Aberrant expression and secretion of heat shock protein 90 in patients with bullous pemphigoid.

The cell stress chaperone heat shock protein 90 (Hsp90) has been implicated in inflammatory responses and its inhibition has proven successful in different mouse models of autoimmune diseases, including epidermolysis bullosa acquisita. Here, we investigated expression levels and secretory responses of Hsp90 in patients with bullous pemphigoid (BP), the most common subepidermal autoimmune blistering skin disease. In comparison to healthy controls, the following observations were made: (i) Hsp90 was highly expressed in the skin of BP patients, whereas its serum levels were decreased and inversely associated with IgG autoantibody levels against the NC16A immunodominant region of the BP180 autoantigen, (ii) in contrast, neither aberrant levels of circulating Hsp90 nor any correlation of this protein with serum autoantibodies was found in a control cohort of autoimmune bullous disease patients with pemphigus vulgaris, (iii) Hsp90 was highly expressed in and restrictedly released from peripheral blood mononuclear cells of BP patients, and (iv) Hsp90 was potently induced in and restrictedly secreted from human keratinocyte (HaCaT) cells by BP serum and isolated anti-BP180 NC16A IgG autoantibodies, respectively. Our results reveal an upregulated Hsp90 expression at the site of inflammation and an autoantibody-mediated dysregulation of the intracellular and extracellular distribution of this chaperone in BP patients. These findings suggest that Hsp90 may play a pathophysiological role and represent a novel potential treatment target in BP

Human IgG specific to laminin γ1 is not pathogenic ex vivo.

<p>Using recombinant forms of the C-terminus of laminin γ1 (hLAMC1-cterm) and full length laminin γ1 (LAMC1-FL) IgG specific for hLAMC1-cterm (a, c; lane 3) and LAMC1-FL (b, lane 3; c lane 5 ) was generated from anti-p200 pemphigoid serum (a, b, c; lane 2), as well as serum depleted from anti-hLAMC1-cterm (a, c, lane 4) and LAMC1-FL reactivity (b, lane 4; c lane 6) reactivity, respectively, as shown by immunoblotting with recombinant hLAMC1-cterm (a), LAMC1-FL (b), and extract of human dermis<u>-</u>(c). Interestingly, serum depleted from anti-hLAMC1-cterm and LAMC1-FL reactivity, respectively, (a, b; lane 4) still labeled the p200 protein in dermal extract (c, lane 4, lane 6). Monoclonal antibody against LAMC1 (a, b, c; lane 1) and serum from a healthy volunteer (a, b; lane 5; c lane 7) were used as controls. Arrows indicate the positions of the proteins and bars the molecular weight markers (a, 34 kD and 26 kD; b, 200 kD; c, 200 kD). hLAMC1-cterm-specific (h, i) and LAMC1-FL-specific patients IgG (l, m) and the monoclonal anti-LAMC1 antibody (d, e) labeled the dermal-epidermal junction (DEJ) by indirect immunofluorescence (IF) microscopy but did not induce dermal-epidermal separation (DES). In contrast, serum depleted from reactivity against hLAMC1-cterm (j, k), and LAMC1-FL (n, o), respectively, as well as patient serum (f, g) resulted in DES (black triangles mark base of the split). While untouched patient serum (f) as well as serum depleted from anti-hLAMC1-cterm (j) and LAMC1-FL reactivity (n), respectively, stained the DEJ of human skin in a linear pattern, the monoclonal anti-hLAMC1-antibody (d), hLAMC1-cterm-specific patient<u>-</u>IgG (h), and hLAMC1-FL-specific patient IgG showed an additional staining of basal keratinocytes. Serum from a healthy volunteer was used as control (p, q). Magnification: x200.</p

Passive transfer of rabbit anti-mLAMC1-cterm IgG into adult mice is not pathogenic.

<p>Rabbit IgG against the murine laminin γ1 C-terminus (mLAMC1-cterm) was not pathogenic when passively transferred into adult C57BL/6 and BALB/c mice. Injection of 15 mg rabbit anti-mLAMC1-cterm IgG every second day for 10 days did not result in clinical or histopathological (a, e) lesions on day 12. Linear deposition of rabbit IgG at the dermal-epidermal junction (DEJ) was only observed in 2 of 5 C57BL/6 (b) and one of 5 BALB/c mice (f), while staining of murine C3 was negative in all mice (c, g). At day 12, in sera of all 10 mice, rabbit IgG labeled the basal keratinocytes at the DEJ of normal mouse skin (d, h) and reacted with recombinant mLAMC1-cterm by ELISA (i) and immunoblotting (j, C57BL/6, lanes 1; BALB/c, lane 2) and the 200 kDa p200 protein in extract of murine dermis (k, C57BL/6 lane 1; BALB/c, lane 2). Normal mouse sera (j and k, C57BL/6, lane 3; BALB/c, lane 4) were used as controls.</p

Passive transfer of rabbit anti-mLAMC1-cterm IgG into neonatal mice does not reproduce the human disease.

