Bone marrow CD34+ progenitor cells from rheumatoid arthritis patients support spontaneous transformation of peripheral blood B cells from healthy individuals

We show that bone marrow (BM) CD34⁺ progenitor cells from rheumatoid arthritis (RA) patients have the capacity to support spontaneous transformation of peripheral blood B cells. CD34⁺ cells purified from BM blood from eight RA patients and eight osteoarthritis (OA) patients were expanded with granulocyte/macrophage colony stimulating factor (GM-CSF) for 4–6 weeks. GM-CSF-stimulated BM CD34⁺ cells from three of eight RA patients, but none from seven OA patients, gave rise to spontaneous transformation of highly purified B cells of Epstein-Barr virus (EBV)- seronegative healthy donors. GM-CSF-stimulated BM CD34⁺ cells from four of six RA patients and from one of four OA patients also supported the spontaneous transformation of peripheral blood B cells from EBV-seropositive healthy donors. All the transformed B cell lines were positive for EBV-DNA as determined by PCR. Neither GM-CSF-stimulated BM CD34⁺ cells alone nor highly purified B cells alone gave rise to spontaneously transformed B cell lines. These results suggest that the capacity of BM CD34⁺ cells to support survival of B cells might contribute to the pathogenesis of RA by sustaining abnormal B cell responses. Received: 8 November 1999 / Accepted: 10 January 2000     

T cell responses to human type II collagen in patients with rheumatoid arthritis and healthy controls

Objective. To study the prevalence of T cell responses to human type II collagen (CII) in patients with rheumatoid arthritis (RA) with or without antibodies to CII, and in healthy controls. Methods. Assays were performed to study T cell proliferative responses to CII in peripheral blood from 69 patients with RA (11 with anti-CII antibodies and 58 without) and 28 healthy controls. Further analysis was made of the time course of the response and the epitopic specificity, using peptides derived from the cyanogen bromide 11 (CB11) fragment of CII. Results. Significant proliferative responses to CII were found in 50% of patients with anti-CII, 5.3% of RA patients without these antibodies, and 35.7% of healthy controls. Responses in RA patients differed from those in healthy controls; the former had kinetics suggestive of a recall response and the latter that of a primary response. Some common epitopes within CB11 were recognized by T cells from patients and controls. Conclusion. Proliferative T cell responses to CII occur in some healthy individuals, suggesting that thymic tolerance for this antigen may be incomplete. Most patients with RA have no evidence of a T cell response to CII, possibly indicating the development of peripheral tolerance to this antigen as a consequence of cartilage breakdown. However, in a minority of patients, T and B cell responses to CII persist, and may contribute to joint damage.     

IgG subclass distribution of antinative type II collagen and antidenatured type II collagen antibodies in patients with rheumatoid arthritis

In 81 patients with rheumatoid arthritis (RA) with antibodies to native type II collagen, the frequencies of each IgG subclass to native type II collagen were IgG1 (70%), IgG2 (12%), IgG3 (84%) and IgG4 (6%) and to denatured type II collagen were IgG1 (86%), IgG2 (23%), IgG3 (86%) and IgG4 (6%). Thus serum antibodies to type II collagen in patients with RA were predominantly of the complement fixing subclasses IgG1 and IgG3.     

The B cell repertoire of patients with rheumatoid arthritis. II. Increased frequencies of IgG+ and IgA+ B cells specific for mycobacterial heat-shock protein 60 or human type II collagen in synovial fluid and tissue

Objective. A qualitative and quantitative analysis of the functional, antigen-specific B cell receptor repertoire of patients with rheumatoid arthritis (RA) in synovial and peripheral compartments. Methods. B cells were activated to grow and differentiate at high efficiency in vitro under limiting-dilution conditions. Isotype and specificity of the secreted Ig were tested by enzyme-linked immunosorbent assay. Results. In contrast to peripheral B cells, most synovial B cells had already switched to IgG/IgA in vivo. The frequencies of B cells specifically recognizing foreign antigens were decreased within the synovial population, whereas the frequencies of B cells specific for type II collagen, mycobacterial heat-shock protein 60 (hsp60), or IgG Fc fragments were significantly increased, revealing a negative correlation in terms of frequencies. Conclusion. B cells specific for human type II collagen, hsp60, and IgG Fc fragments are produced and/or expanded locally within the affected joints of RA patients. Thus, the specific immune system is definitely involved in the local inflammatory and destructive processes.     

