Properties and reactivity of immune complexes in rheumatoid synovial fluid

Summary Fractionation of immune complexes (IC) from rheumatoid synovial fluid revealed the presence of three different fractions of IC. The largest molecular weight form, fraction I (above 1000 Kdaltons) was predominately composed of IgG and IgM and contained both IgM-RF and IgG-RF. The other IC, fraction II (480 Kdaltons) and fraction III (330 Kdaltons), contained predominately IgG with some IgA and only significant amounts of IgG-RF. All three fractions of IC can bind Clq and stimulate human monocyte prostaglandin E (PGE) production. Fraction I IC bound Clq most readily while fraction III IC were the most effective stimulators of monocyte PGE production. IC stimulation of monocyte PGE production was inhibited by staphylococcus protein A suggesting mediation via activation of Fc receptors. It remains to be determined whether this IC reactivity has any pathologic significance.     

The frequency of circulating immune complexes in rheumatoid arthritis and systemic lupus erythematosus

Summary Sera from patients with rheumatoid arthritis and systemic lupus erythematosus have been examined for the presence of complement-fixing immune complexes using three assays, (a) a fluid phase Clq binding assay, (b) a solid phase Clq binding assay and (c) the Raji cell assay.By simultaneously screening all the sera within each disease group we established that circulating immune complexes frequently occur but that there is a discordance between the assays, even between the two assays involving binding to Clq. Distinct clinical profiles emerged with the fluid phase Clq binding assay being most frequently positive in sera from patients with extra-articular rheumatoid arthritis. The solid phase Clq binding assay and the Raji cell assay were more frequently positive in sera from patients with systemic lupus erythematosus. The prevalence of circulating immune complexes and the comparative performance of the three assays in each disease is examined in detail.     

Relation of clinical activity of rheumatoid arthritis to immune complexes,complement components and anti-immunoglobulins

Summary Circulating immune complexes by fluid phase Clq binding assay, complement components and anti-immunoglobulin levels were studied in sera of 35 patients with rheumatoid arthritis (RA). In 23 of the 35 sera (65.7%), circulating immune complexes were positive, and the mean±SD of Clq binding activity (ClqBA), 44.5±19.4%, was significantly high compared to that of healthy persons, 17.4±8.2%. Antigenic determination of complement components revealed that Clq, C3, C5, C9, factor B and Cl esterase inhibitor (CIINH) were significantly high in sera of RA, but C4 and properdin were not. The disease activity correlated with ClqBA, IgG- and IgM-anti-immunoglobulins, C9 and serum IgG. On the other hand, ClqBA correlated with both IgG- and IgM-anti-immunoglobulin levels but not with complement components.     

Immunoglobulin inclusions in rheumatoid arthritis polymorphonuclear cells: Lack of correlation with circulating immune complexes

Summary A discriminating direct immunofluorescent test has been used to identify immunoglobulin inclusions in polymorphonuclear leucocytes (PMNs) isolated from the blood of patients with rheumatoid arthritis. These inclusions are thought to represent phagocytosed immune complexes, since normal PMNs incubated in RA sera known to contain raised levels of immune complexes developed similar immunoglobulin inclusions. Inclusions did not develop in normal PMNs incubated in normal serum.No correlation was found between the percentage of either RA blood PMNs with immunoglobulin inclusions or normal PMNs developing inclusions after incubation in RA sera, and levels of immune complexes in the corresponding sera.Using heat-aggregated IgG as a laboratory model of immune complexes, a simple relationship has been demonstrated between the uptake of IgG aggregates by normal PMNs and the concentrations of IgG aggregates in the test solutions over a concentration range of 12.5–200 g ml^–1.These results indicate that the C1q- PEG test gives no measure of the actual amounts of immune complexes available in serum for phagocytosis.     

