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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.

Objective: Anti-cyclic citrullinated peptide (anti-CCP) antibodies have been detected in patients with juvenile rheumatoid arthritis (JRA), particularly in those with polyarticular, rheumatoid factor (RF)-positive JRA. Our objectives were to determine whether anti-CCP antibodies are associated with HLA-DR4 in children with polyarticular JRA, whether anti-CCP antibodies are associated with clinical features of disease, and whether affected sibling pairs (ASPs) with JRA are concordant for this antibody., Methods: Stored serum samples obtained from 230 HLA-typed patients with JRA (77 with polyarticular-onset disease and 153 with pauciarticular- or systemic-onset disease), 100 JRA ASPs, and 688 healthy children were tested for anti-CCP antibodies and RF., Results: Thirteen percent of the patients with polyarticular-onset JRA and 2% of the other JRA patients exhibited anti-CCP antibodies, compared with only 0.6% of the controls. Fifty-seven percent of RF-positive patients with polyarticular-onset JRA had anti-CCP antibodies. HLA-DR4-positive patients with polyarticular-onset JRA were more likely to have anti-CCP antibodies than were those without HLA-DR4 alleles (odds ratio [OR] 5.20, 95% confidence interval [95% CI] 1.30-20.9). Anti-CCP antibodies were associated with polyarticular onset (OR 7.46, 95% CI 1.99-28.0), a polyarticular disease course (OR 9.78, 95% CI 1.25-76.7), and erosive disease (OR 14.3, 95% CI 3.01-67.9). Concordance rates for anti-CCP antibodies among ASPs were statistically significant., Conclusion: These data demonstrate increased anti-CCP antibody formation in HLA-DR4-positive patients with polyarticular-onset JRA. The overall prevalence of anti-CCP antibodies in JRA is low, but a substantial proportion of RF-positive patients with polyarticular-onset JRA have these antibodies. Anti-CCP antibodies in JRA are associated with polyarticular onset, a polyarticular course, and erosive disease.

Objective: To examine the association of treatment response and disease duration with changes in rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) antibody levels among patients with rheumatoid arthritis (RA)., Methods: The study sample included 66 RA patients who completed double-blind, randomized clinical protocols and for whom baseline and followup serum samples were available. Anti-CCP and RF levels were measured using commercially available assay kits. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were used to describe the association of response and disease duration with declines in antibody levels., Results: Patients had a mean +/- SD age of 49.9 +/- 12.0 years and were predominantly female (n = 51; 77%). The mean +/- SD duration between the times at which the baseline and followup serum samples were obtained was 13.7 +/- 8.6 months. Among the 64 subjects with positive antibody at baseline, 33 (52%) experienced a > or =25% reduction in the anti-CCP antibody level during the course of treatment, and 35 patients (55%) had a > or =25% reduction in RF. After adjustment for the baseline anti-CCP antibody level, only a shorter disease duration (< or =12 months) was significantly associated with a decline in the level of anti-CCP antibody (OR 3.0, 95% CI 1.0-8.8), and no association with treatment response was observed. Conversely, treatment response was the only significant determinant of a decrease in RF levels (OR 3.6, 95% CI 1.2-10.4)., Conclusion: Shorter disease duration predicts greater declines in anti-CCP antibody levels with treatment in RA. Although treatment response is a robust determinant of a decrease in RF, it does not appear to be associated with declines in the anti-CCP antibody level.

