Autoantibodies in the Autoimmune Disease Pemphigus Foliaceus Induce Blistering via p38 Mitogen-Activated Protein Kinase-Dependent Signaling in the Skin
2008; Elsevier BV; Volume: 173; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2008.080391
ISSN1525-2191
AutoresPaula Berkowitz, Michael Chua, Zhi Liu, Luis A. Díaz, David S. Rubenstein,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoPemphigus foliaceus (PF) is a human autoimmune blistering disease in which a humoral immune response targeting the skin results in a loss of keratinocyte cell-cell adhesion in the superficial layers of the epidermal epithelium. In PF, desmoglein-1-specific autoantibodies induce blistering. Evidence is beginning to accumulate that activation of signaling may have an important role in the ability of pathogenic pemphigus IgGs to induce blistering and that both p38 mitogen-activated protein kinase (MAPK) and heat shock protein (HSP) 27 are part of this signaling pathway. This study was undertaken to investigate the ability of PF IgGs to activate signaling as well as the contribution of this signaling pathway to blister induction in an in vivo model of PF. Phosphorylation of both p38 MAPK and HSP25, the murine HSP27 homolog, was observed in the skin of PF IgG-treated mice. Furthermore, inhibition of p38 MAPK blocked the ability of PF IgGs to induce blistering in vivo. These results indicate that PF IgG-induced blistering is dependent on activation of p38 MAPK in the target keratinocyte. Rather than influencing the immune system, limiting the autoantibody-induced intracellular signaling response that leads to target end-organ damage may be a more viable therapeutic strategy for the treatment of autoimmune diseases. Inhibition of p38 MAPK may be an effective strategy for the treatment of PF. Pemphigus foliaceus (PF) is a human autoimmune blistering disease in which a humoral immune response targeting the skin results in a loss of keratinocyte cell-cell adhesion in the superficial layers of the epidermal epithelium. In PF, desmoglein-1-specific autoantibodies induce blistering. Evidence is beginning to accumulate that activation of signaling may have an important role in the ability of pathogenic pemphigus IgGs to induce blistering and that both p38 mitogen-activated protein kinase (MAPK) and heat shock protein (HSP) 27 are part of this signaling pathway. This study was undertaken to investigate the ability of PF IgGs to activate signaling as well as the contribution of this signaling pathway to blister induction in an in vivo model of PF. Phosphorylation of both p38 MAPK and HSP25, the murine HSP27 homolog, was observed in the skin of PF IgG-treated mice. Furthermore, inhibition of p38 MAPK blocked the ability of PF IgGs to induce blistering in vivo. These results indicate that PF IgG-induced blistering is dependent on activation of p38 MAPK in the target keratinocyte. Rather than influencing the immune system, limiting the autoantibody-induced intracellular signaling response that leads to target end-organ damage may be a more viable therapeutic strategy for the treatment of autoimmune diseases. Inhibition of p38 MAPK may be an effective strategy for the treatment of PF. The treatment of immunobullous disorders, in which an autoimmune response targets the skin to cause loss of epidermal integrity, has traditionally used agents that suppress the immune response. With a compromised epithelial barrier, patients are at risk for fluid and electrolyte imbalance and infection. Untreated, these disorders can be lethal. The introduction of corticosteroids revolutionized the treatment of autoimmune skin disease; however, chronic use of systemic corticosteroids carries significant morbidity and