Autoreactivity to mouse C1q in a murine model of SLE

A large proportion of systemic lupus erythematosus (SLE) patients develop glomerulonephritis, coincident with the appearance of autoantibodies to C1q, the Fcrecognizing collagen-like subcomponent of the first component of complement, C1. The MRL/lpr/lpr mouse is an established model for SLE, developing both antinuclear and anti-type II collagen autoantibodies, and rheumatoid factors(s), exhibiting reduced complement levels and later on developing glomerulonephritis and often arthritis. We report here an age-dependent decrease in serum C1q levels coincident with the development of IgG2b autoantibodies reactive with mouse C1q in MRL/lpr/lpr mice. Unlike IgG2b, although high levels of IgM, IgG1 and IgG2a are present in these mice, few, if any, antibodies of these sub-classes reactive with mouse C1q were observed in this study. This is the first report of autoantibodies against autologous C1q in an animal model, and the results should facilitate in clarification of the roles of C1q and autoantibodies reactive with C1q in SLE, as well as their potential connection with glomerulonephritis.     

Superoxide generation by snovial fluid neutrophils enhanced by immune complexes and suppressed by rheumatoid factor in synovial fluid

Summary Synovial fluid (SF) from patients with rheumatoid arthritis (RA) enhanced superoxide generation by neutrophils isolated from RA SF, in contrast to SF from patients with osteoarthritis. These superoxide generation-enhancing substances may be intermediate-sized immune complexes and a complement C5-derived fragment. Rheumatoid factor (RF) isolated from RA SF suppressed superoxide generation-enhancing activity of aggregated IgG. Therefore, biologically active RF may block the interaction of the immune complexes with neutrophils accumulating in RA SF, and protect the joint tissue from the effects of oxygen radicals or proteases.     

Interleukin-1-like activities in synovial fluids of patients with rheumatoid arthritis and traumatic synovitis

Summary Interleukin-1 (Il-1)-like activity in biological fluids was measured by their ability to rectify the Il-1-dependent lymphokine production of highly purified T lymphocytes to a recall antigen. Il-1-like activity was found in 9 of 11 synovial fluid (SF) specimens from patients with rheumatoid arthritis (RA) but only in 2 of 11 paired RA sera. In traumatic synovitis, low Il-1-like activity was recorded in 5 of 9 SF specimens, and a similar low activity was found in sera of 4 of these patients. The Il-1-like activity was partly absorbed by an anti-Il-1 antibody. The presence of Il-1 in the SF of patients with RA suggests in vivo activation of monocytes/macrophages.     

Separation and characterization of complement-fixing immune complexes in juvenile rheumatoid arthritis patients

Summary Immune complexes (IC) in sera from patients with juvenile rheumatoid arthritis (JRA) were isolated by the use of immunoabsorbent columns. Sera from 14 JRA patients (four seropositive for 19S IgM RF and 10 seronegative, but nine having hidden 19S IgM RF) were analyzed by the anti-human Clq (HClq) and anti-human C3 (HC3) columns. The columns were sequentially eluted with veronal buffer, 0.02 M EDTA, 0.5 M NaCl, and 1 M propionic acid. By the HClq column, IgM RF were detected in at least one of the separated IC fractions of 13 of 14 patients and IgG RF in three patients. By the HC3 column, only five patients demonstrated IgM RF and only one IgG RF in the eluted fractions. On sucrose density gradient analysis (SDGA), all IC were demonstrated in the peaks 19S. 19S IgM RF were demonstrated by ELISA in all 14 patients, but IgG RF in only three. These studies demonstrate that complement-fixing 19S IgM RF, IgG, and IgG RF containing IC can be detected in the serum of JRA patients.     

Isotype-specific immune response to a single hepatitis C virus core epitope defined by a human monoclonal antibody: diagnostic value and correlation to PCR

