Overexpression of IL-4 Alters the Homeostasis in the Skin
2002; Elsevier BV; Volume: 118; Issue: 5 Linguagem: Inglês
10.1046/j.1523-1747.2002.01753.x
ISSN1523-1747
AutoresAdelheid Elbe‐Bürger, Sabine Olt, Georg Stingl, Alena Egyed, Radek Klubal, Ulrike Mann, Klemens Rappersberger, Antal Rot,
Tópico(s)Immunotherapy and Immune Responses
ResumoIL-4 has been implicated to play an important role in the pathogenesis of many inflammatory diseases including skin diseases such as atopic dermatitis. Because it is not clear which pathologic features of atopic dermatitis are dependent on IL-4, we assessed the consequences of IL-4 overexpression in the skin, using transgenic mice overexpressing IL-4 ubiquitously. Although transgenic mice display no clinical signs of skin inflammation, IL-4 induced a wide spectrum of pathologies including an increased number of mast cells and Langerhans cells in dermis and epidermis, respectively, focal deposition of collagen and a considerably reduced adipocyte layer in the dermis as well as an increased mitotic activity of keratinocytes, reflected in acanthosis and hyperkeratosis. The increase in Langerhans cell number may be explained in part by the substantially reduced Langerhans cell emigration from the epidermis in transgenic mice. The molecular mechanism behind this phenomenon remains to be clarified. Under in vitro culture conditions, Langerhans cells from transgenic mice undergo a maturation process similar to that of Langerhans cells from control mice, and their immunostimulatory capacity is also comparable. In contrast, transgenic Langerhans cells are superior to control Langerhans cells in their antigen-processing capacity. We conclude that the overexpression of IL-4 in the skin is, by itself, not sufficient for the induction of a full-blown atopic dermatitis phenotype, but several changes seen in the skin of transgenic mice mirror the cardinal pathologic manifestations of this disease. IL-4 has been implicated to play an important role in the pathogenesis of many inflammatory diseases including skin diseases such as atopic dermatitis. Because it is not clear which pathologic features of atopic dermatitis are dependent on IL-4, we assessed the consequences of IL-4 overexpression in the skin, using transgenic mice overexpressing IL-4 ubiquitously. Although transgenic mice display no clinical signs of skin inflammation, IL-4 induced a wide spectrum of pathologies including an increased number of mast cells and Langerhans cells in dermis and epidermis, respectively, focal deposition of collagen and a considerably reduced adipocyte layer in the dermis as well as an increased mitotic activity of keratinocytes, reflected in acanthosis and hyperkeratosis. The increase in Langerhans cell number may be explained in part by the substantially reduced Langerhans cell emigration from the epidermis in transgenic mice. The molecular mechanism behind this phenomenon remains to be clarified. Under in vitro culture conditions, Langerhans cells from transgenic mice undergo a maturation process similar to that of Langerhans cells from control mice, and their immunostimulatory capacity is also comparable. In contrast, transgenic Langerhans cells are superior to control Langerhans cells in their antigen-processing capacity. We conclude that the overexpression of IL-4 in the skin is, by itself, not sufficient for the induction of a full-blown atopic dermatitis phenotype, but several changes seen in the skin of transgenic mice mirror the cardinal pathologic manifestations of this disease. atopic dermatitis hen egg lysozyme ovalbumin transgenic The prevalence of chronic inflammatory skin diseases of atopic nature, such as chronic atopic dermatitis (AD), is still rising. In many cases, it is associated with elevated serum IgE levels, peripheral blood eosinophilia, as well as rhinoconjunctivitis and/or bronchial asthma. Genetic susceptibility, abnormal lipid synthesis with epidermal barrier dysfunction, and an altered inflammatory and immune response to irritants and allergens were suggested to contribute to AD pathogenesis (Leung, 1995Leung D.Y.M. Atopic dermatitis: The skin as a window into the pathogenesis of chronic allergic diseases.J Allergy Clin Immunol. 