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The Journal of Immunology, 2000, 164: 5113-5119.
Copyright © 2000 by The American Association of Immunologists

Granulocyte/Macrophage Colony-Stimulating Factor Inhibits IL-12 production of Mouse Langerhans Cells

Yayoi Tada1,*, Akihiko Asahina*, Koichiro Nakamura*, Michio Tomura{dagger}, Hiromi Fujiwara{dagger} and Kunihiko Tamaki*

* Department of Dermatology, University of Tokyo, Tokyo, Japan; and {dagger} Biomedical Research Center, Osaka University Medical School, Osaka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the capacity of mouse Langerhans cells (LC) to produce IL-12, a central cytokine in a Th1 type of immune responses. We prepared purified LC (>95%) from BALB/c mouse skin by the panning method using anti-I-Ad mAb. An ELISA showed that purified LC spontaneously produced IL-12 p40, and that its production was up-regulated following simultaneous stimulation with anti-CD40 mAb and IFN-{gamma}. Surprisingly, GM-CSF strikingly inhibited IL-12 p40 production by anti-CD40/IFN-{gamma}-stimulated LC (% inhibition = 97.0 ± 0.9% at 1 ng/ml GM-CSF). Supernatants of 48-h cultured keratinocytes (KC) also caused the inhibition of LC IL-12 p40 secretion, and this effect was neutralized by anti-GM-CSF mAb. IL-1{alpha} (1 ng/ml)-stimulated KC produced much more GM-CSF than unstimulated KC (60.9 ± 0.2 pg/ml vs 20.9 ± 1.7 pg/ml), and IL-1{alpha}-stimulated KC supernatants strongly inhibited IL-12 p40 production by anti-CD40/IFN-{gamma}-stimulated LC (% inhibition = 89.4 ± 1.4%). A bioassay using an IL-12-dependent T cell line demonstrated the correlation of the level of IL-12 p40 with the bioactivity of IL-12. These results provide important implications for the pathogenesis of atopic dermatitis, which involves the participation of LC and KC with the capacity to produce IL-12 and GM-CSF, respectively.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Langerhans cells (LC)2 are bone marrow-derived MHC class II positive APC localized in the epidermis. They belong to a dendritic cell (DC) lineage and are crucial for primary and secondary T cell-dependent immune responses (1). Specific skin inflammation, such as contact hypersensitivity and skin graft rejection, is regulated by a complex and sequential mechanism involving LC, keratinocytes (KC), T cells, and a variety of cytokines they produce (2, 3, 4). These cytokines are also known to modulate the function, morphology, and phenotype of LC (5, 6), including Ag processing and presenting capacity, expression of cell surface molecules, and cytokine secretion.

It is important to note that LC secrete various cytokines, such as IL-1ß, IL-6, and macrophage inflammatory protein (7, 8, 9). Recent studies have disclosed that LC are also capable of secreting IL-12 (10), which is produced predominantly by DC and macrophages (11, 12). IL-12 is a heterodimeric cytokine consisting of p35 and p40 subunits. This cytokine is known to exert multiple effects on the activation of T and NK cells, and it increases their proliferation, cytotoxicity, and cytokine production (such as IFN-{gamma}), thereby serving as a powerful mediator for Th1-type differentiation of T helper cells both in vitro and in vivo (13). IL-12 plays a critical role in the induction of immune responses involving Th1 cells, including delayed-type hypersensitivity, contact hypersensitivity reactions (14), atopic dermatitis (AD) (15), and Leishmania infection (16).

To date, the production of IL-12 p40 and bioactive IL-12 has been shown in isolated human LC (10). However, in the mouse system, bioactive IL-12 was detected only in LC-like immature DC prepared from mouse fetal skin (17), but has not been observed in LC except for the detection of IL-12 p40 mRNA by RT-PCR analysis (16). It is possible that difficulty in detecting IL-12 production was due to culture conditions of mouse LC, because LC of low purity (<50%) were used in the previous studies (16). In such cases, the interference of contaminating KC and KC-derived cytokines must also be considered in addition to the purity of LC populations.