Accumulation of T cells reactive to type II collagen in synovial fluid of patients with rheumatoid arthritis

OBJECTIVE: To determine if type II collagen (CII) reactive T lymphocytes selectively accumulate in the inflamed joint of patients with rheumatoid arthritis (RA) and to study the specificity of the CII reactive cells. METHODS: Synovial fluid (SF) cells or peripheral blood lymphocytes were cultured with interleukin 2 (IL-2) for 24 h and then cultured at limiting dilution concentrations in the presence of filler cells and IL-2. The outgrowing cell lines were screened for their responses to CII. The percentages of the CII reactive T cells from SF were compared with those from peripheral blood of patients with RA. The CII reactive T cell lines were tested for their responses to different types of collagen. RESULTS: CII reactive T cell lines were identified in the SF of 3 RA patients; the frequencies were 5.0% (11/219), 3.7% (5/134), and 3.5% (3/86), respectively. In contrast, none of CII-specific T cell lines were identified in peripheral blood of the patients. The T cell lines recognized both human and bovine CII and, to a lesser extent, type I collagen. CONCLUSION: CII reactive T cells are present in high frequency in the inflamed joint of patients with RA, where they may play an important role in the pathogenesis of RA.     

IgG antibodies to type II collagen reflect inflammatory activity in patients with rheumatoid arthritis

OBJECTIVE: To determine the clinical significance of IgG antibodies to type II collagen (CII) and to define any correlation of antibodies to CII with the inflammatory response in patients with rheumatoid arthritis (RA). METHODS: IgG antibodies to native human type II collagen (IgG anti-CII) were measured in sera and synovial fluid (SF) from patients with RA, patients with osteoarthritis (OA), and healthy controls by an improved ELISA. Demographic, clinical, and laboratory data including tumor necrosis factor-alpha (TNF-alpha) and interleukin 6 (IL-6) levels were also obtained at the time of sampling in patients with RA. RESULTS: The median level and positivity for circulating IgG anti-CII were higher in patients with RA (n = 297) than patients with OA (n = 34) and healthy controls (n = 50) (p < 0.001). The titers of IgG anti-CII in SF were also higher in RA (n = 45) than in OA (n = 16) (p < 0.001). In paired samples, the levels of IgG anti-CII were significantly higher in SF compared to the sera in patients with RA (n = 45) (p < 0.001), but levels were not different in patients with OA (n = 16). Circulating IgG anti-CII converted from positive to negative in 13 patients (10.7%) and from negative to positive in 18 patients (14.8%) among 122 patients with RA in whom IgG anti-CII were monitored sequentially at a mean interval of 12.2 months. IgG anti-CII positive patients (n = 98) had shorter disease duration (p = 0.04) and less frequent deformity (p = 0.013), and higher median erythrocyte sedimentation rate (ESR) (p = 0.001) and C-reactive protein (CRP) (p < 0.001) than IgG anti-CII negative patients (n = 120). The levels of IgG anti-CII correlated with CRP (r = 0.270) and ESR (r = 0.253). CRP decreased significantly in patients (n = 13) who converted from IgG anti-CII positive to negative (p = 0.013). IgG anti-CII positive patients (n = 40) had higher levels of TNF-alpha and IL-6 than negative patients (n = 40) (p < 0.001). Levels of IgG anti-CII correlated well with TNF-alpha (r = 0.617) and IL-6 (r = 0.347). CONCLUSION: Increased IgG anti-CII in sera and SF in RA correlated directly with acute phase reactants and the proinflammatory cytokines TNF-alpha and IL-6. Our data suggest that IgG anti-CII could reflect inflammatory activity with a potential to destroy cartilage in the early stages of RA.     

