The Effects Of IgD On T/B Cells And Fibroblast-like Synoviocytes In Rheumatoid Arthritis And The Potential Mechanism

Introduction:Rheumatoid arthritis ?RA? is a chronic systemic inflammatory disease characterized by inflammation of the joint synovial tissue. Immunoglobulin D ?IgD? is an immunoglobulin ?Ig? isotype that can be expressed as membrane IgD ?mIgD? or secreted IgD ?sIgD?. Except for B cell receptor, IgD also serves as a ligand for IgD^-specific receptor ?IgDR? expressed on CD⁴⁺T cells. IgD promotes immune defense which cause inflammation and tissue damage by inducing the activation and infiltration of immune cells. Accumulating evidences have suggested that IgD may contribute to disease pathogenesis. sIgD levels are increased in autoimmune diseases such as RA, systemic lupus erythematosus ?SLE?, Sjogren's syndrome and autoimmune thyroiditis. Recently, researchers also investigated that anti-IgD treatment selectively depletes mature B cells in collagen-induced arthritis ?CIA? mouse model. This finding generated a new perspective that IgD^-IgDR may provide a new therapeutic target for B cell depletion in autoimmune diseases. RA is a chronic inflammatory autoimmune disease that mainly targets the synovial membrane, resulting in destruction of the joint architecture. Synovial hyperplasia results from a marked increase in macrophage-like and fibroblast-like synoviocytes ?FLS?. Accumulating evidence indicates that activated FLS are among the key players in RA joint destruction. IgDR have been described on CD⁴⁺and CD⁸⁺ T cells in mice and human, but have not been previously demonstrated on FLS. Till now there wasn't any report about the expression of IgDR on T/B cells and FLS in RA patients. It is unknown whether the expression of IgD is abnormal in RA? Whether excessive IgD in RA promote T/B cells activation via IgDR? Whether the excessive IgD could promote the proliferation and cytokines secretion in FLS?This paper intends to collect tissue samples of clinical RA patients, using magnetic bead separation, flow cytometry, western blot and laser confocal microscopy. In order to investigate the abnormal level of IgD and IgDR in T/B cells and FLS from RA, and to observe the effect of IgD on the function of T/B cells and FLS. We also try to illustrate the possible mechanisms of IgD and IgDR which involved in T/B cells activation and FLS abnormal proliferation in RA. This research is aimed to find new pathological mechanism of RA, as well as to provide experimental basis for that IgD^-IgDR represents a potentially novel immunotherapeutic target for RAObjective:To investigate the abnormal level of IgD and IgDR in T/B cells and FLS from RA, and to observe the effect of IgD on the function of T/B cells and FLS. To illustrate the possible mechanisms of IgD and IgDR which involved in T/B cells activation and FLS abnormal proliferation in RA. To provide experimental basis for that IgD^-IgDR represents a potentially novel immunotherapeutic target for RA.Methods:Eligible adults included those with RA according to the revised 1987 American Rheumatism Association criteria for< 6 months, and who had active disease despite methotrexate, disease-modifying anti-rheumatic drug, and/or glucocorticoid therapy at the time of screening. All donors were evaluated through medical histories, physical examinations, and routine clinical laboratory tests. Blood samples were obtained from 54 RA patients and 42 healthy controls. The levels of secretory IgD ?sIgD?, human soluble receptor activator of nuclear factor-?B ligand ?sRANKL? were determined in serum samples by ELISA. Peripheral blood mononuclear cells ?PBMCs? were isolated from peripheral blood of donors by ficoll gradient centrifugation. FLS was previously obtained from synovial tissue of a different patient with RA at the time of synovectomy. Membrane IgD ?mIgD? and IgDR on T/B cells and FLS were detected by using flow cytometry. The expression differences in two groups were compared, and the correlation between the