Effect Of IgD On The Activation Of B Cell Function Through IgDR-Lck Signaling On T Cell Of RA Patients And Effect Of IgD-Fc-Ig Fusion Protein

IntroductionRheumatoid arthritis?RA?is a chronic,systemic disease with unknown etiology,mainly characterized with inflammatory synovitis.Inclinical,the drugs for RA treatment roughly divided into three categories:disease-modifying anti-rheumatic drugs?DMARDs?,steroid anti-inflammatory drugs?SAIDs?and non-steroids Anti-inflammatory drugs?NSAIDs?.The adverse drug reactionscome out after long-term treatment of the drugs.With further understanding of the inflammatory immune pathway,some new drugs targeting inflammatory cytokines are used to treat RA,such as B cells,T cell co-stimulation,tumor necrosis factor-??TNF-??,etc.The newdrugs cannot avoidserious adverse reactions either.Immunoglobulin D?IgD?is an immunoglobulin,which was discovered in 1965.It consists of two identical light and heavy chain,including secreted IgD?s IgD?and membrane-bound IgD?m IgD?.IgD acts like a modulator by binding to its Fc receptor IgD receptor?IgDR?.IgDR was first discovered on T cells and non-T cells of human peripheral blood in 1980,but its gene has not been cloned so far,and the gene sequence is still unknown.The interaction of IgD with IgD receptor?IgDR?on T cells can lead to an increase in antigen-specific T cell clones,and the cross-linking of membrane-bound IgD?m IgD?on B cells and IgDR on T cells,which can inhibit T cell apoptosis.Previous reports suggested that IgDR may be a dual role regulator in T-B cell interaction.Our group found that the levels of s IgD and m IgD in peripheral blood of RA patients were higher than those in healthy controls.IgD could increase the percentage of activated T cells in peripheral blood of RA patients in vitro.IgD could promote the abnormal activation of CD4⁺T cells,imbalance of Th1/Th2/Th17/Treg ratio in healthy controls and increase the phosphorylation of lymphocyte-specific protein tyrosine kinase?Lck?,suggesting that IgD may induce IgDR expression through one or more protein tyrosine kinase?PTK?activation pathways.The PTK activation may involve Lck,zeta-chain associated protein kinase?ZAP70?,and phosphatidylinositol 3-kinase?PI3K?of the Src family.Taken together,IgD may participate in the abnormal activations of T and B cells of RA patients through IgDR.IgD-IgDR may become an ideal target for treating RA,but its specific molecular mechanism is unclear and needs further study.In order to block the IgD-IgDR pathway uniquely,our group has successfully linked the human IgD-Fc segment to the human Ig G1-Fc segment and purified the fusion protein IgD-Fc-Ig,which has been authorized by the Chinese invention patent?201510600762X?.We have proposed hypothesis as follow:during the development of RA,does IgD activate the downstream signaling pathway?Lck-ZAP70-PI3K?and T cells through the interaction between IgDR and Lck?Do activated T cells further promoting the activation of B cells by interacting with B cells?Does IgD-Fc-Ig inhibit the activations of T and B cells by blocking IgDR-Lck and downstream signaling pathways?In this study,CD4⁺T cells and CD19⁺B cells of RA patients and healthy controls were collected and seperated.Flow cytometry,cell co-culture,immunoblotting,and laser confocal techniques were used to detect the function of IgD to activate B cells through IgDR-Lck signals on T cells and the effect of IgD-Fc-Ig.Collagen-induced arthritis?CIA?model was established to further explain the effect of IgD-induced CD4⁺T cells on the function of CD19⁺B cells and the role of IgD-Fc-Ig in CIA mice.Our study clarify the pathological role of abnormally highly expressed IgD in RA,and also provide experimental evidence for the development of IgD-Fc-Ig for highly selective treatment of RA.ObjectiveTo study the molecular mechanisms of IgD on T and B cells activations through the