus that is associated with two main diseases: HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T cell leukemia/lymphoma (ATL). Chemokines are highly specialized groups of cytokines that play important roles in organizing, trafficking, homing, and in the migration of immune cells to the bone marrow, lymphoid organs and sites of infection and inflammation. Aberrant expression or function of chemokines, or their receptors, has been linked to the protection against or susceptibility to specific infectious diseases, as well as increased the risk of autoimmune diseases and malignancy. Chemokines and their receptors participate in pathogenesis of HTLV-1 associated diseases from inflammation in the central nervous system (CNS) which occurs in cases of HAM/TSP to T cell immortalization and tissue infiltration observed in ATL patients. Chemokines represent viable effective prognostic biomarkers for HTLV-1-associated diseases which provide the early identification of high-risk, treatment possibilities and high-yielding clinical trials. This review focuses on the emerging roles of these molecules in the outcome of HTLV-1-associated diseases.
Inflammation, is driven by a network comprising cytokines, chemokines, their target receptors and leukocytes, and is a major pathologic mechanism that adversely affects organ function in diverse human diseases. Despite being supported by substantial target validation, no successful anti-chemokine therapeutic to treat inflammatory disease has yet been developed. This is in part because of the robustness of the chemokine network, which emerges from a large total chemokine load in disease, promiscuous expression of receptors on leukocytes, promiscuous and synergistic interactions between chemokines and receptors, and feedforward loops created by secretion of chemokines by leukocytes themselves. Many parasites, including viruses, helminths and ticks, evade the chemokine network by producing proteins that bind promiscuously to chemokines or their receptors. Evasins - three small glycoproteins identified in the saliva of the brown dog tick - bind multiple chemokines, and are active in several animal models of inflammatory disease. Over 50 evasin homologs have recently been identified from diverse tick species. Characterization of the chemokine binding patterns of evasins show that several have anti-chemokine activities that extend substantially beyond those previously described. These studies indicate that evasins function at the site of the tick bite by reducing total chemokine load. This not only reduces chemokine signaling to receptors, but also interrupts feedforward loops, thus disabling the chemokine network. Taking the lead from nature, a goal for the development of new anti-chemokine therapeutics would be to reduce the total chemokine load in disease. This could be achieved by administering appropriate evasin combinations or by smaller peptides that mimic evasin action.
Several chemokines have been shown to act as angiogenic molecules or to modulate the activity of growth factors such as fibroblast growth factor 2 (FGF-2) and vascular endothelial growth factor (VEGF). The detection of the CC chemokine receptor (CCR) 8 message in human umbilical vein endothelial cells (HUVECs) by reverse transcription– polymerase chain reaction (RT-PCR) and RNase protection assay (RPA), prompted us to investigate the potential role exerted by the CC chemokine I-309, a known ligand of such receptor, in both in vitro and in vivo angiogenesis assays. We show here that I-309 binds to endothelial cells, stimulates chemotaxis and invasion of these cells, and enhances HUVEC differentiation into capillary-like structures in an in vitro Matrigel assay. Furthermore, I-309 is an inducer of angiogenesis in vivo in both the rabbit cornea and the chick chorioallantoic membrane assay (CAM).
Chemokines are small peptides that are potent activators and chemoattractants for leukocyte subpopulations and some nonhemopoietic cells. Their actions are mediated by a family of 7-transmembrane G-protein–coupled receptors, the size of which has grown considerably in recent years and now includes 18 members. Chemokine receptor expression on different cell types and their binding and response to specific chemokines are highly variable. Significant advances have been made in understanding the regulation of chemokine receptor expression and the intracellular signaling mechanisms used in bringing about cell activation. Chemokine receptors have also recently been implicated in several disease states including allergy, psoriasis, atherosclerosis, and malaria. However, most fascinating has been the observation that some of these receptors are used by human immunodeficiency virus type 1 in gaining entry into permissive cells. This review will discuss structural and functional aspects of chemokine receptor biology and will consider the roles these receptors play in inflammation and in infectious diseases.
Apart from its involvement in immune functions, the chemokine CCL1 can participate in the modulation of nociceptive processing. Previous studies have demonstrated the hypernociceptive effect produced by CCL1 in the spinal cord, but its possible action on peripheral nociception has not yet been characterized. We describe here that the subcutaneous administration of CCL1 (1–10 µg/kg) produces dose-dependent and long-lasting increases in thermal withdrawal latencies measured by the unilateral hot plate test in mice. The antinociceptive nature of this effect is further supported by the reduction of spinal neurons expressing Fos protein in response to a noxious thermal stimulus observed after the administration of 10 µg/kg of CCL1. CCL1-induced antinociception was inhibited after systemic, but not spinal administration of the selective antagonist R243 (0.1–1 mg/kg), demonstrating the participation of peripheral CCR8 receptors. The absence of this analgesic effect in mice treated with a dose of cyclophosphamide that produces a drastic depletion of leukocytes suggests its dependency on white blood cells. Furthermore, whereas the antinociceptive effect of CCL1 was unaffected after the treatment with either the antagonist of opioid receptors naloxone or the cannabinoid type 1 receptor blocker AM251, it was dose-dependently inhibited after the administration of the CB2 receptor antagonist SR144528 (0.1-1 mg/kg). The detection by ELISA of an increased presence of the endocannabinoid 2-arachidonoylglycerol after the administration of an analgesic dose of CCL1 supports the notion that CCL1 can evoke thermal analgesia through the release of this endocannabinoid from circulating leukocytes.
Rationale:
Eosinophilic granulomatosis with polyangiitis (eGPA) is a small-vessel vasculitis where 40% of patients present with serum anti-neutrophil cytoplasmic antibodies (ANCA). We examined the presence and clinical relevance of ANCA in sputum in the serum-ANCA negative patients with eGPA.
Methods:
ANCA was investigated in matched sputum and blood samples collected from 23 eGPA (n = 10, serum-ANCA+), 19 eosinophilic asthma (prednisone-dependent), and 13 healthy volunteers. IgG reactivity to common target antigens and cytokine profiles in sputum samples were examined. Pathogenicity of detected sputum ANCA was assessed using in vitro degranulation assays.
