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No Evidence of Apoptotic Cells in Pemphigus Acantholysis

2014; Elsevier BV; Volume: 134; Issue: 7 Linguagem: Inglês

10.1038/jid.2014.60

1523-1747

Ineke C. Janse, Gerda van der Wier, Marcel F. Jonkman, Hendri H. Pas, Gilles F.H. Diercks,

Eosinophilic Disorders and Syndromes

antibody cleaved desmoglein hematoxylin and eosin poly (ADP-ribose) polymerase pemphigus vulgaris pemphigus foliaceus Pemphigus is a group of rare mucocutaneous autoimmune bullous diseases that are characterized by intra-epidermal IgG deposition and loss of cohesion between keratinocytes, known as acantholysis. Although the pathogenic relevance of anti-desmosomal IgG has been clearly demonstrated, the exact mechanism by which IgG induces loss of adhesion remains unsolved. Cell signaling has received a lot of attention in the past years, including the death signaling (apoptotic) pathways. Apoptosis can be activated via an extrinsic or intrinsic pathway. In the extrinsic pathway, Fas L binds to the Fas receptor, which leads to the activation of caspase 8. In the intrinsic pathway, subsequently p53, bax, cytochrome c, and caspase 9 are activated. Both caspases 8 and 9 activate the common pathway caspase 3. Caspase 3 induces DNA fragmentation, which can be detected with poly (ADP-ribose) polymerase (PARP), fractin, and TUNEL (de Boer et al., 2000de Boer R.A. van Veldhuisen D.J. van der Wijk J. et al.Additional use of immunostaining for active caspase 3 and cleaved actin and PARP fragments to detect apoptosis in patients with chronic heart failure.J Card Fail. 2000; 6: 330-337Abstract Full Text PDF PubMed Scopus (51) Google Scholar; Hengartner, 2000Hengartner M.O. The biochemistry of apoptosis.Nature. 2000; 407: 770-776Crossref PubMed Scopus (6253) Google Scholar). Apoptosis has been suggested as an upstream event in acantholysis, but alternatively, also as a downstream event after loss of cell–cell adhesion (Schmidt and Waschke, 2009Schmidt E. Waschke J. Apoptosis in pemphigus.Autoimmun Rev. 2009; 8: 533-537Crossref PubMed Scopus (54) Google Scholar, Schmidt et al., 2009Schmidt E. Gutberlet J. Siegmund D. et al.Apoptosis is not required for acantholysis in pemphigus vulgaris.Am J Physiol Cell Physiol. 2009; 296: C162-C172Crossref PubMed Scopus (45) Google Scholar, Lee et al., 2009Lee H.E. Berkowitz P. Jolly P.S. et al.Biphasic activation of p38MAPK suggests that apoptosis is a downstream event in pemphigus acantholysis.J Biol Chem. 2009; 284: 12524-12532Crossref PubMed Scopus (54) Google Scholar). Furthermore, it has been hypothesized that IgG might induce apoptotic enzymes but that these do not lead to cell death but instead to acantholysis, a mechanism referred to as apoptolysis (Grando et al., 2009Grando S.A. Bystryn J.C. Chernyavsky A.I. et al.Apoptolysis: a novel mechanism of skin blistering in pemphigus vulgaris linking the apoptotic pathways to basal cell shrinkage and suprabasal acantholysis.Exp Dermatol. 2009; 18: 764-770Crossref PubMed Scopus (101) Google Scholar). A thorough examination of the literature on apoptosis in pemphigus revealed that the possible involvement of apoptosis in acantholysis was mainly studied in cultured cell and/or mouse models, but rarely in patient skin (Schmidt and Waschke, 2009Schmidt E. Waschke J. Apoptosis in pemphigus.Autoimmun Rev. 2009; 8: 533-537Crossref PubMed Scopus (54) Google Scholar). Despite this, the caspase pathway has already been suggested as a therapeutic target in pemphigus (Pacheco-Tovar et al., 2011Pacheco-Tovar D. Lopez-Luna A. Herrera-Esparza R. et al.The caspase pathway as a possible therapeutic target in experimental pemphigus.Autoimmune Dis. 2011; 2011: 563091PubMed Google Scholar). This, together with, as mentioned before, conflicting results from various studies, propelled us to reinvestigate this topic. We therefore searched for evidence of apoptosis in pemphigus patient skin and in an in vitro skin model, wherein we induced acantholysis by patient IgG. We checked for activation of both the intrinsic and the extrinsic pathway by immunofluorescence and, furthermore, used electron microscopy to look for hallmarks of apoptosis. For