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Annexin I surface binding sites and their regulation on human fibroblast-like synoviocytes
Abstract
Annexin I is a glucocorticoid-inducible protein whose expression in rheumatoid synovium and inhibitory actions in animal models of arthritis suggests its involvement in human arthritis. The present study explored the potential for annexin I to mediate its antiinflammatory actions via specific cell-surface binding sites on human fibroblast-like synoviocytes (FLS).
Annexin I binding sites on cultured FLS from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) were determined by ligand-binding flow cytometry. Phospholipase A2 (PLA2) activity was determined by arachidonic acid release.
FLS exhibited saturable, concentration-dependent cell-surface annexin I binding, with >99% of the OA FLS exhibiting binding at an annexin I concentration of 10 microM. Annexin I binding of RA FLS was significantly lower than that of OA FLS. FLS annexin I binding sites were not affected by elastase or a specific elastase inhibitor, and elastase release did not differ between RA and OA cells. In contrast, collagenase significantly increased annexin I binding sites on OA FLS and approached a significant effect on RA FLS. Tumor necrosis factor alpha increased annexin I binding sites on OA and RA FLS. Similarly, interleukin-1beta significantly increased annexin I binding on OA FLS; but the increased binding on RA FLS was not significant. Dexamethasone exerted no significant effect on OA or RA FLS annexin I binding sites. Treatment of RA FLS with an annexin I N-terminal peptide significantly inhibited RA FLS PLA2 activity.
This is the first description of the expression, regulation, and function of cell surface annexin I binding sites on FLS. Reduced annexin I binding sites in RA FLS may impair the sensitivity of certain proinflammatory events to glucocorticoids.
... It exerts a constitutive inhibitory influence, and mediates the inhibitor effects of glucocorticoids , in rodent models of RA [3, 9, 10, 14–16]. Regulated expression of functionally active cell surface annexin I binding sites has been reported in human RA fibroblastlike synoviocytes (FLS) [17] [18]. Despite this, the expression and regulation of annexin I in FLS has not been re- ported. ...
... The absence of Anx-1 was also associated with insensitivity to the antiinflammatory effects of dexamethasone, indicating a major role for Anx-1 in the pathopharmacology of inflammation [16]. Annexin I has been demonstrated in human peripheral blood leukocytes and in RA synovium [12] [13], and annexin I cell surface binding sites have been demonstrated in RA FLS [17] [18]. Synovial fibroblasts are important contributors to the pathology of RA, but the regulation of annexin I in human synovial cells by pro-and antiinflammatory mediators has not been previously reported. ...
... Annexin I responses to GC may also potentially be associated with GC resistance. Certainly, leukocyte intracellular annexin I protein responses to glucocorticoids are impaired in RA patients [29], leukocytes and synovial fibroblast cell surface annexin I binding sites are reduced in RA patients [17] [18] [30], and sensitivity to GC in arthritic mice is impaired in the absence of annexin I [16]. Annexin I expression is possibly an index for recognition of GC resistance. ...
The glucocorticoid (GC)-induced antiinflammatory molecule annexin I is expressed in leukocytes and has antiinflammatory effects in animal models of arthritis, but the expression of annexin I in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) is unknown. We report the constitutive and dexamethasone (DEX)-inducible expression of annexin I in RA FLS. DEX increased FLS annexin I protein translocation and mRNA expression. Interleukin (IL)-1beta also induced annexin I translocation and mRNA but also increased intracellular protein. DEX and IL-1 had additive effects on annexin I mRNA, but DEX inhibited the inducing effect of IL-1beta on cell surface annexin I. These results indicate that glucocorticoids and IL-1beta upregulate the synthesis and translocation of annexin I in RA FLS, but interdependent signalling pathways are involved.
... 56 Studies performed over a decade ago, before the identification of FPR2 as the receptor for annexin A1, also reported that monocytes, neutrophils and synovial fibroblasts from patients with RA had reduced numbers of specific cell-surface binding sites for annexin A1 than cells taken from healthy individuals or osteoarthritis syno vial fibroblasts. 57,58 More recent studies by D' Acquisto et al. 43,59 demonstrated increased annexin A1 expression by peripheral blood CD4 + T cells from patients with RA in comparison with those from healthy individuals, and that annexin A1 expression was reduced in CD4 + T cells from patients with RA treated with methylprednisolone. Similar findings were reported for CD4 + T cells from healthy individuals treated with de xamethasone in vitro. ...
... Similar findings were reported for CD4 + T cells from healthy individuals treated with de xamethasone in vitro. 58 ...
Glucocorticoids have broad-ranging and powerful anti-inflammatory and immunomodulatory effects. Unsurprisingly, therefore, glucocorticoids are widely and persistently used to treat a large number of inflammatory diseases, including rheumatoid arthritis (RA), despite the well-described adverse effects of these drugs. Annexin A1 is a glucocorticoid-induced molecule that is known to replicate many of the described anti-inflammatory effects of glucocorticoids. In addition to the well-documented roles of this protein in neutrophil function, emerging evidence suggests that annexin A1 is involved in the modulation of T-cell function and the adaptive immune responses relevant to RA. Interest in annexin A1 was renewed after the delineation of the receptors for this protein. This breakthrough also led to advances in our understanding of anti-inflammatory annexin A1 mimetic peptides and agonistic compounds targeting these receptors, particularly those specific for the receptor N-formyl peptide receptor 2 (FPR2). Herein, we review the current knowledge of the biological activities of annexin A1 and their relevance to RA pathogenesis. We also discuss the potential of annexin A1 mimics and strategies aimed at potentiating annexin A1 signalling to become viable approaches to minimizing glucocorticoid use in RA and other inflammatory disorders.
... target the protein for export, both the full-length and truncated forms are often observed in extracellular environments. Moreover, it appears that proteolytic cleavage of ANXA1 is required for protein secretion, because the majority of ANXA1 released from neutrophils is Nterminally cleaved [32][33][34][35][36][37][38]. Based on this information, our characterization experiments continued with the analysis by Western blot of ANXA1 expression in subcellular compartments of MIA PaCa-2 cells. ...
Annexin A1 (ANXA1), a 37 kDa multifunctional protein, is over-expressed in tissues from patients of pancreatic carcinoma (PC) where the protein seems to be associated with malignant transformation and poor prognosis.
The expression and localization of ANXA1 in MIA PaCA-2, PANC-1, BxPC-3 and CAPAN-2 cells were detected by Western Blotting and Immunofluorescence assay. Expression and activation of Formyl Peptide Receptors (FPRs) were shown through flow cytometry/PCR and FURA assay, respectively. To investigate the role of ANXA1 in PC cell migration and invasion, we performed in vitro wound-healing and matrigel invasion assays.
In all the analyzed PC cell lines, a huge expression and a variable localization of ANXA1 in sub-cellular compartments were observed. We confirmed the less aggressive phenotype of BxPC-3 and CAPAN-2 compared with PANC-1 and MIA PaCa-2 cells, through the evaluation of Epithelial-Mesenchymal Transition (EMT) markers. Then, we tested MIA PaCa-2 and PANC-1 cell migration and invasiveness rate which was inhibited by specific ANXA1 siRNAs. Both the cell lines expressed FPR-1 and -2. Ac2-26, an ANXA1 mimetic peptide, induced intracellular calcium release, consistent with FPR activation, and significantly increased cell migration/invasion rate. Interestingly, in MIA PaCa-2 cells we found a cleaved form of ANXA1 (33 kDa) that localizes at cellular membranes and is secreted outside the cells, as confirmed by MS analysis. The importance of the secreted form of ANXA1 in cellular motility was confirmed by the administration of ANXA1 blocking antibody that inhibited migration and invasion rate in MIA PaCa-2 but not in PANC-1 cells that lack the 33 kDa ANXA1 form and show a lower degree of invasiveness. Finally, the treatment of PANC-1 cells with MIA PaCa-2 supernatants significantly increased the migration rate of these cells.