Objective: To examine the influence of superoxide on the severity of collagen-induced arthritis (CIA) in mice., Methods: CIA was induced in DBA/1J mice lacking the extracellular superoxide dismutase (EC-SOD) gene (knockout [KO]) and in normal DBA/1J mice (wild-type [WT])., Results: The clinical disease activity score was significantly higher in EC-SOD-KO mice than in WT mice between days 36 and 53, and the histologic scores for joint damage on day 53 increased 2-fold or more in the EC-SOD-KO mice. There were no significant differences between the 2 groups of mice in proliferation indices of spleen or lymph node cells in vitro after stimulation with type II collagen. Although both IgG1 and IgG2a anticollagen antibody levels increased in both groups of mice between days 21 and 53, there were no significant differences between the 2 groups. Lipopolysaccharide-stimulated spleen cells from EC-SOD-KO mice produced greater levels of tumor necrosis factor alpha (TNFalpha) over 48 hours in culture compared with cells from WT mice. Increased steady-state levels of messenger RNA (mRNA) for interferon-gamma (IFNgamma), TNFalpha, and interleukin-1beta (IL-1beta), and lower levels of IL-1 receptor antagonist (IL-1Ra) mRNA were present in the joints of the EC-SOD-KO mice compared with the WT mice., Conclusion: The absence of EC-SOD leads to more severe CIA, which may be accompanied by enhanced production of the proinflammatory cytokines IFNgamma, TNFalpha, and IL-1beta, and decreased production of the antiinflammatory cytokine IL-1Ra in the joints.

Objective: To determine the mechanisms of amelioration of collagen-induced arthritis (CIA) in DBA/1J mice by inhibition of complement activation., Methods: Mice received 2 intradermal injections of bovine type II collagen (CII), on days 0 and 21. From day 21 (immediately after the second injection of CII) through day 35, mice received intraperitoneal injections of either phosphate buffered saline (PBS), a monoclonal mouse antibody to murine C5 (anti-C5 antibody), or the C3 convertase inhibitor Crry-Ig., Results: On days 30 and 32, the clinical disease activity score was lower in mice treated with anti-C5 antibody than in those treated with Crry-Ig. Histopathologic evidence of joint damage was 75% lower in the mice treated with anti-C5 antibody than in those treated with either PBS or Crry-Ig. Spleen cells from mice receiving either form of complement inhibition exhibited decreased CII-stimulated proliferation, whereas increased proliferative responses were exhibited by lymph node cells from mice treated with Crry-Ig. Treatment with anti-C5 antibody decreased production of IgG1 anticollagen antibody, while production of IgG2a antibody was inhibited by both complement inhibitory treatments. CII-stimulated spleen cells from anti-C5-treated mice produced lower levels of tumor necrosis factor alpha (TNFalpha) and interleukin-10 (IL-10) compared with those from mice treated with Crry-Ig. Lower steady-state messenger RNA (mRNA) levels for TNFalpha, interferon-gamma (IFNgamma), IL-18, and IL-6 were observed in the joints of anti-C5-treated mice, and for IFNgamma and IL-6 in mice receiving Crry-Ig, all in comparison with PBS-treated mice. However, mRNA levels for IL-1beta and TNFalpha were lower in the joints after treatment with anti-C5 compared with Crry-Ig., Conclusion: These results indicate that inhibition of complement in CIA leads to decreased production of IgG2a antibody and suppressed CII-induced spleen cell proliferation. The greater inhibitory effects on CIA of anti-C5 antibody in comparison with Crry-Ig may be attributable primarily to decreased levels of IL-1beta and TNFalpha mRNA in the joints.

This review has summarized the physiology of some cytokine pathways in RA, emphasizing the redundant and synergistic nature of this network. However, it is important to understand that this system is self-regulating through the action of anti-inflammatory cytokines, opposing cytokines, cytokine receptor antagonists, and possibly naturally occurring antibodies to cytokines (Figure 1). Disease results when an imbalance in the cytokine network develops, either from excess production of pro-inflammatory cytokines or from inadequate presence of natural anti-inflammatory mechanisms. The current therapeutic approaches to RA that are aimed at restoring this balance include the use of monoclonal antibodies to TNFalpha, soluble TNFalpha receptors, and IL-1Ra. Other therapeutic agents that interfere with the cytokine network are in various stages of preclinical and clinical evaluation.