immunofluorescence, we included 11 biopsies from 9 mucocutaneous pemphigus vulgaris (PV) patients (5 from healthy skin, 3 from perilesional skin, and 3 from lesional skin), and 11 biopsies from 7 pemphigus foliaceus (PF) patients (4 from healthy skin, 2 from perilesional skin, and 5 from lesional skin). Immunofluorescent analysis was performed for cleaved caspase 3, cleaved caspase 8, cleaved PARP, fractin, and TUNEL. Protocols and other details can be found in Supplementary Text S1 online. The apoptotic markers stained positive in the positive-control tissues but were absent in the negative controls (Supplementary Figure S1 online). None of the pemphigus skin biopsies showed positive staining of cl caspase 3, cl caspase 8, fractin, or nuclear PARP. In PV, TUNEL was positive in 0.46% (range, 0–0.97%) of lesional epidermis and in 1.25% (range, 0–2.94%) of perilesional epidermis. In PF, these numbers were 4.42% (range, 0–17.78%) for lesional epidermis and 0.84% (range, 0–1.91%) for perilesional epidermis (Figure 1). In healthy pemphigus skin, positive cells were only sporadically present. In healthy control skin, 1.11% of the cells were TUNEL positive (range, 0–4.44%). Download .pdf (.53 MB) Help with pdf files Supplementary Material Second, we used eight biopsies from a previously described pemphigus in vitro model experiment (Oktarina et al., 2011Oktarina D.A. van der Wier G. Diercks G.F. et al.IgG-induced clustering of desmogleins 1 and 3 in skin of patients with pemphigus fits with the desmoglein nonassembly depletion hypothesis.Br J Dermatol. 2011; 165: 552-562Crossref PubMed Scopus (54) Google Scholar). In these experiments, healthy breast reduction skin biopsies had been incubated with purified pemphigus IgG (for 4, 16, and 24 hours with PV IgG and for 24, 48, and 72 hours with PF IgG) (for additional details see Supplementary Text S1 online). Acantholysis was present in PV IgG–incubated biopsies after 16 hours and in the PF IgG–incubated biopsies after 48 hours. None of the in vitro model biopsies showed positive staining for cl caspase 3, cl caspase 8, fractin, or nuclear PARP. An intercellular PARP staining was present in the in vitro model biopsies. This intercellular staining pattern was also present in healthy skin and in the additional controls of the in vitro model. Some TUNEL-positive cells were found in the stratum granulosum of all biopsies but were absent in the layers beneath, including in the acantholytic biopsies. The percentages of TUNEL-positive cells found in the PV model were as follows: 7.46% at t=0 (non-incubated skin), 0% at t=4, 4.03% at t=16, and 3.17% at t=24. For the PF model, these percentages were 1.26% at t=0, 2.32% at t=24, 3.46% at t=48, and 1.62% at t=72 (Supplementary Figure S2 online). Finally, six skin biopsies (two lesional, one perilesional, and three non-lesional) from four mucocutaneous PV patients and nine skin biopsies (five lesional, one perilesional, and three non-lesional) from eight PF patients were investigated by electron microscopy for ultrastructural signs of apoptosis. All details including used controls are described in Supplementary Text S1 online. All perilesional and lesional biopsies showed widening of intercellular spaces. The lesional biopsies show a decreased number of desmosomes. None of the biopsies, however, showed any morphological features of apoptosis, i.e., rounding up of the cell, retraction of pseudopods, pyknosis, karyorrhexis, plasma membrane blebbing, and engulfment by resident phagocytes (Figure 2). In conclusion, we found no evidence of apoptotic cells in pemphigus acantholysis. First, apoptotic signaling of both the intrinsic and the extrinsic pathway was absent in 22 skin biopsies of pemphigus patients and in the in vitro model at any time point. Second, although some TUNEL staining was seen in two acantholytic PF and one PV biopsies, and in the pemphigus model, we also found TUNEL-positive cells in normal healthy skin, especially in the granular layers. The expression of TUNEL in the pemphigus skin and model was also seen in the stratum granulosum, above the blisters. If apoptosis would be a cause of acantholysis, then TUNEL positivity should