Essential role for the lectin pathway in collagen antibody-induced arthritis revealed through use of adenovirus programming complement inhibitor MAp44 expression.

Previous studies using mannose-binding lectin (MBL) and complement C4-deficient mice have suggested that the lectin pathway (LP) is not required for the development of inflammatory arthritis in the collagen Ab-induced arthritis (CAIA) model. MBL, ficolins and collectin-11 are key LP pattern recognition molecules that associate with three serine proteases-MASP-1, MASP-2, and MASP-3-and with two MBL-associated proteins designated sMAP and MBL-associated protein of 44kDA (MAp44). Recent studies have shown that MAp44, an alternatively spliced product of the MASP-1/3 gene, is a competitive inhibitor of the binding of the recognition molecules to all three MASPs. In these studies, we examined the effect of treatment of mice with adenovirus (Ad) programmed to express human MAp44 (AdhMAp44) on the development of CAIA. AdhMAp44 and Ad programming GFP (AdGFP) expression were injected i.p. in C57BL/6 wild type mice prior to the induction of CAIA. AdhMAp44 significantly reduced the clinical disease activity (CDA) score by 81% compared with mice injected with AdGFP. Similarly, histopathologic injury scores for inflammation, pannus, cartilage and bone damage, as well as C3 deposition in the cartilage and synovium, were significantly reduced by AdhMAp44 pretreatment. Mice treated with AdmMAp44, programming expression of mouse MAp44, also showed significantly decreased CDA score and histopathologic injury scores. In addition, administration of AdhMAp44 significantly diminished the severity of Ross River virus-induced arthritis, an LP-dependent model. Our study provides conclusive evidence that an intact complement LP is essential to initiate CAIA, and that MAp44 may be an appropriate treatment for inflammatory arthritis.

Essential role of surface-bound complement factor H in controlling immune complex-induced arthritis.

Factor H (fH) is an endogenous negative regulator of the alternative pathway (AP) that binds polyanions as well as complement activation fragments C3b and C3d. The AP is both necessary and sufficient to develop collagen Ab-induced arthritis (CAIA) in mice; the mechanisms whereby normal control of the AP is overcome and injury develops are unknown. Although primarily a soluble circulating protein, fH can also bind to tissues in a manner dependent on the carboxyl-terminal domain containing short consensus repeats 19 and 20. We examined the role of fH in CAIA by blocking its binding to tissues through administration of a recombinant negative inhibitor containing short consensus repeats 19 and 20 (rfH19-20), which impairs fH function and amplifies surface AP activation in vitro. Administration of rfH19-20, but not control rfH3-5, significantly worsened clinical disease activity, histopathologic injury, and C3 deposition in the synovium and cartilage in wild-type and fH(+/-) mice. In vitro studies demonstrated that rfH19-20 increased complement activation on cartilage extracts and injured fibroblast-like synoviocytes, two major targets of complement deposition in the joint. We conclude that endogenous fH makes a significant contribution to inhibition of the AP in CAIA through binding to sites of immune complex formation and complement activation.

Roles of adipocytes and fibroblasts in activation of the alternative pathway of complement in inflammatory arthritis in mice.

The complement system is involved in mediation of joint damage in rheumatoid arthritis, with evidence suggesting activation of both the classical and alternative pathway (AP). The AP is both necessary and sufficient to mediate collagen Ab-induced arthritis, an experimental animal model of immune complex-induced joint disease. The AP in mice is dependent on MASP-1/3 cleavage of pro-factor D (pro-FD) into mature factor D (FD). The objectives of the current study were to determine the cells synthesizing MASP-1/3 and pro-FD in synovial tissue. Collagen Ab-induced arthritis was studied in wild-type C57BL/6 mice, and the localization of mRNA and protein for FD and MASP-1/3 in synovial adipose tissue (SAT) and fibroblast-like synoviocytes (FLS) was determined using various techniques, including laser capture microdissection. SAT was the sole source of mRNA for pro-FD. Cultured differentiated 3T3 adipocytes, a surrogate for SAT, produced pro-FD but no mature FD. FLS were the main source of MASP-1/3 mRNA and protein. Using cartilage microparticles (CMPs) coated with anti-collagen mAb and serum from MASP-1/3(-/-) mice as a source of factor B, pro-FD in 3T3 supernatants was cleaved into mature FD by MASP-1/3 in FLS supernatants. The mature FD was eluted from the CMP, and was not present in the supernatants from the incubation with CMP, indicating that cleavage of pro-FD into mature FD by MASP-1 occurred on the CMP. These results demonstrate that pathogenic activation of the AP can occur in the joint through immune complexes adherent to cartilage and the local production of necessary AP proteins by adipocytes and FLS.