Objective: To examine the patterns of production of interleukin-1 receptor antagonist (IL-1Ra) isoforms and of IL-1beta during arthritis in vivo., Methods: Arthritis was induced in DBA/1 mice by immunization with type II collagen, and the production of IL-1Ra isoforms was examined in whole joints and in dissected synovial tissues by reverse transcription-polymerase chain reaction (RT-PCR), RNase protection assay, Western blotting, immunostaining, and in situ hybridization. Production of IL-1beta also was examined using similar approaches., Results: Production of IL-1Ra increased in the joints during collagen-induced arthritis (CIA). By RT-PCR, secreted IL-1Ra messenger RNA (mRNA) was detected in normal joints, whereas intracellular IL-1Ra type I (icIL-1Ra1) mRNA was only produced in inflamed joints. Western blot studies showed that icIL-1Ra1 protein levels increased in the joints during the course of CIA and that icIL-1Ra3 protein was also present in low amounts. RNase protection assays showed that the IL-1beta:IL-1Ra mRNA ratio was increased in inflamed joints through day 14 of arthritis, whereas a reverse pattern was present at later time points (from day 20 to day 60). Consistent with this finding, immunohistochemistry and in situ hybridization studies confirmed that icIL-1Ra1 was only present in inflamed joints. The histologic evaluation of CIA during the course of the disease indicated a resolution of acute inflammation, since icIL-1Ra1 production increased and the ratio of IL-1beta to total IL-1Ra decreased., Conclusion: Production of IL-1Ra isoforms, particularly icIL-1Ra1, is stimulated in inflamed joints during CIA in mice. The combination of decreased production of IL-1beta and elevated levels of icIL-1Ra1 during the course of CIA was associated with a reduction in inflammatory activity. These results suggest that icIL-1Ra1 may play a role in the resolution of murine CIA.

This review has summarized information published over the last 5 years on the presence and pathophysiologic role of IL-1 and TNF alpha in RA. The evidence to date shows that 5 of 6 criteria for identifying mediators of tissue damage in human autoimmune diseases are satisfied (Table 1). The last criterion, prevention of clinical progression in patients with RA, is currently being evaluated. Many new therapeutic approaches are currently being developed, including the use of soluble receptors to IL-1 or TNF, monoclonal antibodies to TNF alpha, a specific IL-1 receptor antagonist, and gene therapy with the latter molecule. It should be emphasized that both IL-1 and TNF alpha play important roles in normal host defense; the possible complications of blocking their production or effects need to be carefully evaluated in long-term studies. A recent review has emphasized that although IL-1 and TNF alpha have many overlapping biologic properties, each may exhibit distinct effects in joint disease (99). Anti-TNF treatment may be primarily antiinflammatory but blocking IL-1 may be more effective in preventing cartilage destruction (100). The possibility exists that simultaneous inhibition of TNF alpha and IL-1 may be more therapeutically efficacious than blockade of either agent alone, as was recently demonstrated with IL-1ra and soluble TNF receptors in bacterial cell wall-induced arthritis in rats (101). The next level of clinical studies in rheumatoid arthritis should include the use of two biologic response modifiers together, or one agent combined with a more traditional form of therapy.

Objective: To quantify interleukin-1 receptor antagonist (IL-1ra) and IL-1 production and gene expression by rheumatoid arthritis (RA) synovial tissue (ST) cells., Methods: IL-1 alpha, IL-1 beta, and IL-1ra protein levels were measured by enzyme-linked immunosorbent assay in fresh and cultured ST cells, purified synovial macrophages, and fibroblast-like synoviocytes (FLS). The relative expression of the secreted form of IL-1ra (sIL-1ra) and the alternatively spliced intracellular form (icIL-1ra) was determined by reverse transcription polymerase chain reaction (RT-PCR) techniques., Results: IL-1 alpha, IL-1 beta, and IL-1ra were present in fresh and cultured ST cell samples of synovium from RA and osteoarthritis patients. IL-1ra:IL-1 ratios ranged from 1.2 to 3.6, which is below the 10-100-fold excess of IL-1ra needed to inhibit IL-1 bioactivity. Isolated CD14+ synovial macrophages secreted IL-1ra, but the amount was much less than that of alveolar or in vitro-derived macrophages. Cultured FLS contained intracellular IL-1ra but secreted little IL-1ra into the culture supernatants. RT-PCR showed that icIL-1ra mRNA was more abundant than sIL-1ra mRNA in FLS and unfractionated ST cells., Conclusion: IL-1ra production by RA ST cells is deficient relative to total production of IL-1.