Results:
The majority of eGPA patients (17/23, 74%) showed significantly increased sputum-ANCA compared to eosinophilic asthmatics and healthy controls (P < 0.0001), irrespective of their serum-ANCA status. In addition, 16/17 (94%) of sputum-ANCA+ patients had clinical manifestations of severe asthma compared to 3/6 (50%) in the sputum-ANCA- subset (P = 0.04). Microarray analysis of 123 common antigens failed to reveal a specific target for the ANCA-IgG. However, immunoprecipitated immunoglobulins from ANCA+ sputum allowed extensive extracellular trap formations from both neutrophils and eosinophils in vitro, indicating pathogenicity of detected IgG autoantibodies. Cytokine analysis showed lung-localized increases in CXCL8 (neutrophil/eosinophil chemotaxis), CCL24 (eosinophil recruitment) and CXCL12 (lymphocyte recruitment) in the sputa from ANCA+ patients (P < 0.01).
Conclusions:
We report a novel finding of ANCA-reactivity in the sputa of eGPA patients in whom disease severity is driven by respiratory complications. Investigating localized autoimmunity may lead to the discovery of novel pathomechanisms, therapeutic targets, and optimal biomarkers for diagnosing and managing eGPA.
It is well established that chemokines have a major role in the stimulation of cell movement on extracellular matrix (ECM) substrates. However, it is also clear that ECM substrates may influence the ability of cells to undergo migration. Using the migration chamber method, we assessed the migratory response of human embryonic kidney-293 (HEK) transfectant cells expressing the CC chemokine receptor 5 (CCR5) (HEK-CCR5) to stimulation by chemokines (macrophage inflamatory protein (MIP)-α, MIP-β, and regulated on activation normal-T cell expressed and secreted (RANTES) on ECM substrates (collagen type I and fibronectin). Using filters coated with collagen (20 μg/mL), results showed that the chemokines differed in their ability to elicit cell movement according to the order MIP-1β < RANTES ≈ MIP-1α. In contrast, using filters coated with fibronectin (20 μg/mL), all three chemokines were similar in their ability to stimulate migration of HEK-CCR5 cells. In addition, the migratory response with respect to the concentrations of ECM substrates appeared biphasic; thus, chemokine-stimulated cell movement was inhibited at high ECM concentrations (100 μg/mL). To determine the involvement of β1 integrins, results showed that the migratory response to chemokine stimulation on collagen was largely inhibited by monoclonal antibody (mAb) to α2β1; however, complete inhibition required a combination of mAbs to α2β1 and α2β1. In comparison, migration on fibronectin was inhibited by mAb to α3β1 and α5β1. Our results suggest that the migratory response to CCR5 stimulation may vary quantitatively with both the CCR5 ligand (MIP-1α, MIP-1β, and RANTES), as well as the nature and concentration of the ECM substrate involved.
Cellular immunity is governed by a complex network of migratory cues that enable appropriate immune cell responses in a timely and spatially controlled fashion. This review focuses on the chemokines and their receptors regulating the steady-state localisation of immune cells within healthy peripheral tissues. Steady-state immune cell traffic is not well understood but is thought to involve constitutive (homeostatic) chemokines. The recent discovery of tissue-resident memory T cells (TRM cells) illustrates our need for understanding how chemokines control immune cell mobilisation and/or retention. These studies will be critical to unravel novel pathways for preserving tissue function (aging) and preventing tissue disease (vaccination).
A monocyte chemotactic and activating factor (MCAF) has been purified from TNF-stimulated 8387 human fibrosarcoma cell line-conditioned media. The purified MCAF showed microheterogeneity yielding two bands on SDS-PAGE analysis. Fibrosarcoma-derived MCAF specifically competed with THP-1 (a human monocytic cell line)-derived 125I-labeled MCAF in binding to human PBMC, whereas a similar basic heparin-binding leukocyte chemoattractant, IL-8, did not. The purified MCAF stimulated superoxide anion and N-acetyl beta-D glucosaminidase-releasing activity in human monocytes, as well as monocyte cytostatic augmenting activity against tumor cells and chemotactic activity for monocytes. When injected subcutaneously into Lewis rat ears, the purified human MCAF also induced considerable in vivo local monocyte infiltration beginning at 3 h and becoming maximal at 18 h. In conclusion, the data presented in this paper indicate that MCAF is a potent activator of monocytes as well as a monocyte recruitment factor that acts through receptors that are specific for this novel molecule. This novel cytokine might have an important role in tumor growth control due to its ability to attract and activate monocytes.
The rapid, ultrasensitive silver stains that have been developed recently for detecting proteins in polyacrylamide gels show variation in staining from gel to gel and do not stain certain proteins at all. It was found that treatment of gels with dithiothreitol prior to impregnation with silver nitrate results in more reproducible staining patterns that are also qualitatively similar to those obtained with Coomassie blue. In addition, it obviates the need for treatment with intense light, and results in sensitivities at least as high as those obtained with previously published methods.
The bacterial chemotactic peptide fMet-Leu-Phe (fMLP) activates neutrophils upon binding to surface receptors. In a previous communication we reported the functional reconstitution of the fMLP receptor in Xenopus laevis oocytes (Coats, W. D., and Navarro, J. (1990) J. Biol. Chem. 265, 5964-5966). In this work we report the isolation of the cDNA encoding the fMLP receptor from neutrophils. A rabbit neutrophil cDNA library was screened with an oligonucleotide probe deduced from the nucleotide sequence of G-protein-coupled receptors, and a cDNA encoding the fMLP receptor was isolated. This cDNA was characterized according to the following criteria: 1) Analysis of the deduced amino acid sequence revealed that the clone belongs to a G-protein-coupled receptor. 2) Tissue distribution analysis of the mRNA indicated that the message is only found in neutrophils. 3) In vitro translation of the message revealed a protein corresponding in size to the deglycosylated fMLP receptor. 4) X. laevis oocytes injected with the fMLP receptor message exhibited fMLP-dependent calcium mobilization and specific binding to the fMLP analog 125I-labeled fNle-Leu-Phe-Nle-Tyr-Lys (where Nle is norleucine and fNle is formylnorleucine). The molecular cloning of the fMLP receptor should provide the framework to analyze the relationship between structure, function, and regulation of this receptor.