Summary In this study we tested the seroreactivity of 223 selected anti-HCV-reactive blood donors to the human B-cell epitope N-VYLLPR-C (C_34–39) of the hepatitis C virus core antigen. The epitope was recently identified and characterized by the human monoclonal IgG antibody Ul/F10 [23] and is located within the amino acid residues 34–39 of the aminoterminal core region. The blood donor sera were selected from anti-HCV ELISA (Ortho, 2nd generation)-reactive samples. Sixty-seven of these sera were further reactive in RIBA (Ortho, 2nd generation). According to their RIBA pattern, these samples were divided into four groups. Samples in the first group (n=18) reacted to all four recombinant HCV antigens. The samples of the second (n=9) and third group (n=8) reacted to c223/c33c and c22-3/cl00-3, respectively. Sera from group 4 (n=32) showed a RIBA indeterminate pattern with reactivity only to c22-3. All 223 samples were analyzed for anti-C_34–39 antibodies by ELISA, and the 67 RIBA-reactive samples were additionally tested for the presence of HCV RNA by RT/PCR. In groups 1 and 2, over 80% of the samples showed anti-C_34–39 reactivity which was restricted to the IgG₁ isotype. In contrast, in groups 3 and 4, antibodies to the epitope C_34–39 were detected in less than 10% of the samples. Interestingly, the anti-C_34–39 response correlates with the presence of HCV RNA; 95.5% of the samples had coincident results in all subgroups. None of the RIBA-negative sera showed a specific seroreaction to the C_34–39 peptide.     

An inhibitor of complement-mediated prevention of immune precipitation in rheumatoid arthritis — relationship to disease activity and systemic manifestations

Summary In normal serum complement prevents precipitation of antigen-antibody complexes (PIP). However rheumatoid arthritis (RA) serum contains an inhibitor of this complement-mediated function. We have undertaken two prospective studies in order to look for any relationship between the presence and levels of inhibitory activity in sera and synovial fluids (SF) of patients with RA and disease activity (study A), and the presence of systemic manifestations (nodules and vasculitis) of RA (study B). In study A, levels of inhibitory activity were highest in the sera and synovial fluids of patients with seropositive RA. However there was no correlation between the inhibitory levels and indices of generalised disease activity (articular index, erythrocyte sedimentation rate (ESR), haemoglobin, white cell and platelet counts). Local joint tenderness score correlated weakly with the inhibitory level in SF (P<0.05). There was no correlation, however, with either the SF protein concentration or white cell count. In study B, PIP was shown to be lower in patients with the systemic manifestations of RA than in those with purely articular manifestations. PIP was particularly low in those patients with vasculitis compared to those with subcutaneous nodules. Serum levels of inhibitory activity were highest in patients with vasculitis and lowest in those with articular disease only, whereas patients with nodules had intermediate levels. Our conclusion is that inhibition of immune precipitation is not associated with disease activity, but is associated with the extra-articular manifestations of RA. The inhibitory factor may play a role in the pathogenesis of RA.     

Evaluation of plasma C3d and immune complex determinations in the assessment of disease activity of patients with rheumatoid arthritis,systemic lupus erythematosus,and spondylitis ancylopoetica

Summary Patients with rheumatoid arthritis, systemic lupus erythematosus, and spondylitis ancylopoetica were examined, along with healthy controls, for C3d plasma levels, circulating immune complexes, C3 serum levels, and CRP. Immune complexes were determined using a C1q binding assay, a 2.75% PEG precipitation technique, including the analysis of IgG and C3, and a new laser nephelometric latex test. C3d plasma levels were significantly (P<1%) elevated in all groups of patients as compared to controls. With regard to the demonstration of circulating immune complexes, the PEG precipitation method discriminated best between patients and the control population. It was not possible to differentiate between the different disease entities with neither C3d serum levels or immune complexes. Concerning the assessment of disease activity, none of the evaluated parameters alone appears to be of clinical relevance. The individual application of more than one immune complex assay in combination with the measurement of C3d serum levels must be recommended if disease activity is to be assessed.     

