ABSTRACT
BACKGROUND: Hypodermin A (HA) is a serine esterase that degrades complement, a key element of the innate immune system. Immunosuppressive properties of HA have previously been studied in vitro. However, such properties have not been fully demonstrated in vivo. The aim of this study was to evaluate the effect of HA in inhibiting allograft rejection in an HA transgenic mouse model. METHODS: FVB (HA transgenic mice or wild-type mice) to BALB/c mice skin transplantation model were used. Skin grafts were analyzed by histology, immunohistochemistry, and Western blotting. RESULTS: HA overexpression resulted in significantly prolonged skin allograft survival. Histologic changes in the skin allografts paralleled the gross appearance of rejection. ELISA and Western blotting showed that HA significantly reduced the content of complement C3 and C9 in HA skin allografts. The expressions of CD4, B7-2, and MHC class II were all significantly suppressed in HA skin allografts compared with the control group. CONCLUSIONS: These findings suggest that HA effectively prolongs skin allograft survival. The study results provide insight into a promising strategy to improve the survival of grafts in humans.
Subject(s)
Genetic Therapy/methods , Graft Rejection/prevention & control , Graft Survival/immunology , Serine Endopeptidases/immunology , Skin Transplantation , Animals , Biomarkers/metabolism , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Female , Graft Rejection/enzymology , Graft Rejection/genetics , Graft Rejection/immunology , Graft Survival/genetics , Immunohistochemistry , Mice , Mice, Inbred BALB C , Mice, Transgenic , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Transplantation, Homologous , Treatment Outcome , Up-RegulationABSTRACT
Heme oxygenase (HO) represents an intrinsic antiinflammatory system based on its ability to inhibit expression of proinflammatory cytokines. The constitutive isoform heme oxygenase-2 (HO-2) has high expression and activity in cerebral microvascular endothelial cells (CMVEC). This study was undertaken to evaluate the role of HO-2 in regulation of TLR4/MyD88-dependent signaling and to study the effect of HO-2 on the expression and secretion of the proinflammatory cytokines tumor necrosis factor α (TNF-α) and Interleukin-6 (IL6) in CMVEC. HO-2 short hairpin RNA (shRNA) and HO-2 overexpression plasmids were used to observe the effect of HO-2 on proinflammatory cytokines in CMVEC in vitro, and the results showed that the messenger RNA (mRNA) and protein levels of TNF-α and IL6 were increased and decreased, respectively, compared with control groups. LPS-stimulated TNF-α and IL6 mRNA and protein were also reduced in CMVEC treated with an inhibitor of TLR4 signaling, CLI-095, or HO-2 overexpression. CLI-095 and HO-2 overexpression both reduced TLR4 expression in CMVEC, and HO-2 shRNA blocked these effects of CLI-095. CLI-095 and HO-2 overexpression potently suppressed TLR4/MyD88-dependent proinflammatory cytokine expression in CMVEC. These results suggest that HO-2 plays an important role in protecting CMVEC against cytokine-mediated inflammation.
Subject(s)
Endothelial Cells/metabolism , Endothelium, Vascular/metabolism , Heme Oxygenase (Decyclizing)/metabolism , Interleukin-6/metabolism , Signal Transduction/physiology , Toll-Like Receptor 4/metabolism , Tumor Necrosis Factor-alpha/metabolism , Animals , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Endothelial Cells/cytology , Endothelial Cells/drug effects , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Heme Oxygenase (Decyclizing)/genetics , Interleukin-6/genetics , Lipopolysaccharides/pharmacology , Mice , RNA, Small Interfering , Signal Transduction/drug effects , Sulfonamides/pharmacology , Tumor Necrosis Factor-alpha/geneticsABSTRACT
Hypodermin A (HA), a serine protease secreted by first-instar larvae of Hypoderma lineatum (Diptera: Oestridae) is associated with inflammatory and the specific immune responses in cattle hosts. In the present study, the cDNA sequence of HA was synthesized, and found to have fifteen amino acids which differed from the sequence available in GenBank. We then examined the association between recombinant HA and guinea-pig complement component 3 (C3) through a co-immunoprecipitation assay. Cos7 cells stably expressing HA were generated, and were found to be more resistant to lysis by guinea-pig C3 than the controls. HA was also able to degrade the C6 and C5b-9 of guinea-pig C3. The presumed DNA binding site of HA with guinea-pig C3 was detected by an electrophoretic mobility shift assay (EMSA). In contrast, after stable transfection, mHA was unable to reduce the amount of C3 or to inhibit its cytotoxicity, while HA could degrade guinea-pig C3 and inhibit the complement pathway. The findings suggest that recombinant HA could serve as an immunosuppressive agent against organ rejection after xenotransplantation.
