Valerie Corrigall

The Human Endoplasmic Reticulum Molecular Chaperone BiP Is an Autoantigen for Rheumatoid Arthritis and Prevents the Induction of Experimental Arthritis

Rheumatoid arthritis (RA) is the most common, crippling human autoimmune disease. Using Western blotting and tandem mass spectroscopy, we have identified the endoplasmic reticulum chaperone BiP, a 78-kDa glucose-regulated protein, as a possible autoantigen. It preferentially stimulated increased proliferation of synovial T cells from patients with RA but not from patients with other arthritides. Mice with established collagen- or pristane-induced arthritis developed IgG Abs to BiP. Although BiP injected in CFA failed to induce arthritis in several strains of rats and mice, including HLA-DR4+/−- and HLA-DR1+/+-transgenic animals, it completely inhibited the development of arthritis when given i.v. 1 wk before the injection of type II collagen arthritis. Preimmunization with BiP suppressed the development of adjuvant arthritis in Lewis rats in a similar manner. This is the first report of a mammalian chaperone that is an autoantigen in human RA and in experimental arthritis and that can also prevent the induction of experimental arthritis. These findings may stimulate the development of new immunotherapies for the treatment of RA.

Pathogenesis of rheumatoid arthritis. The role of T cells and other beasts

The evidence coming from the different experimental approaches reviewed in this article strongly supports the hypothesis that RA is T-cell driven at all stages of the disease. Although the effector phases responsible for the events that lead to joint destruction involve several different cell types, cytokines, and other mediators, T cells still direct operations behind the scenes. Direct experimental proof of this proposition in patients is still lacking, but the development of nondepleting modulating CD4 monoclonal antibodies may provide new tools to test this hypothesis. In this respect, it is encouraging that using one such reagent, we have recently shown that not only did the activity of the disease improve but, more importantly, the inflammatory indices and production of non-T-cell cytokines were reduced. This is not to dissimilar from the results of experiments described in animals, where by blocking synovial T cells, the production of IL-1 beta and TNF alpha could be decreased by more than 90%. From this perspective, it may be predicted that by modulating T cells in the joint, it is possible to achieve our ultimate goal of permanently switching off the disease.

Monocyte-platelet interaction induces a pro-inflammatory phenotype in circulating monocytes

Background Activated platelets exert a pro-inflammatory action that can be largely ascribed to their ability to interact with leukocytes and modulate their activity. We hypothesized that platelet activation and consequent formation of monocyte-platelet aggregates (MPA) induces a pro-inflammatory phenotype in circulating monocytes. Methodology/Principal Findings CD62P+ platelets and MPA were measured, and monocytes characterized, by whole blood flow cytometry in healthy subjects, before and two days after receiving influenza immunization. Three monocytic subsets were identified: CD14+CD16−, CD14highCD16+and CD14lowCD16+. The increase in high sensitivity C-reactive protein post-immunization was accompanied by increased platelet activation and MPA formation (25.02±12.57 vs 41.48±16.81; p = 0.01), along with enhancement of circulating CD14highCD16+ cells (4.7±3.6 vs 10.4±4.8; p = 0.003), their percentage being linearly related to levels of CD62P+-platelets (r2 = 0.4347; p = 0.0008). In separate in vitro experiments, co-incubation of CD14+CD16− cells, isolated from healthy donor subjects, with autologous platelets gave rise to up-regulation of CD16 on monocytes as compared with those maintained in medium alone (% change in CD14+CD16+ cells following 48 h co-incubation of monocytes with platelets was +106±51% vs monocytes in medium alone; p<0.001). This effect correlated directly with degree of MPA formation (r2 = 0.7731; p<0.0001) and was associated with increased monocyte adhesion to endothelial cells. P-selectin glycoprotein ligand-1 (PSGL-1) blocking antibody, which abrogates MPA formation, abolished these effects, as did the cyclooxygenase (COX)-2 selective inhibitor NS-398, aspirin and the EP1/EP2-selective antagonist AH6809. Conclusions/Significance These data suggest that MPA formation, as occurs in the blood under pro-inflammatory conditions, expands the pool of circulating CD14highCD16+ monocytes in a COX-2 dependent manner, and these monocytes exhibit increased adhesion to endothelium. Our findings delineate a novel mechanism underlying the pro-inflammatory effect of platelet activation.

