The Differential Fate of Cadherins during T-Cell-Induced Keratinocyte Apoptosis Leads to Spongiosis in Eczematous Dermatitis
2001; Elsevier BV; Volume: 117; Issue: 4 Linguagem: Inglês
10.1046/j.0022-202x.2001.01474.x
ISSN1523-1747
AutoresAxel Trautmann, Frank Altznauer, Mübeccel Akdiş, Hans‐Uwe Simon, Kurt Blaser, Cezmi A. Akdiş, Rainer Disch, Eva-B Bröcker,
Tópico(s)Contact Dermatitis and Allergies
ResumoRecently we have shown that T-cell-mediated keratinocyte apoptosis plays a key pathogenetic role in the formation of eczematous dermatitis. Spongiosis, the histologic hallmark of eczematous dermatitis, is characterized by impairment of cohesion between epidermal keratinocytes. It is conceivable that the intercellular junction of keratinocytes is an early target of apoptosis-inducing T cells. In this study, we demonstrate that the induction of keratinocyte apoptosis is accompanied by a rapid cleavage of E-cadherin and loss of coimmunoprecipitated β-catenin. In situ examination of E-cadherin expression and cellular distribution in acute eczematous dermatitis revealed a reduction in keratinocyte membrane E-cadherin in areas of spongiosis. In contrast, the in vitro and in vivo expression of desmosomal cadherins during early apoptosis remained unchanged. Therefore, induction of keratinocyte apoptosis by skin-infiltrating T cells, subseqent cleavage of E-cadherin, and resisting desmosomal cadherins suggests a mechanism for spongiosis formation in eczematous dermatitis. Recently we have shown that T-cell-mediated keratinocyte apoptosis plays a key pathogenetic role in the formation of eczematous dermatitis. Spongiosis, the histologic hallmark of eczematous dermatitis, is characterized by impairment of cohesion between epidermal keratinocytes. It is conceivable that the intercellular junction of keratinocytes is an early target of apoptosis-inducing T cells. In this study, we demonstrate that the induction of keratinocyte apoptosis is accompanied by a rapid cleavage of E-cadherin and loss of coimmunoprecipitated β-catenin. In situ examination of E-cadherin expression and cellular distribution in acute eczematous dermatitis revealed a reduction in keratinocyte membrane E-cadherin in areas of spongiosis. In contrast, the in vitro and in vivo expression of desmosomal cadherins during early apoptosis remained unchanged. Therefore, induction of keratinocyte apoptosis by skin-infiltrating T cells, subseqent cleavage of E-cadherin, and resisting desmosomal cadherins suggests a mechanism for spongiosis formation in eczematous dermatitis. allergic contact dermatitis atopic dermatitis desmocollin desmoglein E-cadherin Apoptosis is a process of fundamental importance to multicellular organisms that enables the removal of damaged cells (Rathmell and Thompson, 1999Rathmell J.C. Thompson C.B. The central effectors of cell death in the immune system.Annu Rev Immunol. 1999; 17: 781-828Crossref PubMed Scopus (344) Google Scholar). We demonstrated that activated T cells infiltrating the skin in atopic dermatitis (AD) and allergic contact dermatitis (ACD) induce keratinocyte apoptosis (Trautmann et al., 2000Trautmann A. Akdis M. Kleemann D. et al.T cell-mediated Fas-induced keratinocyte apoptosis plays a key pathogenetic role in eczematous dermatitis.J Clin Invest. 