biomarkers of disease and the sIgD level in RA serum were investigated. After PBMCs or FLS were cultured and treated with different concentrations of human IgD. The viability of PBMCs or FLS were measured by CCK-8, inflammatory cytokine production were assessed by inflammation antibody array. T cell subsets(CD³⁺/CD⁴⁺?CD³⁺/CD⁸⁺?CD⁴⁺/CD62L⁺?CD⁴⁺/CD154⁺+? CD⁴⁺/CD69⁺?CD³⁺/CD8^-/IL-17⁺?CD⁴⁺/CD25⁺/FoxP3⁺), B cell subsets?CD19+/CD21+? CD19+/CD23+? CD19-/CD138+? CD19+/IgM⁺/IgD^-? CD19+/IgD⁺? CD19+/CD27⁺? CD19+/IgD⁺/CD27-? CD19+/IgD⁺/CD27⁺? CD19+/IgD^-/CD27⁺? and IgDR expression were tested by flow cytometry. PBMCs were separated into CD⁴⁺ T cells using a magnetic cell separator. The protein expression of IgDR ? Lck and P-Lck were analyzed by western blot. The expression of IgDR in FLS was observed by confocal immunofluorescence microscopy. The intrinstic binding affinity of IgD on IgDR in CD⁴⁺ T cells or FLS was observed by fluorescence based receptor binding assay and scatchard analysis.Results:1. The expression of IgD and IgDR in RA patients and healthy controls.A significantly higher level of sIgD and sRANKL were detected in RA patients compared with healthy controls. The concentrations of sIgD were positively correlated with sRANKL, rheumatoid factor ?RF? and C-reactive protein ?CRP? in RA patients, but not correlated with erythrocyte sedimentation rate ?ESR? and anti-cyclic citrullinated peptide ?anti-CCP?. sIgD concentrations were positively correlated with sRANKL concentrations of all biomarkers ?P< 0.01?. Little mIgD was detected on total T cells (CD³⁺) and total helper T cells (CD³⁺CD⁴⁺). The percentage of CD³⁺CD⁴⁺ and mature B cells ?CD19+IgD⁺? was higher, while that of immature B cells ?CD19+IgM⁺IgD^-? was lower in RA patients than healthy controls. We established the detection method for IgDR through flow cytometry, and confirmed that IgDRs exist on not only T cells but also B cells. The expression of IgDR on CD³⁺ T cells and CD⁴⁺ T cells were significantly higher in RA patients ?P< 0.05? than those in healthy controls. There is no significant difference of IgDR expression on CD19+ B cells between RA patients and healthy controls2. The effects of IgD on the function of PBMCs.The proliferation of PBMCs increased in response to IgD in both RA patients and healthy controls in a concentration-dependent manner ?P< 0.05?. The response of PBMCs from RA patients to IgD was obviously stronger than that of healthy controls at the same time point. These results suggested that the optimal stimulation time was 24 h, and the optimal stimulation concentration ranges from 1 to 10 ?g/ml. The percentages of activated T cells (CD⁴⁺CD69⁺, CD⁴⁺CD154⁺; P< 0.05) were increased and the percentage of unactivated T cells (CD⁴⁺CD62L⁺; P< 0.05) was decreased after co-cultured with IgD ?10 ?g/ml? for 24h in RA patients. IgD had no significant effect on total helper T cells (CD³⁺CD⁴⁺) or total cytotoxic T cells (CD³⁺CD⁸⁺). In healthy controls, the effect of IgD on T cell subsets was similar with the results observed in RA patients. Interestingly it was observed that, the percentage of activated T cells (CD⁴⁺CD69⁺) in RA patients was higher than that in healthy controls. IgD could significantly increase the percentages of Th17 cells (CD³⁺CD8^-IL-17⁺) and reduce Treg cells (CD⁴⁺CD25⁺FoxP3⁺) of healthy controls, causing the imbalance of Th17/Treg. The percentages of activated B cells ?CD19+CD23+, CD19+CD21+?, mature B cells ?CD19+IgD⁺?, and plasma cells ?CD19-CD138+; P< 0.05? were increased when co-cultured with IgD ?10 ?g/ml? in RA patients. Consistent with healthy controls, the effect of IgD on B cell subsets was similar with the results observed in RA patients. Interestingly, the percentage of CD19+CD23+ was higher, and that of CD19+IgM⁺IgD" was lower in RA patients than in healthy controls. Also we observed that the percentage of immature B cells in healthy controls when co-cultured with IgD decreased significantly to a level which was similar to RA patients. Compared with