1995; 96: 302-318Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar;Rudikoff and Lebwohl, 1998Rudikoff D. Lebwohl M. Atopic dermatitis.Lancet. 1998; 351: 1715-1721Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). Acute or early lesions of AD are characterized by spongiosis and a sparse epidermal infiltrate of T lymphocytes. There is often marked dermal edema with a prominent infiltrate of inflammatory cells, particularly T lymphocytes. Mast cells are only slightly increased in number, but they are in various stages of degranulation, which indicates activation of these cells. Chronic lesions of AD exhibit hyperkeratosis, epidermal hyperplasia, minimal spongiosis, and upper dermal fibrosis. The dermal inflammatory infiltrate is composed mainly of macrophages and eosinophils, which have been found to release major basic protein (Rudikoff and Lebwohl, 1998Rudikoff D. Lebwohl M. Atopic dermatitis.Lancet. 1998; 351: 1715-1721Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). An increased number of not-activated mast cells, and cells belonging to the dendritic cell (DC) lineage, including dermal DC, epidermal Langerhans cells, and a distinct population of inflammatory dendritic epidermal cells expressing CD1a, CD1b, or CD36, or combinations thereof, has also been observed (Bos et al., 1986Bos J.D. van Garderen I.D. Krieg S.R. et al.Different in situ distribution patterns of dendritic cells having Langerhans (T6+) and interdigitating (RFD1+) cell immunophenotype in psoriasis, atopic dermatitis, and other inflammatory dermatoses.J Invest Dermatol. 1986; 87: 358-361Crossref PubMed Scopus (57) Google Scholar;Bieber et al., 1988Bieber T. Ring J. Braun-Falco O. Comparison of different methods for enumeration of Langerhans cells in vertical cryosections of human skin.Br J Dermatol. 1988; 118: 385-392Crossref PubMed Scopus (59) Google Scholar;Taylor et al., 1991Taylor R.S. Baadsgaard O. Hammerberg C. et al.Hyperstimulatory CD1a+CD1b+CD36+ Langerhans cells are responsible for increased autologous T lymphocyte reactivity to lesional epidermal cells of patients with atopic dermatitis.J Immunol. 1991; 147: 3794-3802PubMed Google Scholar;Horsmanheimo et al., 1994Horsmanheimo L. Harvima I.T. Järvikallio A. et al.Mast cells are one major source of IL-4 in atopic dermatitis.Br J Dermatol. 1994; 131: 348-353Crossref PubMed Scopus (126) Google Scholar;Rudikoff and Lebwohl, 1998Rudikoff D. Lebwohl M. Atopic dermatitis.Lancet. 1998; 351: 1715-1721Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). Additional characteristics of DC in AD are the markedly upregulated expression of the high-affinity receptor for IgE (FcεRI) (Wollenberg et al., 1996Wollenberg A. Kraft S. Hanau D. et al.Immunomorphological and ultrastructural characterization of Langerhans cells and a novel, inflammatory dendritic epidermal cell (IDEC) population in lesional skin of atopic eczema.J Invest Dermatol. 1996; 106: 446-453Crossref PubMed Scopus (327) Google Scholar;Klubal et al., 1997Klubal R. Osterhoff B. Wang B. et al.The high-affinity receptor for IgE is the predominant IgE-binding struture in lesional skin of atopic dermatitis patients.J Invest Dermatol. 