Cytoplasmic antineutrophil cytoplasmic antibodies (cANCA) that accompany the neutrophilic vasculitis seen in Wegener's granulomatosis (WG), are directed against proteinase-3 (PR-3), a serine proteinase which is located in azurophilic granules of neutrophils and monocytes. PR-3, when expressed on the surface of TNF ␣ -primed neutrophils, can directly activate neutrophils by complexing cANCA and promoting concomitant Fc ␥ receptor (Fc ␥ R) cross-linking. Although the neutrophil's pathogenic role in WG has been studied, the role of the monocyte has not been explored. The monocyte, with its ability to release cytokines and regulate neutrophil influx, also expresses PR-3. Therefore, the monocyte may play a significant role in WG via the interaction of surface PR-3 with cANCA, inducing cytokine release by the monocyte. To test this hypothesis, monocytes were studied for PR-3 expression and for IL-8 release in response to cANCA IgG. PBMC obtained from healthy donors displayed dramatic surface PR-3 expression as detected by immunohistochemistry and flow cytometry in response to 0.5-h pulse with TNF ␣ (2 ng/ml). Purified monoclonal anti-PR-3 IgG added to TNF ␣ -primed PBMC induced 45-fold more IL-8 release than an isotype control antibody. Furthermore, alpha 1-antitrypsin ( ␣ 1-AT), the primary PR-3 antiprotease, inhibited the anti-PR-3 induced IL-8 release by 80%. Importantly, Fab and F(ab Ј ) 2 fragments of anti-PR-3 IgG, which do not result in Fc ␥ receptor cross-linking, do not induce IL-8 release. As a correlate, IgG isolated from cANCA positive patients with WG induced six times as much PBMC IL-8 release as compared to IgG isolated from normal healthy volunteers. Consistent with PR-3 associated IL-8 induction, ␣ 1-AT significantly inhibited this effect. These observations suggest that cANCA may recruit and target neutrophils through promoting monocyte IL-8 release. This induction is mediated via Fc ␥ receptor cross-linking and is regulated in part by ␣

The importance of a dermal element when providing permanent wound cover for skin loss has become evident as the shortcomings of pure epidermal grafts are recognized. We are developing a skin composite formed from sterilized human de-epidermized acellular dermis, keratinocytes and fibroblasts with the ultimate aim of using this composite to cover full-thickness excised burn wounds. These composites can be prepared with or without basement membrane (BM) antigens initially present on the dermis. This study investigates the importance of retaining BM antigens on the dermis to the production and appearance of these composites in vitro. Skin composites prepared from dermis with BM antigens either present or absent initially were studied throughout 3 weeks. Composites with BM antigens present initially were significantly better than those initially lacking BM antigens in: (i) the degree of epithelial cell attachment to the underlying dermis (hemidesmosomes were seen only in the former); (ii) the morphology of the epithelial layer; (iii) the consistent presence of collagen IV and laminin and the increasing expression of tenascin; and (iv) the amount of soluble collagen IV and fibronectin detected in the conditioned media. We conclude that an initial BM antigen template is vital in this skin composite model for the attachment and differentiation of the epithelial layer and for the subsequent remodelling of the BM in vitro.

No abstract

Local regulation of alpha1-antitrypsin (alpha1-AT) may have importance in maintenance of the protease-antiprotease balance in the microenvironment of inflammatory cells. We therefore studied whether lipopolysaccharide (LPS), interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNFalpha) affect the pericellular concentration of alpha1-AT in human peripheral blood mononuclear cells (PBMC). PBMC taken from normal healthy volunteers were treated with LPS, IL-1beta, and TNFalpha, and the concentration of human alpha1-AT in conditioned supernatants was measured. When compared with unstimulated control supernatants (147 +/- 19 ng/ml), LPS (439 +/- 66 ng/ml; p < or = 0.001), IL-1beta (263 +/- 37 ng/ml; p < or = 0.01), and TNFalpha (316 +/- 59 ng/ml; p < or = 0.05) induced a 2- to 3-fold increase of alpha1-AT. Up-regulation of alpha1-AT protein correlated with an increase in alpha1-AT mRNA, suggesting a simultaneous increase in alpha1-AT synthesis. Despite the increase in alpha1-AT concentration, functional antiprotease activity could not be detected. Furthermore, protease activity was present in all samples, with the amount of activity being inversely related to the amount of alpha1-AT measured in supernatants. These findings suggest that local inflammatory conditions up-regulate alpha1-AT production by monocytes which complex with a protease derived from the PBMC population.

No abstract

No abstract

No abstract

No abstract