Treatment of Rheumatoid Arthritis with Single Dose or Weekly Pulses of Chimaeric Anti‐CD4 Monoclonal Antibody

The aetiotogy of rheumatoid arthritis is unknown but CD4+ T eells are known to be involved in its pathogenesis. Because of this, anti‐CD4 monoelonal antibody has been used in open studies with clinical benefit in up to 60% of patients. We have used a ehimaeric anti‐CD4 monoclonal antibody (cM‐T4l2, Centocor) in a randomized, double‐blinded, placebo controlled trial as treatment for rheumatoid arthritis. Nine patients with active rheumatoid arthritis resistant to traditional disease‐modifying drugs were recruited. Four received an intravenous 50 mg bolus of antibody, and three received 50 mg weekly for four consecutive weeks. Two patients received placebo. Despite a marked reduction (P<0.001) in peripheral blood CD4+ lymphocytes, there was no significant clinical improvement in any of these patienis. The decrease in CD4+ lymphocyte number lasted one week after a single 50 mg dose of cM‐T412 but was more prolonged in the patients who reeeived four infusions. CD8+ T cells. CD16+ cytoloxic cells and CD14+ monoeytes showed only a transient reduction. It may be concluded that the therapeutic efficacy of anti‐CD4 therapy is not direetly related to CD4 T‐cell lymphopenia.

The role of the chemokines MCP-1, GRO-α, IL-8 and their receptors in the adhesion of monocytic cells to human atherosclerotic plaques

Monocyte adhesion to the arterial endothelium and subsequent migration into the intima are central events in the pathogenesis of atherosclerosis. Previous experimental models have shown that chemokines can enhance monocyte–endothelial adhesion by activating monocyte integrins. Our study assesses the role of chemokines IL-8, MCP-1 and GRO-α, together with their monocyte receptors CCR2 and CXCR2 in monocyte adhesion to human atherosclerotic plaques. In an adhesion assay, a suspension of monocytic U937 cells was incubated with human atherosclerotic artery sections and the levels of endothelial adhesion were quantified. Adhesion performed in the presence of a monoclonal antibody to a chemokine, chemokine receptor or of an isotype matched control immunoglobulin, shows that antibodies to all chemokines tested, as well as their receptors, inhibit adhesion compared to the control immunoglobulins. Immunohistochemistry demonstrated the expression of MCP-1, GRO-α and their receptors in the endothelial cells and intima of all atherosclerotic lesions. These results suggest that all these chemokines and their receptors can play a role in the adhesion of monocytes to human atherosclerotic plaques. Furthermore, they suggest that these chemokine interactions provide potential targets for the therapy of atherosclerosis.

Resolution‐associated molecular patterns (RAMP): RAMParts defending immunological homeostasis?

The resolution of inflammation is central to the maintenance of good health and immune homeostasis. Recently, several intracellular stress proteins have been described as having extracellular properties that are anti‐inflammatory or favour the resolution of inflammation. We propose that these molecules should be defined as resolution‐associated molecular patterns (RAMPs). RAMPs are released at times of cellular stress and help to counterbalance the inflammatory effects of pathogen‐associated (PAMPs) and damage‐associated (DAMPs) molecular patterns. We propose that heat shock protein 10 (HSP10), αB‐crystallin (αBC), HSP27 and binding immunoglobulin protein (BiP) should be considered founding members of the RAMP family. A greater understanding of RAMP biology may herald the development of novel immunotherapies.

Glucose-regulated protein 78 (Grp78/BiP) is secreted by human oviduct epithelial cells and the recombinant protein modulates sperm-zona pellucida binding.

OBJECTIVE To determine the secretion of Grp78 by human oviduct epithelial cells, its association to spermatozoa, and its involvement in gamete interaction. DESIGN Prospective study. SETTING Basic research laboratory. SUBJECT(S) Semen samples obtained from normozoospermic volunteers. Tubal tissue provided by patients undergoing hysterectomies. Oocytes collected from women undergoing IVF-ET. INTERVENTION(S) Analysis of Grp78 expression and secretion by oviductal tissue. Gamete incubation with recombinant Grp78 (rec-Grp78). MAIN OUTCOME MEASURE(S) Assessment of protein expression and secretion by immunohistochemistry and Western immunoblotting, respectively. Evaluation of rec-Grp78 binding to human spermatozoa by immunocytochemistry, and analysis of its effect upon gamete interaction using the hemizona assay. RESULT(S) Grp78 was found in the surface of oviduct epithelial cells. Soluble Grp78 was detected in oviductal fluids from women in the periovulatory period and in oviductal tissue conditioned medium. Rec-Grp78 was able to bind to the sperm acrosomal cap, and its presence during gamete interaction led to a decrease in the number of spermatozoa bound to the zona pellucida (ZP). When calcium ions from the incubation medium were replaced by strontium, rec-Grp78 enhanced sperm-ZP interaction. CONCLUSION(S) Grp78 is expressed and secreted by oviduct epithelial cells. The protein would bind to the gametes and may modulate their interaction in a calcium-dependent manner.