1997; 108: 336-342Crossref PubMed Scopus (55) Google Scholar) and CD86 (Ohki et al., 1997Ohki O. Yokozeki H. Katayama I. et al.Functional CD86 (B7–2/B70) is predominantly expressed on Langerhans cells in atopic dermatitis.Br J Dermatol. 1997; 136: 838-845Crossref PubMed Scopus (38) Google Scholar). Influx of activated T lymphocytes into the skin lesions represents a hallmark in AD (Grewe et al., 1998Grewe M. Bruijnzeel-Koomen C.A.F.M. Schöpf E. et al.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 (654) Google Scholar) and recent results indicate a dynamic T cell-derived cytokine production in AD. In addition to the well-known Th2/Tc2 component of acute lesions (Akdis et al., 1999Akdis A. Simon H.U. Weigl L. et al.Skin homing (cutaneous lymphocyte-associated antigen-positive) CD8+ T cells respond to superantigen and contribute to eosinophilia and IgE production in atopic dermatitis.J Immunol. 1999; 163: 466-475PubMed Google Scholar), chronic lesions are characterized by an Th1/Th0 cytokine pattern (Grewe et al., 1998Grewe M. Bruijnzeel-Koomen C.A.F.M. Schöpf E. et al.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 (654) Google Scholar). To what degree IL-4 contributes to the development and perpetuation of underlying pathologies remains speculative. IL-4 is a pleiotropic cytokine and is produced by CD4+ helper T cells of the Th2 subset, some CD8+ T cells, activated mast cells, basophils, and eosinophils. IL-4 affects a broad spectrum of different cell types including T cells, B cells, natural killer cells, mast cells, monocytes/macrophages, endothelial cells, fibroblasts, adipocytes, DC, Langerhans cells, and keratinocytes and regulates the immune response in a number of ways (Chomarat et al., 1998Chomarat P. Rybak M.E. Banchereau J. Interleukin-4.in: Thomson A.W. The Cytokine Handbook. Academic Press, San Diego1998: 133-174Google Scholar). In T cells, IL-4 directs the development of undifferentiated T cells into IL-4-producing Th2 cells (Brown and Hural, 1997Brown M.A. Hural J. Functions of IL-4 and control of its expression.Crit Rev. 1997; 17: 1-32Google Scholar;Chomarat et al., 1998Chomarat P. Rybak M.E. Banchereau J. Interleukin-4.in: Thomson A.W. The Cytokine Handbook. Academic Press, San Diego1998: 133-174Google Scholar). IL-4 promotes growth and increased survival in B cells, enhances their antigen-presenting capacity by increasing the expression of MHC class II molecules and low-affinity Fcε receptors, and Th2-derived IL-4 induces isotype switching from IgM to IgG1 and IgE production in B cells. As it has long been recognized that IL-4 plays a crucial role in directing the adaptive immune response of T and B cells and that IL-4 is a key factor in the pathogenesis of atopy, therapeutic strategies aiming to block IL-4 activity (e.g., by monoclonal antibodies, soluble receptors, or small molecular compounds) have been developed mostly to prevent asthma and other IgE-mediated diseases (Renz, 1999Renz H. Soluble interleukin-4 receptor (sIL-4R) in allergic diseases.Inflamm Res. 1999; 48: 425-431https://doi.org/10.1007/s000110050482Crossref PubMed Scopus (22) Google Scholar;Wong and Koh, 2000Wong W.S.F. Koh D.S.K. Advances in immunopharmacology of asthma.Biochem Pharmacol. 2000; 59: 1323-1355Crossref PubMed Scopus (38) Google Scholar;Mitchell et al., 2001Mitchell R.N. Abbas A.K. Cytokines regulating immune inflammation: interleukin-4, interleukin-10, and interleukin-12.in: Austen K.F. Burakoff S.J. Rosen F.S. Strom T.B. Therapeutic Immunology. Blackwell Science, Oxford2001: 201-219Google Scholar;Stutz et al., 2001Stutz A.M. Hoeck J. Natt F. et al.Inhibition of interleukin-4- and CD40-induced IgE germline gene promotor activity by 2′-aminoethoxy-modified triplex-forming oligonucleotides.J Biol Chem. 2001; 276: 11759-11765Crossref PubMed Scopus (31) Google Scholar;Tomkinson et al., 2001Tomkinson A. Duez C. Cieslewicz G. et al.A murine IL-4 receptor antagonist that inhibits IL-4- and IL-13-induced responses prevents antigen-induced airway eosinophilia and airway hyperresponsiveness.J Immunol. 