2000; 106: 25-35Crossref PubMed Scopus (346) Google Scholar). Normal human keratinocytes express low levels of Fas that is enhanced by interferon-γ (IFN-γ) and they are rendered susceptible to apoptosis when Fas numbers reach a threshold of approximately 40,000 per keratinocyte. Subsequently, keratinocytes undergo apoptosis induced by supernatants from activated T cells, direct contact with activated T cells, and Fas triggering by anti-Fas monoclonal antibodies (MoAb) or soluble Fas ligand. Keratinocyte apoptosis was demonstrated in situ in lesional skin of AD, ACD, and patch tests. Moreover, studies with normal human skin and cultured skin equivalents demonstrated that keratinocyte apoptosis caused by skin-infiltrating T cells is a key event in the pathogenesis of eczematous dermatitis (Trautmann et al., 2000Trautmann A. Akdis M. Kleemann D. et al.T cell-mediated Fas-induced keratinocyte apoptosis plays a key pathogenetic role in eczematous dermatitis.J Clin Invest. 2000; 106: 25-35Crossref PubMed Scopus (346) Google Scholar). In the dermis of acute AD and ACD, there is a marked perivascular T cell infiltrate consisting predominantly of activated memory/effector T cells bearing cutaneous lymphocyte-associated antigen and CD45RO (Leung et al., 1983Leung D.Y.M. Bhan A.K. Schneeberger E.E. Geha R.S. Characterization of the mononuclear cell infiltrate in atopic dermatitis using monoclonal antibodies.J Allergy Clin Immunol. 1983; 71: 47-56Abstract Full Text PDF PubMed Scopus (195) Google Scholar;Santamaria Babi et al., 1995Santamaria Babi L.F. Picker L.J. Perez-Soler M.T. Drzimalla K. Flohr P. Blaser K. Hauser C. Circulating allergen-reactive T cells from patients with atopic dermatitis and allergic contact dermatitis express the skin-selective homing receptor, the cutaneous lymphocyte-associated antigen.J Exp Med. 1995; 181: 1935-1940Crossref PubMed Scopus (250) Google Scholar;Grabbe and Schwarz, 1998Grabbe S. Schwarz T. Immunoregulatory mechanisms involved in elicitation of allergic contact hypersensitivity.Immunol Today. 1998; 19: 37-44Abstract Full Text PDF PubMed Scopus (460) Google Scholar;Akdis et al., 2000aAkdis C.A. Akdis M. Trautmann A. Blaser K. Immune regulation in atopic dermatitis.Curr Oppin Immunol. 2000; 12: 641-646Crossref PubMed Scopus (154) Google Scholar). ACD is regarded as a type 1 T cell-mediated phenomenon, based on a number of observations (Grabbe and Schwarz, 1998Grabbe S. Schwarz T. Immunoregulatory mechanisms involved in elicitation of allergic contact hypersensitivity.Immunol Today. 1998; 19: 37-44Abstract Full Text PDF PubMed Scopus (460) Google Scholar). A polarized type 2 T cell cytokine pattern was previously regarded as a specific feature reflecting immune dysregulation in AD, but current studies demonstrate that both type 2 T cell and type 2 T cell cytokines play important roles in the skin inflammatory response of AD (Akdis et al., 1997Akdis M. Akdis C.A. Weigl L. Disch R. Blaser K. Skin-homing CLA+ memory T cells are activated in atopic dermatitis and regulate IgE by an IL-13 dominated cytokine pattern: IgG4 counter-regulation by CLA‒ memory T cells.J Immunol. 1997; 159: 4611-4619PubMed Google Scholar,Akdis et al., 1999aAkdis C.A. Akdis M. Simon D. et al.T cells and T cell-derived cytokines as pathogenetic factors in the nonallergic form of atopic dermatitis.J Invest Dermatol. 1999; 113: 628-634Crossref PubMed Scopus (168) Google Scholar;Grewe et al., 1998Grewe M. Bruijnzeel-Koomen CAFM. Schöpf E. Thepen T. Lengeveld-Wildschut E.G. Ruzicka T. Krutman J. A role for Th1 and Th2 cells in the immunopathogenesis of atopic dermatitis.Immunol Today. 1998; 19: 359-361Abstract Full Text Full Text PDF PubMed Scopus (652) Google Scholar;Leung, 1999Leung D.Y.M. Pathogenesis of atopic dermatitis.J Allergy Clin Immunol. 1999; 104: 99-108Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar;Leung, 2000Leung D.Y.M. Atopic dermatitis: new insights and opportunities for therapeutic intervention.J Allergy Clin Immunol. 2000; 105: 860-876Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar;Spergel et al., 1999Spergel J.M. Mizoguchi E. Oettgen H. Bhan A.K. Geha R.S. Roles of Th1 and Th2 cytokines in a murine model of allergic dermatitis.J Clin Invest. 