IgDR-Lck-ZAP70-PI3K signaling pathway.To reveal the effect and part of the mechanism of IgD-Fc-Ig on T and B cells activations in RA patients and CIA mice.MethodsPeripheral blood of healthy controls and RA patients were collected and biochemical reports as well as DAS28 scores of RA patients were recorded.ELISA method was used to detect plasma IgD levels of healthy controls and RA patients.Analysis of the correlation between plasma IgD levels and biochemical indicators,DAS28 scores in RA patients.Peripheral blood mononuclear cells?PBMCs?from healthy controls and RA patients were obtained by density gradient centrifugation,CD4⁺T cells and CD19⁺B cells selected from PBMCs were collected by flow sorting.Laser confocal and co-immunoprecipitation methods were used to detect the effect of IgD-Fc-Ig on the co-expression of IgDR and Lck signal on IgD induced CD4⁺T cells of healthy controls.Western blot method was used to detect the effect of IgD-Fc-Ig on the expression of p-Lck,ZAP-70,p-ZAP70,PI3K,andp-PI3K proteins on IgD induced CD4⁺T cells of healthy controls.CCK-8 method was used to detect the effect of IgD-stimulated CD4⁺T cells on the viability of CD19⁺B cells in healthy controls and RA patients.Western blot method was used to detect the expression of CD40L and CD40 protein on human CD4⁺T and CD19⁺B cells.Flow cytometry was used to detect the effect of IgD-Fc-Ig on the expression of CD86 on IgD induced CD19⁺B cells in RA patients.Establishing CIA mice models,and the DBA/1 mice were randomly divided into9 groups,the normal group,the model group,the four dose groups of IgD-Fc-Ig?1.625,3.25,6.5,13 mg/kg?,and the positive controls IgD antibody?2mg/kg?and Etanercept?rh TNFR:Fc,4.5mg/kg?groups,and Ig G1-Fc?13mg/kg?negative control group,per group had 10 mice.ELISA method was used to detect the plasma IgD levels of normal and CIA mice.Immunohistochemistry was used to detect the effect of IgD-Fc-Ig on the expression of IgD,CD40L and CD40 proteins on spleen tissues of mice.Western blot method was used to detect the effect of IgD-Fc-Ig on the expression of CD40L protein and CD40 protein on mouse T and B cells.ELISA method was used to detect the IL-17 level in the supernatant of CD4⁺T cells after treated with IgD-Fc-Ig.Results1. Correlation of plasma IgD levels with biochemical indicators,DAS28 scores in RA patientsThirty RA patients were collected,24 female patients and 6 male patients,aged29 to 73 years,the clinical normal range of anti-cyclic citrullinated peptide?Anti-CCP,RU/ml?is 0-25,and the Anti-CCP values of 30 RA patients are all beyond the normal range.Plasma IgD level in RA patients was significantly higher than that in healthy controls?60.40mg/L±7.64 vs.7.09mg/L±0.48?;IgD plasma concentration was set to be higher than 80mg/L for IgD high expression population,16 RA patients expressed high IgD,and the plasma IgD level of RA patients had significant correlation with Anti-CCP level and DAS28 scores.2. Effect of IgD-Fc-Ig on co-expression of IgDR and Lck signals on IgD induced CD4⁺T cells of healthy controlsCo-immunoprecipitation and laser confocal results show that,under normal conditions,IgDR and Lck signals were co-expressed on CD4⁺T cells,CD4⁺T cells from healthy controls stimulated with IgD?3?g/ml?up-regulated the co-expression of IgDR and Lck signals in vitro for 24 hours.IgD-Fc-Ig?10?g/ml?group and positive control drug Lck inhibitor A770041?10?g/ml?group inhibited the effect of IgD to up-regulate the co-expression of IgDR and Lck signals.3. Effect of IgD-Fc-Ig on expression of p-Lck,ZAP-70,p-ZAP70,PI3K,p-PI3K proteins on IgD induced CD4⁺T cells of healthy controls CD4⁺T cells from healthy controls stimulated with IgD?3,10?g/ml?significant-ly up-regulated the expressions of p-Lck,p-ZAP70,and p-PI3K proteins in vitro for24 hours.IgD-Fc-Ig?10?g/ml?group and positive control drug Lck inhibitor A770041?10?g/ml?group significantly inhibited