Rahul Purwar

Histamine Upregulates Keratinocyte MMP-9 Production via the Histamine H1 Receptor

Skin inflammation and the migration of cells at the site of the immune response play an important role in allergic skin diseases. It has already been described that matrix metalloproteinase 9 (MMP-9) influences tissue remodeling and facilitates cell migration by proteolytic degradation of basal membrane components. The aim of this study was to investigate MMP-9 expression on human primary keratinocytes (KCs) upon stimulation with histamine, a potent mediator in allergic responses. With ELISA and zymography, we could show that histamine induced dose-dependent upregulation of MMP-9 in cultured KCs and in punch biopsies of human skin. The histamine H(1) receptor (H(1)R) agonist beta-histine-but not agonists for H(2)R, H(3)R, and H(4)R-induced MMP-9, whereas the H(1)R antagonist clemastine blocked the effect in a dose-dependent manner. Immunohistological staining showed that histamine-induced MMP-9 led to destruction of type IV collagen at the basement membrane in healthy skin. In a coculture system of KCs and T cells, migration of T cells through an artificial basement membrane was increased after histamine stimulation of KCs. Our findings demonstrate enhanced MMP-9 production and cell migration after histamine stimulation and may represent a new mechanism by which KCs contribute to the pathology of skin diseases.

Induction of C3 and CCL2 by C3a in keratinocytes: a novel autocrine amplification loop of inflammatory skin reactions.

The complement fragment-3a (C3a) acts via a G protein-coupled C3aR and is of importance in allergic and inflammatory diseases. Recent studies suggest the presence of complement proteins in the epidermal compartment and synthesis of some of these proteins (C3, factor B, and factor H) by human primary keratinocytes (KCs) during inflammation. However, expression of C3aR and its role in human KCs is not elucidated thus far. In this study, we demonstrate the expression of C3aR on KCs as detected by quantitative real-time RT-PCR and flow cytometry. IFN-γ and IFN-α strongly up-regulated the surface expression of C3aR on KCs among all other cytokines tested. After up-regulation of C3aR by IFN-γ and IFN-α, we observed the induction of five genes (CCL2, CCL5, CXCL8, CXCL10, and C3) after stimulation of KCs with C3a in microarray analysis. We confirmed the induction of C3 and CCL2 at RNA and protein levels. Furthermore, incubation of C3 with skin mast cells tryptase resulted in the generation of C3 fragments with C3a activity. In conclusion, our data illustrate that epidermal KCs express functional C3aR. The increases of C3 and CCL2 synthesis by C3a and C3 activation by skin mast cell tryptase delineates a novel amplification loop of complement activation and inflammatory responses that may influence the pathogenesis of allergic/inflammatory skin diseases.

Polymorphisms within the C3 gene are associated with specific IgE levels to common allergens and super-antigens among atopic dermatitis patients.

Abstract:  Atopic dermatitis (AD) is a chronic inflammatory skin disease. Twin and family studies suggest a strong genetic component of the disease. The keratinocytes secrete high amounts of C3 after stimulation with pro‐inflammatory cytokines, which may play a functional role in skin inflammation. In this study, we genotyped four different single nucleotide polymorphisms (SNPs) by melting curve analysis using sequence specific hybridization probes in a well‐characterized cohort of AD patients. Among four SNPs within C3 gene, higher frequencies of rs10410674 (23.5% vs 12.2%) and rs366510 (13.8% vs 6.5%) were observed in AD patients as compared with control group. None of the tested polymorphisms showed significant association with the risk of the disease phenotype. Analysis of rs10402876 SNP revealed its association with less severe AD disease expression (low SCORAD). Total serum IgE levels were not different among AD patients having any of the four SNPs. However, we observed significantly less serum‐specific IgE levels to common allergens (Dermatophagoides pteronyssinus and birch pollens) and Staphylococcal enterotoxin B in AD patients having rs366510 SNP. Thus, associations of polymorphism within C3 gene with less severe AD disease expression and a weaker sensitization to common allergens suggest the role of these SNPs in the development of AD.

A protective role of complement component 3 in T cell-mediated skin inflammation.

Abstract:  Keratinocytes synthesize complement component 3 (C3) constitutively, and increased expression of C3 has been described during skin inflammation. In this study, we investigated the role of C3 in T cell‐mediated allergic contact dermatitis, which is a clinical manifestation of contact sensitivity (CS). C3‐deficient mice (C3KO) showed substantial higher CS responses to haptens, inducing a Th1 cytokine‐mediated skin inflammation (2,4‐dinitrofluorobenzene and dinitrochlorobenzene), and to haptens known to induce a Th2‐polarized inflammatory response (fluoro‐isothiocynate and toluene‐2,4‐diisocyanate) as compared to their wild‐type (WT) controls. There was a higher influx of GR‐1+, CD4+, and CD8+ cells into the skin of hapten‐treated C3KO mice compared with WT mice. Activated splenocytes from C3KO mice immunized with DNCB secreted higher amounts of IFN‐γ compared with WT controls but not of Th2 (IL‐4, IL‐5, and IL‐10) cytokines or IL‐17. A higher secretion of IL‐12 from splenocytes of C3KO mice as compared with WT mice was observed after TLR‐4 ligand (LPS) or TLR‐2 ligand (peptidoglycan) stimulation. Thus, an increased expression of IL‐12 and of IFN‐γ may be responsible for the increased hapten‐induced inflammation in C3 deficiency. Finally, we demonstrated that C3KO mice developed oral tolerance to haptens to a lower degree than WT mice. Our findings provide a new insight into a novel anti‐inflammatory role of C3 in skin inflammation.