In this study, we succeeded in preparing highly purified LC (>95%) from BALB/c mice by applying the panning method using anti-I-Ad mAb (18). Using these purified LC, we show that either ligation of CD40 or addition of IFN-{gamma} enhances IL-12 production by mouse LC, as has been observed for other types of IL-12 producing cells (10, 19), and that simultaneous triggering induces a synergistic effect. We also examined a direct effect of GM-CSF on IL-12 secretion by LC, because this cytokine is essential for functional maturation of LC (20, 21). To our surprise, GM-CSF strongly inhibited IL-12 production by anti-CD40/IFN-{gamma}-stimulated LC in our system. Moreover, addition of KC supernatants to LC culture caused the inhibition of IL-12 production, and GM-CSF was found to be responsible for this inhibition. Thus, this study provides the first demonstration for IL-12 production of mouse LC and its inhibition by KC-derived GM-CSF.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

BALB/c female mice were purchased from Japan SLC (Hamamatsu, Japan) and maintained under specific pathogen-free conditions at the University of Tokyo Animal Facilities until use at the age of 8–12 wk.

Reagents and mAbs

Mouse rGM-CSF and IL-1{alpha} were purchased from PeproTech (London, U.K.). For stimulation of IL-12 production in mouse LC, mouse rIFN-{gamma} was purchased from R&D Systems (Minneapolis, MN), and rat anti-mouse CD40 mAb (NA/LE, clone 3/23) was purchased from PharMingen (San Diego, CA). Neutralizing rat anti-mouse GM-CSF mAb (NA/LE, clone MP1-22E9) was purchased from PharMingen. For flow cytometry, the following mAbs and control isotype-matched irrelevant mAbs were used for immunostaining; FITC-conjugated goat IgG fraction to mouse IgG F(ab')2 (Organon Teknika, Durham, NC), biotin-conjugated hamster anti-mouse CD80 (B7-1), biotin-conjugated rat anti-mouse CD86 (B7-2), FITC-conjugated streptavidin, FITC-conjugated mouse anti-mouse I-Ad, FITC-conjugated hamster anti-mouse CD11c, FITC-conjugated rat anti-mouse CD14, purified rat anti-mouse CD44, FITC-conjugated mouse anti-rat IgG, FITC-conjugated mouse IgG isotype control, FITC-conjugated hamster IgG isotype control, and FITC-conjugated rat IgG (IgG1 and IgG2) isotype control (PharMingen). Purified rat anti-mouse NLDC 145 was purchased from BMA Biomedicals (Augst, Switzerland). For a bioassay of IL-12, mouse rIL-12 was purchased from PeproTech, neutralizing anti-mouse IL-12 mAb (C15.1) was purchased from Biomeda (Foster City, CA), and rat IgG isotype control was purchased from PharMingen.

Purification and culture of LC and splenic DC

LC were prepared by using a previously described panning method (18). Briefly, mouse trunkal skin was separated and treated with dispase (3000 U/ml, Godo Shusei, Tokyo, Japan) in RPMI supplemented with 10% FCS (RPMI 10) for 3 h at 37°C. Epidermis was separated from dermis and incubated in RPMI 10 containing 0.025% deoxyribonuclease I (Sigma, St. Louis, MO) for 20 min at room temperature. Epidermal cell suspension was obtained by vigorous pipetting of the epidermal sheets, and it was treated with mouse anti-mouse I-Ad mAb (1:600; Meiji Seika, Tokyo, Japan) in RPMI 10 for 45 min on ice. The cell suspension in RPMI 10 was then incubated in plates coated with goat anti-mouse IgG (Fc) (1:100; Organon Teknika) for 45 min at 4°C. After washing, adherent cells were collected and designated purified LC. Floating cells were collected in some experiments and designated KC. DC were purified from spleens of BALB/c mice. 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+ 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 >80% purity of CD11c+ DC. LC and DC were incubated in 96-well culture plates (1.5 x 106 cells/ml per 200 µl in each well) for up to 48 h or 72 h, with or without various cytokines and/or Abs in culture medium which consists of RPMI 10, 100 U/ml penicillin G sodium, 100 µg/ml streptomycin sulfate, and 0.25 µg/ml amphotericin B. In some experiments, KC, prepared as mentioned above, were cultured for 48 h with or without IL-1{alpha} (1 ng/ml) in the same way. Supernatants of KC were then collected, and LC were cultured in the supernatants of KC.