This study provides new insights on the role of ANXA1 protein in PC progression. Our findings suggest that ANXA1 protein could regulate metastasis by favouring cell migration/invasion intracellularly, as cytoskeleton remodelling factor, and extracellularly like FPR ligand.
... A role for AnxA1 in the regulation of inflammatory arthritis has been supported by several studies. AnxA1 is expressed in human RA synovial tissue and cells (Goulding et al., 1995;Sampey et al., 2000;Morand et al., 2006) and has been identified as an important endogenous anti-inflammatory mediator in several animal models of RA. For example, our previous studies demonstrated that deficiency of AnxA1 significantly exacerbated antigen-induced arthritis in mice, and reduced the inhibitory effect of glucocorticoids in this model . ...
Background and purpose:
Annexin A1 (AnxA1) is an endogenous anti-inflammatory protein and agonist of the formyl peptide receptor 2 (FPR2). However, the potential for therapeutic FPR ligands to modify immune-mediated disease has been little explored. We investigated the effects of a synthetic FPR agonist on joint disease in the K/BxN model of rheumatoid arthritis (RA) and RA fibroblast-like synoviocytes (FLS).
Experimental approach:
Arthritis was induced by injection of K/BxN serum at day 0 and 2 in wild-type (WT) or AnxA1(-/-) mice and clinical and histopathological manifestations measured 8-11 days later. WT mice were given the FPR agonist compound 43 (Cpd43) (6 or 30 mg·kg(-1) i.p.) for 4 days. Effects of AnxA1 and Cpd43 on RANKL-induced osteoclastogenesis were assessed in RAW 264.7 cells and human RA FLS and macrophages.
Key results:
Treatment with Cpd43 before or after the onset of arthritis reduced clinical disease severity and attenuated synovial TNF-α and osteoclast-associated gene expression. Deletion of AnxA1 in mice exacerbated arthritis severity in the K/BxN model. In vitro, Cpd43 suppressed osteoclastogenesis and NFAT activity elicited by RANKL, and inhibited IL-6 secretion by mouse macrophages. In human RA joint-derived FLS and monocyte-derived macrophages, Cpd43 treatment inhibited IL-6 release, while blocking FPR2 or silencing AnxA1 increased this release.
Conclusions and implications:
The FPR agonist Cpd43 reduced osteoclastogenesis and inflammation in a mouse model of RA and exhibited anti-inflammatory effects in relevant human cells. These data suggest that FPR ligands may represent novel therapeutic agents capable of ameliorating inflammation and bone damage in RA.
... ANXA1 has been shown to localize to the cell surface of various cell types where it is thought to be important in biological function [15][16][17][18][19][20]. ...
Annexin A1 (ANXA1, lipocortin-1) is a glucocorticoid-regulated 37-kDa protein, so called since its main property is to bind (i.e. to annex) to cellular membranes in a Ca(2+)-dependent manner. Although ANXA1 has predominantly been studied in the context of immune responses and cancer, the protein can affect a larger variety of biological phenomena, including cell proliferation and migration. Our previous results show that endogenous ANXA1 positively modulates myoblast cell differentiation by promoting migration of satellite cells and, consequently, skeletal muscle differentiation. In this work, we have evaluated the hypothesis that ANXA1 is able to exert effects on myoblast cell migration acting through formyl peptide receptors (FPRs) following changes in its subcellular localization as in other cell types and tissues. The analysis of the subcellular localization of ANXA1 in C2C12 myoblasts during myogenic differentiation showed an interesting increase of extracellular ANXA1 starting from the initial phases of skeletal muscle cell differentiation. The investigation of intracellular Ca(2+) perturbation following exogenous administration of the ANXA1 N-terminal derived peptide Ac2-26 established the engagement of the FPRs which expression in C2C12 cells was assessed by qualitative PCR. Wound healing assay experiments showed that Ac2-26 peptide is able to increase migration of C2C12 skeletal muscle cells and to induce cell surface translocation and secretion of ANXA1. Our results suggest a role for ANXA1 as a highly versatile component in the signaling chains triggered by the proper calcium perturbation that takes place during active migration and differentiation or membrane repair since the protein is strongly redistributed onto the plasma membranes after an rapid increase of intracellular levels of Ca(2+). These properties indicate that ANXA1 may be involved in a novel repair mechanism for skeletal muscle and may have therapeutic implications with respect to the development of ANXA1 mimetics.
... Annexin A1 is widely expressed in many cell types including Mφ. Immunohistochemical analysis has demonstrated an increased expression of annexin A1 in the RA synovial lining layer macrophages and fibroblasts compared to OA and normal joints. This may, however, be due to the increased lining layer thickness in this condition (Goulding et al., 1995) as other studies have shown decreased binding of annexin A1 to several cell types in RA Sampey et al., 2000). Glucocorticoid stimulation causes annexin A1 mobilization to the cell surface and secretion where it mediates glucocorticoid induced anti-inflammatory effects. ...
Synovial macrophages are one of the resident cell types in synovial tissue and while they remain relatively quiescent in the healthy joint, they become activated in the inflamed joint and, along with infiltrating monocytes/macrophages, regulate secretion of pro-inflammatory cytokines and enzymes involved in driving the inflammatory response and joint destruction. Synovial macrophages are positioned throughout the sub-lining layer and lining layer at the cartilage–pannus junction and mediate articular destruction. Sub-lining macrophages are now also considered as the most reliable biomarker for disease severity and response to therapy in rheumatoid arthritis (RA). There is a growing understanding of the molecular drivers of inflammation and an appreciation that the resolution of inflammation is an active process rather than a passive return to homeostasis, and this has implications for our understanding of the role of macrophages in inflammation. Macrophage phenotype determines the cytokine secretion profile and tissue destruction capabilities of these cells. Whereas inflammatory synovial macrophages have not yet been classified into one phenotype or another it is widely known that TNFα and IL-l, characteristically released by M1 macrophages, are abundant in RA while IL-10 activity, characteristic of M2 macrophages, is somewhat diminished. Here we will briefly review our current understanding of macrophages and macrophage polarization in RA as well as the elements implicated in controlling polarization, such as cytokines and transcription factors like NFκB, IRFs and NR4A, and pro-resolving factors, such as LXA4 and other lipid mediators which may promote a non-inflammatory, pro-resolving phenotype, and may represent a novel therapeutic paradigm.
... Altered expression profiles, phosphorylation, subcellular localization, and/or specific modulation of mitogenic signals are all possible mechanisms by which ANXA1 protein could explicate its biological effects (Lim and Pervaiz, 2007). ANXA1 has been shown to localize to the cell surface of various cell types including leukocytes, endothelial cells, lung epithelial cells, and synoviocytes where it is thought to be important in biological function (Hullin et al., 1989;Ambrose and Hunninghake, 1990;Croxtall et al., 1996;Perretti et al., 1996;Rhee et al., 2000;Sampey et al., 2000). The regulatory action of cell surface or extracellular ANXA1 has been shown to be mediated by signaling through formyl peptide receptors (FPRs) (Perretti et al., 2001;Gavins et al., 2003;Ernst et al., 2004). ...
