Effects Of CD40-CD40L Costimulatory Pathway In Human Orbital Fibroblasts

ObjectivesCD40-CD40L is important protein tyrosine kinase in cellular signal transduction. Studies have confirmed that the CD40-CD40L cotimulatory molecules play an important role in the pathogenesis of Graves'ophthalmopathy and the abnormally expressive ICAM-1 in orbital fibroblasts of Graves'ophthalmopathy. However, the molecule mechanism and signal transduction pathways in the regulation of ICAM-1 by CD40-CD40L in Graves'ophthalmopathy are uncertain. This research was designed to study the expression of ICAM-1 and key enzymes of mitogen activated protein kinase (MAPK), Src family protein (Syk,Lyn)and nuclear factor-kappa B (NF-κB) signal transduction pathways in orbital fibroblasts after CD40L stimulation and the correlation between ICAM-1 and the above signal transduction pathways, to investigation the expression changes and significance of ICAM-1 in OF and to explore its upstream signal transduction pathways and their interrelationship in above process.Methods1 . To detect the expression of CD40-CD40L, ICAM-1-LFA-1 with immunohistochemical staining in human orbital tissues of GO patients. Orbital fibroblasts were isolated and cultured from patients of Graves'ophthalmopathy. The cells were identified as OF by the immunohistochemical method and passaged.2.To determine whether the secretion of ICAM-1 after stimulation by sCD40L occurred at transcriptional and translational levels, steady-state ICAM-1 mRNA was quantified using PT-PCR and ICAM-1 protein was examined by ELISA.3. To assess the signal transduction pathways involved in CD40L-induced ICAM-1 gene expression in OF cells, various inhibitors(PD98059, an ERK1/2 inhibitor; SB203580, a p38 inhibitor; SP600125, a JNK inhibitor; PDTC, a NF-κB inhibitor; and PP1, a Src inhibitor) specific to signal mediators were employed. Activation of MAPKs proteins (phosphorylation) was assessed by Western blot using antibodies against the active (phospho) forms of MAPKs proteins. Nuclear extracts were prepared and subjected to EMSA to test DNA binding activity of the nuclear factor.4.Activation of Src family (Syk,Lyn) proteins (phosphorylation) was assessed by Western blot after stimulation of CD40L in OF. Piceatannol, Syk inhibitor was employed to assess the Syk signal transduction pathways involved in MAPKs and NF-κB pathways in OF after stimulation of CD40L.5.To construct the recombinant eukaryotic expression vector pEGFP-C2-TSHR, was indentified by restricting enzyme digestion analysis and DNA sequencing. The female BALB/c mice (6weeks) were randomly divided into group A (10 mice) and B (15 mice) which were immuned blank plasmid pEGFP-C2 with and recombinant plasmid pEGFP-C2-TSHR for 3 times at 3-week intervals respectively. Mice in 2 groups were sacrificed 18 weeks after immunization. The orbital tissues and thyroid tissues were harvested for pathological examination and serum was collected for TT4 and TRAb measurements.Results1.The positive staining of CD40 and ICAM-1 protein mainly localized in orbital fibroblasts of GO and ICAM-1 protein intensively expressed in the vascular endothelial cells. CD4+ and CD8+ inflammatory cells were distributed diffusely and highly expressioned LFA-1 protein.2.Results of RT-PCR and Western blot showed that both ICAM-1 mRNA expression and protein production were higher in GO group than those in control group (P<0.05). Results of RT-PCR showed the kinetics and dose response of CD40L-inducing effects on ICAM-1 mRNA expression.3.CD40L-induced ICAM-1 mRNA production was inhibited by PDTC, PD98059, SB203580, SB600125 and PP1(P<0.01). The results of Western blot revealed that all three MAPK subfamilies(ERK1/2, JNK, p38)examined were rapidly and strongly activated by CD40L treatment in a time-dependent pattern; CD40L induced phosphorylation of IκB-αand a moderate activation of NF-κB, which increased in a time-dependent manner. PD98059 and SB600125 significantly decreased CD40L-induced binding activity of NF-κB to DNA and the phosphorylation of IκB-α(P<0.05). But simultaneous use of SB203580 did not result in a significant potential effect(P>0.05).4.The results of Western blot revealed that Src families (Syk,Lyn) examined were rapidly and strongly activated by CD40L treatment. CD40L-induced ICAM-1 mRNA production was inhibited by Piceatannol, Syk inhibitor(P<0.05). Piceatannol significantly decreased CD40L-induced phosphorylation of MAPK(ERK1/2, JNK, p38), IκB-αand binding activity of NF-kB to DNA(P<0.05). 5.After immunization with recombinant plasmid pEGFP-C2-TSHR, orbital tissue microscopically displayed proliferation of adipose tissues and fibrous tissues, degeneration and disruption of muscular fibers in 66.7% of mice (group B). The thyroid tissue microscopically displayed diffuse hypercellularity with colloid reduced and the follicular epithelial being tall cuboidal cells with irregular papillary poles protruding into the follicular space; there were many immune cells in the interstitium with slight hyperaemia and edema. Serum total T4 levels were significantly elevated (P<0.05) in mice of group B(64.58±7.61) when compared with group A (56.42±4.19), and serum TRAb levels were significantly elevated (P<0.05) in mice of group B (11.00±9.91)when compared with group A (2.40±3.24). Two mice displayed serology characteristics of hyperthyroidism with high total T4 and high TRAb levels.Conclusions1.The positive staining of CD40 and ICAM-1 protein mainly localized in orbital fibroblasts of Graves'ophthalmopathy.2.ICAM-1 mRNA and protein were expressed higher in orbital fibroblasts of Graves'ophthalmopathy after stimulation by CD40L.3.Our results indicate that MAPK (ERK1/2, p38, JNK) and NF-κB pathways were all involved in ICAM-1 expression induced by CD40L. CD40L-induced NF-κB binding was modulated by ERK1/2 and JNK, but not by p38. p38 MAPK may be involved in processes leading to other nuclear translocation (as NF-κB) that regulate ICAM-1 gene expression.4.Syk and Lyn protein were activated by CD40L treatment. The activated Syk induced phosphorylation of MAPK(ERK1/2, JNK, p38), IκB-αand increased the binding activity of NF-kB to DNA.5.The GO animal model established by immuning homoimmune BALB/c mice with pEGFP-C2-TSHR was very similar to human GO in histological characteristics and was a feasible and reliable experimental method.