2001; 166: 5792-5800Crossref PubMed Scopus (145) Google Scholar). Conceivably, such an approach could also be useful in the treatment of AD, because IL-4 also influences cells known to be phenotypically and/or functionally altered in AD, i.e., keratinocytes, fibroblasts, endothelial cells, and Langerhans cells. While this report was in progress, a study was published where transgenic (tg) mice were generated expressing epidermal IL-4 (Chan et al., 2001Chan L.S. Robinson N. Xu L. Expression of interleukin-4 in the epidermis of transgenic mice results in a pruritic inflammatory skin disease: an experimental animal model to study atopic dermatitis.J Invest Dermatol. 2001; 117: 977-983Crossref PubMed Google Scholar). Interestingly, the normal appearing skin of these mice revealed no pathology; however, a large percentage of these mice spontaneously developed a pruritic inflammatory skin disease with many of the key features of AD. We studied the structural, phenotypic, and functional properties of skin and individual skin cell populations in tg mice overexpressing IL-4 ubiquitously (Erb et al., 1994Erb K.J. Holtschke T. Muth K. et al.T cell subset distribution and B cell hyperreactivity in mice expressing interleukin-4 under the control of major histocompatibility complex class I regulatory sequences.Eur J Immunol. 1994; 24: 1143-1147Crossref PubMed Scopus (26) Google Scholar), and found that IL-4 failed to induce a spontaneous skin disease but induced a wide spectrum of pathologies including an increased number of Langerhans cells, mast cells, focal deposition of collagen, a reduced adipocyte layer, acanthosis, and hyperkeratosis. Newborn to 42-wk-old female IL-4 tg mice [B6C3HF1 (H-2b/k)] were used in the experiments (Erb et al., 1994Erb K.J. Holtschke T. Muth K. et al.T cell subset distribution and B cell hyperreactivity in mice expressing interleukin-4 under the control of major histocompatibility complex class I regulatory sequences.Eur J Immunol. 1994; 24: 1143-1147Crossref PubMed Scopus (26) Google Scholar). The non-tg offspring served as age-matched, littermate controls. Six to twelve-wk-old female inbred mice [BALB/c (H-2d)] were obtained from Charles River Wiga GmbH (Sulzfeld, Germany). RPMI 1640 medium was supplemented with 10% heat-inactivated fetal calf serum (PAA Laboratories GmbH, Linz, Austria), 25 mM HEPES, 10 µg gentamycin per ml, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 50 µM 2-ME, and 1 × antibiotic-antimycotic solution (all from Gibco Life Technologies, Grand Island, NY). Unlabeled or FITC-conjugated monoclonal antibodies (MoAb) 145–2C11 and 500A2 (anti-CD3), GK1.5 and RM4-4 (anti-CD4), 53–6.72 (anti-CD8), B3B4 (anti-CD23), J11d and M1/69 (anti-CD24), 3C7 and 7D4 (anti-CD25), Ha2/5 (anti-CD29), 3/23 (anti-CD40), IM7.8.1 (anti-CD44), 30F11.1 (anti-CD45), 23G2 (anti-CD45RB), R1-2 (CD49d), RA3–6B2 (anti-CD45R/B220), 3E2 (anti-CD54), MEL-14 (anti-CD62L), 16–10A1 (anti-CD80), GL1 (anti-CD86), and G7 (anti-CD90) were purchased from PharMingen (San Diego, CA). The MoAb NLDC-145 (anti-DEC-205), ECCD-2 (anti-E-cadherin), F7D5 (anti-CD90), and the polyclonal antibody Ki-67 were obtained from Serotec (Oxford, U.K.), Zymed (South San Francisco, CA), Biosource (Camarillo, CA), and Novocastra (Newcastle, U.K.), respectively. Hybridomas N418 (anti-CD11c, HB 224), M5/114.15.2 (anti-I-Ab,d,q & I-Ed,k, TIB 120), RA3–3A1/6.1 (anti-CD45R/B220, TIB 146), and F4/80 (antimacrophage, HB 198) were obtained from the American Type Culture Collection (Manassas, VA). The