In situ detection of inflammatory cytokines and apoptosis in pemphigus foliaceus patients.

Endemic pemphigus foliaceus (EPF), or fogo selvagem, is a chronic autoimmune disease (1)fc characterized by the formation of intraepidermal blisters, which are attributed to the presence of immunoglobulin G4 (IgG4) and IgG1 autoantibodies against desmoglein 1 (2) that reduce the adhesion between keratinocytes. (3) In addition to the presence of autoantibodies, a high production of cytokines involved in the inflammatory response, such as interleukin 1 (IL-1) and tumor necrosis factor [alpha] (TNF-[alpha]), by blood cells has been demonstrated in both pemphigus foliaceus and pemphigus vulgaris. (4,5)

Recently, the role of apoptosis, a physiologicmechanism of cell death, in blister formation has been investigated. Studies analyzing skin samples from patients with pemphigus vulgaris have demonstrated the presence of apoptosis, which can be induced in vitro in keratinocytes by patient antibodies. (6) Apoptosis can also be induced by pathways that depend on the activation of the cellular immune response through Fas/Fas-L and TNFR, pathways that are involved in both tissue injuries and control mechanisms of the immune response. (7,8)

In the present study, we evaluated the in situ expression of the proinflammatory cytokines interleukin 1 (IL-1), interferon [gamma] (IFN-[gamma]), and TNF-[alpha], the proapoptotic inducers Fas and inducible nitric oxide synthase (iNOS), and the apoptosis inhibitor Bcl-2, as well as the presence of apoptosis in skin biopsies from patients with EPF.


Skin biopsies were obtained from 13 patients presenting with lesions compatible with EPF who were hospitalized at Hospital do Penfigo de Uberaba (Uberaba, Brazil). The biopsies were performed in regions close to the blisters with a 5-mm punch. The fragments were fixed, embedded in paraffin, and cut into serial 5-[micro]m-thick sections that were used for confirmation of the diagnosis, immunohistochemistry for the detection of TNF-[alpha], IFN-[gamma], Bcl-2, Fas, and iNOS, and detection of apoptosis by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique. Control samples were obtained from patients undergoing plastic surgery. The study protocol was approved by the Ethics Committee of Universidade Federal do Triangulo Mineiro (Uberaba, Brazil).

For immunohistochemistry, deparaffinized sections were treated with 3% hydrogen peroxide in methanol for 10 minutes, incubated for 30 minutes at 90[degrees]C for antigen retrieval, and then incubated with phosphate-buffered saline 2% bovine serum albumin to reduce nonspecific binding. Next, the sections were incubated with an anti-cytokine monoclonal antibody specific for human IL-1 (1:100; R&D Systems, Minneapolis, Minn), TNF-[alpha] (1: 200; R&D Systems), IFN-[gamma] (1:200; R&D Systems), [NOS.sub.2] (1:100; Santa Cruz Biotechnology, Santa Cruz, Calif), and Bcl-2 and Fas Pharmingen, San Diego, Calif) diluted in phosphate-buffered saline 2% bovine serum albumin for 2 hours at 37[degrees]C. In the second step, a biotinylated Link System (002488, Dako, Carpinteria, Calif) was used according to the manufacturer's instructions. The reaction was visualized by incubating the sections with diaminobenzidine (Sigma, St Louis, Mo) and counterstaining with hematoxylin.


For immunohistochemical analysis, the number of cells positive for each cytokine was evaluated semiquantitatively at a magnification of x400, and immunostaining was classified as absent, discrete, moderate, or intense.

Apoptosis was investigated in situ by the TUNEL technique using the Boehringer Mannheim (Mannheim, Germany) ApopTag kit according to manufacturer's instructions. The slides were examined under an immunofluorescence microscope, and the presence of apoptosis was identified by fluorescent staining of the nucleus.


Proinflammatory cytokines were only detected in cells of the inflammatory exudate, including IL-1 and TNF-[alpha] in 11 cases (85.4%) and IFN-[gamma] in 12 cases (92.3%). Immunostaining for the apoptosis inducers was observed in both epithelial and inflammatory cells, with iNOS being detected in 10 cases (80%). Bcl-2 was expressed by epithelial cells in 4 cases (31%) and by inflammatory cells in 9 cases (69.2%). Fas was detected in epithelial cells in 3 cases (23%) and in inflammatory cells in 10 cases (80%). Epithelial apoptosis was observed in 12 cases (92.3%) and subepithelial apoptosis in 11 cases (85%; Figure; Table).


In the present study, apoptosis and the expression of proinflammatory cytokines and apoptosis inducers were observed in biopsies of patients with EPF lesions. The presence of TNF-[alpha], IFN-[gamma], and IL-1 in the inflammatory exudate of lesions in patients with EPF suggests that these cytokines play a role in the mechanisms of tissue injury. These findings agree with studies demonstrating a high production of IL-1 in peripheral blood mononuclear cells from patients with active EPF lesions, (4) and with those investigating other blistering diseases, such as pemphigus vulgaris, in which the expression of proinflammatory cytokines, such as TNF-[alpha] and IL-6, around the blister was demonstrated by in situ hybridization. (5) These inflammatory mediators might contribute to tissue injury by their ability to directly or indirectly induce apoptosis.

