A Mannose-Binding Receptor is Expressed on Human Keratinocytes and Mediates Killing of Candida albicans
2001; Elsevier BV; Volume: 117; Issue: 2 Linguagem: Inglês
10.1046/j.1523-1747.2001.14071.x
ISSN1523-1747
AutoresGyõzõ Szolnoky, Zsuzsanna Bata‐Csörgő, Anna Sz. Kenderessy, Mária Kiss, Andor Pivarcsi, Zoltán Novàk, Katalin Nagy, Günther Michel, T. Ruzicka, László Maródi, A. Dobozy, Lajos Kemény,
Tópico(s)Nicotinic Acetylcholine Receptors Study
ResumoHuman keratinocytes are known to kill Candida albicans in vitro, but the mechanism of killing is not yet understood. Here, we demonstrate that spontaneous, ultraviolet-B-light-induced, α-melanocyte-stimulating-hormone-induced, and interleukin-8-induced Candida killing by keratinocytes can be inhibited with mannan and mannosylated bovine serum albumin (Man-BSA). A polyclonal goat serum raised against the human macrophage mannose receptor stained suprabasal keratinocytes, but no staining was observed on keratinocytes with a monoclonal antibody (mAb15) specific for the human macrophage mannose receptor. Mannose-affinity chromatography of keratinocyte extract isolated a 200 kDa protein, and on the Western blot the goat antiserum reacted with a 200 kDa protein. In radioligand binding studies, the binding of 125I-Man-BSA to human keratinocytes was inhibited by mannan in a concentration-dependent manner. Analysis of the binding revealed a single class keratinocyte mannose receptor with a KD of 1.4 × 10-8 M and a Bmax of 1 × 104 binding sites per cell. The binding of 125I-Man-BSA to keratinocytes proved to be time-dependent, acid-precipitable, and Ca2+- and trypsin-sensitive. After trypsinization the receptors underwent a rapid recovery at 37°C. These results demonstrate the presence of mannose receptor on human keratinocytes, and its active involvement in the killing of Candida albicans. Human keratinocytes are known to kill Candida albicans in vitro, but the mechanism of killing is not yet understood. Here, we demonstrate that spontaneous, ultraviolet-B-light-induced, α-melanocyte-stimulating-hormone-induced, and interleukin-8-induced Candida killing by keratinocytes can be inhibited with mannan and mannosylated bovine serum albumin (Man-BSA). A polyclonal goat serum raised against the human macrophage mannose receptor stained suprabasal keratinocytes, but no staining was observed on keratinocytes with a monoclonal antibody (mAb15) specific for the human macrophage mannose receptor. Mannose-affinity chromatography of keratinocyte extract isolated a 200 kDa protein, and on the Western blot the goat antiserum reacted with a 200 kDa protein. In radioligand binding studies, the binding of 125I-Man-BSA to human keratinocytes was inhibited by mannan in a concentration-dependent manner. Analysis of the binding revealed a single class keratinocyte mannose receptor with a KD of 1.4 × 10-8 M and a Bmax of 1 × 104 binding sites per cell. The binding of 125I-Man-BSA to keratinocytes proved to be time-dependent, acid-precipitable, and Ca2+- and trypsin-sensitive. After trypsinization the receptors underwent a rapid recovery at 37°C. These results demonstrate the presence of mannose receptor on human keratinocytes, and its active involvement in the killing of Candida albicans. galactose bovine serum albumin keratinocyte mannose-binding receptor macrophage mannose receptor mannosylated bovine serum albumin α-melanocyte stimulating hormone In both experimental and naturally occurring cutaneous Candida albicans infections, the infecting Candida albicans organisms are confined to the upper portion of the epidermis (Kirkpatrick et al., 1971Kirkpatrick C.H. Rich R.R. Bennett J.E. Chronic mucocutaneous candidiasis: model-building in cellular immunity.Ann Intern Med. 1971; 74: 955-978Crossref PubMed Scopus (245) Google Scholar;Scherwitz, 1982Scherwitz C. Ultrastructure of human cutaneous candidosis.J Invest Dermatol. 