Identification and Quantitation of Interferon-γ Producing T Cells in Psoriatic Lesions: Localization to Both CD4+ and CD8+ Subsets
1998; Elsevier BV; Volume: 111; Issue: 6 Linguagem: Inglês
10.1046/j.1523-1747.1998.00419.x
ISSN1523-1747
AutoresSarolta K. Szabo, Craig Hammerberg, Yuichi Yoshida, Zsuzsanna Bata‐Csörgõ, Kevin D. Cooper,
Tópico(s)Dermatology and Skin Diseases
ResumoInterferon-γ (IFN-γ) produced by lesional T cell clones is critical for the induction into G1 of the cell cycle by psoriatic keratinocyte stem cells; however, direct data demonstrating psoriatic lesional T cell subset IFN-γ expression, and quantitation at a single cell level to calculatein vivo proportions, are lacking. In this study, using flow cytometry of freshly isolated normal and psoriatic lesional T cells from keratome biopsies, we found elevated CD3+, CD4+, and CD8+ T cells in all compartments of psoriatic skin, compared with normals. Using Brefeldin A to induce short-term intracellular accumulation of IFN-γ in T cells capable of IFN-γ production, we found that 90% of psoriatic patients have IFN-γ-producing T cells at a greater proportion of their CD3+ cells than normals, with a mean of 16%±3%, as compared with 4%±2% in normal epidermis (p = 0.01). Expressed as density in the tissue, the IFN-γ+ CD3+ cell number in psoriatic epidermis was 97 ± 22 per mm2 surface area, as compared with 4.4 ± 1.8 per mm2 of normal epidermis (p = 0.002). Thus, the total number of IFN-γ+CD3+ T cells in the skin of a patient with 20% involvement is estimated to be 3.9 × 108. CD4+ and CD8+ IFN-γ+ T cells were both elevated in psoriatic epidermis (p = 0.04 and p = 0.008, respectively) relative to normal skin. In the dermis, only 44% of patients demonstrated a higher percentage of IFN-γ-producing T cells than did normals (p = 0.1), possibly indicating dilution, in some patients, by fresh infiltrating T cells. Interleukin-4 was not found by a combination of flow cytometry, reverse transcriptase-polymerase chain reaction, western blot, and immunoprecipitation. In conclusion, a significant portion of lesional T cells in psoriasis are IFN-γ producing, without interleukin-4. The increased numbers of both IFN-γ+CD4+ and IFN-γ+CD8+ T cells indicate that both CD4+ and CD8+ IFN-γ+ T cells are present in appropriate anatomic locations to sustain the lesional pathology. Interferon-γ (IFN-γ) produced by lesional T cell clones is critical for the induction into G1 of the cell cycle by psoriatic keratinocyte stem cells; however, direct data demonstrating psoriatic lesional T cell subset IFN-γ expression, and quantitation at a single cell level to calculatein vivo proportions, are lacking. In this study, using flow cytometry of freshly isolated normal and psoriatic lesional T cells from keratome biopsies, we found elevated CD3+, CD4+, and CD8+ T cells in all compartments of psoriatic skin, compared with normals. Using Brefeldin A to induce short-term intracellular accumulation of IFN-γ in T cells capable of IFN-γ production, we found that 90% of psoriatic patients have IFN-γ-producing T cells at a greater proportion of their CD3+ cells than normals, with a mean of 16%±3%, as compared with 4%±2% in normal epidermis (p = 0.01). Expressed as density in the tissue, the IFN-γ+ CD3+ cell number in psoriatic epidermis was 97 ± 22 per mm2 surface area, as compared with 4.4 ± 1.8 per mm2 of normal epidermis (p = 0.002). Thus, the total number of IFN-γ+CD3+ T cells in the skin of a patient with 20% involvement is estimated to be 3.9 × 108. CD4+ and CD8+ IFN-γ+ T cells were both elevated in psoriatic epidermis (p = 0.04 and p = 0.008, respectively) relative to normal skin. In the dermis, only 44% of patients demonstrated a higher percentage of IFN-γ-producing T cells than did normals (p = 0.1), possibly