1999; 103: 1103-1111Crossref PubMed Scopus (305) Google Scholar;Trautmann et al., 2000Trautmann A. Akdis M. Kleemann D. et al.T cell-mediated Fas-induced keratinocyte apoptosis plays a key pathogenetic role in eczematous dermatitis.J Clin Invest. 2000; 106: 25-35Crossref PubMed Scopus (346) Google Scholar). Spongiosis is a characteristic histopathologic appearance in diseases of the skin, such as eczematous dermatitis and pemphigus (Wolff et al., 1993Wolff K. Kibbi A.G. Mihm M.C. Basic pathologic reactions of the skin.in: Fitzpatrick TB. Dermatology in General Medicine. McGraw-Hill, New York1993: 66-84Google Scholar;Machado-Pinto et al., 1996Machado-Pinto J. McCalmont T.H. Golitz L.E. Eosinophilic and neutrophilic spongiosis: clues to the diagnosis of immunobullous diseases and other inflammatory disorders.Semin Cutan Med Surg. 1996; 15: 308-316Crossref PubMed Scopus (42) Google Scholar). It is characterized by condensation of cells, widening of the intercellular space, and stretching of remaining intercellular contacts, resulting in a spongelike appearance of the tissue. Specific adhesiveness of keratinocytes is provided by homophilic interactions of the cadherin superfamily (Amagai, 1995Amagai M. Adhesion molecules. I. Keratinocyte–keratinocyte interactions; cadherins and pemphigus.J Invest Dermatol. 1995; 104: 146-152Crossref PubMed Scopus (141) Google Scholar;Koch et al., 1999Koch A.W. Bozic D. Pertz O. Engel J. Homophilic adhesion by cadherins.Curr Opin Struct Biol. 1999; 9: 275-281Crossref PubMed Scopus (123) Google Scholar). Adherens junctions anchor actin microfilaments and contain E-cadherin (E-cad) as their transmembrane glycoprotein. The intracellular segment of E-cad associates with α-catenin, β-catenin, and γ-catenin (plakoglobin) (Amagai, 1995Amagai M. Adhesion molecules. I. Keratinocyte–keratinocyte interactions; cadherins and pemphigus.J Invest Dermatol. 1995; 104: 146-152Crossref PubMed Scopus (141) Google Scholar;Yap et al., 1997Yap A.S. Brieher W.M. Gumbiner B.M. Molecular and functional analysis of cadherin-based adherens junctions.Annu Rev Cell Dev Biol. 1997; 13: 119-146Crossref PubMed Scopus (669) Google Scholar;Koch et al., 1999Koch A.W. Bozic D. Pertz O. Engel J. Homophilic adhesion by cadherins.Curr Opin Struct Biol. 1999; 9: 275-281Crossref PubMed Scopus (123) Google Scholar). The major transmembrane components of desmosomes are desmogleins (Dsg) and desmocollins (Dsc). These desmosomal cadherins bind to the cytoplasmic proteins plakoglobin and desmoplakin, and are linked to keratin intermediate filaments (Amagai, 1995Amagai M. Adhesion molecules. I. Keratinocyte–keratinocyte interactions; cadherins and pemphigus.J Invest Dermatol. 1995; 104: 146-152Crossref PubMed Scopus (141) Google Scholar). Although central to cellular adhesion, cadherins display physiologic functions beyond the mechanical interconnection of cells. Recent studies suggested that cadherins play a crucial role in regulatory pathways involved in various aspects of cell fate including developmental decisions, cell differentiation, and cell survival (Zhu and Watt, 1996Zhu A.J. Watt F.M. Expression of a dominant negative cadherin mutant inhibits proliferation and stimulates terminal differentiation of human epidermal keratinocytes.J Cell Sci. 1996; 109: 3013-3023Crossref PubMed Google Scholar;Yap et al., 1997Yap A.S. Brieher W.M. Gumbiner B.M. Molecular and functional analysis of cadherin-based adherens junctions.Annu Rev Cell Dev Biol. 1997; 13: 119-146Crossref PubMed Scopus (669) Google Scholar;Hakimelahi et al., 2000Hakimelahi S. Parker H.R. Gilchrist A.J. Barry M. Li Z. Bleackley C. Pasdar M. Plakoglobin regulates the expression of the anti-apoptotic protein BCL-2.J Biol Chem. 2000; 275: 10905-10911Crossref PubMed Scopus (80) Google Scholar). In this context, we