Human Epidermal Langerhans Cells Express the Immunoregulatory Enzyme Indoleamine 2,3-Dioxygenase
2004; Elsevier BV; Volume: 123; Issue: 2 Linguagem: Inglês
10.1111/j.0022-202x.2004.23217.x
ISSN1523-1747
AutoresDagmar von Bubnoff, Huguette Bausinger, Heike Matz, Susanne Koch, Georg Häcker, Osamu Takikawa, Thomas Bieber, Daniel Hanau, Henri de la Salle,
Tópico(s)Immunotherapy and Immune Responses
ResumoLangerhans cells (LC) are a special subset of dendritic cells integrating cutaneous immunity. The study of LC function is of major interest not only for efforts of vaccine design and immunotherapy but also for gaining an insight into the pathogenesis of immune-mediated cutaneous diseases and neoplasias. Recently, defined antigen-presenting cells were described that express indoleamine 2,3-dioxygenase (IDO) and inhibit T cell proliferation in vitro and in vivo. Here, we show that stimulation with interferon-γ (IFN-γ) induces the expression of functionally active IDO in highly purified human epidermal LC. The induction of IDO after stimulation of LC with IFN-γ seems to follow a defined kinetic with rapid upregulation followed by a downregulation after about 24 h of culture. Accordingly, proliferation of T cells induced by anti-CD3 antibodies was modulated by supernatants of IFN-γ-activated human epidermal LC. Importantly, downregulation of T cell proliferation by supernatants of 24 h IFN-γ-activated LC was prevented by inhibition of IDO. These results indicate that LC not only have the capacity to stimulate but also to inhibit T cells, and suggest that LC possess an immunoregulatory function in promoting T cell tolerance by production of IDO. Langerhans cells (LC) are a special subset of dendritic cells integrating cutaneous immunity. The study of LC function is of major interest not only for efforts of vaccine design and immunotherapy but also for gaining an insight into the pathogenesis of immune-mediated cutaneous diseases and neoplasias. Recently, defined antigen-presenting cells were described that express indoleamine 2,3-dioxygenase (IDO) and inhibit T cell proliferation in vitro and in vivo. Here, we show that stimulation with interferon-γ (IFN-γ) induces the expression of functionally active IDO in highly purified human epidermal LC. The induction of IDO after stimulation of LC with IFN-γ seems to follow a defined kinetic with rapid upregulation followed by a downregulation after about 24 h of culture. Accordingly, proliferation of T cells induced by anti-CD3 antibodies was modulated by supernatants of IFN-γ-activated human epidermal LC. Importantly, downregulation of T cell proliferation by supernatants of 24 h IFN-γ-activated LC was prevented by inhibition of IDO. These results indicate that LC not only have the capacity to stimulate but also to inhibit T cells, and suggest that LC possess an immunoregulatory function in promoting T cell tolerance by production of IDO. dendritic cells high-performance liquid chromatography indoleamine 2,3-dioxygenaseIFN-γinterferon-γ keratinocytes Langerhans cells1-MT1-methyltryptophan The development of an adaptive immune response requires presentation of antigens to T cells by specialized cells. Besides the mere presentation of MHC–peptide complexes, the crucial role of antigen-presenting cells (APC) entails the delivery of co-stimulatory signals to T cells. Co-stimulatory signals can be either provided by membrane-bound ligands or by soluble factors secreted by APC, and the sum of these signals will determine the outcome of the T cell response, ranging from a productive response to T cell unresponsiveness (Guermonprez et al., 2002Guermonprez P. Valladeau J. Zitvogel L. Thery C. Amigorena S. Antigen presentation and T cell stimulation by dendritic cells.Annu Rev Immunol. 2002; 20: 621-667Crossref PubMed Scopus (1340) Google Scholar;Lutz and Schuler, 2002Lutz M.B. Schuler G. Immature, semi-mature and fully mature dendritic cells: Which signals induce tolerance or immunity?.Trends Immunol. 2002; 20: 445-449Abstract Full Text Full Text PDF Scopus (1121) Google Scholar). It is now clear that dendritic cells (DC) are the most important APC (Steinman, 2003Steinman R.M. The control of immunity and tolerance by dendritic cell.Pathol Biol (Paris). 