1982; 78: 200-205Crossref PubMed Scopus (48) Google Scholar;Sohnle and Hahn, 1992Sohnle P.G. Hahn B.L. The fate of individual organisms during clearance of experimental cutaneous Candida albicans infections in mice.Acta Derm Venereol. 1992; 72: 241-244PubMed Google Scholar). Deep or systemic Candida infections are rare in patients with chronic mucocutaneous candidiasis, despite extensive superficial infections (Ray and Wuepper, 1978Ray T.L. Wuepper K.D. Experimental cutaneous candidiasis in rodents. II. Role of the stratum corneum barrier and serum complement as a mediator of a protective inflammatory response.Arch Dermatol. 1978; 114: 539-543Crossref PubMed Scopus (42) Google Scholar;Kauffman et al., 1981Kauffman C.A. Shea M.J. Frame P.T. Invasive fungal infections in patients with chronic mucocutaneous candidiasis.Arch Intern Med. 1981; 141: 1076-1079Crossref PubMed Scopus (22) Google Scholar). The formation of neutrophilic infiltrates in the epidermis and epidermal hyperproliferation are characteristic cutaneous changes in Candida albicans infections of the skin. Each of these cutaneous responses has been suggested to be important for the defense against superficial fungal infections (Sohnle and Kirkpatrick, 1978Sohnle P.G. Kirkpatrick C.H. Epidermal proliferation in the defense against experimental cutaneous candidiasis.J Invest Dermatol. 1978; 70: 130-133Crossref PubMed Scopus (43) Google Scholar;Sohnle and Hahn, 1989Sohnle P.G. Hahn B.L. Epidermal proliferation and the neutrophilic infiltrates of experimental cutaneous candidiasis in mice.Arch Dermatol Res. 1989; 281: 279-283Crossref PubMed Scopus (16) Google Scholar). Although polymorphonuclear (PMN) leukocytes easily phagocytize Candida albicans in vitro, ultrastructural examination of Candida-infected skin failed to show fungal elements in regions of microabscesses (Scherwitz, 1982Scherwitz C. Ultrastructure of human cutaneous candidosis.J Invest Dermatol. 1982; 78: 200-205Crossref PubMed Scopus (48) Google Scholar). On the other hand, in that same study, the majority of the fungal cells were found among epithelial cells of the stratum corneum and they could not be detected in noncornified cells of the malphigian layer. In a more recent investigation using cyclophosphamide-treated mice it was shown that even in the absence of epidermal neutrophil infiltrates Candida albicans is confined to the epidermis. Removal of the epidermis by scraping prior to inoculation with the yeast, however, resulted in Candida pseudohyphae invasion of the dermis, indicating that suprabasal keratinocytes may play a role in the defense against cutaneous Candida infection (Hahn and Sohnle, 1988Hahn B.L. Sohnle P.G. Characteristics of dermal invasion in experimental cutaneous candidiasis of leucopenic mice.J Invest Dermatol. 1988; 91: 233-237Abstract Full Text PDF PubMed Google Scholar). Vaginal epithelial cells of both rodents and macaques have recently been shown to be predominantly responsible for innate immunity against yeast cell colonization (Steele et al., 1999aSteele C. Ozenci H. Luo W. Scott M. Fidel Jr, P.L. Growth inhibition of Candida albicans by vaginal cells from naive mice.Med Mycol. 1999; 37: 251-259Crossref PubMed Google Scholar,Steele et al., 1999bSteele C. Ratterree M. Fidel Jr, P.L. Differential susceptibility of two species of macaques to experimental vaginal candidiasis.J Infect Dis. 1999; 180: 802-810Crossref PubMed Scopus (47) Google Scholar). In previous work we have shown that epidermal cells have direct candidacidal activity and this activity can be increased through stimulation of epidermal cells with ultraviolet (UV) light (Csato et al., 1986Csato M. Bozoky B. Hunyadi J. Dobozy A. Candida albicans phagocytosis by separated human epidermal cells.Arch Dermatol Res. 1986; 279: 136-139Crossref PubMed Scopus (29) Google Scholar,Csato et al., 1987Csato M. Kenderessy A.S. Dobozy A. Enhancement of Candida albicans killing activity of separated human epidermal cells by ultraviolet radiation.Br J Dermatol. 