Complement and immunoglobulins in synovial fluid from synovectomized patients with rheumatoid arthritis

In order to see if the complement (C) consumption and conversion, which are typical of rheumatoid joints, continue after synovectomy, 23 knee joints in which synovectomy had been performed from 4-5 to 6-5 years previously, were studied. The mean ratio of the concentration of C3, C4, and C5 in synovial fluid to that in palsma of the same patient was significantly lower than the corresponding ratio for the total protein content. This was found both in joints with active arthritis and in joints without clinical signs of arthritis, and in both seropositive and seronegative patients. Conversion products of C3 were found in 7 of the synovial fluids. The study thus indicated that the complement alterations in synovectomized joints are very similar to those in nonsynovectomized rheumatoid joints. In one synovial fluid agarose electrophoresis showed multiple sharp bands in the gamma region. By crossed immunoelectrophoresis some bands seemed to contain IgG with one type of light chains only. In plasma of the same patients the bands were much weaker, indicating local production of oligoclonal IgG in the joint.     

Analysis of type 1 and type 2 T cells in synovial fluid and peripheral blood of patients with rheumatoid arthritis

OBJECTIVE: It has been reported that CD4+ helper T cells play an important role in the pathogenesis of rheumatoid arthritis (RA). We evaluated the presence of intracellular cytokines interleukin 4 (IL-4) and interferon-gamma (IFN-gamma) produced by CD4+ and CD8+ T cells in the synovial fluid and peripheral blood of patients with RA at the single cell level. METHODS: We used 3 color flow cytometric analysis. Synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC) were stimulated with phorbol myristate acetate (PMA) and calcium ionophore. The stimulated SFMC and PBMC were triple stained with conjugated mononuclear antibodies (Mab) against cytokines and surface antigens after fixation and permeabilization with a saponine buffer solution. The cells were analyzed for intracellular cytokines (IFN-gamma, IL-4) and surface antigens (CD3, CD4, CD8) using a flow cytometer. RESULTS: The CD4/CD8 ratio was significantly lower in SFMC than in PBMC. The positive rates of IFN-gamma producing cells among CD4+ T cells were significantly higher than those of IL-4 producing cells in both the SFMC and the PBMC of patients with active RA. In the SF of these patients, we also found CD8+ T cells that produce IL-4 alone, or both IL-4 and IFN-gamma. CONCLUSION: In the SF of patients with RA, CD4+ type 1 T cells, which may infiltrate into the synovium and cause pathogenic immune responses in the tissue, are predominant. We believe this cell type also induces migration and activation of CD8+ type 2 T cells into the active site of inflammation, which appears to downregulate the activity of CD4+ type 1 T cells, modulating the excess immune response.     

Functional analysis of rheumatoid factor-producing B cells from the synovial fluid of rheumatoid arthritis patients

OBJECTIVE: To understand the regulation of rheumatoid factor (RF) production in rheumatoid arthritis (RA), we studied IgM-RF production by B cells isolated from the synovial fluid (SF). METHODS: Highly purified SF and peripheral blood (PB) B cells were isolated by negative selection in a fluorescence-activated cell sorter (FACS) and then cultured with either L cells, CD40 ligand (CD40L)-transfected L cells, or type B synoviocytes in the presence or absence of interleukin-2 (IL-2), IL-4, or IL-10. Total IgM and IgM-RF were detected after 14 days by enzyme-linked immunosorbent assay. Enzyme-linked immunospot assays were performed to detect cells that spontaneously produced immunoglobulin. SF B cells were also phenotypically characterized by FACS analysis. RESULTS: Terminally differentiated CD20-,CD38+ synovial plasma cells (PC) present in the SF of RA patients secreted IgM-RF in the absence of a stimulus. IgM-RF production markedly increased when SF B cells were cultured in the presence of type B RA synoviocytes together with IL-10, but independently of CD40-CD40L interaction. Although CD20-,CD38+ PC could also be demonstrated in SF B cells from patients with other forms of arthritis, IgM-RF production was restricted to the SF B cell cultures of patients with seropositive RA. The frequency of IgM-RF-producing cells among IgM-producing PC in patients with seropositive RA was estimated to be as much as 50%. CONCLUSION: These data demonstrate that terminally differentiated CD20-,CD38+ IgM-RF-producing B cells are specifically present in the inflamed joints of patients with seropositive RA. There is evidence that the local environment in the rheumatoid joint favors RF production. The relatively high frequency of IgM-RF PC in the SF B cell population provides evidence of a dominant RA-specific antigen-driven response in the development of the synovial PC repertoire.     