Objective: To measure synovial fluid (SF) levels of interleukin-1 receptor antagonist (IL-1ra) and to determine the capacity of SF neutrophils (PMN) to synthesize and release IL-1ra., Methods: A sensitive and specific enzyme-linked immunosorbent assay was used to measure SF IL-1ra protein concentrations and IL-1ra production by isolated SF PMN:, Results: SF IL-1ra levels were elevated in 13 of 16 samples from patients with rheumatoid arthritis (RA) (mean 17.1 ng/ml), in 6 of 18 samples from patients with infectious or inflammatory, non-RA arthropathies (mean 10.6 ng/ml), and in none of 11 noninflammatory SF samples. SF IL-1ra levels correlated with SF PMN concentrations (r = 0.680, P < 0.00001). Isolated SF PMN contained preexisting IL-1ra protein in the absence of messenger RNA (mRNA). In addition, both lipopolysaccharide and granulocyte-macrophage colony-stimulating factor induced modest increases in IL-1ra mRNA by cultured SF-PMN., Conclusion: IL-1ra levels are increased in > 80% of RA SF samples. SF PMN produce IL-1ra, possibly contributing to the levels of IL-1ra present within the SF.

Criteria for identifying Henoch-Schönlein Purpura (HSP) and distinguishing HSP from other forms of systemic arteritis were developed by comparing the manifestations in 85 patients who had HSP with those of 722 control patients with other forms of vasculitis. By the traditional format of choosing different combinations of candidate criteria and comparing the combinations for their ability to separate HSP cases from controls, 4 criteria were identified: age less than or equal to 20 years at disease onset, palpable purpura, acute abdominal pain, and biopsy showing granulocytes in the walls of small arterioles or venules. The presence of any 2 or more of these criteria distinguish HSP from other forms of vasculitis with a sensitivity of 87.1% and a specificity of 87.7%. The criteria selected by a classification tree method were similar: palpable purpura, age less than or equal to 20 years at disease onset, biopsy showing granulocytes around arterioles or venules, and gastrointestinal bleeding. These were able to distinguish HSP from other forms of vasculitis with a sensitivity of 89.4% and a specificity of 88.1%.

Criteria for the classification of Churg-Strauss syndrome (CSS) were developed by comparing 20 patients who had this diagnosis with 787 control patients with other forms of vasculitis. For the traditional format classification, 6 criteria were selected: asthma, eosinophilia greater than 10% on differential white blood cell count, mononeuropathy (including multiplex) or polyneuropathy, non-fixed pulmonary infiltrates on roentgenography, paranasal sinus abnormality, and biopsy containing a blood vessel with extravascular eosinophils. The presence of 4 or more of these 6 criteria yielded a sensitivity of 85% and a specificity of 99.7%. A classification tree was also constructed with 3 selected criteria: asthma, eosinophilia greater than 10% on differential white blood cell count, and history of documented allergy other than asthma or drug sensitivity. If a subject has eosinophilia and a documented history of either asthma or allergy, then that subject is classified as having CSS. For the tree classification, the sensitivity was 95% and the specificity was 99.2%. Advantages of the traditional format compared with the classification tree format, when applied to patients with systemic vasculitis, and their comparison with earlier work on CSS are discussed.

