The Mechanism Of Hyper-immune Response In Tonsillar Mononuclear Cells In Patients With IgA Nephropathy

IgA nephropathy (IgAN), which is characterized by mesangial deposits of IgA, is the most common form of primary glomerulonephritis worldwide.The course of IgAN was initially considered benign, but now it is recognized that progress to end-stage renal failure often occurs in these patients over a period of 20 years.The disease often gets worse after and/or during upper respiratory tract infections, particularly, acute tonsillitis. Tonsillectomy has been reported to be an effective treatment for IgAN patients.Recently, clinical comparison studies between tonsillectomy patients and non-tonsillectomy patients observed for over 10 years showed that renal survival rates of tonsillectomy patients were statistically higher than those of non-tonsillectomy patients, concluding that tonsillectomy was an independent factor for long-term renal survival in patients with IgAN. Therefore, IgAN is now recognized as a tonsil-related diseases.Several alterations related to IgA production in IgAN have been reported:namely, elevation of serum IgA, increases in circulating IgA immune complexes, increases in IgA-bearing cells in peripheral blood, and enhancement of in vitro IgA production by peripheral blood mononuclear cells.Thus, it is believed that hyperproduction of IgA plays a key role in the pathogenesis of IgAN.Initial occurrence of IgAN pathogenesis is known to occur by aberrant mucosal immune response of upper respiratorytracts, especially of tonsils, to chronic environmental antigens from bacteria and viruses.In IgAN patients, streptococcal M proteins, which are the most common pathogens of acute tonsillitis, provoke cytokine TGF-βproduction by lymphocyte that enhances IgA producing plasma cells increased. H. parainfluenzae antigens also provoke IgA production of tonsillar lymphocytes. A variety of viruses have been related to exacerbation of IgAN.In mice models,coxsackie B4 virus inoculated repeatedly into mice induces depositions of IgA and viral gene in the mesangium similar to IgA nephropathy.These facts emphasize that the innate immune response may be involved in the onset of IgAN.In our previous research,we found that, a-hemolytic streptococci is the most common bacterium in tonsils crypt. And J chain mRNA-positive IgA-secreting cells increased stimulated with heat-inactivated a-Hemolytic streptococcus in patients with IgA nephropathy.So we choose Lipopolysaccharide and a-Hemolytic streptococcus to mimic the immunostimulatory activity to tonsil mononuclear cells.The objective of the present study was to observe IgA production and cell proliferation stimulated with Lipopolysaccharide and a-Hemolytic streptococcus in IgAN.Then we observed expression of IgA class switching gene in IgAN.For explore the mechanism of IgA hyperproduction in tonsils,we investigated cytokine IFN-γ,IL-4 and FcaRI expression in Tonsillar mononuclear cells in IgAN. ObjectiveTo observe the effect of Lipopolysaccharide and a-Hemolytic streptococcus on tonsillar mononuclear cells proliferation in IgAN.To observe IgA and IgA1 production stimulated with Lipopolysaccharide and a-Hemolytic streptococcus on tonsillar mononuclear cells in IgAN.MethodsTonsillar mononuclear cells were isolated by the gradient centrifugation method from 26 cases with IgA nephropathy and 25 cases with chronic tonsillitis. Tonsillar mononuclear cells (1×106 per ml)were cultured for 72h with Lipopolysaccharide or a-Hemolytic streptococcus. Cell proliferation level determined by MTT. IgA and IgA1 production in cultured supernatants were measured in enzyme-linked immunosorbent assay(ELISA).ResultsTonsillar mononuclear cells proliferation significantly increased in IgAN patients compared to non-IgAN patients.P<0.05.Tonsillar mononuclear cells proliferation stimulated with 10μg/ml of Lipopolysaccharide significantly increased in IgAN patients compared to non-IgAN patients.P<0.05.Tonsillar mononuclear cells proliferation increased stimulated with Lipopolysaccharide orα-Hemolytic streptococcus on concentration-dependent way in IgAN patients.IgA production on tonsillar mononuclear cells significantly increased in IgAN patients.(32.48±9.81ng/ml vs 23.33±7.20ng/ml P<0.05).Compared to non-IgAN patients,IgA production stimulated with Lipopolysaccharide or a-Hemolytic streptococcus on tonsillar mononuclear cells significantly increased in IgAN patients. (36.24±6.51ng/ml vs 25.87±7.52ng/ml P<0.05;43.76±10.36ng/ml vs 31.24±7.17ng/ml P<0.05).Compared to non-IgAN patients, IgA1 production stimulated with Lipopolysaccharide or Hemolytic streptococcus on tonsillar mononuclear cells significantly increased in IgAN patients.