The activating effect of synovial fluid and washings of synovial membrane on autologous lymphocytes in rheumatoid arthritis

The effect of synovial fluid and washings of synovial membrane on autologous lymphocytes from patients with rheumatoid arthritis and other diseases has been studied using a rapid method based upon the increase in intranuclear birefringence occurring in the early stages of lymphocyte activation. Retardation of polarized light indicating increased lymphocyte activation was seen in lymphocytes from patients with rheumatoid arthritis but not in lymphocytes from patients with other diseases.     

T-cell receptor V-gene usage in synovial fluid lymphocytes of patients with chronic arthritis

In this study we analyzed the usage frequencies of the TCR V-gene segments by alpha beta+ T cells present in synovial fluid of 17 patients with chronic arthritis, including rheumatoid arthritis. The results of this study, obtained from semiquantitative PCR analyses, showed that in all patients most of the TCR V alpha- and V beta-gene segments could be detected both in fresh PBMCs and in fresh SFMCs. The relative frequencies of use of these V-region genes were variable between the different patients. Although there was some skewing of increased usage frequencies of particular TCR V alpha and V beta genes among SFMC-derived TCRs when compared with PBMCs, we could not correlate such increased TCR V-gene usage with the inflammation in the joints as a disease-specific marker.     

Ferritin subunits in sera and synovial fluids from patients with rheumatoid arthritis

OBJECTIVE: To determine the proportion of glycosylated ferritin [ferritin bound to concanavalin A (Con-A)] and ferritin subunits in sera and synovial fluids (SF) from patients with rheumatoid arthritis (RA). METHODS: Ferritin concentrations were measured by a sandwich ELISA using rabbit IgG F(ab')2 anti-human ferritin antibody as a coating antibody. Proportions of glycosylated ferritin were examined using Con-A Sepharose 4B. Ferritin subunits were tested by Western blot analysis. RESULTS: Ferritin concentrations in RA SF were significantly elevated compared to those in osteoarthritis (OA) SF (p < 0.01) and those in RA sera (p < 0.01). Percentages of glycosylated ferritin in SF were low in both RA and OA (RA 11.9 +/- 10.7, n = 41; OA 6.9 +/- 11.0, n = 10). However, percentages of glycosylated ferritin in RA sera (65.9 +/- 15.0, n = 20) were significantly higher than in RA SF (p < 0.01). Western blot analysis revealed both G subunit (23 kDa) and L subunit ( 19 kDa) in RA sera, although SF ferritin was composed mostly of L subunit. CONCLUSION: Significant differences in ferritin molecule composition were observed between sera and SF from patients with RA, which suggests that in RA most SF ferritin is synthesized locally in the affected joint.     

Flow cytometric single-cell analysis of cytokine production by CD4+ T cells in synovial tissue and peripheral blood from patients with rheumatoid arthritis

