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Immunohistochemical localization of TGF-β in the skin of patients with alopecia areata
ELSEVIER
Journal of the European
Academy of Dermatology 7 (1996) 75-90
and Venereology
Letters to the Editor Immunohistochemical localization of TGF-P the skin of patients with alopecia areata
in
To the Editor: Alopecia areata (AA) is a pathological process in which the initial etiopathogenic mechanisms are unknown. Two distinctive features are present: an inflammatory component and microvascular abnormalities [I]. Recently evidence has demonstrated the importance of the expression of adhesion molecules on the cell surface, and the leucocyte traffic in the inflammatory tissue as well as in the alopecic sites. Transforming growth factor B (TGF-B) is an important cytokine widely involved in the modulation of the inflammatory process. TGFB’s effects in vascular structures are also known [2]. Using immunohistochemistry, we demonstrated the immunocytochemical ;staining pattern of the intracellular and extracellular forms of TGF-(3 in alopecic skin and compared it to the distribution of inflammatory cells revealed by specific cell surface markers. Five subjects with patchy alopecia areata, aged 1 l-34 years, with recent onset and active lesions of AA were biopsied. Each biopsy was frozen in liquid nitrogen, stored at - 80°C and cut into 5 p,rn sections on a cryostat. The sections were incubated with mouse monoclonal antibodies (OK-HLADR, 84H10 (anti-ICAM 11, OK-panB, OK-panT, OKT4, OKT8) and rabbit antibodies against the extracellular and intracellular forms of TGF-B. The streptoavidin-biotin peroxidase complex was used for the immunohistochemical staining of all the sections. In AA patients evident staining for an intracellular form of TGF-B in follicular keratinocytes was found. Intracellular TGF-B was also present in the endothelial cells of perifollicular and papillary dermis (Fig. I), but not in the control skin. Moreover, biopsies 0926-9939/96/$15.00
Copyright
0 1996 Elsevier
from AA patients showed a moderate staining for an extracellular form of TGF-B in the bulbar cells and endothelial cells. In the perifollicular and interfollicular derma of the alopecic skin HLA-DR+ lymphocytes were significantly increased, as was the ICAMI’ inflammatory cell infiltrate, compared to the control. We studied the role of TGF-B in the development of AA and we compared this with inflammatory cell expression. TGF-B’s effect on endothelial cell function may be critical to the microvascular damage seen in this disease [3]. There are several studies dealing with the action of TGF-(3 on endothelial cells with conflicting results; TGF-B sometimes stimulated, sometimes inhibited proliferation [3,4]. Recently, TGF-B has been shown to have a profound effect on the vascular endothelium, inhibiting the proliferation of endothelial cells in response to cell damage, however produced. When an endothelial lesion occurs, the exposed subendothelium interacts with the platelets, the latter strongly stimulating the myocells of the vascular wall [5]. This event could explain the obliterative lesions reported by some authors [6]. We also saw in active AA an exhaustion of microvascular tPA-dependent fibrinolytic activity, leading to perivascular deposits of fibrin-like material [7]. It is known that in the presence of TGF-B matrix degradation is inhibited by increased synthesis of protease inhibitors, such as tPA inhibitor or its decreased synthesis [8]. Concerning the lymphomonocytic infiltrate, a strong effect of the TGF-B on immunocompetent cells by leucocyte recruitment and activation, essential to the development of inflammatory lesions, is reported [9]. Once at the inflammatory site, mononuclear cells are stimulated by TGF-B to release important cytokines in the network of molecules regulating the host response to
Science B.V. All rights reserved
Letters
Fig. 1. Immunohistochemical TCF-P positive endothelial
localization of intracellular TGF-P cells (immunoperoxidase, X 63).
