The Expression And Proliferation Effect Of IgD In B Cell Non-Hodgkin Lymphoma And Partial Mechanism

Non-Hodgkin's lymphoma ?NHL? divides into B-cell lymphoma and T cell/NK cell lymphoma. B cell Non-Hodgkin's lymphoma ?B-NHL? is a type of lymphoma affecting B cells, more than 85% of NHL in the world is the mature B-NHL. B-NHL is classified into several different subtypes, including diffuse large B-cell lymphoma ?DLBCL? and Burkitt lymphoma ?BL?. DLBCL is the most common B-NHL, accounting for 30%-40% of adult NHL and less than 5% of children NHL. BL mostly occurs in children and adolescents, accounting for 3%-5% of all NHL and 40% of children NHL. Both DLBCL and BL are aggressive BCL, with increased incidence rates in immunocompromised patients. Combined chemotherapy is currently the most effective treatment for DLBCL and BL patients. However, some patients are unable to tolerate the numerous adverse events associated with immune chemotherapy, and others even die with tumor progression. Therefore, looking for more innovative methods of treatment and discovering new therapeutic targets have become important in recent studies. There are two forms of immunoglobulin D ?IgD?:secretory IgD ?sIgD? and membrane-bound IgD ?mIgD?. IgD plays an important role in antigen recognition, B cell activation and differentiation and immune regulation response. It also has some connection with the clearance of outer periphery B lymphocytes and interferes with immune tolerance. IgD receptor ?IgDR? binding with IgD can mediate the enhancement of autoreactive B cells, the activation of B cell, as well as the interaction of B and T cells. Due to these significant roles, IgD and IgDR may become new therapeutic targets in malignant B cell proliferation diseases. Consequently, blocking the interaction between IgD-IgDR may become a new strategy of B-NHL immunotherapy.Pathological lymph node and blood samples of newly diagnosed DLBCL patients were collected for this research. We then detected the expressions of IgD and IgDR using different methods, such as immunohistochemistry ?IHC?, flow cytometry ?FCM?, and enzyme-linked immunosorbent assay ?ELISA?. We chose Daudi, MEC-2 and REC-1 cells for in vitro experiments to investigate the proliferative effect of human IgD ?hIgD? protein, explore the role of IgD and IgDR in the development of lymphoma, and to provide experimental evidence in support of IgD as a possible new target.Purpose:We applied pathological lymph node and blood samples of DLBCL patients to observe the expressions of IgD and IgDR, and to analyze the relationship of these expressions with the clinicopathological parameters. We chose Daudi, MEC-2 and REC-1 cells for in vitro experiments to investigate the proliferative effect of human IgD ?hIgD? protein, explore the role of IgD and IgDR in the development of lymphoma, and to provide experimental evidence in support of IgD as a possible new target.Methods:In order to observe the expressions of IgD and IgDR in DLBCL patients,34 cases pathological lymph node tissue samples of DLBCL patients and 14 cases pathological lymph node tissue samples of reactive lymphoid hyperplasia patients,16 cases blood samples of DLBCL patients and 10 cases blood samples of healthy volunteers were collected. All clinical data were completed. The expressions of IgD and IgDR on pathological tissue slices were detected by IHC. Serum sIgD levels were detected by ELISA. The expressions of IgD and IgDR on peripheral blood mononuclear cells ?PBMCs? were detected by FCM. Chi-square test ?Fisher exact test? and Spearman correlation analysis were used to evaluate the relationship between IgD, IgDR and clinicopathological parameters.In order to observe the promoting proliferation effect of hIgD protein on Daudi, MEC-2 and REC-1 cells and study the partial mechanism of hIgD on Daudi cells, logarithmically growing cells were collected. Cell proliferation was evaluated by cell counting kit-8 ?CCK-8? assay. The expression of IgDR, surface markers for Daudi, as well as cell cycle and apoptosis were measured by FCM. The expressions of c-myc, cyclin D3, CDK6, and p16^INK4a mRNA of Daudi cells were measured by real-time quantitative PCR ?QRT-PCR?, and the protein expressions of c-myc, cyclin D3, CDK6, p16^INK4a,tyrosine phosphorylation of 70kD protein, p-Lyn and Lyn of Daudi cells were measured by Western blot assay.Results:1. The expressions of IgD and IgDR in DLBCL patients were correlated with clinicopathological parameters.The IHC results showed that:IgD and IgDR in pathological lymph node tissue samples of DLBCL patients localized in the tumor cell membrane/pulp. Positive expression of IgD in 34 cases of DLBCL patients' pathological lymph nodes was 47.1% ?16/34?, with the IgDR positive expression rate of 55.9%?19/34?. Compared with reactive hyperplasia of lymph node, the expressions of IgD and IgDR in DLBCL patients were up-regulated ?P<0.05? and IgD expression was significantly associated with IgDR expression ?r=0.426, P=0.02?. Further correlation analysis