(26.56±5.76ng/ml vs 19.72±4.16ng/ml P<0.05; 35.68±7.84ng/ml vs 23.61±5.06ng/ml P<0.05).IgAl/IgA ratio stimulated with a-Hemolytic streptococcus by Tonsillar mononuclear cells(TMCs) significantly increased in IgAN.(52.46±15.17% vs 66.00±24.98% P<0.05).ConclusionsIt reveals that hyper-immune response to antigen and hyperproduction of IgA by Tonsillar mononuclear cells is present in IgAN patients. ObjectiveTo observe the expression of IgA class switching gene in Tonsillar mononuclear cells in IgANMethodsTonsillar mononuclear cells were isolated by the gradient centrifugation method from IgAN and non-IgAN group.Expression of Iα-Cαgermline transcript,AID(activation induced cytidine deaminase) mRNA were examined by Reverse Transcription Real-time PCR.ResultsExpression of Iα-Cαgermline transcript mRNA in Tonsillar mononuclear cells significantly increased in IgAN patients compared to non-IgAN patients.(100±22.72% vs 6.80±7.28% P<0.05).Expression of AID mRNA in Tonsillar mononuclear cells significantly increased in IgAN patients compared to non-IgAN patients.(100±5.44% vs 26.62±6.19% P<0.05)ConclusionsExpression of Iα-Cαgermline transcript,AID mRNA in Tonsillar mononuclear cells significantly increased in IgAN.it may relate to hyperproduction of IgA in TMCs in IgAN. ObjectiveTo observe IFN-γ,IL-4 production stimulated with Lipopolysaccharide and a-Hemolytic streptococcus on tonsillar mononuclear cells in IgAN.To observe the expression of IFN-γ,IL-4mRNA in Tonsillar mononuclear cells in IgANMethodsTonsillar mononuclear cells were isolated by the gradient centrifugation method from IgAN and non-IgAN group. IFN-y and IL-4 in cultured supernatants were measured in enzyme-linked immunosorbent assay(ELISA). Expression of IFN-γ,IL-4 mRNA were examined by Reverse Transcription Real-time PCR.ResultsThe IFN-y production of Tonsillar mononuclear cells without stimulation were higher in IgAN patients than non-IgAN patients.(638.98±178.41pg/ml vs 400.76±136.43pg/ml P<0.05).The IFN-y production of Tonsillar mononuclear cells stimulated with Lipopolysaccharide was significantly higher in IgAN patients than non-IgAN patients.(655.04±223.97pg/ml vs 398.33±180.63pg/ml P<0.05).However, The IFN-y production of Tonsillar mononuclear cells stimulated with a-Hemolytic streptococcus decreased significantly in IgAN patients compared to non-IgAN patients.(466.96±221.69pg/ml vs 671.84±232.31pg/ml P<0.05).The IL-4 production of Tonsillar mononuclear cells without stimulation were higher in IgAN patients than non-IgAN patients.(17.72±6.29pg/ml vs 7.29±3.04pg/ml P<0.05).The IL-4 production of Tonsillar mononuclear cells stimulated with Lipopolysaccharide or a-Hemolytic streptococcus was significantly higher in IgAN patients than non-IgAN patients.(30.65±7.38pg/ml vs 8.67±3.15pg/ml P<0.05; 21.55±5.16pg/ml vs 8.0±3.78pg/ml P<0.01).Expression of IFN-γmRNA in Tonsillar mononuclear cells significantly increased in IgAN patients compared to non-IgAN patients.(100±0.69% vs 6.85±4.07% P<0.05).Expression of IL-4 mRNA in Tonsillar mononuclear cells significantly increased in IgAN patients compared to non-IgAN patients. (100±1.25% vs 36.92±0.95% P<0.05)ConclusionsIFN-γ,IL-4 gene expression and cytokine production by Tonsillar mononuclear cells increased in IgAN patients.Cytokine IFN-y downregulated and IL-4 upregulated stimulated with a-Hemolytic streptococcus.it suggests that Th cell cytokine especially Th2 predominance may play a role in pathgenesis of IgAN. ObjectiveTo observe the effect of Lipopolysaccharide or a-Hemolytic streptococcus on FcaRI expression in Tonsillar mononuclear cells in patients with IgA nephropathyMethodsTonsillar mononuclear cells were isolated by the gradient centrifugation method from IgAN and non-IgAN group.surface FcαRI expression in Tonsillar mononuclear cells was detected by flow cytometric analysis.Expression of FcaRI mRNA were examined by Reverse Transcription Real-time PCR.ResultsThe surface FcαRI expression in Tonsillar mononuclear cells was significantly higher in IgAN patients than non-IgAN patients.(2.27±1.16% vs 1.11±0.5%P<0.05).Under stimulation with a-Hemolytic streptococcus, The surface FcaRI expression in Tonsillar mononuclear cells was significantly higher in IgAN patients than non-IgAN patients. 4.08±1.39% vs 1.460.69% P<0.01).In IgAN group,The surface FcαRI expression in Tonsillar mononuclear cells is positively correlated with IFN-γlevel in cultured supernatants(r=0.641 P<0.05),negatively correlated with IL-4 in cultured supernatants(r=-0.721 P<0.05) Expression of FcaRI mRNA increased significantly in IgAN patients compared to non-IgAN patients.(100±1.19%vs 5.30±7.70% P<0.05).ConclusionsExpression of FcαRI in Tonsillar mononuclear cells increased significantly in IgAN patients may play a role in hyper-immune response in tonsil.