2003; 51: 59-60Crossref PubMed Scopus (131) Google Scholar). DC are found in lymphatic and some non-lymphatic tissues, and various subgroups of DC have been recognized based, for instance, on surface molecule expression, their varying capacity to secrete cytokines, or their responsiveness to microbial ligands such as CpG-DNA (Kaisho and Akira, 2003Kaisho T. Akira S. Regulation of dendritic cell function through Toll-like receptors.Curr Mol Med. 2003; 3: 373-385Crossref PubMed Scopus (155) Google Scholar). Besides their well-established function in picking up antigens and initiating an adaptive immune response, recent evidence indicates that DC may also respond to complex information from sites of chronic infections, such as autoimmune disorders, and may be able to downregulate an immune response (Zhang et al., 2003Zhang G.X. Yu S. Gran B. et al.Role of IL-12 receptor beta1 in regulation of T cell response by APC in experimental autoimmune encephalomyelitis.J Immunol. 2003; 171: 4485-4492Crossref PubMed Scopus (75) Google Scholar). One way in which how such a regulatory signal can be delivered is the expression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) (Grohmann et al., 2003Grohmann U. Fallarino F. Puccetti P. Tolerance, DCs and tryptophan: Much ado about IDO.Trends Immunol. 2003; 24: 242-248Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar;Mellor et al., 2003Mellor A.L. Baban B. Chandler P. et al.Cutting edge: Induced indoleamine 2, 3 dioxygenase expression in dendritic cell subsets suppresses T cell clonal expansion.J Immunol. 2003; 171: 1652-1655Crossref PubMed Scopus (384) Google Scholar). IDO activity results in tryptophan depletion and the accumulation of its metabolites in the extracellular space (Terness et al., 2002Terness P. Bauer T.M. Röse L. Dufter C. Watzlik A. Simon H. Opelz H. Inhibition of allogeneic T cell proliferation by indoleamine 2, 3-dioxygenase-expressing dendritic cells: Mediation of suppression by tryptophan metabolites.J Exp Med. 2002; 196: 447-457Crossref PubMed Scopus (741) Google Scholar). The expression of IDO by various cells has been demonstrated to block T cell responses in a number of situations, such as immune tolerance toward allogeneic fetuses (Munn et al., 1998Munn D.H. Zhou M. Attwood J.T. et al.Prevention of allogeneic fetal rejection by tryptophan catabolism.Science. 1998; 281: 1191-1193Crossref PubMed Scopus (2034) Google Scholar), the inhibition of graft rejection (Grohmann et al., 2002Grohmann U. Orabona C. Fallarino F. et al.CTLA-4-Ig regulates tryptophan catabolism in vivo.Nat Immunol. 2002; 3: 1097-1101Crossref PubMed Scopus (953) Google Scholar), or, possibly, the prevention of allergic disease in some individuals (von Bubnoff et al., 2002von Bubnoff D. Matz H. Frahnert C. Rao M.L. Hanau D. de la Salle H. Bieber T. FcεRI induces the tryptophan degradation pathway involved in regulating T cell responses.J Immunol. 2002; 169: 1810-1816Crossref PubMed Scopus (82) Google Scholar). A recent study shows that a subset of monocyte-derived human DC (defined by the expression of CD123 and CCR6) constitutively expresses IDO (Munn et al., 2002Munn D.H. Sharma M.D. Lee J.R. et al.Potential regulatory function of human dendritic cells expressing indoleamine 2, 3-dioxygenase.Science. 2002; 297: 1867-1870Crossref PubMed Scopus (846) Google Scholar). Furthermore, IDO expression by DC has been shown to have consequences for the development of an immune response in vivo. Injection of the immunomodulatory reagent CTLA4-Ig into mice induced IDO expression in specific DC subsets of the spleen and prevented the development of a specific CD8 T cell response (Mellor et al., 2003Mellor A.L. Baban B. Chandler P. et al.Cutting edge: Induced indoleamine 2, 3 dioxygenase expression in dendritic cell subsets suppresses T cell clonal expansion.J Immunol. 2003; 171: 1652-1655Crossref PubMed Scopus (384) Google Scholar). Therefore, specialized DC subsets appear to exist, which can, by virtue of their capacity to produce IDO, negatively affect a T cell response. This is probably due to the induction of apoptosis as a consequence of tryptophan depletion and/or metabolite accumulation (Fallarino et al., 2002Fallarino F. Grohmann U. Vacca C. et al.T cell apoptosis by tryptophan catabolism.Cell Death Differ. 2002; 9: 1069-1077Crossref PubMed Scopus (686) Google Scholar). Langerhans cells (LC) are a subset of DC that are typically localized in the basal and suprabasal layers of the epidermis and along mucosal surfaces (Allam et al., 2003Allam J.P. Novak N. Fuchs C. et al.Characterization of dendritic cells from human oral mucosa: A new Langerhans' cell type with high constitutive FcepsilonRI expression.J Allergy Clin Immunol. 