Human phagocytic cells express receptors for bacterial N-formyl peptides (formyl peptide receptor or FPR) which mediate chemotaxis, degranulation, and the respiratory burst. Although cDNA encoding a human phagocyte formyl peptide-binding protein has been reported recently (Boulay, F., Tardif, M., Brouchon, L., and Vignais, P. (1990) Biochem. Biophys. Res. Commun. 168, 1103-1109), functional coupling to signal transduction processes was not demonstrated. We describe corresponding full-length cDNA clones and prove that they encode the calcium-mobilizing human formyl peptide receptor by demonstrating functional reconstitution in the Xenopus oocyte. We further demonstrate that in contrast to all other cloned guanine nucleotide-binding regulatory protein (G-protein) coupled receptors expressed in this system, microinjection of FPR transcripts is not sufficient to confer ligand responsiveness to the oocyte: co-injection of phagocyte RNA encoding a complementary human factor that is not the alpha subunit of the heterotrimeric G-proteins Gi1, Gi2 or Gi3 is also required. Whereas a 1.4-kilobase FPR transcript is expressed exclusively in differentiated phagocytic cells, the complementary factor activity localizes to a 3.5-kilobase RNA fraction and is expressed in both differentiated and undifferentiated myeloid cells as well as in liver. The deduced human FPR protein possesses seven hydrophobic putative membrane spanning segments, three sites for N-linked glycosylation, and a short 18-amino acid predicted third cytoplasmic loop. Surprisingly, the human FPR possesses only 28% amino acid identity with the rabbit FPR reported recently by Thomas and co-workers (Thomas, K. M., Pyun, H. Y., and Navarro, J. (1990) J. Biol. Chem. 265, 20061-20065). Moreover, the rabbit FPR does not require a complementary factor for calcium mobilization in the oocyte. Structural alignment reveals at most 20% amino acid identity of the human FPR with other G-protein coupled receptors, indicating a common ancestral gene. Functional reconstitution of the recombinant FPR will now permit precise delineation of its functional and regulatory domains. Moreover, discovery of a complementary factor for oocyte expression of the human FPR establishes a novel approach to the qualification by ligand screening of cDNA encoding other suspected G-protein coupled receptors.
A model of the three-dimensional structure of the monocyte chemo-attractant and activating protein MCAF/MCP-1 is presented. The model is predicted based on the previously determined solution structure of interleukin-8 (IL-8/NAP-1) [Clore, G.M., Appella, E., Yamada, M., Matsushima, K. and Gronenborn, A.M. (1990) Biochemistry 29, 1689-1696]. Both proteins belong to a superfamily of cytokine proteins involved in cell-specific chemotaxis, host defense and the inflammatory response. The amino acid sequence identity between the two proteins is 24%. It is shown that the regular secondary structure elements of the parent structure can be retained in the modeled structure, such that the backbone hydrogen bonding pattern is very similar in the two structures. The polypeptide backbone is superimposable with an atomic r.m.s. difference of 0.9 A and all side chains can be modeled by transferring the parent side chain conformation to the new structure. Thus, the deduced structure, like the parent one, is a dimer and consists of a six-stranded antiparallel beta-sheet, formed by two three-stranded Greek keys, one from each monomer, upon which lie two symmetry-related antiparallel alpha-helices, approximately 24 A long and separated by approximately 14 A. All amino acid sequence changes can be accommodated within the parent polypeptide framework without major rearrangements. This is borne out by the fact that the IL-8/NAP-1 and modeled MCAF/MCP-1 structures have similar non-bonding energies. These results strongly suggest that both proteins and all other members of the superfamily most likely have the same tertiary structure.(ABSTRACT TRUNCATED AT 250 WORDS)
The crystal structure of a host defense system chemotactic factor, interleukin 8, has been solved by molecular replacement
using as a model the solution structure derived from nuclear magnetic resonance experiments. The structure was refined with
2 A x-ray data to an R factor of 0.187 (0.217 at 1.6 A). A comparison indicates some potential differences between the structure
in solution and in the crystalline state. Our analysis also predicts that residues 4 through 9 on the amino terminus and the
beta-bend, which includes His-33, may be important for receptor binding.
A monocyte chemotactic and activating factor (MCAF) has been purified from TNF-stimulated 8387 human fibrosarcoma cell line-conditioned media. The purified MCAF showed microheterogeneity yielding two bands on SDS-PAGE analysis. Fibrosarcoma-derived MCAF specifically competed with THP-1 (a human monocytic cell line)-derived 125I-labeled MCAF in binding to human PBMC, whereas a similar basic heparin-binding leukocyte chemoattractant, IL-8, did not. The purified MCAF stimulated superoxide anion and N-acetyl beta-D glucosaminidase-releasing activity in human monocytes, as well as monocyte cytostatic augmenting activity against tumor cells and chemotactic activity for monocytes. When injected subcutaneously into Lewis rat ears, the purified human MCAF also induced considerable in vivo local monocyte infiltration beginning at 3 h and becoming maximal at 18 h. In conclusion, the data presented in this paper indicate that MCAF is a potent activator of monocytes as well as a monocyte recruitment factor that acts through receptors that are specific for this novel molecule. This novel cytokine might have an important role in tumor growth control due to its ability to attract and activate monocytes.
We have identified cell surface receptors for Act-2, a secreted protein expressed upon activation of T cells, B cells, and monocytes. Although 125I-Act-2 showed little, if any, specific binding to resting peripheral blood lymphocytes (PBL) receptors were readily detected on PHA/PMA-activated PBL and a variety of cell lines including MT-2, HL60, DMSO differentiated HL60, HeLa, and K562 cells. The equilibrium dissociation constant (Kd) is 3-12 nM for MT-2, K562, and PBL activated with PHA/PMA for 40-80 h. We have also identified a rabbit polyclonal antiserum that can block Act-2 binding to its receptors. The ability to detect specific Act-2 receptors and the development of a blocking antiserum should prove valuable in efforts to molecularly clone the Act-2 receptor and to dissect the biological actions of Act-2.
We have used a cDNA library enriched for T cell-specific sequences to isolate genes expressed by T cells but not by other cell types. We report here one such gene, designated RANTES, which encodes a novel T cell-specific molecule. The RANTES gene product is predicted to be 10 kDa and, after cleavage of the signal peptide, approximately 8 kDa. Of the 68 residues, 4 are cysteines, and there are no sites for N-linked glycosylation. RANTES is expressed by cultured T cell lines that are Ag specific and growth factor dependent. RANTES expression is inducible in PBL by Ag or mitogen. In CTL, expression of RANTES decreases after stimulation with Ag and growth factors. Interestingly, RANTES was not expressed by any T cell tumor line tested. There is significant homology between the RANTES sequence and several other T cell genes, suggesting that they comprise a previously undescribed family of small T cell molecules.