Role of C3a receptors, C5a receptors, and complement protein C6 deficiency in collagen antibody-induced arthritis in mice.

The complement system, especially the alternative pathway, plays essential roles in the induction of injury in collagen Ab-induced arthritis (CAIA) in mice. The goal of the current study was to directly compare the roles of receptors for C3a and C5a, as well as the membrane attack complex, as effector mechanisms in the pathogenesis of CAIA. Clinical disease activity in C3aR(-/-), C5aR(-/-), and C6-deficient (C6-def) mice was decreased by 52, 94, and 65%, respectively, as compared with wild-type mice. Decreases in histopathologic injury as well as in IgG and C3 deposition paralleled the clinical disease activity. A decrease in the percentage of synovial neutrophils was observed in C3aR(-/-), C5aR(-/-), and C6-def mice, and a decrease in macrophages was observed in C3aR(-/-) and C5aR(-/-), but not in C6-def, mice. Synovial mRNA obtained by laser capture microdissection exhibited a decrease in TNF-α in C5aR(-/-) mice and in IL-1ß in both C5aR(-/-) and C6-def mice, whereas C3aR(-/-) mice demonstrated no change in either cytokine. Our findings show that absent C3aR-, C5aR-, or membrane attack complex-initiated effector mechanisms each decrease susceptibility to CAIA, with clinical effects most pronounced in C5aR-deficient mice. Although the absence of C3aR, C5aR, or C6 led to differential deficiencies in effector mechanisms, decreased proximal joint IgG and C3 deposition was common to all three genotypes in comparison with wild-type mice. These data suggest the existence of positive-feedback amplification pathways downstream of all three effectors that promote additional IgG deposition and C3 activation in the joint.

N-α-benzoyl-N5-(2-chloro-1-iminoethyl)-L-ornithine amide, a protein arginine deiminase inhibitor, reduces the severity of murine collagen-induced arthritis.

Rheumatoid arthritis is associated with the development of autoantibodies to citrullinated self-proteins. Citrullinated synovial proteins, which are generated via the actions of the protein arginine deiminases (PADs), are known to develop in the murine collagen-induced arthritis (CIA) model of inflammatory arthritis. Given these findings, we evaluated whether N-α-benzoyl-N5-(2-chloro-1-iminoethyl)-L-ornithine amide (Cl-amidine), a recently described pan-PAD inhibitor, could affect the development of arthritis and autoimmunity by treating mice in the CIA model with Cl-amidine on days 0-35. Cl-amidine treatment reduced total synovial and serum citrullination, decreased clinical disease activity by ∼50%, and significantly decreased IgG2a anti-mouse type II collagen Abs. Additionally, histopathology scores and total complement C3 deposition were significantly lower in Cl-amidine-treated mice compared with vehicle controls. Synovial microarray analyses demonstrated decreased IgG reactivity to several native and citrullinated epitopes compared with vehicle controls. Cl-amidine treatment had no ameliorative effect on collagen Ab-induced arthritis, suggesting its primary protective mechanism was not mediated through effector pathways. Reduced levels of citrullinated synovial proteins observed in mice treated with Cl-amidine are consistent with the notion that Cl-amidine derives its efficacy from its ability to inhibit the deiminating activity of PADs. In total, these results suggested that PADs are necessary participants in the autoimmune and subsequent inflammatory processes in CIA. Cl-amidine may represent a novel class of disease-modifying agents that modulate aberrant citrullination, and perhaps other immune processes, necessary for the development of inflammatory arthritis.