2003; 112: 141-148Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). In the healthy skin, immature LC reside in a "steady state" for a number of months without recycling and have been regarded as sentinels of the immune system (Merad et al., 2002Merad M. Manz M.G. Karsunky H. et al.Langerhans cells renew in the skin throughout life under steady-state conditions.Nat Immunol. 2002; 3: 1135-1141Crossref PubMed Scopus (752) Google Scholar). The traditional view is that LC, in response to danger signals, capture, process, and present antigens. According to this model, upon microbial contact, LC undergo maturation and migrate from the epidermis to regional lymph nodes where they stimulate T cells (Stingl et al., 1980Stingl G. Tamaki K. Katz S.I. Origin and function of epidermal Langerhans cells.Immunol Rev. 1980; 53: 149-174Crossref PubMed Scopus (337) Google Scholar). This scenario has experimental support from data obtained in the study of contact allergic reactions, where contact-sensitizing agents can be visualized in LC of lymph nodes (Krasteva et al., 1996Krasteva M. Moulon C. Peguet-Navarro J. Courtellemont P. Redziniak G. Schmitt D. In vitro sensitization of human T cells with hapten-treated Langerhans cells: A screening test for the identification of contact allergens.Curr Probl Dermatol. 1996; 25: 28-36Crossref PubMed Google Scholar;Wang et al., 2002Wang B. Feliciani C. Howell B.G. Freed I. Cai Q. Watanabe H. Sauder D.N. Contribution of Langerhans cell-derived IL-18 to contact hypersensitivity.J Immunol. 2002; 168: 3303-3308Crossref PubMed Scopus (56) Google Scholar). In addition, LC play a critical role in the pathogenesis of atopic dermatitis (Bieber, 1995Bieber T. Role of Langerhans cells in the physiopathology of atopic dermatitis.Pathol Biol (Paris). 1995; 43: 871-875PubMed Google Scholar;Reich et al., 2002Reich K. Hugo S. Middel P. et al.Evidence for a role of Langerhans cell-derived IL-16 in atopic dermatitis.J Allergy Clin Immunol. 2002; 109: 681-687Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In this condition, LC are activated by immune complexes of allergens and specific IgE that bind to the high-affinity receptor for IgE, FcεRI, on LC (Jurgens et al., 1995Jurgens M. Wollenberg A. Hanau D. de la Salle H. Bieber T. Activation of human epidermal Langerhans cells by engagement of the high affinity receptor for IgE, Fc epsilon RI.J Immunol. 1995; 155: 5184-5189PubMed Google Scholar;Kraft et al., 2002Kraft S. Wessendorf J.H. Haberstok J. Novak N. Wollenberg A. Bieber T. Enhanced expression and activity of protein-tyrosine kinases establishes a functional signaling pathway only in FcepsilonRI high Langerhans cells from atopic individuals.J Invest Dermatol. 2002; 119: 804-811Crossref PubMed Scopus (15) Google Scholar). These LC are able to present foreign antigens in vitro and probably interact with T cells in vivo (Mudde et al., 1990Mudde G.C. Van Reijsen F.C. Boland G.J. de Gast G.C. Bruijnzeel P.L. Bruijnzeel-Koomen C.A. Allergen presentation by epidermal Langerhans' cells from patients with atopic dermatitis is mediated by IgE.Immunology. 1990; 69: 335-341PubMed Google Scholar). Two recent studies challenge this long-held opinion of LC function by the demonstration that LC do not play a leading role in activating T cell-mediated defenses against viral infections of the skin or mucosa (Allan et al., 2003Allan R.S. Smith C.M. Belz G.T. van Lint A.L. Wakim L.M. Heath W.R. Carbone F.R. Epidermal viral immunity induced by CD8alpha+ dendritic cells but not by Langerhans cells.Science. 2003; 301: 1925-1928Crossref PubMed Scopus (482) Google Scholar;Zhao et al., 2003Zhao X. Deak E. Soderberg K. et al.Vaginal submucosal dendritic cells, but not Langerhans cells, induce protective Th1 responses to herpes simplex virus-2.J Exp Med. 2003; 197: 153-162Crossref PubMed Scopus (330) Google Scholar). Since the main role of LC as being primary stimulators of T cells has thus been contested, we reasoned that LC might possess regulatory, i.e., inhibitory, functions. Therefore, we investigated the potential of human epidermal LC to express functional IDO and to suppress T cell responses. Highly purified human epidermal LC and KC were investigated for IDO transcripts. The cells were cultured for 24 h in the presence or absence of interferon-γ (IFN-γ). Semiquantitative analysis