HLA DRB1* alleles in rheumatoid nodulosis: a comparative study with rheumatoid arthritis with and without nodules

Rheumatoid nodulosis (RN) is a rare condition associating rheumatoid nodules, episodes of arthritis, cystic bone lesions and, generally, positive rheumatoid factors (RF). It is considered a benign variant of rheumatoid arthritis (RA). In this study, we determined the HLA DRB1^* alleles of our RN patients and compared the distribution of these alleles to those of 74 healthy controls and 104 RA patients with and without nodules. Four RN patients were observed. All had subcutaneous nodules and RF were negative in three patients. Of the 104 RA patients, 18 had nodules (nodRA). Systemic manifestation (including vasculitis, peripheral neuropathy or lung involvement) were found in seven of these nodRA cases (33.8%) and most had positive RF and erosive changes on X-rays. Only one RN patient had a RA-associated allele (DRB1^*0101). The frequencies of the HLA DRB1^* alleles encompassing the “rheumatoid” shared epitope were similar to those of other RA series: ^*0101, 34.6% (P=0.03 compared with controls); ^*0401, 26.9% (P<0.0001); ^*0404, 12.5% (P=0.04); ^*0405, 4.8% (P=0.8); ^*1001, 8.6% (P=0.5). Of the nodRA and seronegative RA patients, 77.7% and 53.3%, respectively, presented the shared epitope. Thus, there was a tendency to decreased expression of the RA-associated alleles in RN (25%) compared with nodRA and seronegative RA patients. This study is restricted by the small number of tested RN patients, but the results suggest that the RA-associated alleles are poorly expressed in RN. Received: 29 September 1997 / Accepted: 13 February 1998     

系统性红斑狼疮患者C1q受体和C1q抗体的表达及其与发病相关性的研究

目的 检测系统性红斑狼疮（SEE）患者外周血单个核细胞C1qRp和gC1qR的表达及与C1q抗体、补体C1q水平的关系，并进一步探讨其在SLE发病中的意义。方法 用流式细胞计数法测定58例SLE和30名正常人外周血中性粒细胞、单核细胞、淋巴细胞的C1qRp和gc1qR的表达，同时测血清C1q抗体、C1q水平．并将其与C3、c4、抗核抗体（ANA）、抗dsDNA抗体、SLEDAI积分做相关性分析。结果 SLE患者外周血中性粒细胞、单核细胞的C1qRp表达为（7．2±2-3）％和（3．4±2．1）％，均较正常人[（10．6±2．1）％和（9．0±8．7）％]降低（P〈0．05），淋巴细胞不表达C1qRp，但可表达gC1qR。C1qRp在SLE活动期表达略低于稳定期，但差异无统计学意义。SLE患者的gC1qR表达略高于正常人．gC1qR在活动期高于稳定期，但差异无统计学意义。SLE患者血清C1q抗体水平（98±41）U／ml，明显高于正常，C1q为（0．13±0．08）dE，低于正常值。外周血单个核细胞的C1qRp表达与血清C1qAb水平（Pearsonr=-0．574，P〈0．05）、双链DNA（ds—DNA）抗体滴度呈负相关，与补体C1q（Pearsonr=0．673．P〈0．01）、C3、C4水平呈正相关。gc1qR与C1qAb和补体C1q无明显相关性。结论 SLE患者外周血中性粒细胞、单个核细胞C1qR的表达缺陷，导致SLE吞噬功能受损，凋亡细胞清除障碍，诱导产生自身抗体，在SLE的免疫发病机制中起重要的作用。     

IgG and IgA antibody titers against human heat-shock protein (hsp60) in sera of rheumatoid arthritis and osteoarthritis patients

Abstract

To learn whether heat-shock proteins (HSP) are involved in the pathogenesis of rheumatoid arthritis (RA), antirecombinant human heat-shock protein 60 (hsp60) IgG and IgA in sera of RA and osteoarthritis (OA) patients were investigated. Only the anti-hsp60 IgG titer of seropositive (RF-positive) patients was found to be elevated. Although RF titers of the sera of seropositive RA patients were increased, there was no correlation between the individual anti-hsp60 IgG titer and the corresponding RF titer. In contrast, all the anti-hsp60 IgA titers of the sera of OA, seronegative RA, and seropositive RA patients were found to be elevated. Among them, only the serum IgA concentration of seropositive RA patients was increased. Thus, it was suggested that the increased anti-hsp60 IgG reflects the pathogenesis of RA and its activity. It was also suggested that the increased anti-hsp60 IgA response reflects an involvement of hsp60 in the pathogenesis of arthritides rather than the pathogenesis of RA.     