Essential role of complement mannose-binding lectin-associated serine proteases-1/3 in the murine collagen antibody-induced model of inflammatory arthritis.

Gene-targeted mice deficient in the complement mannose-binding lectin-associated serine protease-1 and -3 (MASP1/3(-/-)) express only the zymogen of factor D (pro-factor D [pro-Df]), a necessary component of the alternative pathway (AP). We used the murine collagen Ab-induced arthritis (CAIA) model, in which the AP is unique among complement pathways in being both necessary and sufficient for disease induction, to determine whether MASP-1/3 are required in vivo for the development of tissue injury. Disease activity scores, complement C3 tissue deposition in the joint, and histopathologic injury scores were markedly decreased in MASP1/3(-/-) as compared with wild-type (WT) mice. MASP-1 protein was immunochemically localized to synovial cells of knees of WT mice with arthritis. Pro-Df was present in both synovial cells and chondrocytes of knees of WT and MASP1/3(-/-) mice without arthritis, with increased amounts present in synovial cells of WT mice with CAIA. No conversion of pro-Df to mature Df was detectable in the serum of MASP1/3(-/-) mice during the evolution of CAIA. C3 activation and deposition as well as C5a generation induced in vitro by adherent anti-type II collagen mAbs were absent using sera from MASP1/3(-/-) mice under conditions in which only the AP was active. The addition of human Df fully reconstituted in vitro C3 activation and C5a generation using sera from MASP1/3(-/-) mice. Our studies demonstrate for the first time, to our knowledge, the absolute requirement for the activity of MASP-1 protein in autoimmune-associated inflammatory tissue injury in vivo through activation of the AP of complement by cleavage of pro-Df to mature Df.

IL-1, IL-18, and IL-33 families of cytokines.

SUMMARY: The interleukin-1 (IL-1), IL-18, and IL-33 families of cytokines are related by mechanism of origin, receptor structure, and signal transduction pathways utilized. All three cytokines are synthesized as precursor molecules and cleaved by the enzyme caspase-1 before or during release from the cell. The NALP-3 inflammasome is of crucial importance in generating active caspase-1. The IL-1 family contains two agonists, IL-1alpha and IL-1beta, a specific inhibitor, IL-1 receptor antagonist (IL-1Ra), and two receptors, the biologically active type IL-1R and inactive type II IL-1R. Both IL-1RI and IL-33R utilize the same interacting accessory protein (IL-1RAcP). The balance between IL-1 and IL-1Ra is important in preventing disease in various organs, and excess production of IL-1 has been implicated in many human diseases. The IL-18 family also contains a specific inhibitor, the IL-18-binding protein (IL-18BP), which binds IL-18 in the fluid phase. The IL-18 receptor is similar to the IL-1 receptor complex, including a single ligand-binding chain and a different interacting accessory protein. IL-18 provides an important link between the innate and adaptive immune responses. Newly described IL-33 binds to the orphan IL-1 family receptor T1/ST2 and stimulates T-helper 2 responses as well as mast cells.

Targeted inhibition of the complement alternative pathway with complement receptor 2 and factor H attenuates collagen antibody-induced arthritis in mice.