Criteria for the classification of Wegener's granulomatosis (WG) were developed by comparing 85 patients who had this disease with 722 control patients with other forms of vasculitis. For the traditional format classification, 4 criteria were selected: abnormal urinary sediment (red cell casts or greater than 5 red blood cells per high power field), abnormal findings on chest radiograph (nodules, cavities, or fixed infiltrates), oral ulcers or nasal discharge, and granulomatous inflammation on biopsy. The presence of 2 or more of these 4 criteria was associated with a sensitivity of 88.2% and a specificity of 92.0%. A classification tree was also constructed with 5 criteria being selected. These criteria were the same as for the traditional format, but included hemoptysis. The classification tree was associated with a sensitivity of 87.1% and a specificity of 93.6%. We describe criteria which distinguish patients with WG from patients with other forms of vasculitis with a high level of sensitivity and specificity. This distinction is important because WG requires cyclophosphamide therapy, whereas many other forms of vasculitis can be treated with corticosteroids alone.

Criteria for the classification of Takayasu arteritis were developed by comparing 63 patients who had this disease with 744 control patients with other forms of vasculitis. Six criteria were selected for the traditional format classification: onset at age less than or equal to 40 years, claudication of an extremity, decreased brachial artery pulse, greater than 10 mm Hg difference in systolic blood pressure between arms, a bruit over the subclavian arteries or the aorta, and arteriographic evidence of narrowing or occlusion of the entire aorta, its primary branches, or large arteries in the proximal upper or lower extremities. The presence of 3 or more of these 6 criteria demonstrated a sensitivity of 90.5% and a specificity of 97.8%. A classification tree also was constructed with 5 of these 6 criteria, omitting claudication of an extremity. The classification tree demonstrated a sensitivity of 92.1% and a specificity of 97.0%.

Criteria for the classification of hypersensitivity vasculitis were developed by comparing 93 patients who had this disease with 714 control patients with other forms of vasculitis. For the traditional format classification, 5 criteria were selected: age greater than 16 at disease onset, history of taking a medication at onset that may have been a precipitating factor, the presence of palpable purpura, the presence of maculopapular rash, and a biopsy demonstrating granulocytes around an arteriole or venule. The presence of 3 or more of these 5 criteria was associated with a sensitivity of 71.0% and a specificity of 83.9%. A classification tree was also constructed. The criteria appearing in the tree structure were the same as for the traditional format, except there were 2 pathology criteria: one required the presence of granulocytes in the wall of an arteriole or venule, and the other required the presence of eosinophils in the inflammatory exudate. The classification tree was associated with a sensitivity of 78.5% and a specificity of 78.7%.

Criteria for the classification of polyarteritis nodosa were developed by comparing 118 patients who had this disease with 689 control patients who had other forms of vasculitis. For the traditional format classification, 10 criteria were selected: weight loss greater than or equal to 4 kg, livedo reticularis, testicular pain or tenderness, myalgias, mononeuropathy or polyneuropathy, diastolic blood pressure greater than 90 mm Hg, elevated blood urea nitrogen or serum creatinine levels, presence of hepatitis B reactants in serum, arteriographic abnormality, and presence of granulocyte or mixed leukocyte infiltrate in an arterial wall on biopsy. The presence of 3 or more of these 10 criteria was associated with a sensitivity of 82.2% and specificity of 86.6%. A classification tree was also constructed, with 6 criteria being selected. Three of these, angiographic abnormality, biopsy-proven granulocyte or mixed leukocyte infiltrate in arterial wall, and neuropathy, were criteria used in the traditional format. The other 3 criteria used in the tree format included the patient's sex, weight loss greater than 6.5 kg, and elevated serum aspartate aminotransferase or alanine aminotransferase levels above the range of normal. The classification tree yielded a sensitivity of 87.3% and a specificity of 89.3%.

