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Interleukin-16 Supports the Migration of Langerhans Cells, Partly in a CD4-Independent Way

2001; Elsevier BV; Volume: 116; Issue: 5 Linguagem: Inglês

10.1046/j.1523-1747.2001.01328.x

1523-1747

Patrizia Stoitzner, Gudrun Ratzinger, Franz Koch, Katrin Janke, Peter Fritsch, Nikolaus Romani, Thomas Schöller, Arthur Kaser, Herbert Tilg, William W. Cruikshank,

Immune Cell Function and Interaction

Migration of cutaneous dendritic cells is essential for the induction of primary immune responses. Chemotaxis plays an important part in guiding migrating cells through the skin. Therefore, we investigated the influence of interleukin-16, a potent chemoattractant, on the migratory properties of cutaneous dendritic cells. Interleukin-16 added to murine and human skin explant cultures, enhanced emigration of Langerhans cells as well as dermal dendritic cells out of the skin. In contrast to tumor necrosis factor-α, intradermally injected interleukin-16 did not reduce the density of Langerhans cells suggesting a chemotactic rather than a mechanistic migration-inducing effect of interleukin-16. In support of these findings, the known migration-promoting effect of tumor necrosis factor-α in skin explant cultures could be neutralized by anti-interleukin-16 antibody and vice versa, indicating different but cooperative ways of action for both cytokines. In whole skin explant cultures blocking of the interleukin-16 effect was also achieved with a monoclonal antibody against CD4, the receptor for interleukin-16. In contrast, in cultures of murine epidermis alone no blocking by anti-CD4 became obvious and in CD4-deficient mice Langerhans cell migration in response to interleukin-16 was maintained. This suggests that another receptor for interleukin-16 might be operative for Langerhans cells in the mouse epidermis. Finally, we detected interleukin-16-positive cells in the dermis of skin explants, tumor necrosis factor-α-treated and contact allergen-treated skin. Taken together, it seems likely that locally secreted interleukin-16 might serve to enhance the migration of cutaneous dendritic cells and optimize the response to foreign antigen encountering the skin. Migration of cutaneous dendritic cells is essential for the induction of primary immune responses. Chemotaxis plays an important part in guiding migrating cells through the skin. Therefore, we investigated the influence of interleukin-16, a potent chemoattractant, on the migratory properties of cutaneous dendritic cells. Interleukin-16 added to murine and human skin explant cultures, enhanced emigration of Langerhans cells as well as dermal dendritic cells out of the skin. In contrast to tumor necrosis factor-α, intradermally injected interleukin-16 did not reduce the density of Langerhans cells suggesting a chemotactic rather than a mechanistic migration-inducing effect of interleukin-16. In support of these findings, the known migration-promoting effect of tumor necrosis factor-α in skin explant cultures could be neutralized by anti-interleukin-16 antibody and vice versa, indicating different but cooperative ways of action for both cytokines. In whole skin explant cultures blocking of the interleukin-16 effect was also achieved with a monoclonal antibody against CD4, the receptor for interleukin-16. In contrast, in cultures of murine epidermis alone no blocking by anti-CD4 became obvious and in CD4-deficient mice Langerhans cell migration in response to interleukin-16 was maintained. This suggests that another receptor for interleukin-16 might be operative for Langerhans cells in the mouse epidermis. Finally, we detected interleukin-16-positive cells in the dermis of skin explants, tumor necrosis factor-α-treated and contact allergen-treated skin. Taken together, it seems likely that locally secreted interleukin-16 might serve to enhance the migration of cutaneous dendritic cells and optimize the response to foreign antigen encountering the skin. dendritic cell macrophage inflammatory protein-3β secondary lymphoid tissue chemokine lymphocyte activation antigen-3 As skin is the first place to encounter antigen it needs a well-functioning immune defense to act immediately against penetrating pathogenic agents. Dendritic cells (DC) of the skin (Langerhans cells and dermal DC), as sentinels in the peripheral tissues, have the capacity to take up and process cutaneously applied antigen and present immunogenic peptides on their cell surface. As professional antigen-presenting cells they are able to migrate from peripheral sites to the draining lymph nodes where they efficiently stimulate resting or memory antigen-specific T cells (Schuler et al., 1997Schuler G. Thurner B. Romani N. Dendritic cells: From ignored cells to major players in T-cell-mediated immunity.Int Arch Allergy Immunol. 