The solution conformation of interleukin-8 (IL-8), a small protein of 72 residues with a wide range of proinflammatory activities, has been investigated by two-dimensional NMR spectroscopy. The 1H-NMR spectrum of IL-8 is assigned in a sequential manner and regular elements of secondary structure are identified on the basis of a qualitative interpretation of the nuclear Overhauser, coupling constant and amide exchange data. The IL-8 monomer contains a triple stranded anti-parallel beta-sheet arranged in a Greek key and a long C-terminal helix (residues 57-72). It is shown that IL-8 is a dimer in solution in which the interface is principally formed by six backbone hydrogen bonds between residues 25, 27, and 29 of one monomer and residues 29, 27, and 25, respectively, of the other. As a result, the two units of the dimer form a contiguous six-stranded anti-parallel beta-sheet. The secondary structure of IL-8 is similar to that found in the crystal structure of the sequence related protein platelet factor 4.
The biological properties of a neutrophil-activating factor (NAF), which was recently identified as a novel peptide of approximately 6,000 mol wt, are described. NAF is produced de novo by human blood monocytes upon stimulation with LPS, PHA, and Con A. It induces two main responses in human neutrophils, i.e., exocytosis (release from specific granules in normal, and from specific and azurophil granules in cytochalasin B-treated cells) and the respiratory burst (formation of superoxide and hydrogen peroxide). The action of NAF appears to be mediated by a surface receptor as shown by the following observations. (a) NAF induces a rapid and transient rise in cytosolic free Ca2+; (b) interaction with NAF results in desensitization, since the cells do not respond to a second NAF challenge; and (c) the respiratory burst elicited by NAF is similar in onset, and time course to that induced by C5a or FMLP. The NAF receptor can be distinguished from the receptors of C5a, FMLP, platelet-activating factor, and leukotriene B4 by the lack of cross-desensitization. Unlike C5a, the other host-derived neutrophil-activating peptide, NAF is not inactivated by serum and thus presumably accumulates in inflamed tissue.
Recent studies have focused on the potential role of transforming growth factor type beta (TGF-beta) as an immunoregulatory peptide. In this context, we demonstrate that TGF-beta is a potent chemoattractant for human peripheral blood monocytes. At concentrations from 0.1 to 10 pg/ml, TGF-beta induces directed monocyte migration in vitro. Consistent with this observation is the expression of high-affinity TGF-beta receptors on the monocytes with a Kd of 1-10 pM. At higher concentrations of TGF-beta (greater than or equal to 1 ng/ml), monocytes are stimulated to generate biologically active mediator(s) that enhance fibroblast growth. Gene expression for one of these growth factors, interleukin 1, is induced in monocytes within hours after exposure to TGF-beta. Thus, TGF-beta may provide an important signal for monocyte recruitment and for regulation of their synthesis of mediators of fibroblast growth and activity in wound healing.
The platelet-derived growth factor-inducible gene JE was found to encode a 148-residue basic (pI = 10.4) secretory protein which shows striking similarity to the gene products of a family of small inducible genes (SIG), LD78, TCA3, IP10, 3-10C, 9E3/pCEF4, and gro/MGSA, and to several of the proteins secreted from platelet alpha-granules. Members of the SIG family have spatially conserved cysteine residues that vary in distance by only one amino acid residue as well as conserved proline residues at analogous sites. Hydrophilicity plots show alternating hydrophobic and hydrophilic domains which are similar for all members of the SIG family except IP10 and platelet factor 4, which show similarities to each other. The genomic organization of SIG family members is similar in the location of the splice junctions and the number of introns and exons, suggesting that they were derived from a common ancestor. The collective evidence suggests that a family of inducible cytokines, which are mitogenic or chemotactic, may act as intercellular coordinators of diverse responses designed to combat infection and promote the healing and regeneration of injured tissue.
Platelet factor 4 (PF4), which is released by platelets during coagulation, binds very tightly to negatively charged oligosaccharides such as heparin. To date, six other proteins are known that are homologous in sequence with PF4 but have quite different functions. The structure of a tetramer of bovine PF4 complexed with one Ni(CN)4²⁻ molecule has been determined at 3.0 Å resolution and refined to an R factor of 0.28. The current model contains residues 24–85, no solvent, and one overall temperature factor. Residues 1–13, which carried an oligosaccharide chain, were removed with elastase to induce crystallization; residues 14–23 and presumably 86–88 are disordered in the electron density map. Because no heavy atom derivative was isomorphous with the native crystals, the complex of PF4 with one Ni(CN)4²⁻ molecule was solved using a single, highly isomorphous Ni(CN)4²⁻ derivative and the iterative, single isomorphous replacement method.
The secondary structure of the PF4 subunit, from amino- to carboxyl-terminal end, consists of an extended loop, three strands of antiparallel β-sheet arranged in a Greek key, and one α-helix. The tetramer contains two extended, six-stranded β-sheets, each formed by two subunits, which are arranged back-to-back to form a “β-bilayer” structure with two buried salt bridges sandwiched in the middle. The carboxyl-terminal α-helices, which contain lysine residues that are thought to be intimately involved in binding heparin, are arranged as antiparallel pairs on the surface of each extended β-sheet.
A number of macrophage-derived mediators have been implicated in the vascular changes of inflammation. We recently reported the isolation of a novel monokine, macrophage inflammatory protein 1 (MIP-1), which causes local inflammatory responses in vivo, and induces superoxide production by neutrophils in vitro. Purified native MIP-1 comprises two peptides with very similar physical characteristics. We report here the resolution of MIP-1 into component peptides by SDS-hydroxylapatite chromatography, and compare the NH2-terminal sequences of the two peptides, now referred to as MIP-1 alpha and MIP-1 beta. A synthetic oligonucleotide probe pool corresponding to the NH2-terminal amino acid sequence of MIP-1 beta was used to isolate a cDNA clone containing its coding sequence. The sequence codes for a 109 amino acid-long polypeptide, of which 69 amino acids correspond to the mature product. Comparison of this MIP-1 beta cDNA with our previously cloned MIP-1 alpha sequence reveals that the MIP-1 peptides, members of a growing family of potential inflammatory mediators, are distinct but highly homologous (58.9% sequence identity) products of different genes.
We report the identification and purification of a new inflammatory monokine synthesized by the macrophage tumor cell line RAW 264.7 in response to endotoxin. This monokine, which we term "macrophage inflammatory protein" (MIP), is a doublet with an apparent molecular mass of approximately 8,000 daltons on SDS-PAGE but forms aggregates of greater than 2 x 10(6) daltons as assessed by gel filtration. Partial NH2-terminal amino acid sequence data reveal no significant homology with any previously described protein. Although the monokine is anionic under physiological conditions, it is one of two major macrophage-secreted proteins that bind to heparin at high salt concentrations. At 100 ng/ml or greater, MIP is chemokinetic for human polymorphonuclear cells and triggers hydrogen peroxide production. Subcutaneous injection of 10 ng or greater of MIP into footpads of C3H/HeJ mice elicits an inflammatory response, characterized by neutrophil infiltration. These findings suggest that MIP is an endogenous mediator that may play a role in the host responses that occur during endotoxemia and other inflammatory events.