mortality. Steroid-sparing agents and therapies that target specific components of the immune response have been used to minimize these complications; however, immune suppression itself carries risk, including the potential for infectious complications. Rather than inhibit the immune system, an alternative strategy would be to block the ability of the autoimmune response to damage the target end- organ. The pemphigus family of human autoimmune blistering diseases offers several advantages in the study of autoimmunity and the development of specific mechanism based therapies: i) end-organ damage is readily visible as a blister, ii) the pathogenic target autoantigens are defined,1Amagai M Klaus-Kovtun V Stanley JR Autoantibodies against a novel epithelial cadherin in pemphigus vulgaris, a disease of cell adhesion.Cell. 1991; 67: 869-877Abstract Full Text PDF PubMed Scopus (886) Google Scholar, 2Eyre RW Stanley JR Human autoantibodies against a desmosomal protein complex with a calcium-sensitive epitope are characteristic of pemphigus foliaceus patients.Journal of Experimental Medicine. 1987; 165: 1719-1724Crossref PubMed Scopus (138) Google Scholar, 3Eyre RW Stanley JR Identification of pemphigus vulgaris antigen extracted from normal human epidermis and comparison with pemphigus foliaceus antigen.Journal of Clinical Investigation. 1988; 81: 807-812Crossref PubMed Scopus (198) Google Scholar iii) in vivo models are available,4Anhalt GJ Labib RS Voorhees JJ Beals TF Diaz LA Induction of pemphigus in neonatal mice by passive transfer of IgG from patients with the disease.New England Journal of Medicine. 1982; 306: 1189-1196Crossref PubMed Scopus (594) Google Scholar, 5Takahashi Y Patel HP Labib RS Diaz LA Anhalt GJ Experimentally induced pemphigus vulgaris in neonatal BALB/c mice: a time-course study of clinical, immunologic, ultrastructural, and cytochemical changes.Journal of Investigative Dermatology. 1985; 84: 41-46Crossref PubMed Scopus (87) Google Scholar, 6Amagai M Tsunoda K Suzuki H Nishifuji K Koyasu S Nishikawa T Use of autoantigen-knockout mice in developing an active autoimmune disease model for pemphigus.Journal of Clinical Investigation. 2000; 105: 625-631Crossref PubMed Scopus (230) Google Scholar and iv) the skin is accessible to topical as well as systemically delivered drugs. In pemphigus, a humoral immune response targets epithelial structures leading to loss of cell-cell adhesion and blister formation. The two major variants are pemphigus foliaceus (PF) and pemphigus vulgaris (PV). Desmogleins, transmembrane nonclassical cadherin cell adhesion proteins, are the major pathogenic autoantibody targets of both pemphigus variants. Clinically, PV is characterized by flaccid vesicles and erosions of mucosa (mucosal PV) and subsequently mucosa and skin (mucocutaneous PV), whereas PF is characterized by superficial crusted vesicles and blisters. Histologically, loss of cell-cell adhesion (eg, acantholysis) occurs in the suprabasal epidermis in PV and in the subcorneal epidermis in PF. In PF, the autoantibody response is directed against desmoglein-1 (dsg1)2Eyre RW Stanley JR Human autoantibodies against a desmosomal protein complex with a calcium-sensitive epitope are characteristic of pemphigus foliaceus patients.Journal of Experimental Medicine. 1987; 165: 1719-1724Crossref PubMed Scopus (138) Google Scholar and found in suprabasilar desmosomes of epidermal epithelia, whereas in PV, the autoantibody response is initially directed against desmoglein-3 (dsg3)1Amagai M Klaus-Kovtun V Stanley JR Autoantibodies against a novel epithelial cadherin in pemphigus vulgaris, a disease of cell adhesion.Cell. 1991; 67: 869-877Abstract Full Text PDF PubMed Scopus (886) Google Scholar, 3Eyre RW Stanley JR Identification of pemphigus vulgaris antigen extracted from normal human epidermis and comparison with pemphigus foliaceus antigen.Journal of Clinical Investigation. 