The IL-8 release from cultured human keratinocytes, mediated by antibodies to bullous pemphigoid autoantigen 180, is inhibited by dapsone

Abstract

SUMMARYBullous pemphigoid (BP) is a subepidermal blistering disease associated with autoantibodies to the hemidesmosomal 180 kD BP autoantigen (BP180). However, the binding of autoantibodies to BP180 alone is not sufficient for blister formation in this disease and the infiltration of neutrophils into the skin is required. Dapsone and nicotinamide inhibit neutrophil chemotaxis and are used effectively in treating BP. IL-8 is a known chemoattractant for neutrophils and has been implicated in the inflammatory process of both human and experimental murine BP. We have recently shown that antibodies to BP180 mediate a dose and time-dependent release of IL-6 and IL-8 from cultured normal human epidermal keratinocytes (NHEK). In the present study, we addressed the question whether dapsone or nicotinamide influence this cytokine release. We demonstrate that dapsone, but not nicotinamide, in its pharmacological range, inhibits the IL-8, but not the IL-6 release from NHEK, induced by anti-BP180 IgG, in a dose-dependent fashion as detected by ELISA. IL-8 mRNA levels, as determined by RT-PCR, were the same in cells treated with BP IgG alone compared to cells treated with BP IgG plus dapsone. This observation suggests that dapsone inhibits the BP IgG-induced IL-8 release from cultured NHEK by mechanisms at the post-transcriptional level. Our findings contribute to the understanding how dapsone leads to a reduced influx of neutrophils into BP lesions and, finally, to the cessation of blister formation in this disease.

Hsp90 blockade modulates bullous pemphigoid IgG-induced IL-8 production by keratinocytes

“…*P<0.05, **P<0.01, ***P<0.001 2013). Together with these previous observations and the recent finding that BP180-deficient epidermal keratinocytes display an abnormally high IL-8 response under various inflammatory stimuli (Van den Bergh et al 2012), our current results support the assumption that targeting BP180 directly affects the epidermal keratinocyte proinflammatory cytokine response (Schmidt et al 2000(Schmidt et al , 2001Messingham et al 2011;Van den Bergh et al 2012).…”

“…Similar to previous experiments, which showed that IgG or IgE autoantibodies from BP patients stimulated Fc receptorindependent production of IL-6 and IL-8 in primary cultures of normal human epithelial keratinocytes (NHEK) (Schmidt et al 2000(Schmidt et al , 2001Messingham et al 2011), we also observed a significant BP IgG-induced secretion of both cytokines compared to normal IgG using the HaCaT cell line. Cytokine background levels that were seen in medium containing normal IgG and which have also been reported by previous investigators may reflect the culture conditions on a plastic surface (and not human dermis) or may result from unspecific stimuli produced by keratinocytes (Schmidt et al 2000(Schmidt et al , 2001. Like in the previous studies, the purified BP IgG reacted with the NC16A domain of BP180 that is known to contain the major antigenic sites for most BP sera (Schmidt and Zillikens Fig.…”

“…Inhibition of IL-8 secretion by 17-DMAG was also found in cells not treated with the autoantibody, which may reflect suppression of constitutively expressed IL-8 in HaCaT cells rather than cytotoxic effects of this Hsp90 inhibitor as no such cytokine release was observed for IL-6 and cytotoxicity of 17-DMAG in the concentrations used was ruled out using the LDH assay. Similar results were previously obtained by Schmidt and colleagues using another immunomodulatory agent, the sulfone derivative dapsone (Schmidt et al 2001). This drug is applied successfully in the treatment of different autoimmune bullous diseases associated with neutrophil or eosinophil skin Fig.…”

“…The rationale of our study to probe the effects of 17-DMAG on BP IgG-induced IL-6 and IL-8 production by HaCaT cells was mainly based on three documented findings: (i) both inflammatory cytokines play a key role in the pathophysiology of BP (Schmidt et al 1996a, b;Liu et al 1997;Inaoki and Takehara 1998;Schmidt et al 2000;Chen et al 2001;Schmidt et al 2001;Nelson et al 2006;Messingham et al 2011), (ii) the extensive list of Hsp90 client proteins includes many proinflammatory molecules (Salminen et al 2008;Taipale et al 2010;Henderson and Kaiser 2013), and (iii) recent evidence suggests that Hsp90 represents a novel treatment target in autoimmune bullous diseases (Kasperkiewicz et al 2011;Tukaj et al 2013).…”

“…Insights into the functionally relevant pathogenic mechanisms in BP, which include complement activation, mast cell degranulation, recruitment and activation of neutrophils and eosinophils, and release of basal membrane zone-degradading proteinases from these effector cells, have been provided by in vitro studies using cultured human keratinocytes and ex vivo studies using cryosections of human skin as well as various mouse models, although the role of mast cells in models of antibody-mediated autoimmunity has recently been questioned (Kasperkiewicz and Zillikens 2007;Feyerabend et al 2011;Schmidt and Zillikens 2013). Although the precise sequence of these events is largely unknown, it has been proposed that one of the first steps leading to blister formation in BP comprise the binding of autoantibodies to BP180, thereby initiating Fc receptor-independent events leading to the release of interleukin 6 (IL-6) and IL-8 from basal keratinocytes (Schmidt et al 2000(Schmidt et al , 2001Messingham et al 2011). IL-8 is a potent chemoattractant for pathophysiologically relevant leukocyte effector cells and together with IL-6 known to be elevated in sera and blister fluids of BP patients and critical for blister formation in mouse models (Schmidt et al 1996a, b;Liu et al 1997;Inaoki and Takehara 1998;Chen et al 2001;Nelson et al 2006;Kobayashi 2008).…”