indicating dilution, in some patients, by fresh infiltrating T cells. Interleukin-4 was not found by a combination of flow cytometry, reverse transcriptase-polymerase chain reaction, western blot, and immunoprecipitation. In conclusion, a significant portion of lesional T cells in psoriasis are IFN-γ producing, without interleukin-4. The increased numbers of both IFN-γ+CD4+ and IFN-γ+CD8+ T cells indicate that both CD4+ and CD8+ IFN-γ+ T cells are present in appropriate anatomic locations to sustain the lesional pathology. Psoriasis is characterized by hyperproliferation of the normally slowly cycling epidermal progenitors (Bata-Csorgo et al., 1993Bata-Csorgo Z. Hammerberg C. Voorhees J.J. Cooper K.D. Flow cytometric identification of proliferative subpopulations within normal human epidermis and the localization of the primary hyperproliferative population in psoriasis.J Exp Med. 1993; 178: 1271-1281Crossref PubMed Scopus (114) Google Scholar), and acute and subsequently chronic accumulation of immunocompetent cells (Christophers et al., 1973Christophers E. Parzefall R. Braun-Falco O. Initial events in psoriasis: Quantitative assessment.Br J Dermatol. 1973; 89: 327-334Crossref PubMed Scopus (44) Google Scholar;Baker and Fry, 1992Baker B.S. Fry L. The immunology of psoriasis.Br J Dermatol. 1992; 126: 1-9Crossref PubMed Scopus (178) Google Scholar;Snijdewint et al., 1993Snijdewint Fgm Kalinski P. Wierenga E.A. Bos J.D. Kapsenberg M.L. Prostaglandin E2 differentially modulates cytokine secretion profiles of human T helper lymphocytes.J Immunol. 1993; 150: 5321-5329PubMed Google Scholar). Although the initiating factor(s) in the chronic, self-sustaining cycle of psoriatic lesions have not yet been established, increasing attention is being devoted to intralesional T cell activation (Valdimarsson et al., 1986Valdimarsson H. Baker B.S. Jonsdottir I. Fry L. Psoriasis: a disease of abnormal keratinocyte proliferation induced by T lymphocytes.Immunol Today. 1986; 7: 256-259Abstract Full Text PDF PubMed Scopus (273) Google Scholar;Morganroth et al., 1991Morganroth G.S. Chan L.S. Weinstein G.D. Voorhees J.J. Cooper K.D. Proliferating cells in psoriatic dermis are comprised primarily of T cells, endothelial cells, and factor XIIIa+ perivascular dendritic cells.J Invest Dermatol. 1991; 96: 333-340Abstract Full Text PDF PubMed Google Scholar;Bata-Csorgo et al., 1995aBata-Csorgo Z. Hammerberg C. Voorhees J.J. Cooper K.D. Intralesional T-lymphocyte activation as a mediator of psoriatic hyperplasia.J Invest Dermatol. 1995 a; 105: 89S-94SAbstract Full Text PDF PubMed Scopus (94) Google Scholar;Wrone-Smith and Nickoloff, 1996Wrone-Smith T. Nickoloff B.J. Dermal injection of immunocytes induces psoriasis.J Clin Invest. 1996; 98: 1878-1887Crossref PubMed Scopus (381) Google Scholar;Gilhar et al., 1997Gilhar A. David M. Ullmann Y. Berkutski T. Kalish R.S. T-lymphocyte dependence of psoriatic pathology in human psoriatic skin grafted to SCID mice.J Invest Dermatol. 1997; 109: 283-288Abstract Full Text PDF PubMed Scopus (114) Google Scholar), in the context of a disturbed psoriatic environment, via superantigens (Baker et al., 1993Baker B.S. Bokth S. Powles A. Garioch J.J. Lewis H. Valdimarsson H. Fry L. Group A streptococcal antigen-specific T lymphocytes in guttate psoriatic lesions.Br J Dermatol. 1993; 128: 493-499Crossref PubMed Scopus (99) Google Scholar;Leung et al., 1993Leung Dym Walsh P. Giorno R. Norris D.A. A potential role for superantigens in the pathogenesis of psoriasis.J Invest Dermatol. 1993; 100: 225-228Abstract Full Text PDF PubMed Google Scholar;Boehncke et al., 1997Boehncke W.-H. Zollner T.M. Dressel D. Kaufmann R. Induction of psoriasiform inflammation by a bacterial superantigen in the SCID-hu xenogeneic transplantation model.J Cutan Pathol. 