1988; 81: 807-812Crossref PubMed Scopus (198) Google Scholar, 7Amagai M Karpati S Prussick R Klaus-Kovtun V Stanley JR Autoantibodies against the amino-terminal cadherin-like binding domain of pemphigus vulgaris antigen are pathogenic.Journal of Clinical Investigation. 1992; 90: 919-926Crossref PubMed Scopus (312) Google Scholar and found in desmosomes of the basal layer of stratified epidermal epithelia and in mucosal epithelia. In PV, the autoantibody response subsequently evolves to include dsg1 as the disease transitions from mucosal to mucocutaneous PV.8Ding X Aoki V Mascaro Jr, JM Lopez-Swiderski A Diaz LA Fairley JA Mucosal and mucocutaneous (generalized) pemphigus vulgaris show distinct autoantibody profiles.Journal of Investigative Dermatology. 1997; 109: 592-596Crossref PubMed Scopus (224) Google Scholar, 9Amagai M Tsunoda K Zillikens D Nagai T Nishikawa T The clinical phenotype of pemphigus is defined by the anti-desmoglein autoantibody profile.Journal of the American Academy of Dermatology. 1999; 40: 167-170Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 10Miyagawa S Amagai M Iida T Yamamoto Y Nishikawa T Shirai T Late development of antidesmoglein 1 antibodies in pemphigus vulgaris: correlation with disease progression.British Journal of Dermatology. 1999; 141: 1084-1087Crossref PubMed Scopus (63) Google Scholar Dsg1 and dsg3 are similar in that they both are transmembrane proteins of the cadherin superfamily and are components of desmosome cell-cell adhesion junctions.11Green KJ Simpson CL Desmosomes: new perspectives on a classic.J Invest Dermatol. 2007; 127: 2499-2515Crossref PubMed Scopus (307) Google Scholar The compensation hypothesis, the ability of dsg1 to compensate for loss of dsg3 function with the different distribution of dsg1 and dsg3 in epidermal epithelia and mucosa, has been proposed to explain the tissue distribution and location of the cleavage plane in PV and PF.12Mahoney MG Wang Z Rothenberger K Koch PJ Amagai M Stanley JR Explanations for the clinical and microscopic localization of lesions in pemphigus foliaceus and vulgaris.Journal of Clinical Investigation. 1999; 103: 461-468Crossref PubMed Scopus (421) Google Scholar Different mechanisms have been proposed to explain blister induction by Ig in autoimmune blistering diseases of the skin. For example, in the human subepidermal blistering disease bullous pemphigoid (BP), IgGs bind to the hemidesmosome-associated protein BP180 and trigger a complement-, mast cell-, and neutrophil-dependent inflammatory cascade culminating in human neutrophil elastase-dependent proteolytic cleavage of BP180 and separation at the dermal-epidermal junction.13Nelson KC Zhao M Schroeder PR Li N Wetsel RA Diaz LA Liu Z Role of different pathways of the complement cascade in experimental bullous pemphigoid.J Clin Invest. 2006; 116: 2892-2900Crossref PubMed Scopus (93) Google Scholar, 14Liu Z Li N Diaz LA Shipley M Senior RM Werb Z Synergy between a plasminogen cascade and MMP-9 in autoimmune disease.J Clin Invest. 2005; 115: 879-887Crossref PubMed Scopus (94) Google Scholar, 15Chen R Ning G Zhao ML Fleming MG Diaz LA Werb Z Liu Z Mast cells play a key role in neutrophil recruitment in experimental bullous pemphigoid.Journal of Clinical Investigation. 2001; 108: 1151-1158Crossref PubMed Scopus (209) Google Scholar, 16Liu Z Giudice GJ Swartz SJ Fairley JA Till GO Troy JL Diaz LA The role of complement in experimental bullous pemphigoid.Journal of Clinical Investigation. 