BACKGROUND AND PURPOSE: To detect areas of cerebral perfusion from bypass arteries after vascular reconstruction, we administered selective intraarterial microsphere tracer into the external carotid arteries and determined (via single-photon emission computed tomography [IA-SPECT]) whether the distribution of radiotracer matched the arteriographic distribution of contrast material as shown on external carotid angiograms. METHODS: We compared the extent of regional distribution of tracer after external carotid artery injection of 20 to 40 MBq of 99mTc-HMPAO or 99mTc-ECD with that of contrast medium on the external carotid angiograms in 582 cortical regions in 12 patients with atherosclerotic occlusive disease and in 18 patients with moyamoya disease. RESULTS: Marked accumulation of tracer was found only in the expected, specific, newly developed areas of cerebral perfusion from bypass arteries. The regional distribution of tracer corresponded to that of contrast medium in 523 regions (90%) and did not correspond in 59 regions (10%). Significant overestimation of the distribution of contrast material relative to that of tracer was observed in the patients with moyamoya disease. CONCLUSION: SPECT showed slightly different distribution of tracer from that predicted by conventional angiography. IA-SPECT should enhance the analysis of newly developed areas of cerebral perfusion from the bypass arteries.     

Immunological findings in serum and synovial fluid in patients with rheumatoid arthritis (author's transl)

Sera and synovial fluid were investigated in 45 patients with rheumatoid Arthritis and 50 patients with osteoarthritis in inflammatory exacerbation (control group). The following tests were performed: IgG, IgM, IgA determinations, complement components C3, C3, C4, C3-proactivator, ceruloplasmin, electrophoresis, LDH and total acid phosphatase. 1. Serum levels of the ceruloplasmin, alpha 1, alpha 2 and gamma fractions of electrophoresis are significantly higher in patients with rheumatoid arthritis than in patients with osteoarthritis. 2. Synovial fluid: a) There is a significantly higher concentration of IgG, IgM, IgA, C3-proactivator and total acid phosphatase in the synovial fluid of patients with rheumatoid arthritis. b) C4 is significantly lower in patients with rheumatoid arthritis. c) Both groups were also compared with the help of a point system. Every patient received a plus point when the following criteria were seen: IgM greater than 150 mg/100 ml, C3 greater than 50 mg/100 ml, ceruloplasmin greater than 35 mg/100 ml, alpha 1 greater than 0.21 g%, alpha 2 greater than 0.44 g%, beta greater than 0.60 g% and gamma fraction on electrophoresis greater than 0.90 g%. Another point was added if the criteria ceruloplasmin greater than 22 mg/100 ml and C4 less than 17 mg/100 ml were simultaneously seen. With the help of this points system 48 out of the 50 osteoarthritis patients (96%) received zero points, one received 1 point and one 2 points, as opposed to the patients with rheumatoid arthritis where 35 out of 45 (78%) received one or more points. d) The differentation is not improved through additional testing of the rheumatic factors.     

Composition and function of peripheral blood stem and progenitor cell harvests from patients with severe active rheumatoid arthritis

High-dose chemotherapy with autologous stem cell rescue has been proposed as an intensive therapy for severe rheumatoid arthritis (RA). In view of previous observations of abnormal haemopoiesis in RA patients, the composition and function of peripheral blood stem cell harvests (PBSCH) was investigated. Compared with PBSCH from healthy allogeneic donors mobilized with the same dose of G-CSF (filgrastim; 10 microg/kg/d, n = 14), RA PBSCH (n = 9) contained significantly fewer mononuclear cells (375 v 569 x 10(6)/kg, P = 0.03) and CD34+ cells (2.7 v 5.8 x 10(6)/kg, P = 0.003). However, there were increased proportions of CD14+ cells (P = 0.006) and CD14+ CD15+ cells (the phenotype of previously described 'abnormal' myeloid cells, P = 0.002) in the RA PBSCH which translated into 3.5- and 7-fold increases respectively on a per CD34+ cell basis. There were no differences in T-cell activation status as judged by proportions of CD4+ and CD8+ expressing CD45RA, CD45RO, HLA-DR and CD28 (RA PBSCH, n = 7, donor PBSCH, n = 5, P = 0.2-0.7). Phytohaemagglutinin responses determined fluorocytometrically with induction of CD69 expression were reduced in CD4+ and CD8+ cells following filgrastim administration in 3/3 RA patients tested. Compared with bone marrow as a potential source of CD34+ cells, PBSCH contained 11-fold more T cells (P < 0.0005), 8-fold more B cells (P < 0.0005) and 4-fold more monocytes (P = 0.02). In short-term methylcellulose culture there were no differences in colony counts (CFU-GM, CFU-GEMM, BFU-E) per CD34+ cell from PBSCH from RA patients (n = 11) and healthy donors (n = 10). Long-term culture initiator cells were cultured successfully from cryopreserved PBSCH from RA patients (n = 9). In conclusion, PBSCH from RA patients differed significantly in composition from normal individuals, but in vitro studies support normal stem and progenitor cell function. Changes in T-cell function occur during mobilization in RA patients. This work provides reassurance for the use of PBSCH as haematological rescue and baseline data for clinical trials of graft manipulation strategies in patients with RA.     