of transcripts for IDO was performed by PCR. The density of the β-actin PCR band on the gel was used as internal standardization for equal cDNA loading. Transcripts for IDO could be clearly detected in unstimulated LC (Figure 1a). In IFN-γ-stimulated LC, PCR for IDO gave a strong signal. In 24 h cultured unstimulated KC, a faint band after PCR for IDO was present. IDO mRNA was clearly detectable when cells had been IFN-γ stimulated, but the level of IDO transcripts appeared weaker than with LC (Figure 1b). Significant contamination of KC with LC is unlikely since specific fragments for Langerin, an LC-specific cell protein, could not be amplified after 27 cycles, whereas under the same conditions, RT-PCR using RNA from LC gave maximal amplification products (data not shown). Together, these results demonstrate that in human epidermal LC and KC, IDO mRNA expression is induced after stimulation with IFN-γ. To detect IDO protein expression, immunofluorescence staining was performed on freshly isolated and on 24 h cultured epidermal cells either stimulated with IFN-γ or not. Immunolabeling was performed with anti-IDO monoclonal antibodies (mAb), anti-CD1a mAb, and the respective isotype controls. In freshly isolated epidermal cells after density centrifugation (15%–30% LC), IDO protein expression was not detected in LC and in KC (Figure 2a). After stimulation of highly purified LC for 24 h with IFN-γ, IDO protein expression could be clearly found in all cells, and some LC displayed a very strong staining (Figure 2b). IDO protein expression could also be detected in about 20%–50% of 24 h cultured unstimulated, highly purified CD1a+ cells, but the intensity of the staining was weaker than with stimulated cells. Highly purified, 24 h cultured unstimulated KC did not show IDO protein expression, whereas in 24 h IFN-γ-stimulated KC, a weak expression of IDO protein could be seen (Figure 2c). The same pattern of IDO protein expression in 24 h cultured stimulated and unstimulated LC and KC was observed after density centrifugation (data not shown). Together, these results demonstrate strong IDO protein expression in 24 h IFN-γ-activated, human epidermal LC, and a weak IDO expression in some unstimulated cultured LC and IFN-γ-stimulated KC. Freshly isolated LC and KC and unstimulated 24 h cultured KC do not seem to express IDO protein. The next question we addressed was whether IFN-γ-induced IDO was functionally active in LC, i.e., capable of degrading tryptophan. Therefore, IDO activity was measured as the level of tryptophan consumed and kynurenine generated in the culture supernatants, a well-defined system of IDO activity (von Bubnoff et al., 2002von Bubnoff D. Matz H. Frahnert C. Rao M.L. Hanau D. de la Salle H. Bieber T. FcεRI induces the tryptophan degradation pathway involved in regulating T cell responses.J Immunol. 2002; 169: 1810-1816Crossref PubMed Scopus (82) Google Scholar). Highly purified LC were stimulated or not in increasing cell numbers with IFN-γ (50 ng per mL) for 24 h in tryptophan-free medium supplemented with 100 μM tryptophan (Figure 3). Supernatants were harvested and the degradation of tryptophan and the presence of kynurenine were analyzed by high-performance liquid chromatography (HPLC). In unstimulated, 24 h cultured LC, no significant functional IDO activity could be detected. Interestingly, in 24 h IFN-γ-stimulated LC, significant IDO activity could only be observed at high cell numbers, i.e., with 1.5 × 106 cells per mL, as revealed by the concomitant degradation of tryptophan and production of kynurenine. In unstimulated and IFN-γ-stimulated CD1a- KC, no degradation of tryptophan and no increase in kynurenine was observed throughout the range of increasing cell numbers up to 1.5 × 106 per mL (data not shown). These results demonstrate that IFN-γ stimulation induces the expression of functionally active IDO in human epidermal LC. To determine the amount of tryptophan degradation and kynurenine production in normal medium over time, cultures of highly enriched IFN-γ-activated and non-activated LC and, for comparison, of monocytes, were investigated for functional IDO after 24 and 48 h of culture (Figure 4). Supernatants were harvested after the first 24 h of incubation, the cells were washed once, and incubated for an additional 24 h in the absence of IFN-γ. Interestingly, IDO activity was strongly induced in LC during the first 24 h of IFN-γ stimulation, but was markedly decreased thereafter. In monocytes, IDO activity followed a different time course. Although only moderate IDO activity was induced during the first 24 h of culture, a marked increase in IDO activity was seen during the second 24 h of incubation. The viability and cell numbers of LC and monocytes did not decrease significantly as confirmed by trypan blue exclusion at 48 h of culture. These results suggest that in human epidermal LC, the upregulation of IDO by IFN-γ is only transient, followed by a rapid decline in activity. Another possibility is that LC need, in contrast to monocytes, sustained stimulation with IFN-γ to maintain IDO activity. The stimulatory capacity of APC toward T cells depends on the expression of surface molecules and the secretion of soluble factors. The accessory function of freshly isolated LC is weak, but increases dramatically during culture for 48 h (Picut et al., 1988Picut C.A. Lee C.S. Dougherty E.P. Anderson K.L. Lewis R.M. Immunostimulatory capabilities of highly enriched Langerhans cells in vitro.J Invest Dermatol. 1988; 90: 201-206Abstract Full Text PDF PubMed Google Scholar). We addressed the question of what the effect of IFN-γ-induced IDO activity was on the overall stimulatory activity of LC-derived supernatants. First, supernatants from LC cultures were tested in a time course experiment for their effect on supporting T cell proliferation. Highly enriched human LC were stimulated for 48 h with IFN-γ or not at a cell concentration of 1 × 106cells per mL. Supernatants were collected after 24, 36, and 48 h and tested for their effect on CD3-driven T cell proliferation (Figure 5). Importantly, compared with T cell proliferation in supernatants from unstimulated LC, T cell proliferation was markedly reduced in supernatants from 24 h- and 36 h-IFN-γ-stimulated LC. In contrast, by 48 h of culture, supernatants from IFN-γ-stimulated LC showed increased stimulatory capacity toward T cells compared with supernatants from unstimulated LC. These results suggest that LC exert activation-dependent dual contributions, i.e., suppressive and immunogenic functions to T cells after activation with IFN-γ. The lower proliferation of T cells stimulated in the presence of supernatants from IFN-γ-treated LC compared with untreated LC correlated in time with the detected IDO activity. Therefore, we tested whether IDO activity was responsible for this "suppression". LC were left unstimulated, or were stimulated with IFN-γ in normal culture medium and in culture medium substituted with the IDO-inhibitor 1-methyltryptophan (1-MT) for 24 h. Supernatants were collected and induction of T cell proliferation by surface-bound anti-CD3 mAb in these supernatants was determined (Figure 6). As before, T cell proliferation in supernatants from 24 h IFN-γ-stimulated LC was reduced compared with T cell proliferation in supernatants from unstimulated cells. Interestingly, this reduction in T cell proliferation was completely prevented in two independent experiments and partially prevented in one experiment (data not shown) when supernatants from LC were used, whose activation had been performed in the presence of 1000 μM 1-MT. Together, these results identify LC-derived IDO activity as a T cell inhibitory factor. In this study, we show that highly purified human epidermal CD1a+ LC have the potential to express IDO in response to stimulation with IFN-γ. Although it is known that epidermal KC exert considerable stimulatory and regulatory functions on LC (Cumberbatch et al., 1997Cumberbatch M. Dearman R.J. Kimber I. Interleukin 1 beta and the stimulation of Langerhans cell migration: Comparisons with tumour necrosis factor alpha.Arch Dermatol Res. 1997; 289: 277-284Crossref PubMed Scopus (105) Google Scholar), IDO induction in human epidermal LC seems to be independent of KC. Using a method that combines the techniques of density centrifugation and immunomagnetic bead isolation, highly purified CD1a+ LC (>90%) were obtained. In this cell fraction, and not in the CD1a- fraction of KC, functional IDO-protein expression was greatly enhanced after stimulation with IFN-γ. The functional activities of DC largely depend on their state of activation and differentiation. In particular, freshly isolated LC are relatively poor T cell stimulators but gain high stimulatory capacity after about 48 h of culture (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 (848) Google Scholar). Unlike mature DC, immature DC may have functions to induce and maintain T cell tolerance in the periphery (Steinman et al., 2003Steinman R.M. Hawiger D. Liu K. et al.Dendritic cell function in vivo during the steady state: A role in peripheral tolerance.Ann NY Acad Sci. 2003; 987: 15-25Crossref PubMed Scopus (361) Google Scholar). How this maintenance could be achieved on a cellular or molecular level is uncertain. Our study suggests that the induction of IDO may play a key role in silencing T cell responses by immature LC. High IDO activity after IFN-γ stimulation of LC was short-lived and was seen only during the first 24 h of culture. This suggests that after IFN-γ stimulation, rapid induction of IDO may be abrogated by factors that promote LC maturation and IDO downregulation. LC maturation has been characterized mostly in vitro and can be induced by proinflammatory cytokines and/or the increase of defined costimulatory molecules (Symington et al., 1993Symington F.W. Brady W. Linsley P.S. Expression and function of B7 on human epidermal Langerhans cells.J Immunol. 1993; 150: 1286-1295PubMed Google Scholar;Wang et al., 1999Wang B. Amerio P. Sauder D.N. Role of cytokines in epidermal Langerhans cell migration.J Leukoc Biol. 1999; 66: 33-39Crossref PubMed Scopus (141) Google Scholar). To correlate IDO-dependent regulatory functions to LC maturation, we compared the expression of CD83, HLA-DR, and CD86 on freshly isolated LC with the expression on LC after 48 h of culture by FACS analysis (data not shown). Whereas only about 25% of freshly isolated LC expressed the maturation marker CD83, all LC stained positive for CD83 after 48 h of culture. In addition, compared with freshly isolated LC, the costimulatory molecule CD86 was strongly increased on 48 h cultured, IFN-γ-stimulated, and unstimulated LC. MHC class II was slightly decreased on the cell surface of unstimulated LC after 48 h but was preserved at high expression levels on IFN-γ-stimulated cells. It was difficult to quantify the difference of IDO protein expression between 24 and 48 h IFN-γ-stimulated LC by immunofluorescence because cells at 48 h had a completely different cell shape as a reflection of their maturity. Nevertheless, IDO protein expression appeared to be markedly weaker, although not completely downregulated, in mature IFN-γ-stimulated LC by immunofluorescence (data not shown). Together, these experiments do not rule out the possibility that functional IDO activity is even sustained in mature LC under defined circumstances. Functionally, supernatants from short-term-cultured and stimulated LC, but not from longer cultures, had suppressive effects on T cell proliferation. In this context, we could demonstrate that increased IDO activity was the main suppressive mediator of T cell proliferation in these supernatants since the IDO-inhibitor 1-MT prevented T cell suppression in supernatants from 24 h IFN-γ-activated LC. These data clearly show that activated immature LC have the potential to affect T cell responses negatively. The kinetics suggest that the observed immunosuppressive effect of IFN-γ-stimulated LC-culture supernatants results from short-lived products derived from the tryptophan degradation pathway and not from tryptophan deprivation itself. In this regard, it is well known that the metabolic product 3-hydroxykynurenine is very unstable in the culture medium and it can be toxic to various cells (Okuda et al., 1996Okuda S. Nishiyama N. Saito H. Katsuki H. Hydrogen peroxide-mediated neuronal cell death induced by an endogenous neurotoxin, 3-hydroxykynurenine.Proc Natl Acad Sci USA. 1996; 93: 12553-12558Crossref PubMed Scopus (235) Google Scholar). Our observations in LC parallel the findings from other studies with CD123+CCR6+IDO+DC, where IFN-γ-induced functional IDO in immature DC, but completely downregulates the enzymatic activity of IDO in mature DC (Munn et al., 2002Munn D.H. Sharma M.D. Lee J.R. et al.Potential regulatory function of human dendritic cells expressing indoleamine 2, 3-dioxygenase.Science. 