the control group, the concentrations of IL-1?, IL-1?, TNF-?, IL-6, IL-8 and IL-10 were increased significantly in IgD ?10 ?g/ml? group of RA patients ?P<0.05?. The level of TNF-a in PBMCs supernatants of RA patients was significantly higher than that of healthy controls.3. The effects of IgD on the expression of IgDR and protein tyrosine kinase ?PTK? signaling.The percentage of CD³⁺IgDR⁺ T cells, CD³⁺CD⁴⁺IgDR⁺ T and CD19+IgDR⁺B cells showed a significant increase ?P<0.05? in PBMCs from both RA patients and healthy controls, which was caused by IgD. In particular, the percentage of CD³⁺IgDR⁺T cells and CD19+IgDR⁺B cells were increased by nearly 12 folds and 2.5 folds by IgD in RA patients respectively. CD⁴⁺ T cells were separated from healthy control and stimulated by IgD ?3 ?g/ml?. The expression of IgDR was increased in response to treatment with sIgD in a time-dependent and concentration-dependent manner, which was consistent with the finding by flow cytometry. Stimulating by sIgD resulted in enhanced phosphorylation of Lck compared with that in the medium control sample. The expression of Lck was not changed. As inhibitor of PTK, Herbimycin A ?1 ?M? or A770041 ?0.15 ?M?, which combined with IgD could significantly inhibit phosphorylation of Lck. Compared with control, the prolifertion promoting effect of IgD was blocked by Herbimycin A or A770041.4. The effects of IgD on the function of FLS.IgDR was constitutively expressed on FLS. We did not find the existence of mIgD on the surface of FLS. We further characterized the subcellular localization of IgDR in FLS from RA patients ?RA-FLS? and healthy controls ?HC-FLS? by confocal immunofluorescence microscopy. IgDR was principally localized in the cell membrane and cytoplasm. The Mean Fluorescent Intensity ?MFI? of IgDR was higher on FLS of RA patient compared with healthy control. IgD could slightly enhance FLS proliferation from RA patients after 24h, while it had no significant effect on FLS from healthy controls. To a lesser degree, the proliferation was also increased by IgD in healthy controls after 48h co-culture. The results described above clearly demonstrate the response of FLS from RA patients to IgD was obviously stronger than that of healthy controls at the same time point. Compared with unstimulated cells, secretion of IL-1?, IL-6, MCP-1 and TNF-a were highly induced by IgD ?10 ?g/ml? ?P?0.05?. The expression of IgDR on FLS was stimulated by IgD after 48 h co-culture in RA patients which analyzed by flow cytometry and western blotting. To determine whether the observed binding of IgD to FLS or CD⁴⁺T cells occurred with the characteristics of receptor-ligand interaction, experiments were designed to demonstrate receptor saturation and affinity of binding. Scatchard line of IgD^-IgDR binding on CD⁴⁺T cells was Y=-25.125X+137428 ?r=-0.937?. The KD value was 0.216 nM, Bmax value was 5470 arbitrary unit/104 cells. Scatchard line of IgD^-IgDR binding on FLS was Y=-80.224X+498634 ?r=-0.908?. The KD value was 0.067 nM, Bmax value was 6215 arbitrary unit/10⁴ cells. The binding affinity was higher on FLS.Conclusions:1. The expression of sIgD in RA patients were significantly higher than those in healthy controls. The concentrations of sIgD were positively correlated with sRANKL, RF and CRP in RA patients.2. mIgD was mostly expressed on B cells, it was not existed on T cell and FLS. IgDR were constitutively expressed on T/B cells and FLS, the expression of IgDR in RA patients were significantly higher. The KD values of IgD^-IgDR binding on CD⁴⁺T cells and FLS were 0.216 n M and 0.067 n M, seperately. The binding affinity was higher on FLS.3. IgD could enhance the proliferation of PBMCs, induce the production of cytokines, and activate T and B cells and simultaneously promoted the expression of IgDR and p-Lck, which were more sensitive in RA patients.4. IgD-IgDR crosslinking contributes to the activation of RA-FLS. IgD represents a potentially novel immunotherapeutic target for the management of RA.