Annexin A1 (ANXA1, lipocortin-1) is the first characterized member of the annexin superfamily of proteins, so called since their main property is to bind (i.e., to annex) to cellular membranes in a Ca(2+) -dependent manner. ANXA1 has been involved in a broad range of molecular and cellular processes, including anti-inflammatory signalling, kinase activities in signal transduction, maintenance of cytoskeleton and extracellular matrix integrity, tissue growth, apoptosis, and differentiation. New insights show that endogenous ANXA1 positively modulates myoblast cell differentiation by promoting migration of satellite cells and, consequently, skeletal muscle differentiation. This suggests that ANXA1 may contribute to the regeneration of skeletal muscle tissue and may have therapeutic implications with respect to the development of ANXA1 mimetics.
... Anx-A1 is seen in several locations within samples of human synovial tissue . Anx-A1-binding sites have been detected in synoviocytes taken from patients with rheumatoid arthritis (Sampey et al., 2000), but the actual number of sites was less than that in cells taken from patients with osteoarthritis. Administration of the N-acetyl 2-26 peptide to cultured synoviocytes inhibited PLA 2 activation. ...
The glucocorticoids are the most potent anti‐inflammatory drugs that we possess and are effective in a wide variety of diseases. Although their action is known to involve receptor mediated changes in gene transcription, the exact mechanisms whereby these bring about their pleiotropic action in inflammation are yet to be totally understood. Whilst many different genes are regulated by the glucocorticoids, we have identified one particular protein—annexin A1 (Anx‐A1)—whose synthesis and release is strongly regulated by the glucocorticoids in many cell types. The biology of this protein, as revealed by studies using transgenic animals, peptide mimetics and neutralizing antibodies, speaks to its role as a key modulator of both of the innate and adaptive immune systems. The mechanism whereby this protein exerts its effects is likely to be through the FPR receptor family—a hitherto rather enigmatic family of G protein coupled receptors, which are increasingly implicated in the regulation of many inflammatory processes. Here we review some of the key findings that have led up to the elucidation of this key pathway in inflammatory resolution.
British Journal of Pharmacology (2008) 155 , 152–169; doi: 10.1038/bjp.2008.252 ; published online 21 July 2008
... Recent studies using different cell types, such as pancreatic islets, leukemia cell line, anterior pituitary cells, osteoclast, human fibroblast-like synovioeytes, and leukocytes, have shown that annexins might bind to the cell surface binding sites [24][25][26][27][28][29][30][31][32][33][34]. In particular, receptors of 15 and 18 kDa have been identified in the plasma membrane of human monocytes [29]. ...
This study investigated the effect of extracellular annexin I on regulating insulin secretion in MIN6N8a (an insulin secreting cell line) cells. The properties of annexin I receptor in MIN6N8a cells were also determined. Annexin I stimulated insulin release in MIN6N8a cells, regardless of the presence or absence of extracellular Ca(2+). Confocal microscopy revealed that annexin I bound to the surface of MIN6N8a cells. In addition, FACs analysis showed that annexin I bound to the surface of MIN6N8a cells in a dose-dependent manner. However, the annexin I-stimulated insulin secretion and the annexin I binding were abolished in MIN6N8a cells treated with proteases. Annexin I receptors were regenerated time-dependently. Furthermore, annexin I-stimulated insulin secretion was inhibited by cycloheximide but not by actinomycin D. These results showed that annexin I binds to the surface receptor in order to regulate the stimulation of insulin release in MIN6N8a cells.
... Finally, in line with the pharmacological data, it is likely that ANXA1 receptor(s) also exist on fibroblasts, since binding of the protein is modulated by cell activation as seen during active disease, e.g. rheumatoid, compared to cells prepared from the synovium of osteoarthritic patients (Sampey et al. 2000). ...
The concept of anti-inflammation is currently evolving with the definition of several endogenous inhibitory circuits that are important in the control of the host inflammatory response. Here we focus on one of these pathways, the annexin 1 (ANXA1) system. Originally identified as a 37 kDa glucocorticoid-inducible protein, ANXA1 has emerged over the last decade as an important endogenous modulator of inflammation. We review the pharmacological effects of ANXA1 on cell types involved in inflammation, from blood-borne leukocytes to resident cells. This review reveals that there is scope for more research, since most of the studies have so far focused on the effects of the protein and its peptido-mimetics on neutrophil recruitment and activation. However, many other cells central to inflammation, e.g. endothelial cells or mast cells, also express ANXA1: it is foreseen that a better definition of the role(s) of the endogenous protein in these cells will open the way to further pharmacological studies. We propose that a more systematic analysis of ANXA1 physio-pharmacology in cells involved in the host inflammatory reaction could aid in the design of novel anti-inflammatory therapeutics based on this endogenous mediator.
... We subsequently characterized its expression in both stationary and migrating cells to determine how the establishment of cell migration affected its distribution, thereby providing insight into how AnxA1 might regulate epithelial cell migration. AnxA1 has been shown to localize to the cell surface of various cell types including leukocytes, endothelial cells, lung epithelial cells, and synoviocytes where it is thought to be important in its biological function (67)(68)(69)(70)(71)(72). We therefore explored cell surface expres- sion of AnxA1 in SKCO-15 cells. ...
Annexin 1 (AnxA1) is a multifunctional phospholipid-binding protein associated with the development of metastasis in some invasive epithelial malignancies. However, the role of AnxA1 in the migration/invasion of epithelial cells is not known. In this study, experiments were performed to investigate the role of AnxA1 in the invasion of a model epithelial cell line, SKCO-15, derived from colorectal adenocarcinoma. Small interfering RNA-mediated knockdown of AnxA1 expression resulted in a significant reduction in invasion through Matrigel-coated filters. Localization studies revealed a translocation of AnxA1 to the cell surface upon the induction of cell migration, and functional inhibition of cell surface AnxA1 using antiserum (LCO1) significantly reduced cell invasion. Conversely, SKCO-15 cell invasion was increased by approximately 2-fold in the presence of recombinant full-length AnxA1 and the AnxA1 N-terminal-derived peptide mimetic, Ac2-26. Because extracellular AnxA1 has been shown to regulate leukocyte migratory events through interactions with n-formyl peptide receptors (nFPRs), we examined the expression of FPR-1, FPRL-1, and FPRL-2 in SKCO-15 cells by reverse transcriptase-PCR and identified expression of all three receptors in this cell line. Treatment of SKCO-15 cells with AnxA1, Ac2-26, and the classical nFPR agonist, formylmethionylleucylphenylalanine, induced intracellular calcium release consistent with nFPR activation. Furthermore, the nFPR antagonist, Boc2, abrogated the AnxA1 and Ac2-26-induced intracellular calcium release and increase in SKCO-15 cell invasion. Together, these results support an autocrine/paracrine role for membrane AnxA1 in stimulating SKCO-15 cell migration through nFPR activation. The findings in this study suggest that activation of nFPRs stimulates epithelial cell motility important in the development of metastasis as well as wound healing.
These anti-inflammatory drugs that are used to treat acute gout are discussed in detail. Their administration, pharmacology, and toxicity are considered. Then, urate-lowering therapy is thoroughly described, again considering the administration, pharmacology, and toxicity of these agents. The widespread mismanagement of gout in general and even specialty medical practice makes this information important for patients and their physicians.