The alternative pathway (AP) of complement is required for the induction of collagen Ab-induced arthritis (CAIA) in mice. The objective of this study was to examine the effect of a recombinant AP inhibitor containing complement receptor 2 and factor H (CR2-fH) on CAIA in mice. CR2 binds to tissue-fixed activation fragments of C3, and the linked fH is a potent local inhibitor of the AP. CAIA was induced in C57BL/6 mice by i.p. injections of 4 mAb to type II collagen (CII) on day 0 and LPS on day 3. PBS or CR2-fH (250 or 500 microg) were injected i.p. 15 min after the mAb to CII on day 0 and 15 min after LPS on day 3; the mice were sacrificed on day 10. The disease activity score (DAS) was decreased significantly (p < 0.001) in both groups receiving CR2-fH compared with the PBS. Histology scores for inflammation, pannus, bone damage, and cartilage damage decreased in parallel with the DAS. C3 deposition in the synovium and cartilage was significantly reduced (p < 0.0001) in the mice treated with CR2-fH. In vitro studies with immune complexes containing type II collagen and mAb to CII showed that CR2-fH specifically inhibited the AP with minimal effect on the classical pathway (CP) and no effect on the lectin pathway (LP). The relative potency of CR2-fH in vitro was superior to mAbs to factor B and C5. Thus, CR2-fH specifically targets and inhibits the AP of complement in vitro and is effective in CAIA in vivo.

Antibodies against citrullinated proteins enhance tissue injury in experimental autoimmune arthritis.

Antibodies against citrullinated proteins are specific and predictive markers for rheumatoid arthritis although the pathologic relevance of these antibodies remains unclear. To investigate the significance of these autoantibodies, collagen-induced arthritis (CIA) in mice was used to establish an animal model of antibody reactivity to citrullinated proteins. DBA/1J mice were immunized with bovine type II collagen (CII) at days 0 and 21, and serum was collected every 7 days for analysis. Antibodies against both CII and cyclic citrullinated peptide, one such citrullinated antigen, appeared early after immunization, before joint swelling was observed. Further, these antibodies demonstrated specific binding to citrullinated filaggrin in rat esophagus by indirect immunofluorescence and citrullinated fibrinogen by Western blot. To evaluate the role of immune responses to citrullinated proteins in CIA, mice were tolerized with a citrulline-containing peptide, followed by antigen challenge with CII. Tolerized mice demonstrated significantly reduced disease severity and incidence compared with controls. We also identified novel murine monoclonal antibodies specific to citrullinated fibrinogen that enhanced arthritis when coadministered with a submaximal dose of anti-CII antibodies and bound targets within the inflamed synovium of mice with CIA. These results demonstrate that antibodies against citrullinated proteins are centrally involved in the pathogenesis of autoimmune arthritis.

The balance between IL-1 and IL-1Ra in disease.

IL-1 is an important mediator of inflammation and tissue damage in multiple organs, both in experimental animal models of disease and in human diseases. The IL-1 family consists of two agonists, IL-1alpha and IL-1beta, two receptors, biologically active IL-1RI and inert IL-1RII, and a specific receptor antagonist, IL-1Ra. The balance between IL-1 and IL-1Ra in local tissues plays an important role in the susceptibility to and severity of many diseases. An allelic polymorphism in the IL-1Ra gene has been associated with a variety of human diseases primarily of epithelial and endothelial cell origin. This association may be secondary to an imbalance in the IL-1 system with enhanced production of IL-1beta and reduced production of the major intracellular isoform of IL-1Ra. Treatment of RA with daily subcutaneous injections of recombinant IL-1Ra protein has been shown to be efficacious. Gene therapy approaches with IL-1Ra are being evaluated for the treatment of RA and other human diseases.

Generation and characterization of mice transgenic for human IL-18-binding protein isoform a.