immunologic challenge [lo]. Through these cytokines, TGF-P is able to increase DR antigen expression by the endothelial cells and keratinocytes, involving them in the inflammatory process. Our immunohistochemical data on AA areas show a parallel behaviour of TGF-l3 and HLA-DR and ICAMpositivity. Such co-expression of TGF-(3, as well as activated DR + inflammatory cells, may indicate an accelerated sequence of events leading to an inflammatory process. Furthermore ICAMpositivity seems to indicate increase in endothelial cell activation. Therefore, our results suggest a role and complex action of TGF-l3 in AA but also point out a synergic influence of different factors modulating the alopecic process, resulting in a cascade of interdependent events. Moreover, our results open interesting possibilities for the treatment of AA, sustaining a therapeutic role of anti-TGF-l3 antibodies, as observed in other pathologies, or using soluble TGFP-binding proteins and receptors as well as approaches directed at reducing TGF-I3 gene expression, In AA TGF-l3 therapeutic approaches might
to the Editor
in an alopecic
follicle
(immunoperoxidase,
X25).
Insert:
intracellular
have to be associated with other pharmaceutical therapy programs such as guard immunotherapy. A. Offldani *, G. Lucarini a, A. Cellini, 0. Simonetti, M. Nicolini, G. Biagini “, C. Castaldini b Institute of Dermatology and a Institute of Normal Human Morphology, University of Ancona, Ospedale Umberto P, Piazza Cuppelli 1, 60100 Ancona, Italy b Institute of Histology, University of Bologna, Bologna, Italy
References [l]
Teofoli cal and infiltrate different [2] Heimark dothelial tor from
P, Ghersetih I, Benci T, Lotti T. Immunophenotypiultrastructural study of perivascular and peribulbar and adhesion molecules expression in alopecia areata clinical phases. J Invest Dermatol 1992;98:4, 599. RL, Twardzik DR, Shwartz SM. Inibition of enregeneration by type-beta transforming growth facplatelets. Science 1986;233:1078-1080.
Letters [3] Roberts AB, Spom MB, Assoian RK, Smith JM, Roche NS, Wakelield LM, Heine UI, Liotta LA, Falanga V, Kerl JH, Fauci A. Transforming growth factor type: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc Nat1 Acad Sci USA 1986;83:41674171. [4] Takeara K, Leroy EC, Grotendorst GR. TGF inhibition of endothelial cell proliferation: alteration of EGF binding and EGF induced growth-regulatory (competence) gene expression Cell 1987;49:415-422. [5] Messague .I. The TGF-Bl family of growth and differentiation factors. Cell 1987;49:437-438. [6] Meo AL, Fedi AM, L.otti T. Microvessel alterations in alopecia areata. In: Dermatology in Europe. Proceedings of the 1st Congress of European Academy of Dermatology and Venereology. Panconesi, 1991:829-830. [7] Lotti T, Teofoli P. The role of plasminogen activators in alopecia areata. Int J Dermatol 1991;30:19-21.
Gammalinolenic dermatitis
acid in the treatment
of atopic
To the Editor: Treatment of atopic dermatitis (AD) with essential fatty acids (EFAs) is still controversial and has yielded conflicting results [l-6]. Hansen [7] was the first to suggest that AD might be related to abnormal EFA intake or metabolism by demonstrating a reduced level of unsaturated fatty acids in the blood of the patients. Moreover, he stated that dietary supplements of linoleic acid improved the clinical conditions. It has been suggested that a defect in IS-6-desaturase enzyme function might explain the findings and some clinical signs and symptoms of AD, and that GLA and arachidonic acid are of great importance in the maintenance of a normal epidermal barrier as membrane stabilizing factors. The possible mechanism by which EFAs might improve eczematous lesions includes modification of eicosanoid metabolism so as to favour synthesis of non-inflammatory prostaglandins (PGE 1) and leukotrienes [8]. To evaluate the effects of GLA in AD, a group of patients was treated with this compound in the form of borage oil obtained from seeds of the plant Borago ofJicinaZis. Thirty-one consecutive patients suffering from AD took part in our study; it lasted 6 months during the winter, from November to April,
to the Editor [8] Edward DR, Murphy G, Reynolds JJ et al. Transforming growth factor-beta modulates the expression of collagenase and metallo-proteinase inhibitor. EMBO J 1987;6: 18991904. [9] Kehrl JH, Wakefield LM, Roberts AB, Jakowlew S, AlvarezMon M, Derynck R, Spom MB, Fauci AS. Production of transforming growth factor by human T lymphocytes and its potential role in the regulation of T cell growth. J Exp Med 1986;163:1037-1050. [IO] Darley R, Morris A. Interactions between interferon gamma and retinoic induction of immune recognition molecules. Cancer Immunol Immunother 1993;37: I1 2- 118. PZZ SO926-9959(96)0001
* Corresponding
1-6
author.