1995; 95: 1539-1544Crossref PubMed Scopus (268) Google Scholar, 17Liu Z Giudice GJ Zhou X Swartz SJ Troy JL Fairley JA Till GO Diaz LA A major role for neutrophils in experimental bullous pemphigoid.Journal of Clinical Investigation. 1997; 100: 1256-1263Crossref PubMed Scopus (231) Google Scholar, 18Liu Z Zhou X Shapiro SD Shipley JM Twining SS Diaz LA Senior RM Werb Z The serpin alpha1-proteinase inhibitor is a critical substrate for gelatinase B/MMP-9 in vivo.Cell. 2000; 102: 647-655Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar In contrast, pemphigus antibody binding to dsg does not require inflammatory components to mediate blister formation; pemphigus IgG induced epidermal cell detachment is neither Fc-,19Mascaro Jr, JM Espana A Liu Z Ding X Swartz SJ Fairley JA Diaz LA Mechanisms of acantholysis in pemphigus vulgaris: role of IgG valence.Clinical Immunology & Immunopathology. 1997; 85: 90-96Crossref PubMed Scopus (69) Google Scholar complement-,20Anhalt GJ Till GO Diaz LA Labib RS Patel HP Eaglstein NF Defining the role of complement in experimental pemphigus vulgaris in mice.Journal of Immunology. 1986; 137: 2835-2840PubMed Google Scholar nor proteinase-dependent.21Mahoney MG Wang ZH Stanley JR Pemphigus vulgaris and pemphigus foliaceus antibodies are pathogenic in plasminogen activator knockout mice.Journal of Investigative Dermatology. 1999; 113: 22-25Crossref PubMed Scopus (86) Google Scholar Steric hindrance, the direct blocking of desmosome cadherin-adhesive interactions by pemphigus IgG, has also been suggested as a mechanism for the adhesive disrupting ability of these immunoglobulins22Shimizu A Ishiko A Ota T Tsunoda K Amagai M Nishikawa T IgG binds to desmoglein 3 in desmosomes and causes a desmosomal split without keratin retraction in a pemphigus mouse model.J Invest Dermatol. 2004; 122: 1145-1153Crossref PubMed Scopus (79) Google Scholar; however, steric hindrance alone may not be sufficient to explain the pathogenic effects because energy-requiring cellular events are required for pemphigus IgG to induce acantholysis.23Waschke J Bruggeman P Baumgartner W Zillikens D Drenckhahn D Pemphigus foliaceus IgG causes dissociation of desmoglein 1-containing junctions without blocking desmoglein 1 transinteraction.J Clin Invest. 2005; 115: 3157-3165Crossref PubMed Scopus (140) Google Scholar, 24Calkins CC Setzer SV Jennings JM Summers S Tsunoda K Amagai M Kowalczyk AP Desmoglein endocytosis and desmosome disassembly are coordinated responses to pemphigus autoantibodies.J Biol Chem. 2006; 281: 7623-7634Crossref PubMed Scopus (190) Google Scholar Binding of pemphigus IgG to the keratinocyte has been proposed to activate intracellular signaling within the target keratinocyte, and this may contribute to the loss of cell-cell adhesion.25Seishima M Esaki C Osada K Mori S Hashimoto T Kitajima Y Pemphigus IgG, but not bullous pemphigoid IgG, causes a transient increase in intracellular calcium and inositol 1,4,5-triphosphate in DJM-1 cells, a squamous cell carcinoma line.J Invest Dermatol. 1995; 104: 33-37Crossref PubMed Scopus (108) Google Scholar In our previous work on PV, we identified a series of intracellular phosphorylation events initiated by binding of PV IgG to keratinocytes. Incubation of cultured human keratinocytes with IgG purified from PV patient sera resulted in activation of p38 mitogen-activated protein kinase (MAPK), phosphorylation of the small heat shock protein (HSP) 27, reorganization of the actin cytoskeleton, and intermediate filament collapse. In tissue culture, inhibitors of p38MAPK prevented PV IgG-induced phosphorylation of HSP27 and reorganization of the cytoskeleton, events preceding the loss of cell-cell adhesion induced by PV IgG.26Berkowitz P Hu P Liu Z Diaz LA Enghild JJ Chua MP