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Differential Production of Th1- and Th2-Type Chemokines by Mouse Langerhans Cells and Splenic Dendritic Cells
Differential Production of Th1- and Th2-Type Chemokines by Mouse Langerhans Cells and Splenic Dendritic Cells Hideki Fujita, Akihiko Asahina, Makoto Sugaya, Koichiro Nakamura,w Ping Gao,z Hiromi Fujiwara,z and Kunihiko Tamaki Department of Dermatology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan; wDepartment of Dermatology, School of Medicine, Fukushima
Medical University, Fukushima, Japan; zDepartment of Oncology, Graduate School of Medicine, Osaka University, Osaka, Japan
Some chemokines specifically attract T helper 1 (Th1) cells, whereas others attract T helper 2 (Th2) cells. In this study, we investigated the capacity of Langerhans cells (LC) to produce Th1- and Th2-type chemokines in comparison with that of splenic CD11c þ dendritic cells (DC). We prepared highly purified (495%) LC from BALB/c mouse skin using the panning method. With regard to Th1-type chemokines, exogenous stimulus, such as interferon-c (IFN-c), lipopolysaccharide, or polyinosinic–polycytidylic acid, was mandatory for the production of substantial amounts of CXCL10, CXCL9, and CXCL11 both in LC and splenic DC. LC, as a whole, exhibited low ability to produce Th1-type chemokines in comparison with splenic DC. As for Th2-type chemokines, LC, but not splenic DC, produced high levels of CCL22 and CCL17 constitutively during culture even without exogenous stimuli. The production of Th2-type chemokines was regulated in a complicated manner. In particular, interleukin-4 upregulated, and IFN-c downregulated both CCL22 and CCL17 production by LC. Of note, LC produced much more amounts of Th2-type chemokines than splenic DC under any conditions tested. Finally, Th1- and Th2-type chemokines produced by LC were shown to be functional using chemokine receptor-transfected-2B4 T cells. The high production of CC chemokine receptor 4 ligands by LC in the absence of IFN-c may be an important character discriminating LC from other DC.
Key words: CCL17/CCL22/CXCL10/IFN-g/skin J Invest Dermatol 124:343–350, 2005
Chemokines are chemotactic cytokines that play an important role in regulating leukocyte migration (Baggiolini, 1998; Zlotnik and Yoshie, 2000). T helper 1 (Th1) and T helper 2 (Th2) cells express different types of chemokine receptors and respond to distinct types of chemokines. CXC chemokine receptor 3 (CXCR3) is predominantly expressed on Th1 cells (Bonecchi et al, 1998; Sallusto et al, 1998; Zlotnik and Yoshie, 2000). Interferon (IFN-g)-inducible protein-10 (IP10)/CXCL10 (Zlotnik and Yoshie, 2000), monokine induced by IFN-g(Mig)/CXCL9 (Zlotnik and Yoshie, 2000), and IFNinducible T cell a chemoattractant (I-TAC)/CXCL11 (Zlotnik and Yoshie, 2000) all bind to CXCR3, and thus promote the migration of Th1 cells. On the other hand, CC chemokine receptor 4 (CCR4) is predominantly expressed on Th2 cells (Sallusto et al, 1998; Imai et al, 1999; Zlotnik and Yoshie, 2000), CD4 þ CD25 þ T cells with regulatory activity (Iellem et al, 2001; Sebastiani et al, 2001), and also on skin homing memory T cells positive for cutaneous lymphocyte antigen
(CLA) (Campbell et al, 1999). Thymus and activation-regulated chemokine (TARC)/CCL17 (Zlotnik and Yoshie, 2000) as well as macrophage-derived chemokine (MDC)/CCL22 (Zlotnik and Yoshie, 2000) promote the migration of these cells via CCR4. Dendritic cells (DC) are professional antigen-presenting cells with a unique ability to stimulate naı¨ve T cells and initiate primary immune responses (Liu, 2001). Distinct DC subpopulations exhibit distinct functions leading to different immune regulations (Banchereau et al, 2000; Liu, 2001), and their heterogeneity reflects the organs they reside in (Reis e Sousa et al, 1999). Langerhans cells (LC) are a specific subtype of DC localized in the epidermis (Banchereau and Steinman, 1998). LC differ from other DC in possessing Birbeck granules, lacking functional mannose receptors, expressing E-cadherin, and being dependent on transforming growth factor-b (TGF-b) for their differentiation (Banchereau and Steinman, 1998; Banchereau et al, 2000; Wu et al, 2001). As for functional characteristics of LC in comparison with those of splenic DC, we have reported the unique modality in the regulation of interleukin-12 (IL-12) production (Tada et al, 2000, 2001) and inability of IFN-g production (Tada et al, 2003) in LC. Furthermore, we have found substantial differences in responsiveness to microorganisms through Toll-like receptors between LC and splenic DC (Fujita et al, 2004b; Mitsui et al, 2004; Tada et al, 2004).
Abbreviations: Ab, antibody; DC, dendritic cells; LC, Langerhans cells; LN, lymph nodes; LPS, lipopolysaccharide; GM-CSF, granulocyte macrophage colony-stimulating factor; IFN, interferon; IL, interleukin; mAb, monoclonal antibody; Poly(IC),polyinosinic–polycytidylic acid; SAC, Staphylococcus aureus Cowen 1; Th1, T helper 1; Th2, T helper 2; TNF-a, tumor necrosis factor-a; TGF-b, transforming growth factor-b
Copyright r 2005 by The Society for Investigative Dermatology, Inc.
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Not only are DC attracted by chemokines but DC are also an abundant and strategic source of various chemokines (Sallusto et al, 1999). In addition, a recent report disclosed that the property of chemokine production is diverse among DC subsets (Penna et al, 2002a, b). We have recently reported the expression and regulation of inflammatory chemokines, such as CCL3, CCL4, and CCL5, in mouse LC (Fujita et al, 2004a). Apart from this study, however, chemokine production by resident LC has not been well described. In particular, it is unknown whether LC are capable of producing the Th1-type chemokines, CXCL10, CXCL9, and CXCL11. In this study, in order to further define functional properties of LC to shape immune responses, we focused on the expression and regulation of Th1- and Th2-type chemokines in mouse LC. For this purpose, we prepared highly purified LC population (495%) from mouse skin, which was phenotypically immature and acquired mature phenotypes during culture in vitro (Salgado et al, 1999a, b; Tada et al, 2000). Furthermore, we used splenic CD11c þ DC for comparison with LC, and illustrated distinct characteristics of LC in chemokine production.
Results Messenger RNA for Th1-type chemokines in LC and splenic DC CXCL10, CXCL9, and CXCL11 are Th1-type chemokines that are structurally related and share the common receptor CXCR3 (Olson and Ley, 2002). To investigate whether LC and splenic DC express their mRNA, semiquantitative RT-PCR analysis was performed. The mRNA of none of these Th1-type chemokines was detected in fresh LC (Fig 1). After 48 h culture, LC came to express CXCL10
Figure 1 RT-PCR analysis of CXCL10, CXCL9, and CXCL11 gene expression in freshly isolated Langerhans cells (fLC), 48 h cultured LC (cLC), interferon-c (IFN-c)-stimulated cLC (c-cLC), freshly isolated splenic dendritic cells (fDC), 48 h cultured splenic DC (cDC), and IFN-cstimulated cDC (c-cDC). LC and splenic DC were purified and cultured for 48 h in the absence or presence of 100 ng per mL of IFN-g. Samples from fresh and cultured LC and splenic DC were collected and mRNA expression was analyzed using specific primers for each chemokine. CXCL10, CXCL9, and CXCL11 mRNA expression was hardly detectable in fLC. In cLC, mRNA expression of CXCL10 and CXCL11, but not CXCL9, was induced. CXCL9 mRNA was strongly expressed only in g-cLC. In addition, mRNA for CXCL10 and CXCL11 was also strongly expressed in g-cLC. In splenic DC, mRNA for these T helper 1(Th1)-type chemokines was almost undetectable both in fDC and cDC. When stimulated with IFN-g, CXCL10, CXCL9, and CXCL11 mRNA expression was induced. Data are representative of three independent experiments.