hybridomas 2B6.2D8 (anti-CD4) and 3.168.81 (anti-CD8) were kindly provided by E. M. Shevach (National Institute of Allergy and Infectious Diseases, Bethesda, MD), and H. R. MacDonald (Ludwig Institute for Cancer Research, Epalinges, Switzerland), respectively. Second step reagents were polyclonal FITC-labeled F(ab′)2 goat antirat IgG (H + L) (Immunotech, Marseille, France), FITC-conjugated goat antihamster IgG (H + L) (Caltag, South San Francisco, CA), Streptavidin-Phycoerythrin (SAv-PE) (PharMingen) or Streptavidin Texas Red (Amersham Pharmacia Biotech, Vienna, Austria). Irrelevant, isotype-matched MoAb were used as negative controls. Some of the MoAb used were purified from supernatants of the corresponding hybridomas, and protein concentrations were adjusted to 1 mg per ml before they were FITC-conjugated or biotinylated. Hen egg lysozyme (HEL) and ovalbumin (OVA) were purchased from Sigma (St. Louis, MO). Ears from tg mice and controls were washed in 70% ethanol, dried, split, placed dermal side down on a dispase II/PBS solution (2.4 U per ml; Boehringer Mannheim, Mannheim, Germany) for 45 min at 37°C. In some experiments trunk skin was used. Epidermal sheets were peeled from the dermis and further digested by stirring in a trypsin-GNK solution (0.1% trypsin, sodium bicarbonate, NaCl, KCl, and glucose) containing DNase (200 µg per ml) for 20 min at 37°C. The single cell suspension was filtered through a cell strainer and total cell number [number of isolated cells per ear: control: 7 × 105; tg: 8 × 105 (n = 8)] and viability (> 95%) were assessed. EC either were used for flow cytometry analyzes and antigen-processing assays or were cultured (1.5 × 106 cells per ml) in 75 cm2 flasks (Costar, Cambridge, MA) at 37°C. After 3 d, nonadherent EC were harvested and dead cells were largely eliminated by density gradient centrifugation (Lympholyte-M, Cedarlane Laboratories, Hornby, Ontario, Canada). The resulting cell suspension was either analyzed by flow cytometry or prepared for FACS-sorting experiments. Briefly, cultured EC were incubated with a FITC-labeled anti-DEC-205 MoAb and subsequently sorted with a FACS Vantage (Becton Dickinson, Mountain View, CA), yielding a highly viable (> 98%) suspension of >95% DEC-205+ cells. Keratinocytes from control and tg mice were isolated and cultured (1 × 106 cells per well) in 24-well plates as described (Carroll et al., 1995Carroll J.M. Romero M.R. Watt F.M. Suprabasal integrin expression in the epidermis of transgenic mice results in developmental defects and a phenotype resembling psoriasis.Cell. 1995; 83: 957-968Abstract Full Text PDF PubMed Scopus (277) Google Scholar). At selected time points, adherent and nonadherent keratinocytes were harvested from both cultures, and cell numbers and viability were assessed. EC suspensions of control and tg mice were cultured (2 × 106 cells per ml) in 24-well plates (Costar). At selected time points, supernatants were collected and stored at -20°C until use. IL-4 concentrations were determined by ELISA (Endogen, Woburn, MA) according to the manufacturer's instructions. Ear thickness of anesthesized control and tg mice (7–15 mice per group) was measured using an engineer's micrometer (Hahn und Kolb, Stuttgart, Germany). Alternatively, adult mice were killed and the ears and skin of the midback region were removed and cut in half. One half was embedded and snap frozen in liquid nitrogen, the other half was fixed in 4% neutral-buffered formalin, routinely processed and embedded either in paraffin or in Technovit 7100 (Kulzer, Wehrheim, Germany). Sections (3 µm) were stained using (i) Masson trichrome, for the identification of collagen fibers; (ii) 10% Giemsa solution (Merck, Darmstadt, Germany) at pH 