1997; 24: 1-7Crossref PubMed Scopus (43) Google Scholar), autoantigens (Baker and Fry, 1991Baker B.S. Fry L. Autoreactive proliferative responses by T-cell lines isolated from psoriatic lesions.J Invest Dermatol. 1991; 97: 606Crossref PubMed Scopus (3) Google Scholar;Nikaein et al., 1991Nikaein A. Phillips C. Gilbert S.C. Savino D. Silverman A. Stone M.J. Menter A. Characterization of skin-infiltrating lymphocytes in patients with psoriasis.J Invest Dermatol. 1991; 96: 3-9Abstract Full Text PDF PubMed Google Scholar), or conventional antigens (Moss et al., 1997Moss P. Charmley P. Mulvihill E. et al.The repertoire of T cell antigen receptor b-chain variable regions associated with psoriasis.Vulgaris. J Invest Dermatol. 1997; 109: 14-19Abstract Full Text PDF PubMed Scopus (22) Google Scholar). The efficacy of the immunosuppressive agents in clinical use demonstrates that T cells are essential in the maintenance of psoriatic lesions.In vivo treatment with cyclosporine A (Ellis et al., 1986Ellis C.N. Gorsulowsky D.C. Hamilton T.A. et al.Cyclosporine improves psoriasis in a double-blind study.JAMA. 1986; 256: 3110-3116Crossref PubMed Scopus (462) Google Scholar;Griffiths et al., 1986Griffiths C.E. Powles A.V. Leonard J.N. Fry L. Baker B.S. Valdimarsson H. Clearance of psoriasis with low dose cyclosporin.Br Med J Clin Res. 1986; 293: 731-732Crossref PubMed Scopus (184) Google Scholar), anti-CD3 (Weinshenker et al., 1989Weinshenker B.G. Bass B.H. Ebers G.C. Rice GpA Remission of psoriatic lesions with muromonab-CD3 (Orthoclone OKT3) treatment.J Am Acad Dermatol. 1989; 20: 1132-1133Abstract Full Text PDF PubMed Scopus (73) Google Scholar), and anti-CD4 (Prinz et al., 1991Prinz J. Braun-Falco O. Meurer M. Daddona P. Reiter C. Rieber P. Riethmuller G. Chimaeric CD4 monoclonal antibody in treatment of generalised pustular psoriasis.Lancet. 1991; 338: 320-321Abstract PubMed Scopus (150) Google Scholar) monoclonal antibodies, FK 506 (tacrolimus) (Yoshimura and Oka, 1990Yoshimura N. Oka T. FK 506, a new immunosuppressive agent: a review.J Immunol Immunopharmacol. 1990; 10: 32-36Google Scholar), Rapamycin (sirolimus) (Javier et al., 1997Javier A.F. Bata-Csorgo Z. Ellis C.N. Kang S. Voorhees J.J. Cooper K.D. Rapamycin (sirolimus) inhibits proliferating cell nuclear antigen expression and blocks cell cycle in the G1 phase in human keratinocyte stem cells.J Clin Invest. 1997; 99: 2094-2099Crossref PubMed Scopus (54) Google Scholar), interleukin (IL)-2-diphteria toxin fusion protein (Gottlieb et al., 1995Gottlieb S.L. Gilleaudeau P. Johnson R. Estes L. Woodworth T.G. Gottlieb A.B. Krueger J.G. Response of psoriasis to a lymphocyte-selective toxin (DAB389IL-2) suggests a primary immune, but not keratinocyte, pathogenic basis.Nature Med. 1995; 1: 442-447Crossref PubMed Scopus (566) Google Scholar), and CTLA4Ig 1Krueger JG, Hayes E, Brown Met al. Blockade of T cell costimulation with CTLA4Ig (BMS-188667) reverses pathologic inflammation and keratinocyte activation in psoriatic plaques.J Invest Derm 108:555, 1997 (abstr.)1Krueger JG, Hayes E, Brown Met al. Blockade of T cell costimulation with CTLA4Ig (BMS-188667) reverses pathologic inflammation and keratinocyte activation in psoriatic plaques.J Invest Derm 108:555, 1997 (abstr.)provided evidence of T cell involvement. Koebner-initiated and spontaneous eruptions of new psoriatic lesions are associated with the migration of CD4+ T cells into the epidermis (Baker et al., 1988aBaker B.S. Powles A.V. Lambert S. Valdimarsson H. Fry L. A prospective study of the Koebner reaction and T lymphocytes in uninvolved psoriatic skin.Acta Derm Venereol (Stockh). 