1987; 116: 469-475Crossref PubMed Scopus (10) Google Scholar), α-melanocyte stimulating hormone (α-MSH) (Csato et al., 1989Csato M. Kenderessy A.S. Dobozy A. Enhancement of Candida albicans killing activity of separated human epidermal cells by alpha-melanocyte stimulating hormone [letter].Br J Dermatol. 1989; 121: 145-147Crossref PubMed Scopus (8) Google Scholar), and interleukin-8 (IL-8). 1Kemeny L, Kenderessy AS, Arenberger P, Peter RU, Dobozy A, Ruzicka A: Interleukin-8 induced chemotaxis and increased Candida albicans killing activity of normal human epidermal cells. Arch Dermatol Res 284:26, 1992 (abstr.)1Kemeny L, Kenderessy AS, Arenberger P, Peter RU, Dobozy A, Ruzicka A: Interleukin-8 induced chemotaxis and increased Candida albicans killing activity of normal human epidermal cells. Arch Dermatol Res 284:26, 1992 (abstr.) IL-1, prostaglandin E2 (PGE2), and platelet-activating factor have also been demonstrated to be involved in Candida killing by human epidermal cells (Csato et al., 1990Csato M. Kenderessy A.S. Judak R. Dobozy A. Inflammatory mediators are involved in the Candida albicans killing activity of human epidermal cells.Arch Dermatol Res. 1990; 282: 348-350Crossref PubMed Scopus (12) Google Scholar), but the mechanism of killing remains unknown. Mononuclear phagocytes are believed to play an important role in combating fungal infections. Optimal phagocytosis of Candida albicans requires opsonization, but unopsonized yeast can also be internalized by macrophages through the mannose receptor (Karbassi et al., 1987Karbassi A. Becker J.M. Foster J.S. Moore R.N. Enhanced killing of Candida albicans by murine macrophages treated with macrophage colony-stimulating factor: evidence for augmented expression of mannose receptors.J Immunol. 1987; 139: 417-421PubMed Google Scholar). The major components of the cell wall of Candida are mannan (an α-linked polymer of mannose), glucan (a β-linked branched chain polysaccharide of glucose), and chitin (a cellulose-like biopolymer consisting predominantly of N-acetyl-D-glucosamine) (Cassone, 1989Cassone A. Cell wall of Candida albicans: its functions and its impact on the host.Curr Top Med Mycol. 1989; 3: 248-314Crossref PubMed Scopus (78) Google Scholar). The macrophage mannose receptor (MMR) recognizes carbohydrates expressed on the surfaces of microorganisms and is particularly well suited to direct particles to phagolysosomes and trigger a respiratory burst (Marodi et al., 1993Marodi L. Schreiber S. Anderson D.C. MacDermott R.P. Korchak H.M. Johnston Jr, R.B. Enhancement of macrophage candidacidal activity by interferon-gamma. Increased phagocytosis, killing, and calcium signal mediated by a decreased number of mannose receptors.J Clin Invest. 1993; 91: 2596-2601Crossref PubMed Scopus (143) Google Scholar). It has been shown that human keratinocytic cells from squamous carcinoma (SCL-1) and multiply passaged cultured keratinocytes from normal skin express sugar-binding proteins on their surface (Cerdan et al., 1991Cerdan D. Grillon C. Monsigny M. Redziniak G. Kieda C. Human keratinocyte membrane lectins: characterization and modulation of their expression by cytokines.Biol Cell. 1991; 73: 35-42Crossref PubMed Scopus (32) Google Scholar;Condaminet et al., 1997Condaminet B. Redziniak G. Monsigny M. Kieda C. Ultraviolet rays induced expression of lectins on the surface of a squamous carcinoma keratinocyte cell line.Exp Cell Res. 1997; 232: 216-224https://doi.org/10.1006/excr.1997.3518Crossref