and CXCL11, but not CXCL9, mRNA. Expression of CXCL9 mRNA was strongly induced by IFN-g. These results indicate that mRNA expression of CXCL9 is regulated differently from that of CXCL10 and CXCL11 during the maturation of LC. In the case of splenic DC, the expression of mRNA for all three chemokines was negligible in fresh and unstimulated cultured cells. mRNA for CXCL10, CXCL9, and CXCL11 was, however, induced by IFN-g. Time-course measurement of the production of Th1type chemokines by LC and splenic DC To determine whether Th1-type chemokines were produced at protein level by cultured LC and splenic DC and secreted into culture media, ELISA was carried out using media cultured for 0, 12, 24, 36, and 48 h. As shown in Fig 2, without IFN-g treatment, LC and splenic DC produced small amounts of Th1-type chemokines. In contrast, LC and splenic DC produced substantial amounts of CXCL10, CXCL9, and CXCL11 time dependently by stimulation with IFN-g (100 ng per mL). Splenic DC have, on the whole, an ability to produce larger amounts of Th1-type chemokines than LC. Biological activity of Th1-type chemokines produced by LC and splenic DC To ascertain whether Th1-type chemokines produced by LC and splenic DC were functional, culture supernatants were tested for their capacity to induce the migration of CXCR3-transfected 2B4 T cells (Gao et al, 2003). As demonstrated above, CXCR3 ligands are produced at large amounts by stimulation with IFN-g both in LC and splenic DC. Indeed, the supernatants of 48 h cultured unstimulated LC and splenic DC induced only minimal migration of these cells (data not shown). Therefore, we used the supernatants of 48 h cultured IFN-g-treated LC and splenic DC for the chemotaxis of CXCR3 transfectant. As shown in Fig 3, the supernatant of LC exhibited considerable chemotactic activity to CXCR3 transfectant. Neutralizing anti-CXCL10 antibody (Ab) and anti-CXCL9 Ab partially blocked the migration, respectively. When both of the Abs were added simultaneously, the migration of transfectant was almost completely inhibited. The supernatant of splenic DC induced more prominent migration of the transfectant than that of LC. In the same way, this migration was partially blocked by anti-CXCL10 Ab or antiCXCL9 Ab, respectively, and almost completely blocked by both of them. Isotype-matched control of neutralizing Ab did not affect the migration of transfectant (data not shown). These results indicate that at least CXCL10 and CXCL9 produced by LC and splenic DC actually possess chemotactic activity, and the contribution of CXCL11 for the migration of the transfectant seems to be minimal both in LC and splenic DC. Messenger RNA for Th2-type chemokines in LC and splenic DC CCL22 and CCL17 are Th2-type chemokines that share the receptor CCR4. Therefore, we analyzed the mRNA expression of CCL22 and CCL17 in LC and splenic DC by semi-quantitative RT-PCR. As shown in Fig 4, CCL22 and CCL17 showed almost the same pattern of mRNA expression in fresh and cultured LC. The mRNA of both chemokines was only faintly detected in fresh LC, and strongly expressed after 48 h culture. The results obtained
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Figure 2 Production of T helper 1-type chemokines during culture of Langerhans cells (LC) and splenic dendritic cells (DC). Purified LC and splenic DC were cultured with or without interferon-g (IFN-g), and the concentration of CXCL10, CXCL9, and CXCL11 was measured at different time points (0, 12, 24, 36, 48 h) in the supernatants by ELISA. Representative data of three independent experiments.
for CCL17 were consistent with those reported previously (Xiao et al, 2003a). In sharp contrast, fresh splenic DC did not express mRNA for Th2-type chemokines. Of note, mRNA for CCL22 and CCL17 was not induced in splenic DC even after culture. Time-course measurement of the production of Th2type chemokines by LC and splenic DC CCL22 and CCL17 production by LC and splenic DC was determined
Figure 3 Chemotaxis of mCXCR3-transfected 2B4 T cells. Langerhans cells (LC) and splenic dendritic cells (DC) were cultured for 48 h with 100 ng per mL of interferon-g (IFN-g) and the supernatants were collected. The culture supernatants were preincubated with or without neutralizing anti-chemokine monoclonal antibody (mAb) indicated in the figure for 30 min, and assessed for chemotactic activity to CXCR3 transfectant. RPMI 10 medium alone served as a negative control. RPMI 10 medium containing recombinant chemokine served as a positive control. The supernatants of IFN-g-stimulated LC and splenic DC exhibited chemotactic activity to CXCR3 transfectant, which is mediated at least by CXCL10 and CXCL9. Mean (SD) (n ¼ 3). Data are representative of three independent experiments.
by ELISA using media cultured for 0, 12, 24, 36, and 48 h. As shown in Fig 5, LC produced substantial levels of these chemokines in a time-dependent manner even in the absence of exogenous stimulation. Conversely, splenic DC in the same condition failed to produce Th2-type chemokines at any time point up to 48 h. These results were in parallel with mRNA expression analyzed by RT-PCR. Biological activity of Th2-type chemokines produced by LC and splenic DC To investigate whether Th2-type chemokines produced by LC and splenic DC have chemotactic activity to T cells, we carried out a chemotaxis assay using CCR4-transfected 2B4 T cells (Gao et al, 2003). The results shown in Fig 6 demonstrate that the supernatant of 48 h cultured unstimulated LC induced the migration of a considerable number of CCR4 transfectant. This migration was partially blocked by neutralizing anti-CCL17 Ab and anti-CCL22 Ab, respectively. When both of the Ab were
Figure 4 RT-PCR analysis of CCL22 and CCL17 gene expression in freshly isolated Langerhans cells (fLC), 48 h cultured LC (cLC), freshly isolated splenic dendritic cells (fDC), and 48 h cultured splenic DC (cDC). LC and splenic DC were purified and cultured for 48 h without exogenous stimulation. mRNA of fresh and cultured LC and splenic DC was extracted, and mRNA expression was analyzed using specific primers for each chemokine. CCL22 and CCL17 mRNA was expressed weakly in fLC, and strongly in cLC. In splenic DC, mRNA for CCL22 and CCL17 was not detected both in fDC and cDC. Data are representative of two independent experiments.
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(1:10000), and polyinosinic–polycytidylic acid (Poly(I:C)) (20 mg per mL) induced CXCL10 production in LC. In the case of CXCL9 and CXCL11, of all the stimuli tested, only IFN-g induced the production of these chemokines in LC (Fig
Figure 5 Production of T helper 2-type chemokines during culture of Langerhans cells (LC) and splenic dendritic cells (DC). Purified LC and splenic DC were cultured without exogenous stimulation, and the concentration of CCL22 and CCL17 was measured at different time points (0, 12, 24, 36, 48 h) in the supernatants by ELISA. Data are representative of three independent experiments.
added simultaneously, the migration of the transfectant was inhibited almost completely. On the other hand, the supernatant of 48 h cultured splenic DC exhibited minimal chemotactic activity to CCR4 transfectant. These results confirm that CCL22 and CCL17 produced by LC are functional. Production of Th1-type chemokines and its regulation in LC and splenic DC As mentioned above, IFN-g-stimulated LC and splenic DC produced a substantial level of Th1-type chemokines. To investigate the production of Th1-type chemokines and their regulation in LC and splenic DC, we cultured them with various stimuli. As shown in Fig 7a, in addition to IFN-g, IL-12 (2 ng per mL), lipopolysaccharide (LPS) (1 mg per mL), Staphylococcus aureus Cowen 1 (SAC)
Figure 6 Chemotaxis of mCCR4-transfected 2B4 T cells. Langerhans cells (LC) and splenic dendritic cells (DC) were cultured for 48 h without stimulation and the supernatants were collected. The culture supernatants were preincubated with or without neutralizing anti-chemokine monoclonal antibody (mAb) indicated in the figure for 30 min, and assessed for chemotactic activity to CCR4 transfectant. RPMI 10 medium alone served as a negative control. RPMI 10 medium containing recombinant chemokine served as a positive control. The supernatant of LC, but not of splenic DC, exhibited CCL22- and CCL17-dependent chemotactic activity to CCR4 transfectant. Mean (SD) (n ¼ 3). Data are representative of three independent experiments.
Figure 7 Regulation of T helper 1-type chemokines produced by Langerhans cells (LC) and splenic dendritic cells (DC). LC (&) and splenic DC (’) were purified and cultured (1.5 106 cells per mL per 200 mL in each well) for 48 h with or without various stimuli. The supernatants were collected and the concentration of CXCL10 (a), CXCL9 (b), and CXCL11 (c) was measured by ELISA. CXCL10 production by LC was induced by interferon-g (IFN-g), interleukin (IL)-12, lipopolysaccharide (LPS), Staphylococcus aureus Cowen 1 (SAC), and polyinosinic–polycytidylic acid (Poly(I:C)). In the case of CXCL9 and CXCL11, only IFN-g induced their production by LC. In the case of splenic DC, the production of CXCL10, CXCL9, and CXCL11 was induced by IFN-g, IL-18, LPS, and Poly(I:C). Results are the mean (SD) (n ¼ 4). Significant increase (po0.05) compared with the unstimulated group. Data are representative of four independent experiments.
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7b, c). As for splenic DC, the mode of the regulation of Th1-type chemokines was identical among CXCL10, CXCL9, and CXCL11 (Fig 7a–c). Splenic DC produced CXCL10, CXCL9, and CXCL11 in response to IFN-g, IL-18 (100 ng per mL), LPS, and Poly(I:C). Interestingly, splenic DC, as a whole, exhibited high ability to produce Th1-type chemokines in comparison with LC. Production of Th2-type chemokines and its regulation in LC and splenic DC As demonstrated above, LC, but not splenic DC, produced Th2-type chemokines constitutively during culture even without stimulation. Here, we further
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examined the production of CCL22 and CCL17 in response to various stimuli. The patterns of the regulation of Th2-type chemokines were rather complicated. As shown in Fig 8a, CCL22 production by LC was upregulated by tumor necrosis factor-a (TNF-a) (10 ng per mL), granulocyte macrophage colony-stimulating factor (GM-CSF) (1 ng per mL), IL-1b (10 ng per mL), IL-4 (10 ng per mL), and agonistic antiCD40 Ab (20 mg per mL). On the other hand, IFN-g, IL-10 (10 ng per mL), and Poly(I:C) had a significant inhibitory effect on its production. Other stimuli, such as TGF-b (1 ng per mL), IL-12, IL-13 (10 ng per mL), IL-18, LPS, and SAC, failed to affect the production of CCL22 in cultured LC. The mode of the regulation of CCL17 production in LC was similar to that of CCL22. Stimulation with TGF-b, TNF-a, IL-4, IL-13, and anti-CD40 Ab enhanced CCL17 production significantly. In contrast, its production level decreased significantly in the presence of GM-CSF, IFN-g, IL-10, LPS, SAC, and Poly (I:C). IL-12 and IL-18 did not cause significant changes in CCL17 production (Fig 8b). In the case of splenic DC, the production of CCL22 and CCL17 was found to be regulated in almost the same way (Fig 8a, b). TNF-a, GM-CSF, IL-1b, IL-4, IL-12, IL-13, IL-18, and anti-CD40 Ab induced CCL22 and CCL17 production to some extent. When splenic DC were stimulated with microbial components, such as LPS, SAC, and Poly(I:C), however, the production of CCL22 and CCL17 was induced differently. LPS and SAC induced a high level of CCL22 production, but they did not affect CCL17 production. Poly(I:C) induced not only CCL22 but also CCL17 production, although to a much lesser extent. Another striking finding was that the amounts of Th2-type chemokines produced by splenic DC were much smaller than those produced by LC under any conditions tested.
