Autoantibodies to BP180 Associated with Bullous Pemphigoid Release Interleukin-6 and Interleukin-8 from Cultured Human Keratinocytes

Abstract

Bullous pemphigoid is an inflammatory subepidermal blistering disease that is associated with auto- antibodies to the keratinocyte surface protein, BP180. In addition to the binding of autoantibodies, the infiltration of inflammatory cells is necessary for blister formation. Cytokines, including interleukin-6 and interleukin-8, have been implicated in the disease process of both human and experimental murine bullous pemphigoid. This study was aimed at testing the hypothesis that the binding of anti-BP180 antibodies to their target antigen triggers a signal transduction event that results in the secretion of these pro-inflammatory cytokines. Consistent with this hypothesis, treatment of cultured normal human epidermal keratinocytes with bullous pemphigoid IgG, but not control IgG, led to increased levels of interleukin-6 and interleukin-8, but not interleukin-1alpha, interleukin-1beta, tumor necrosis factor-alpha, interleukin-10, or monocyte chemoattractant protein-1, in the culture medium. This effect was concentration- and time-dependent and was abolished by depleting the bullous pemphigoid IgG of reactivity to two distinct epitopes on the BP180 NC16A domain. Upregulation of interleukin-6 and interleukin-8 was found at both protein and mRNA levels. In addition, bullous pemphigoid IgG did not induce the release of interleukin-6 and interleukin-8 from BP180-deficient keratinocytes obtained from a patient with generalized atrophic benign epidermolysis bullosa. These data indicate that bullous pemphigoid-associated autoantibodies to the human BP180 ectodomain trigger a signal transducing event that leads to expression and secretion of interleukin-6 and interleukin-8 from human keratinocytes.

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.…”

“…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).…”

Bullous Pemphigoid, Mucous Membrane Pemphigoid and Pemphigus Vulgaris: An Update on Pathobiology

“…Additional potential direct mechanisms of blister induction may involve intracellular signaling pathways, resulting in the disassembly of hemidesmosomes and cytokine release. BP180 autoantibodies have been shown to modulate the expression of IL-6 and IL-8 in cultured human keratinocytes, the relevance of which is the subject of future investigation [55].…”