Circulating and intra-articular immune complexes in rheumatoid arthritis: a comparative study of the C1q binding and monoclonal rheumatoid factor assays.

The C1q binding assay and the nephelometric monoclonal rheumatoid factor assay were able to discriminate 79% and 57% respectively of rheumatoid arthritis (RA) patients from healthy blood donors. In addition these assays could distinguish those patients with active arthritis from those with inactive disease, and the C1q binding assay correlated significantly with other laboratory indices of the rheumatoid process, including the erythrocyte sedimentation rate, low molecular weight or 7S IgM, and the rheumatoid factor titre. High levels of C1q binding were also seen in rheumatoid vasculitis. Both assays gave higher mean values in synovial fluid compared with the corresponding serum, but it appeared from ultracentrifugal analysis and from a lack of a consistent correlation between these assays that each assay was measuring different forms of immunecomplex-like material which may be involved in the immunopathogenesis of this disease. The C1q binding assay is of some value in the laboratory assessment of rheumatoid arthritis and appears to offer greater advantages than the monoclonal rheumatoid factor assay, although the usefulness of this latter assay may be very dependent on the monoclonal rheumatoid factor used.     

Induction of a lymphotoxin-like mediator in peripheral blood and synovial fluid lymphocytes by incubation with synovial fluid from patients with rheumatoid arthritis

Summary A significantly increased spontaneous cell-mediated cytotoxicity (SCMC) has been reported in synovial fluid lymphocytes (SFL) as compared to peripheral blood lymphocytes (PBL) of patients with rheumatoid arthritis (RA) and that of normal controls [1–3]. To determine whether this increased SCMC activity is due to the production of a lymphokine and related to the production of a lymphotoxin (LT)-like mediator, PBL from normal controls and PBL and SFL from RA patients were incubated either with a human melanoma cell line (IGR 3) or with cell-free synovial fluid (SF) from RA patients. The SF and the cell-free supernatants of the different cultures were tested for LT activity by estimation of inhibition of DNA synthesis of HeLa cell monolayers and they were added to a SCMC assay system using normal PBL and IGR 3 as target.In the supernatants from cocultures of either PBL from controls or PBL and SFL from RA patients with IGR 3 cells, there was no significant difference in LT activity. An LT-like mediator was observed in the supernatants of all lymphocytes cocultured with SF, whereas SF alone and supernatants of lymphocytes alone exhibited little or no LT activity. In a control experiment, LT induction was not observed when normal lymphocytes were cultured with the serum of RA patients. Absorption of the culture supernatants with an insolubilised goat anti-human Ig did not remove LT activity. The demonstrated release of an LT-like mediator from lymphocytes incubated with SF might be one contributing mechanism to the inflammatory joint reaction in RA patients.     

The clearance of heat-damaged erythrocytes by the reticulo-endothelial system in systemic lupus erythematosus and rheumatoid arthritis

Summary Reticulo-endothelial function was assessed in 20 patients with active rheumatoid arthritis and 11 patients with systemic lupus erythematosus with regard to the clearance of heat-damaged erythrocytes (HDE). In contrast to previous reports, no correlations were found between disease activity, levels of circulating immune complexes and splenic function. There was no evidence of an obvious hypofunction in the reticulo-endothelial system of the spleen in patients with rheumatoid arthritis or systemic lupus erythematosus. Moreover, a splenic hyperfunction is suggested to be present in some patients. A method for measuring the specific uptake by the liver, spleen and the clearance rate (T 1/2) of the HDE is also described.     