Discussion
Figure 8 Regulation of T helper 2-type chemokines produced by Langerhans cells (LC) and splenic dendritic cells (DC). LC (&) and splenic DC (’) were purified and cultured (1.5 106 cells per mL per 200 mL in each well) for 48 h with or without various stimuli. The supernatants were collected and assessed for the concentration of CCL22 (a), CCL17 (b) by ELISA. (a) CCL22 production by LC was upregulated by tumor necrosis factor-a (TNF-a), granulocyte macrophage colonystimulating factor (GM-CSF), interleukin (IL)-1b, IL-4, and anti-CD40 antibody (Ab), and downregulated by interferon-g (IFN-g), IL-10, and polyinosinic–polycytidylic acid (Poly(I:C)). In splenic DC, its production was induced by TNF-a, GM-CSF, IL-1b, IL-4, IL-12, IL-13, IL-18, antiCD40 Ab, lipopolysaccharide (LPS), Staphylococcus aureus Cowen 1 (SAC), and Poly(I:C). (b) CCL17 production by LC was upregulated by transforming growth factor-b (TGF-b), TNF-a, IL-1b, IL-4, IL-13, and anti-CD40 Ab, and downregulated by GM-CSF, IFN-g, IL-10, LPS, SAC, and Poly(I:C). In splenic DC, its production was induced by TNFa, GM-CSF, IL-1b, IL-4, IL-12, IL-13, IL-18, anti-CD40 Ab, and Poly(I:C). Results are the mean (SD) (n ¼ 4).Significant increase (po0.05) compared with the unstimulated group. #, Significant decrease (po0.05) compared with the unstimulated group. Data are representative of four independent experiments.
In this study, we demonstrated that the chemokine production represents an additional diversity between the two discrete populations of mouse DC, epidermal LC, and splenic DC. LC indeed have the ability to produce functional Th1and Th2-type chemokines. With regard to Th1-type chemokines, exogenous stimulation was mandatory for their production both in LC and splenic DC. Only limited stimuli, such as IFN-g, LPS, and Poly(I:C), induced the production of Th1-type chemokines, namely, CXCL10, CXCL9, and CXCL11, both in LC and splenic DC. But the amounts of CXCL10, CXCL9, and CXCL11, produced by LC in the presence of such stimuli, were lower than those produced by splenic DC. Among the Th1-type chemokines, LC produced CXCL10 in response to IFN-g, IL-12, and microbial components. Interestingly, only IFN-g, but not microbial components, was able to induce CXCL9 and CXCL11 production in LC. These findings further confirmed our previous observations that LC exhibited low responsiveness to microbial components (Fujita et al, 2004b; Mitsui et al, 2004; Tada et al, 2004). As for Th2-type chemokines, the mode of the regulation was different from that of Th1-type chemokines. First, even without exogenous stimulation, LC, but not splenic DC, were able to produce CCL22 and CCL17 constitutively during culture. Although some of the stimuli induced the production of CCL22 and CCL17 in splenic DC,
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they were produced in much smaller amounts by splenic DC than by LC under any conditions tested. Other investigators also reported that mouse splenic DC failed to express CCL22 and CCL17 at least under some experimental settings in vivo (Tang and Cyster, 1999; Alferink et al, 2003). Second, the production of Th2-type chemokines was regulated in a complicated manner. Many factors influenced the production of CCL22 and CCL17 by LC either positively or negatively. In particular, IL-4 upregulated and IFN-g downregulated their production. The regulation of Th2-type chemokines in LC observed in this study was not simply because of the changes of cell viability or maturation status. For example, although GM-CSF increased cell viability and the expression of co-stimulatory molecules (Salgado et al, 1999a), it downregulated CCL17 production by LC. Moreover, TGF-b decreased cell viability and the expression of co-stimulatory molecules (Tada et al, 2001; and data not shown), but it upregulated CCL17 production by LC. Following cell maturation, LC emigrate from skin to draining lymph nodes (LN), but some of them are likely to remain in the skin to interact with T cells there (Katou et al, 2000, 2003). Cytokine availability in the microenvironment of LC may thus modify the balance of the production of Th1and Th2-type chemokines by LC in vivo. But reports on chemokine expression of LC or skin-derived DC in vivo are presently limited. Katou et al (2001) reported that DC-derived CCL22 and CCR4 on T cells may be involved in the formation of in vivo T cell–DC clusters in inflamed skin. Further, in vivo observations have so far suggested that LC may produce CCL22 and CCL17 as they migrate into T cell zones of LN (Tang and Cyster, 1999; Merad et al, 2000). As for the inflammatory human skin diseases such as atopic dermatitis (AD), DC with the mature phenotype are often found in the dermis of AD patients, and they are shown to express CCL22 (Vulcano et al, 2001). Moreover, CCL22 is expressed by dermal DC in AD-like skin lesions of NC/Nga mice, a model of human AD (Vestergaard et al, 1999). AD is characterized by enhancement of the expression of Th2 cytokines such as IL-4, as well as suppression of that of IFN-g in the acute phase (Vestergaard et al, 1999, 2000; Galli et al, 2000; Kakinuma et al, 2001, 2002a; Wakugawa et al, 2001). Interestingly, in line with this, our study showed an enhancing effect of IL-4 on the production of CCL22 and CCL17, together with an inhibitory effect of IFN-g on the production of both of them. In conclusion, this study strengthens the notion that LC are a unique subset of DC exhibiting some distinct characteristics. The fact that LC readily produce Th2-type chemokines in vitro suggests that LC may be an essential source of these chemokines in the skin in contrast to splenic DC, which produce them only in small amounts. In addition, the observation that CLA þ skin homing T cells typically coexpress CCR4 (Campbell et al, 1999) is in accordance with our finding. Neighboring keratinocytes are also capable of producing Th1- and Th2-type chemokines. Their production, however, is often regulated differently between keratinocytes and LC (Kakinuma et al, 2002b; Xiao et al, 2003b), further indicating a critical role for LC as the chemokine producer. Given that LC-derived Th1- and Th2- type chemokines exhibited functional activity, LC may indeed contribute to the recruitment of CXCR3- or CCR4-bearing T
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cells in vivo. Further investigations are necessary to elucidate in vivo relevance of our novel observations in human skin diseases. Materials and Methods Mice BALB/c female mice were purchased from Charles River Japan (Yokohama, Japan) and maintained until 8–12 weeks of age under specific pathogen-free conditions in the Animal Facilities of the University of Tokyo. All animal experiments were approved by the Animal research Committee of the University of Tokyo. Reagents and antibodies Mouse rIFN-g, rIL-1b, rIL-4, rIL-10, rIL13, rTNF-a, rTGF-b1, rCCL17, rCCL22, and rCXCL9 were purchased from R&D Systems (Minneapolis, Minnesota). rGM-CSF and rIL-12 were obtained from PeproTech (London, UK). rIL-18 was purchased from MBL (Nagoya, Japan). SAC (pansorbin) was obtained from Calbiochem (San Diego, California), and LPS (serotype 011: B4) was from Sigma-Aldrich (St Louis, Missouri). Poly(I:C) was obtained from Amersham Biosciences (Piscatway, New Jersey). rCXCL10 was provided by DAKO Japan (Kyoto, Japan). Goat anti-CXCL10 Ab, goat anti-CXCL9 Ab, goat anti-CCL17 Ab, goat anti-CCL22 Ab, and control goat IgG were obtained from GenzymeTechne (Cambridge, Massachusetts). Rat anti-mouse CD40 monoclonal antibody (mAb) (NA/LE, clone 3/23) was purchased from BD Biosciences (San Diego, California). Peroxidase-conjugated rabbit anti-goat IgG Ab was from MBL. Purification and culture of LC and splenic DC LC were prepared using the previously described panning method, and had a purity of over 95% (Salgado et al, 1999b; Tada et al, 2000). Briefly, mouse trunkal skin was separated and treated with dispase (3000 U per mL, Godo Shusei, Tokyo, Japan) in RPMI medium supplemented with 10% FCS (RPMI 10 medium) for 3 h at 371C. The epidermis was separated and incubated in RPMI 10 medium containing 0.025% deoxyribonuclease I (Sigma) for 20 min at room temperature. An epidermal cell suspension was obtained by vigorously pipetting the epidermal sheets, and was treated with mouse anti-mouse I-Ad mAb (1:600; Meiji Seika, Tokyo, Japan) in RPMI 10 medium for 45 min on ice. The cell suspension in RPMI 10 medium was then incubated in plates coated with goat anti-mouse IgG (Fc) (1:100; MP Biomedicals, Aurora, Ohio) for 45 min at 41C. After a wash, adherent cells were collected and designated purified LC. Splenic DC were purified from spleens of BALB/c mice. A single cell suspension was obtained by gentle teasing with forceps and rubbers and filtered through a nylon mesh. Erythrocytes were lysed by treatment with ammonium chloride. From the remaining unfractionated cell populations, CD11c þ DC were separated by positive magnetic selection using microbeads-conjugated hamster anti-mouse CD11c (Miltenyi Biotec, Belgisch Gladbach, Germany) and a magnetic cell separator according to the manufacturer’s instructions, routinely resulting in 490% purity of CD11c þ DC. LC and splenic DC were incubated in 96-well culture plates (1.5 106 cells per mL per 200 mL in each well) for up to 48 h, with or without various cytokines, Ab, or microbial components in a culture medium that consisted of RPMI 10 medium, 100 U per mL of penicillin G sodium, 100 mg per mL of streptomycin sulfate, and 0.25 mg per mL of amphotericin B. Semi-quantitative RT-PCR A semi-quantitative RT-PCR was used to assess the gene expression of CXCL10, CXCL9, CXCL11, CCL22, and CCL17 in murine LC and splenic DC. LC and splenic DC were prepared and cultured for 48 h as described above, and from the lysate of fresh and cultured LC and splenic DC, mRNA was isolated using the Quickprep Micro mRNA Purification Kit (Pharmacia Biotech, Uppsala, Sweden). Complementary DNA was synthesized by RT using random hexamers and mouse reverse transcriptase (First-Strand cDNA Synthesis Kit; Pharmacia Biotech). The PCR was performed in a programmable thermal cycler
124 : 2 FEBRUARY 2005 (Perkin-Elmer Co., Norwalk, Connecticut). The primers used for amplification of CXCL10 (Kuroda et al, 2001), CXCL9 (Park et al, 2001), CXCL11 (Lacroix-Lamande et al, 2002), CCL22 (Kuroda et al, 2001), CCL17 (Xiao et al, 2003a), and glyceraldehyde-3phosphate dehydrogenase (Xiao et al, 2003a) were described elsewhere. All reactions were run at 941C per 1 min, 561C per 1 min, 721C per 2 min for 35 cycles. The PCR was within the linear range. The PCR products were analyzed on 2.5% agarose gels and visualized by ethidium bromide staining and UV illumination.