1997; 112: 317-322Crossref PubMed Scopus (56) Google Scholar;Banchereau and Steinman, 1998Banchereau J. Steinman R.M. Dendritic cells and the control of immunity.Nature. 1998; 392: 245-252https://doi.org/10.1038/32588Crossref PubMed Scopus (11826) Google Scholar;Romani et al., 2001Romani N. Ratzinger G. Pfaller K. Salvenmoser W. Stössel H. Koch F. Stoitzner P. Migration of dendritic cells into lymphatics—the Langerhans cells example. Routes, regulation, relevance.Int Rev Cytol. 2001; 207 (in press)PubMed Google Scholar). The most commonly used system to study DC migration from skin to lymph nodes is the murine contact hypersensitivity model. Upon application of contact sensitizers, major histocompatibility complex (MHC) class II positive cells migrate out of the skin into regional lymph nodes (Botham et al., 1987Botham P.A. Rattray N.J. Walsh S.T. Riley E.J. Control of the immune response to contact sensitizing chemicals by cutaneous antigen-presenting cells.Br J Dermatol. 1987; 117: 1-9Crossref PubMed Scopus (28) Google Scholar;Macatonia et al., 1987Macatonia S.E. Knight S.C. Edwards A.J. Griffiths S. Fryer P. Localization of antigen on lymph node dendritic cells after exposure to the contact sensitizer fluorescein isothiocyanate.J Exp Med. 1987; 166: 1654-1667Crossref PubMed Scopus (510) Google Scholar). A proportion of those contain Birbeck granules and seem to be Langerhans cells. Emigrated cells can sensitize naive T cells in syngeneic recipients (Kripke et al., 1990Kripke M.L. Munn C.G. Jeevan A. Tang J.-M. Bucana C. Evidence that cutaneous antigen-presenting cells migrate to regional lymph nodes during contact sensitization.J Immunol. 1990; 145: 2833-2838PubMed Google Scholar).Larsen et al., 1990Larsen C.P. Steinman R.M. Witmer-Pack M. Hankins D.F. Morris P.J. Austyn J.M. Migration and maturation of Langerhans cells in skin transplants and explants.J Exp Med. 1990; 172: 1483-1493Crossref PubMed Scopus (569) Google Scholar and our group (Ortner et al., 1996Ortner U. Inaba K. Koch F. Heine M. Miwa M. Schuler G. Romani N. An improved isolation method for murine migratory cutaneous dendritic cells.J Immunol Methods. 1996; 193: 71-79https://doi.org/10.1016/0022-1759(96)00058-0Crossref PubMed Scopus (55) Google Scholar) have established and refined a skin explant culture that is a useful tool to define further the migratory pathways of cutaneous DC. At the onset of the cultures Langerhans cells and dermal DC start to migrate. In the epidermis the Langerhans cells downregulate E-cadherin expression and loosen their connections to the surrounding keratinocytes (Tang et al., 1993Tang A. Amagai M. Granger L.G. Stanley J.R. Udey M.C. Adhesion of epidermal Langerhans cells to keratinocytes mediated by E-cadherin.Nature. 1993; 361: 82-85Crossref PubMed Scopus (406) Google Scholar). After passage of the basement membrane the emigrating DC invade the lymphatic vessels in the dermis and end up in the culture medium (Lukas et al., 1996Lukas M. Stössel H. Hefel L. et al.Human cutaneous dendritic cells migrate through dermal lymphatic vessel culture model.J Invest Dermatol. 1996; 106: 1293-1299Crossref PubMed Scopus (99) Google Scholar;Weinlich et al., 1998Weinlich G. Heine M. Stössel H. et al.Entry into afferent lymphatics and maturation in situ of migrating cutaneous dendritic cells.J Invest Dermatol. 1998; 110: 441-448https://doi.org/10.1046/j.1523-1747.1998.00161.xCrossref PubMed Scopus (99) Google Scholar). The emigration of cutaneous DC is regulated by inflammatory mediators, such as lipopolysaccharide (LPS), tumor necrosis factor (TNF)-α and interleukin (IL)-1β. After subcutaneous application of LPS and TNF-α a loss of Langerhans cells in the epidermis, and lymph vessels filled with emigrating DC became visible (Roake et al., 1995Roake J.A. Rao A.S. Morris P.J. Larsen C.P. Hankins D.F. Austyn J.M. Dendritic cell loss from nonlymphoid tissues after systemic administration of lipopolysaccharide, tumor necrosis factor, and interleukin 1.J Exp Med. 1995; 181: 2237-2248Crossref PubMed Scopus (416) Google Scholar). Similar to that,Cumberbatch et al., 1997Cumberbatch M. Dearman R.J. Kimber I. Langerhans cells require signals from both tumour necrosis factor-α and interleukin-1β for migration.Immunology. 1997; 92: 388-395Crossref PubMed Scopus (284) Google Scholar showed that TNF-α and IL-1β injected intradermally could reduce the number of remaining Langerhans cells in the epidermis and increase the number of DC in draining lymph nodes. We have recently confirmed and further dissected the effects of TNF-α and IL-1β in the murine skin explant model (Stoitzner et al., 1999Stoitzner P. Zanella M. Ortner U. et al.Migration of Langerhans cells and dermal dendritic cells in skin organ cultures: augmentation by TNF-α and IL-1β.J Leukocyte Biol. 1999; 66: 462-470PubMed Google Scholar). Furthermore, it was demonstrated that in response to the application of contact allergens on to murine and human skin, the mRNA in epidermal cells for IL-1β was detected after 15 min, and that of TNF-α and IL-1α 45 min later (Enk et al., 1993Enk A.H. Angeloni V.L. Udey M.C. Katz S.I. An essential role for Langerhans cell-derived IL-1β in the initiation of primary immune responses in skin.J Immunol. 1993; 150: 3698-3704PubMed Google Scholar;Rambukkana et al., 1996Rambukkana A. Pistoor F.H.M. Bos J.D. Kapsenberg M.L. Das P.K. Effects of contact allergens on human Langerhans cells in skin organ culture: Migration, modulation of cell surface molecules, and early expression of interleukin-1β protein.Lab Invest. 1996; 74: 422-436PubMed Google Scholar). There is evidence that chemokines, such as macrophage inflammatory protein-3β, MIP-3β/CCL19 (Kellermann et al., 1999Kellermann S.A. Hudak S. Oldham E.R. Liu Y.J. McEvoy L.M. The CC chemokine receptor-7 ligands 6Ckine and macrophage inflammatory protein-3β are potent chemoattractants for in vitro- and in vivo-derived dendritic cells.J Immunol. 1999; 162: 3859-3864PubMed Google Scholar), secondary lymphoid tissue chemokine, SLC/CCL21 (Saeki et al., 1999Saeki H. Moore A.M. Brown M.J. Hwang S.T. Cutting edge: Secondary lymphoid-tissue chemokine (SLC) and CC chemokine receptor 7 (CCR7) participate in the emigration pathway of mature dendritic cells from the skin to regional lymph nodes.J Immunol. 1999; 162: 2472-2475PubMed Google Scholar), and anti-inflammatory cytokines, such as IL-10 (Wang et al., 1999Wang B. Zhuang L.H. Fujisawa H. et al.Enhanced epidermal Langerhans cell migration in IL-10 knockout mice.J Immunol. 1999; 162: 277-283PubMed Google Scholar), can interfere in this emigration process in a regulatory way. IL-16 is synthesized and released by CD8+ and CD4+ T cells, mast cells, eosinophils, and epithelial cells of asthmatics in response to antigen, mitogen, histamine, and serotonin. This cytokine induces chemotactic responses in T cells, eosinophils, and monocytes via its receptor CD4 (Center et al., 1996Center D.M. Kornfeld H. Cruikshank W.W. Interleukin 16 and its function as a CD4 ligand.Immunol Today. 1996; 17: 476-481https://doi.org/10.1016/0167-5699(96)10052-iAbstract Full Text PDF PubMed Scopus (0) Google Scholar). The best known function of IL-16 is the recruitment of CD4-positive effector cells into inflammatory sites, as in asthma (Laberge et al., 1997Laberge S. Ernst P. Ghaffar O. Cruikshank W.W. Kornfeld H. Center D.M. Hamid Q. Increased expression of interleukin-16 in bronchial mucosa of subjects with atopic asthma.Am J Respir Cell Mol Biol. 1997; 17: 193-202Crossref PubMed Scopus (132) Google Scholar) and atopic dermatitis (Laberge et al., 1998Laberge S. Ghaffar O. Boguniewicz M. Center D.M. Leung D.Y. Hamid Q. Association of increased CD4+ T-cell infiltration with increased IL-16 gene expression in atopic dermatitis.J Allergy Clin Immunol. 1998; 102: 645-650Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Recent data show that human monocyte-derived DC produce IL-16, but are also able to migrate along an IL-16 gradient (Kaser et al., 1999Kaser A. Dunzendorfer S. Offner F.A. et al.A role for IL-16 in the cross-talk between dendritic cells and T cells.J Immunol. 