Autoimmunity to citrullinated type II collagen in rheumatoid arthritis

Abstract

The production of autoantibodies to citrullinated type II collagen and the citrullination of type II collagen were analyzed in rheumatoid arthritis. Autoantibodies to citrullinated type II collagen were detected in 78.5% of serum samples from 130 rheumatoid arthritis patients. Autoantibodies to native noncitrullinated type II collagen were detected in 14.6% of serum samples, all of which were positive for anti-citrullinated type II collagen antibodies. Serum samples were also positive for anti-citrullinated type II collagen antibodies in 1 of 31 systemic lupus erythematosus patients and 2 of 55 patients with osteoarthritis of the knee. In contrast, sera samples from 24 systemic sclerosis patients, 21 dermatomyositis/polymyositis patients, 21 ankylosing spondylitis patients, and 18 psoriatic arthritis patients were all negative for anti-citrullinated type II collagen antibodies. Anti-citrullinated type II collagen antibodies and fragments of citrullinated type II collagen were found in the synovial fluid obtained from affected knee joints of 15 rheumatoid arthritis patients. Moreover, anti-citrullinated type II collagen antibodies were isolated from the synovium of affected knee joints in 8 rheumatoid arthritis patients using antigen/antibody immunocomplex dissociation buffer but not by using standard buffers. These findings indicate that autoantibodies that react with citrullinated type II collagen are specifically produced and that immunocomplexes composed of fragments of citrullinated type II collagen and autoantibodies are deposited in the inflamed articular synovium in rheumatoid arthritis patients. Assaying for the presence of anti-citrullinated type II collagen antibodies may therefore be useful for diagnosing rheumatoid arthritis, and the deposition of these immunocomplexes in the articular synovium may be involved in pathogenesis.     

Specificity of antibodies to type II collagen in rheumatoid arthritis.

To reassess the role of autoantibodies to type II collagen in the pathogenesis of diseases, we studied antibodies from patients with rheumatoid arthritis (RA) and from patients with relapsing polychondritis for species specificity and collagen type specificity, using an improved enzyme-linked immunosorbent assay. Antibodies were found in the sera of 15% of the RA patients and 50% of the relapsing polychondritis patients, as well as in the cartilage of 69% of the RA patients examined. Reaction with both homologous and heterologous type II collagens was common. Analysis of 19 selected RA sera revealed that autoantibodies were generally associated with specific antibodies to some species of heterologous type II collagen. In contrast, antibodies found in 4% of the non-RA controls were specific for either bovine or chick type II collagen. These findings indicate that autoantibody formation in RA and relapsing polychondritis may occur as a result of an immune response to heterologous type II collagen. However, since RA and relapsing polychondritis patient sera differed in their reactivity with the cyanogen bromide-digested peptides, it is possible that the clinical manifestation of collagen autoimmunity might be influenced by the epitope specificity of the antibodies.     

Gm allotypes and HLA in rheumatoid arthritis patients with circulating antibodies to native type II collagen.

HLA antigens and immunoglobulin heavy chain allotypes (Gm) were determined in 166 unrelated patients with rheumatoid arthritis (RA), 44 of whom had circulating antibodies to native type II collagen. Collagen antibody positive patients showed an association with HLA-DR3 and DR7 (68% compared with 39% of collagen antibody negative RA, p less than 0.005), and with the Gm phenotype, Gm(zafngb). This contrasted with the collagen antibody negative RA patients where there was an association with HLA-DR4 and, in DR4 positive disease only, with the Gm allotype, G1m(x). The Gm(zafngb) phenotype was found in 26% of DR3 or DR7 positive patients overall and only 9% of RA patients negative for these DR antigens (p less than 0.005), suggesting an interaction between HLA-DR3/7 and Gm(zafngb). The differing Gm associations for collagen antibody positive and negative RA provide further evidence for genetic heterogeneity in susceptibility to RA.     