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Manuscript received July 30, 2004; revised September 24, 2004; accepted for publication October 13, 2004 Address correspondence to: Akihiko Asahina, MD, Department of Dermatology, Faculty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan. Email: [email protected]. ac.jp
References Measurement of CXCL10 and CCL22 Culture supernatants were collected, stored at 201C, and subjected to the quantification of protein levels of CXCL10 and CCL22 by competitive ELISA inhibition assay as previously described (Kuroda et al, 2001). Briefly, ELISA plates were coated with 100 mL of rCXCL10 or rCCL22 at 250 ng per mL in phosphate-buffered saline (PBS) containing 0.1% BSA at 41C for 18 h. Then the wells were washed and blocked with 300 mL of RPMI 10 medium at 371C for 30 min. Standard solutions (40,000–625 pg per mL in RPMI 10 medium) or culture supernatants and equal volumes of 100 ng per mL of anti-CXCL10 Ab or 400 ng per mL of anti-CCL22 Ab were mixed in microtubes and incubated at 371C for 1 h. After the incubation, 200 mL of prepared standard or sample solution was transferred to each well of CXCL10- or CCL22-coated plates, and the plates were kept at 41C for 18 h. The wells were washed and 100 mL of peroxidase-conjugated rabbit anti-goat IgG Ab (diluted 1:2000 in PBS containing 0.05% Tween 20 and 1% BSA) was added. The plates were incubated at 371C for 2 h. Then they were washed and 100 mL of substrate (0.04% o-phenylenediamine, 0.01% H2O2 in 50 mM disodium hydrogen phosphate, and 150 mM citric acid, pH 5) was added to the wells. The plates were incubated at room temperature for 30 min. The enzyme reaction was stopped by the addition of 25 mL of 8 N H2SO4, and the absorbance at 490 nm was measured. Measurement of CXCL9, CXCL11, and CCL17 Culture supernatants were collected, stored at 201C, and subjected to the quantification of protein levels of CXCL9, CXCL11, and CCL17 by ELISA using commercially available mouse CXCL9, CXCL11, and CCL17 immunoassay kits (R&D Systems), respectively, according to the manufacturer’s instructions. Each sample was tested in duplicate. Chemotaxis assay Chemotaxis assay was carried out using mouse CCR4- or CXCR3-transfected 2B4 T cells (Gao et al, 2003). Chemotaxis of the transfectants was assessed in 24-well transwells equipped with 5 mM pore polycarbonate membranes (Transwell, Corning Costar Corp., Cambridge, Masschusetts). Cells (1 106) suspended in 100 mL of RPMI 10 medium were transferred to the upper chamber. For the chemotaxis of CCR4 transfectant, the supernatants of 48 h cultured unstimulated LC and splenic DC were used. Culture supernatants and RPMI 10 medium with or without recombinant chemokine were preincubated with or without 2 mg per mL of anti-CCL17 mAb, or 10 mg per mL of anti-CCL22 mAb, or both for 30 min at room temperature. They were then transferred to the lower chamber in 600 mL for each group. After an incubation period of 3 h at 371C, the upper chambers were removed, and the cells in the lower chamber of each well were collected and counted using a Coulter Counter (Beckman Coulter, Jersey City, New Jersey). Chemotaxis of CXCR3 transfectant was carried out in the same way using the supernatants of 48 h IFN-g-stimulated LC and splenic DC. In this case, to neutralize the activity of chemokines, 4 mg per mL of anti-CXCL10 mAb and 2 mg per mL of anti-CXCL9 mAb were used. All assays were carried out in triplicate. Statistical analysis Student’s t-test was used to analyze the results, and a p-value of less than 0.05 was considered statistically significant. DOI: 10.1111/j.0022-202X.2004.23607.x
Alferink J, Lieberam I, Reindl W, et al: Compartmentalized production of CCL17 in vivo: Strong inducibility in peripheral dendritic cells contrasts selective absence from the spleen. J Exp Med 197:585–599, 2003 Baggiolini M: Chemokines and leukocyte traffic. Nature 392:565–568, 1998 Banchereau J, Steinman RM: Dendritic cells and the control of immunity. Nature 392:245–252, 1998 Banchereau J, Briere F, Caux C, et al: Immunobiology of dendritic cells. Annu Rev Immunol 18:767–811, 2000 Bonecchi BR, Bianchi G, Bordignon PP, et al: Differential expression of chemokine receptors and chemotactic responsiveness of Type 1 T helper cells (Th1s) and Th2s. J Exp Med 187:129–134, 1998 Campbell JJ, Haraldsen G, Pan J, et al: The chemokine receptor CCR4 in vascular recognition by cutaneous but not intestinal memory T cells. Nature 400:776–780, 1999 Fujita H, Asahina A, Gao P, Fujiwara H, Tamaki K: Expression and regulation of RANTES/CCL5, MIP-1a/CCL3, and MIP-1b/CCL4 in mouse Langerhans cells. J Invest Dermatol 122:1331–1333, 2004a Fujita H, Asahina A, Mitsui H, Tamaki K: Langerhans cells exhibit low responsiveness to double-stranded RNA. Biochem Biophys Res Commun 319: 832–839, 2004b Galli G, Chantry D, Annunziato F, et al: Macrophage-derived chemokine production by activated human T cells in vitro and in vivo: Preferential association with the production of type 2 cytokines. Eur J Immunol 30: 204–210, 2000 Gao P, Zhou X-Y, Yashiro-Ohtani Y, et al: The unique target specificity of a nonpeptide chemokine receptor antagonist: Selective blockade of two Th1 chemokine receptors CCR5 and CXCR3. J Leukoc Biol 73:273–280, 2003 Iellem A, Mariani M, Lang R, Recalde H, Panina-Bordignon P, Sinigaglia F, D’Ambrosio D: Unique chemotactic response profile and specific expression of chemokine receptors CCR4 and CCR8 by CD4 þ CD25 þ regulatory T cells. J Exp Med 194:847–854, 2001 Imai T, Nagata M, Takagi S, et al: Selective recruitment of CCR4-bearing Th2 cells toward antigen-presenting cells by the CC chemokines thymus and activation-regulated chemokine and macrophage-derived chemokine. Int Immunol 11:81–88, 1999 Kakinuma T, Nakamura K, Wakugawa M, et al: Thymus and activation-regulated chemokine in atopic dermatitis: Serum thymus and activation-regulated chemokine level is related with disease activity. J Allergy Clin Immunol 107:535–541, 2001 Kakinuma T, Nakamura K, Wakugawa M, et al: Serum macrophage-derived chemokine (MDC) levels are closely related with the activity of atopic dermatitis. Clin Exp Immunol 127:270–273, 2002a Kakinuma T, Nakamura K, Wakugawa M, et al: IL-4, but not IL-13, modulates TARC(thymus and activation-regulated chemokine)/CCL17 and IP-10(interferon-induced protein of 10 kDa)/CXCL10 release by TNF-a and IFN-g in HaCaT cell line. Cytokine 20:1–6, 2002b Katou F, Ohtani H, Saaristo A, Nagura H, Motegi K: Immunological activation of dermal Langerhans cells in contact with lymphocytes in a model of human inflamed skin. Am J Pathol 156:519–527, 2000 Katou F, Ohtani H, Nakayama T, et al: Macrophage-derived chemokine (MDC/ CCL22) and CCR4 are involved in the formation of T lymphocyte-dendritic cell clusters in human inflamed skin and secondary lymphoid tissue. Am J Pathol 158:1263–1270, 2001 Katou F, Ohtani H, Nakayama T, Nagura H, Yoshie O, Motegi K: Differential expression of CCL19 by DC-lamp þ mature dendritic cells in human lymph node versus chronically inflamed skin. J Pathol 199:98–106, 2003 Kuroda E, Sugiura T, Okada K, Zeki K, Yamashita U: Prostaglandin E2 up-regulates macrophage-derived chemokine production but suppresses IFNinducible protein-10 production by APC. J Immunol 166:1650–1658, 2001 Lacroix-Lamande S, Mancassola R, Naciri M, Laurent F: Role of gamma interferon in chemokine expression in the ileum of mice and in a murine intestinal epithelial cell line after Crystosporidium parvum infection. Infect Immun 70:2090–2099, 2002 Liu Y-J: Dendritic cell subsets and lineages, and their functions in innate and adaptive immunity. Cell 106:259–262, 2001