in order to exclude the positive effect of sunshine on eczema. Eighteen subjects were children, aged from 2 to 14 years, and 13 were adults, aged from 15 to 38 years. The diagnosis of AD was made according to the criteria of Hanifin and Raika [9]. Criteria for patients’ admission to the study included having active lesions and avoiding use of systemic and/or topical steroids in the 3 weeks prior. All patients were given the same emollient cream in unlimited quantities. At the start of the study and every 4th week thereafter the same clinician evaluated the extent of body involvement and the activity of the disease. The body surface was divided in 20 areas according to the method of Atherton et al. [lo]. The symptoms itching, erythema, vesicles, papules, excoriation, scales and lichenification were scored for each skin area according to the following scale: 1 = absent. 2 = mild, 3 = moderate, 4 = severe. Subsequently a quantitative analysis (area score) was performed and the patients were classified into the following groups: mild AD (score < 2101, moderate AD (score = 210-280) and severe AD (score > 280). After clinical assessment, the patients were randomly divided into 2 groups: one group received GLA and the other group placebo. Active capsules contained 500 mg of borage oil, largely made up of GLA (80 mg/a capsule) and linoleic acid, as well as small amounts of palmitic, oleic and stearic acids.
Journal of the European
Academy of Dermatology 7 (1996) 75-90
and Venereology
Letters to the Editor Immunohistochemical localization of TGF-P the skin of patients with alopecia areata
in
To the Editor: Alopecia areata (AA) is a pathological process in which the initial etiopathogenic mechanisms are unknown. Two distinctive features are present: an inflammatory component and microvascular abnormalities [I]. Recently evidence has demonstrated the importance of the expression of adhesion molecules on the cell surface, and the leucocyte traffic in the inflammatory tissue as well as in the alopecic sites. Transforming growth factor B (TGF-B) is an important cytokine widely involved in the modulation of the inflammatory process. TGFB’s effects in vascular structures are also known [2]. Using immunohistochemistry, we demonstrated the immunocytochemical ;staining pattern of the intracellular and extracellular forms of TGF-(3 in alopecic skin and compared it to the distribution of inflammatory cells revealed by specific cell surface markers. Five subjects with patchy alopecia areata, aged 1 l-34 years, with recent onset and active lesions of AA were biopsied. Each biopsy was frozen in liquid nitrogen, stored at - 80°C and cut into 5 p,rn sections on a cryostat. The sections were incubated with mouse monoclonal antibodies (OK-HLADR, 84H10 (anti-ICAM 11, OK-panB, OK-panT, OKT4, OKT8) and rabbit antibodies against the extracellular and intracellular forms of TGF-B. The streptoavidin-biotin peroxidase complex was used for the immunohistochemical staining of all the sections. In AA patients evident staining for an intracellular form of TGF-B in follicular keratinocytes was found. Intracellular TGF-B was also present in the endothelial cells of perifollicular and papillary dermis (Fig. I), but not in the control skin. Moreover, biopsies 0926-9939/96/$15.00
Copyright
0 1996 Elsevier
