Molecular Mechanism Of Targeted IgA/FcαRI Anti Henoch-schonlein Purpura Vascular Endothelial Cell Inflammation

PART Ⅰ In Vivo Expression of FcαRI in Children with Henoch-Schonlein PurpuraObjective: to observe the expression of Ig A receptor FcαRI in peripheral blood and lesional skin in Henoch-Schonlein Purpura, to learn the correlation between serum s FcαRI-Ig A level and lesional skin protein level, to explore relationship with inflammatory cytokines and its role in the pathogenesis of Henoch-Schonlein Purpura with acute stage.Methods: Immunohistochemistry analyzes FcαRI expression in lesional skin. Direct immunofluorescence detects Ig A, C3, Fib expression; Western blot analyzes FcαRI in protein level of lesional skin; Sandwich solid-phase ELISA detects s FcαRI-Ig A expression in serum; The serum and PBMC supernatant concentration of inflammatory cytokine IL-6, TNF-α were detected by ELISA.Results: 1. FcαRI deposits in the lesional skin of HSP. Strong positive expression was found in basement membrane, granular layer, sweat gland ducts, also in neutrophils, but not in corneum; The staining intensity of FcαRI in HSP group is obviously darker than control group. Immunohistochemical HSCORE scoring is average of 205 points in HSP group, significantly higher than that of the control(p<0.01). The CD89/beta actin level of HSP group is higher than the control by Western blot, while there is no difference between HSP and HSPN(p>0.05).2. The serum soluble s FcαRI-Ig A level of HSP and HSPN group with acute stage were higher than control group obviously(p<0.01), while no significant statistical differences between HSP group and HSPN(p>0.05). Serum levels of s FcαRI-Ig A and FcαRI expression levels in lesional skin were positively correlated(r=0.51, p=0.02). Serum IL-6 levels in acute stage of HSP is higher than that of control group(p<0.01), There is no statistical significance between HSP and HSPN(p>0.05). The PBMC supernatant concentration of TNF-ɑ of HSP in acute stage is much higher than control group(p < 0.05), and HSPN is much higher than HSP(p<0.05).Conclusion: FcαRI plays an important role in the systemic vascular inflammation in HSP, Serum level of s FcαRI-Ig A in acte stage of HSP has significantly increased, and positively correlated with the expression in lesional skin. FcαRI is thought to be involved in the immune pathogenesis of HSP.PARTⅡEffects of HSP Neutrophil Activation on vascular endothelial cellsObjective: To investigate the effects of neutrophil activation on vascular endothelial cells apoptosis and on production of proinflammatory cytokines such as IL-8 and TNF-α in HSP. To explore the effect of different type of Ig A via FcαRI to regulate neutrophil induced vascular endothelial cell inflammation.Methods: Flow cytometry analyzes CD11 b expression on neutrophil. QPCR detects peripheral blood neutrophils FcαRI m RNA expression, Western blot analyzes the expression of peripheral blood neutrophils FcαRI protein level. Cell co-culture technique to establish inflammation cell model of HSP neutrophil and HUVEC. Stimulated with m Ig A and p Ig A, HUVEC apoptosis were detected by Flow cytometry. Supernatant concentration of IL-8 and TNF-α were detected by ELISA.Results:1. There is no significantly difference of neutrophil FcαRI m RNA expression between HSP and control group(p=0.98), but protein level of FcαRI is significantly decreased(p<0.05). CD89/beta actin of HSP group is lower than the control by Western blot. Mean fluorescence intensity(MFI) of neutrophil CD11 b of HSP is higher than control(p<0.01).2. Mean apoptosis rate of HUVEC+HSP Neu+p Ig A group is 63.99%, which is significantly higher than