2002; 297: 1867-1870Crossref PubMed Scopus (846) Google Scholar). There, the expression of functional IDO in mature DC was sustained in the presence of the regulatory cytokines interleukin-10 or transforming growth factor-β. Our data clearly show that LC can express both stimulatory and inhibitory (i.e., IDO) activities. The extent to which IDO expression by LC impacts on the balance between tolerance and immunity in vivo remains to be determined. T cell proliferation may be inhibited or regulatory T cells may be activated by the degradation of tryptophan/increase of metabolites. Now that recent evidence disputes that LC are the initiators of the immune response upon microbial infection, the maintenance of tolerance in certain situations is a possible function for these cells that will have to be further explored. FITC- or PE-labeled mAb to human CD1a, CD14, HLA-DR, CD83, CD86, and isotype controls were used to stain LC and monocytes for immunofluorescence and FACS procedures (Pharmingen, San Diego, California). An mAb against human IDO (mouse IgG1) was provided byTakikawa et al., 1988Takikawa O. Kuroiwa T. Yamazaki F. Kido R. Mechanism of interferon-gamma action. Characterization of indoleamine 2, 3-dioxygenase in cultured human cells induced by interferon-gamma and evaluation of the enzyme-mediated tryptophan degradation in its anticellular activity.J Biol Chem. 1988; 263: 2041-2048Abstract Full Text PDF PubMed Google Scholar). Cy3-labeled IgG goat-anti-mouse Ab was purchased from Beckman Coulter (Paris, France). Poly-L-lysine, DNAse, trypsine, saponin (S-7900), and tryptophan were purchased from Sigma (St Louis, Missouri). IFN-γ was used to activate LC, monocytes, and KC (R&D, Wiesbaden, Germany). RPMI-VLE (very low endotoxin) 1640 medium, tryptophan-free RPMI-VLE 1640 medium and tryptophan-free fetal calf serum (FCS) were purchased from Biochrom (Berlin, Germany). Normal FCS, L-glutamine, antibiotics/antimycotics were from Invitrogen (Karlsruhe, Germany). Normal human split skin was obtained from patients undergoing abdominal plastic surgery, as previously described (Bieber et al., 1992Bieber T. Rieger A. Stingl G. Sander E. Wanek P. Strobel I. CD69, an early activation antigen on lymphocytes, is constitutively expressed by human epidermal Langerhans cells.J Invest Dermatol. 1992; 98: 771-776Crossref PubMed Scopus (34) Google Scholar). Written informed consent was obtained from all patients and the protocol was approved by the local ethic committe. Cell suspensions were prepared as described (Hanau et al., 1987Hanau D. Fabre M. Schmitt D.A. et al.Human epidermal Langerhans cells internalize by receptor-mediated endocytosis T6 (CD1, NA1/34) surface antigen: Birbeck granules are involved in the intracellular traffic of the T6 antigen.J Invest Dermatol. 1987; 89: 172-177Abstract Full Text PDF PubMed Google Scholar). These suspensions contained 0.5%–2% freshly isolated CD1a+ LC and were enriched for LC by density gradient centrifugation on ficoll (Lymphocyte separation medium, Eurobio, Les Ullis, France). The interface (15%–30% LC) was collected, washed, and subjected to further positive immunomagnetic isolation with Miltenyi beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Then, the cells were separated into CD1a+ LC and CD1a- KC with a Positive Selection MS Column (Miltenyi Biotec) according to the manufacturer's instructions. The enrichment for LC (>90%) was controlled by CD1a immunolabeling. Peripheral blood monocytes were isolated as described (von Bubnoff et al., 2003von Bubnoff D. Bezold G. Matz H. Hanau D. de la Salle H. Bieber T. Quantification of indoleamine 2, 3-doixygenase gene induction in atopic and non-atopic monocytes after ligation of the high-affinity receptor for IgE, FcεRI and interferon-γ stimulation.Clin Exp Immunol. 2003; 132: 247-253Crossref PubMed Scopus (15) Google Scholar). CD14 expression was determined by flow cytometry and was >90% of isolated cells. For quantitative evaluation of surface antigens, dead cells were excluded by 7-amino-actinomycin D staining, and the CD1a+ population was gated out manually. Epidermal cell suspensions after density centrifugation (15%–30% LC), highly enriched LC, and monocytes (both >90% purity) were stimulated or not for 24 h with IFN-γ (50 ng per mL) in 14 mL polypropylene round-bottom Falcon tubes (BD Biosciences, Heidelberg, Germany) at the indicated densities. As control cells, unstimulated cells were incubated for the same time. The CD1a- KC fraction was stimulated or not for 24 h with IFN-γ (50 ng per mL) in six-well plates (Nalge Nunc International, Roskilde, Denmark) at a density of 1 × 106 cells per mL. Cells were cultured in KC-SFM (serum-free medium) supplemented with bovine pituitary extract (20 μg per mL) and recombinant epidermal growth factor (0.2 ng per mL; all from Invitrogen, Cergy-Pontoise, France). The KC were recovered by a cell scraper for subsequent isolation of RNA and immunofluorescence studies. The supernatants were harvested for HPLC analysis of tryptophan and kynurenine. RT-PCR reactions were carried out as described (von Bubnoff et al., 2002von Bubnoff D. Matz H. Frahnert C. Rao M.L. Hanau D. de la Salle H. Bieber T. FcεRI induces the tryptophan degradation pathway involved in regulating T cell responses.J Immunol. 