PubMed Scopus (22) Google Scholar). Human keratinocytes also possess the ability to synthesize and express cell surface moieties characteristic of effector and/or accessory cells of the immune system (Hunyadi et al., 1992Hunyadi J. Simon Jr, M. Dobozy A. Immune-associated surface markers of human keratinocytes.Immunol Lett. 1992; 31: 209-216Crossref PubMed Scopus (20) Google Scholar;Hunyadi et al., 1993Hunyadi J. Simon Jr, M. Kenderessy A.S. Dobozy A. Expression of monocyte/macrophage markers (CD13, CD14, CD68) on human keratinocytes in healthy and diseased skin.J Dermatol. 1993; 20: 341-345Crossref PubMed Scopus (22) Google Scholar). Here, we demonstrate the presence of mannose-binding receptors on normal human keratinocytes and provide data indicating their involvement in the killing of Candida. The keratinocyte mannose-binding receptor (KcMR), isolated by mannose-affinity chromatography, has a different molecular mass (200 kDa) from that of MMR (175 kDa). The fact that the KcMR differs from the MMR is also indicated by the absence of cross-reactivity to a monoclonal MMR antibody (mAb15). At the same time, a goat immune serum raised against the human MMR cross-reacts with KcMR, showing membrane staining of suprabasally localized cells on skin sections. The same goat antiserum also reacted with a band of 200 kDa on the Western blot of keratinocyte extracts. The KcMR resembles the MMR in that it is Ca2+-dependent, sensitive to proteolysis, and has a KD of 1.4 × 10-8 M and a Bmax of 104 per cell, but unlike the MMR it does not internalize mannose efficiently. Human epidermal cells were obtained from healthy individuals undergoing plastic surgery after they had given their informed consent in conformity with the requirements of the Institutional Review Board of the Albert Szent-Györgyi Medical Center, University of Szeged. After removal of the subcutaneous tissue and much of the reticular dermis, the tissue samples were cut into small strips and incubated with 0.25% trypsin (Sigma, Budapest, Hungary) overnight at 4°C. Subsequently, the epidermis was peeled off from the dermis. Dissociation of the epidermal layer into a single cell suspension was accomplished by gentle aspiration with a Pasteur pipette. The cell suspension was filtered through a 100 µm cell filter (BioDesign of New York, Carmel, NY). A suspension of single cells (106) was prepared in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Eggenstein, Germany) supplemented with 20% fetal bovine serum, 20 mM HEPES buffer, 100 U per ml penicillin, and 100 µg per ml streptomycin (each from Gibco). The viability of the cells was always >85%, as determined by trypan blue exclusion. The separated cells were characterized by immunohistochemistry on cytospin prepar ations (Cytospin, Shandon-Elliot, Frankfurt, Germany). Keratinocytes were labeled with the monoclonal antibody Lu 5 (Boehringer Mannheim Biochemicals, Mannheim, Germany), which reacts with an epitope common to all cytokeratins (1–19); Langerhans cells were labeled with anti-CD1a antibody (Boehringer); and endothelial cells were labeled with the monoclonal antibody BMA 120 (Behring Werke, Marburg, Germany), which reacts with a 200 kDa antigen that is different from the von Willebrand factor molecule but is also highly specific for endothelial cells (Alles and Bosslet, 1988Alles J.U. Bosslet K. Immunocytochemistry of angiosarcomas. A study of 19 cases with special emphasis on the applicability of endothelial cell specific markers to routinely prepared tissues.Am J Clin Pathol. 