have been present below the blister cavity. Third, ultrastructurally no morphological features of apoptosis were present. It should be noted that EM is considered to be the gold standard for the identification of apoptotic cells (Taatjes et al., 2008Taatjes D.J. Sobel B.E. Budd R.C. Morphological and cytochemical determination of cell death by apoptosis.Histochem Cell Biol. 2008; 129: 33-43Crossref PubMed Scopus (161) Google Scholar). Our results are in line with the findings of Schmidt et al., 2009Schmidt E. Gutberlet J. Siegmund D. et al.Apoptosis is not required for acantholysis in pemphigus vulgaris.Am J Physiol Cell Physiol. 2009; 296: C162-C172Crossref PubMed Scopus (45) Google Scholar who conclude that apoptosis is not required for pemphigus acantholysis. Like us, they found no evidence of positive cl caspase 3 and TUNEL when analyzing lesional PV patient skin. Moreover, in cultures of human keratinocytes treated with PV IgG, they also found acantholysis in absence of positive TUNEL staining as well as caspase 3 cleavage. Lee et al., 2009Lee H.E. Berkowitz P. Jolly P.S. et al.Biphasic activation of p38MAPK suggests that apoptosis is a downstream event in pemphigus acantholysis.J Biol Chem. 2009; 284: 12524-12532Crossref PubMed Scopus (54) Google Scholar also disagreed that apoptosis is an upstream event in pemphigus acantholysis. In PF IgG–treated mice and PV IgG–treated keratinocyte cultures, they found activation of cl PARP, cl caspase 3, and TUNEL, but only after acantholysis. The belief that apoptosis is an upstream event in acantholysis is largely based on studies in model systems (Schmidt and Waschke, 2009Schmidt E. Waschke J. Apoptosis in pemphigus.Autoimmun Rev. 2009; 8: 533-537Crossref PubMed Scopus (54) Google Scholar). We question whether these model systems are suitable for answering such questions on pemphigus pathogenesis, as they differ from the in vivo situation. For instance, in cultured keratinocytes the desmosomal makeup in terms of molecular composition does not accurately reflect the in vivo situation (van der Wier et al., 2010van der Wier G. Pas H.H. Jonkman M.F. Experimental human cell and tissue models of pemphigus.Dermatol Res Pract. 2010; 2010: 143871PubMed Google Scholar). As for mouse models, the repertoire of expressed genes involved in apoptosis in humans and mice is different (Reed et al., 2003Reed J.C. Doctor K. Rojas A. et al.Comparative analysis of apoptosis and inflammation genes of mice and humans.Genome Res. 2003; 13: 1376-1388Crossref PubMed Scopus (99) Google Scholar). Furthermore, previous data on pemphigus skin are scarce and based on a few lesional biopsies only (Wang et al., 2004Wang X. Bregegere F. Frusic-Zlotkin M. et al.Possible apoptotic mechanism in epidermal cell acantholysis induced by pemphigus vulgaris autoimmunoglobulins.Apoptosis. 2004; 9: 131-143Crossref PubMed Scopus (106) Google Scholar; Pacheco-Tovar et al., 2009Pacheco-Tovar M.G. Avalos-Diaz E. Vega-Memije E. et al.The final destiny of acantholytic cells in pemphigus is fas mediated.J Eur Acad Dermatol Venereol. 2009; 23: 697-701Crossref PubMed Scopus (20) Google Scholar, Deyhimi and Tavakoli, 2012Deyhimi P. Tavakoli P. Study of apoptosis in oral pemphigus vulgaris using immunohistochemical marker bax and TUNEL technique.J Oral Pathol Med. 2012; 42: 409-414Crossref PubMed Scopus (12) Google Scholar). In conclusion, this study does not support the hypothesis that apoptosis is involved in pemphigus acantholysis. However, although we studied morphological hallmarks of apoptosis, the involvement of certain apoptotic caspases or signaling pathways, which might be involved in dissociation of (inter)desmosomal adhesion complexes, cannot be excluded (Grando et al., 2009Grando S.A. Bystryn J.C. Chernyavsky A.I. et al.Apoptolysis: a novel mechanism of skin blistering in pemphigus vulgaris linking the apoptotic pathways to basal cell shrinkage and suprabasal acantholysis.Exp Dermatol. 2009; 18: 764-770Crossref PubMed Scopus (101) Google Scholar). We thank J Zuiderveen, G Meijer, M Mastik, and A Poot for their valuable help. Supplementary material is linked to the online version of the paper at http://www.nature.com/jid