Rubenstein DS Desmosome Signaling: INHIBITION OF p38MAPK PREVENTS PEMPHIGUS VULGARIS IgG-INDUCED CYTOSKELETON REORGANIZATION.J Biol Chem. 2005; 280: 23778-23784Crossref PubMed Scopus (212) Google Scholar These observations in tissue culture suggested that p38MAPK could be part of the acantholytic mechanism of PV IgG and that blocking p38MAPK activity might have a role in preventing PV IgG-induced blistering. Indeed, we then demonstrated that pharmacological inhibitors of p38MAPK prevented in vivo blister formation in the PV passive transfer mouse model.27Berkowitz P Hu P Warren S Liu Z Diaz LA Rubenstein DS p38MAPK inhibition prevents disease in pemphigus vulgaris mice.Proceedings of the National Academy of Sciences of the United States of America. 2006; 103: 12855-12860Crossref PubMed Scopus (194) Google Scholar Furthermore, phosphorylation of both p38MAPK and HSP27 has been observed in perilesional epidermis of pemphigus patients.28Berkowitz P Diaz LA Hall RP Rubenstein DS Induction of p38MAPK and HSP27 phosphorylation in pemphigus patient skin.J Invest Dermatol. 2008; 128: 738-740Crossref PubMed Scopus (56) Google Scholar In PF, IgGs that target dsg1 are pathogenic. The similar immunobiology of PF and PV led us to hypothesize that like PV, PF IgGs might activate intracellular signaling via p38MAPK and HSP27. Using the passive transfer mouse model, this study was undertaken to determine if PF IgG induced activation of p38MAPK and HSP25, the murine HSP27 homolog, in the skin, and if pharmacological inhibition of p38MAPK could prevent blister formation in vivo. The results demonstrate that keratinocyte p38MAPK is activated in response to binding of pathogenic PF IgG to the skin and that inhibiting p38MAPK blocks the ability of the pathogenic IgG to cause end-organ damage, eg, blistering, in the skin. These observations suggest that targeting the mechanism by which the autoimmune response damages the end-organ may have utility in the treatment of autoimmune disorders. Rabbit polyclonal anti-HSP25 antibodies were from StressGen (Ann Arbor, MI). Rabbit monoclonal anti-phospho HSP27 antibodies, rabbit monoclonal anti-phospho-p38MAPK (Thr180/Tyr182) antibodies, and horseradish peroxidase-linked anti-rabbit secondary antibodies were from Cell Signaling Technologies (Beverly, MA). Rabbit polyclonal anti-p38MAPK antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA) and polyclonal anti-lactate dehydrogenase V antibodies were from Cortex Biochem (San Leandro, CA). Rabbit anti-sheep horseradish peroxidase-conjugated secondary antibodies were from Cortex Biochem. The p38MAPK inhibitors SB202190 and SB203580, and the inactive analog SB202474 were from Calbiochem (La Jolla, CA). PF sera were previously described.29Rock B Labib RS Diaz LA Monovalent Fab' immunoglobulin fragments from endemic pemphigus foliaceus autoantibodies reproduce the human disease in neonatal Balb/c mice.Journal of Clinical Investigation. 1990; 85: 296-299Crossref PubMed Scopus (120) Google Scholar, 30Warren SJ Lin MS Giudice GJ Hoffmann RG Hans-Filho G Aoki V Rivitti EA Santos V Diaz LA The prevalence of antibodies against desmoglein 1 in endemic pemphigus foliaceus in Brazil. Cooperative Group on Fogo Selvagem Research.New England Journal of Medicine. 