Autoimmunity to citrullinated type II collagen in rheumatoid arthritis

The production of autoantibodies to citrullinated type II collagen and the citrullination of type II collagen were analyzed in rheumatoid arthritis. Autoantibodies to citrullinated type II collagen were detected in 78.5% of serum samples from 130 rheumatoid arthritis patients. Autoantibodies to native noncitrullinated type II collagen were detected in 14.6% of serum samples, all of which were positive for anti-citrullinated type II collagen antibodies. Serum samples were also positive for anti-citrullinated type II collagen antibodies in 1 of 31 systemic lupus erythematosus patients and 2 of 55 patients with osteoarthritis of the knee. In contrast, sera samples from 24 systemic sclerosis patients, 21 dermatomyositis/polymyositis patients, 21 ankylosing spondylitis patients, and 18 psoriatic arthritis patients were all negative for anti-citrullinated type II collagen antibodies. Anti-citrullinated type II collagen antibodies and fragments of citrullinated type II collagen were found in the synovial fluid obtained from affected knee joints of 15 rheumatoid arthritis patients. Moreover, anti-citrullinated type II collagen antibodies were isolated from the synovium of affected knee joints in 8 rheumatoid arthritis patients using antigen/antibody immunocomplex dissociation buffer but not by using standard buffers. These findings indicate that autoantibodies that react with citrullinated type II collagen are specifically produced and that immunocomplexes composed of fragments of citrullinated type II collagen and autoantibodies are deposited in the inflamed articular synovium in rheumatoid arthritis patients. Assaying for the presence of anti-citrullinated type II collagen antibodies may therefore be useful for diagnosing rheumatoid arthritis, and the deposition of these immunocomplexes in the articular synovium may be involved in pathogenesis.     

Mechanisms of CD23 hyperexpression on B cells from patients with rheumatoid arthritis.

OBJECTIVE: To analyze the mechanisms involved in the characteristic hyperexpression of CD23 on peripheral blood B cells from patients with rheumatoid arthritis (RA). METHODS: Peripheral blood mononuclear cells (PBMC) were obtained from patients with active disease and activated during 18 h with an anti-CD3 monoclonal antibody in the presence or absence of blocking antibodies to CD154 or CD40. PBMC were further purified by rosetting and CD23 expression was assessed on B cells by flow cytometry after double staining (CD19/CD23). Lymphocytes were also isolated from synovial fluid (SF). CD154 expression was analyzed on PB or SF CD4+ T cells after double staining (CD4/CD154) by flow cytometry at basal conditions and after different stimuli [anti-CD3 or phorbol myristic acetate (PMA) plus ionomycin]. Co-culture experiments between SF and PB cells were performed to analyze the involvement of the CD40-CD154 interaction on CD23 expression. CD154 and CD23 expression was also analyzed on synovial membrane by immunohistochemical techniques. RESULTS: A high proportion of activated CD23 B cells was detected in patients with RA. Blocking experiments with both anti-CD40 and anti-CD154 Mab showed a significant reduction in the proportion of PB B cells expressing CD23. Following activation with anti-CD3 Mab or PMA plus ionomycin, CD154 expression was mainly induced on PB CD4+ T cells. In co-culture experiments, SF T cells were more efficient than PB T cells in inducing CD40 dependent CD23 expression on PB B cells. In addition, CD4+ T cells from synovial membrane clearly expressed CD154. CONCLUSION: Our results establish a link between CD154-CD40 pathway and CD23 expression on PB B cells from patients with RA. T cells from the synovial microenvironment were active participants in this CD23 expression, presumably in the context of cell recirculation.     

The role of CD19+CD24highCD38high and CD19+CD24highCD27+ regulatory B cells in patients with type 1 autoimmune pancreatitis