Semiquantitative RT-PCR

Semiquantitative RT-PCR was employed to assess IL-12 p40 gene expression in cultured LC. LC were prepared and cultured for 14 h as described above, and from the lysate of these LC, mRNA was isolated using the Quickprep Micro mRNA Purification Kit (Pharmacia Biotech, Uppsala, Sweden). One hundred nanograms of mRNA was obtained from 1 x 106 LC. Reverse transcription was achieved at 37°C for 60 min, followed by heat inactivation (65°C for 10 min). Complementary DNA was synthesized by reverse transcription of the mRNA elute (20 µl) using random hexamers and mouse reverse transcriptase (First-Strand cDNA Synthesis Kit; Pharmacia Biotech). The PCR was performed in a programmable thermal cycler (Perkin-Elmer, Norwalk, CT) as follows for each primer: IL-12 p40: denaturation at 94°C for 0.75 min, annealing at 60°C for 1 min and polymerization at 72°C for 1 min for 42 cycles; and for G3PDH: 30 cycles. The PCR mixture consisted of 2 µl of the cDNA solution in 48 µl of PCR buffer containing 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each dNTP, 100 pmol of each primer, and 2.5 U of Taq DNA polymerase (Toyobo, Tokyo, Japan). The PCR for G3PDH evaluated the quality of the cDNA templates. The primers used for amplification of IL-12 p40 (22) were sense, 5'-AACCTCACCTGTGACACGCC-3', and antisense, 5'-CAAGTCCATGTTTCTTTGCACC-3'; and those used for amplification of G3PDH were sense, 5'-TGAAGGTCGGTGTGAACGGATTTGGC-3' and antisense, 5'-CATGTAGGCCATGAGGTCCACCAC-3'. The expected size of the PCR product was 309 bp for IL-12 p40 and 983 bp for G3PDH. The PCR was within the linear range. The PCR products were fractionated by 10% PAGE in a volume of 10 µl and were visualized by ethidium bromide staining and UV illumination. A 100-bp ladder served as the standard.

Flow cytometric analysis

Fresh LC or cultured LC treated according to the experimental protocols were resuspended in PBS supplemented with 0.1% BSA and 0.1% NaN3 and aliquoted into tubes for Ab staining. The cells were first incubated with the respective mAb at 1:50 dilution for 30 min at 4°C, and then with the respective second mAb at 1:50 dilution for the next 30 min. They were washed twice by centrifugation after each incubation. The final suspension was made in 300 µl of PBS supplemented with 0.1% BSA and 0.1% NaN3 containing propidium iodide to exclude nonviable cells. Samples were analyzed on a fluorescence-activated cell sorter (FACSCalibur; Becton Dickinson, San Diego, CA).

Measurement of IL-12 p40 and GM-CSF by ELISA

Culture supernatants of LC, KC, and DC were collected, stored at -20°C, and subjected to the quantification of protein levels of IL-12 p40 and GM-CSF by ELISA using commercially available mouse IL-12 p40 and GM-CSF immunoassay kits (R&D Systems) according to the manufacturer’s instructions. Each sample was tested in duplicate. Percent inhibition of IL-12 p40 was calculated as follows: % inhibition = [1 - (IL-12 p40 production of LC in each treatment group)/(IL-12 p40 production of LC stimulated with anti-CD40 plus IFN-{gamma})] x 100(%).

Bioassay for IL-12 p70 heterodimer

Proliferative assay of IL-12-responsive T cell clone, 2D6 (23), was used as a bioassay for the active p70 heterodimer. In each well of a 96-well microculture plate, 2D6 cells (2.0 x 104/well) were incubated in the supernatants of cultured LC or serially diluted standard mouse rIL-12 solutions for 48 h at 37°C. Either 10 µg/ml of rat anti-mouse IL-12 mAb (C17.8) or rat IgG isotype control (PharMingen) was added to each well. The cells were pulse-labeled with 20 kBq/well of [3H]TdR for the final 6 h. All samples were cultured in duplicate. IL-12 bioactivity of the culture supernatants was determined by subtracting the [3H]TdR uptake of cells incubated with anti-mouse IL-12 mAb (C17.8) from the [3H]TdR uptake of cells incubated with rat IgG isotype control. The maximum inhibition limit of anti-mouse IL-12 mAb was 100 pg/ml. The standard curve was generated using known amounts of standard mouse rIL-12 solutions, up to 100 pg/ml. The detection limit was found to be 10 pg/ml. IL-12 concentration of culture supernatants were estimated by the standard curve.