Apoptosis is a phenomenon that occurs naturally in the epithelium and is observed at a rare to discrete frequency in normal controls. The higher intensity of apoptosis in EPF might contribute to the pathogenesis of these lesions. This view is supported by the demonstration that in vitro induction of apoptosis in keratinocytes leads to the cleavage of desmoglein 1. (3) On the other hand, apoptosis was absent or discrete in cells of the inflammatory infiltrate in 85% of cases, emphasizing the absence of this mechanism in the control of inflammation in EPF lesions. In biopsies of patients with pemphigus vulgaris, apoptosis was abundantly detected by the TUNEL technique in keratinocytes present in acantholytic regions and in the epidermal adherence under the blisters. (6) The system regulating the activation and mechanisms of protection against apoptosis is complex. In the present study, we investigated the expression of some molecules, including Bcl-2, involved in the protection against apoptosis, and the apoptosis inducers Fas and iNOS. (9-11)

Bcl-2 was detected in the epithelium of 42% of the samples examined. On the other hand, this marker was present in 92% of cells in the inflammatory exudate. These results may indicate that epithelial cells are poorly protected against death by apoptosis, whereas inflammatory cells, which express more Bcl-2, are more protected, a fact contributing to the persistence of inflammation and epithelial injury.

Inducible NOS was detected in the inflammatory exudate in all cases. The expression of iNOS in all keratinocytes suggests that NO might be associated with themechanisms of epithelial injury, since this radical is related to the induction of programmed cell death. Nitric oxide also plays an important role in the regulation of collagen biosynthesis and in the healing of skin lesions. (9) The expression of iNOS and the production of NO have been demonstrated in keratinocyte cultures exposed to serum of pemphigus patients and have been associated with the induction of apoptosis in these cells. (12)

Fas, a member of the TNF receptor family, is involved in the activation of apoptosis, and its activation through Fas-L triggers intracellular signals of apoptosis. (10,11) In the present study, Fas expression predominated in cells of the inflammatory exudate and may represent a control mechanism of inflammation. The expression of Fas has been demonstrated in the epithelium of pemphigus vulgaris patients, and the presence of circulating Fas-L suggests that this pathway is involved in the induction of apoptosis. (13) In addition, cytokines such as IFN-[gamma] increase the expression of Fas which, in turn, contributes to apoptosis signaling. (14)

The observation of apoptotic cells in the epidermis suggests that this process of cell death is involved in the pathogenesis of EPF lesions. Apoptosis might be triggered by the loss of contact between cells caused by autoantibodies or induced by inflammatory mediators present at the site of injury. In contrast to Zuccolotto et al, (15) this study suggests that apoptosis is involved in EPF lesions and indicates that the local production of proinflammatory cytokines is one of the events that induce apoptosis. To our knowledge, this is the first study to investigate molecules involved in the regulation/induction of apoptotic pathways. These results may contribute to the development of new therapeutic approaches to EPF.

This work was supported by Fundacao de amparo a pesquisa de Minas Gerais and Fundacao de ensino e pesquisa de Uberaba.

Accepted for publication July 7, 2008.


(1.) Stanley JR, Koulu L, Thivolet C. Distinction between epidermal antigens binding pemphigus vulgaris and pemphigus foliaceous autoantibodies. J Clin Invest. 1984;74:313-320.

(2.) Allen EM, Giudice GJ, Diaz LA. Subclass reactivity of pemphigus foliaceus autoantibodies with recombinant human desmoglein. J Invest Dermatol. 1993; 100:685-691.

(3.) Lanza A, Cirillo N. Caspase-dependent cleavage of desmoglein 1 depends on the apoptotic stimulus. Br J Dermatol. 2007;156:400-402.

(4.) Rocha-Rodrigues DB, Paschoini G, Pereira SAL, dos Reis MA, Teixeira V, Rodrigues V Jr. High levels of interleukin-1 in patients with endemic pemphigus foliaceus. Clin Diagn Lab Immunol. 2003;10:741-743.

(5.) D'Auria L, Bonifati C, Mussi A, et al. Cytokines in the sera of patients with pemphigus vulgaris: interleukin-6 and tumour necrosis factor-alpha levels are significantly increased as compared to healthy subjects and correlate with disease activity. Eur Cytokine Netw. 1997;8:383-387.

(6.) Gniadecki R, Jemec GB, Thomsen BM, Hansen M. Relationship between keratinocyte adhesion and death: anoikis in acantolytic diseases. Arch Dermatol Res. 1998;10:528-532.