1988; 89: 463-471PubMed Google Scholar). Fibroblasts and melanocytes were identified with anti-vimentin antibody (Boehringer) (Arenberger et al., 1993Arenberger P. Kemeny L. Ruzicka T. Characterization of high-affinity 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) binding sites on normal human keratinocytes.Epithelial Cell Biol. 1993; 2: 1-6PubMed Google Scholar). Immunohistochemical analyses of the separated cells demonstrated that over 95% were keratinocytes; the rest were contaminating fibroblasts and melanocytes; no endothelial or Langerhans cells were detected. Candida albicans (0656 CBS Delft) was cultured on Sabouraud agar and transferred onto fresh agar 24 h prior to the killing assay. Candida cells were harvested with phosphate-buffered saline (PBS) and counted in a hemocytometer. Under these conditions sufficient numbers of Candida cells can be obtained without germ tubes and hyphae. Their viability was checked with trypan blue. For the adherence assay, Candida albicans cells were resuspended in PBS at a final concentration of 107 blastospore cells per ml and then fixed with 70% ethanol for 1 h at room temperature and washed twice in PBS. The Candida cells were next incubated with 0.1 mg per ml fluorescein isothiocyanate (FITC) (Sigma, Budapest) in a 0.5 M carbonate/bicarbonate buffer (pH 9.5) for 30 min on a moving plate in the dark and finally washed five times in PBS (Sarasella et al., 1997Sarasella M. Roda K. Speciale L. Taramelli D. Mendozzi E. Guerini F. Ferrante P. A rapid evaluation of phagocytosis and killing of Candida albicans by CD13+ leukocytes.J Immunol Meth. 1997; 210: 227-234Crossref PubMed Scopus (27) Google Scholar). Human PMN leukocytes were isolated from venous blood, freshly drawn from healthy donors after obtaining their informed consent in accordance with the requirements of the Institutional Review Board of the Medical and Health Science Center, University of Debrecen, by dextran sedimentation and centrifugation over Ficoll-Hypaque (Pharmacia, Uppsala, Sweden), as previously described (Arenberger et al., 1993Arenberger P. Kemeny L. Ruzicka T. Characterization of high-affinity 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) binding sites on normal human keratinocytes.Epithelial Cell Biol. 1993; 2: 1-6PubMed Google Scholar). Contaminating erythrocytes were lysed with hypotonic saline and the PMN leukocytes were then washed with PBS and counted under a microscope. A modified method originally introduced for blood leukocytes was employed to evaluate Candida killing by keratinocytes (Csato et al., 1987Csato M. Kenderessy A.S. Dobozy A. Enhancement of Candida albicans killing activity of separated human epidermal cells by ultraviolet radiation.Br J Dermatol. 1987; 116: 469-475Crossref PubMed Scopus (10) Google Scholar). Briefly, 106 human keratinocytes and 2 × 106 Candida cells, or Candida alone in DMEM, were incubated for 4 h at 37°C in an atmosphere of 5% CO2 in a volume of 1 ml. After the incubation period Triton X-100 (1%) (Sigma) was added to the mixture and to the Candida control (Candida alone) for 5 min at 4°C in order to lyse the keratinocytes. The Candida albicans cells were then stained with methylene blue (0.1%) for 45 min at 4°C and the killed, non-viable Candida cells were counted in a hemocytometer under the microscope. The number of Candida killed by the keratinocytes was calculated by subtracting the number of dead Candida cells in the control from the number of dead Candida cells in the reaction mixture. The percentage of non-viable Candida cells was defined as the killing activity. To ascertain the accuracy of our methylene blue assay we also determined the colony forming units (Marodi et al., 1991bMarodi L. Korchak H.M. Johnston Jr, R.B. Mechanisms of host defense against Candida species. I. Phagocytosis by monocytes and monocyte-derived macrophages.J Immunol. 1991; 146: 2783-2789PubMed Google Scholar;Sawyer et al., 1992Sawyer R.T. Garner R.E. Hudson J.A. Effect of lectins on hepatic clearance and killing of Candida albicans by the isolated perfused mouse liver.Infect Immun. 