found that IgD positive expression was correlated with serum ?2-microglobulin ??2-MG? levels ?r=0.408, P=0.035?, IgDR positive expression correlated with serum LDH levels ?r=0.46,P=0.013? and International Lymphoma Prognostic Index ?IPI? score ?r=0.407, P=0.025?. ELISA results showed that, compared with healthy volunteers, serum IgD in DLBCL patients ?26.15±1.7 ?g/ml? was significantly increased ?P=0.014?, and correlated with serum p2-MG ?r=0.574, P=0.02? and LDH levels ?r=0.558, P=0.024?. FCM showed that IgD mean fluorescence intensity ?MFI? ?16.49 ± 2.08 vs.10.44±1.89? and IgDR ?13.33±1.83 vs.6.74±1.01? MFI of peripheral blood PBMCs from DLBCL patients were significantly higher than that of healthy controls. From the results, the expressions of IgD and IgDR in DLBCL patients were up-regulated, and were associated with adverse outcomes. In brief, IgD and IgDR may be in an abnormal activation state in DLBCL patients and may be involved in the disease processes of DLBCL.2. hlgD promoted the proliferation of Daudi, MEC-2 and REC-1 cells, accelerated G1/S transition and the cell cycle process of Daudi cells.Daudi cells in exponential growth were stimulated with different concentrations of hIgD ?0.1,0.3,1,3,10 u?g/ml? at different times ?12,24,48,72h?. Meanwhile MEC-2 and REC-1 cells were stimulated with hlgD ?3 ?g/ml? for 48 h in exponential phase. The results showed that, compared with the control group, hIgD significantly promoted Daudi, MEC-2 and REC-1 cell proliferation; Annexin-V/PI staining showed hIgD ?3 ?g/ml? reduced the apoptosis rate of Daudi cells; Cell cycle detection with PI staining showed hIgD played a role in proliferation via acting on G1 and S phases of the cell cycle. Compared with the control group, the proportion of Daudi cells in G1 phase decreased, while the cells in S-phase gradually increased after hIgD stimulation, however, the proportion of cells in G2 phase had no significant change. These results suggest that hIgD promotes Daudi cell cycle progression by accelerating G1/S phase transition.We further detected mRNA and protein expression of c-myc, cyclin D3, CDK6 and p16^INK4a factor of Daudi cells. Q-PCR and Western results showed that compared with the control group, c-myc, cyclin D3, CDK6 mRNA and protein expressions in Daudi cells were significantly increased, and p16^INK4a expression was significantly reduced after hIgD stimulation. These results suggest that hIgD accelerates G1/S phase transition, induces cell cycle progression, promotes Daudi cell proliferation, and these functions may be exerted by regulating c-myc and cyclin D3-CDK6-p16^INK4a expressions.3. Inducing IgDR expression and starting tyrosine phosphorylation pathway may be the important mechanisms of hIgD in promoting Daudi cell proliferationFCM analysis showed that Daudi cells are immature B cells with CD19+, IgD-IgM+and IgDR+. Compared with the control group, the expression of IgDR on Daudi cells was induced after hIgD ?1,3,10 ?g/ml? stimulation for 24 h. Western blot analysis showed that, compared with the control group, a 70kD protein was tyrosine phosphorylated in Daudi cells after hIgD stimulation. As a result, we hypothesized that this protein would be a component of IgDR.Finally, we detected the downstream Lyn signal and found that p-Lyn expression was significantly increased while total Lyn protein did not change markedly. Therefore, we speculate that hIgD promotes Daudi cell proliferation by combining with IgDR and activate tyrosine phosphorylation pathway.Conclusion:1. The expressions of IgD and IgDR in pathological lymph node samples of DLBCL patients were significantly up-regulated and significantly associated with each other. Similar results were recorded for serum levels of ?₂-MG, LDH or IPI score. Serum IgD level in DLBCL patients were significantly increased and correlated with serum ?2-MG and LDH levels. The expressions of IgD and IgDR in PBMCs of DLBCL patients were up-regulated. These results suggest that IgD and IgDR may be abnormally activated in DLBCL patients and may be involved in the pathogenesis of DLBCL.2. hIgD promoted the proliferation of Daudi, MEC-2 and REC-1 cells, inhibited the apoptosis of Daudi cells, reduced the proportion of Daudi cells in G1 phase, and increased the proportion of cells in S phase. Meanwhile, hIgD increased the mRNA and protein expressions of c-myc, cyclin D3, CDK6, and decreased pi6INK4a expression. These results suggest that hIgD accelerates G1/S phase transition, induces cell cycle progression, and promotes Daudi cell proliferation. These functions may be exerted by regulation of c-myc and cyclin D3-CDK6-p16^INK4a expressions.3. IgDR expression on Daudi cell membrane was obviously up-regulated and 70kD protein was tyrosine phosphorylated with hIgD stimulation. p-Lyn expression was significantly increased while total Lyn protein level did not change significantly. Therefore we propose that hIgD may promote Daudi cell proliferation by combining with IgDR and activate tyrosine phosphorylation pathway.