Statistical analysis

The Student’s t test was used to analyze the results, and a p value <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Enrichment of LC by the panning method

We prepared highly purified LC from BALB/c mice using the panning method with anti-I-Ad mAb. The purity of LC was consistently over 95% as determined by flow cytometry using FITC-conjugated goat anti-mouse IgG (Fig. 1Go). The same result was obtained by flow cytometry using FITC-conjugated mouse anti-mouse I-Ad (data not shown). By RT-PCR experiments, they expressed mRNAs for IL-1ß and TNF-{alpha}, but not for IL-1{alpha}, GM-CSF and TCR{gamma}{delta} (24). In the following experiments, we cultured LC in complete medium containing FCS to keep LC viability in the absence of KC.



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  		FIGURE 1. Preparation of highly purified LC by the panning method. LC were purified from BALB/c mouse skin by using the panning method with mouse anti-mouse I-Ad mAb as described in Materials and Methods. The cells were stained with FITC-conjugated goat anti-mouse IgG (solid line) or with an FITC-conjugated mouse IgG isotype control (dashed line). In this experiment, LC were purified to 96% as determined by I-Ad+ cells by flow cytometry.

 
Stimulation of LC with anti-CD40 and IFN-{gamma} up-regulates IL-12 p40 production

As shown in Fig. 2Go, mouse LC spontaneously secreted IL-12 p40 in a time-dependent manner for up to 48 h in our system. In contrast, KC cultured in the same way produced only a small level of this protein (data not shown), although stimulated KC are also capable of producing IL-12 (25).



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  		FIGURE 2. Time-course of IL-12 p40 production by LC. LC (1.5 x 106/ml) were cultured for the time periods indicated. The supernatants were then collected and IL-12 p40 contents were measured by a mouse IL-12 p40 ELISA kit. Results are mean ± SE (n = 3).

 
We next studied the regulation of IL-12 p40 production by mouse LC. We stimulated LC by incubation with anti-CD40 mAb (20 µg/ml) and observed up-regulation of IL-12 p40 secretion from LC (Table IGo). In addition, stimulation of LC with a serial concentrations of IFN-{gamma} (0.1–100 ng/ml) caused up-regulation of IL-12 p40 secretion in a dose-dependent manner (data not shown), and 100 ng/ml IFN-{gamma} was as potent as anti-CD40 mAb (Table IGo). Furthermore, IFN-{gamma} and anti-CD40 mAb synergistically up-regulated the secretion of IL-12 p40 (Table IGo).


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  		Table I. Effect of anti-CD40 mAb, IFN-{gamma}, and GM-CSF on IL-12 p40 production by LC1

 
GM-CSF inhibits IL-12 p40 production by mouse LC

We then examined the effect of GM-CSF, a cytokine known to potentiate LC maturation (20, 21), on IL-12 p40 production. As shown in Table IGo, IL-12 p40 production was significantly inhibited in GM-CSF (1 ng/ml)-treated LC as compared with LC incubated without this cytokine. We next stimulated LC with both anti-CD40 mAb and IFN-{gamma} for maximal IL-12 p40 production, and an increasing concentrations of GM-CSF (0.01–10 ng/ml) was added to the culture. GM-CSF dose-dependently inhibited IL-12 p40 production by anti-CD40/IFN-{gamma}-stimulated LC, and its inhibition was significant at the concentration of 0.1 ng/ml and higher (Table IGo). Percent inhibition was 97.0 ± 0.9% at 1 ng/ml and this inhibition was abrogated when anti-GM-CSF mAb (20 µg/ml) was added with 1 ng/ml GM-CSF.