investigated the fate of different cadherins in keratinocytes that are undergoing apoptosis in cultures and skin biopsies. This study demonstrates that E-cad is cleaved during early apoptosis of keratinocytes, whereas the desmosomal cadherins Dsg and Dsc remain intact. We conclude that loss of E-cad and sustained expression of demosomal cadherins result in epidermal spongiosis and may represent an important mechanism for the development of eczematous dermatitis. Incisional skin biopsies were taken from lesional skin of five patients with acute AD and from positive epicutaneous patch test sites of six patients with ACD. AD was diagnosed according to the standard criteria (Hanifin, 1982Hanifin J.M. Atopic dermatitis.J Am Acad Dermatol. 1982; 6: 1-13Abstract Full Text PDF PubMed Scopus (132) Google Scholar). ACD for nickel was diagnosed by epicutaneous patch testing. Control skin was obtained from three healthy, nonatopic individuals. T cells were purified from nine healthy donors. Informed consent was obtained from all subjects, and the study was approved by the Ethical Committee of Davos. Ethidium bromide was purchased from Sigma (St. Louis, MO). Anti-CD14, anti-CD16, anti-CD19, and anti-CD45RA magnetic microbeads for magnetic activated cell sorting (MACS) were obtained from Miltenyi Biotec (Bergisch Gladbach, Germany). Anti-CD2 (4B2, 6G4) and anti-CD28 (15E4) MoAb were purchased from the Red Cross Blood Transfusion Service (Amsterdam, The Netherlands). Anti-CD3 MoAb was provided by clone CRL8001 obtained from ATCC (Manassas, VA). For immunoprecipitation, immunoblotting, immunocytology, and immunohistology the following antibodies were used: anti-pan-Dsg (AHP321, Serotec, Oxford, U.K.), anti-Dsg1+2 (DG3.10), anti-Dsg3 (Dsg-G194), anti-Dsc1 (Dsc1-U100), anti-Dsc3 (Dsc3-U114) (all from Progen Biotechnik, Heidelberg, Germany), anti-E-cad (H-108, Santa Cruz Biotechnology, Santa Cruz, CA; 67A4, Immunotech, Marseilles, France; HECD-1, Alexis, San Diego, CA; SHE78-7, Alexis), and anti-β-catenin (C-18, Santa Cruz Biotechnology). The apoptosis-inducing anti-Fas MoAb (CH-11) was purchased from Immunotech, and the blocking anti-IFN-γ receptor MoAb from Genzyme Pharmaceuticals (Cambridge, MA). Recombinant soluble Fas ligand and Fas-Fc protein were purchased from Alexis. The caspase inhibitors Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-FMK) and Ac-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) were from Bachem (Bubendorf, Switzerland). Human keratinocytes were obtained from neonatal foreskins. The skin was split overnight in sucrose/trypsin solution [0.1% sucrose, 0.25% trypsin, 1 mM ethylenediamine tetraacetic acid (EDTA), all from Sigma] at 4°C. Epidermal sheets were removed from the dermis and keratinocyte cell suspensions were cultured in a fully supplemented (5 µg per ml bovine insulin, 0.5 µg per ml hydrocortisone, 0.1 ng per ml human epidermal growth factor, 30 µg per ml bovine pituitary extract, 100 µg per ml gentamicin, 100 ng per ml amphotericin B), low calcium (0.15 mM Ca2+), serum-free keratinocyte growth medium (Clonetics, San Diego, CA). Hydrocortisone and the antibiotics were removed from the culture medium during experiments. Mononuclear cells were isolated by Ficoll (Biochrom, Berlin, Germany) density gradient centrifugation of peripheral venous blood. CD45RO+ T cells were isolated with the MACS system according to the instructions of the manufacturer (Miltenyi Biotec). In brief, monocytes, B cells, natural killer cells, and naive T cells were depleted by means of microbead-conjugated anti-CD14, anti-CD19, anti-CD16, and anti-CD45RA. CD45RO+ T cells grown in RPMI 1640 (supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U per ml penicillin, and 100 µg per ml streptomycin; all from Life Technologies, Basel, Switzerland) were stimulated with a combination of soluble anti-CD2 (0.5 µg per ml), anti-CD3 (1.0 µg per ml), and anti-CD28 (0.5 µg per ml) MoAb (Akdis et al., 1999aAkdis C.A. Akdis M. Simon D. et al.T cells and T cell-derived cytokines as pathogenetic factors in the nonallergic form of atopic dermatitis.J Invest Dermatol. 