2000; 343 ([comment]): 23-30Crossref PubMed Scopus (139) Google Scholar Data presented in Figure 1, Figure 2, Figure 3 are from IgGs purified from a single PF patient, PF1, whose serum was available in sufficient quantities to perform the described studies; the activity of this serum was determined by indirect immunofluorescence on human skin with a titer of 1:160. A second independent series of experiments (Supplementary Figure S1, see http://ajp.amjpathol.org) used PF IgGs purified from sera of a second patient, PF2, (whose activity was determined by indirect immunofluorescence on human skin with a titer of 1:80) and showed similar results. Both sera were positive by dsg1 enzyme-linked immunosorbent assay (ELISA) and negative by dsg3 ELISA. PF IgGs were purified from PF patient sera by ammonium sulfate precipitation followed by affinity chromatography on Protein G (HiTrap; Pharmacia, Piscataway, NJ) as previously described.26Berkowitz P Hu P Liu Z Diaz LA Enghild JJ Chua MP Rubenstein DS Desmosome Signaling: INHIBITION OF p38MAPK PREVENTS PEMPHIGUS VULGARIS IgG-INDUCED CYTOSKELETON REORGANIZATION.J Biol Chem. 2005; 280: 23778-23784Crossref PubMed Scopus (212) Google Scholar IgG fractions were dialyzed against phosphate-buffered saline (PBS) and sterile-filtered. Purity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and activity assayed by indirect immunofluorescence and ELISA. Control IgGs (no activity by indirect immunofluorescence) were prepared in parallel from normal human sera.Figure 2Inhibiting p38MAPK prevents clinical blistering in PF passive transfer mice. Neonatal C57BL/6J mice were injected intradermally with either PF IgG (0.1 mg of IgG/g body weight) (A) or PF IgG (0.1 mg of IgG/g body weight) plus SB2023580 (B). After 18 hours, the skin of neonatal mice from the test and control groups was examined clinically. PF IgG-treated mice have a positive Nikolsky's sign (white arrows), demonstrating loss of epithelial cell-cell adhesion. In contrast, mice treated with the SB203580 and PF IgG have a negative Nikolsky's sign, indicating that epithelial adhesion remains intact. Inhibiting p38MAPK prevents histological blistering in PF passive transfer mice. Representative skin biopsies of mice treated with control IgG (0.015 mg of IgG/g body weight) (C), PF IgG (0.015 mg of IgG/g body weight) (D), SB203580 and then PF IgG (E), and SB202474 and then PF IgG (F), were fixed in formalin and stained with H&E. Acantholysis leading to blister formation is seen in PF IgG-treated mice, but is blocked in mice treated with the p38MAPK inhibitor SB203580, but not the inactive analog SB202474.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3The inhibitors do not prevent systemic absorption of the injected IgG: anti-dsg1 IgG serum levels. Serum samples obtained from control IgG, PF IgG-treated, PF IgG plus SB203580-treated, and PF IgG plus SB202470-treated mice were examined for the presence of anti-dsg1 autoantibodies by using a dsg1 ectodomain-based ELISA. (A) No significant differences in circulating anti-dsg1 IgG were found in mice pretreated with the active p38MAPK inhibitor or the inactive analog when compared to mice treated with PF IgG alone. P values are as follows: i) PF IgG compared to control, P = 0.004; ii) PF IgG + SB203580 compared to PF IgG, P = 0.27; iii) PF IgG + SB202474 compared to PF IgG, P = 0.4. SD is shown by error bars (n = 3 mice per treatment group); P values were calculated by using the Student's t-test. The inhibitors do not prevent the diffusion of IgG to the epidermis. Perilesional skin biopsies from control IgG (B), PF IgG-treated (C), PF IgG plus SB203580-treated (D), and PF IgG plus SB202470-treated (E) mice were examined for the presence of human anti-dsg1 PF IgG by direct immunofluorescence by using a mouse anti-human Cy-2-conjugated monoclonal antibody. A honeycomb pattern of staining in the epidermis is seen in mice treated with PF IgG, PF IgG plus SB203580, and PF IgG plus SB202474, demonstrating that the inhibitors do notprevent binding of PF autoantibodies to the keratinocyte cell surface.