Induction of invasive and degradative phenotype in normal synovial fibroblasts exposed to synovial fluid from patients with juvenile rheumatoid arthritis: role of mononuclear cell population

OBJECTIVE: To investigate the effect of synovial fluid (SF) from patients with juvenile rheumatoid arthritis (JRA) on proliferation and induction of degradative and invasive phenotype in normal synovial fibroblasts, and to elucidate the contribution of SF cells to this activity. METHODS: SF and/or conditioned medium (CM) from SF cells were evaluated for their ability to (1) stimulate a proliferative response, (2) induce the "activated phenotype" capable of invading cartilage matrix, and (3) promote the release of key matrix metalloproteinases (MMP) in normal synovial fibroblasts. RESULTS: Proliferation of normal synovial fibroblasts exposed to SF or CM from SF cells of patients with JRA was up to 3 times greater than untreated controls. Concomitant with induction of an activated phenotype in the treated synovial fibroblasts, the activated form exhibited up to 250% invasiveness of cartilage matrix compared to untreated synovial fibroblasts (100%), in addition to releasing increased MMP activity, not normally associated with these quiescent cells. This induction was not solely due to tumor necrosis factor-alpha, transforming growth factor-beta, interleukin 1beta (IL-1beta), and IL-6, as SF and/or CM depleted of these cytokines sustained about 40% of their invasive and inducing ability. We observed that the mononuclear cell (MNC) population that infiltrated into the joint cavity secretes this "inducing activity," which can be maintained in culture up to several weeks. CONCLUSION: Our data suggest that the cellular component of SF releases soluble factor(s) that directly or indirectly contribute to (a) proliferation of synovial fibroblasts, and (b) production and release of extracellular MMP by synovial fibroblasts, thereby inducing a degradative and invasive phenotype culminating in cartilage and bone destruction.     

Differential expression of two major cytokines produced by neutrophils, interleukin-8 and the interleukin-1 receptor antagonist, in neutrophils isolated from the synovial fluid and peripheral blood of patients with rheumatoid arthritis

OBJECTIVE: Neutrophils have been shown to produce interleukin-8 (IL-8) and interleukin-1 receptor antagonist (IL-1ra) in large amounts compared with other cytokines. Since IL-8 has a proinflammatory action whereas IL-1ra is antiinflammatory, our objective was to examine the relative levels of production of these cytokines by synovial fluid (SF) neutrophils isolated from patients with rheumatoid arthritis (RA). METHODS: We measured cytokine production using an enzyme-linked immunosorbent assay and analyzed messenger RNA accumulation in cells by Northern blot. RESULTS: SF neutrophils produced significantly more IL-8 and IL-1 beta, but significantly less IL-1ra, than peripheral blood neutrophils. CONCLUSION: These observations provide new information on the production of pro- and antiinflammatory molecules by neutrophils in the SF environment, and their possible role in RA.     