1999; 113: 628-634Crossref PubMed Scopus (168) Google Scholar). The supernatants were harvested after 3 d and were added 1:2 diluted to keratinocytes. CD45RO+ T cells were stimulated with a combination of soluble anti-CD2 (0.5 µg per ml), anti-CD3 (1.0 µg per ml), and anti-CD28 (0.5 µg per ml) MoAb (Akdis et al., 1999aAkdis C.A. Akdis M. Simon D. et al.T cells and T cell-derived cytokines as pathogenetic factors in the nonallergic form of atopic dermatitis.J Invest Dermatol. 1999; 113: 628-634Crossref PubMed Scopus (168) Google Scholar) for 24 h and washed twice before coculturing with keratinocytes in six-well Transwell plates (Corning-Costar, Cambridge, U.K.). The Transwells consist of a lower and an upper compartment, which are separated by a polycarbonate membrane with pores of 0.4 µm size and 10 µm thickness. Keratinocytes (3 × 105) were transferred into the lower culture well, T cells (1 × 105) were added to the upper compartment. Keratinocyte viability was evaluated by means of ethidium bromide (1 µM) uptake and flow cytometry (EPICS™ XL-MCL flow cytometer, Beckmann Coulter, Nyon, Switzerland). Apoptotic cells were identified in situ by staining double-strand DNA breaks. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) was employed, as described previously (Gavrieli et al., 1992Gavrieli Y. Sherman Y. Ben-Sasson S.A. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.J Cell Biol. 1992; 119: 493-501Crossref PubMed Scopus (8961) Google Scholar). TUNEL reaction mixture (Boehringer Mannheim, Mannheim, Germany) was added to the samples, which were incubated for 60 min at 37°C. Incorporated dUTP was detected by Fab fragments from sheep, conjugated with alkaline phosphatase, and substrate reaction with Fast Red (Boehringer Mannheim). Hoechst staining was done as described previously (Norris et al., 1997Norris D.A. Middleton M.H. Whang K. et al.Human keratinocytes maintain reversible anti-apoptotic defenses in vivo and in vitro.Apoptosis. 1997; 2: 136-148Crossref PubMed Scopus (36) Google Scholar). After fixation with 4% paraformaldehyde in 200 mM dihydrogen phosphate (pH 7.0) overnight at 4°C, staining was performed with Hoechst 33342 dye (1 µg per ml; Sigma) for 5 min. Stained sections were evaluated with an ultraviolet microscope (Axiovert 405 M, Carl Zeiss, Feldbach, Switzerland). For labeling with [35S]-methionine, keratinocytes were incubated in methionine-free labeling medium (containing 0.1–0.2 mCi per ml [35S]-methionine) for 6 h. Immunoprecipitation and immunoblots were performed as described previously (Akdis et al., 2000bAkdis C.A. Joss A. Akdis M. Faith A. Blaser K. A molecular basis for T cell suppression by IL-10. CD28-associated IL-10 receptor inhibits CD28 tyrosine phosphorylation and phosphatidylinositol 3-kinase binding.FASEB J. 2000; 14: 1666-1668PubMed Google Scholar). Keratinocytes were lysed with lysis buffer (20 mM 3-[N-morpholino]propanesulfonic acid, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% sodium lauryl sulfate, 1 mM phenylmethylsulfonyl fluoride, 10 µg per ml leupeptin, 10 µg per ml aprotinin, 1 mM orthovanadate; all reagents from Fluka Chemie, Buchs, Switzerland) for 1 h at 4°C. Following a pre-clearing step lysates were quantitated with the Bio-Rad Protein Assay (Bio-Rad Laboratories, Gattbrugg, Switzerland). Immunoprecipitations with 500–1000 µg of total cellular protein were performed for 2 h at 4°C with appropriate primary antibodies. Complexes