1988 a; 68: 430-434PubMed Google Scholar;Paukkonen et al., 1992Paukkonen K. Naukkarinen A. Horsmanheimo M. The development of manifest psoriatic lesions is linked with the invasion of CD8 + T Cells and CD11c + macrophages into the epidermis.Arch Dermatol Res. 1992; 284: 375-379Crossref PubMed Scopus (68) Google Scholar), whereas CD8+ T cells appear to traffic in prior to resolution (Baker et al., 1984Baker B.S. Swain A.F. Fry L. Valdimarsson H. Epidermal T lymphocytes and HLA-DR expression in psoriasis.Br J Dermatol. 1984; 110: 555-564Crossref PubMed Scopus (240) Google Scholar). Some, however, view the CD8+ population as the key player in the maintenance of chronic psoriatic lesions, due to the class I major histocompatibility complex association with psoriasis (Henseler and Christophers, 1985Henseler T. Christophers E. Psoriasis of early and late onset: Characterization of two types of psoriasis vulgaris.J Am Acad Dermatol. 1985; 13: 450-456Abstract Full Text PDF PubMed Scopus (735) Google Scholar), the clonality (Chang et al., 1994Chang Jcc Smith L.R. Froning K.J. et al.CD8+ T cells in psoriatic lesions preferentially use T-cell receptor V beta 3 and/or V beta 13.1 genes.Proc Natl Acad Sci USA. 1994; 91: 9282-9286Crossref PubMed Scopus (210) Google Scholar), the association with HIV (Duvic, 1990Duvic M. Immunology of AIDS related to psoriasis.J Invest Dermatol. 1990; 95: 38S-40SAbstract Full Text PDF Google Scholar), and the rapid and selective clearing of CD8+ T cells with successful UVB therapy (Krueger et al., 1995Krueger J.G. Wolfe J.T. Nabeya R.T. et al.Successful ultraviolet B treatment of psoriasis is accompanied by a reversal of keratinocyte pathology and by selective depletion of intraepidermal T cells.J Exp Med. 1995; 182: 2057-2068Crossref PubMed Scopus (221) Google Scholar) (although CD4+ T cells also clear with UVB) 2Szabo SK, Hammerberg C, Bata-Csorgo ZS, Cooper KD: The mechanism of action of UVB in psoriasis: rapidly decreased T cell numbers and preferential reduction of interferon γ (IFNγ) production.J Invest Dermatol 108:558 1997 (abstr.)2Szabo SK, Hammerberg C, Bata-Csorgo ZS, Cooper KD: The mechanism of action of UVB in psoriasis: rapidly decreased T cell numbers and preferential reduction of interferon γ (IFNγ) production.J Invest Dermatol 108:558 1997 (abstr.)in association with the clinical resolution of the lesion. Moreover, a patient with idiopathic, profound CD4+ lymphocytopenia has recently been reported who developed active psoriatic lesions, had plaques that displayed dramatically elevated total and activated epidermal CD8+ T cells, and had similar CD4+ T cell numbers of a group of psoriatic patients with normal peripheral blood CD4+ T cell counts (Hardman et al., 1997Hardman C.M. Baker B.S. Lortan J. Breuer J. Surentheran T. Powles A. Fry L. Active psoriasis and profound CD4+ lymphocytopenia.Br J Dermatol. 1997; 136: 930-932Crossref PubMed Scopus (17) Google Scholar). Despite the fact that interferon (IFN)-γ is a potent inhibitor of keratinocyte proliferationin vitro on its own (Nickoloff et al., 1984Nickoloff B.J. Basham T.Y. Merigan T.C. Morhenn V.B. Antiproliferative effects of recombinant alpha- and gamma-interferons on cultured human keratinocytes.Lab Invest. 1984; 51: 697-701PubMed Google Scholar,Nickoloff et al., 1989Nickoloff B.J. Mitra R.S. Elder J.T. Fisher G.J. Voorhees J.J. Decreased growth inhibition by recombinant gamma interferon is associated with increased transforming growth factor-alpha production in keratinocytes cultured from psoriatic lesions.Br J Dermatol. 1989; 121: 161-174Crossref PubMed Scopus (99) Google Scholar), accumulated evidence from bothin vivo (Baker et al., 1988bBaker B.S. Powles A.V. Valdimarsson H. Fry L. An altered response by psoriatic keratinocytes to gamma interferon.Scan J Immunol. 