Stimulated human monocytes release several proteins thought to play a role in inflammation, including interleukin 1, tumor necrosis factor, and plasminogen activator. We have purified another proinflammatory protein that is chemotactic for human neutrophils from conditioned medium of lipopolysaccharide-stimulated monocytes. After a series of steps that included anion-exchange chromatography, gel filtration, and HPLC on cation-exchange and reverse-phase columns, an apparently pure protein was obtained that migrated as a single 7-kDa band on NaDodSO4/polyacrylamide gels under reducing or nonreducing conditions. The amino acid composition of this monocyte-derived neutrophil chemotactic factor was different from that of interleukin 1 and tumor necrosis factor. N-terminal amino acid sequence of the first 42 residues was determined. This portion of the molecule has up to 56% sequence similarity with several proteins that may be involved in host responses to infection or tissue injury. It is identical to a portion of a sequence deduced from an mRNA induced by staphylococcal enterotoxin treatment of human leukocytes. At the optimal concentration of 10 nM, 50% of neutrophils added to chemotaxis assay wells migrated toward the pure attractant. Potency and efficacy are comparable to that of fMet-Leu-Phe, which is often used as a reference. In contrast to many attractants, the protein was not chemotactic for human monocytes.
A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, "quin2", the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.
The biological properties of a neutrophil-activating factor (NAF), which was recently identified as a novel peptide of approximately 6,000 mol wt, are described. NAF is produced de novo by human blood monocytes upon stimulation with LPS, PHA, and Con A. It induces two main responses in human neutrophils, i.e., exocytosis (release from specific granules in normal, and from specific and azurophil granules in cytochalasin B-treated cells) and the respiratory burst (formation of superoxide and hydrogen peroxide). The action of NAF appears to be mediated by a surface receptor as shown by the following observations. (a) NAF induces a rapid and transient rise in cytosolic free Ca2+; (b) interaction with NAF results in desensitization, since the cells do not respond to a second NAF challenge; and (c) the respiratory burst elicited by NAF is similar in onset, and time course to that induced by C5a or FMLP. The NAF receptor can be distinguished from the receptors of C5a, FMLP, platelet-activating factor, and leukotriene B4 by the lack of cross-desensitization. Unlike C5a, the other host-derived neutrophil-activating peptide, NAF is not inactivated by serum and thus presumably accumulates in inflamed tissue.
A number of macrophage-derived mediators have been implicated in the vascular changes of inflammation. We recently reported the isolation of a novel monokine, macrophage inflammatory protein 1 (MIP-1), which causes local inflammatory responses in vivo, and induces superoxide production by neutrophils in vitro. Purified native MIP-1 comprises two peptides with very similar physical characteristics. We report here the resolution of MIP-1 into component peptides by SDS-hydroxylapatite chromatography, and compare the NH2-terminal sequences of the two peptides, now referred to as MIP-1 alpha and MIP-1 beta. A synthetic oligonucleotide probe pool corresponding to the NH2-terminal amino acid sequence of MIP-1 beta was used to isolate a cDNA clone containing its coding sequence. The sequence codes for a 109 amino acid-long polypeptide, of which 69 amino acids correspond to the mature product. Comparison of this MIP-1 beta cDNA with our previously cloned MIP-1 alpha sequence reveals that the MIP-1 peptides, members of a growing family of potential inflammatory mediators, are distinct but highly homologous (58.9% sequence identity) products of different genes.
Interleukin-8 (IL-8) is a member of a family of pro-inflammatory cytokines. Although the best characterized activities of IL-8 include the chemoattraction and activation of neutrophils, other members of this family have a wide range of specific actions including the chemotaxis and activation of monocytes, the selective chemotaxis of memory T cells, the inhibition of hematopoietic stem cell proliferation, and the induction of neutrophil infiltration in vivo. A complementary DNA encoding the IL-8 receptor from human neutrophils has now been isolated. The amino acid sequence shows that the receptor is a member of the superfamily of receptors that couple to guanine nucleotide binding proteins (G proteins). The sequence is 29% identical to that of receptors for the other neutrophil chemoattractants, fMet-Leu-Phe and C5a. Mammalian cells transfected with the IL-8 receptor cDNA clone bind IL-8 with high affinity and respond specifically to IL-8 by transiently mobilizing calcium. The IL-8 receptor may be part of a subfamily of related G protein-coupled receptors that transduce signals for the IL-8 family of pro-inflammatory cytokines.
Host defence and inflammatory responses are controlled and amplified by receptor-mediated events often initiated by a chemotactic factor that directs the approach of phagocytic cells. Complement receptors CR1 and CR3 are responsible for the phagocytic and adhesive properties of neutrophils, whereas the C5a receptor mediates the pro-inflammatory and chemotactic actions of the complement anaphylatoxin C5a. In addition to stimulating chemotaxis, granule enzyme release and superoxide anion production, this receptor stimulates upregulation of expression and activity of the adhesion molecule MAC-1, and of CR1, and a decrease in cell-surface glycoprotein 100MEL-14 on neutrophils. In vivo, the C5a receptor may participate in anaphylactoid and septic shock. The human C5a receptor was cloned from U937 and HL-60 cells and identified by high affinity binding when expressed in COS-7 cells. The deduced amino-acid sequence of the receptor reveals the expected motifs befitting its interaction with cellular GTP-binding proteins.
Interleukin-8 (IL-8) is an inflammatory cytokine that activates neutrophil chemotaxis, degranulation, and the respiratory
burst. Neutrophils express receptors for IL-8 that are coupled to guanine nucleotide-binding proteins (G proteins); binding
of IL-8 to its receptor induces the mobilization of intracellular calcium stores. A cDNA clone from HL-60 neutrophils, designated
p2, has now been isolated that encodes a human IL-8 receptor. When p2 is expressed in oocytes from Xenopus laevis, the oocytes
bind 125I-labeled IL-8 specifically and respond to IL-8 by mobilizing calcium stores with an EC50 of 20 nM. This IL-8 receptor
has 77% amino acid identity with a second human neutrophil receptor isotype that binds IL-8 with higher affinity. It also
exhibits 69% amino acid identity with a protein reported to be an N-formyl peptide receptor from rabbit neutrophils, but less
than 30% identity with all other known G protein-coupled receptors, including the human N-formyl peptide receptor.