1999; 163: 3232-3238PubMed Google Scholar). We, therefore, wondered whether IL-16 would also exert effects on the migratory capacity of DC in the skin. BALB/C mice were purchased from Charles River (Sulzfeld, Germany) and used at the age of 8–15 wk. CD4-knock-out mice, originating from the laboratory of Dr. T.W. Mak, Toronto, ON, Canada (Rahemtulla et al., 1991Rahemtulla A. Fung-Leung W.-P. Schilham M.W. et al.Normal development and function of CD8+ cells but markedly decreased helper cell activity in mice lacking CD4.Nature. 1991; 353: 180-184Crossref PubMed Scopus (598) Google Scholar), were kindly provided by Dr. B. Ludewig, Zürich, CH. Culture medium was RPMI-1640 (Biochrom KG, Berlin, Germany) supplemented with 10% fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel), 2 µM L-glutamine (Sebac, Stuben, Austria), 0.05 µM 2-mercaptoethanol (Sigma, St Louis, MO) and 20 µg gentamycin per ml (Amimed AG, Allschwil, CH). Recombinant human IL-16 was obtained from Pharmingen (San Diego, CA). As human and mouse IL-16 are mutually cross-reactive to a very high degree (Keane et al., 1998Keane J. Nicoll J. Kim S. et al.Conservation of structure and function between human and murine IL-16.J Immunol. 1998; 160: 5945-5954PubMed Google Scholar) we used the human cytokine for both human and mouse experiments. Recombinant murine TNF-α (specific activity 1.2 × 107 U per mg) was a generous gift of Dr. G.R. Adolf (Bender, Vienna, Austria) and SLC/CCL21 was from Peprotech (London, U.K.). The antibody against human IL-16 (14.1;Cruikshank et al., 1994Cruikshank W.W. Center D.M. Nisar N. Wu M. Natke B. Theodore A.C. Kornfeld H. Molecular and functional analysis of a lymphocyte chemoattractant factor: association of biologic function with CD4 expression.Proc Natl Acad Sci USA. 1994; 91: 5109-5113Crossref PubMed Scopus (223) Google Scholar) and the appropriate control antibody (mouse IgG2a) were also from Pharmingen. A neutralizing polyclonal antibody against TNF-α was purchased from Biosource (Camarillo, CA) and a MoAb against TNF-α (V1q;Echtenacher et al., 1993Echtenacher B. Falk W. Männel D.N. Krammer P.H. Requirement of endogenous tumor necrosis factor/cachectin for recovery from experimental peritonitis.J Exp Med. 1993; 177: 1391-1398Crossref PubMed Scopus (186) Google Scholar) was a kind gift of Dr. B. Echtenacher (Regensburg, Germany). Antibodies against CD4 (GK1.5;Dialynas et al., 1983Dialynas D.P. Wilde D.B. Marrack P. et al.Characterization of the murine antigenic determinant, designated L3T4a, recognized by monoclonal antibody GK1.5: expression of L3T4a by functional T cell clones appears to correlate primarily with class II MHC antigen-reactivity.Immunol Rev. 1983; 74: 29-56Crossref PubMed Scopus (895) Google Scholar) and the isotype-matched control (LO-DNP-11, rat IgG2b) were used as hybridoma supernatants. Ears from mice were disinfected in 70% ethanol and separated into dorsal and ventral halves with forceps. The dorsal halves (cartilage-free) were incubated in 24-well tissue culture plates (one ear per well) as described (Larsen et al., 1990Larsen C.P. Steinman R.M. Witmer-Pack M. Hankins D.F. Morris P.J. Austyn J.M. 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. Heine M. Miwa M. Schuler G. Romani N. An improved isolation method for murine migratory cutaneous dendritic cells.J Immunol Methods. 1996; 193: 71-79https://doi.org/10.1016/0022-1759(96)00058-0Crossref PubMed Scopus (55) Google Scholar). Before the onset of culture the ears were rested at + 4°C for 28 h in an attempt to attenuate endogenous cytokine production. Human skin explant cultures were carried out as described before (Lenz et al., 1993Lenz A. Heine M. Schuler G. Romani N. 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;Pope et al., 1995Pope M. Betjes M.G.H. Hirmand H. Hoffman L. Steinman R.M. Both dendritic cells and memory T lymphocytes emigrate from organ cultures of human skin and form distinctive dendritic-T-cell conjugates.J Invest Dermatol. 