1992; 60: 1041-1046PubMed Google Scholar) in parallel with the Candida albicans cell counts in a separate experiment. A strong correlation was found between the results of the two assays: r = 0.984, p < 0.05. In several experiments we used human PMN leukocytes to measure their Candida killing activity. For the killing assay of PMN leukocytes, Candida yeasts were opsonized with 2.5% normal human serum for 30 min prior to each assay and then washed with PBS. 106 leukocytes were incubated with 2 × 106 opsonized Candida cells, or Candida alone in DMEM was incubated for 30 min at 37°C in an atmosphere of 5% CO2 in a volume of 1 ml. After the incubation period, Triton X-100 (1%) (Sigma) was added to the mixture and to the Candida control (Candida alone) for 5 min at 4°C in order to lyse the granulocytes. The Candida albicans cells were then stained with methylene blue (0.1%) for 45 min at 4°C and the killed, non-viable Candida cells were counted in a hemocytometer under the microscope. The number of Candida cells killed by the PMN leukocytes was calculated by subtracting the number of dead Candida cells in the control from the number of dead Candida cells in the reaction mixture. The percentage of non-viable Candida cells was defined as the killing activity. Keratinocytes in suspension were exposed to UV radiation up to 220 mJ per cm2 using a Saalman-SUP lamp (Gerhard Saalman, Herford, Germany) with a spectrum of 290–330 nm without any specific filtration subsequent to a 20 min warm-up period. Spectroradiometry was employed to determine the spectral output before each experiment. The viability of the cells after UV irradiation never dropped below 80%. After UV exposure, keratinocytes were incubated in DMEM at 37°C at 5% CO2 for 3 h before use in the above-described Candida killing assay. In other experiments keratinocytes were stimulated for 12 h with 2 × 10-8 M recombinant human IL-8 (Hermann Biermann, Bad Nauheim, Germany) or with 20 µg per ml α-MSH (Sigma) for 1 h (37°C, 5% CO2) prior to the killing assay. 100 or 500 µg per ml mannan (Sigma, >99% pure) or mannosylated-BSA (Man-BSA) (E/Y Labs, San Mateo, CA) or 300 µg per ml galactose-BSA (Gal-BSA) (Sigma) dissolved in DMEM was added to a mixture of human keratinocytes and Candida cells to investigate the inhibition of killing activity. To probe the hypothesis that Man-BSA may interfere with the killing of Candida by cells that do not express mannose-binding receptors, the killing of Candida albicans by PMN leukocytes was studied in the presence or absence of 1 mg per ml Man-BSA. To examine a possible non-specific protective effect of mannan on Candida albicans, making it more resistant to chemically induced cell death, 5 × 105 yeast cells were pretreated with 3 mg per ml mannan for 120 min prior to incubation with the fungicide ciclopirox olamine (Hoechst, Budapest, Hungary) in a concentration of 2.5 or 5 mg per ml for 120 min in a final volume of 1 ml PBS. Dead, non-viable Candida cells were determined as described above. In the adherence assay, 5 × 105 freshly separated human keratinocytes were mixed with 2 × 106 FITC-conjugated Candida cells in a final volume of 1 ml PBS and incubated for 120 min on a rotator at 37°C. The cells were then examined by fluorescence microscopy (Opton, Germany) and by flow cytometric analysis using a FACStar Plus Flow Cytometer (Becton Dickinson). Data were collected by using a linear amplifier for side scatter (SSC) and logarithmic amplifiers for forward scatter (FSC) and fluorescence intensity (FL1). FSC/SSC proved to be efficient in discriminating between human keratinocytes and Candida albicans cells, due to the substantial differences in structure and size. To determine the extent of