from AA patients showed a moderate staining for an extracellular form of TGF-B in the bulbar cells and endothelial cells. In the perifollicular and interfollicular derma of the alopecic skin HLA-DR+ lymphocytes were significantly increased, as was the ICAMI’ inflammatory cell infiltrate, compared to the control. We studied the role of TGF-B in the development of AA and we compared this with inflammatory cell expression. TGF-B’s effect on endothelial cell function may be critical to the microvascular damage seen in this disease [3]. There are several studies dealing with the action of TGF-(3 on endothelial cells with conflicting results; TGF-B sometimes stimulated, sometimes inhibited proliferation [3,4]. Recently, TGF-B has been shown to have a profound effect on the vascular endothelium, inhibiting the proliferation of endothelial cells in response to cell damage, however produced. When an endothelial lesion occurs, the exposed subendothelium interacts with the platelets, the latter strongly stimulating the myocells of the vascular wall [5]. This event could explain the obliterative lesions reported by some authors [6]. We also saw in active AA an exhaustion of microvascular tPA-dependent fibrinolytic activity, leading to perivascular deposits of fibrin-like material [7]. It is known that in the presence of TGF-B matrix degradation is inhibited by increased synthesis of protease inhibitors, such as tPA inhibitor or its decreased synthesis [8]. Concerning the lymphomonocytic infiltrate, a strong effect of the TGF-B on immunocompetent cells by leucocyte recruitment and activation, essential to the development of inflammatory lesions, is reported [9]. Once at the inflammatory site, mononuclear cells are stimulated by TGF-B to release important cytokines in the network of molecules regulating the host response to
Science B.V. All rights reserved
Letters
Fig. 1. Immunohistochemical TCF-P positive endothelial
localization of intracellular TGF-P cells (immunoperoxidase, X 63).
immunologic challenge [lo]. Through these cytokines, TGF-P is able to increase DR antigen expression by the endothelial cells and keratinocytes, involving them in the inflammatory process. Our immunohistochemical data on AA areas show a parallel behaviour of TGF-l3 and HLA-DR and ICAMpositivity. Such co-expression of TGF-(3, as well as activated DR + inflammatory cells, may indicate an accelerated sequence of events leading to an inflammatory process. Furthermore ICAMpositivity seems to indicate increase in endothelial cell activation. Therefore, our results suggest a role and complex action of TGF-l3 in AA but also point out a synergic influence of different factors modulating the alopecic process, resulting in a cascade of interdependent events. Moreover, our results open interesting possibilities for the treatment of AA, sustaining a therapeutic role of anti-TGF-l3 antibodies, as observed in other pathologies, or using soluble TGFP-binding proteins and receptors as well as approaches directed at reducing TGF-I3 gene expression, In AA TGF-l3 therapeutic approaches might
to the Editor
in an alopecic
follicle
(immunoperoxidase,
X25).
Insert:
intracellular
have to be associated with other pharmaceutical therapy programs such as guard immunotherapy. A. Offldani *, G. Lucarini a, A. Cellini, 0. Simonetti, M. Nicolini, G. Biagini “, C. Castaldini b Institute of Dermatology and a Institute of Normal Human Morphology, University of Ancona, Ospedale Umberto P, Piazza Cuppelli 1, 60100 Ancona, Italy b Institute of Histology, University of Bologna, Bologna, Italy
References [l]
Teofoli cal and infiltrate different [2] Heimark dothelial tor from
P, Ghersetih I, Benci T, Lotti T. Immunophenotypiultrastructural study of perivascular and peribulbar and adhesion molecules expression in alopecia areata clinical phases. J Invest Dermatol 1992;98:4, 599. RL, Twardzik DR, Shwartz SM. Inibition of enregeneration by type-beta transforming growth facplatelets. Science 1986;233:1078-1080.