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Breeding pairs of C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained at the University of North Carolina DLAM Facility in accordance with Institutional Animal Care and Use Committee protocols. Neonatal mice (24 to 36 hours old with body weights between 1.4 g and 1.6 g) were used for passive transfer experiments. Neonates were injected intradermally with a sterile solution of either control IgGs or PF IgGs as described previously.4Anhalt GJ Labib RS Voorhees JJ Beals TF Diaz LA Induction of pemphigus in neonatal mice by passive transfer of IgG from patients with the disease.New England Journal of Medicine. 1982; 306: 1189-1196Crossref PubMed Scopus (594) Google Scholar, 31Roscoe JT Diaz L Sampaio SA Castro RM Labib RS Takahashi Y Patel H Anhalt GJ Brazilian pemphigus foliaceus autoantibodies are pathogenic to BALB/c mice by passive transfer.Journal of Investigative Dermatology. 1985; 85: 538-541Crossref PubMed Scopus (175) Google Scholar, 32Rock B Martins CR Theofilopoulos AN Balderas RS Anhalt GJ Labib RS Futamura S Rivitti EA Diaz LA The pathogenic effect of IgG4 autoantibodies in endemic pemphigus foliaceus (fogo selvagem).New England Journal of Medicine. 1989; 320: 1463-1469Crossref PubMed Scopus (252) Google Scholar For direct clinical examination, mice (n = 3) were injected with PF IgGs (PF1: 0.10 mg IgG/g animal weight) in a total volume of 50 μl of PBS. This dose of PF IgGs resulted in gross sloughing of the skin. Alternatively, mice (n = 3) were preinjected with 18.75 μg of SB203580 in 50 μl intradermally then at 2 hours re-injected intradermally with 18.75 μg of SB203580 + PF IgG/50 μl (total of 37.5 μg of SB203580). The skin of neonatal mice from PF IgG and PF IgG + inhibitor-treated groups was examined 18 hours after the injection of IgG for the presence of Nikolsky's sign, in which gentle friction of perilesional skin causes sheet-like sloughing of the epidermis. A second group of animals (n = 3 mice) received a lower dose of PF IgGs (PF1, 0.015 mg/g body weight in 50 μl of PBS) to preserve the cutaneous architecture lost by epithelial sloughing at the higher dose. After clinical examination, the animals were sacrificed, and skin and serum specimens obtained for routine histological examination using light microscopy (hematoxylin and eosin staining) and direct immunofluorescence assays to detect keratinocyte cell surface-bound pemphigus IgGs. Serum samples were assayed for the presence of circulating human anti-dsg1 IgGs by ELISA against baculovirus expressed ectodomains of human dsg1 as previously described.33Arteaga LA Prisayanh PS Warren SJ Liu Z Diaz LA Lin MS A subset of pemphigus foliaceus patients exhibits pathogenic autoantibodies against both desmoglein-1 and desmoglein-3.J Invest Dermatol. 2002; 118: 806-811Crossref PubMed Scopus (60) Google Scholar Additional skin samples were harvested to prepare protein extracts for SDS-PAGE and two-dimensional gel electrophoresis. After transfer to polyvinylidene difluoride, membranes were probed by immunoblot for proteins of interest. For in vivo inhibitor studies, mice (n = 3 mice per treatment group) were preinjected with 6.25 μg of SB202190 in 50 μl intradermally then at 2 hours re-injected intradermally with 6.25 μg of SB202190 + PF IgG/50 μl (total of 12.5 μg of SB202190). No increased mortality was observed in the inhibitor versus control mice. As a control, a separate group of mice (n = 3) were pretreated with the inactive analog SB202474 (total of 12.5 μg of SB202470)34Lee JC Laydon JT McDonnell PC Gallagher TF Kumar S Green D McNulty D Blumenthal MJ Heys JR Landvatter SW et al.A protein kinase involved in the regulation of inflammatory cytokine biosynthesis.Nature. 