1995; 104: 11-17Crossref PubMed Scopus (89) Google Scholar). Split-thickness skin (0.3 mm) from corrective plastic surgery of mammae and abdomina was trimmed to pieces with a punch (8 mm diameter). Alternatively, epidermis and dermis were separated from each other by means of the bacterial enzyme dispase (Kitano and Okado, 1983Kitano Y. Okado N. Separation of the epidermal sheet by dispase.Br J Dermatol. 1983; 108: 555-560Crossref PubMed Scopus (149) Google Scholar), and the dermis-free epidermal sheets were placed in culture. Culture medium was supplemented with different concentrations of cytokines. Whenever antibodies were used the skin explants were preincubated with the MoAbs alone for 1–2 h. In the neutralization assays IL-16 and MoAb 14.1 were preincubated in culture wells for 2 h before skin explants were placed on to the culture medium. The skin organ cultures were incubated for 48 h at 37°C. DC, which had emigrated into the culture medium, could be identified by their typical “veiled” appearance and were enumerated with the hemocytometer. For each experimental condition six explants were pooled. Organ culture on culture medium alone was used as a control. DC numbers from this control were set equal to 100%. Each experimental condition was expressed in percent of the control. This needed to be done to allow for a better comparison of data from different experiments because the absolute numbers of DC that migrated from the explants varied considerably between different individual experiments. This approach had been described previously (Randolph et al., 1998Randolph G.J. Beaulieu S. Pope M. Sugawara I. Hoffman L. Steinman R.M. Muller W.A. A physiologic function for p-glycoprotein (MDR-1) during the migration of dendritic cells from skin via afferent lymphatic vessels.Proc Natl Acad Sci USA. 1998; 95: 6924-6929Crossref PubMed Scopus (218) Google Scholar). All experiments done were included into the analysis and the numbers are indicated as “n” in the figure legends. Student's t test for paired samples was applied to determine the significance of the differences of mean values. Cytocentrifuge smears were prepared from emigrated DC of murine skin organ cultures and fixed in acetone. The phenotype of the cutaneous DC was determined by staining with MoAb against invariant chain (clone IN-1, rat IgG2b;Koch and Hämmerling, 1982Koch N. Hämmerling G.J. Ia invariant chain detected on lymphocyte surfaces by monoclonal antibody.Nature. 1982; 299: 644-646Crossref PubMed Scopus (122) Google Scholar), a maturation marker (clone 2A1, rat IgG2a, from R.M. Steinman, New York, NY;Inaba et al., 1992Inaba K. Inaba M. Romani N. et al.Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor.J Exp Med. 1992; 176: 1693-1702Crossref PubMed Scopus (3209) Google Scholar;Steinman et al., 1997Steinman R.M. Pack M. Inaba K. Dendritic cells in the T-cell areas of lymphoid organs.Immunol Rev. 1997; 156: 25-37Crossref PubMed Scopus (483) Google Scholar) and the costimulatory molecule B7-2/CD86 (Pharmingen) for 30 min at 37°C. The subsequent incubations were carried out with biotinylated anti-rat immunoglobulin, streptavidin–Texas Red (both Amersham Life Science, Amersham, U.K.), rat immunoglobulin for blocking residual free binding sites (Jackson Immuno Research Laboratory, Avondale, PA) and anti-I-A/Ediverse (clone 2G9, fluoresceinated, Pharmingen) for counterstaining the DC. Cyto kines and antibodies were diluted in phosphate-buffered saline/10% fetal bovine serum and injected with 1 ml tuberculin syringes equipped with a 30-gauge needle. Mice received 50 µl of cytokine dilution intradermally into the pinna of one ear and the carrier protein alone into the contralateral ear. After different time points (45 min, 1 h, and 24 h) epidermal sheets were prepared. The skin was floated dermal side down on 0.5 M ammonium thiocyanate for 20 min at 37°C. The epidermis was peeled off the dermis and both parts were cut into 5 × 5 mm pieces and fixed in acetone for 20 min. The sheets were stained with anti-I-Ab,d (clone B21.2/TIB229, rat IgG2b, from R.M. Steinman, New York, NY) followed by biotinylated anti-rat immunoglobulin and streptavidin-fluorescein isothiocyanate for 90 min at 37°C. The density of Langerhans cells in the epidermis was counted under the microscope using × 40 objective lenses and a calibrated grid (at least 20 fields). Student's t test for paired samples was applied to