Inflammation is essential to protect the host from exogenous and endogenous dangers that ultimately lead to tissue injury. The consequent tissue repair is intimately associated with the fate of the inflammatory response. Restoration of tissue homeostasis is achieved through a balance between pro-inflammatory and anti-inflammatory/pro-resolving mediators. In chronic inflammatory diseases such balance is compromised resulting in persistent inflammation and impaired healing. During the last two decades the glucocorticoid-regulated protein Annexin A1 (AnxA1) has emerged as a potent pro-resolving mediator acting on several facets of the innate immune system. Here, we review the therapeutic effects of AnxA1 on tissue healing and repairing together with the molecular targets responsible for these complex biological properties.
Rheumatoid arthritis (RA) is a chronic and systemic inflammatory disease that attacks the joints and other organs, such as blood vessels, skin, and lungs. Different cellular types and inflammatory molecules are involved in the events of joint destruction and pain and are currently the main targets for the management of RA. However, several patients are unresponsive or intolerant to the available treatments. Furthermore, the development of secondary immunosuppression culminates in the high rates of opportunistic infections and malignancies in RA patients. Here, we overview the current therapies for RA and explore new approaches that could be used in the future for the management of RA.
Annxine-1 (Anx-1), a member of the annexin superfamily of calcium- and phospholipid- binding proteins, is induced by glucocorticoids (GC) and functions as a mediator of their anti-inflammatory effects. The wide range of effects of Anx-1 includes inhibition of leukocyte recruitment, suppression of the production of inflammatory mediators and cytokines, and induction of apoptosis in inflammatory cells. This profile of activity suggests that the inhibitory effects of Anx-1 would be beneficial in the pathological context of rheumatoid arthritis (RA). Anx-1 is expressed in human RA synovial tissue and cells, and has recently been identified as an important endogenous anti-inflammatory mediator in multiple animal models of RA. Emerging data on the mechanisms of action of Anx-1 suggest it acts to inhibit mitogen activated protein (MAP) kinases, through as yet unidentified mechanisms, thereby inhibiting pro-inflammatory pathways known to be important in RA. Anx-1 treatment strategies, especially as an alternative to GC therapy, may be valuable in RA and other inflammatory diseases.
This study investigates the effect of extracellular annexin I (Anx I) on regulating insulin secretion in isolated rat pancreatic islets. Results show that Anx I stimulates insulin release in pancreatic islets regardless of the presence or absence of extracellular Ca2+. In particular, confocal microscopy shows that Anx I binds to the surface of islet cells in the absence of extracellular Ca2+. However, insulin secretion through Anx I significantly decreases in trypsin-treated islets. Likewise, there is minimal binding of Anx I to the surface of trypsin-treated islets. Anti-Anx I polyclonal antibody also inhibits the stimulating effect of Anx I on insulin secretion. These results indicate that Anx I is capable of binding to the cell surface receptor, in order to regulate the stimulation of insulin release in rat pancreatic islets.
Annexin 1 (Anx-1) is a putative mediator of the antiinflammatory actions of glucocorticoids (GCs). This study investigated the role of Anx-1 in experimental arthritis and in GC-mediated inhibition of inflammation, using antigen-induced arthritis (AIA) in Anx-1 knockout (Anx-1(-/-)) mice.
Arthritis was induced by intraarticular injection of methylated BSA (mBSA) in mice preimmunized with mBSA. Disease was assessed after 7 days by histologic examination of the knee joints. Serum levels of anti-mBSA IgG were determined by enzyme-linked immunosorbent assay. Cytokine messenger RNA (mRNA) expression was detected by real-time polymerase chain reaction.
A significant exacerbation of arthritis was observed in the Anx-1(-/-) mice compared with wild-type (WT) mice. This was associated with increased mRNA expression of synovial interleukin-1 beta, tumor necrosis factor alpha, interleukin-6, and macrophage migration inhibitory factor. Dexamethasone significantly reduced the histologic severity of synovitis and bone damage in the WT mice, but exerted no inhibitory effects in the Anx-1(-/-) mice, and also significantly reduced the serum levels of anti-mBSA IgG and the numbers of peripheral blood neutrophils and lymphocytes in WT mice, but had no such effect in Anx-1(-/-) mice.
Anx-1 exerts endogenous antiinflammatory effects on AIA via the regulation of cytokine gene expression, and also mediates the antiinflammatory actions of dexamethasone in AIA.
Clinical criteria for the classification of patients with hip pain associated with osteoarthritis (OA) were developed through a multicenter study. Data from 201 patients who had experienced hip pain for most days of the prior month were analyzed. The comparison group of patients had other causes of hip pain, such as rheumatoid arthritis or spondylarthropathy. Variables from the medical history, physical examination, laboratory tests, and radiographs were used to develop different sets of criteria to serve different investigative purposes. Multivariate methods included the traditional “number of criteria present” format and “classification tree” techniques.
Clinical criteria: A classification tree was developed, without radiographs, for clinical and laboratory criteria or for clinical criteria alone. A patient was classified as having hip OA if pain was present in combination with either 1) hip internal rotation ≥15º, pain present on internal rotation of the hip, morning stiffness of the hip for ≤60 minutes, and age >50 years, or 2) hip internal rotation <15º and an erythrocyte sedimentation rate (ESR) ≤45 mm/hour; if no ESR was obtained, hip flexion ≤115º was substituted (sensitivity 86%; specificity 75%).
Clinical plus radiographic criteria: The traditional format combined pain with at least 2 of the following 3 criteria: osteophytes (femoral or acetabular), joint space narrowing (superior, axial, and/or medial), and ESR <20 mm/hour (sensitivity 89%; specificity 91%). The radiographic presence of osteophytes best separated OA patients and controls by the classification tree method (sensitivity 89%; specificity 91%).
The “number of criteria present” format yielded criteria and levels of sensitivity and specificity similar to those of the classification tree for the combined clinical and radiographic criteria set. For the clinical criteria set, the classification tree provided much greater specificity. The value of the radiographic presence of an ostophyte in separating patients with OA of the hip from those with hip pain of other causes is emphasized.
To examine the distribution of four annexins in non-inflamed rheumatoid arthritic and osteoarthritic synovial tissue.
Frozen sections were stained with monoclonal antibodies (MAb) specific for annexins-I, -II, -IV, and -VI, and for cell lineage related markers including CD68 and CD14 (macrophages), prolyl hydroxylase (fibroblasts), and CD3 (T cells).
Each of the annexins was present in synovial tissues in significant amounts in the three groups studied. Annexin-I was predominantly found within the synovial lining layer and double labelling showed it to be present predominantly in cells of the macrophage lineage. In rheumatoid specimens there was increased staining within the lining layer, perivascularly and on macrophages within the tissue stroma. Annexin-II was present in a distribution similar to that of annexin-I, but with more prominent perivascular staining. Annexins-IV and -VI were seen chiefly in association with areas of lymphocyte infiltration in rheumatoid tissue, whereas annexins-I and -II were absent from these areas. Endothelial cells stained weakly positive for annexins-I and -II, and more strongly for -IV and -VI.
This study demonstrates that annexins (particularly annexin-I, a putative mediator of the anti-inflammatory activities of glucocorticoids) are abundant in rheumatoid and non-rheumatoid synovial tissue, annexins-IV and -VI having a distribution distinct from that of -I and -II.