1994; 372: 739-746Crossref PubMed Scopus (3169) Google Scholar following the split dose protocol used for SB202190. Additionally, a third group of mice (n = 3) were pretreated with the p38MAPK inhibitor SB203580 (total of 25 μg of SB203580) following the split dose protocol used for SB202190. Typically, neonatal litters were groups of five to six mice. Because of the large numbers of mice used for this study, not all mice were injected at the same time, but rather, based on availability of neonatal litters. Extracts were prepared from skin biopsies by Dounce homogenization in IEF lysis buffer (8 mol/L urea, 4% CHAPS, 2.5 mmol/L dithiothreitol, 40 mmol/L Tris, 10 μmol/L pepstatin, 100 μmol/L leupeptin, 10 μmol/L E-64, 1 mmol/L phenylmethyl sulfonyl fluoride). Protein concentration was by modified Bradford as described.35Hu P O'Keefe EJ Rubenstein DS Tyrosine phosphorylation of human keratinocyte beta-catenin and plakoglobin reversibly regulates their binding to E-cadherin and alpha-catenin.Journal of Investigative Dermatology. 2001; 117: 1059-1067Crossref PubMed Google Scholar IPG buffer (pH 4 to 7, Pharmacia) was added to each sample to a final concentration of 0.5% before isoelectric focusing. Samples were separated in the first dimension using 7-cm, pH 4 to 7, linear IPGphor strips (Pharmacia) and in the second dimension by 10% SDS-PAGE. Gels were transferred to polyvinylidene difluoride membranes for immunoblot analysis. Western blots were developed by enhanced chemiluminescence (ECL) reaction and the signal intensity quantified by scanning chemiluminescence on a GeneGnome scanner (Syngene Bio Imaging, Frederick, MD) using GeneSnap software. The signal intensity for each HSP25 isoform was expressed as a percentage of total HSP25 using the formula Pn/(P0 + P1 + P2), where, n corresponds to the signal intensity for spot 0, 1, or 2, and P0 + P1 + P2 is the summed signal intensity for all three HSP25 isoforms. Statistical significance was determined using the Student's t-test. Neonatal C57BL/6J mice were injected intradermally with PF IgGs purified from PF patient sera or control IgG from nonaffected individuals. After 18 hours, mice were examined clinically for the presence of blister formation. Additionally, skin biopsies were obtained for histological examination, immunohistochemical staining, and for biochemical analyses. Pretreatment by intradermal injection of the p38MAPK inhibitors SB203580, SB202190, or the inactive analog SB202474, followed by passive transfer of PF IgGs was used to determine whether inhibition of p38MAPK blocked PF IgG-mediated acantholysis in vivo. Extracts from biopsies were prepared by tissue homogenization in IEF lysis buffer and separated by SDS-PAGE and two-dimensional gel electrophoresis. After electrotransfer, polyvinylidene difluoride membranes were probed with antibodies to p38MAPK, phospho-p38MAPK, and HSP25. Compared to control IgG-treated mice, increased levels of phospho-p38MAPK were detected in skin biopsies from PF IgG-treated mice. The increase in phosphorylation was approximately twofold greater than baseline levels in controls. PF IgG-induced phosphorylation of p38MAPK was blocked when mice were pretreated with the p38MAPK inhibitors SB203580 (Figure 1, A and B) or SB202190 (data not shown), but not in mice pretreated with the inactive analog SB202474. In skin biopsies from PF IgG-treated mice, the blister roof and floor could be readily separated from one another and independently analyzed. As seen in Figure 1, phopsho-p38MAPK was found predominantly in the blister roof. Phosphorylation of HSP25 occurs as a downstream consequence of p38MAPK-mediated kinase activity. Predicting that PF IgG-induced phosphorylation of p38MAPK would result in phosphorylation of HSP25, we next examined the phospho
Referência(s)