5.5, for the identification of eosinophils and mast cells; (iii) Luna's Biebrich red for identification of eosinophils; or (iv) immunocytochemical techniques (e.g., Ki67), and were then examined by light microscopy. Immunohistochemical staining for Ki67 antigens was performed using the 3-Amino-9-Ethylcarbazole staining kit (Dako, Vienna, Austria) according to the manufacturer's recommendations. Ear and back skin sections were analyzed for the presence of mast cells using Olympus BX60 (Olympus, Austria, Vienna) and analySIS, an imaging and analysis system (Soft Imaging Systems, Munich, Germany), which provided direct morphometric measurements from digitized images obtained by a video camera. The entire section areas were analyzed and the total number of mast cells per mm2 of dermis was calculated. Epidermal sheets from ear and back skin were prepared using the ammonium-thiocyanate separation technique and either incubated with the FITC-labeled anti-MHC class II MoAb M5/114 for 60 min at 37°C or stained for ADPase activity (Elbe et al., 1989Elbe A. Tschachler E. Steiner G. et al.Maturational steps of bone marrow-derived dendritic murine epidermal cells. Phenotypic and functional studies on Langerhans cells and Thy-1+ dendritic epidermal cells in the perinatal period.J Immunol. 1989; 143: 2431-2438PubMed Google Scholar). Sheets were mounted on glass slides in PBS/glycerol (Difco, Detroit, MI), cover slipped, and viewed under a fluorescence microscope (Leitz Diaplan, Wetzlar, Germany). Observed staining patterns were documented with a Leitz Orthomate E system (Wetzlar) using 29 DIN artificial light color film (Scotch Chrome, 640-T, 3M, Milan, Italy). Labeled cells in the epidermal sheets were enumerated at 400× magnification using a rectangular grid. Eighty to a hundred fields were randomly chosen and the density of positive cells was determined and expressed as the number of cells (±SD) per mm2 of skin surface. Experimental groups consisted of four mice each. Transmission electron microscopy of single EC suspensions was performed as described (Berger et al., 1992Berger R. Gartner S. Rappersberger K. et al.Isolation of human immunodeficiency virus type 1 from human epidermis: virus replication and transmission studies.J Invest Dermatol. 1992; 99: 271-277Crossref PubMed Scopus (33) Google Scholar). Ears from tg mice and controls were dissected, rinsed with 70% ethanol, air-dried, and split with forceps into dorsal (i.e., cartilage-free) and ventral halves. In some experiments, skin was separated into dermis and epidermis by means of dispase before the onset of culture (Kitano and Okada, 1983Kitano Y. Okada N. Separation of the epidermal sheet by dispase.Br J Dermatol. 1983; 108: 555-560Crossref PubMed Scopus (149) Google Scholar;Lenz et al., 1993Lenz A. Heine M. Schuler G. et al.Human and murine dermis contain dendritic cells. Isolation by means of a novel method and phenotypical and functional characterization.J Clin Invest. 1993; 92: 2587-2596Crossref PubMed Scopus (252) Google Scholar). Dorsal ear halves were floated dermal side down on 2 ml of culture medium in 24 well tissue culture plates (Costar) for 3–7 d, one ear half per well (Larsen et al., 1990Larsen C.P. Steinman R.M. Witmer-Pack M. et al.Migration and maturation of Langerhans cells in skin transplants and explants.J Exp Med. 1990; 172: 1483-1493Crossref PubMed Scopus (569) Google Scholar;Ortner et al., 1996Ortner U. Inaba K. Koch F. et al.An improved isolation method for murine migratory cutaneous dendritic cells.J Immunol Meth. 1996; 193: 71-79Crossref PubMed Scopus (55) Google Scholar). Depending on the duration of the culture period, they were fed every 3 d by carefully aspirating 500 µl of spent medium and adding back the same