By semiquantitative RT-PCR, we studied whether IL-12 p40 production was regulated at the transcription level. We isolated mRNA from the lysate of 14-h cultured LC using the mRNA purification kit and RT-PCR was performed. As shown in Fig. 3Go, stimulation with anti-CD40 mAb and IFN-{gamma} up-regulated IL-12 p40 mRNA and GM-CSF (1 ng/ml) down-regulated IL-12 p40 mRNA of anti-CD40/IFN-{gamma}-simulated LC. This result was compatible with our result of IL-12 p40 ELISA.



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  		FIGURE 3. GM-CSF down-regulates IL-12 p40 mRNA expression of anti-CD40 mAb/IFN-{gamma}-stimulated LC. LC were cultured for 14 h with anti-CD40 mAb (20 µg/ml) plus IFN-{gamma} (100 ng/ml) with or without the presence of GM-CSF (1 ng/ml). From the lysate of these LC, mRNA was isolated using the mRNA purification kit and semiquantitative RT-PCR was performed. PCR products are shown. The size of the PCR product was 309 bp for IL-12 p40 and 983 bp for G3PDH.

 
We have previously shown that increasing concentrations of GM-CSF do not alter the cell viability of 36-h cultured LC (24). Thus, GM-CSF was not toxic to LC in our system. We also examined the effect of M-CSF on anti-CD40/IFN-{gamma}-stimulated LC as a control cytokine. Compared with GM-CSF, M-CSF (100 ng/ml) showed no such inhibitory effect on IL-12 p40 production of anti-CD40/IFN-{gamma}-stimulated LC (661.0 ± 189.5 pg/ml vs 731.5 ± 147.8 pg/ml (n = 3); mean ± SD).

GM-CSF produced by KC inhibits IL-12 p40 production by mouse LC

In the skin, KC are a major source of cytokines, and GM-CSF is predominantly produced by KC (8). We therefore investigated whether KC-derived factors could indeed inhibit IL-12 production by LC. For this purpose, we collected supernatants of KC cultured for 48 h in complete medium. The supernatants contained virtually no IL-12 p40 by ELISA (data not shown). LC incubated in the supernatants for 48 h showed lower IL-12 p40 production than LC incubated in fresh complete medium (52.1 ± 33.2 pg/ml vs 107 ± 13 pg/ml; p = 0.03). This indicated that KC supernatants contained factors that inhibited IL-12 p40 production by unstimulated LC. We then stimulated LC with both anti-CD40 mAb and IFN-{gamma} for 48 h in the presence or absence of KC supernatants. As shown in Table IIGo, IL-12 p40 production was lower when LC were incubated in the KC supernatants (% inhibition = 58.2 ± 8.3%). Importantly, addition of anti-GM-CSF mAb (20 µg/ml) to the supernatants neutralized this effect, indicating that endogenous GM-CSF in the supernatants is responsible for this inhibition (Table IIGo). To confirm the production of GM-CSF by KC, we measured the content of this cytokine in the supernatants. Concentration of GM-CSF in the KC supernatants was 20.9 ± 1.7 pg/ml, and the degree of inhibition of IL-12 p40 secretion was compatible with that predicted by the dose-dependent response shown in Table IGo. In contrast, GM-CSF was not detected in the supernatants of LC incubated for 48 h in fresh complete medium (data not shown).


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  		Table II. GM-CSF produced by KC inhibits IL-12 p40 production by stimulated LC1

 
IL-1{alpha} is an important cytokine in the regulation of inflammatory, proliferative, and immunologic events (26). Because it is also known to stimulate GM-CSF production by KC (27), we examined an inhibitory potential for LC IL-12 p40 secretion by endogenous GM-CSF derived from IL-1{alpha}-stimulated KC. In our study, the supernatants of IL-1{alpha} (1 ng/ml)-stimulated KC contained higher amounts of GM-CSF than those of unstimulated KC (60.9 ± 0.2 pg/ml vs 20.9 ± 1.7 pg/ml) as reported previously (27, 28). We then stimulated LC with both anti-CD40 mAb and IFN-{gamma} for 48 h in the presence of IL-1{alpha}-stimulated KC supernatants. As shown in Table IIGo, IL-12 p40 production was much lower when LC were incubated in the supernatants of stimulated KC (% inhibition = 89.4 ± 1.4%). Addition of IL-1{alpha} (1 ng/ml) alone did not have such an inhibitory effect (Table IIGo).