We have developed a novel expression system that allows the fission yeast, Schizosaccharomyces pombe, to be used for the efficient overproduction of heterologous proteins. As an example of the utility of this system, human lipocortin I was expressed to 50 percent of soluble protein, and 150 mg of highly purified material was obtained from 10 grams of wet cell paste. Expression of lipocortin I was driven by the human cytomegalovirus (hCMV) promoter in a vector that also contains a neomycin resistance gene (neo) under the control of the SV40 early promoter, permitting selection for increasing copy-number with increasing concentrations of the antibiotic G418. The purified protein was equivalent to its native counterpart with respect to antigenicity and biochemical properties such as phospholipase A2 inhibition, actin binding and N-terminal acetylation. We have also used this system to produce comparable amounts of other proteins including rat arginase, rat NDP-kinase and human interleukin-6.
Recombinant human annexin I and a monoclonal antibody specific for this protein (mAb 1B) were used to investigate surface binding of this member of the annexin family of proteins to peripheral blood monocytes. Flow cytometric analysis demonstrated trypsin-sensitive, saturable binding of annexin I to human peripheral blood monocytes but not to admixed lymphocytes. A monoclonal antibody that blocks the anti-phospholipase activity of annexin I also blocked its binding to monocytes. These findings suggest the presence of specific binding sites on monocytes. Furthermore, surface iodination, immunoprecipitation and SDS/PAGE analysis were used to identify two annexin I-binding proteins on the surface of monocytes with molecular masses of 15 kDa and 18 kDa respectively. The identification and characterization of these annexin I-binding molecules should help us to better understand the specific interactions of annexin I with monocytes that lead to down-regulation of pro-inflammatory cell functions.
Lipocortin 1 (LC1) is an important mediator of glucocorticoid action in the anterior pituitary gland, where it appears to act via cell surface binding sites to suppress peptide release. We have exploited a combination of fluorescence-activated cell (FAC) analysis/sorting and electron microscopy to detect, characterize, and localize LC1-binding sites on the surface of dispersed rat anterior pituitary cells, using human recombinant LC1 (hu-r-LC1) as a probe. High affinity (Kd = 14 +/- 3 nM) hu-r-LC1-binding sites were detected on approximately 80% of anterior pituitary cells dispersed with collagenase. The binding characteristics of the ligand resembled those observed in leukocytes, in that it was saturable; concentration, Ca2+, and temperature dependent; and abolished by trypsin. Functional studies demonstrated an excellent correlation between the presence of the cell surface binding protein and the capacity of an anti-LC1 monoclonal antibody to abrogate the inhibitory actions of dexamethasone (10 nM) on the release of ACTH initiated in vitro by CRH-41 (1 nM). Morphological analysis of cells harvested by FAC sorting showed that 1) somatotrophs, corticotrophs, lactotrophs, thyrotrophs, and gonadotrophs were all included in the population expressing LC1 binding sites; and 2) the LC1-binding sites assume a punctate distribution across the cell surface. These data show that anterior pituitary cells express high affinity surface LC1-binding protein(s); they thus provide further evidence for a specific membrane mechanism of action of LC1 in regulating the endocrine function of the anterior pituitary.
Specific binding sites for the anti-inflammatory protein annexin I have been detected on the surface of human monocytes and polymorphonuclear leukocytes (PMN). These binding sites are proteinaceous in nature and are sensitive to cleavage by the proteolytic enzymes trypsin, collagenase, elastase and cathepsin G. When monocytes and PMN were isolated independently from peripheral blood, only the monocytes exhibited constitutive annexin I binding. However PMN acquired the capacity to bind annexin I following co-culture with monocytes. PMN incubation with sodium azide, but not protease inhibitors, partially blocked this process. A similar increase in annexin I binding capacity was also detected in PMN following adhesion to endothelial monolayers. We propose that a juxtacrine activation rather than a cleavage-mediated transfer is involved in this process. Removal of annexin I binding sites from monocytes with elastase rendered monocytes functionally insensitive to full length annexin I or to the annexin I-derived pharmacophore, peptide Ac2-26, assessed as suppression of the respiratory burst. These data indicate that the annexin I binding site on phagocytic cells may have an important function in the feedback control of the inflammatory response and their loss through cleavage could potentiate such responses.
Lipocortin 1 (LC1) is an important mediator of glucocorticoid action in the anterior pituitary gland, where it appears to act via cell surface binding sites to suppress peptide release. We have exploited a combination of fluorescence-activated cell (FAC) analysis/sorting and electron microscopy to detect, characterize, and localize LC1-binding sites on the surface of dispersed rat anterior pituitary cells, using human recombinant LC1 (hu-r-LC1) as a probe. High affinity (Kd = 14 ± 3 nm) hu-r-LC1-binding sites were detected on approximately 80% of anterior pituitary cells dispersed with collagenase. The binding characteristics of the ligand resembled those observed in leukocytes, in that it was saturable; concentration, Ca²⁺, and temperature dependent; and abolished by trypsin. Functional studies demonstrated an excellent correlation between the presence of the cell surface binding protein and the capacity of an anti-LC1 monoclonal antibody to abrogate the inhibitory actions of dexamethasone (10 nm) on the release of ACTH initiated in vitro by CRH-41 (1 nm). Morphological analysis of cells harvested by FAC sorting showed that 1) somatotrophs, corticotrophs, lactotrophs, thyrotrophs, and gonadotrophs were all included in the population expressing LC1 binding sites; and 2) the LC1-binding sites assume a punctate distribution across the cell surface. These data show that anterior pituitary cells express high affinity surface LC1-binding protein(s); they thus provide further evidence for a specific membrane mechanism of action of LC1 in regulating the endocrine function of the anterior pituitary.
The revised criteria for the classification of rheumatoid arthritis (RA) were formulated from a computerized analysis of 262 contemporary, consecutively studied patients with RA and 262 control subjects with rheumatic diseases other than RA (non-RA). The new criteria are as follows: 1) morning stiffness in and around joints lasting at least 1 hour before maximal improvement; 2) soft tissue swelling (arthritis) of 3 or more joint areas observed by a physician; 3) swelling (arthritis) of the proximal interphalangeal, metacarpophalangeal, or wrist joints; 4) symmetric swelling (arthritis); 5) rheumatoid nodules; 6) the presence of rheumatoid factor; and 7) radiographic erosions and/or periarticular osteopenia in hand and/or wrist joints. Criteria 1 through 4 must have been present for at least 6 weeks. Rheumatoid arthritis is defined by the presence of 4 or more criteria, and no further qualifications (classic, definite, or probable) or list of exclusions are required. In addition, a “classification tree” schema is presented which performs equally as well as the traditional (4 of 7) format. The new criteria demonstrated 91–94% sensitivity and 89% specificity for RA when compared with non-RA rheumatic disease control subjects.