were captured with Sepharose 4B-coupled protein G (Sigma). Precipitates were washed with phosphate-buffered saline (pH 7.4) and boiled in 2 × reducing sample buffer before electrophoresis. Immunoprecipitates were electrophoresed and proteins were transferred to nitrocellulose membranes. Immunoblots were probed with appropriate antibodies followed by peroxidase-conjugated antimouse or antirabbit IgG (Dako, Glostrup, Denmark). Detection was performed with the ECL system (Amersham Pharmacia Biotech, Buckinghamshire, U.K.). In the case of [35S]-methionine labeling bands were evaluated with phosphorus imaging (FLA-3000, Raytest Schweiz, Urdorf, Switzerland). Keratinocytes cultured in Transwell plates or on culture slides (Becton Dickinson, Meylan, France) were fixed in acetone for 10 min at 4°C. The tissue samples were placed in Tissue-Tek OCT compound (Sakura-Finetek Europe, Zoeterwoude, The Netherlands). Five micrometer cryostat sections were prepared on gelatin-coated slides. After air-drying sections were fixed in acetone for 10 min at 4°C. Sections or cultured keratinocytes were incubated with the primary antibodies at 4°C overnight, followed by incubation with biotin-conjugated antimouse or antirabbit IgG and preformed streptAB-Complex-peroxidase (all from Dako) for 1 h. The peroxidase-specific substrate 3-amino-9-ethylcarbazole (Sigma) was used for visualization, with hematoxylin counterstaining. Results are shown as mean ± SD. The paired Student's t test was used for comparison of paired conditions. Features of T-cell-induced keratino cyte apoptosis were recently reported in detail (Trautmann et al., 2000Trautmann A. Akdis M. Kleemann D. et al.T cell-mediated Fas-induced keratinocyte apoptosis plays a key pathogenetic role in eczematous dermatitis.J Clin Invest. 2000; 106: 25-35Crossref PubMed Scopus (346) Google Scholar). Spongiosis, the histologic hallmark of eczematous disorders, is characterized by impairment of cohesion between epidermal keratinocytes Figure 1a, b. T cells constitute the major cells in dermal infiltrates and some of the CD4+ and CD8+ T cells invade the spongiotic epidermis Figure 1c. Apoptotic cells may be identified in situ by histochemical techniques staining double-strand DNA breaks (Gavrieli et al., 1992Gavrieli Y. Sherman Y. Ben-Sasson S.A. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.J Cell Biol. 1992; 119: 493-501Crossref PubMed Scopus (8961) Google Scholar). As shown in Figures 1(d, e)1(f), TUNEL-stained keratinocytes are visible in acute lesions of AD and ACD. In keratinocyte-T cell cocultures, we observed killing of keratinocytes by stimulated CD45RO+ memory/effector T cells Figure 1g. Keratinocyte apoptosis could be inhibited by pretreatment with either anti-IFN-γ receptor blocking MoAb or with the Fas-Fc protein, a competitive inhibitor of Fas ligand–Fas interactions. In summary, T cells infiltrating the skin render keratinocytes susceptible to apoptosis by IFN-γ and induce apoptosis by Fas ligand expression. Coculture of keratinocytes and stimulated T cells induces apoptosis of keratinocytes with characteristic morphologic features Figure 2a, b. The first morphologic changes are retraction and reduction of keratinocyte contacts that ultimately result in cell detachment. The apoptotic type of keratinocyte death was investigated by demonstration of apoptosis features such as chromatin condensation and fragmented nuclei with Hoechst staining Figure 2c, d. To eliminate that the apoptotic bodies are derived from T cells, keratinocytes and T cells were separated in Transwell plates. Viable keratinocyte monolayers are characterized by an intense cell membrane staining with anti-E-cad, Dsg, and Dsc MoAb Figure 2e, f. Induction of apoptosis destroys the keratinocyte monolayer