IgG and IgA antibody titers against human heat-shock protein (hsp60) in sera of rheumatoid arthritis and osteoarthritis patients

 To learn whether heat-shock proteins (HSP) are involved in the pathogenesis of rheumatoid arthritis (RA), antirecombinant human heat-shock protein 60 (hsp60) IgG and IgA in sera of RA and osteoarthritis (OA) patients were investigated. Only the anti-hsp60 IgG titer of seropositive (RF-positive) patients was found to be elevated. Although RF titers of the sera of seropositive RA patients were increased, there was no correlation between the individual anti-hsp60 IgG titer and the corresponding RF titer. In contrast, all the anti-hsp60 IgA titers of the sera of OA, seronegative RA, and seropositive RA patients were found to be elevated. Among them, only the serum IgA concentration of seropositive RA patients was increased. Thus, it was suggested that the increased anti-hsp60 IgG reflects the pathogenesis of RA and its activity. It was also suggested that the increased anti-hsp60 IgA response reflects an involvement of hsp60 in the pathogenesis of arthritides rather than the pathogenesis of RA. Received: August 8, 2001 / Accepted: June 5, 2002 Acknowledgments This study was supported in part by a grant-in-aid from the Japanese Ministry of Education, Science, and Culture. We thank Dr. Yashima for kindly providing normal adult serum samples. We thank Drs. Nishimiya and Hiraoka for technical help. Correspondence to:S. Watanabe     

Only weak association between disease severity and HLA-DRB 1 genes in a Swiss population of rheumatoid arthritis patients

This study analyses the prognostic value of HLA-DRB 1 genes for Swiss patients with rheumatoid arthritis (RA). HLA-DRB 1 genotyping was performed in 83 patients using the polymerase chain reaction and subsequent oligonucleotide hybridisation. They were categorised according to the presence of one or two putatively relevant genes (DRB 1^*01 and/or DRB 1^*04) and retrospectively evaluated for sex, age at disease onset, seropositivity, erosive disease and extraarticular manifestations. Sixty-one patients (73%) had disease-associated alleles. Twenty-four patients showed HLA-DRB 1^*04 variants on both alleles or combined an HLA-DRB 1^*04 variant with HLA-DRB 1^*01, while 37 patients expressed only one relevant allele. Interestingly, 22 patients did not express any relevant allele. Some 52% of patients had nodular disease, 88% were seropositive, 96% had joint erosions and I I% expressed vasculitis and/or rheumatoid organ disease. A significant difference was observed only for the number of seropositive individuals, which was slightly higher in the group of patients expressing a double dose of disease-associated alleles than in patients who had no relevant alleles. Moreover, patients expressing homozygous DRB 1 alleles had a significantly earlier onset of disease than those who were heterozygous. We conclude from these findings that HLA-DRB 1 genotyping in a Swiss population of RA patients only weakly identifies clinical subsets with distinct profiles of disease manifestations and is not of strong prognostic value to determine disease severity in individual patients.     

Impaired NFKBIE gene function decreases cellular uptake of methotrexate by down-regulating SLC19A1 expression in a human rheumatoid arthritis cell line

Objective: A non-synonymous single nucleotide polymorphism (nsSNP, rs2233434, Val194Ala) in the NFKBIE (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, epsilon) gene is known to be a rheumatoid arthritis (RA) susceptibility polymorphism in the Japanese RA population and could be closely associated with nuclear factor kappaB (NF-κB) activity. Inflammation caused by RA is sometimes associated with changes in expression levels of MTX (methotrexate) pathway-related genes. It is of interest to examine whether the NFKBIE gene had any influences on the mode of MTX action.

Methods: Both knockdown of NFKBIE gene expression and overexpression of wild-type NFKBIE and Val194Ala mutation were performed. A transfected human RA synovial cell line was cultured and then gene expressions in the MTX pathway were measured. In addition, we measured the uptake and efflux of MTX derivatives under the NFKBIE knockdown condition.

Results: Knockdown of NFKBIE reduced the mRNA for SLC19A1, a main MTX membrane transporter, and the intracellular accumulations of MTX derivatives. Moreover, our experiments also confirmed that overexpression of Val194Ala mutant NFKBIE decreased the SLC19A1 mRNA when compared to that of wild-type NFKBIE.

Conclusions: We suggest that the impairment of NFKBIE gene function can reduce the uptake of MTX into cells, suggesting that the gene is an important factor for the RA outcome.