volume of fresh medium. Non-adherent migratory cells were recovered from the bottom of tissue culture wells after 3, 5, and 7 d by gentle rinsing and total cell numbers and viability were determined by counting in a hematocytometer in the presence of trypan blue to assess cell viability. Five × 103 viable cells were placed on each reaction field of an adhesion slide (Bio-Rad, Richmond, CA) and incubated in a humidified chamber for 30 min at room temperature to allow their sedimentation. Anchoring of viable cells to the positively charged reaction field was monitored under an inverted microscope and nonattached cells were rinsed off with PBS. Attached cells were fixed with acetone for 10 min at room temperature and consecutively reacted with biotinylated anti-MHC class II-Streptavidin Texas Red/FITC-CD45 MoAb or isotype-matched control MoAb, washed, and embedded in Glycergel (Dakopatts). Data represent the mean ± SD of DC per well over four wells. For two-color analyzes, cells (3 × 105 per sample) were resuspended in cold PBS/1%FCS/0.1%NaN3 and serially incubated with FITC-conjugated MoAb directed against selected mouse antigens and biotinylated anti-CD45 MoAb followed by SAv-PE. For the detection of E-cadherin molecules, EC were prepared as described (Tang et al., 1993Tang A. Amagai M. Granger L.G. et al.Adhesion of epidermal Langerhans cells to keratinocytes mediated by E-cadherin.Nature. 1993; 361: 82-85Crossref PubMed Scopus (406) Google Scholar). Specificity of staining was confirmed using isotype-matched control MoAb. Fluorescence was measured using a FACScan flow cytometer, and data were analyzed with Cell Quest software (both from Becton Dickinson). Dead cells were excluded by 7-aminoactinomycin D (Sigma) uptake. T cells were prepared from mesenteric lymph nodes of BALB/c mice using Ab- and C-mediated lysis as described (Elbe et al., 1994Elbe A. Schleischitz S. Strunk D. et al.Fetal skin-derived MHC class I+, MHC class II– dendritic cells stimulate MHC class I-restricted responses of unprimed CD8+ T cells.J Immunol. 1994; 153: 2878-2889PubMed Google Scholar). Briefly, cell suspensions were passed through nylon wool columns, and nonadherent cells were treated with a cocktail of the following MoAb: 3C7, 7D4, J11d.2, M5/114, IM7, RA3–3A1 for 30 min at 4°C. Subsequently, cells were incubated with Low-tox-M rabbit C′ (Cedarlane) for 45 min at 37°C; 97%-99% of these cells were CD3+ as determined by flow cytometry. To purify CD4+ or CD8+ T cells, MoAb 3.168.81 or GK1.5 plus 2B6.2D8 were added to the Ab cocktail. T cells were > 97% CD44low, CD45RBhigh, CD62Lhigh as determined by flow cytometry (data not shown). Control and IL-4 tg EC were irradiated (X-ray, 15 Gray; 1.5 Gray per min, Philips RT 305, Philips, Vienna, Austria) and cultured in 96 well round-bottom microtiter plates (5 × 104 per well). After 3 d, purified lymph node T cells (2 × 105 per well) were added to the cultures. In other experiments, cultured, FACS-sorted Langerhans cells were incubated with either allogeneic CD4+ (>98%) or CD8+ (>99%) lymph node T cells, or alone in 96 well round bottom culture plates (Costar) at 37°C. At the indicated time points, 37 KBq (3H)-TdR was added to each well for 10–12 h. Thereafter, cells were harvested and (3H)-TdR incorporation was measured in a liquid scintillation counter (Packard Instruments, Meriden, CT). Data are expressed as mean cpm ± SD of triplicate unless indicated otherwise. The processing activity of control and tg Langerhans cells (H-2b/k) was measured using MHC class II-restricted T cell hybridomas (C10.9, HEL-specific, I-Ak-restricted, E8, OVA-specific, I-Ek-restricted). The activation of hybridoma cells was determined by measuring IL-2 production by ELISA (Endogen). Briefly, freshly