GM-CSF inhibits IL-12 p40 production by mouse spleen-derived DC

We next studied the regulation of IL-12 p40 production by mouse spleen-derived CD11c+ DC obtained by positive magnetic selection. We also treated DC with the same anti-I-Ad, after DC isolation by magnetic cell separator, either with or without the panning method on the plate. However, cell viability of the anti-I-Ad-treated DC fell below 20% at 72 h, and this procedure seemed noxious to DC prepared in this way. For this reason, we used untreated DC as the control cells in the following experiments.

Both untreated and anti-CD40-mAb stimulated DC produced IL-12 p40 in a time-dependent manner for up to 72 h (data not shown). We stimulated DC by incubation with anti-CD40 mAb (20 µg/ml) and/or IFN-{gamma} (100 ng/ml), and observed up-regulation of DC IL-12 p40 production (Table IIIGo). GM-CSF also down-regulated IL-12 p40 production by anti-CD40/IFN-{gamma}-treated DC, but the inhibition rate was much smaller than that observed in LC. These results suggest that the inhibitory effect of GM-CSF is not specific to LC.


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  		Table III. Effect of anti-CD40 mAb, IFN-{gamma}, and GM-CSF on IL-12 p40 production by DC1

 
GM-CSF inhibits bioactive IL-12 production by primed LC

Although we have demonstrated the production of IL-12 p40 by mouse LC, it is known that the level of IL-12 p40 does not necessarily correlate with the bioactivity of IL-12 (29). Therefore, we employed a bioassay using an IL-12-dependent cell line 2D6 to detect the level of bioactive IL-12 p70 heterodimer (23). 2D6 cells were cultured for 48 h in the supernatants of LC stimulated with anti-CD40 mAb and IFN-{gamma}. IL-12 bioactivity was expressed as anti-IL-12 mAb (C17.8; 10 µg/ml)-inhibitable [3H]TdR uptake of 2D6. The detection range of this assay was 10 pg/ml to 100 pg/ml. Each culture supernatant of LC was within this range. Isotype-matched control mAb did not influence 2D6 proliferation at the same concentration in this assay (data not shown). GM-CSF, IFN-{gamma}, or anti-CD40 mAb did not induce the proliferation of unstimulated or rIL-12-stimulated 2D6 cells (data not shown). The proliferation rate of 2D6 as assessed by the uptake of [3H]TdR correlated with the level of IL-12 p40 measured by ELISA (data not shown). As shown in Fig. 4Go, this IL-12 bioactivity was enhanced when LC were stimulated with anti-CD40 mAb and IFN-{gamma}, and GM-CSF (1 ng/ml) added to the mixture strongly inhibited IL-12 production. We estimate that the LC secreted 55 ± 24 pg/ml of IL-12 p70 when stimulated with anti-CD40 mAb and IFN-{gamma}, whereas LC secreted 10 ± 2 pg/ml when GM-CSF (1 ng/ml) was added with anti-CD40 mAb and IFN-{gamma}.



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  		FIGURE 4. GM-CSF inhibits bioactive IL-12 production by stimulated LC. LC (1.5 x 106/ml) were stimulated with anti-CD40 mAb (20 µg/ml) plus IFN-{gamma} (100 ng/ml) with or without the presence of GM-CSF (1 ng/ml) for 48 h. Culture supernatants were obtained and tested for IL-12 activity in a bioassay using a IL-12-dependent cell line 2D6 (see Materials and Methods). IL-12 bioactivity was expressed as anti-IL-12 (C17.8)-inhibitable [3H]TdR uptake of 2D6. Results are mean ± SE (n = 3). *, p < 0.05 compared with [3H]TdR uptake of 2D6 cultured with supernatants of LC stimulated with anti-CD40 mAb (20 µg/ml) plus IFN-{gamma} (100 ng/ml).