A family consisting of at least ten distinct novel 8-10 kd cytokines has been identified over the past 12 years. These cytokines exhibit from 20 to 45% homology in amino acid sequence, are probably all basic heparin-binding polypeptides, and have proinflammatory and reparative activities. The cDNA for these cytokines are characterized by conserved single open reading frames, typical signal sequences in the 5' region, and AT rich sequences in the 3' untranslated regions. Those human cytokines known as interleukin 8, platelet factor 4, beta thromboglobulin, IP-10 and melanoma growth stimulating factor or GRO can be assigned to a subfamily based on their location on chromosome 4 and unique structural features, whereas the second subset consisting of LD78, ACT-2, I-309, RANTES, and macrophage chemotactic and activating factor (MCAF) are all closely linked on human chromosome 17. In this review we have summarized and discussed the available information concerning the regulation and structure of the genes, the structure and biochemical properties of the polypeptide products, their receptors, signal transduction, cell sources, and in vitro as well as in vivo activities of these cytokines.
The inflammatory response involves the recruitment and activation of various types of cells from the systemic circulation and from local tissues. One important component of the inflammatory response is the activation of platelets at sites of tissue injury and inflammation. In particular, activated platelets release large amounts of two proteins, platelet factor 4 (PF4) and beta-thromboglobulin (beta TG), which mediate several inflammatory processes. Recently, many novel proteins that are structurally related to PF4 and beta TG have been identified. The PF4-related proteins are secreted by white blood cells, endothelial cells, and fibroblasts in response to various inflammatory and mitogenic stimuli. Like PF4, these proteins appear to be inflammatory response mediators; several of them are potent chemoattractants, activating agents, or mitogens for specific cell types that are involved in the inflammatory response. The study of PF4-related proteins provides new insight into the mechanisms of the immune response, and may result in the development of new therapeutic agents.
The binding of human monocyte chemoattractant protein-1 (MCP-1) to human monocytes was studied. MCP-1 was radioiodinated with Iodo-beads (Pierce Chemical Co., Rockford, IL) without significant loss of biologic activity. 125I-MCP-1 binding to PBMC occurred within 5 min at 0 degrees C and the binding was inhibited by unlabeled MCP-1 dose dependently but not by neutrophil attractant/activation protein-1 or FMLP. 125I-MCP-1 bound to monocytes; no significant binding to either neutrophils or lymphocytes was observed. Scatchard plot analysis indicated that monocytes had a minimum of 1700 +/- 600 binding sites per cell with a Kd of 1.9 +/- 0.2 x 10(-9) M. For analysis of binding by flow cytometry, MCP-1 was biotinylated. In contrast to radioiodination, biotinylation resulted in loss of activity; potency was 10-fold less, but the efficacy was retained. Detection by flow cytometry of bound biotinylated MCP-1 with avidin-FITC confirmed results obtained with 125I-MCP-1. Biotinylated MCP-1 bound to monocytes but not to lymphocytes; and the binding was inhibited by a 100-fold excess of unlabeled MCP-1.
Two variants of the human N-formylpeptide chemoattractant receptor have been isolated from a CDM8 expression library prepared from mRNA of human myeloid HL-60 cells differentiated to the granulocyte phenotype with Bt2cAMP. Both recombinant receptors, fMLP-R26 and fMLP-R98, are 350 amino acids long (Mr 38,420); they differ from each other by two residue changes at positions 101 and 346 and by significant differences in the 5' and 3' untranslated regions. Both clones were able to transfer to COS-7 cells the capacity to specifically bind a new and highly efficient hydrophilic derivative of N-formyl-Met-Leu-Phe-Lys, referred to as fMLPK-Pep12. Photolabeling experiments revealed that the glycosylated form of the fMLP receptor in COS cells has a molecular weight (Mr 50,000-70,000) similar to that observed for the native receptor in differentiated HL-60 cells. Northern blot analysis revealed a major transcript of 1.6-1.7 kb and two minor hybridization signals of 2.3 and 3.1 kb, suggesting a related family of receptors. The complex hybridization pattern obtained with restricted genomic DNA was consistent with either two genes encoding fMLP receptor isoforms or a single gene with at least one intron in the coding sequence. Sequence comparison established that the fMLP receptor belongs to the G-protein-coupled receptor superfamily. The structural similarities observed with RDC1, a receptor isolated from a dog thyroid cDNA library, which shares weak homologies with other members of the family, suggests that the fMLP receptor is representative of a new subfamily.
TCA3 is a cDNA originally isolated from activated T cells. Transcription of this gene has been shown to correlate with Ag-induced cellular activation of both T cells and mast cells. Based on the predicted amino acid sequence encoded by the cDNA, we previously proposed that TCA3 represents a cytokine. In this report we have used rDNA technology to express TCA3 in two mammalian cell lines. In both cases, TCA3 was expressed as a secreted molecule with an apparent molecular mass of 16 kDa. Digestion of the (rTCA3) with the enzyme N-glycanase revealed that approximately 8 kDa is caused by N-linked glycosylation. Intradermal injection of rTCA3 into mouse footpads resulted in a rapid swelling response. The sites of injection were characterized histologically by a local accumulation of neutrophils. These findings are discussed with particular attention to a family of related proteins, some of whose members also have inflammatory properties.
Neutrophil attractant/activation protein-1 (NAP-1) was previously shown to attract human neutrophils, but not monocytes. The purpose of this study was to determine if NAP-1 interacted with other types of blood leukocytes. In addition to its chemotactic activity for neutrophils, NAP-1 induced chemotactic responses by T lymphocytes and basophils. Chemotactic potency (10(-8) M for an optimal response) was the same for all three cell types. However, NAP-1 caused a chemotactic response in excess of random migration of 7% or 16% of basophils (depending on the medium used) and only 9% of T lymphocytes, in contrast to 30% of neutrophils. This agonist was not chemotactic for partially purified normal human eosinophils. The symmetrical histogram obtained by flow cytometry of neutrophils equilibrated at 0 degree C with fluoresceinated NAP-1 indicates that all neutrophils bound the ligand. A dose-response curve plateau, and inhibition of binding of NAP-1-FITC by unlabeled ligand are evidence for saturable binding to receptors, estimated to be 7000 per cell. Our results suggest that, for induction of an acute inflammatory response, the quantitatively significant action of NAP-1 is on neutrophils.
A discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system for the separation of proteins in the range from 1 to 100 kDa is described. Tricine, used as the trailing ion, allows a resolution of small proteins at lower acrylamide concentrations than in glycine-SDS-PAGE systems. A superior resolution of proteins, especially in the range between 5 and 20 kDa, is achieved without the necessity to use urea. Proteins above 30 kDa are already destacked within the sample gel. Thus a smooth passage of these proteins from sample to separating gel is warranted and overloading effects are reduced. This is of special importance when large amounts of protein are to be loaded onto preparative gels. The omission of glycine and urea prevents disturbances which might occur in the course of subsequent amino acid sequencing.
A method for the direct visualization of Coomassie blue-stained polypeptide bands during electrophoresis with subsequent elution of polypeptides and removal of sodium dodecyl sulfate (SDS) and Coomassie blue is described. Primarily it is intended as a means for easy and--because there is no protein fixation step--nearly quantitative recovery of separated polypeptides for amino acid sequencing. It may also be used to obtain rapid information about the protein patterns during a run. Together with our new high resolution SDS-polyacrylamide gel electrophoresis system for small proteins and polypeptides (H. Schägger and G. Von Jagow (1987) Anal. Biochem. 166, 368-379) the method described allows the preparative separation of protein fragments as even protein fragments between 1 and 3.5 kDa are easily detected.
We have isolated and characterized four cDNA clones that encode mRNA expressed more abundantly in Con A-activated mouse helper T cells than by resting T cells. One mRNA encoded a approximately 14-kDa protein with a hydrophobic N-terminal sequence and was abundantly expressed by the Th 2 subset of Th cells, but was not expressed by Th 1 cells. The remaining three mRNA encoded related approximately 8-kDa secreted proteins that are part of a family of small, secreted, and inducible mouse and human proteins. This family of proteins is itself distantly related to another family of growth and inflammatory factors that are associated with various lymphoid and fibroblast activation phenomena. One of the small, inducible, secreted proteins has a predicted mature N terminus identical to that of the previously described macrophage inflammatory protein.
As a function of protein concentration, proton NMR spectra of human platelet factor 4 (PF4) differ. Correlation with low-angle laser light scattering data has allowed identification of concentration-dependent NMR spectral changes to PF4 aggregation, with tetramers being the largest aggregates formed. Well-resolved aromatic ring proton NMR resonances were assigned to Tyr-60, His-I, and His-II in monomer, dimer, and tetramer states. Since Tyr-60 3.5 ring proton resonances are well resolved from state to state, estimation of fractional populations in each state was possible. By varying the PF4 concentration, changes in these populations when plotted according to the Hill equation show a bimolecular mechanism of aggregation which proceeds from monomers to tetramers through a dimer intermediate. Equilibrium constants for dimer association (KD) and tetramer association (KT) have been estimated as a function of pH and ionic strength. At pH 4, where KD and KT approach the same value, resonances associated with all three aggregate states are observed. Lowering the pH shifts the equilibrium to the monomer state, while raising the pH shifts the equilibrium to dimer and tetramer states. Analysis of the pH dependence of KD and KT suggests that electrostatic interactions, probably arising from Glu/Asp and Lys/Arg side chains, play a role in the binding process. Increasing the solvent ionic strength stabilizes the tetramer state especially at low pH, suggesting that intersubunit, repulsive electrostatic interactions probably between/among cationic side chains (Lys/Arg) attenuate the aggregation process. Information based primarily on histidine pKa values and photo-CIDNP 1H NMR data suggests that Tyr-60 and His-I, but not His-II, are significantly affected by the aggregation process.
In a study of the structural basis for leukocyte specificity of chemoattractants, we determined the complete amino acid sequence of human glioma-derived monocyte chemotactic factor (GDCF-2), a peptide that attracts human monocytes but not neutrophils. The choice of a tumor cell product for analysis was dictated by its relative abundance and an amino acid composition indistinguishable from that of lymphocyte-derived chemotactic factor (LDCF), the agonist thought to account for monocyte accumulation in cellular immune reactions. By a combination of Edman degradation and mass spectrometry, it was established that GDCF-2 comprises 76 amino acid residues, commencing at the N terminus with pyroglutamic acid. The peptide contains four half-cystines, at positions 11, 12, 36, and 52, which create a pair of loops, clustered at the disulfide bridges. The relative positions of the half-cystines are almost identical to those of monocyte-derived neutrophil chemotactic factor (MDNCF), a peptide of similar mass but with only 24% sequence identity to GDCF. Thus, GDCF and MDNCF have a similar gross secondary structure because of the loops formed by the clustered disulfides, and their different leukocyte specificities are most likely determined by the large differences in primary sequence.
Certain inflammatory stimuli render cultured human vascular endothelial cells hyperadhesive for neutrophils. This state is transient and reversible, in part because activated endothelial cells secrete a leukocyte adhesion inhibitor (LAI). LAI was identified as endothelial interleukin-8 (IL-8), the predominant species of which is an extended amino-terminal IL-8 variant. At nanomolar concentrations, purified endothelial IL-8 and recombinant human IL-8 inhibit neutrophil adhesion to cytokine-activated endothelial monolayers and protect these monolayers from neutrophil-mediated damage. These findings suggest that endothelial-derived IL-8 may function to attenuate inflammatory events at the interface between vessel wall and blood.
We have identified two cDNA clones, I-309 and G-26, which define genes expressed abundantly in activated human PBMC, but at low or undetectable levels in resting PBMC. Based upon nucleotide sequence analysis, both clones are predicted to encode small, structurally related polypeptides, each containing a hydrophobic leader sequence characteristic of secreted proteins and a motif of four conserved cysteine residues. Further, I-309 and G-26 are structurally related to a growing family of genes that apparently encode small polypeptides whose secretion is induced upon cell activation. I-309 represents a previously undescribed human gene. We have generated an anti-peptide antiserum to the I-309 gene product which recognizes proteins in culture supernatants of an activated T cell clone and of COS cells transfected with the I-309 cDNA, supporting the idea that I-309 encodes a secreted protein. Because I-309 encodes a small protein secreted by activated T cells that displays structural features similar to other cytokines, we believe that it defines a novel cytokine with as yet unknown function.