1988 b; 28: 735-740Crossref PubMed Scopus (57) Google Scholar) andin vitro (Bata-Csorgoet al. 1995b) studies show that in the context of other lymphokines, IFN-γ is a critical element in the induction of keratinocyte hyperproliferation in psoriasis. Cloned T cells from psoriatic lesions, in general, showed characteristics of Th-1-type lymphocytes by their production of IFN-γ, but not IL-4 or IL-5 (Schlaak et al., 1994Schlaak J.F. Buslau M. Jochum W. et al.T cells involved in psoriasis vulgaris belong to the Th1 subset.J Invest Dermatol. 1994; 102: 145-149Abstract Full Text PDF PubMed Google Scholar;Vollmer et al., 1994Vollmer S. Menssen A. Trommler P. Schendel D. Prinz J.C. T lymphocytes derived from skin lesions of patients with psoriasis vulgaris express a novel cytokine pattern that is distinct from that of T helper type 1 and T helper type 2 cells.Eur J Immunol. 1994; 24: 2377-2382Crossref PubMed Scopus (130) Google Scholar;Bata-Csorgo et al., 1995aBata-Csorgo Z. Hammerberg C. Voorhees J.J. Cooper K.D. Intralesional T-lymphocyte activation as a mediator of psoriatic hyperplasia.J Invest Dermatol. 1995 a; 105: 89S-94SAbstract Full Text PDF PubMed Scopus (94) Google Scholar). T cell clones derived from psoriatic lesions release IFN-γin vitro, which together with growth factors (IL-3, GM-CSF) (Bata-Csorgo et al., 1995aBata-Csorgo Z. Hammerberg C. Voorhees J.J. Cooper K.D. Intralesional T-lymphocyte activation as a mediator of psoriatic hyperplasia.J Invest Dermatol. 1995 a; 105: 89S-94SAbstract Full Text PDF PubMed Scopus (94) Google Scholar) and fibronectin (Bata-Csorgo et al., 1998Bata-Csorgo Z. Cooper K.D. Ting K. Voorhees J.J. Hammerberg C. Fibronectin and α5 integrin regulate keratinocyte cell cycling: a mechanism for increased fibronectin potentiation of T cell lymphokine-driven keratinocyte hyperproliferation in psoriasis.J Clin Invest. 1998; 101: 1509-1518Crossref PubMed Scopus (59) Google Scholar) are essential for the increased cell cycle induction that occurin vivo among K1/K10– keratinocyte stem cells (Bata-Csorgo et al., 1993Bata-Csorgo Z. Hammerberg C. Voorhees J.J. Cooper K.D. Flow cytometric identification of proliferative subpopulations within normal human epidermis and the localization of the primary hyperproliferative population in psoriasis.J Exp Med. 1993; 178: 1271-1281Crossref PubMed Scopus (114) Google Scholar). IFN-γ produced by T cell clonesin vitro is key not only for proliferation but also for inducing anin vivo psoriatic keratinocyte phenotype (Baadsgaard et al., 1990Baadsgaard O. Tong P. Elder J.T. et al.UM4D4+ (CDw60) T cells are compartmentalized into psoriatic skin and release lymphokines that induce a keratinocyte phenotype expressed in psoriatic lesions.J Invest Dermatol. 1990; 95: 275-282Abstract Full Text PDF PubMed Google Scholar). IFN-γ has also been shown to be responsible for initiating psoriatic lesionsin vivo (Fierlbeck et al., 1990Fierlbeck G. Rassner G. Muller C. Psoriasis induced at the injection site of recombinant interferon gamma. Results of immunohistologic investigations.Arch Dermatol. 1990; 126: 351-355Crossref PubMed Scopus (165) Google Scholar). Locally produced IFN-γ may have profound effects on keratinocytes. Psoriatic keratinocytes express class II major histocompatibility complex proteins (Morhenn et al., 1982Morhenn V.B. Abel E.A. Mahrle G. Expression of HLA-DR antigen in skin from patients with psoriasis.J Invest Dermatol. 1982; 78: 165-168Abstract Full Text PDF PubMed Scopus (69) Google Scholar), intercellular adhesion molecule-1 (Griffiths et al., 1989aGriffiths Cem Voorhees J.J. Nickoloff B.J. Characterization of intercellular adhesion molecule-1 and HLA-DR expression in normal and inflamed skin: modulation by recombinant gamma interferon and tumor necrosis factor.J Am Acad Dermatol. 