CD100 Contributes To Antibody Production In Bullous Pemphigoid And The Underlying Mechanism

BackgroundBullous pemphigoid(BP)is a common autoimmune skin disease,which is characterized by epidermis blisters,the infiltration of eosinophils,neutrophils and other inflammatory cells and deposition of autoantibodies and complement in basement membranes.Specific autoantibodies against basal membrane antigens(BP180 and BP230)of the epidermis basement membrane after activation of autoreactive B cells are critical for the pathogenesis of BP.At present,the production of autoantibodies relies on the activation of B cells with the help of helper T cells(Th)followed by differentiation into plasma cells to synthesize various immunoglobulins in humoral immunity.In the process of autoantibody production,on the one hand,the B cell surface receptor(BCR)captures antigen providing the first signal for B cell activation.On the other hand,the interaction of Th cell and B cell providesthe second essential signal for B cell activation.At the same time,on the surface of B-cell membranes,there are co-stimulatory molecules involving in the regulation of B-cell activation.Therefore,exploring the co-stimulatory molecules that affect the activation of B-cells is helpful for deep analysis of the key links of B-cell activation and antibody production,and is of great significance for elucidating the pathogenesis of BP and exploring new therapeutic strategies.CD100(Semaphorin 4D)a type I membrane molecule,is a member of the Semaphorin family and is the firstsemaphorin protein found to have immunomodulatory properties.The CD100 molecule is a cell membrane surface protein with a full length of 150-kDa that can be cleaved by a variety of proteolytic enzymes and detached from the cell membrane surface to form a 120-kDa free-form molecule(sCD100),and this free form of CD100 has the same biological activity as membrane type CD100.CD100 receptors include CD72,Plexin-B1and Plexin-B2,of which CD72 is mainly expressed on the surface of B lymphocytes.Studies have shown that CD72 is one of the important negative co-stimulatory molecules on the surface of B lymphocytes and can inhibit B lymphocyte activation and proliferation by negatively regulating BCR signaling.The binding of CD100 to CD72 on the surface of B cells can turn off the inhibitory signal of CD72 on BCR and participate in B cell activation,proliferation,differentiation and maturation.Therefore,we propose the following hypothesis:The sCD100 in serum and blisters of BP patients can promote the activation,proliferation and differentiation of B cells through the negative co-stimulatory molecule CD72 on the surface of B cells,contributing to antibody production in BP.Objective:1.The expression of CD100 in serum,blister,and local lesions of BP patients was analyzed to determine the correlation between CD100 and BP;2.To clarify the role of CD100 in pathogenic autoantibody production in patients with BP and to elucidate its specific mechanism.Methods:1.The serum,blister,and skin lesions of BP patients were collected.The expression of CD100 in serum,blister,and local lesions of BP patients was detected by ELISA,Western blot,immunohistochemistry,and tissue immunofluorescence.2.To evaluate the BPDAI score of BP patients and determine the anti-BP180 and anti-BP230 antibody titers in BP patients as a clinical basis to reflect the patient's disease activity and severity,and then to analyze the correlation with sCD100 concentrations in serum and sputum of BP patients.3.Different concentrations of CD100 recombinant protein were used to stimulate PBMC of BP patients.After stimulation,cell culture supernatants were collected at different time points.ELISA was used to detect the titer of anti-BP180 antibody in cell culture supernatants,and the optimal concentration and stimulation time of antibodies to PBMC stimulated by CD100 recombinant protein were selected.On this basis,PBMC from BP patients were stimulated with the optimal concentration of CD100 recombinant protein.The anti-BP180 and antibody BP230 antibody titer of the cell culture supernatant was measured by ELISA at the best stimulation time,and it was clarified that CD100 produced pathogenic autoantibodies in BP patients.4.The PBMC of BP patients were stimulated with CD100 recombinant protein.The cell pellet was collected by centrifugation.The differentiation of B cells before and after CD100 stimulation was detected by flow cytometry.The intracellular cytokine staining and flow cytometry were used to screen the downstream pathway of CD100-stimulated PBMC activation.And use inhibitors verify the downstream pathway of CD100-activated B cells.5.PBMC,granulocytes and blister cells from patients with BP were collected.Flow cytometry was used to detect the expression of CD100 on the surface of different cell subpopulations.The cell origin of free sCD100 in BP patients was determined.Granulocytes were treated with different proteolytic enzymes to clearly cleave the cell membrane.Surface CD100 proteolytic enzymes and proteolytic enzyme specific inhibitors were used to verify their cleavage function.Conclusion:During the pathogenesis of BP,ADAM10 can cleavage CD100 on the surface of CD15+cells in patients with BP,which is an important source of high levels of sCD100 in BP patients'serum and blister.Elevated sCD100 activates the Akt/NF-?B/Erk signaling pathway by binding to CD72 on the surface of B cells and induces CD19~+CD138~-priming B cells developing into CD19~+CD138~+plasmablasts with antibody secretion function,leading to autoimmune skin damage.Our study found that there was a high level of sCD100in serum and blister of BP patients,and it was clarified that sCD100 was derived from the shearing effect of ADAM10 on CD15+granulocytes,confirming that CD100 is involved in the production of BP pathogenic autoantibodies.The results enrich the immunological mechanism of BP pathogenic autoantibody production and provide a theoretical basis for exploring new therapeutic strategies for BP.Result:1.The sCD100 levels in serum and blister of BP patients were significantly higher than those in healthy controls,and were positively correlated with the severity and activity of BP patients.Immunohistochemistry and tissue immunofluorescence showed subepidermal blisters and superficial dermis in BP patients,and a large number of inflammatory cells such as eosinophils and neutrophils were infiltrated around subepidermal blisters and the layer of blood vessels.2.CD100 recombinant protein can induce BP180~+BP230~-PBMCs producing anti-BP180 antibodies,and BP180~+BP230~+PBMCs producing anti-BP180 and BP230 antibodies,while CD100 antibody can significantly inhibit CD100 protein stimulation of PBMCs to produce pathogenic autoantibodies.3.CD100 recombinant protein activates PBMC via Akt/NF-?B/Erk pathway to produce pathogenic autoantibodies.Small molecule inhibitors of Akt/NF-?B p-65/Erk can significantly inhibit CD100 recombinant protein stimulation of PBMC to produce pathogenic autoantibodies.4.CD100 recombinant protein can stimulate the differentiation of CD19~+CD138~-B cells into CD19~+CD138~+plasmablasts in PBMCs.5.High levels of sCD100 in serum and blister from patients with BP were derived from the cleavage of CD100 on the CD15+granulocyte membrane by ADAM10 proteolytic enzymes.