and a cell population appears to float in the medium. The remaining islets of keratinocytes still attached to the cell culture plate exhibit reduction in E-cad membrane staining Figure 2g. In contrast, the desmosomal cadherins Dsg1+2 and Dsc3 of still adhering keratinocytes sustained their surface staining Figure 2h, j. Using the anti-Dsg3 MoAb we observed a slight decrease of immunoreactivity Figure 2i. A double label assay of the same keratinocyte monolayer using Hoechst staining together with E-cad immunohistology 1 d after induction of keratinocyte apoptosis demonstrated loss of E-cad Figure 2k and chromatin condensation and fragmentation as signs of ongoing apoptosis Figure 2l simultaneously. Primary human keratinocytes expressed full-length E-cad (120 kDa) at reasonably high levels. Keratinocytes were induced to undergo apoptosis by preincubation with IFN-γ followed by anti-Fas MoAb Figure 3a or by coculture with stimulated T cells Figure 3b. After 24 h in both conditions a significant reduction of keratinocyte viability was observed. E-cad cleavage started after 6 h and the 85 kDa degradation product appeared as early as 6 h, as demonstrated by immunoprecipitation and immunoblotting with anti-E-cad MoAb (H-108 recognizes both full-length 120 kDa E-cad and cleaved 85 kDa E-cad; epitope corresponding to aminoacids 600–707 within the extracellular domain) (Vallorosi et al., 2000Vallorosi C.J. Day K.C. Zhao X. et al.Truncation of the β-catenin binding domain of E-cadherin precedes epithelial apoptosis during prostate and mammary involution.J Biol Chem. 2000; 275: 3328-3334Crossref PubMed Scopus (83) Google Scholar). E-cad was fully degraded after 24 h, when keratinocyte viability substantially decreased to 47%. Comparison of viability and E-cad expression at the 6, 12, and 24 h time points demonstrated that the E-cad cleavage and accumulation of the degradation product preceded the onset of cell death and therefore represents an early event in keratinocyte apoptosis. In contrast, β-catenin was not cleaved during early keratinocyte apoptosis Figure 3a. We further used biosynthetic labeling to study the synthetic processing and fate of E-cad Figure 3a. After 6 h [35S]-methionine labeling we observed a reduction of E-cad at the 12 and 24 h time points. To rule out that the E-cad fragmentation is generated through transcriptional mechanisms, we examined the levels of E-cad mRNA to determine whether the fragments resulted from alternatively spliced transcripts. E-cad mRNA isolated from IFN-γ and anti-Fas MoAb treated keratinocytes revealed the accumulation of a single 535 bp transcript by reverse transcriptase polymerase chain reaction (data not shown). Blocking of E-cad by anti-E-cad MoAb results in reduced keratinocyte contacts mimicking epidermal spongiosis in vitro Figure 3c. This functional blockade of E-cad alone did not alter the viability of cultured keratinocytes (data not shown), probably because integrin-mediated adhesion to the cell culture plate provides essential signals that promote survival and growth of cultured keratinocytes (Zhu and Watt, 1996Zhu A.J. Watt F.M. Expression of a dominant negative cadherin mutant inhibits proliferation and stimulates terminal differentiation of human epidermal keratinocytes.J Cell Sci. 1996; 109: 3013-3023Crossref PubMed Google Scholar). The intracellular domain of E-cad is linked to the actin cytoskeleton through its interaction with the cytoplasmic adaptor proteins α-catenin, β-catenin, and γ-catenin. This interaction is essential for the adhesive function of E-cad (Amagai, 1995Amagai M. Adhesion molecules. I. Keratinocyte–keratinocyte interactions; cadherins and pemphigus.J Invest Dermatol. 