2002; 169: 1810-1816Crossref PubMed Scopus (82) Google Scholar). Specific primer sequences for the genes were as follows: human β-actin: forward 5′-GAGCGGGAAATCGT GCGT-GACATT-3′ and reverse 5′-GATGGAGTTGAAGGTAGTTTCGTG-3′ (240 bp); human IDO: forward 5′-CTTCCTGGTCTCTCTATTGG-3′ and reverse 5′-GAAGTTCCTG TGAGCTGGTG-3′ (430 bp). The PCR cycle number for the detection of IDO and Langerin was 27 and 25 for β-actin. Immunofluorescence microscopy of fixed permeabilized highly purified LC (>90% CD1a+ LC) and KC, or epidermal cells (LC and KC) after density centrifugation (about 15%–30% LC), was carried out as previously described (Saudrais et al., 1998Saudrais C. Spehner D. de la Salle H. et al.Intracellular pathway for the generation of functional MHC class II peptide complexes in immature human dendritic cells.J Immunol. 1998; 160: 2597-2607PubMed Google Scholar). The concentration for unconjugated anti-IDO mAb was 2.5 μg per mL, for the secondary Ab goat-anti-mouse-Cy3 1/1200, and for anti-human-CD1a-FITC-labeled Ab 2.5 μg per mL. Immunofluorescence analysis was performed using a Leica DMR microscope (Microsystems, Heidelberg, Germany) interfaced with a 12V100W halogen lamp (Osram, Munich, Germany). Double fluorescence acquisition was performed using a × 63 oil immersion Plan Apo objective (Merck, Darmstadt, Germany) (numerical aperture=1). For HPLC analyses of tryptophan and kynurenine in cell supernatants with increasing amounts of LC numbers, cells were incubated for 24 h in tryptophan-free medium supplemented with 100 μM tryptophan. KC were cultured for 24 h in KC-specific medium as described above. For kinetic studies, cells were incubated in normal culture medium and either stimulated or not. The baseline concentration of tryptophan in normal culture medium was determined by HPLC and was relatively constant over time (20–21 μM). Supernatants were collected after 24 h of culture and cells were further incubated with fresh medium for another 24 h without addition of further IFN-γ. Tryptophan and kynurenine levels were quantified by HPLC from the first 24 h of incubation and after 48 h (=second 24 h) of culture. Samples for the determination of L-tryptophan and L-kynurenine were prepared and assayed as described (von Bubnoff et al., 2002von Bubnoff D. Matz H. Frahnert C. Rao M.L. Hanau D. de la Salle H. Bieber T. FcεRI induces the tryptophan degradation pathway involved in regulating T cell responses.J Immunol. 2002; 169: 1810-1816Crossref PubMed Scopus (82) Google Scholar). T cell isolations from peripheral blood of donors were carried out as described (von Bubnoff et al., 2002von Bubnoff D. Matz H. Frahnert C. Rao M.L. Hanau D. de la Salle H. Bieber T. FcεRI induces the tryptophan degradation pathway involved in regulating T cell responses.J Immunol. 2002; 169: 1810-1816Crossref PubMed Scopus (82) Google Scholar). Allogeneic T cell proliferation assays were carried out as follows. Highly purified LC (>90% CD1a+) were isolated from human skin as described above. Freshly isolated LC were added in increasing cell numbers (from 0 to 10,000 cells) to 2 × 105 pure allogeneic T cells. Cocultures were either stimulated with IFN-γ (50 ng per mL) or not. Seventy-two hours later, T cell proliferation was measured using overnight incubation with [3H]thymidine (0.5 μCi per well). To study T cell proliferation in supernatants of LC, an anti-CD3 mAb (R&D) was immobilized onto 96-flat bottom tissue culture wells (1 μg per 100 μL per well) according to the manufacturer's protocol. T cells (1 × 105) were resuspended in 100 μL of supernatant and seeded onto anti-CD3 mAb-coated wells. After 72 h, T cell proliferation was measured using overnight incubation with [3H]thymidine (0.5 μCi per well). This work was supported by a grant from the University of Bonn, Germany, and the Etablissement Français du Sang-Alsace, Strasbourg, France, INSERM, and ARMESA.
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