HUVEC+HSP Neu+m Ig A group(27.07%)(p<0.01). Apoptosis rate of HUVEC+HSP Neu+p Ig A group is higher than HUVEC+HSP Neu+PBS group(34.9%)(p=0.01). Mean apoptosis rate of HUVEC+HSP Neu+m Ig A group is lower than HUVEC+HSP Neu+PBS group, but there is no statistical difference(p>0.05).3. Supernatant concentration of IL-8 and TNF-α of HUVEC+HSP Neu+p Ig A group were significantly higher than HUVEC+HSP Neu+m Ig A group(p < 0.01,p < 0.01), also higher than HUVEC+HSP Neu+PBS group(p<0.05,p<0.05). Supernatant concentration of IL-8 and TNF-α of HUVEC+HSP Neu+m Ig A group is lower than HUVEC+HSP Neu+PBS group, there is no statistical difference between them(p>0.05,p>0.05).Conclusion:1. The transcription level of FcαRI expressed on neutrophil is normal in HSP patients, while the protein level of FcαRI is decreased vs control. Increased adhesion molecule CD11 b of early activation stage of neutrophil can be observed. CD11 b can promote neutrophil adhesion and migration to vascular endothelial cell.2. In acute stage of HSP, neutrophil can induce human unbilical vein endothelial cell apoptosis. PIg A has proinflammatory effect on HUVEC apoptosis mediated by neutrophils. MIg A may have an inhibitory effect on HUVEC apoptosis mediated by neutrophils. PIg A can improve secretion of inflammatory cytokine IL-8 and TNF-α. MIg A may have inhibitory effect on IL-8 and TNF-α secretion.PARTⅢ molecular mechanisms of Ig A/ FcαRI mediated neutrophils effect on vascular endothelial cellObjective: To investigate the molecular mechanism of Ig A/FcαRI mediated neutrophil activation to damage vascular endothelial cell in HSP children. After monoclonal antibody MIP8 a blocked, to discuss regulation function of different kinds of ligands Ig A in the signaling pathway.Methods: Establish cell co-culture technique of neutrophil-HUVEC inflammation model. Intervention with different type of ligands Ig A and monoclonal antibody MIP8 a, Syk, PI3 K m RNA expression in neutrophils were detected by q PCR. Western blot analyzed Syk and p-Syk, PI3 K, p-PI3 K protein levels in neutrophils; Flow cytometry detects apoptosis rate of HUVEC.Results:1. Neutrophils Syk, PI3 K m RNA expression were decreased with increment of MIP8 a concentration in p Ig A groups, MIP8 a 0ug group is significantly higher than the control group(p<0.01), MIP8 a 5ug and 10 ug groups were both lower than 0ug group(p<0.05,p<0.05), while there is no statistical difference between 3ug group and 0ug group(p>0.05).2. Along with MIP8 a concentration increased from 0ug to 10 ug, there were no statistical difference of non phosphorylated Syk and PI3 K protein level among p Ig A groups, while the phosphorylated Syk(p-Syk) and phosphorylated PI3K(p-PI3K) level is gradually reduced(p<0.01). Along with MIP8 a increased from 0ug to 10 ug, p-Syk and p-PI3 K protein level were gradually increased Among m Ig A groups(p<0.01).3. HUVEC apoptosis rate of control group was 8.26%. In p Ig A groups, HUVEC apoptosis rate of MIP8 a 0ug was 73.63%. Along with the MIP8 a concentration increased from 3ug to 10 ug, HUVEC apoptosis rate was gradually decreased(p<0.01). In m Ig A groups, along with MIP8 a concentration increased from 3ug to 10 ug, HUVEC apoptosis rate increased gradually(p<0.01).Conclusion: Intracellular signal transduction molecules Syk, PI3 K is important signal pathways of neutrophils FcαRI activation. PIg A and m Ig A crosslinking FcαRI through signaling pathway Syk, PI3 K may promote or inhibit neutrophil activation. MIP8 a can competitively combine with receptor Fc domain to play a blocking role. Targeted intervention of Ig A/FcαRI can regulate neutrophils mediated immune inflammation of HSP.