Multiple T cell expansions are found in the blood and synovial fluid of patients with reactive arthritis

OBJECTIVE: To look for evidence of T lymphocyte expansions in the blood and synovial fluid (SF) of patients with reactive arthritis (ReA). METHODS: Paired peripheral blood and synovial samples from 10 patients with ReA were studied by dual color flow cytometry using T cell receptor (TCR) V beta specific and CD4 or CD8 specific antibodies. Two synovial CD8 expansions were studied by 3 color flow cytometry. Peripheral blood samples from 13 healthy, age matched individuals were studied as controls. RESULTS: Statistically significant expansions were observed in all patients, occurring in blood and SF CD4 and CD8 compartments, but were most common in the synovial CD8 compartment. Expansions studied in further detail displayed an activated "memory" phenotype. A synovial BV22S1/CD8 expansion was seen in 5/6 patients with sexually acquired ReA. CONCLUSION: Multiple T lymphocyte expansions are found in both the blood and SF of patients with ReA. Expansions were most commonly found in the synovial CD8 compartment, where they appeared to express both activation and memory markers. This indicates that T lymphocytes (and in particular cytotoxic T cells) may play a pathogenic role in ReA. These findings are consistent with either an antigen or a superantigen driven response.     

Coexpression of CD69 and HLADR activation markers on synovial fluid T lymphocytes of patients affected by rheumatoid arthritis: a three-colour cytometric analysis

Haploid and diploid strains of Aspergillus nidulans have been repeatedly treated with the strong mutagen 6-N-hydroxylaminopurine (HAP) which causes only base substitutions. An enormous amount of variability may be rapidly accumulated in haploid or diploid strains of A. nidulans. In particular, in the diploids the analysis of the results shows that after 12 cycles of treatment the conidia differ from each other for about ten recessive lethals and therefore probably for several hundreds of mutations. The viability of the heterozygous multiply mutant diploids is not appreciably different from that of untreated controls. In the diploid strains the accumulated variability was very high. The treatment of a haploid strain during vegetative growth also caused a strong accumulation of mutations, even though deleterious, because they can be maintained in the heterokaryotic condition.     

T helper 1 response is dominant and localized to the synovial fluid in patients with Lyme arthritis

Cytokines produced by subsets of CD4+ T helper cells responding to an infection influences the efficiency with which the host is able to mount a protective immune response. In an attempt to elucidate the population of active cells involved in the propagation of Lyme arthritis we have utilized intracellular cytokine staining to analyze the polyclonal immune response at the single cell level. We have determined the Th phenotype in the synovial fluid of patients with a variety of chronic inflammatory arthritides, including patients representative of the spectrum of Lyme arthritis. Th1 cells dominate the immune response in the synovial fluid of patients with Lyme as well as those with rheumatoid or other types of chronic inflammatory arthritis. In addition, the severity of Lyme arthritis directly correlates with the ratio of Th1 to Th2 cells in the synovial fluid, such that the larger the effusion, the higher the ratio (r = 0.67, p < 0.05). These results suggest that Th1 cells play a direct role in the pathogenesis of the inflammatory process seen in Lyme arthritis, and that Th2 cells modulate the pro-inflammatory response generated by Th1 cells in the joint. Finally, we identify Th1 cells specific for outer surface protein A of Borrelia burgdorferi, the agent of Lyme disease. These cells are restricted to patients with Lyme arthritis and are localized to the joint. Furthermore, they persist in patients with prolonged antibiotic treatment-resistant Lyme arthritis, suggesting the possibility of an autoimmune process.     

The response of peripheral blood lymphocytes from lung cancer patients to in-vitro treatment with recombinant interleukin-2

The cytotoxic activity of peripheral blood lymphocytes of 135 lung cancer patients was studied. A wide range of cytotoxicity values was shown--from 0 to 88%. During the randomized investigation of these patients no strong correlation was found between the levels of cytotoxicity and the cancer stages. A certain tendency of increasing the number of patients with weak cytotoxic activity upon progressing disease was followed. The lymphocytes with a strong natural killer activity did not react on the in vitro incubation with interleukin-2. The lymphocytes with a strong was obtained when the lymphocytes with the lowest cytotoxicity levels were incubated with this immunomodulator.     