prepared control and tg EC were treated with anti-Thy-1.2 MoAb for 30 min at 4°C, followed by Low-Tox-M-rabbit C′ for 40 min at 37°C to deplete some keratinocytes and epidermal T cells. After removal of dead cells by density gradient centrifugation, resulting cells (1.5 × 106 cells per ml) were cultured either with or without HEL (1 mg per ml) and OVA (2 mg per ml) for 20 h in 24 well plates. Thereafter, nonadherent cells were adjusted to equal numbers of Langerhans cells as determined by flow cytometry. Antigen-pulsed and, for control purposes, unpulsed control and tg Langerhans cells (5 × 103 per well) were cultured either with hybridoma cells (105 per well) or alone in 96 well flat-bottom microtiter plates. Supernatants were harvested after 24 h and stored at -20°C until use. Two-tailed t test was used to evaluate the significance of experimental versus control groups. To test whether functional protein is produced in tg epidermis, we examined the supernatants of EC cultures for the presence of IL-4 by ELISA. As shown in Figure 1, supernatants from cultures derived from tg mice contained at all time points elevated amounts of IL-4 relative to cultures derived from control mice. Tg mouse skin appeared dry with signs of scaling but exhibited no macroscopic signs of skin disease. Light microscopic examination of ear sections (Figures 2a, b) and ear measurements (Table I) revealed a significantly thickened skin in tg mice compared with controls. This was partly due to acanthosis and hyperkeratosis and partly to deposition of collagenous material in the superficial and deep dermis as demonstrated by trichrome staining (Figures 2a, b). Giemsa-stained sections revealed an increase in mast cell numbers in the ear and back skin dermis of tg mice by a factor of approximately two (Figures 2c, d,Table II). In contrast to control mice, considerable numbers of mast cells in the tg skin were located between fat cells and in the muscle layer. The observation that the tg dermis had less and smaller sized adipocytes than control dermis was striking (Figures 3a, b). In some tg mice the dermal fat tissue disappeared completely (Figure 3c). (Immuno)histochemistry of the tg dermis showed no evidence of acute inflammation (no eosinophil, T cell, macrophage and neutrophil infiltration) (data not shown). Two approaches were chosen to study the hyperplastic epidermal morphogenesis. First, proliferating keratinocytes were identified by immunohistochemistry. Although Ki-67+ nuclei were observed in the basal skin layer of both control and tg mice (Figures 4a, b), the proportion of Ki-67+ cells was greater in tg mice, indicating accelerated proliferation of epidermal keratinocytes. Scattered Ki-67+ cells were also identified in the suprabasal layers of the tg epidermis (data not shown). In a second series of experiments, we compared the yields of viable cells in keratinocyte cultures from control and tg mice at selected time points. Over the entire observation period of 10 d, we found a 2–3-fold increase of keratinocyte cell numbers in cultures isolated from tg mice (Figure 4c). Thus, we conclude that IL-4 causes keratinocyte hyperproliferation, induces mast cell accumulation and dermal collagen deposition, and leads to a loss of fat tissue in the dermis.Table IEar thickness in IL-4 tg miceEar thickness (×10-2 mm) ± SDap < 0.05Age (wk)No. of miceControlIL-4 tg%vs. control91125.3 ± 2.429.2 ± 4.7*p < 0.05159825.8 ± 1.530.4 ± 2.3***p < 0.001.1891525.4 ± 1.328.1 ± 3.8**p < 0.0111101524.3 ± 1.229.7 ± 3***p < 0.001.22101526 ± 1.529.1 ± 2***p < 0.001.1210726.1 ± 1.732.4 ± 2.6***p < 0.001.24a p < 0.05* p < 0.05** p < 0.01*** p < 0.001. Open table in a new tab
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