Langerhans Cells Do Not Produce Interferon-c

Interferon-g (IFN-g) is the predominant cytokine during Th1dominated immune reactions, participates importantly during antigen presentation, and is the prototypical macrophage-activating cytokine. Consequently, a pivotal role of IFN-g in the clearance of various intracellular pathogens has been demonstrated (Boehm et al, 1997). IFN-g had historically been regarded as a product solely of T cells and natural killer cells; however, it was discovered recently that, with appropriate stimulation, B cells, macrophages (Mf), and even dendritic cells (DC) could produce IFN-g (Di Marzio et al, 1994; Yoshimoto et al, 1997; Ohteki et al, 1999). In the case of DC, IL-18 acts in synergy with IL-12 on IFN-g production (Fukao et al, 2000). In addition, IL-4 unexpectedly enhances DC IFN-g production, synergistically with IL-12 or with DC stimulation through CD40 or major histocompatibility complex class II molecules triggering endogenous IL-12 production (Fukao et al, 2000). Therefore, we were interested in examining whether Langerhans cells, a special subset of DC localized in the epidermis, could produce IFN-g in response to these stimulations. Recently, we succeeded in preparing high purity Langerhans cells (495%) from BALB/c mouse skin (Tada et al, 2000), and this allowed us to examine direct e¡ects of several cytokines on Langerhans cell IFN-g production, without interference of keratinocytes and keratinocyte-derived cytokines. First, we employed reverse transcription polymerase chain reaction (RT-PCR) to assess IFN-g gene expression in Langerhans cells exposed to various combined stimulations including IL-12 (2 ng per ml), IL-18 (20 ng per ml), IL-4 (10 ng per ml), and IFN-g (100 ng per ml), as shown in Fig 1. These stimulations were formerly reported to induce IFN-g production in both spleenand bone-marrow-derived DC or Mf (Di Marzio et al, 1994; Fukao et al, 2000; Stober et al, 2001). As shown in Fig 1, no IFN-g mRNAwas detected in any of the RNA extracted from fresh Langerhans cells and 24 h incubated Langerhans cells with and without stimulation. As expected, IFN-g mRNA was detected in spleen-derived CD11c DC stimulated with IL-12 and IL-18. Next, supernatants of 48 h cultured Langerhans cells and DC were subjected to the measurement of IFN-g protein levels using enzyme-linked immunosorbent assay (ELISA). The synergistic e¡ects of IL-4 and IL-18 on IL-12-dependent IFN-g production of spleen-derived CD11c DC previously reported by Fukao et al (2000) were con¢rmed (Fig 2A). In contrast to spleen-derived CD11c DC, simultaneous addition of IL-12 and IL-18 failed to induce Langerhans cell IFN-g secretion (Fig 2B). Further administration of IL-4 to the combination of IL-12 and IL-18 did not a¡ect this Langerhans cell unresponsiveness (Fig 2B). IL-15, recently characterized as another IFN-g-inducing cytokine from DC (Mattei et al, 2001), also failed to stimulate Langerhans cells for IFN-g production (data not shown). We also evaluated the e¡ect of other stimulations closely related to Langerhans cell maturation and IL-12 production (Tada et al, 2001), such as anti-CD40 monoclonal antibody (20 mg per ml), granulocyte macrophage colony stimulating factor (1 ng per ml), tumor necrosis factor a (10 ng per ml), IFN-b (1000 U per ml), anti-IL-10 monoclonal antibody (20 mg per ml), transforming growth factor b (TGF-b; 1 ng per ml), and the combination of anti-CD40 monoclonal antibody and TGF-b. None of these reagents caused Langerhans cell IFN-g production, however, even when added together with IL-12, IL-18, and IL-4 (data not shown). The same results were obtained with both 48 h and 72 h cultured Langerhans cells. We used only dispase for epidermal sheet separation from the mouse skin, and did not use trypsin throughout the process of Langerhans cell isolation.With this isolation technique, receptors of cytokines are less likely to be degraded on Langerhans cells. The whole procedure may have a¡ected expression of cell surface receptors, however, causing Langerhans cells to be unresponsive to several cytokines in comparison to spleen-derived CD11c DC. To address this issue, we treated DC in the same condition that was employed for Langerhans cell isolation, but the viability of treated DC then fell signi¢cantly and an exact comparison was not possible (Tada et al, 2000). As another approach, we left isolated Langerhans cells untreated in culture medium for 24 h before the addition of stimuli, in order to allow reappearance of cell surface receptors putatively degraded by the isolation procedure. This experiment also failed to stimulate Langerhans cells for IFN-g production (data not shown). Finally, we have recently found out that IL-12, an important IFN-g inducer of splenic DC, strongly enhances the level of IFN-inducible protein 10 (IP-10) production of isolated Langerhans cells prepared in the same way (Fujita et al, submitted), indicating that IL-12 indeed has a biologic activity on Langerhans cells.