350 FUJITA ET AL Merad M, Fong L, Bogenberger J, Engleman EG: Differentiation of myeloid dendritic cells into CD8a-positive dendritic cells in vivo. Blood 96:1865–1872, 2000 Mitsui H, Watanabe T, Saeki H, et al: Differential expression and function of Tolllike receptors in Langerhans cells: Comparison with splenic dendritic cells. J Invest Dermatol 122:95–102, 2004 Olson TS, Ley K: Chemokines and chemokine receptors in leukocyte trafficking. Am J Physiol Regul Integr Comp Physiol 283:R7–R28, 2002 Park MK, Hoffmann KF, Cheever AW, Amichay D, Wynn TA, Farber JM: Patterns of chemokine expression in models of Schistosoma mansoni inflammation and infection reveal relationships between type 1 and type 2 responses chemokines in vivo. Infect Immun 69:6755–6768, 2001 Penna G, Vulcano M, Roncari A, Facchetti F, Sozzani S, Adorini L: Differential chemokine production by myeloid and plasmacytoid dendritic cells. J Immunol 169:6673–6676, 2002a Penna G, Vulcano M, Sozzani S, Adorini L: Differential migration behavior and chemokine production by myeloid and plasmacytoid dendritic cells. Hum Immunol 63:1164–1171, 2002b Reis e Sousa C, Sher A, Kaye P: The role of dendritic cells in the induction and regulation of immunity to microbial infection. Curr Opin Immunol 11: 392–399, 1999 Salgado CG, Nakamura K, Sugaya M, et al: Differential effects of cytokines and immunosuppressive drugs on CD40, B7-1, B7-2 expression on purified epidermal Langerhans cells. J Invest Dermatol 113:1021–1027, 1999a Salgado CG, Nakamura K, Sugaya M, et al: Functional CD40 ligand is expressed on epidermal Langerhans cells. J Leukoc Biol 66:281–285, 1999b Sallusto F, Lanzavecchia A, Mackay CR: Chemokines and chemokine receptors in T-cell priming and Th1/Th2-mediated responses. Immunol Today 19:568–574, 1998 Sallusto F, Palermo B, Lenig D, et al: Distinct patterns and kinetics of chemokine production regulate dendritic cell function. Eur J Immunol 29:1617–1625, 1999 Sebastiani S, Allavena P, Albanesi C, et al: Chemokine receptor expression and function in CD4 þ T lymphocytes with regulatory activity. J Immunol 166:996–1002, 2001 Tada Y, Asahina A, Fujita H, Mitsui H, Torii H, Watanabe T, Tamaki K: Differential effects of LPS and TGF-b on the production of IL-6 and IL-12 by La-
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY ngerhans cells, splenic dendritic cells, and macrophages. Cytokine 25:155–161, 2004 Tada Y, Asahina A, Nakamura K, Tomura M, Fujiwara H, Tamaki K: Granulocyte/ macrophage colony-stimulating factor inhibits IL-12 production of mouse Langerhans cells. J Immunol 164:5113–5119, 2000 Tada Y, Asahina A, Nakamura K, Miyazono K, Tomura M, Fujiwara H, Tamaki K: Transforming growth factor-b up-regulates CD40-engaged IL-12 production of mouse Langerhans Cells. Eur J Immunol 31:294–300, 2001 Tada Y, Asahina A, Fujita H, Sugaya M, Tamaki K: Langerhans cells do not produce IFN-g. J Invest Dermatol 120:891–892, 2003 Tang HL, Cyster JG: Chemokine up-regulation and activated T cell attraction by maturing dendritic cells. Science 284:819–822, 1999 Vestergaard C, Bang K, Gesser B, Yoneyama H, Matsushima K, Larsen CG: A Th2 chemokine, TARC, produced by keratinocytes may recruit CLA þ CCR4 þ lymphocytes into lesional atopic dermatitis skin. J Invest Dermatol 115: 640–646, 2000 Vestergaard C, Yoneyama H, Murai M, et al: Overproduction of Th2-specific chemokines in NC/Nga mice exhibiting atopic dermatitis-like lesions. J Clin Invest 104:1097–1105, 1999 Vulcano M, Albansei C, Stoppacciaro A, et al: Dendritic cells as a major source of macrophage-derived chemokine/CCL22 in vitro and in vivo. Eur J Immunol 31:812–822, 2001 Wakugawa M, Nakamura K, Kakinuma T, Onai N, Matsushima K, Tamaki K: CC chemokine receptor 4 expression on peripheral blood CD4 þ T cells reflects disease activity of atopic dermatitis. J Invest Dermatol 117: 188–196, 2001 Wu L, Vandenabeele S, Georgopoulos K: Derivation of dendritic cells from myeloid and lymphoid precursors. Int Rev Immunol 20:117–135, 2001 Xiao T, Fujita H, Saeki H, et al: Thymus and activation-regulated chemokine (TARC/CCL17) produced by mouse epidermal Langerhans cells is upregulated by TNF-a and IL-4 and downregulated by IFN-g. Cytokine 23: 126–132, 2003a Xiao T, Kagami S, Saeki H, et al: Both IL-4 and IL-13 inhibit the TNF-a and IFN-g enhanced MDC production in a human keratinocyte cell line, HaCaT cells. J Dermatol Sci 31:111–117, 2003b Zlotnik A, Yoshie O: Chemokines: A new classification system and their role in immunity. Immunity 12:121–127, 2000
Medical University, Fukushima, Japan; zDepartment of Oncology, Graduate School of Medicine, Osaka University, Osaka, Japan
Some chemokines specifically attract T helper 1 (Th1) cells, whereas others attract T helper 2 (Th2) cells. In this study, we investigated the capacity of Langerhans cells (LC) to produce Th1- and Th2-type chemokines in comparison with that of splenic CD11c þ dendritic cells (DC). We prepared highly purified (495%) LC from BALB/c mouse skin using the panning method. With regard to Th1-type chemokines, exogenous stimulus, such as interferon-c (IFN-c), lipopolysaccharide, or polyinosinic–polycytidylic acid, was mandatory for the production of substantial amounts of CXCL10, CXCL9, and CXCL11 both in LC and splenic DC. LC, as a whole, exhibited low ability to produce Th1-type chemokines in comparison with splenic DC. As for Th2-type chemokines, LC, but not splenic DC, produced high levels of CCL22 and CCL17 constitutively during culture even without exogenous stimuli. The production of Th2-type chemokines was regulated in a complicated manner. In particular, interleukin-4 upregulated, and IFN-c downregulated both CCL22 and CCL17 production by LC. Of note, LC produced much more amounts of Th2-type chemokines than splenic DC under any conditions tested. Finally, Th1- and Th2-type chemokines produced by LC were shown to be functional using chemokine receptor-transfected-2B4 T cells. The high production of CC chemokine receptor 4 ligands by LC in the absence of IFN-c may be an important character discriminating LC from other DC.
Key words: CCL17/CCL22/CXCL10/IFN-g/skin J Invest Dermatol 124:343–350, 2005
Chemokines are chemotactic cytokines that play an important role in regulating leukocyte migration (Baggiolini, 1998; Zlotnik and Yoshie, 2000). T helper 1 (Th1) and T helper 2 (Th2) cells express different types of chemokine receptors and respond to distinct types of chemokines. CXC chemokine receptor 3 (CXCR3) is predominantly expressed on Th1 cells (Bonecchi et al, 1998; Sallusto et al, 1998; Zlotnik and Yoshie, 2000). Interferon (IFN-g)-inducible protein-10 (IP10)/CXCL10 (Zlotnik and Yoshie, 2000), monokine induced by IFN-g(Mig)/CXCL9 (Zlotnik and Yoshie, 2000), and IFNinducible T cell a chemoattractant (I-TAC)/CXCL11 (Zlotnik and Yoshie, 2000) all bind to CXCR3, and thus promote the migration of Th1 cells. On the other hand, CC chemokine receptor 4 (CCR4) is predominantly expressed on Th2 cells (Sallusto et al, 1998; Imai et al, 1999; Zlotnik and Yoshie, 2000), CD4 þ CD25 þ T cells with regulatory activity (Iellem et al, 2001; Sebastiani et al, 2001), and also on skin homing memory T cells positive for cutaneous lymphocyte antigen
(CLA) (Campbell et al, 1999). Thymus and activation-regulated chemokine (TARC)/CCL17 (Zlotnik and Yoshie, 2000) as well as macrophage-derived chemokine (MDC)/CCL22 (Zlotnik and Yoshie, 2000) promote the migration of these cells via CCR4. Dendritic cells (DC) are professional antigen-presenting cells with a unique ability to stimulate naı¨ve T cells and initiate primary immune responses (Liu, 2001). Distinct DC subpopulations exhibit distinct functions leading to different immune regulations (Banchereau et al, 2000; Liu, 2001), and their heterogeneity reflects the organs they reside in (Reis e Sousa et al, 1999). Langerhans cells (LC) are a specific subtype of DC localized in the epidermis (Banchereau and Steinman, 1998). LC differ from other DC in possessing Birbeck granules, lacking functional mannose receptors, expressing E-cadherin, and being dependent on transforming growth factor-b (TGF-b) for their differentiation (Banchereau and Steinman, 1998; Banchereau et al, 2000; Wu et al, 2001). As for functional characteristics of LC in comparison with those of splenic DC, we have reported the unique modality in the regulation of interleukin-12 (IL-12) production (Tada et al, 2000, 2001) and inability of IFN-g production (Tada et al, 2003) in LC. Furthermore, we have found substantial differences in responsiveness to microorganisms through Toll-like receptors between LC and splenic DC (Fujita et al, 2004b; Mitsui et al, 2004; Tada et al, 2004).