(7.) Nagata S, Golstein P. The Fas death factor. Science. 1995;267:1449-1456.

(8.) Laucella SA, Rottenberg ME, Titto EH. Role of cytokines in resistance and pathology in Trypanosoma cruzi infection. Rev Arg Microbiol. 1996;28:99-109.

(9.) Shukla A, Rasik AM, Shankar R. Nitric oxide inhibits wounds collagen synthesis. Mol Cell Biochem. 1999;200:27-33.

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(11.) Brunner T, Mogil RJ, Laface D, et al. Cell-autonomous Fas (CD95)/Fasligand interaction mediates activation-induced apoptosis in T-cell hybridomas. Nature. 1995;373:441-444.

(12.) Baroni A, Buommino E, Paoletti I, Orlando M, Ruocco E, Ruocco V. Pemphigus serum and captopril induce heat shock protein 70 and inducible nitric oxide syntase overexpression, triggering apoptosis in human keratinocytes. Br J Dermatol. 2004;150:1070-1080.

(13.) Puviani M, Marconi A, Cozzani E, Pincelli C. Fas ligant in pemphigus sera induces keratinocyte apoptosis through the activation of caspase-8. J Invest Dermatol. 2003;120:164-167.

(14.) Sayama K, Yonehara S, Watanabe Y, Miki Y. Expression of Fas antigen on keratinocytes in vivo and induction of apoptosis in cultured keratinocytes. J Invest Dermatol. 1994;103:330-334.

(15.) Zuccolotto I, Roselino AM, Ramalho LN, Zucoloto S. Apoptosis and p63 expression in the pathogenesis of bullous lesions of endemic pemphigus foliaceus. Arch Dermatol Res. 2003;295:284-286.

Denise Bertulucci Rocha Rodrigues, PhD; Sanivia Aparecida Lima Pereira, PhD; Marlene Antonia dos Reis, MD, PhD; Sheila Jorge Adad, MD, PhD; Joao Eduardo Caixeta, MD, PhD; Angelica Maeda Chiba, MD; Richard Atila Sousa; Virmondes Rodrigues, Jr, MD, PhD

From the Laboratory of Immunology (Drs D. B. R. Rodrigues and V. Rodrigues) and the Department of Pathology (Drs dos Reis and Adad, and Mr Sousa), Universidade Federal do Tria^ngulo Mineiro, Uberaba, Minas Gerais, Brazil; and the Laboratory of Immunology (Drs D. B. R. Rodrigues, Chiba, and Caixeta) and the Department of Pathology (Dr Pereira), Universidade de Uberaba, Uberaba, Minas Gerais, Brazil.

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Virmondes Rodrigues, Jr, MD, PhD, Laboratory of Immunology, Universidade Federal do Triangulo Mineiro, 38025-180 Uberaba, Minas Gerais, Brazil (e-mail:
In Situ Expression of the Proinflammatory Cytokines Interleukin 1
(IL-1), Interferon [gamma] (IFN-[gamma]), and Tumor Necrosis Factor
[alpha] (TNF-[alpha]), the Apoptosis Inducers Fas and Inducible
Nitric Oxide Synthase (iNOS), and the Apoptosis Inhibitor Bcl-2, and
the Presence of Apoptosis in Skin Biopsies From Patients With Endemic
Pemphigus Foliaceus *

 Proinflammatory Cytokines:
 Exudate, No. (%)

Intensity IL-1 IFN-[gamma] TNF-[gamma]]

Absent 2 (15) 1 (7.6) 2 (15)
Discrete 6 (46) 6 (46) 5 (38)
Moderate 3 (23) 4 (31) 4 (31)
Intense 2 (15) 2 (15) 2 (15)
Total 13 13 13

 Apoptosis Inducers and Inhibitor, No. (%)

 iNOS Fas

Intensity Epid Exud Epid Exud

Absent 3 (23) 3 (23) 10 (77) 3 (23)
Discrete 0 (0) 2 (15) 2 (15) 5 (38)
Moderate 0 (0) 4 (31) 1 (7.6) 2 (15)
Intense 10 (77) 4 (31) 0 (0) 3 (23)
Total 13 13 13 13

 Apoptosis Inducers and Inhibitor, No. (%)


Intensity Epid Exud

Absent 9 (69) 2 (15)
Discrete 1 (7.6) 5 (38)
Moderate 1 (7.6) 4 (23)
Intense 2 (15) 2 (7.6)
Total 13 13

 Apoptosis, No. (%)


Intensity Epid Exud

Absent 1 (7.6) 2 (15)
Discrete 5 (38) 9 (69)
Moderate 3 (23) 1 (7.6)
Intense 4 (31) 1 (7.6)
Total 13 13

* Sections were classified according to the degree of expression of
absent, discrete, moderate, or intense. TUNEL indicates terminal
deoxynucleotidyl transferase-mediated dUTP nick-end labeling;
Epid, epidermal; and Exud, exudate.

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