In pre-labelled A549 cells epidermal growth factor (EGF) (10 nM) stimulates the release of [5,6,8,9,11,12,14,15-3H(N)]-arachidonic acid (3H-AA) by approximately 70%. Increasing Cai2+ with thapsigargin (50 nM) stimulates 3H-AA release by approximately 120%. However, the combined use of these two agents results in a synergistic stimulation of 3H-AA release by over 700%. The EGF stimulated release is sensitive to pertussis toxin (10 ng/mL) and guanosine 5′-O-(2-thiodiphosphate) suggesting a G protein-mediated event. This is supported by the fact that the G protein activators AlF4− and guanosine 5′-O-(2-thiotriphosphate) both stimulate 3H-AA release. The stimulation of 3H-AA release by both EGF or direct G protein activation is completely blocked following pre-treatment for 3 hr with 1 nM dexamethasone. This effect is reversed with a neutralizing antibody to lipocortin-1 (1 μg/mL) suggesting that this protein mediates the inhibitory effects of glucocorticoids on agonist activated 3H-AA release. Thapsigargin stimulation of 3H-AA release is insensitive to dexamethasone treatment. A peptide fragment from the N-terminus of lipocortin-1-Lc13–25 (20–200 μg/mL) mimics the effect of glucocorticoid in suppressing both EGF and G protein activated 3H-AA release. A peptide with Me-Tyr substituting Tyr21 is much reduced in activity suggesting that the presence of this residue is essential. As peptide Lc13–25 is not derived from the Ca2+/phospholipid binding domain of the native protein then sequestration of phospholipid substrate for PLA2 remains an unlikely mechanism of action for this peptide.
Objective
Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine whose involvement in tumor necrosis factor α (TNFα) synthesis and T cell activation suggests a role in the pathogenesis of rheumatoid arthritis (RA). Antagonism of MIF is associated with marked inhibition of animal models of RA. Uniquely, MIF is inducible by low concentrations of glucocorticoids. We sought to investigate the expression of MIF in RA synovial tissue.MethodsMIF was demonstrated in human RA synovium by immunohistochemistry, flow cytometry, enzyme-linked immunosorbent assay (ELISA), and reverse transcription–polymerase chain reaction (RT-PCR). Regulation of MIF expression was investigated by treatment of cultured fibroblast-like synoviocytes (FLS) with interleukin-1β (IL-1β), TNFα, or interferon-γ (IFNγ), and dexamethasone (DEX). Mononuclear cell TNFα release after exposure to FLS-conditioned medium was measured by ELISA.ResultsMIF was present in RA synovial lining CD14+ macrophages and FLS. Constitutive MIF messenger RNA (mRNA) expression was demonstrated by RT-PCR of RNA from unstimulated cultured RA FLS, which also released abundant MIF. Serum, synovial fluid, and FLS intracellular MIF were significantly higher in RA patients than in controls. Synoviocyte MIF was not increased by IL-1β, TNFα, or IFNγ. In contrast, DEX 10−7M significantly reduced synoviocyte MIF, while DEX 10−10–10−12M induced a significant increase in MIF and MIF mRNA. Peripheral blood mononuclear cell TNFα release was induced by culture in RA FLS-conditioned medium, and this induction was significantly abrogated by monoclonal anti-MIF antibody, suggesting that MIF is an upstream regulator of TNFα release.Conclusion
These data represent the first demonstration of the cytokine MIF in human autoimmune disease and suggest MIF as a potential therapeutic target in RA.
For the purposes of classification, it should be specified whether osteoarthritis (OA) of the knee is of unknown origin (idiopathic, primary) or is related to a known medical condition or event (secondary). Clinical criteria for the classification of idiopathic OA of the knee were developed through a multicenter study group. Comparison diagnoses included rheumatoid arthritis and other painful conditions of the knee, exclusive of referred or paraarticular pain. Variables from the medical history, physical examination, laboratory tests, and radiographs were used to develop sets of criteria that serve different investigative purposes. In contrast to prior criteria, these proposed criteria utilize classification trees, or algorithms.
A multi-faceted approach was used to investigate the effect of an anti-inflammatory peptide derived from human lipocortin 1 N-terminus region (amino acid 2–26; termed human Ac2–26) on human neutrophil activation in vitro. When incubated with purified human neutrophils. human Ac2–26 produced a concentration-dependent inhibition of elastase release stimulated by formyl-Met-Leu-Phe (fMLP), platelet-activating factor, or leukotriene B4, with an approximate EC50 of 33 μM (100 μg/ml). At this concentration, human Ac2–26 also inhibited (77%) the release of [3H]-arachidonic acid from neutrophils stimulated with fMLP. The peptide, however, did not inhibit the up-regulation of the β2-integrin CD11b and the concomitant shedding of L-selectin from neutrophil plasma membrane induced by fMLP. In adhesion experiments, human Ac2–26 inhibited neutrophil adhesion to endothelial monolayers when this was stimulated with fMLP, but not when this followed endothelial cell activation with histamine or platelet-activating factor. Again, the effect of the peptide was concentration-dependent, and an approximate EC50 of 33 μM was calculated. When a preparation of 125I-labeled human Ac2–26 was incubated with the neutrophils, the peptide was internalised in an energy-dependent fashion. All together, these observations lead us to propose a model in which this peptide derived from the N-terminus of human lipocortin 1 alters a common cellular mechanism producing a selective inhibition of neutrophil activation.
The effect of dexamethasone on mRNA and protein synthesis of lipocortins (LCT) 1, 2 and 5 has been investigated in U-937 cells. A constitutive expression of both mRNAs and proteins was detected in undifferentiated U-937 cells. This constitutive level was increased time- and dose-dependently by incubation with phorbol myristate acetate (PMA). In U-937 cells differentiated by 24 h incubation with 6 ng/ml PMA, dexamethasone (DEX) (1 microM for 16 h) caused an increased synthesis of the mRNA level of LCT-1 and 2, but not of LCT-5, over the level induced by PMA. DEX had no effect in undifferentiated cells. Moreover, DEX stimulated the extracellular release of LCT-1 and 5, but not of LCT-2, and inhibited the release of PGE2 and TXB2 only in the differentiated U-937 cells. These results suggest that the responsiveness of these cells to glucocorticoids is dependent on the phase of cell differentiation. The selective release of lipocortins by differentiated U-937 cells may explain, at least in part, the inhibition by DEX of the prostanoid release.
Clinical criteria for the classification of patients with hip pain associated with osteoarthritis (OA) were developed through a multicenter study. Data from 201 patients who had experienced hip pain for most days of the prior month were analyzed. The comparison group of patients had other causes of hip pain, such as rheumatoid arthritis or spondylarthropathy. Variables from the medical history, physical examination, laboratory tests, and radiographs were used to develop different sets of criteria to serve different investigative purposes. Multivariate methods included the traditional "number of criteria present" format and "classification tree" techniques. Clinical criteria: A classification tree was developed, without radiographs, for clinical and laboratory criteria or for clinical criteria alone. A patient was classified as having hip OA if pain was present in combination with either 1) hip internal rotation greater than or equal to 15 degrees, pain present on internal rotation of the hip, morning stiffness of the hip for less than or equal to 60 minutes, and age greater than 50 years, or 2) hip internal rotation less than 15 degrees and an erythrocyte sedimentation rate (ESR) less than or equal to 45 mm/hour; if no ESR was obtained, hip flexion less than or equal to 115 degrees was substituted (sensitivity 86%; specificity 75%). Clinical plus radiographic criteria: The traditional format combined pain with at least 2 of the following 3 criteria: osteophytes (femoral or acetabular), joint space narrowing (superior, axial, and/or medial), and ESR less than 20 mm/hour (sensitivity 89%; specificity 91%). The radiographic presence of osteophytes best separated OA patients and controls by the classification tree method (sensitivity 89%; specificity 91%). The "number of criteria present" format yielded criteria and levels of sensitivity and specificity similar to those of the classification tree for the combined clinical and radiographic criteria set. For the clinical criteria set, the classification tree provided much greater specificity. The value of the radiographic presence of an osteophyte in separating patients with OA of the hip from those with hip pain of other causes is emphasized.