Abbreviations: Ab, antibody; DC, dendritic cells; LC, Langerhans cells; LN, lymph nodes; LPS, lipopolysaccharide; GM-CSF, granulocyte macrophage colony-stimulating factor; IFN, interferon; IL, interleukin; mAb, monoclonal antibody; Poly(IC),polyinosinic–polycytidylic acid; SAC, Staphylococcus aureus Cowen 1; Th1, T helper 1; Th2, T helper 2; TNF-a, tumor necrosis factor-a; TGF-b, transforming growth factor-b
Copyright r 2005 by The Society for Investigative Dermatology, Inc.
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Not only are DC attracted by chemokines but DC are also an abundant and strategic source of various chemokines (Sallusto et al, 1999). In addition, a recent report disclosed that the property of chemokine production is diverse among DC subsets (Penna et al, 2002a, b). We have recently reported the expression and regulation of inflammatory chemokines, such as CCL3, CCL4, and CCL5, in mouse LC (Fujita et al, 2004a). Apart from this study, however, chemokine production by resident LC has not been well described. In particular, it is unknown whether LC are capable of producing the Th1-type chemokines, CXCL10, CXCL9, and CXCL11. In this study, in order to further define functional properties of LC to shape immune responses, we focused on the expression and regulation of Th1- and Th2-type chemokines in mouse LC. For this purpose, we prepared highly purified LC population (495%) from mouse skin, which was phenotypically immature and acquired mature phenotypes during culture in vitro (Salgado et al, 1999a, b; Tada et al, 2000). Furthermore, we used splenic CD11c þ DC for comparison with LC, and illustrated distinct characteristics of LC in chemokine production.
Results Messenger RNA for Th1-type chemokines in LC and splenic DC CXCL10, CXCL9, and CXCL11 are Th1-type chemokines that are structurally related and share the common receptor CXCR3 (Olson and Ley, 2002). To investigate whether LC and splenic DC express their mRNA, semiquantitative RT-PCR analysis was performed. The mRNA of none of these Th1-type chemokines was detected in fresh LC (Fig 1). After 48 h culture, LC came to express CXCL10
Figure 1 RT-PCR analysis of CXCL10, CXCL9, and CXCL11 gene expression in freshly isolated Langerhans cells (fLC), 48 h cultured LC (cLC), interferon-c (IFN-c)-stimulated cLC (c-cLC), freshly isolated splenic dendritic cells (fDC), 48 h cultured splenic DC (cDC), and IFN-cstimulated cDC (c-cDC). LC and splenic DC were purified and cultured for 48 h in the absence or presence of 100 ng per mL of IFN-g. Samples from fresh and cultured LC and splenic DC were collected and mRNA expression was analyzed using specific primers for each chemokine. CXCL10, CXCL9, and CXCL11 mRNA expression was hardly detectable in fLC. In cLC, mRNA expression of CXCL10 and CXCL11, but not CXCL9, was induced. CXCL9 mRNA was strongly expressed only in g-cLC. In addition, mRNA for CXCL10 and CXCL11 was also strongly expressed in g-cLC. In splenic DC, mRNA for these T helper 1(Th1)-type chemokines was almost undetectable both in fDC and cDC. When stimulated with IFN-g, CXCL10, CXCL9, and CXCL11 mRNA expression was induced. Data are representative of three independent experiments.
and CXCL11, but not CXCL9, mRNA. Expression of CXCL9 mRNA was strongly induced by IFN-g. These results indicate that mRNA expression of CXCL9 is regulated differently from that of CXCL10 and CXCL11 during the maturation of LC. In the case of splenic DC, the expression of mRNA for all three chemokines was negligible in fresh and unstimulated cultured cells. mRNA for CXCL10, CXCL9, and CXCL11 was, however, induced by IFN-g. Time-course measurement of the production of Th1type chemokines by LC and splenic DC To determine whether Th1-type chemokines were produced at protein level by cultured LC and splenic DC and secreted into culture media, ELISA was carried out using media cultured for 0, 12, 24, 36, and 48 h. As shown in Fig 2, without IFN-g treatment, LC and splenic DC produced small amounts of Th1-type chemokines. In contrast, LC and splenic DC produced substantial amounts of CXCL10, CXCL9, and CXCL11 time dependently by stimulation with IFN-g (100 ng per mL). Splenic DC have, on the whole, an ability to produce larger amounts of Th1-type chemokines than LC. Biological activity of Th1-type chemokines produced by LC and splenic DC To ascertain whether Th1-type chemokines produced by LC and splenic DC were functional, culture supernatants were tested for their capacity to induce the migration of CXCR3-transfected 2B4 T cells (Gao et al, 2003). As demonstrated above, CXCR3 ligands are produced at large amounts by stimulation with IFN-g both in LC and splenic DC. Indeed, the supernatants of 48 h cultured unstimulated LC and splenic DC induced only minimal migration of these cells (data not shown). Therefore, we used the supernatants of 48 h cultured IFN-g-treated LC and splenic DC for the chemotaxis of CXCR3 transfectant. As shown in Fig 3, the supernatant of LC exhibited considerable chemotactic activity to CXCR3 transfectant. Neutralizing anti-CXCL10 antibody (Ab) and anti-CXCL9 Ab partially blocked the migration, respectively. When both of the Abs were added simultaneously, the migration of transfectant was almost completely inhibited. The supernatant of splenic DC induced more prominent migration of the transfectant than that of LC. In the same way, this migration was partially blocked by anti-CXCL10 Ab or antiCXCL9 Ab, respectively, and almost completely blocked by both of them. Isotype-matched control of neutralizing Ab did not affect the migration of transfectant (data not shown). These results indicate that at least CXCL10 and CXCL9 produced by LC and splenic DC actually possess chemotactic activity, and the contribution of CXCL11 for the migration of the transfectant seems to be minimal both in LC and splenic DC. Messenger RNA for Th2-type chemokines in LC and splenic DC CCL22 and CCL17 are Th2-type chemokines that share the receptor CCR4. Therefore, we analyzed the mRNA expression of CCL22 and CCL17 in LC and splenic DC by semi-quantitative RT-PCR. As shown in Fig 4, CCL22 and CCL17 showed almost the same pattern of mRNA expression in fresh and cultured LC. The mRNA of both chemokines was only faintly detected in fresh LC, and strongly expressed after 48 h culture. The results obtained
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Figure 2 Production of T helper 1-type chemokines during culture of Langerhans cells (LC) and splenic dendritic cells (DC). Purified LC and splenic DC were cultured with or without interferon-g (IFN-g), and the concentration of CXCL10, CXCL9, and CXCL11 was measured at different time points (0, 12, 24, 36, 48 h) in the supernatants by ELISA. Representative data of three independent experiments.
for CCL17 were consistent with those reported previously (Xiao et al, 2003a). In sharp contrast, fresh splenic DC did not express mRNA for Th2-type chemokines. Of note, mRNA for CCL22 and CCL17 was not induced in splenic DC even after culture. Time-course measurement of the production of Th2type chemokines by LC and splenic DC CCL22 and CCL17 production by LC and splenic DC was determined
Figure 3 Chemotaxis of mCXCR3-transfected 2B4 T cells. Langerhans cells (LC) and splenic dendritic cells (DC) were cultured for 48 h with 100 ng per mL of interferon-g (IFN-g) and the supernatants were collected. The culture supernatants were preincubated with or without neutralizing anti-chemokine monoclonal antibody (mAb) indicated in the figure for 30 min, and assessed for chemotactic activity to CXCR3 transfectant. RPMI 10 medium alone served as a negative control. RPMI 10 medium containing recombinant chemokine served as a positive control. The supernatants of IFN-g-stimulated LC and splenic DC exhibited chemotactic activity to CXCR3 transfectant, which is mediated at least by CXCL10 and CXCL9. Mean (SD) (n ¼ 3). Data are representative of three independent experiments.
by ELISA using media cultured for 0, 12, 24, 36, and 48 h. As shown in Fig 5, LC produced substantial levels of these chemokines in a time-dependent manner even in the absence of exogenous stimulation. Conversely, splenic DC in the same condition failed to produce Th2-type chemokines at any time point up to 48 h. These results were in parallel with mRNA expression analyzed by RT-PCR. Biological activity of Th2-type chemokines produced by LC and splenic DC To investigate whether Th2-type chemokines produced by LC and splenic DC have chemotactic activity to T cells, we carried out a chemotaxis assay using CCR4-transfected 2B4 T cells (Gao et al, 2003). The results shown in Fig 6 demonstrate that the supernatant of 48 h cultured unstimulated LC induced the migration of a considerable number of CCR4 transfectant. This migration was partially blocked by neutralizing anti-CCL17 Ab and anti-CCL22 Ab, respectively. When both of the Ab were
Figure 4 RT-PCR analysis of CCL22 and CCL17 gene expression in freshly isolated Langerhans cells (fLC), 48 h cultured LC (cLC), freshly isolated splenic dendritic cells (fDC), and 48 h cultured splenic DC (cDC). LC and splenic DC were purified and cultured for 48 h without exogenous stimulation. mRNA of fresh and cultured LC and splenic DC was extracted, and mRNA expression was analyzed using specific primers for each chemokine. CCL22 and CCL17 mRNA was expressed weakly in fLC, and strongly in cLC. In splenic DC, mRNA for CCL22 and CCL17 was not detected both in fDC and cDC. Data are representative of two independent experiments.
