High expression of Hsp90 and its compromised secretion by peripheral blood mononuclear cells of bullous pemphigoid patients.

<p>Hsp90 was detected in whole cell lysates of freshly isolated peripheral blood mononuclear cells (PBMCs) as well as in conditioned medium of PBMC cultures of bullous pemphigoid (BP) patients and healthy controls by enzyme-linked immunosorbent assay. (<b>a</b>) The mean intracellular Hsp90 content of PBMCs was comparatively higher in BP patients. (<b>b</b>) The mean extracellular Hsp90 content of PBMC cultures was comparatively lower in BP patients after 18-hour stimulation with LPS/CpG and TNF-α. Values are mean ± SEM of 5 BP patients and 5 controls. *<i>P</i><0.05.</p

High expression of Hsp90 in the epidermis of bullous pemphigoid patients.

<p>Hsp90 expression was analyzed in perilesional skin biopsies of bullous pemphigoid (BP) patients and in skin sections of healthy controls by fluorescent immunohistochemistry. (<b>a, b</b>) Representative immunofluorescent and DAPI-counterstained (in blue) skin samples showed a stronger intracellular Hsp90 expression (in green) mainly localized to the epidermal layer of the skin of a BP patient compared to control skin. Magnification: x200. (<b>c, d</b>) Higher magnification (x600) of the respective skin samples. (<b>e</b>) Relative mean epidermal Hsp90 fluorescence intensity by densitometry measurements. Values are mean ± SEM of 6 BP patients and 6 controls. ***<i>P</i><0.001.</p

Potent induction of Hsp90 expression in and its compromised release from keratinocytes by BP serum and isolated anti-BP180 NC16A IgG, respectively.

<p>(<b>a</b>) Human keratinocyte (HaCaT) cells were incubated with normal human serum (NHS) of healthy donors and serum of bullous pemphigoid (BP) patients over 24 hours. With increasing serum concentration a comparatively stronger intracellular upregulation of Hsp90 was induced by BP serum as measured by enzyme-linked immunosorbent assay (ELISA) with HaCaT cell lysates. Values are mean ± SEM of 2 independent experiments of 3 BP patients and 3 controls. *<i>P</i><0.05. (<b>b</b>) This finding was not dependent on the presence of BP autoantibodies, as a 24-hour stimulation of HaCaT cells with total BP IgG or affinity-purified anti-BP180 NC16A IgG from a BP patient failed to reproduce such effect when compared with NHS IgG from a healthy donor as measured by ELISA with HaCaT cell lysates. Values are mean ± SEM of 4 independent experiments. (<b>c</b>) Total BP IgG or affinity-purified anti-BP180 NC16A IgG from a BP patient, but not BP180 NC16A-depleted IgG, significantly inhibited the secretion of Hsp90 compared to NHS IgG from a healthy donor as measured by ELISA with conditioned medium of 24-hour stimulated HaCaT cells. Values are mean ± SEM of 4 independent experiments. *<i>P</i><0.05, ***<i>P</i><0.001.</p

No contribution of TGF-α, protein phosphatase 5 or protein kinase A to decreased levels of secreted Hsp90.

<p>Expression of (<b>a</b>) TGF-α, (<b>b</b>) protein phosphatase 5 (PP5) and (<b>c</b>) protein kinase A (PKA) was analyzed in serum (TGF-α) and peripheral blood mononuclear cells (PBMC) lysates (PP5 and PKA) of bullous pemphigoid (BP) patients and healthy controls by enzyme-linked immunosorbent assay and western blotting, respectively, and revealed no significant difference between both study groups. (<b>d</b>) Mean PP5 and PKA concentrations were expressed relative to the β-Actin level using densitometry measurements of immunoblots. Values are mean ± SEM of 3–9 BP patients and 3–6 controls.</p

Decreased Hsp90 serum levels and their inverse association with anti-BP180 NC16A autoantibodies in bullous pemphigoid patients.

<p>Circulating Hsp90 was evaluated in serum of bullous pemphigoid (BP) patients and controls (healthy subjects and pemphigus vulgaris [PV] patients) by enzyme-linked immunosorbent assay. (<b>a</b>) Mean serum Hsp90 levels were comparatively lower in BP patients than in healthy controls. (<b>b</b>) Intraindividual serum levels of Hsp90 in BP patients showed an increase after a mean follow-up time of 29 weeks of immunosuppressive treatment. (<b>c</b>) Rising levels of circulating Hsp90 were paralleled by a reduction in individual serum anti-BP180 NC16A IgG autoantibodies. (<b>d</b>) A significant inverse correlation was observed between Hsp90 serum levels and circulating BP autoantibodies. (<b>e</b>) Mean serum Hsp90 levels were comparable between PV patients and corresponding healthy controls. (<b>f</b>) Intraindividual serum levels of Hsp90 in PV patients showed no consistent trend towards an increase or decrease, while serum (<b>g</b>) anti-desmoglein 3 (Dsg3) and (<b>h</b>) 1 (Dsg1) IgG autoantibodies declined after a mean follow-up time of 41 weeks of immunosuppressive treatment. (<b>i, j</b>) No significant correlation between Hsp90 serum levels and circulating PV autoantibodies could be recorded. Values are mean ± SEM of 12 BP patients, 10 PV patients and 12–29 healthy controls. *<i>P</i><0.05, ***<i>P</i><0.001.</p