determine differences and significance of the results. To detect endogenous IL-16 and its ligand CD4, we prepared epidermal and dermal sheets from murine ear skin before and after culture and after intradermal injection of TNF-α. Additionally, mouse ear skin was sensitized with 10 µl 1%-TNCB (2,4,6-trinitro-1-chlorobenzene, picryl-chloride, Kodak Eastman, Rochester, NY) on the dorsal and ventral side. Five days later the contralateral ear skin was challenged in the same way with 5 µL 1%-TNCB and used for sheet preparation after the development of a contact hypersensitivity reaction (24 h or 48 h later). Also sheets obtained 12 and 24 h after the first picryl-chloride application (i.e., sensitization phase) were analyzed. The sheets were stained for 90 min at 37°C with antibodies against IL-16 (MoAb 14.1 and biotinylated polyclonal antibody anti-mouse and anti-human IL-16, both from Pharmingen) or anti-CD4 (GK1.5), followed by biotinylated-second step antibodies and/or streptavidin-fluorescein isothiocyanate/–Texas Red and in some cases by anti-I-A/Ediverse (clone 2G9, fluoresceinated). For the intracellular detection of IL-16 the sheets were additionally permeabilized with 0.1% saponin during the whole staining procedure. Epidermal cells were isolated as described before (Schuler and Steinman, 1985Schuler G. Steinman R.M. Murine epidermal Langerhans cells mature into potent immunostimulatory dendritic cells in vitro.J Exp Med. 1985; 161: 526-546Crossref PubMed Scopus (847) Google Scholar;Romani et al., 1997Romani N. Bhardwaj N. Pope M. et al.Dendritic cells.in: Herzenberg L.A. Weir D.M. Herzenberg L. Blackwell C. Weir's Handbook of Experimental Immunology. Blackwell Science, Oxford1997: 1561-15614Google Scholar). After fixation with 1% paraformaldehyde (PFA) and permeabilization with 0.1% saponin, the DC were stained with anti-IL-16 (14.1), biotinylated anti-mouse immunoglobulin and streptavidin–fluorescein isothiocyanate for 30 min on ice. Emigrated DC from mouse skin explant cultures were incubated with anti-CD4 (GK1.5), biotinylated anti-rat immunoglobulin and streptavidin–fluorescein isothiocyanate for 30 min on ice. The cells were analyzed by flow cytometry. An additional way to detect small amounts of surface antigen is complement-mediated lysis using specific MoAb (Crowley et al., 1989Crowley M.T. Inaba K. Witmer-Pack M. Steinman R.M. The cell surface of mouse dendritic cells: FACS analyses of dendritic cells from different tissues including thymus.Cell Immunol. 1989; 118: 108-125Crossref PubMed Scopus (279) Google Scholar). Epidermal cells were isolated and incubated for 1 h with anti-I-Ab,d (clone B21.2) as positive control, anti-CD4 (GK1.5) and isotype-matched MoAb (LO-DNP11, rat IgG2b) and rabbit-low-tox complement (Cedarlane, Ontario, Canada) for depleting Langerhans cells expressing these antigens. The remaining cells were cultured in medium supplemented with 5 ng per ml granulocyte-monocyte colony-stimulating factor for 72 h at 37°C. The fully mature, and therefore clearly recognizable “veiled” Langerhans cells, which had survived the killing procedure, were counted in the hemocytometer. Additionally, in one experiment we added anti-Thy-1.2 (TIB 99, clone HO-13.4, mouse IgM) to the above described settings to deplete a large part of keratinocytes and analyzed the number of surviving Langerhans cells by flow cytometry immediately thereafter. To investigate the influence of IL-16 on the migration of cutaneous DC we cultured murine skin explants with graded concentrations of IL-16 (0.01–10 ng per ml) and counted the emigrated DC in the culture medium. Spontaneous emigration into culture medium in the absence of exogenous cytokines was set equal to 100%. Between 5000 and 20,000 DC emigrated from one whole skin explant (i.e., one dorsal ear half) and between 10,000 and 35,000 Langerhans cells could be recovered from one cultured epidermal explant (i.e., epidermal sheet from one dorsal ear half). Each experimental condition was set in relation to and expressed in percent of the control. Even very low amounts of IL-16 (0.01 ng per ml) enhanced the migration rate up to 126%. Intermediate concentrations (0.1–2 ng per ml) increased migration to 128–136% and the high doses (5 and 10 ng per ml) had weaker effects on cutaneous DC (Figure 1a). Data for 1 ng per ml (where most experiments were done) were highly significant (136 ± 29%, n = 37, p < 0.0001, range: 77–203%) when compared with the spontaneous emigration in the absence of exogenously added cytokines. To set these values in relation to published data (Saeki et al., 1999Saeki H. Moore A.M. Brown M.J. Hwang S.T. Cutting edge: Secondary lymphoid-tissue chemokine (SLC) and CC chemokine receptor 7 (CCR7) participate in the emigration pathway of mature dendritic cells from the skin to regional lymph nodes.J Immunol. 