Clinical criteria for the classification of symptomatic idiopathic (primary) osteoarthritis (OA) of the hands were developed from data collected in a multicenter study. Patients with OA were compared with a group of patients who had hand symptoms from other causes, such as rheumatoid arthritis and the spondylarthropathies. Variables from the medical history, physical examination, laboratory tests, and radiographs were analyzed. All patients had pain, aching, or stiffness in the hands. Patients were classified as having clinical OA if on examination there was hard tissue enlargement involving at least 2 of 10 selected joints, swelling of fewer than 3 metacarpophalangeal joints, and hard tissue enlargement of at least 2 distal interphalangeal (DIP) joints. If the patient had fewer than 2 enlarged DIP joints, then deformity of at least 1 of the 10 selected joints was necessary in order to classify the symptoms as being due to OA. The 10 selected joints were the second and third DIP, the second and third proximal interphalangeal, and the trapeziometacarpal (base of the thumb) joints of both hands. Criteria derived using the "classification tree" method were 92% sensitive and 98% specific. The "traditional format" classification method required that at least 3 of these 4 criteria be present to classify a patient as having OA of the hand. The latter sensitivity was 94% and the specificity was 87%. Radiography was of less value than clinical examination in the classification of symptomatic OA of the hands.
Synovial tissue from patients with rheumatoid arthritis was enzymatically dissociated, and single cell suspensions were fractionated into subpopulations by centrifugation on continuous Percoll gradients. Five fractions (F1-F5) with densities of 0.991-0.998 gm/ml, 0.998-1.042 gm/ml, 1.042-1.062 gm/ml, 1.062-1.082 gm/ml, and 1.082-1.180 gm/ml, respectively, were prepared. F3 consistently contained the highest number of macrophages, while F2 and F4 contained substantially fewer macrophages. Macrophages present in F2, F3, and F4 were enriched by differential adherence to fibronectin-coated collagen gels. These macrophage-enriched cell preparations were found to be Fc and C3 positive, esterase positive, and peroxidase negative, to stain positively with anti-HLA-DR, anti-Leu-M3, OKM1, and OKM5 monoclonal antibodies, and to show characteristic features of macrophages by electron microscopy. Macrophages from F3 consistently induced neovascularization in rat corneas, while equal numbers of macrophages from F2 and F4 did not. Fibroblastic synovial cells and cells that did not adhere to fibronectin-coated collagen gels did not induce neovascularization. Within the rheumatoid synovium, there appears to be a major subpopulation of macrophages capable of inducing neovascularization, a process vital to the development of the rheumatoid synovial pannus.
For the purposes of classification, it should be specified whether osteoarthritis (OA) of the knee is of unknown origin (idiopathic, primary) or is related to a known medical condition or event (secondary). Clinical criteria for the classification of idiopathic OA of the knee were developed through a multicenter study group. Comparison diagnoses included rheumatoid arthritis and other painful conditions of the knee, exclusive of referred or para-articular pain. Variables from the medical history, physical examination, laboratory tests, and radiographs were used to develop sets of criteria that serve different investigative purposes. In contrast to prior criteria, these proposed criteria utilize classification trees, or algorithms.
In pre-labelled A549 cells epidermal growth factor (EGF) (10 nM) stimulates the release of [5,6,8,9,11,12,14,15-3H(N)]-arachidonic acid (3H-AA) by approximately 70%. Increasing Ca2+i with thapsigargin (50 nM) stimulates 3H-AA release by approximately 120%. However, the combined use of these two agents results in a synergistic stimulation of 3H-AA release by over 700%. The EGF stimulated release is sensitive to pertussis toxin (10 ng/mL) and guanosine 5'-O-(2-thiodiphosphate) suggesting a G protein-mediated event. This is supported by the fact that the G protein activators AlF-4 and guanosine 5'-O-(2-thiotriphosphate) both stimulate 3H-AA release. The stimulation of 3H-AA release by both EGF or direct G protein activation is completely blocked following pre-treatment for 3 hr with 1 nM dexamethasone. This effect is reversed with a neutralizing antibody to lipocortin-1 (1 microgram/mL) suggesting that this protein mediates the inhibitory effects of glucocorticoids on agonist activated 3H-AA release. Thapsigargin stimulation of 3H-AA release is insensitive to dexamethasone treatment. A peptide fragment from the N-terminus of lipocortin-1-Lc13-25 (20-200 micrograms/mL) mimics the effect of glucocorticoid in suppressing both EGF and G protein activated 3H-AA release. A peptide with Me-Tyr substituting Tyr21 is much reduced in activity suggesting that the presence of this residue is essential. As peptide Lc13-25 is not derived from the Ca2+/phospholipid binding domain of the native protein then sequestration of phospholipid substrate for PLA2 remains an unlikely mechanism of action for this peptide.
Lipocortin (annexin) 1 is a putative mediator of the inflammatory effects of glucocorticoids. By flow cytometric analysis (FACS) we have studied the effect of dexamethasone on the cellular localization of lipocortin 1. U-937 cells were incubated with or without 10 nM phorbol 12-myristate 13-acetate (PMA) to induce cell differentiation. Then 1 microM dexamethasone was added and incubation carried out for increasing times (1-24 h). Dexamethasone caused a time-dependent biphasic translocation of lipocortin 1 from the intracellular compartment to the cell membrane with maximal membrane expression at 4 and 24 h. In differentiated U-937 cells the steroid-induced membrane accumulation of lipocortin 1 was significantly higher than that of undifferentiated cells. The accumulation of the protein in the cell membrane may precede its release which is stimulated by dexamethasone in differentiated U-937 cells. Since extracellular lipocortin 1 has anti-inflammatory properties the modulation of the translocation/secretion process of the protein by glucocorticoids may be part of their mechanism of action.
Inflammation normally results in enhanced synthesis and secretion of hypothalamic corticotropin-releasing hormone (CRH) which, in turn, exerts antiinflammatory effects by virtue of increased adrenal glucocorticoid production. CRH and CRH binding sites are also expressed in the peripheral nervous and immune systems. Our groups have recently shown that CRH is secreted locally in acute carrageenin-induced inflammation in rats and has predominantly proinflammatory effects. We have also shown that CRH is expressed in the joints of Lewis rats with experimental arthritis. To determine if CRH is present in human inflammatory arthritis, we examined synovial fluids and tissues from patients with rheumatoid arthritis (RA) or osteoarthritis (OA) and normal individuals. We found markedly enhanced expression of immunoreactive CRH in situ in synovium from patients, which was significantly greater in RA than in OA (p < 0.01). CRH concentrations were also significantly higher in RA (140 +/- 33 pg/ml, mean +/- SEM; n = 10) than OA (25 +/- 4 pg/ml; n = 6) synovial fluids (p < 0.005). HPLC showed immunoreactive CRH extracted from RA and OA synovial tissues and fluids coeluted with CRH 1-41. CRH mRNA was present in low levels in synovial tissue from patients with RA and, to a lesser extent, OA. In summary, immunoreactive CRH is locally secreted in the synovium of patients with RA and, at lower levels, OA. These data support the view that CRH functions as an autocrine and/or paracrine mediator of inflammation in humans.