adherence, non-adherent yeasts were gated out and the green fluorescence of the keratinocytes was measured by using the FL1 channel. This allows a distinction of Candida-binding (FL1-positive) and non-binding FL1-negative keratinocytes. Flow cytometric data (an average of 5 × 103 events) were analyzed by the Cell Quest 3.1 F software (Becton Dickinson). To study the effect of mannan on the ability of keratinocytes to bind Candida cells, keratinocytes were incubated with 3 mg per ml mannan dissolved in PBS for 60 min before the adherence assay. Skin biopsies were taken from disposed tissues of healthy individuals undergoing plastic surgery operations. The samples were embedded in cryomatrix (Shandon, Life Sciences International, U.K.), 7 µm sections were cut and fixed in acetone (10 min, 4°C), and the sections were stained with a monoclonal antibody (mAb15) against the human MMR (mouse IgG1) and with the goat antiserum against the purified human MMR (both kindly provided by Philip D. Stahl, Washington University, St. Louis, MO). The incubation step with the primary antibodies (1:50 dilutions) and the appropriate controls (mouse IgG1, preimmune goat serum) for 60 min at room temperature was followed with incubation of biotin-conjugated secondary antibodies, and then streptavidin-biotin peroxidase, using the DAKO Strept ABC complex kit and its protocol (DAKO, Denmark). 3-Amino-9-ethylcarbazole (AEC, Sigma) was used as a peroxidase substrate. 2.5 ml AEC (4 mg AEC dissolved in 1 ml N, N-dimethylformamide) was added to 17 ml 0.1 M acetate buffer (pH 5.2) and 10 µl 30% H2O2. The sections were counterstained with hematoxylin. Freshly separated human keratinocytes were collected from healthy individuals undergoing plastic surgery operations as described above. 2.4 × 108 keratinocytes were homogenized with a handheld Polytron homogenizer (Brinkmann Instruments, Westbury, NY) in loading buffer containing 0.5% Triton X-100, 10 mM Tris-HCl (pH 7.4), 1.25 M NaCl, 15 mM CaCl2, and 0.1 mM phenyl methylsulfonyl fluoride (PMSF) (all from Sigma). Debris was removed by centrifugation (12,000g, 35 min). The receptor was bound to a mannose-coupled epoxy-activated mannose-Sepharose 6B column (Pharmacia Biotech, Uppsala, Sweden) in loading buffer and subsequently eluted with the same buffer containing 0.1 M mannose. The partially purified mannose-binding protein was subjected to electrophoresis in 7.5% polyacrylamide gel (PAGE) in sodium dodecyl sulfate (SDS) under non-reducing conditions. Protein was visualized with silver staining (Sammons et al., 1981Sammons D.W. Adams L.D. Nishizawa E.E. Ultrasensitive silver-based color staining of polypeptides in polyacrylamide gels.Electrophoresis. 1981; 2: 135Crossref Scopus (462) Google Scholar). Protein extracts from freshly separated human keratinocytes and the HaCaT human keratinocyte cell line (Boukamp et al., 1988Boukamp P. Petrussevska R.T. Breitkreutz D. Hornung J. Markham A. Fusenig N.E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.J Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3278) Google Scholar) were prepared in a lysis buffer of 1.5% SDS, 62.5 mM Tris-HCl pH 6.8, 5 mM ethylenediamine tetraacetic acid, 5% 2-mercapto ethanol, 1 µg per ml PMSF, 1 µg per ml antipain, 1 µg per ml chymostatin, and 1 µg per ml leupeptin (all from Sigma). Lysates were centrifuged and supernatants were stored at -20°C. The extracted proteins of keratinocytes were separated by SDS-PAGE (with 9% separating gel) and then transferred to nitrocellulose membrane (Protran BA 83). Blots were blocked in Tris-buffered saline (150 mM NaCl, 25 mM Tris-HCl pH 7.4) containing 0.05% Tween-20 and 3% non-fat dry milk (all from Sigma). Blots were incubated overnight at 4°C with a 1:200 dilution of goat antiserum for the human MMR. Alkaline phosphatase conjugated mouse anti-goat immunoglobulin (Sigma) was used as secondary antibody at a 1:4500 dilution for 2 h at room temperature. Blots were developed by using 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium substrate (Sigma). Man-BSA was trace-labeled with 125I by the chloramine T method (Salacinski et al., 1981Salacinski P.R. McLean C. Sykes J.E. Clement-Jones V.V. Lowry P.J. Iodination of proteins, glycoproteins, and peptides using a solid-phase oxidizing agent, 1,3,4,6-tetrachloro-3α,6α-diphenyl glycoluril (Iodogen).Anal Biochem. 