1989 a; 20: 617-629Abstract Full Text PDF PubMed Scopus (427) Google Scholar), IP-10 (Gottlieb et al., 1988Gottlieb A.B. Luster A.D. Posnett D.N. Carter D.M. Detection of a gamma interferon-induced protein IP-10 in psoriatic plaques.J Exp Med. 1988; 168: 941-948Crossref PubMed Scopus (195) Google Scholar), and UM4D4 (Baadsgaard et al., 1990Baadsgaard O. Tong P. Elder J.T. et al.UM4D4+ (CDw60) T cells are compartmentalized into psoriatic skin and release lymphokines that induce a keratinocyte phenotype expressed in psoriatic lesions.J Invest Dermatol. 1990; 95: 275-282Abstract Full Text PDF PubMed Google Scholar)in vivo and IFN-γ induces each of these molecules on human cultured keratinocytes (Baker et al., 1984Baker B.S. Swain A.F. Fry L. Valdimarsson H. Epidermal T lymphocytes and HLA-DR expression in psoriasis.Br J Dermatol. 1984; 110: 555-564Crossref PubMed Scopus (240) Google Scholar;Basham et al., 1984Basham T.Y. Nickoloff B.J. Merigan T.C. Recombinant gamma interferon induces HLA-DR expression on cultured keratinocytes.J Invest Dermatol. 1984; 83: 88-92Abstract Full Text PDF PubMed Scopus (293) Google Scholar;Griffiths et al., 1989bGriffiths Cem Voorhees J.J. Nickoloff B.J. Gamma interferon induces different keratinocyte cellular patterns of expression of HLA-DR and DQ and intercellular adhesion molecule-I (ICAM-I) antigens.Br J Dermatol. 1989 b; 120: 1-7Crossref PubMed Scopus (72) Google Scholar). Cyclosporine therapy causes rapid loss of these molecules’ expression on keratinocytes, even prior to depletion of T cells in treated lesions, indicating loss of support of synthesis due to decreased production of IFN-γ by T cells still present but inhibited (Cooper et al., 1990Cooper K.D. Voorhees J.J. Fisher G.J. Chan L.S. Gupta A.K. Baadsgaard O. Effects of cyclosporine on immunologic mechanisms in psoriasis.J Am Acad Dermatol. 1990; 23: 1318-1328Abstract Full Text PDF PubMed Scopus (32) Google Scholar). IFN-γ has been detected in psoriatic lesions by immunohistochemistry (Livden et al., 1989Livden J.K. Nilsen R. Bjerke J.R. Matre R. Situ Localization of Interferons in Psoriatic Lesions.Arch Dermatol Res. 1989; 281: :392-397Crossref PubMed Scopus (54) Google Scholar) and polymerase chain reaction (Barker et al., 1991Barker Jnwn Karabin G.D. Stoof T.J. Sarma V.J. Dixit V.M. Nickoloff B.J. Detection of interferon-gamma mRNA in psoriatic epidermis by polymerase chain reaction.J Dermatol Sci. 1991; 2: 106-111Abstract Full Text PDF PubMed Scopus (87) Google Scholar). Interestingly, in fully developed lesions, HLA-DR+ keratinocytes are located in close microanatomical proximity with the IFN-γ deposits, suggesting a positive, influencing relationship of IFN-γ on keratinocytes (Kaneko et al., 1990Kaneko F. Suzuki M. Takiguchi Y. Itoh N. Minagawa T. Immunohistopathologic studies in the development of psoriatic lesion influenced by gamma-interferon and the producing cells.J Dermatol Sci. 1990; 1: 425-434Abstract Full Text PDF PubMed Scopus (15) Google Scholar). There are no data concerning the cell origin of IFNγ in lesions, and whether a single subset (CD4 or CD8) or both is capable of producing IFN-γ in lesions and what the actual percentage of IFN-γ producing cells are in chronic psoriatic lesions. Recent progress in the simultaneous identification of individual cells, and their function by intracellular cytokine staining (Picker et al., 1995Picker L.J. Singh M.K. Zdraveski Z. Treer J.R. Waldrop S.L. Bergstresser P.R. Maino V.C. Direct demonstration of cytokine synthesis heterogeneity among human memory/effector T cells by flow cytometry.Blood. 1995; 86: 1408-1419PubMed Google Scholar), with analysis by flow cytometry now allow for the characterization of cells isolated from the epidermis (Spetz et al., 1996Spetz A.-L. Strominger J. Groh-Spies V. T cell subsets in normal human epidermis.Am J Pathol. 