Large granular lymphocyte expansions in patients with Felty's syndrome: analysis using anti‐T cell receptor Vβ‐specific monoclonal antibodies

Feltys syndrome (FS), the association of rheumatoid arthritis (RA) and idiopathic neutropenia, remains an unexplained phenomenon. HLA‐DR4 is found in over 90% of cases. Patients with FS may have a T cell lymphocytosis of CD3+CD8+CD57+ large granular lymphocytes (LGL syndrome). In this study of 47 patients with FS, 19% had clear evidence for LGL expansions, while in total 42% had variable evidence for the LGL syndrome using currently available techniques. Of these T cell expansions, 76% were clonal, as demonstrated by Southern blotting and analysis with T cell receptor (TCR) β chain constant region probes. This technique may fail to detect clonal populations in some patients. Cytofluorographic analysis using antibodies specific for TCR β chains identified patients with clonal LGL expansions with results comparable to those obtained with Southern blotting. No evidence for shared Vβ usage among expansions from different patients was seen. The role of LGL in RA and FS is currently unclear, but this technique offers a practical and accessible means of identifying patients with LGL expansions, as a starting point for further investigation.

Lack of CD80 expression by fibroblast-like synoviocytes leading to anergy in T lymphocytes.

OBJECTIVE To investigate whether contact with HLA-DR+, but CD80-, fibroblast-like synoviocytes (FLS) in the presence of antigen leads to the induction of anergy in, rather than stimulation of, T cells. METHODS Cell surface expression of activation and costimulatory markers on FLS were studied by flow cytometry. Functional changes were investigated by T cell proliferation to tuberculin purified protein derivative or allogeneic responses to FLS, in the presence or absence of DAP3.B7 cells, a human CD80-transfected mouse fibroblast cell line. Induction of anergy was investigated by a 2-stage culture system. T cells were cocultured with allogeneic FLS in the primary culture, rested, and restimulated in the secondary culture by FLS in the presence or absence of DAP3.B7 cells or interleukin-2 (IL-2). RESULTS Direct contact between T cells and FLS caused up-regulation of CD69 on T cells and HLA-DR on FLS in both the allogeneic and autologous cultures. The addition of DAP3.B7 cells to FLS-T cell cocultures restored the depressed allogeneic responses of T cells. The allogeneic response by T cells to FLS in the presence of DAP3.B7 cells could be completely inhibited by blocking CD80 with CTLA-4 Ig. Indirect evidence that T cells cocultured with FLS were anergic was the up-regulation of CD25, negligible T cell proliferation, and the restoration of proliferation by the addition of exogenous IL-2. Direct evidence of anergy was obtained when T cells from the primary cultures with FLS remained unresponsive to secondary culture with FLS even in the presence of DAP3.B7 cells. In contrast, primary culture of T cells with FLS plus DAP3.B7 cells initiated a good allogeneic response in all subsequent cultures. CONCLUSION It is possible that T cells within the synovium may be anergized by contact with HLA-DR+ CD80- FLS.

Autoantibodies to Posttranslationally Modified Type II Collagen as Potential Biomarkers for Rheumatoid Arthritis

OBJECTIVE Type II collagen (CII) posttranslationally modified by reactive oxygen species (ROS-CII) that are present in the inflamed joint is an autoantigen in rheumatoid arthritis (RA). The aim of this study was to investigate the potential use of anti-ROS-CII autoantibodies as a biomarker of RA. METHODS CII was exposed to oxidants that are present in the rheumatoid joint. Autoreactivity to ROS-CII was assessed by enzyme-linked immunosorbent assays in synovial fluid (SF) and serum samples obtained from patients during various phases of RA. This group included disease-modifying antirheumatic drug (DMARD)-naive patients with early RA (n = 85 serum samples) and patients with established RA (n = 80 serum and 50 SF samples), who were categorized as either DMARD responders or DMARD nonresponders. Control subjects included anti-citrullinated protein antibody (ACPA)-positive patients with arthralgia (n = 58 serum samples), patients with osteoarthritis (OA; n = 49 serum and 52 SF samples), and healthy individuals (n = 51 serum samples). RESULTS Reactivity to ROS-CII among DMARD-naive patients with early RA was significantly higher than that among patients with ACPA-positive arthralgia, patients with OA, and healthy control subjects (P < 0.0001), with 92.9% of serum samples from the patients with early RA binding to anti-ROS-II. There was no significant difference in anti-ROS-CII reactivity between ACPA-positive and ACPA-negative patients with RA, with 93.8% and 91.6% of serum samples, respectively, binding to ROS-CII. The sensitivity and specificity of binding to ROS-CII in patients with early RA were 92% and 98%, respectively. Among patients with established RA, serum reactivity in DMARD nonresponders was significantly higher than that in DMARD responders (P < 0.01); 58.3% of serum samples from nonresponders and 7.6% of serum samples from responders bound to HOCl-ROS, while the respective values for SF were 70% and 60%. In patients with longstanding RA, autoreactivity to ROS-CII changed longitudinally. CONCLUSION Autoantibodies to ROS-CII have the potential to become diagnostic biomarkers of RA.