1981; 117: 136-146Crossref PubMed Scopus (1009) Google Scholar). 5 × 105 keratinocytes were incubated with increasing concentrations of 125I-Man-BSA (0.25–5 µg per ml, 1 × 106 cpm per µg) in DMEM containing 1% bovine serum albumin (BSA), 10 mM HEPES, and 1.2 mM CaCl2 in a final volume of 400 µl. The incubation lasted for 90 min at 4°C under continuous shaking, and was terminated by washing the cells twice with ice-cold PBS. Cell-associated radioactivity was detected in a Packard gamma-spectrometer (Packard Instrument, Downers Grove, IL). Mannan-independent binding was determined in the presence of 3 mg per ml unlabeled mannan. The mannan-specific binding was defined as the difference between the total and the mannan-independent 125I-Man-BSA binding (Sato and Beutler, 1993Sato Y. Beutler E. Binding internalization, and degradation of mannose-terminated glucocerebrosidase by macrophages.J Clin Invest. 1993; 91: 1909-1917Crossref PubMed Scopus (58) Google Scholar). Each assay was carried out in duplicate in three separate experiments. For determination of the dissociation constant (KD) and the number of binding sites per cell (Bmax), saturation curves were analyzed with the computerized nonlinear curve-fitting program MxN-FIT (Bürgisser, 1988Bürgisser E.J. MxN-FIT.An IBM-PC program for the analysis of complex binding data according to the law of mass action. Buerco AG, Rheienfeld, Germany1988Google Scholar). In competition studies the cells were incubated with 0.15 µg per ml 125I-Man-BSA and with increasing concentrations of unlabeled ligand in the range 10–3000 µg per ml. Competition studies were performed under the same conditions as the saturation assays. Man-BSA was again trace-labeled with 125I by the chloramine T method. To determine the kinetics of internalization of 125I-Man-BSA, a modified radioligand binding study was performed at 37°C (Ezekowitz et al., 1981Ezekowitz R.A. Austyn J. Stahl P.D. Gordon S. Surface properties of bacillus Calmette-Guerin-activated mouse macrophages. Reduced expression of mannose-specific endocytosis, Fc receptors, and antigen F4/80 accompanies induction of Ia.J Exp Med. 1981; 154: 60-76Crossref PubMed Scopus (150) Google Scholar). Briefly, keratinocytes (5 × 105 per well) were incubated with 10 µg (1.0 × 106 cpm per µg) 125I-Man-BSA in the presence or absence of 3 mg per ml mannan in a final volume of 400 µl. After different time intervals (10, 20, and 30 min), the binding was stopped by washing the cells four times with ice-cold PBS, and the cell-bound radioactivity was measured with a gamma-counter. The mannan-specific binding was defined as the total binding minus the mannan-independent binding. To examine how much of the cell-bound radioactivity was internalized, cells were treated for 3 min with acetic acid (pH 3) and then washed twice with ice-cold PBS. The acid-stable radioactivity represents internalized ligand, whereas the acid-labile radioactivity is equivalent to surface-bound ligand (Arenberger et al., 1993Arenberger P. Kemeny L. Ruzicka T. Characterization of high-affinity 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) binding sites on normal human keratinocytes.Epithelial Cell Biol. 1
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