Physicochemical characteristics of monocyte chemotactic activity in the culture fluid of PHA-stimulated human mononuclear leukocytes (MNL) were investigated. Among several chemotactic activity peaks eluted from a TSK-2000 gel filtration column, one peak, corresponding to a molecular mass of 17 kDa, accounted for about 40% of total chemotactic activity. On a chromatofocusing column, most of the 17-kDa activity eluted in a pH range of 9.4 to 7.9. It could bind to Orange-A Sepharose. These three characteristics--molecular mass, basic isoelectric point, and dye column binding--were similar to those of human glioma-derived monocyte chemotactic factor (GDCF), recently purified in our laboratory. Therefore, the MNL-derived chemoattractant was purified by the same procedures used for purification of GDCF, namely Orange-A Sepharose chromatography, carboxymethyl (CM)-HPLC, and reverse phase (RP) HPLC. About 50% of the culture fluid chemotactic activity bound to Orange-A Sepharose and was eluted in a single peak by a NaCl gradient. The active pool from the Orange-A column was separated into two sharp peaks by CM-HPLC, each of which eluted at identical acetonitrile concentrations from a RP HPLC column. By SDS-PAGE, the peptides had apparent molecular masses of 15 and 13 kDa and appeared homogeneous. Amino acid analysis showed that the composition of the two peptides was almost identical; and the N terminus of each peptide was apparently blocked. Shared characteristics of these peptides and the GDCF peptides include identical elution patterns from CM- and RP HPLC columns, identical SDS-PAGE migration, almost identical amino acid composition, and blocked N terminus. This suggests that the monocyte attractants isolated from culture fluid of PHA-stimulated MNL are identical to those derived from human glioma cells.
Recently the human T cell erythrocyte receptor CD2 has been shown to bind human erythrocytes through LFA-3, a heavily glycosylated surface protein of broad tissue distribution. CD2-LFA-3 interactions are important for cytolytic conjugate formation, for thymocyte adhesion, and for T cell activation. A complementary DNA clone encoding LFA-3 was isolated using a complementary DNA clone encoding LFA-3 was isolated using a novel transient expression system of mouse cells. The cDNA encodes a phospholipid-linked membrane protein whose extracellular domain shares significant homology with CD2. As CD2 is homologous with the neural cell adhesion molecule NCAM in immunoglobulin-like domains, cellular adhesion molecules in both neural and lymphoid tissues could have a common ancestor.
A cDNA clone encoding a novel hematopoietic growth factor activity produced by a gibbon T cell line has been identified using a mammalian cell expression cloning system. The sequence of this cDNA proved to have significant homology to the sequence encoding murine interleukin 3 (IL-3). The human gene, which was readily identified because of its high degree of homology to the gibbon sequence, also displayed significant homology with the murine IL-3 sequence. The recombinant gibbon IL-3 protein proved to have multipotent colony stimulating activity when tested with normal human bone marrow cells, proving that this primate hematopoietin is not only structurally but also functionally related to murine IL-3.
We describe a simple calcium phosphate transfection protocol and neo marker vectors that achieve highly efficient transformation
of mammalian cells. In this protocol, the calcium phosphate-DNA complex is formed gradually in the medium during incubation
with cells and precipitates on the cells. The crucial factors for obtaining efficient transformation are the pH (6.95) of
the buffer used for the calcium phosphate precipitation, the CO2 level (3%) during the incubation of the DNA with the cells,
and the amount (20 to 30 micrograms) and the form (circular) of DNA. In sharp contrast to the results with circular DNA, linear
DNA is almost inactive. Under these conditions, 50% of mouse L(A9) cells can be stably transformed with pcDneo, a simian virus
40-based neo (neomycin resistance) marker vector. The NIH3T3, C127, CV1, BHK, CHO, and HeLa cell lines were transformed at
efficiencies of 10 to 50% with this vector and the neo marker-incorporated pcD vectors that were used for the construction
and transduction of cDNA expression libraries as well as for the expression of cloned cDNA in mammalian cells.
Framework monoclonal antibodies have identified a population of human lymphocytes that express the T3 glycoprotein but not the T-cell receptor (TCR) alpha- and beta-subunits. Chemical crosslinking experiments reveal that these lymphocytes express novel T3-associated polypeptides, one of which appears to be the product of the T gamma gene. The other polypeptide may represent a fourth TCR subunit, designated T delta.
A simple method is described for detecting 3H in polyacrylamide gels by scintillation autography (fluorography) using X-ray film. The gel is dehydrated in dimethyl sulphoxide, soaked in a solution of 2,5-diphenyloxazole (PPO) in dimethylsulphoxide, dried and exposed to RP Royal “X-Omat” film at -70 °C. Optimal conditions for each step are described. β-particles from 3H interact with the 2,5-diphenyloxazole emitting light which causes local blackening of an X-ray film. The image produced resembles that obtained by conventional autoradiography of isotopes with higher emission energies such as 14C. 3000 dis. 3H/min in a band in a gel can be detected in a 24-h exposure. Similarly 500 dis./min can be detected in one week.
When applied to the detection of 35S and 14C in polyacrylamide gels, this method is ten times more sensitive than conventional autoradiography. 130 dis. 35S or 14C/min in a band in a gel can be detected in 24 h.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
Mutants of Chinese hamster ovary cells lacking dihydrofolate reductase (tetrahydrofolate dehydrogenase, 7,8-dihydrofolate:NADP+ oxidoreductase; EC 1.5.1.3) activity were isolated after mutagenesis and exposure to high-specific-activity [3H]deoxyuridine as a selective agent. Fully deficient mutants could not be isolated starting with wild-type cells, but could readily be selected from a putative heterozygote that contains half of the wild-type level of dihydrofolate reductase activity. The heterozygote itself was selected from wild-type cells by using [3H]deoxyuridine together with methotrexate to reduce intracellular dihydrofolate reductase activity. Fully deficient mutants require glycine, a purine, and thymidine for growth; this phenotype is recessive to wild type in cell hybrids. Revertants have been isolated, one of which produces a heat-labile dihydrofolate reductase activity. These mutants may be useful for metabolic studies relating to cancer chemotherapy and for fine-structure genetic mapping of mutations by using available molecular probes for this gene.
We designed a 48-well chemotaxis chamber to minimize manipulation time and amount of material required by the larger blindwell or Boyden chemotaxis chamber. Cell and chemoattractant dose-response curves showed that results were comparable to our better than those obtained with blindwell chambers. The volume of chemoattractant per well is 25 microliter; the number of cells can be as low as 10,000. The time needed for setting up this multiwell unit and for staining the membrane filter sheet is negligible. Combined with the use of an image analyzer to count the number of migrated cells, the method is suitable for clinical research on the functional state of monocytes in large groups of patients.
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