the effect of IgD to up-regulate the expressions of these three proteins.There were no significant changes on the expressions of ZAP70 and PI3K proteins in each treatment group.4. Effect of IgD-stimulated CD4⁺T cells on CD19⁺B cell proliferation in healthy controlsCD4⁺T cells from healthy controls stimulated with IgD?1,3,10?g/ml?induced the proliferation of CD19⁺B cells after co-cultured with CD19⁺B cells in vitro for 24or 48 hours.5. Effects of IgD on expression of CD40L protein on CD4⁺T cells and CD40protein on CD19⁺B cells in healthy controlsCD4⁺T cells from healthy controls stimulated with IgD?3,10?g/ml?up-regulated the expressions of CD40L and CD40 proteins after co-cultured with CD19⁺B cells in vitro for 48 hours.6. Effect of IgD-stimulated CD4⁺T cells on CD19⁺B cell proliferation in RA patientsCD4⁺T cells from RA patients stimulated with IgD?1,3,10?g/ml?induced the proliferation of CD19⁺B cells after co-cultured with CD19⁺B cells in vitro for 24 or48 hours.7. Effects of IgD-Fc-Ig on expression of CD40L protein on IgD induced CD4⁺T cells and CD40 protein on CD19⁺B cells in RA patientsCD4⁺T cells from RA patients stimulated with IgD?3,10?g/ml?up-regulated the expressions of CD40L and CD40 proteins after co-cultured with CD19⁺B cells in vitro for 48 hours.IgD-Fc-Ig?3,10?g/ml?group inhibited the effect of IgD to up-regulate the expressions of CD40L and CD40 proteins.8. Effect of IgD-Fc-Ig on expression of CD86 protein on IgD induced CD19⁺B cells in RA patientsCD4⁺T cells from RA patients stimulated with IgD?10?g/ml?up-regulated the expression of stimulatory factor CD86 after co-cultured with CD19⁺B cells in vitro for 48 hours.IgD-Fc-Ig?3,10?g/ml?group inhibited the effect of IgD to up-regulate the expression of stimulatory factor CD86.9. Plasma IgD level in normal and CIA miceCompared with normal mice,plasma IgD level in CIA mice was significantly higher?50.19mg/L±15.58 vs.25.58mg/L±7.31?.1 0. Effects of IgD-Fc-Ig on expression of IgD,CD40L and CD40 proteins in spleen tissues of miceThe expressions of IgD,CD40L and CD40 proteins in spleen tissues of model group were significantly higher than those of normal group.Positive control drug Etanercept?4.5mg/kg?and anti-IgD antibody?2mg/kg?group down-regulated the expressions of IgD,CD40L and CD40 proteins in spleen tissues.While,IgD-Fc-Ig?3.25,6.5,13mg/kg?down-regulated the expressions of IgD,CD40L and CD40proteins in spleen tissues,the negative control group Ig G₁-Fc?13mg/kg?had no significant effect.11.Effects of IgD-Fc-Ig on expression of CD40L protein on IgD induced CD4⁺T cells and CD40 protein on CD19⁺B cells in CIA miceCD4⁺T cells from CIA mice stimulated with IgD?3?g/ml?up-regulated the expressions of CD40L and CD40 proteins after co-cultured with CD19⁺B cells in vitro for 48 hours.IgD-Fc-Ig?10?g/ml?group and positive control drug Lck inhibitor A770041?10?g/ml?group inhibited the effect of IgD to up-regulate the expressions of CD40L protein and CD40 proteins.Ig G₁-Fc protein?10?g/ml?group had no significant effect on IgD.12.Effect of IgD-Fc-Ig on the level of cytokine IL-17 in the supernatant of IgD induced mice cellsCD4⁺T cells from CIA mice stimulated with IgD?3?g/ml?up-regulated the level of cytokine IL-17 after co-cultured with CD19⁺B cells in vitro for 48 hours.IgD-Fc-Ig?3,10?g/ml?group and positive control drug Lck inhibitor A770041?10?g/ml?group inhibited the effect of IgD to up-regulate the level of IL-17.Ig G₁-Fc protein?10?g/ml?group had no significant effect.Conclusions1.IgD can induce the activation of T cells by affecting the IgDR-Lck-ZAP70-PI3K signaling pathways on T cells.2.IgD-activated T cells can induce the activation of B cells via increasing the expression of co-stimulatory molecules CD40-CD40L after T-B cell co-culture.3.IgD-Fc-Ig fusion protein can inhibit the activations of T and B cells via blocking the IgD-IgDR-Lck-ZAP70-PI3K signaling pathways.