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(1:10000), and polyinosinic–polycytidylic acid (Poly(I:C)) (20 mg per mL) induced CXCL10 production in LC. In the case of CXCL9 and CXCL11, of all the stimuli tested, only IFN-g induced the production of these chemokines in LC (Fig
Figure 5 Production of T helper 2-type chemokines during culture of Langerhans cells (LC) and splenic dendritic cells (DC). Purified LC and splenic DC were cultured without exogenous stimulation, and the concentration of CCL22 and CCL17 was measured at different time points (0, 12, 24, 36, 48 h) in the supernatants by ELISA. Data are representative of three independent experiments.
added simultaneously, the migration of the transfectant was inhibited almost completely. On the other hand, the supernatant of 48 h cultured splenic DC exhibited minimal chemotactic activity to CCR4 transfectant. These results confirm that CCL22 and CCL17 produced by LC are functional. Production of Th1-type chemokines and its regulation in LC and splenic DC As mentioned above, IFN-g-stimulated LC and splenic DC produced a substantial level of Th1-type chemokines. To investigate the production of Th1-type chemokines and their regulation in LC and splenic DC, we cultured them with various stimuli. As shown in Fig 7a, in addition to IFN-g, IL-12 (2 ng per mL), lipopolysaccharide (LPS) (1 mg per mL), Staphylococcus aureus Cowen 1 (SAC)
Figure 6 Chemotaxis of mCCR4-transfected 2B4 T cells. Langerhans cells (LC) and splenic dendritic cells (DC) were cultured for 48 h without stimulation and the supernatants were collected. The culture supernatants were preincubated with or without neutralizing anti-chemokine monoclonal antibody (mAb) indicated in the figure for 30 min, and assessed for chemotactic activity to CCR4 transfectant. RPMI 10 medium alone served as a negative control. RPMI 10 medium containing recombinant chemokine served as a positive control. The supernatant of LC, but not of splenic DC, exhibited CCL22- and CCL17-dependent chemotactic activity to CCR4 transfectant. Mean (SD) (n ¼ 3). Data are representative of three independent experiments.
Figure 7 Regulation of T helper 1-type chemokines produced by Langerhans cells (LC) and splenic dendritic cells (DC). LC (&) and splenic DC (’) were purified and cultured (1.5 106 cells per mL per 200 mL in each well) for 48 h with or without various stimuli. The supernatants were collected and the concentration of CXCL10 (a), CXCL9 (b), and CXCL11 (c) was measured by ELISA. CXCL10 production by LC was induced by interferon-g (IFN-g), interleukin (IL)-12, lipopolysaccharide (LPS), Staphylococcus aureus Cowen 1 (SAC), and polyinosinic–polycytidylic acid (Poly(I:C)). In the case of CXCL9 and CXCL11, only IFN-g induced their production by LC. In the case of splenic DC, the production of CXCL10, CXCL9, and CXCL11 was induced by IFN-g, IL-18, LPS, and Poly(I:C). Results are the mean (SD) (n ¼ 4). Significant increase (po0.05) compared with the unstimulated group. Data are representative of four independent experiments.
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7b, c). As for splenic DC, the mode of the regulation of Th1-type chemokines was identical among CXCL10, CXCL9, and CXCL11 (Fig 7a–c). Splenic DC produced CXCL10, CXCL9, and CXCL11 in response to IFN-g, IL-18 (100 ng per mL), LPS, and Poly(I:C). Interestingly, splenic DC, as a whole, exhibited high ability to produce Th1-type chemokines in comparison with LC. Production of Th2-type chemokines and its regulation in LC and splenic DC As demonstrated above, LC, but not splenic DC, produced Th2-type chemokines constitutively during culture even without stimulation. Here, we further
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examined the production of CCL22 and CCL17 in response to various stimuli. The patterns of the regulation of Th2-type chemokines were rather complicated. As shown in Fig 8a, CCL22 production by LC was upregulated by tumor necrosis factor-a (TNF-a) (10 ng per mL), granulocyte macrophage colony-stimulating factor (GM-CSF) (1 ng per mL), IL-1b (10 ng per mL), IL-4 (10 ng per mL), and agonistic antiCD40 Ab (20 mg per mL). On the other hand, IFN-g, IL-10 (10 ng per mL), and Poly(I:C) had a significant inhibitory effect on its production. Other stimuli, such as TGF-b (1 ng per mL), IL-12, IL-13 (10 ng per mL), IL-18, LPS, and SAC, failed to affect the production of CCL22 in cultured LC. The mode of the regulation of CCL17 production in LC was similar to that of CCL22. Stimulation with TGF-b, TNF-a, IL-4, IL-13, and anti-CD40 Ab enhanced CCL17 production significantly. In contrast, its production level decreased significantly in the presence of GM-CSF, IFN-g, IL-10, LPS, SAC, and Poly (I:C). IL-12 and IL-18 did not cause significant changes in CCL17 production (Fig 8b). In the case of splenic DC, the production of CCL22 and CCL17 was found to be regulated in almost the same way (Fig 8a, b). TNF-a, GM-CSF, IL-1b, IL-4, IL-12, IL-13, IL-18, and anti-CD40 Ab induced CCL22 and CCL17 production to some extent. When splenic DC were stimulated with microbial components, such as LPS, SAC, and Poly(I:C), however, the production of CCL22 and CCL17 was induced differently. LPS and SAC induced a high level of CCL22 production, but they did not affect CCL17 production. Poly(I:C) induced not only CCL22 but also CCL17 production, although to a much lesser extent. Another striking finding was that the amounts of Th2-type chemokines produced by splenic DC were much smaller than those produced by LC under any conditions tested.
Discussion
Figure 8 Regulation of T helper 2-type chemokines produced by Langerhans cells (LC) and splenic dendritic cells (DC). LC (&) and splenic DC (’) were purified and cultured (1.5 106 cells per mL per 200 mL in each well) for 48 h with or without various stimuli. The supernatants were collected and assessed for the concentration of CCL22 (a), CCL17 (b) by ELISA. (a) CCL22 production by LC was upregulated by tumor necrosis factor-a (TNF-a), granulocyte macrophage colonystimulating factor (GM-CSF), interleukin (IL)-1b, IL-4, and anti-CD40 antibody (Ab), and downregulated by interferon-g (IFN-g), IL-10, and polyinosinic–polycytidylic acid (Poly(I:C)). In splenic DC, its production was induced by TNF-a, GM-CSF, IL-1b, IL-4, IL-12, IL-13, IL-18, antiCD40 Ab, lipopolysaccharide (LPS), Staphylococcus aureus Cowen 1 (SAC), and Poly(I:C). (b) CCL17 production by LC was upregulated by transforming growth factor-b (TGF-b), TNF-a, IL-1b, IL-4, IL-13, and anti-CD40 Ab, and downregulated by GM-CSF, IFN-g, IL-10, LPS, SAC, and Poly(I:C). In splenic DC, its production was induced by TNFa, GM-CSF, IL-1b, IL-4, IL-12, IL-13, IL-18, anti-CD40 Ab, and Poly(I:C). Results are the mean (SD) (n ¼ 4).Significant increase (po0.05) compared with the unstimulated group. #, Significant decrease (po0.05) compared with the unstimulated group. Data are representative of four independent experiments.
In this study, we demonstrated that the chemokine production represents an additional diversity between the two discrete populations of mouse DC, epidermal LC, and splenic DC. LC indeed have the ability to produce functional Th1and Th2-type chemokines. With regard to Th1-type chemokines, exogenous stimulation was mandatory for their production both in LC and splenic DC. Only limited stimuli, such as IFN-g, LPS, and Poly(I:C), induced the production of Th1-type chemokines, namely, CXCL10, CXCL9, and CXCL11, both in LC and splenic DC. But the amounts of CXCL10, CXCL9, and CXCL11, produced by LC in the presence of such stimuli, were lower than those produced by splenic DC. Among the Th1-type chemokines, LC produced CXCL10 in response to IFN-g, IL-12, and microbial components. Interestingly, only IFN-g, but not microbial components, was able to induce CXCL9 and CXCL11 production in LC. These findings further confirmed our previous observations that LC exhibited low responsiveness to microbial components (Fujita et al, 2004b; Mitsui et al, 2004; Tada et al, 2004). As for Th2-type chemokines, the mode of the regulation was different from that of Th1-type chemokines. First, even without exogenous stimulation, LC, but not splenic DC, were able to produce CCL22 and CCL17 constitutively during culture. Although some of the stimuli induced the production of CCL22 and CCL17 in splenic DC,
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they were produced in much smaller amounts by splenic DC than by LC under any conditions tested. Other investigators also reported that mouse splenic DC failed to express CCL22 and CCL17 at least under some experimental settings in vivo (Tang and Cyster, 1999; Alferink et al, 2003). Second, the production of Th2-type chemokines was regulated in a complicated manner. Many factors influenced the production of CCL22 and CCL17 by LC either positively or negatively. In particular, IL-4 upregulated and IFN-g downregulated their production. The regulation of Th2-type chemokines in LC observed in this study was not simply because of the changes of cell viability or maturation status. For example, although GM-CSF increased cell viability and the expression of co-stimulatory molecules (Salgado et al, 1999a), it downregulated CCL17 production by LC. Moreover, TGF-b decreased cell viability and the expression of co-stimulatory molecules (Tada et al, 2001; and data not shown), but it upregulated CCL17 production by LC. Following cell maturation, LC emigrate from skin to draining lymph nodes (LN), but some of them are likely to remain in the skin to interact with T cells there (Katou et al, 2000, 2003). Cytokine availability in the microenvironment of LC may thus modify the balance of the production of Th1and Th2-type chemokines by LC in vivo. But reports on chemokine expression of LC or skin-derived DC in vivo are presently limited. Katou et al (2001) reported that DC-derived CCL22 and CCR4 on T cells may be involved in the formation of in vivo T cell–DC clusters in inflamed