BackgroundPatients with type 1 autoimmune pancreatitis (AIP) have several immunologic and histologic abnormalities. It is known that depletion of B cells by rituximab is effective for treatment of IgG4-related disease (IgG4-RD) such as type 1 AIP, suggesting that B cells may be a key player in IgG4-RD. However, the role of regulatory B cells (Bregs) in type 1 AIP is unclear, and the objective of this paper is to clarify the role of Bregs in the pathophysiology of type 1 AIP by analyzing circulating Bregs.MethodWe recruited 21 patients with type 1 AIP as determined by the International Consensus Diagnostic Criteria for AIP (ICDC). No patients received corticosteroid treatments. For comparison, we recruited 14 patients with chronic pancreatitis (CP), 20 patients with pancreatic cancer, and 25 healthy subjects as controls. We analyzed Bregs as CD19⁺CD24^highCD38^high and CD19⁺CD24^highCD27⁺ from peripheral blood by flow cytometry.ResultsIn peripheral blood, CD19⁺CD24^highCD38^high Bregs were significantly increased in type 1 AIP patients compared with CP, pancreatic cancer, and healthy controls. Although not significant different, CD19⁺CD24^highCD27⁺ Bregs of type 1 AIP were decreased compared to those of other groups. IL-10⁺ B cells were not significantly different from type 1 AIP patients and healthy controls. In untreated type 1 AIP patients, the number of CD19⁺CD24^highCD38^high Bregs and IgG4 were not correlated.ConclusionsOur data suggested that CD19⁺CD24^highCD38^high Bregs seemed to increase reactively to suppress the disease activity, and are consistent with the hypothesis that CD19⁺CD24^highCD27⁺ Bregs might be involved in the development of type 1 AIP, although it still remains unclear whether the decrease of CD19⁺CD24^highCD27⁺ cells is cause or effect of AIP.     

Antibodies to type II collagen in early rheumatoid arthritis. Correlation with disease progression

Objective. To establish that frequencies and levels of IgG antibodies to type II collagen are higher in early rheumatoid arthritis (RA), and to correlate these results with disease activity. Methods. Forty-four patients were characterized as having early RA. Patient sera obtained at initial presentation and at 12 months were tested by enzyme-linked immunosorbent assay for IgG antibodies to native and denatured type II collagen. Results. IgG antibodies to native and denatured type II collagen were detected at initial presentation in 27% and 82% of patients, respectively, and after 12 months in 14% and 50%, respectively. The presence of antibodies to native collagen was associated with activity of RA and severity of symptoms, and loss of antibodies at 12 months was associated with initially erosive RA and the DRB1 disease susceptibility motif. Conclusion. Levels of serum IgG antibodies to collagen in RA decrease over time and, therefore, are not attributable simply to cartilage destruction. The presence of early positivity for these antibodies, together with the RA susceptibility motif, appears to be predictive of rapidly progressive RA.     

Limited proliferative response to type II collagen in rheumatoid arthritis

In order to define the potential importance of type II collagen in the activation of rheumatoid arthritis (RA) synovial fluid (SF) lymphocytes, we examined the proliferative response of matched peripheral blood and SF lymphocytes to type II collagen. The mean proliferative response to the collagen was somewhat greater (p less than 0.05) with SF, compared to peripheral blood lymphocytes. However, the magnitude of this response was relatively weak as determined by stimulation indices, and it did not approach that observed with peripheral blood lymphocytes in response to tetanus toxoid. Sixty-seven percent of peripheral bloods and 50% of SF demonstrated positive responses to the control antigen, tetanus toxoid. In contrast, only 6 and 28%, respectively, were positive in response to type II collagen. The addition of exogenous interleukin 2 augmented responses to the tetanus toxoid, however, no such enhancement with type II collagen was noted in our patients. Our collagen was arthritogenic in experimental animals. These observations do not support the existence of T cell specificity toward type II collagen as a common mechanism for the expansion of synovial lymphocytes in RA.     