<p>Rabbit IgG against the murine laminin γ1 C-terminus (mLAMC1-cterm) was not pathogenic when passively transferred into neonatal C57BL/6 mice. Injection of rabbit anti-mLAMC1-cterm IgG at a concentration of 10 mg/g body weight every second day for 10 days did not <u>induce</u> histopathological lesions on day 12 (<u>a</u>). Linear deposition of rabbit IgG at the DEJ was only observed in 2 of 8 mice (b), while staining of murine C3 was always negative (c). At day 12, in sera of all mice, rabbit IgG stained the basal keratinocytes at the DEJ of normal mouse skin (d), reacted with recombinant mLAMC1-cterm by ELISA (e) and immunoblotting (f, lanes 2–6) and with the 200 kDa p200 protein in extract of murine dermis (g, lanes 2–6). Polyclonal rabbit antibody H-190 against mLAMC1 (f, g, lane 1) and normal mouse serum (f, g, lane 7) was used as controls.</p

Primer sequences for PCR amplification of cDNA fragments of human.

<p>F, forward primer; R, reverse primer.</p

Serum autoantibody reactivity in anti-p200 pemphigoid patients with overlapping fragments of laminin γ1 covering the whole molecule.

<p>Serum autoantibody reactivity in anti-p200 pemphigoid patients with overlapping fragments of laminin γ1 covering the whole molecule.</p

Schematic diagram of the 6 recombinant fragments of human laminin γ1 (hLAMC1) used in this study.

<p>The recombinant C-terminus (hLAMC1-cterm) was previously used as antigenic target in an ELISA for the diagnosis of anti-p200 pemphigoid <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041769#pone.0041769-Groth1" target="_blank">[8]</a>. Each recombinant fragment is fused with an N-terminal His-tag. Amino acid numbers are shown next to the fragments.</p

Anti-p200 pemphigoid patients’ sera induce dermal-epidermal separation ex vivo.

<p>Sera from anti-p200 pemphigoid patients recruit neutrophils to the dermal-epidermal junction (DEJ) and induce dermal-epidermal separation (DES) in cryosections of human skin. Anti-p200 pemphigoid sera (anti-p200 pemphigoid; b, e), but not from sera of healthy volunteers (HV; c, f), recruited leukocytes to the DEJ after 1 h of incubation (b, c) and induced DES after 3 h (e, f). As positive control for DES, IgG from a patient with bullous pemphigoid was used (BP a, d). Recruited neutrophils are marked by arrows, base of the split is marked by black triangles. All sections were stained with hematoxylin and eosin. Magnification, x200.</p

A high concentration of rabbit anti-mLAMC1-cterm IgG is not pathogenic ex vivo.

<p>Rabbit IgG generated against the murine laminin γ1 C-terminus (mLAMC1-cterm) did not induce DES in cryosections of mouse skin. Rabbit anti-mLAMC1-cterm (a, lane 2; c, g; dilution 1∶1000) and commercial rabbit antibody H-190 against mLAMC1 (a, lane 1; b, f, dilution 1∶200) recognized the p200 protein by immunoblotting with extract of murine dermis (a) and labeled the DEJ of murine skin (b, c) but did not induce DES in cryosections of mouse skin (f, g). Of note, anti-mLAMC1-cterm rabbit IgG stained the basal layer of keratinocytes at the murine DEJ (b, c, inserts) in a similar pattern as seen with monoclonal anti-hLAMC1-antibody and hLAMC1-cterm-specific patient IgG (Fig. 3, c-f). Rabbit IgG against murine BP180 NC15A (e) and preimmune rabbit IgG (d, h) were used as positive and negative controls, respectively. Magnification: x400.</p

Immunization of mice with mLAMC1-cterm induces high serum levels of anti-mLAMC1-cterm antibodies but no skin lesions.

<p>Immunization of different mouse strains with the recombinant murine laminin γ1 C-terminus (mLAMC1-cterm) did induce high levels of anti-mLAMC1-cterm antibodies but no macro- or microscopic disease. Three different mouse strains (B57BL/6, BALB/c, and SJL; n = 5/strain) were immunized 4 times with 60 µg of recombinant mLAMC1-cterm in conjunction with TiterMax®. After 16 weeks, no clinical or histopathological changes (a, e, i) were seen. Deposition of mouse IgG at the dermal-epidermal junction (DEJ) was found in only 2 C56BL/6 mice (b). In all other mice, no IgG or C3 deposition was detected at the DEJ (f, j, c, g, k). In all mice, serum autoantibodies labeled the basal layer of keratinocytes at the murine DEJ (d, h, l) and reacted with recombinant mLAMC1-cterm by ELISA (m) and immunoblotting (n; C57BL/6, lane 1; BALB/c, lane 2; SJL, lane 3) and the 200 kDa p200 protein in extract of murine dermis (o; C57BL/6, lane 1; BALB/c, lane 2; SJL, lane 3). Serum of a mouse immunized with TiterMax® alone served as control (n and o; lane 4).</p