Selective accumulation of related CD4+ T cell clones in the synovial fluid of patients with rheumatoid arthritis

The role of T cells in the pathogenesis of rheumatoid arthritis (RA), especially in the perpetuation of advanced disease, remains unclear. Previous studies have focused on the TCR repertoire of synovial T cells in an attempt to determine whether the pattern of expression is characteristic of Ag-stimulated populations. However, the results of past studies have been conflicting. In the present work, we have undertaken an extensive analysis of the TCRs expressed by CD4+ T cells freshly isolated from synovial fluid of different joints and blood in three patients with established RA. Despite marked heterogeneity of synovial TCR expression, the results showed that 20 to 30% of the TCR beta-chain gene (TCRB) sequences found in one joint were also expressed in a second joint, but not in peripheral blood T cells of the same individual. Analysis of expressed TCRB complementarity-determining region 3 sequences showed the presence of multiple expanded clonal populations that were not predicted by quantitation of beta-chain variable region (Vbeta) expression by immunofluorescence staining. These studies also demonstrated sets of related, but different, complementarity-determining region 3 nucleotide sequences that encoded identical or highly homologous beta-chain amino acid sequences. Analysis of matching T cell clones derived from the joint by limiting dilution culture confirmed coexpression of highly homologous TCR alpha-chain gene (TCRA) and TCRB sequences. Together, these studies suggest that a significant proportion of synovial CD4+ T cells has been selected and expanded by conventional Ag(s) in this disease.     

Increase in adhesion molecules on CD4+ cells and CD4+ cell subsets in synovial fluid from patients with rheumatoid arthritis

OBJECTIVE: To elucidate the role of adhesion molecules in the pathogenesis of rheumatoid arthritis (RA). METHODS: We evaluated their expression and that of an activation marker on CD4+ cell populations and CD4+ cell subsets in specimens of peripheral blood (PB) and synovial fluid (SF) obtained from 10 patients with RA and 7 with osteoarthritis (OA). A 2 or 3-color immunofluorescent method was used for analysis. RESULTS: The SF from both groups of patients showed a greater density of adhesion molecules including LFA-1 alpha, LFA-1 beta, CD2, VLA-4 alpha and VLA-5 alpha on CD4+ cells, and a higher percentage of CD4+HLA-DR+ cells compared with their PB. IN PB-CD4+ cell subsets from the arthritic and healthy subjects, the CD4+CD45RO+ cell population showed an increased expression of adhesion molecules compared with CD4+CD45RA+ cell population. The expression of adhesion molecules on circulating CD4+ cell population and CD4+ cell subsets from the patients with RA and OA was comparable to that from healthy subjects. SF from both groups of patients showed a higher percentage of CD4+CD45RO+ cells and a lower percentage of CD4+CD45RA+ cells. In SF-CD4+ cell subsets from patients with RA, the CD4+CD45RO+ cell population had an increased expression of VLA-4 alpha compared to the CD4+CD45RA+ cell population; however, there was no significant difference in other adhesion molecule expression and the percentage of HLA-DR+ cells between the 2 cell subsets. Furthermore, the expression of VLA-4 alpha on the CD4+CD45RO+ cell population in SF from patients with RA was significantly higher than that in matched PB. In CD4+CD45RA+ cell population from both groups of patients, SF showed an enhanced expression of adhesion molecules and an increased percentage of HLA-DR+ cells compared with matched PB. CONCLUSION: Our results suggest that increased expression of adhesion molecules and increased percentage of HLA-DR+ cells on CD4+ cells in SF may be responsible for cellular interactions between these cells and synovial cells or extracellular matrix.     

Expression of macrophage markers by a population of T cells obtained from synovial fluid of a subgroup of patients with juvenile rheumatoid arthritis