Letters [3] Roberts AB, Spom MB, Assoian RK, Smith JM, Roche NS, Wakelield LM, Heine UI, Liotta LA, Falanga V, Kerl JH, Fauci A. Transforming growth factor type: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc Nat1 Acad Sci USA 1986;83:41674171. [4] Takeara K, Leroy EC, Grotendorst GR. TGF inhibition of endothelial cell proliferation: alteration of EGF binding and EGF induced growth-regulatory (competence) gene expression Cell 1987;49:415-422. [5] Messague .I. The TGF-Bl family of growth and differentiation factors. Cell 1987;49:437-438. [6] Meo AL, Fedi AM, L.otti T. Microvessel alterations in alopecia areata. In: Dermatology in Europe. Proceedings of the 1st Congress of European Academy of Dermatology and Venereology. Panconesi, 1991:829-830. [7] Lotti T, Teofoli P. The role of plasminogen activators in alopecia areata. Int J Dermatol 1991;30:19-21.
Gammalinolenic dermatitis
acid in the treatment
of atopic
To the Editor: Treatment of atopic dermatitis (AD) with essential fatty acids (EFAs) is still controversial and has yielded conflicting results [l-6]. Hansen [7] was the first to suggest that AD might be related to abnormal EFA intake or metabolism by demonstrating a reduced level of unsaturated fatty acids in the blood of the patients. Moreover, he stated that dietary supplements of linoleic acid improved the clinical conditions. It has been suggested that a defect in IS-6-desaturase enzyme function might explain the findings and some clinical signs and symptoms of AD, and that GLA and arachidonic acid are of great importance in the maintenance of a normal epidermal barrier as membrane stabilizing factors. The possible mechanism by which EFAs might improve eczematous lesions includes modification of eicosanoid metabolism so as to favour synthesis of non-inflammatory prostaglandins (PGE 1) and leukotrienes [8]. To evaluate the effects of GLA in AD, a group of patients was treated with this compound in the form of borage oil obtained from seeds of the plant Borago ofJicinaZis. Thirty-one consecutive patients suffering from AD took part in our study; it lasted 6 months during the winter, from November to April,
to the Editor [8] Edward DR, Murphy G, Reynolds JJ et al. Transforming growth factor-beta modulates the expression of collagenase and metallo-proteinase inhibitor. EMBO J 1987;6: 18991904. [9] Kehrl JH, Wakefield LM, Roberts AB, Jakowlew S, AlvarezMon M, Derynck R, Spom MB, Fauci AS. Production of transforming growth factor by human T lymphocytes and its potential role in the regulation of T cell growth. J Exp Med 1986;163:1037-1050. [IO] Darley R, Morris A. Interactions between interferon gamma and retinoic induction of immune recognition molecules. Cancer Immunol Immunother 1993;37: I1 2- 118. PZZ SO926-9959(96)0001
* Corresponding
1-6
author.
in order to exclude the positive effect of sunshine on eczema. Eighteen subjects were children, aged from 2 to 14 years, and 13 were adults, aged from 15 to 38 years. The diagnosis of AD was made according to the criteria of Hanifin and Raika [9]. Criteria for patients’ admission to the study included having active lesions and avoiding use of systemic and/or topical steroids in the 3 weeks prior. All patients were given the same emollient cream in unlimited quantities. At the start of the study and every 4th week thereafter the same clinician evaluated the extent of body involvement and the activity of the disease. The body surface was divided in 20 areas according to the method of Atherton et al. [lo]. The symptoms itching, erythema, vesicles, papules, excoriation, scales and lichenification were scored for each skin area according to the following scale: 1 = absent. 2 = mild, 3 = moderate, 4 = severe. Subsequently a quantitative analysis (area score) was performed and the patients were classified into the following groups: mild AD (score < 2101, moderate AD (score = 210-280) and severe AD (score > 280). After clinical assessment, the patients were randomly divided into 2 groups: one group received GLA and the other group placebo. Active capsules contained 500 mg of borage oil, largely made up of GLA (80 mg/a capsule) and linoleic acid, as well as small amounts of palmitic, oleic and stearic acids.