1999; 162: 2472-2475PubMed Google Scholar) we also added the chemokine SLC/CLL21 (10 ng per ml) to the explants. We could observe a similar enhancement (139%) of the DC emigration rate. In time course experiments we noted that IL-16 is already operative in the initial stage of migration: already after an overnight and a 24 h whole skin organ culture the numbers of emigrated DC were increased in response to IL-16 (Table I). In cultures of epidermal sheets we could observe a migration-promoting effect of IL-16 similar to the whole skin explants, but less prominent (Figure 1b, for 1 ng per ml: 124 ± 19%, n = 10, p < 0.01, range: 101–169%). To confirm the specificity of cytokine action, a neutralizing MoAb against IL-16 (14.1) was added into the skin explant cultures. Anti-IL-16 (10 µg per ml) clearly abrogated the migration-promoting effect of IL-16 (1 ng per ml) in cultures of both whole skin and epidermal sheets (Figure 1c).Table IKinetics of the migration-enhancing effect of IL-16Overnight cultureaMouse whole skin explants were cultured for the indicated times. Skin explants were removed and the emigrated cells were further cultured in the presence of granulocyte-monocyte colony-stimulating factor up to 48 h. This needed to be done because after the short culture periods DC cannot be reliably identified in the hemocytometer. Note that, as expected, numbers of emigrant cells increase with time and, more importantly, IL-16 augments these numbers in most experiments also during short-term cultures.24 h cultureaMouse whole skin explants were cultured for the indicated times. Skin explants were removed and the emigrated cells were further cultured in the presence of granulocyte-monocyte colony-stimulating factor up to 48 h. This needed to be done because after the short culture periods DC cannot be reliably identified in the hemocytometer. Note that, as expected, numbers of emigrant cells increase with time and, more importantly, IL-16 augments these numbers in most experiments also during short-term cultures.48 h standard cultureaMouse whole skin explants were cultured for the indicated times. Skin explants were removed and the emigrated cells were further cultured in the presence of granulocyte-monocyte colony-stimulating factor up to 48 h. This needed to be done because after the short culture periods DC cannot be reliably identified in the hemocytometer. Note that, as expected, numbers of emigrant cells increase with time and, more importantly, IL-16 augments these numbers in most experiments also during short-term cultures.DC per one ear explantbAbsolute number of DC emigrated per one explant.Relative cell numberscRelative numbers of emigrated DC in relation to “medium only” controls that were set equal to 100%.DC per one ear explantRelative cell numbersDC per one ear explantRelative cell numbersExperiment no. 1 Medium only3210100%3405100%9740100% IL-16 100 pg per ml225070%5365158%10850112% IL-16 1 ng per ml275086%4750140%14575149%Experiment no. 2 Medium only1000100%1665100%3955100% IL-16 100 pg per ml1275128%2325140%350088% IL-16 1 ng per ml1065107%106564%4935125%Experiment no. 3 Medium only1100100%4125100%5280100% IL-16 100 pg per ml1135103%329580%7400140% IL-16 1 ng per ml1835167%353586%7695146%Experiment no. 4 Medium only1405100%2520100%6745100% IL-16 100 pg per ml2210157%3795151%7280108% IL-16 1 ng per ml2155153%3225128%7695140%a Mouse whole skin explants were cultured for the indicated times. Skin explants were removed and the emigrated cells were further cultured in the presence of granulocyte-monocyte colony-stimulating factor up to 48 h. This needed to be done because after the short culture periods DC cannot be reliably identified in the hemocy