Interleukin (IL)-18 binding protein (IL-18BP) is a natural inhibitor of the pleiotropic cytokine IL-18. To study the role of IL-18BP in modulating inflammatory responses in vivo, mice transgenic for human IL-18BP isoform a (IL-18BP-Tg) were generated. The transgene was expressed at high levels in each organ examined. High levels of bioactive human IL-18BPa were detectable in the circulation of IL-18BP-Tg mice, which were viable, fertile, and had no tissue or organ abnormality. The high levels of IL-18BP in the transgenic mice were able to completely neutralize the interferon-gamma (IFN-gamma)-inducing activity of exogenously administered IL-18. Following administration of endotoxin, with or without prior sensitization with heat-inactivated Propionibacterium acnes, IL-18BP-Tg mice produced significantly lower serum levels of IFN-gamma and macrophage-inflammatory protein-2 compared with nontransgenic littermates. Significantly reduced production of IFN-gamma in response to endotoxin was also observed in cultures of IL-18BP-Tg splenocytes. Finally, IL-18BP-Tg mice were completely protected in a model of hepatotoxicity induced by administration of concanavalin A. These results indicate that high endogenous levels of IL-18BP in trangenic mice effectively neutralize IL-18 and are protective in response to different inflammatory stimuli.

Cytokines in the rheumatic diseases.

Extensive data has accumulated over the last 10 to 15 years to implicate various cytokines in pathways of pathophysiology in rheumatic diseases. Abnormalities in cytokine production are not the cause of these diseases, but reflect continual production by immune and inflammatory cells. Cytokines are heterogeneous and function in an overlapping and redundant network. An important principle to emerge is that the net biologic response in a diseased organ or tissue reflects a balance between the local levels of proinflammatory and anti-inflammatory cytokines and factors. Thus, a chronic disease may result from the excess production of proinflammatory cytokines or the inadequate production of anti-inflammatory cytokines. This article summarizes the role of cytokines in rheumatic diseases by focusing on each disease and the involved pathways of pathophysiology.

The mode of action of cytokine inhibitors.

Tumor necrosis factor-alpha (TNF-alpha) and interleukin 1 (IL-1) are important mediators of inflammation and tissue damage in animal models of inflammatory arthritis and in patients with active rheumatoid arthritis (RA). Several inhibitors of these cytokines are now available for RA treatment, each having a different mode of action. Etanercept is a recombinant fusion protein of the soluble type II TNF receptor on a human IgG1 backbone, whereas infliximab is a chimeric anti-TNF-alpha monoclonal antibody containing a murine TNF-alpha binding region and human IgG1 backbone. Both agents potently and selectively bind TNF-alpha in the cellular microenvironment, thereby preventing TNF-alpha from interacting with membrane-bound TNF receptors on target cells. In comparison, anakinra is a recombinant human IL-1 receptor antagonist (IL-1Ra) that binds avidly to type 1 IL-1 receptors but does not stimulate any intracellular responses. Studies of these agents in animal models of inflammatory arthritis suggest that TNF-alpha plays a more important role in promoting inflammation, whereas IL-1 is more important in causing cartilage and bone destruction. However, these differential actions have not been borne out in clinical trials, where TNF-alpha blockers and anakinra similarly reduce clinical signs and symptoms of RA as well as slow radiographic evidence of disease progression.

Pre-rheumatoid arthritis: predisposition and transition to clinical synovitis.

Multiple proven and potential risk factors for the development of rheumatoid arthritis (RA) have been identified, and represent interactions between genes and the environment. Proven risk factors include genetic influences on the function of the innate and adaptive immune systems, smoking, anti-citrullinated protein antibodies (ACPAs), and rheumatoid factors (RF). Potential risk factors include epigenetic control of gene expression, the microbiome and other environmental factors, Toll-like receptors, cytokines, and Fc receptors. Preclinical abnormalities such as circulating RF and ACPAs may occur more than 10 years prior to the onset of clinical disease. However, the precise mechanisms whereby these risk factors lead to clinical disease remain unclear. It is possible that, combined with activation of the innate immune system, a subset of ACPAs initiates the disease in the cartilage or synovium after binding to endogenous citrullinated proteins. Subsequent engagement of Fc receptors and complement activation would lead to secondary inflammation in the synovium with induction of a perpetuating cycle of chronic synovitis.

Mechanisms of mannose-binding lectin-associated serine proteases-1/3 activation of the alternative pathway of complement.