OBJECTIVE: To characterize distinctive lymphoid cell populations in the synovial fluid (SF) of patients with juvenile rheumatoid arthritis (JRA) that have the specific ability to display monocytic markers when cultured in vitro. METHODS: Mononuclear cells obtained from SF of patients with JRA and depleted of adherent macrophages were cultured in vitro in RPMI 1640 medium supplemented with only fetal calf serum (FCS). Phenotypic evaluation of these cells was by flow cytometry and immunohistochemical analysis was by specific fluorochrome labeled antibodies. RESULTS: T cells from a JRA subgroup displayed some typical macrophage attributes, i.e., abundant cytoplasm, adherence to plastic, and phagocytosis of latex beads when cultured in vitro. These cells have the ability to survive in culture for several weeks in RPMI 1640 medium containing only 10% FCS. The macrophage-like T cells rosetted with sheep red blood cells and proliferated when stimulated with phytohemagglutinin or anti-CD3, indicating functional T cell responses. CONCLUSION: Our data indicate that a population of T cells obtained from the SF of a subgroup of patients with JRA exhibited characteristics of macrophages, yet retained their CD3 and T cell receptor expression. Whether this promiscuous behavior is caused by malignant transformation of lymphoid precursor cells or is induced by the concerted effect of a myriad of cytokines and growth factors present in the SF remains unknown. The presence of these cells in the SF of 2 patients with JRA with different onset types raises the question of their function and significance in an autoimmune disorder such as JRA.     

Anti-Fc gamma receptor III autoantibody is associated with soluble receptor in rheumatoid arthritis serum and synovial fluid

We sought to detect anti-Fc gamma receptor (Fc gamma R) autoantibodies and soluble Fc gamma R in the serum and synovial fluid (SF) from patients with rheumatoid arthritis (RA) and to correlate these serological abnormalities to the polymorphonuclear cell (PMN) activation state. Paired samples of blood and SF were obtained from 33 RA patients as well as blood from 25 normal adults from SF from 20 non-RA patients. Anti-Fc gamma R autoantibodies were assessed by an enzyme-linked immunosorbent assay (ELISA) using recombinant human Fc gamma R as the substrate. Soluble Fc gamma RIII was determined by an ELISA based on the combination of two monoclonal antibodies (MAb). The mean fluorescence intensity (MFI) of complement receptor 1 (CD35) and 3 (CD11b) and Fc gamma RIII (CD16) was evaluated by flow cytometry on the membrane of PMN. IgM anti-Fc gamma RIII activity was present in seven RA sera and five SF, and IgG in eight RA sera and six SF. The average concentration of soluble Fc gamma RIII was 1.80 +/- 3.50 micrograms/ml in RA patients and 0.33 +/- 0.06 micrograms/ml in normal adults (P < 0.05). This was elevated in the SF of 15 RA, while normal in that of all the non-RA patients. There was an inverse correlation between the CD16 MFI on the PMN and the serum/SF soluble Fc gamma RIII level, whereas the density of CD35 and CD11b was markedly augmented. Anti-Fc gamma RIII activity exists in RA patients, associated with soluble Fc gamma RIII. PMN activation could be due to these autoantibodies and thereby obviate the clearance of immune complexes.     

VH usage and somatic hypermutation in peripheral blood B cells of patients with rheumatoid arthritis (RA)

The human antibody repertoire has been demonstrated to have a marked V-gene-dependent bias that is conserved between individuals. In RA patients, certain heavy chain V genes (VH) have been found to be preferentially used for encoding autoantibodies. To determine if such preferential use of VH genes in autoantibodies is associated with a general distortion of the V gene repertoire in RA patients, the VH composition of peripheral blood B cells was analysed among four RA patients and four age- and sex-matched healthy controls. Usage of individual VH genes (eight VH3 and three VH4 genes tested by hybridization with a set of gene-specific oligonucleotide probes) was highly biased among RA patients, but no evidence of a distortion in the bias was observed compared with healthy controls. However, the occurrence of somatic mutations in these VH genes (estimated by differential hybridization with motif-specific oligonucleotide probes targeted to CDR and FR of the tested genes, and by DNA sequence analysis) was strikingly different between patients and healthy subjects. The number of VH3 rearrangements that had accumulated somatic mutations and the number of mutations per rearrangement were significantly elevated in three of the four RA patients. A slight but not significant elevation in mutations among rearranged VH4 genes was also observed in these patients. These data suggest that although usage of individual VH genes among peripheral blood B cells is not affected by the disease, the autoimmune process may involve a significant fraction of the B cell compartment.