Parvovirus B19 infection in patients with rheumatoid arthritis in Taiwan

OBJECTIVE: To investigate the role of human parvovirus B19 infection in the pathogenesis of rheumatoid arthritis (RA) in Taiwan. METHODS: Seventy-eight patients with RA and 55 unrelated controls (51 trauma and 4 osteoarthritis) were enrolled. Anti-parvovirus B19 IgG and IgM antibodies were detected in plasma of patients with RA and controls by the enzyme immunoassay method. These antibodies were also detected in the synovial fluid of 18 RA patients and 52 controls. B19 DNA was measured in the plasma of 72 patients with RA and 45 controls by nested polymerase chain reaction (PCR). It was also measured in the synovial fluid of 14 RA patients and 39 controls. Immunohistochemistry was performed to detect viral capsid protein VP1 of B19 in the synovial membrane of 7 RA patients and 32 controls. HLA-DR genotyping was performed by the sequence-specific primer PCR method. The interactions between B19 infection and HLA-DR genotype and susceptibility to RA were also analyzed. RESULTS: The prevalence of B19 infection was significantly increased in patients with RA compared with controls. The positive rates of B19 DNA in plasma and synovial fluid were significantly higher in RA patients than in controls. The odds ratio of DR4(+) B19 infection(+) was higher than that in DR4(+) B19 infection(-) or DR4(-) B19 infection(+) in comparison with DR4(-) B19 infection(-). A significant association was found between RA and DR4(+) B19 infection(+) in comparison with DR4(+) B19 infection(-). The odds ratio of DR4(+) plasma B19 DNA(+) was also higher than that of DR4(+) plasma B19 DNA(-) or DR4(-) plasma B19 DNA(+) in comparison with DR4(-) plasma B19 DNA(-). RA tended to be associated with DR4(+) plasma B19 DNA(+) compared with DR4(+) plasma B19 DNA(-). CONCLUSION: The prevalence of parvovirus B19 infection was significantly higher in patients with RA than in controls. Synergistic effects were present between HLA-DR4 and parvovirus B19 infection or plasma B19 DNA for susceptibility to RA. Parvovirus B19 infection may play a role in susceptibility to RA.     

CD5+ B lymphocytes in systemic lupus erythematosus and rheumatoid arthritis.

B lymphocytes which express the CD5 antigen on their cell surface have the ability to produce autoantibodies in vitro. We describe 2 patients, one with systemic lupus erythematosus and another with rheumatoid arthritis, whose percentages of peripheral blood CD5+ B lymphocytes were dramatically increased to 68 and 100%, respectively. A reduction of this subpopulation of B cells was associated with clinical improvement after therapy. This association suggests a relationship between CD5+ B cells and autoimmune disease activity.     

Expression of CD22 on peripheral B cells in patients with rheumatoid arthritis: relation to CD5-positive B cells

B cells in patients with rheumatoid arthritis (RA) are hyperactivated. Although B cell receptor signal transduction may be affected by various response regulators, CD22 plays an important role as a response regulator of B cells. Therefore, we investigated and examined CD22 expression on peripheral blood B cells of patients with RA. Thirty-two RA patients and 16 controls were enrolled in this study, and CD22 expressions on B cells were analyzed by flow cytometry. In patients with RA, CD22⁺ B cells significantly decreased in comparison to the controls (ratio: P < 0.05). However, there was no correlation between this decrease and the clinical data. Interestingly, CD5⁺ CD22⁻ B cells significantly increased in RA patients. The decrease in CD22⁺ B cells and increased in CD5⁺CD22⁻ B cells play critical roles in the pathogenesis of RA mediated by the activation of B cells.     

Tristetraprolin (TTP) gene polymorphisms in patients with rheumatoid arthritis and healthy individuals

Abstract

Tristetraprolin (TTP) is an intracellular protein that modulates the production of cytokines, including TNFα, by binding to and destabilizing the mRNAs of these cytokines. Therefore, differences in TTP gene expression may affect the severity of inflammatory diseases, such as rheumatoid arthritis (RA). We searched for polymorphisms in the human TTP gene and for this purpose, we sequenced the entire TTP gene in 20 Japanese individuals (ten with RA and ten healthy volunteers) and found one single nucleotide polymorphism (SNP) in the promoter region. We analyzed this SNP (A/G) by restriction fragment length polymorphism method in 155 RA patients and 100 control subjects. While the frequency of A allele in this SNP was similar in RA patients (74.5%) and controls (76.0%), the disease duration in RA patients with genotype GG was shorter than that of patients with genotypes AA/AG and RA patients with genotype GG had a higher probability of being treated with infliximab. We studied the difference in promoter activity between the two alleles by luciferase assay and found that the promoter activity of TTP promoter region with allele A was around two-fold higher than that with allele G. We conclude that this SNP in the promoter region of the TTP gene mildly affects promoter activity, and thus, may influence the disease activity of inflammatory disorders including RA.