Mannose-binding lectin-associated serine proteases-1/3 (MASP-1/3) are essential in activating the alternative pathway (AP) of complement through cleaving pro-factor D (pro-Df) into mature Df. MASP are believed to require binding to mannose binding lectins (MBL) or ficolins (FCN) to carry out their biological activities. Murine sera have been reported to contain MBL-A, MBL-C, and FCN-A, but not FCN-B that exists endogenously in monocytes and is thought not to bind MASP-1. We examined some possible mechanisms whereby MASP-1/3 might activate the AP. Collagen antibody-induced arthritis, a murine model of inflammatory arthritis dependent on the AP, was unchanged in mice lacking MBL-A, MBL-C, and FCN-A (MBL(-/-)/FCN A(-/-) mice) in comparison to wild-type mice. The in vitro induction of the AP by adherent mAb to collagen II was intact using sera from MBL(-/-)/FCN A(-/-) mice. Furthermore, sera from MBL(-/-)/FCN A(-/-) mice lacked pro-Df and possessed only mature Df. Gel filtration of sera from MBL(-/-)/FCN A(-/-) mice showed the presence of MASP-1 protein in fractions containing proteins smaller than the migration of MBL-A and MBL-C in sera from C4(-/-) mice, suggesting possible binding of MASP-1 to an unknown protein. Lastly, we show that FCN-B was present in the sera of MBL(-/-)/FCN A(-/-) mice and that it was bound to MASP-1. We conclude that MASP-1 does not require binding to MBL-A, MBL-C, or FCN-A to activate the AP. MASP-1 may cleave pro-Df into mature Df through binding to FCN-B or to an unknown protein, or may function as an unbound soluble protein.

Initiation of the alternative pathway of murine complement by immune complexes is dependent on N-glycans in IgG antibodies.

OBJECTIVE: Collagen antibody-induced arthritis in mice exhibits a requirement for amplification by the alternative pathway of complement. Although the alternative pathway is activated by spontaneous hydrolysis, it is not known whether this pathway can also be initiated directly by IgG antibodies in immune complexes (ICs). IgG lacking terminal sialic acid and galactose (G0 IgG) can activate the lectin pathway of complement, but it is not known if G0 IgG can also activate the classical or alternative pathway. The purpose of this study was to examine the mechanism of initiation of the alternative pathway of complement by ICs. METHODS: We used adherent ICs containing bovine type II collagen (CII) and 4 monoclonal antibodies (mAb) to CII (adCII-IC). C3 activation was measured in the presence of sera from wild-type C57BL/6 mice or from mice deficient in informative complement components. The mAb were used intact or after enzyme digestion to create G0 IgG or to completely remove the N-glycan. RESULTS: Both the classical and alternative pathways, but not the lectin pathway, mediated C3 activation induced by the adCII-IC. Mannose inhibited the alternative pathway-mediated C3 activation but had no effect on the classical pathway, and N-glycans in IgG were required by the alternative pathway but not the classical pathway. Both the classical and alternative pathways mediated C3 activation induced by G0 IgG. Mannose-binding lectin bound avidly to G0 IgG, but lectin pathway-mediated C3 activation was only slightly increased by G0 IgG. CONCLUSION: The alternative pathway of complement is capable of initiating C3 activation induced by adCII-IC and requires the presence of N-glycans on the IgG. G0 IgG activates both the classical and alternative pathways more strongly than the lectin pathway.

Anti-dsDNA antibody assay: high specificity and sensitivity with a filtration radioassay in comparison to low specificity with the standard ELISA.