skin. Further, in vivo observations have so far suggested that LC may produce CCL22 and CCL17 as they migrate into T cell zones of LN (Tang and Cyster, 1999; Merad et al, 2000). As for the inflammatory human skin diseases such as atopic dermatitis (AD), DC with the mature phenotype are often found in the dermis of AD patients, and they are shown to express CCL22 (Vulcano et al, 2001). Moreover, CCL22 is expressed by dermal DC in AD-like skin lesions of NC/Nga mice, a model of human AD (Vestergaard et al, 1999). AD is characterized by enhancement of the expression of Th2 cytokines such as IL-4, as well as suppression of that of IFN-g in the acute phase (Vestergaard et al, 1999, 2000; Galli et al, 2000; Kakinuma et al, 2001, 2002a; Wakugawa et al, 2001). Interestingly, in line with this, our study showed an enhancing effect of IL-4 on the production of CCL22 and CCL17, together with an inhibitory effect of IFN-g on the production of both of them. In conclusion, this study strengthens the notion that LC are a unique subset of DC exhibiting some distinct characteristics. The fact that LC readily produce Th2-type chemokines in vitro suggests that LC may be an essential source of these chemokines in the skin in contrast to splenic DC, which produce them only in small amounts. In addition, the observation that CLA þ skin homing T cells typically coexpress CCR4 (Campbell et al, 1999) is in accordance with our finding. Neighboring keratinocytes are also capable of producing Th1- and Th2-type chemokines. Their production, however, is often regulated differently between keratinocytes and LC (Kakinuma et al, 2002b; Xiao et al, 2003b), further indicating a critical role for LC as the chemokine producer. Given that LC-derived Th1- and Th2- type chemokines exhibited functional activity, LC may indeed contribute to the recruitment of CXCR3- or CCR4-bearing T
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cells in vivo. Further investigations are necessary to elucidate in vivo relevance of our novel observations in human skin diseases. Materials and Methods Mice BALB/c female mice were purchased from Charles River Japan (Yokohama, Japan) and maintained until 8–12 weeks of age under specific pathogen-free conditions in the Animal Facilities of the University of Tokyo. All animal experiments were approved by the Animal research Committee of the University of Tokyo. Reagents and antibodies Mouse rIFN-g, rIL-1b, rIL-4, rIL-10, rIL13, rTNF-a, rTGF-b1, rCCL17, rCCL22, and rCXCL9 were purchased from R&D Systems (Minneapolis, Minnesota). rGM-CSF and rIL-12 were obtained from PeproTech (London, UK). rIL-18 was purchased from MBL (Nagoya, Japan). SAC (pansorbin) was obtained from Calbiochem (San Diego, California), and LPS (serotype 011: B4) was from Sigma-Aldrich (St Louis, Missouri). Poly(I:C) was obtained from Amersham Biosciences (Piscatway, New Jersey). rCXCL10 was provided by DAKO Japan (Kyoto, Japan). Goat anti-CXCL10 Ab, goat anti-CXCL9 Ab, goat anti-CCL17 Ab, goat anti-CCL22 Ab, and control goat IgG were obtained from GenzymeTechne (Cambridge, Massachusetts). Rat anti-mouse CD40 monoclonal antibody (mAb) (NA/LE, clone 3/23) was purchased from BD Biosciences (San Diego, California). Peroxidase-conjugated rabbit anti-goat IgG Ab was from MBL. Purification and culture of LC and splenic DC LC were prepared using the previously described panning method, and had a purity of over 95% (Salgado et al, 1999b; Tada et al, 2000). Briefly, mouse trunkal skin was separated and treated with dispase (3000 U per mL, Godo Shusei, Tokyo, Japan) in RPMI medium supplemented with 10% FCS (RPMI 10 medium) for 3 h at 371C. The epidermis was separated and incubated in RPMI 10 medium containing 0.025% deoxyribonuclease I (Sigma) for 20 min at room temperature. An epidermal cell suspension was obtained by vigorously pipetting the epidermal sheets, and was treated with mouse anti-mouse I-Ad mAb (1:600; Meiji Seika, Tokyo, Japan) in RPMI 10 medium for 45 min on ice. The cell suspension in RPMI 10 medium was then incubated in plates coated with goat anti-mouse IgG (Fc) (1:100; MP Biomedicals, Aurora, Ohio) for 45 min at 41C. After a wash, adherent cells were collected and designated purified LC. Splenic DC were purified from spleens of BALB/c mice. A single cell suspension was obtained by gentle teasing with forceps and rubbers and filtered through a nylon mesh. Erythrocytes were lysed by treatment with ammonium chloride. From the remaining unfractionated cell populations, CD11c þ DC were separated by positive magnetic selection using microbeads-conjugated hamster anti-mouse CD11c (Miltenyi Biotec, Belgisch Gladbach, Germany) and a magnetic cell separator according to the manufacturer’s instructions, routinely resulting in 490% purity of CD11c þ DC. LC and splenic DC were incubated in 96-well culture plates (1.5 106 cells per mL per 200 mL in each well) for up to 48 h, with or without various cytokines, Ab, or microbial components in a culture medium that consisted of RPMI 10 medium, 100 U per mL of penicillin G sodium, 100 mg per mL of streptomycin sulfate, and 0.25 mg per mL of amphotericin B. Semi-quantitative RT-PCR A semi-quantitative RT-PCR was used to assess the gene expression of CXCL10, CXCL9, CXCL11, CCL22, and CCL17 in murine LC and splenic DC. LC and splenic DC were prepared and cultured for 48 h as described above, and from the lysate of fresh and cultured LC and splenic DC, mRNA was isolated using the Quickprep Micro mRNA Purification Kit (Pharmacia Biotech, Uppsala, Sweden). Complementary DNA was synthesized by RT using random hexamers and mouse reverse transcriptase (First-Strand cDNA Synthesis Kit; Pharmacia Biotech). The PCR was performed in a programmable thermal cycler
124 : 2 FEBRUARY 2005 (Perkin-Elmer Co., Norwalk, Connecticut). The primers used for amplification of CXCL10 (Kuroda et al, 2001), CXCL9 (Park et al, 2001), CXCL11 (Lacroix-Lamande et al, 2002), CCL22 (Kuroda et al, 2001), CCL17 (Xiao et al, 2003a), and glyceraldehyde-3phosphate dehydrogenase (Xiao et al, 2003a) were described elsewhere. All reactions were run at 941C per 1 min, 561C per 1 min, 721C per 2 min for 35 cycles. The PCR was within the linear range. The PCR products were analyzed on 2.5% agarose gels and visualized by ethidium bromide staining and UV illumination.
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Manuscript received July 30, 2004; revised September 24, 2004; accepted for publication October 13, 2004 Address correspondence to: Akihiko Asahina, MD, Department of Dermatology, Faculty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan. Email: [email protected]. ac.jp
References Measurement of CXCL10 and CCL22 Culture supernatants were collected, stored at 201C, and subjected to the quantification of protein levels of CXCL10 and CCL22 by competitive ELISA inhibition assay as previously described (Kuroda et al, 2001). Briefly, ELISA plates were coated with 100 mL of rCXCL10 or rCCL22 at 250 ng per mL in phosphate-buffered saline (PBS) containing 0.1% BSA at 41C for 18 h. Then the wells were washed and blocked with 300 mL of RPMI 10 medium at 371C for 30 min. Standard solutions (40,000–625 pg per mL in RPMI 10 medium) or culture supernatants and equal volumes of 100 ng per mL of anti-CXCL10 Ab or 400 ng per mL of anti-CCL22 Ab were mixed in microtubes and incubated at 371C for 1 h. After the incubation, 200 mL of prepared standard or sample solution was transferred to each well of CXCL10- or CCL22-coated plates, and the plates were kept at 41C for 18 h. The wells were washed and 100 mL of peroxidase-conjugated rabbit anti-goat IgG Ab (diluted 1:2000 in PBS containing 0.05% Tween 20 and 1% BSA) was added. The plates were incubated at 371C for 2 h. Then they were washed and 100 mL of substrate (0.04% o-phenylenediamine, 0.01% H2O2 in 50 mM disodium hydrogen phosphate, and 150 mM citric acid, pH 5) was added to the wells. The plates were incubated at room temperature for 30 min. The enzyme reaction was stopped by the addition of 25 mL of 8 N H2SO4, and the absorbance at 490 nm was measured. Measurement of CXCL9, CXCL11, and CCL17 Culture supernatants were collected, stored at 201C, and subjected to the quantification of protein levels of CXCL9, CXCL11, and CCL17 by ELISA using commercially available mouse CXCL9, CXCL11, and CCL17 immunoassay kits (R&D Systems), respectively, according to the manufacturer’s instructions. Each sample was tested in duplicate. Chemotaxis assay Chemotaxis assay was carried out using mouse CCR4- or CXCR3-transfected 2B4 T cells (Gao et al, 2003). Chemotaxis of the transfectants was assessed in 24-well transwells equipped with 5 mM pore polycarbonate membranes (Transwell, Corning Costar Corp., Cambridge, Masschusetts). Cells (1 106) suspended in 100 mL of RPMI 10 medium were transferred to the upper chamber. For the chemotaxis of CCR4 transfectant, the supernatants of 48 h cultured unstimulated LC and splenic DC were used. Culture supernatants and RPMI 10 medium with or without recombinant chemokine were preincubated with or without 2 mg per mL of anti-CCL17 mAb, or 10 mg per mL of anti-CCL22 mAb, or both for 30 min at room temperature. They were then transferred to the lower chamber in 600 mL for each group. After an incubation period of 3 h at 371C, the upper chambers were removed, and the cells in the lower chamber of each well were collected and counted using a Coulter Counter (Beckman Coulter, Jersey City, New Jersey). Chemotaxis of CXCR3 transfectant was carried out in the same way using the supernatants of 48 h IFN-g-stimulated LC and splenic DC. In this case, to neutralize the activity of chemokines, 4 mg per mL of anti-CXCL10 mAb and 2 mg per mL of anti-CXCL9 mAb were used. All assays were carried out in triplicate. Statistical analysis Student’s t-test was used to analyze the results, and a p-value of less than 0.05 was considered statistically significant. DOI: 10.1111/j.0022-202X.2004.23607.x
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