1995; 104: 146-152Crossref PubMed Scopus (141) Google Scholar;Yap et al., 1997Yap A.S. Brieher W.M. Gumbiner B.M. Molecular and functional analysis of cadherin-based adherens junctions.Annu Rev Cell Dev Biol. 1997; 13: 119-146Crossref PubMed Scopus (669) Google Scholar;Koch et al., 1999Koch A.W. Bozic D. Pertz O. Engel J. Homophilic adhesion by cadherins.Curr Opin Struct Biol. 1999; 9: 275-281Crossref PubMed Scopus (123) Google Scholar). We employed coimmunoprecipitation to examine the loss of β-catenin binding by the cleaved E-cad. When keratinocyte lysates were immunoprecipitated with an anti-E-cad MoAb, the coimmunoprecipitated (i.e., bound) β-catenin could be detected by immunoblotting with an anti-β-catenin MoAb. Significant apoptosis in keratinocytes leads to a reduction (12 h) and finally loss (24 h) of coimmunoprecipitated β-catenin protein Figure 3b. Dsg and Dsc exist as closely related isoforms, designated Dsg1, Dsg2, Dsg3, Dsc1, Dsc2, and Dsc3 (Schäfer et al., 1994Schäfer S. Koch P.J. Franke W.W. Identification of the ubiquitous human desmoglein, Dsg2, and the expression catalogue of the desmoglein subfamily of desmosomal cadherins.Exp Cell Res. 1994; 211: 391-399Crossref PubMed Scopus (196) Google Scholar;Amagai, 1995Amagai M. Adhesion molecules. I. Keratinocyte–keratinocyte interactions; cadherins and pemphigus.J Invest Dermatol. 1995; 104: 146-152Crossref PubMed Scopus (141) Google Scholar;King et al., 1995King I.A. Sullivan K.H. Bennett R. Buxton R.S. The desmocollins of human foreskin epidermis: identification and chromosomal assignment of a third gene and expression patterns of the three isoforms.J Invest Dermatol. 1995; 105: 314-321Crossref PubMed Scopus (69) Google Scholar). Therefore MoAb that recognize different isoforms of either molecule were used for immunoprecipitation and immunoblotting (Schäfer et al., 1994Schäfer S. Koch P.J. Franke W.W. Identification of the ubiquitous human desmoglein, Dsg2, and the expression catalogue of the desmoglein subfamily of desmosomal cadherins.Exp Cell Res. 1994; 211: 391-399Crossref PubMed Scopus (196) Google Scholar;Schäfer et al., 1996Schäfer S. Stumpp S. Franke W.W. Immunological identification and characterization of the desmosomal cadherin Dsg2 in coupled and uncoupled epithelial cells and in human tissues.Differentiation. 1996; 60: 99-108Crossref PubMed Google Scholar;Nuber et al., 1996Nuber U.A. Schäfer S. Stehr S. Rackwitz H.R. Franke W.W. Patterns of desmocollin synthesis in human epithelia: immunolocalization of desmocollins 1 and 3 in special epithelia and cultured cells.Eur J Cell Biol. 1996; 71: 1-13PubMed Google Scholar). In cultured primary keratinocytes Dsc1 is restricted to local piles of differentiated cells (Nuber et al., 1996Nuber U.A. Schäfer S. Stehr S. Rackwitz H.R. Franke W.W. Patterns of desmocollin synthesis in human epithelia: immunolocalization of desmocollins 1 and 3 in special epithelia and cultured cells.Eur J Cell Biol. 1996; 71: 1-13PubMed Google Scholar). In contrast to the marked reduction of E-cad expression during early apoptosis, immunoprecipitation and immunoblotting with anti-Dsg1+2, anti-Dsg3, anti-Dsc1, and anti-Dsc3 MoAb demonstrated that the desmosomal cadherins were not degraded in the first 24 h of keratinocyte apoptosis Figure 3a. E-cad was not detectable in lysates of apoptotic keratinocytes after 24 h of treatment with supernatants from stimulated T cells or IFN-γ and anti-Fas MoAb Figure 4a, b. Inhibition of apoptosis with Fas-Fc protein and blocking of the IFN-γ receptor results in viable keratinocytes with native E-cad expression Figure 4a. Activation of caspases and caspase-mediated cleavage of intracellular proteins are crucial steps in apoptosis (Rathmell and Thompson, 1999Rathmell J.C. Thompson C.B. The central effectors of cell de
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