The activity of the steroid-inducible protein lipocortin-1 (LC1; with a primary sequence of 346 amino acids; also called annexin 1), a fragment corresponding to amino acids 1-188 and a short peptide from the N-terminus (amino acid 2-26) were tested for anti-inflammatory actions in three models of acute inflammation in the mouse in comparison with a mAb anti-CD11b (αCD11b). In the mouse air-pouch model LC1, fragment 1-188 and peptide Ac2- 26 exhibited powerful inhibitory effects (ED50 ≃ 5.2, 38 and 88 μg/mouse, respectively) on leukocyte migration elicited by IL-1. LC1 was approximately 200 times more potent than Ac2-26 on a molar basis although both gave maximal inhibitions, in contrast fragment 1-188 only produced a partial dose-response curve. LC1 was approximately 20 times more potent on a molar basis in this assay than the αCD11b mAb. Peptide Ac2-26 and the mAb αCD11b also blocked cell migration into the air-pouch induced by IL-8 with approximately the same potency. In the mouse skin edema and zymosan peritonitis assays Ac2-26 was inhibitory (ED50 of 200 μg/mouse) but less so than the αCD11b antibody (ED50 ≃ 0.5 mg/mouse). Both LC1 (10 μg) and Ac2-26 (200 μg) completely blocked FMLP-induced neutropenia in the mouse. Studies using an inactivated LC1 preparation, which binds to the same high affinity binding sites as the biologically active material, indicated that the short peptide acts on the same sites as the native LC1. This study confirms the activity of LC1 in another model of experimental inflammation and suggests that it acts partly through inhibition of leukocyte activation with an overall effect qualitatively comparable to the blocking of CD11b portion of a β2-integrin complex. It also shows that peptides derived from the N-terminal domain of LC1 may mimic the activity of the full length molecule and points the way for a new family of anti-inflammatory substances that inhibit leukocyte trafficking.
Lipocortin 1, a member of the annexin superfamily of calcium and phospholipid binding proteins, mediates some of the anti-inflammatory actions of the glucocorticoid hormones. Lipocortin 1 binds to the surface of murine peripheral blood monocytes and polymorphonuclear leukocytes (Kd estimate 2 x 10(-8) M) but not lymphocytes. Resident peritoneal macrophages exhibit binding (Kd estimate 1.3 x 10(-8) M) but lymphocytes do not. A 95-98% reduction in lipocortin 1 binding was observed to leukocytes obtained from air pouch or peritonitis models of inflammation. When given intravenously, lipocortin 1 binds rapidly to murine leukocytes within 5 min but disappears before 10 min, leaving the binding capacity of the cells unaltered. Modulation of lipocortin 1 binding sites could be an important step in regulating the function of inflammatory cells.
The glucocorticoid-induced antiinflammatory protein lipocortin 1 is present in arthritic synovium but its ability to regulate joint inflammation has not previously been studied. We investigated the role of lipocortin 1 in the antiinflammatory activity of glucocorticoids in an acute arthritis model induced by intraarticular injection of carrageenan. Compared to control joints (0.09 +/- 0.08 x 10(6) synovial fluid cell count), carrageenan injected joints exhibited marked infiltration of PMN (10.2 +/- 0.7 x 10(6), p < 0.001). Both intraperitoneal (1.0 mg/kg) and intraarticular administration (5 micrograms) of dexamethasone (DEX) significantly suppressed arthritis severity (p < 0.001 and 0.005, respectively), and the effects of DEX were significantly prevented by intra-articular injection of antilipocortin 1 mAb (p < 0.05). Carrageenan arthritis was also significantly inhibited by intraarticular administration of the N-terminal lipocortin 1 peptide Ac2-26 at doses of 1 or 2 mg/kg (p < 0.01). Intraarticular injection antilipocortin 1 mAb in the absence of DEX also significantly exacerbated arthritis severity (p < 0.005). In vitro treatment of PMN with DEX was associated with significant inhibition of phagocytosis (p < 0.005) and reactive oxygen species (ROS) generation (p < 0.001). Antilipocortin 1 mAb significantly reduced the inhibitory effects of DEX (p < 0.01 and 0.005, respectively). These results demonstrate that lipocortin 1 mediates the effects of exogenous glucocorticoids on neutrophil migration in carrageenan-induced acute arthritis, exerts an endogenous antiinflammatory influence, and mediates glucocorticoid inhibition of neutrophil activation.
Nitric oxide (NO) is a mediator of inflammatory injury which is inhibited by glucocorticoids and is implicated in rheumatoid (RA) and adjuvant arthritis (AA). The glucocorticoid-induced anti-inflammatory molecule lipocortin 1 is expressed in RA synovium, but the effects of lipocortin 1 on synovial inflammation have been little studied. We investigated the effects of glucocorticoids and lipocortin 1 on inducible NO synthase (iNOS) and glucocorticoids on the induction of lipocortin 1 in AA synovial macrophages. NO production was measured by Griess assay in supernatants of day 14 AA rat synovial explants and of synovial macrophages purified from enzyme-digested synovium and treated with lipopolysaccharide (LPS) 1 microg/ml, dexamethasone (DEX) 10(-7) M, and anti-lipocortin 1 MoAb. iNOS and lipocortin 1 expression were detected by flow cytometry using specific MoAb. Cell surface lipocortin was determined by Western blot. NO was produced by all AA synovial explants and NO was released by cultured synovial macrophages (14.5 +/- 2.1 micromol/24 h). iNOS was detected in synovial macrophages (ED-1+) by permeabilization flow cytometry. LPS increased synovial macrophage NO release (P < 0.0001) and iNOS expression (P = 0.04). DEX inhibited constitutive (P = 0.002) and LPS-induced (P < 0.001) NO release and iNOS expression (P = 0.03). DEX inhibition of synovial macrophage NO was associated with induction of cell surface and intracellular lipocortin 1. Anti-lipocortin 1 MoAb treatment reduced the inhibition of NO release by DEX (P = 0.002), but had no effect on iNOS expression. These findings demonstrate a role for lipocortin I in the inhibition by glucocorticoids of AA synovial macrophage iNOS activity.
Annexin I is an endogenous antiinflammatory mediator, expressed in rheumatoid arthritis (RA) synovium, the contribution of which to autoregulation of the synovial inflammatory response has not been examined in models of RA. We investigated the antiinflammatory role of annexin I in rat adjuvant arthritis.
Rats with adjuvant-induced arthritis (AIA) were treated with a specific anti-annexin I monoclonal antibody (mAb), isotype control IgG, and/or dexamethasone. Clinical outcomes and synovial synthesis of tumor necrosis factor alpha (TNFalpha), prostaglandin E2 (PGE2), and nitric oxide were examined, and annexin I expression was assessed by flow cytometry and reverse transcription-polymerase chain reaction.
Anti-annexin I mAb reversed the effects of dexamethasone on the clinical features of AIA and exacerbated AIA in the absence of exogenous glucocorticoid. Clinical exacerbation of AIA by anti-annexin I mAb was accompanied by significantly increased synovial TNFalpha and PGE2, suggesting that annexin I tonically inhibits the production of these mediators. Anti-annexin I mAb treatment was associated with significantly reduced leukocyte intracellular annexin I, despite increased annexin I messenger RNA expression, consistent with a depletion effect of extracellular mAb via the cell surface.
Annexin I is a key endogenous inhibitory mediator of arthritis via mechanisms that include inhibition of cytokine and effector molecule production. Moreover, a synthesis-independent depletion of intracellular annexin I by extracellular antibody supports the hypothesis that externalization of annexin I is involved in its mode of action.