CP-25 Attenuates the Activation of CD4+ T Cells Stimulated with Immunoglobulin D in Human.

Researchers have shown that the level of immunoglobulin D (IgD) is often elevated in patients with autoimmune diseases. The possible roles of IgD on the function of human T cell activation are still unclear. Paeoniflorin-6'-O-benzene sulfonate (code: CP-25), the chemistry structural modifications of paeoniflorin, was a novel drug of anti-inflammation and immunomodulation. The aims of this study were to determine if human CD4+ T cells could be activated by IgD via the IgD receptor (IgDR)-Lck pathway and whether the novel compound CP-25 could affect the activation of T cells by regulating Lck. Human CD4+ T cells were purified from peripheral blood mononuclear cells using microbeads. T cell viability and proliferation were detected by Cell Counting Kit-8 and CFSE Cell Proliferation Kit. Cytokines secreted by T cells were assessed with the Quantibody Human Inflammation Array. The binding affinity and expression of IgDR on T cells were detected by flow cytometry, and protein expression of IgDR, Lck, and P-Lck were analyzed by western blot. IgD was shown to bind to IgDR on CD4+ T cells in a concentration-dependent manner and stimulate the activation and proliferation of these cells by enhancing phosphorylation of the activating tyrosine residue of Lck (Tyr394). CP-25 inhibited the IgD-stimulated activation and proliferation of CD4+ T cells, as well as the production of inflammatory cytokines; it was thus suggested that this process might be related to the downregulation of Lck (Tyr394) phosphorylation. These results demonstrate that IgD amplifies the activation of CD4+ T cells, which could be mediated by Lck phosphorylation. Further, CP-25, via its ability to modulate Lck, is a novel potential therapeutic agent for the treatment of human autoimmune diseases.

The immunoglobulin D Fc receptor expressed on fibroblast-like synoviocytes from patients with rheumatoid arthritis contributes to the cell activation.

Immunoglobulin IgD might play an important role in autoimmune diseases, but the function of IgD has remained elusive, despite multiple attempts to define its biological function. Fibroblast-like synoviocytes (FLSs) are specialized cells of the synovium that play a key role in the pathogenesis of rheumatoid arthritis (RA). In this study we explored the possible roles of excessive IgD expression on the function of FLSs from RA patients (RA-FLSs). We showed that IgD Fc receptor (IgDR) was constitutively expressed on FLSs, and was significantly elevated in RA-FLSs compared with FLSs prepared from synovial tissues of healthy controls (HC-FLSs). Furthermore, IgDR was mainly detected on the cell surface and in the cytoplasm. We further detected the intrinsic binding affinity of IgD to IgDR on HC-FLSs with an equilibrium dissociation constant (KD) of 0.067 nmol/L. Incubation of RA-FLSs with IgD (1-10 µg/mL) for 48 h dose-dependently promoted the expression of IgDR, and stimulated the production of inflammatory cytokines and chemokines, such as IL-1ß, IL-6, monocyte chemotactic protein (MCP)-1, TNF-α and receptor activator of nuclear factor-κB ligand (RANKL), thus potentially contributing to IgD-IgDR crosslinking. Moreover, incubation with IgD (0.1-10 µg/mL) for 48 h dose-dependently enhanced viability for both HC-FLSs and RA-FLSs. Our results demonstrate that IgDR is expressed on RA-FLSs and contributes to the activation of FLSs, and suggest that IgD-IgDR is a potential novel immunotherapeutic target for the management of RA.

Effects of immunoglobulin D on expression of IgD receptor and protein tyrosine kinase signaling in human CD4+ T cells / 中国药理学与毒理学杂志

OBJECTIVE To observe whether human CD4 + T cells could be activated by immuno-globulin D (IgD) via IgD receptor(IgDR)-Lck. METHODS Human CD4+ T cells were purified from peripheral blood mononuclear cells (PBMCs) with microbeads. The viability of T cells were detected by CCK-8. The binding affinity and expression of IgDR on T cells were detected by flow cytometry. The protein expression of IgDR, Lck and P-Lck were analyzed by western blot. RESULTS IgD could concentration-dependent bind to IgDR on CD4+ T cells. The expression of IgDR was increased in response to treatment with IgD in a time- dependent and concentration- dependent manner. Stimulating by IgD 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 or A770041, which combined with IgD could significantly inhibit phosphorylation of Lck(Tyr394). The proliferation promoting effect of IgD was blocked by Herbimycin A or A770041. IgD could stimulate CD4+ T cell activation and proliferation through upreg?ulating activating tyrosine residue of Lck (Tyr394) phosphorylation. CONCLUSION These results demon?strate that IgD exaggerates CD4+T cell activities, which may be through promoting Lck phosphorylation.

Function of IgD on lymphocyte activation and effect of hIgD-Fc-Ig fusion protein on human PBMC proliferation / 中国药理学与毒理学杂志

OBJECTIVE This study aimed to investigate the influence of IgD on T/B cell activationand construct hIgD-Fc-Ig fusion protein to competitive inhibition IgD binding with IgDR. METHODS T/B cells were sorted by magnetic cell sorting. The differences of mIgD and IgD-R level between different T/B cell subtypes were detected by FCM. Serum IgD level was detected by ELISA. Human IgD-Fc-IgG1- Fc sequence was amplified by cross- PCR and then subcloned into PET28a(+ ) empty vector. After prokaryotic expression through escherichia coli, we obtained the hIgD-Fc-Ig fusion protein by affinity chromatograph. Western blot was used to identify the hIgD- Fc- Ig fusion protein. Human peripheral blood monouclear cells (PBMC) and fibroblast like synoviocytes (FLS) proliferation were detected using a cell counting kit-8 (CCK-8). RESULTS The percentage of CD3+/CD4+, CD3+/IgD+, CD3+/CD4+/IgD+, CD3+/IgD-R+ and CD3+/CD4+/IgD-R+ cells increased significantly in RA patients comparing to healthy people. IgD can stimulate PBMC proliferation. IgD (1, 3, 10, 30 μg·mL-1) stimulate PBMC proliferation significantly after 24 h. We obtained stable and active hIgD-Fc-Ig fusion protein. The hIgD-Fc-Ig fusion protein showed no effect on PBMC proliferation. But it could downregulate human IgD protein promoting proliferation effects in human PBMC. CONCLUSION This result suggests that IgD and IgDR play an important role on T/B cell activation in RA patients and the hIgD-Fc-Ig fusion protein may competitively inhibit IgD's function and may play an therapeutic role in autoimmune diseases.