OBJECTIVE: To evaluate whether a new fluid-phase filtration radioassay possesses both high sensitivity and specificity compared with the currently used ELISA and Farr assays. METHODS: Sequential sera (25 samples) from 9 patients with systemic lupus erythematosus (SLE), sera from 20 patients with SLE possessing anti-dsDNA antibodies by the Crithidia assay, 75 patients with rheumatoid arthritis possessing rheumatoid factors, 50 healthy control subjects, 767 from patients with type 1 diabetes, and a commercial standard serum sample were tested for anti-dsDNA antibodies with the 3 different assays. RESULTS: Of serial dilutions of a standard anti-dsDNA antibody sample, only the highest positive sample (50 IU/ml) in the ELISA and the highest 2 positive samples (50 and 25 IU/ml) in the Farr assay were above the normal range. In contrast, all dilutions (to 2.5 IU/ml) of the standard anti-dsDNA antibody sample were above the normal range in the filtration radioassay. Using the values of 50 healthy control subjects in each assay to define the normal range, all 25 sequential sera from 9 patients with SLE were positive. In addition, 20/20 of the SLE individual sera, 2/75 (2.7%) of the RA sera, and 12/767 (1.6%) of the diabetes sera were positive (signal above normal range) in the filtration radioassay. The SLE sera were further examined in 2 additional assays, ELISA and Farr assay, and both assays were less sensitive and specific compared with the filtration radioassay. CONCLUSION: The fluid-phase filtration radioassay demonstrated high sensitivity and specificity for the detection of anti-dsDNA antibodies in SLE, with the standard ELISA exhibiting lower specificity. We suggest that testing for anti-dsDNA antibodies can be improved using a fluid-phase filtration radioassay in comparison to commercial assays.

Pathogenic complement activation in collagen antibody-induced arthritis in mice requires amplification by the alternative pathway.

Immune complex-induced inflammation can be mediated by the classical pathway of complement. However, using mice genetically deficient in factor B or C4, we have shown that the collagen Ab-induced model of arthritis requires the alternative pathway of complement and is not dependent on the classical pathway. We now demonstrate that collagen Ab-induced arthritis is not altered in mice genetically deficient in either C1q or mannose-binding lectins A and C, or in both C1q and mannose-binding lectins. These in vivo results prove the ability of the alternative pathway to carry out pathologic complement activation in the combined absence of intact classical and lectin pathways. C3 activation was also examined in vitro by adherent collagen-anti-collagen immune complexes using sera from normal or complement-deficient mice. These results confirm the ability of the alternative pathway to mediate immune complex-induced C3 activation when C4 or C1q, or both C1q and mannose-binding lectins, are absent. However, when all three activation pathways of complement are intact, initiation by immune complexes occurs primarily by the classical pathway. These results indicate that the alternative pathway amplification loop, with its ability to greatly enhance C3 activation, is necessary to mediate inflammatory arthritis induced by adherent immune complexes.

Rheumatoid factor seropositivity is inversely associated with oral contraceptive use in women without rheumatoid arthritis.

OBJECTIVES: To examine whether oral contraceptive use is associated with the presence of serum rheumatoid factor in women of reproductive age without rheumatoid arthritis. METHODS: 304 women selected from parents of children who were at increased risk of developing type 1 diabetes were studied, because they were enriched with the human leucocyte antigen-DR4 allele, a susceptibility marker for both type 1 diabetes and rheumatoid arthritis. Participants visited a clinic where blood was drawn for rheumatoid factor testing, and exposure data were collected via questionnaires. A medical history and joint examination were performed to rule out rheumatoid arthritis. Participants and examiners were unaware of the participants' rheumatoid factor status at the time of examination and questionnaire. RESULTS: Use of oral contraceptives at any time was inversely associated with rheumatoid factor positivity (adjusted odds ratio (OR) 0.2, 95% confidence interval (CI) 0.07 to 0.52) independent of age, education and smoking. Smoking > or = 20 pack-years was also associated with rheumatoid factor positivity (adjusted OR 56.38, 95% CI 4.31 to 736.98) compared with never smoking. Smoking 1-19 pack-years was not associated with a positive rheumatoid factor. CONCLUSIONS: Our results suggest that oral contraceptive use, and possibly cigarette smoking, act early in the development of the immune dysregulation that occurs in rheumatoid arthritis.