1996; 149: 665-674PubMed Google Scholar) and dermis. By using multiparameter flow cytometry with intracellular staining, we analyzed T cells of normal skin and psoriatic lesions at a single cell level to identify changes in specific T cell subsets in the epidermis and the dermis of psoriatic skin, and to delineate the IFN-γ-producing capability of psoriatic T cells. Keratome biopsies (1.5 × 5 cm) were taken from the buttock area of normal volunteers (0.6 mm deep), and patients with chronic psoriasis vulgaris (0.8 mm deep) after informed consent according to IRB protocol. Six female and six male psoriatic patients involved in the study were aged between 26 and 57 y, with an average of 41 y. Five females and one male healthy volunteers participated in this study; they were 42 y old on average (between 25 and 57). Except for one healthy and one psoriatic African-American volunteer, all volunteers were Caucasians. For either group, oral medication for 1 mo prior to obtaining the specimens was discontinued. In addition, the volunteers with psoriasis discontinued external treatment 2 wk before the procedure was performed. Epidermal and dermal sheets were separated with Dispase (Collaborative Biomedical Products, Bedford, MA) (20 min, 37°C). To obtain a single-cell suspension, epidermis was treated with Enzyme-Free/Hanks′-based cell dissociation buffer (Gibco Laboratories, Grand Island, NY) with 0.1% DNase (Sigma, St. Louis, MO) (45 min, 37°C). Dermis was incubated (2–3 h, 37°C) in 15 ml RPMI 1640 (Gibco) containing 40.5 mg collagenase, 18.75 mg hyaluronidase, 0.01% DNase, 0.01% sodium pyruvate, 0.01% HEPES buffer (all from Sigma). Epidermal and dermal cell suspensions were filtered through a 100 μm or 50 μm and 100 μm nylon mesh, respectively (BioDesign, Camel, NY). Cells were washed in Hank’s balanced salt solution (Gibco) containing 1% heat-inactivated fetal bovine serum (HyClone Laboratories, Logan, UT). Viability (average 70%–90%) was determined by trypan blue exclusion. Cell culture Cells were incubated for 12–14 h at 37° C (106 cells per ml) in RPMI 1640 (Gibco) supplemented with L-glutamine, penicillin, streptomycin, and 10% human AB serum (NORMLCERA-Plus, NABI, Miami, FL) In the last 4 h of the incubation, 25 ng phorbol myristate acetate (PMA) per ml and 1 μmol ionomycin per liter (both from Sigma) were added to detect IFN-γ, in the recently in vivo-activated T cells (Picker et al., 1995Picker L.J. Singh M.K. Zdraveski Z. Treer J.R. Waldrop S.L. Bergstresser P.R. Maino V.C. Direct demonstration of cytokine synthesis heterogeneity among human memory/effector T cells by flow cytometry.Blood. 1995; 86: 1408-1419PubMed Google Scholar) Ten micrograms Brefeldin A (Sigma) per ml was also added at this time to block the cytokines transport through the Golgi apparatus (Picker et al., 1995Picker L.J. Singh M.K. Zdraveski Z. Treer J.R. Waldrop S.L. Bergstresser P.R. Maino V.C. Direct demonstration of cytokine synthesis heterogeneity among human memory/effector T cells by flow cytometry.Blood. 1995; 86: 1408-1419PubMed Google Scholar) In all our experiments, for the detection of CD 4 molecules on T cells, several samples had no PMA and ionomycin in the culture media. Staining procedureHuman IgG (Sigma) was used to block nonspecific binding, then Cy-Chrome labeled anti-CD 3, anti-CD 4, anti-CD 8 (IgG 1; all from PharMingen, San Diego, CA) or isotype control monoclonal antibodies were used, respectively, with normal mouse serum (SERA-LAB, Sussex, UK) Following fixation 4% paraformaldehyde, cells were permeabilized with phosphate-buffered saline, 1% fetal bovine serum, 01% Saponin (Sigma), and 01% sodium azide (Curtin Matheson Scientific, Houston, Texas), and anti-IFN-γ-FITC (IgG 1; PharMingen), anti-IL- 4-PE (IgG 1; PharMingen), or isotype control was added to stain intracellular cytokine Cells were kept