Criteria for the classification of giant cell (temporal) arteritis were developed by comparing 214 patients who had this disease with 593 patients with other forms of vasculitis. For the traditional format classification, 5 criteria were selected: age greater than or equal to 50 years at disease onset, new onset of localized headache, temporal artery tenderness or decreased temporal artery pulse, elevated erythrocyte sedimentation rate (Westergren) greater than or equal to 50 mm/hour, and biopsy sample including an artery, showing necrotizing arteritis, characterized by a predominance of mononuclear cell infiltrates or a granulomatous process with multinucleated giant cells. The presence of 3 or more of these 5 criteria was associated with a sensitivity of 93.5% and a specificity of 91.2%. A classification tree was also constructed using 6 criteria. These criteria were the same as for the traditional format, except that elevated erythrocyte sedimentation rate was excluded, and 2 other variables were included: scalp tenderness and claudication of the jaw or tongue or on deglutition. The classification tree was associated with a sensitivity of 95.3% and specificity of 90.7%.

The American College of Rheumatology Subcommittee on Classification of Vasculitis of the Diagnostic and Therapeutic Criteria Committee developed classification criteria for 7 forms of vasculitis: polyarteritis nodosa, Churg-Strauss syndrome, Wegener's granulomatosis, hypersensitivity vasculitis, Henoch-Schönlein purpura, giant cell (temporal) arteritis, and Takayasu arteritis. The data collection methods, quality control, and analytic procedures used to derive the classification rules are discussed herein.

This review has summarized some of the evidence suggesting that cytokines may play an important role in mediating pathophysiologic events in RA. However, these proteins are capable of mediating both stimulatory (agonist) and inhibitory (antagonist) effects in the rheumatoid synovium. GM-CSF, IL-1, TNF alpha, and PDGF are all produced in the rheumatoid synovium and may function to induce inflammation, enzyme release, fibroblast proliferation, and tissue destruction. Local release of IL-6 may alter the effects of IL-1 and TNF alpha, as well as induce Ig production and hepatic synthesis of acute-phase proteins. However, specific inhibitors of IL-1 and TNF alpha exist, which, if also released into the synovium, may antagonize the proinflammatory effects of these cytokines. In addition, IL-1 may have antiinflammatory effects, such as the induction of the synthesis of collagen and enzyme inhibitors by chondrocytes and synovial fibroblasts. Stimulation of these latter cells by TGF beta also may result in decreased matrix degradation and increased formation of scar tissue. The developing scenario is one of cell-cell interactions that are influenced in positive and negative manners by the local release of various mediators. A further understanding of cytokines and cytokine inhibitors in the rheumatoid synovium may lead to the development of more specific and effective therapeutic agents.

The mechanism of granulocyte depletion in a patient with systemic lupus erythematosus and neutropenia was investigated. Neutrophil kinetic studies showed a shortened intravascular survival (t1/2 of 1.6 hours) in the face of an increased marrow neutrophil pool. IgG bound to the patient's neutrophils, measured by the Fab antiF(ab')2 assay, was nearly three times normal. The IgG neutrophil-binding activity of the patient's serum was elevated in serial samples obtained over two years. In addition, his serum was able to opsonize normal neutrophils for ingestion by other neutrophils as detected by 14C-1-glucose oxidation. Enhanced IgG PMN-binding activity was observed with sucrose density gradient fractions of the patient's serum containing either large complexes (19S or greater in size), intermediate complexes (between 7S and 19S), or monomeric IgG. Only the momomeric IgG fraction from the patient's serum, however, opsonized normal neutrophils for ingestion by other neutrophils. These results support the hypothesis that anti-cell antibodies were responsible for the neutropenia in this patient by opsonizing neutrophils for ingestion by other phagocytic cells.

Cold-reactive antibodies cytotoxic for peripheral monocytes from more than half of normal donors were found in the sera of 2 of 25 patients with systemic lupus erythematosus (SLE) and 1 of 26 with rheumatoid arthritis (RA), and they were absent in 25 normal sera. In contrast, lymphocytotoxic activity for T or B lymphocytes was found in over half of the lupus sera. The antibodies to monocytes were primarily IgM and exhibited varying specificities. Some of the antibodies were directed against antigenic determinants common to monocytes, T and B cells, or against determinants shared between monocytes and one lymphocyte type. One serum possessed a high titer of antibodies that were specific for monocytes. The clinical significance of antimonocyte antibodies remains to be established.