in phosphate-buffered saline containing 1% bovine serum albumine (Sigma), and 002% sodium azide until flow cytometry Flow cytometryFour parameter analysis was performed on a FACScan flow cytometer (Coulter, Hialeah, FL) Forward and 90° light scatter was used to gate out cell aggregates and debris Data were analyzed by using ELITE Software (Coulter) For the detection of IL-4, reverse transcriptase-polymerase chain reaction (Uyemura et al., 1993Uyemura K. Yamamura M. Fivenson D.F. Modlin R.L. Nickoloff B.J. The cytokine network in lesional and lesion-free psoriatic skin is characterized by a T-helper type 1 cell-mediated response.J Invest Dermatol. 1993; 101: 701-705Abstract Full Text PDF PubMed Google Scholar;Atamas et al., 1996Atamas S.P. Choi J. Yurovsky V.V. White B. An alternative splice variant of human IL-4, IL-4δ2, inhibits IL-4 stimulated T cell proliferation.J Immunol. 1996; 156: 435-441PubMed Google Scholar), western blot, immunoprecipitation were performed according to standard procedures (recommended by PharMingen, San Diego, CA). A portion of the normal and psoriatic keratome specimens were cut (6 μm) and stained with anti-CD3 (UCHT-1, IgG1; Immunotech, Westbrock, ME) monoclonal antibody or its isotype control (Sigma) after incubation with goat serum. Biotinylated anti-mouse IgG1 was used as secondary antibody (Caltag, San Francisco, CA), followed by peroxidase-conjugated streptavidin (Kirkegaard & Perry, Gaithersburg, MD), then detection with Diaminobenzidine Reagent Set (DAB; Kirkegaard & Perry), and counterstaining with hematoxylin. Computer-based image analysis was done by Optima 6.1 program (OPTIMAS, Bothell, WA). Comparison of the normal and psoriatic values were performed with Student t test. p values < 0.05 were considered significant. To determine what portion of the skin is present in our keratomes for T cell analysis, sections from normal and lesional keratomes were stained for CD3 surface markers. We found an increased distribution of the CD3+ cells in psoriatic skinFigure 1a similar to that of previous reports (Baker et al., 1984Baker B.S. Swain A.F. Fry L. Valdimarsson H. Epidermal T lymphocytes and HLA-DR expression in psoriasis.Br J Dermatol. 1984; 110: 555-564Crossref PubMed Scopus (240) Google Scholar;de Boer et al., 1994de Boer O.J. van der Loos C.M. Hamerlinck F. Bos J.D. Das P.K. Reappraisal of in situ immunophenotypic analysis of psoriasis skin: interaction of activated HLA-DR+ immunocompetent cells and endothelial cells is a major feature of psoriatic lesions.Arch Dermatol Res. 1994; 286: 87-96Crossref PubMed Scopus (38) Google Scholar). As shown inFigure 1, the depth of the normal epidermis (E) and dermis (D) was 0.1 mm and 0.5 mm, respectively. In psoriasis, the epidermis was 0.4 mm and the dermis was 0.4 mmFigure 1b. Applying the schematic horizontal layers used byBos et al., 1987Bos J.D. Zonneveld I. Das P.K. Krieg S.R. van der Loos C.M. Kapsenberg M.L. The skin immune system (SIS): Distribution and immunophenotype of lymphocyte subpopulation in normal human skin.J Invest Dermatol. 1987; 88: 569-573Abstract Full Text PDF PubMed Google Scholar we established that in our flow analysis we are detecting T cell populations of the papillary dermis, including the subepidermal and “free” areas, but excluding the “deep” perivascular compartment, in both normal and psoriatic dermis. Direct flow cytometric quantitation of T cells within psoriatic epidermis: increased CD 3+ cells due to infiltration by both CD 4+ and CD 8+ T cellsFlow cytometric analysis was used to quantitate the actual differences between normal and psoriatic T cellsin vivo and to examine the IFN-γ production at the single cell level. Because preparation of dermal cell suspensions invo
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