 
Modulation of the expression of cell surface molecules by GM-CSF

To assess the effect of GM-CSF (1 ng/ml) on the expression of costimulatory molecules on cultured LC, we examined cell surface expression of B7-1 and B7-2 molecules by flow cytometry. As shown in Table IVGo, GM-CSF up-regulated both B7-1 and B7-2 expression on 24 h-cultured LC. This potentiating effect by GM-CSF was also observed when anti-CD40 mAb (20 µg/ml) and IFN-{gamma} (100 ng/ml) were added, whereas anti-CD40 mAb and IFN-{gamma} alone had no effect. A similar result was observed in 48 h-cultured LC (data not shown). The expression of B7 molecules is known to be involved in immunostimulatory functions of LC (6). Our result is consistent with the previous report (6) and supports the concept that GM-CSF is a major factor for functional maturation of LC. In addition, we studied the effect of GM-CSF (1 ng/ml) on other cell surface molecules of 48-h cultured LC, such as I-Ad, CD44, CD11c, NLDC 145, and CD14. We have previously shown that fresh LC do not express CD14, either by RT-PCR or by flow cytometry (18). GM-CSF further up-regulated I-Ad and NLDC 145 expression compared with 48-h cultured untreated LC, but CD11c and CD14 expression remained negative (Fig. 5Go). CD44 was up-regulated in 48-h cultured untreated LC as previously described (30), and GM-CSF had no effect on its expression (Fig. 5Go). These results suggest a phenotypic shift of LC population toward mature DC but not toward macrophages, by GM-CSF.


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  		Table IV. Percentage modulation of B7-1 and B7-2 expression on LC by GM-CSF and anti-CD40 + IFN-{gamma}1

 


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  		FIGURE 5. The effect of GM-CSF on cell surface molecules of 48-h cultured LC. Purified LC were cultured with GM-CSF (1 ng/ml) for 48 h. I-Ad, NLDC 145, CD44, CD11c, and CD14 levels of untreated and treated LC were analyzed after 48 h using flow cytometry. The expression of the cell surface molecules of fresh LC is also shown. Solid lines, mAbs of interest; dashed lines, isotype control (representative of three independent experiments).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated for the first time that mouse LC secrete bioactive IL-12 spontaneously and that its production is up-regulated following simultaneous stimulation with anti-CD40 mAb and IFN-{gamma}. This spontaneous IL-12 induction in cultured LC may be caused by ligation of MHC class II molecules by the Ab used in the panning method, because IL-12 production can be up-regulated by ligation of MHC class II molecules in mouse DC (31). Indeed, a number of studies suggest CD40 ligation as an important signal for IL-12 induction by APC, including human and mouse DC (31, 32). In addition, secretion of IL-12 was synergistically up-regulated by IFN-{gamma} and anti-CD40 mAb, which is consistent with the previous reports that these two signals mimic a mode of physiological DC stimulation by T cells and lead to high level of IL-12 secretion in both mouse LC-like DC (17) and human DC (33).

Surprisingly, this induction of LC IL-12 p40 production was strongly down-regulated by GM-CSF (% inhibition = 97.0 ± 0.9% at 1 ng/ml of GM-CSF). GM-CSF also down-regulated IL-12 p40 production by anti-CD40 and IFN-{gamma}-treated DC, suggesting that this inhibitory effect may not be necessarily specific to LC. However, this effect was much smaller than that on LC (% inhibition = 97.0 ± 0.9% vs 34.5 ± 2.1% at 1 ng/ml GM-CSF). It might be possible that LC is more sensitive than DC in terms of the effect of GM-CSF on IL-12 secretion. This is consistent with the observation that bone marrow- and spleen-derived DC cultured in GM-CSF are capable of producing sufficient IL-12 (34, 35). In the skin, KC are known to be a major source of GM-CSF (8). In our system, LC were highly purified (>95%) and we were able to minimize the interference by KC and KC-derived cytokines. Based on our findings, highly purified LC without addition of GM-CSF in culture are necessary for sufficient IL-12 production in vitro.

The skin of AD patient is known to have impaired epidermal permeability barrier. This may be causally linked to skin inflammation, because acute barrier perturbation can stimulate cytokine production, including IL-1{alpha} and GM-CSF, in both mouse and human skin (36, 37, 38). Indeed, Pastore et al. (28) have reported that GM-CSF is overproduced by KC in AD, and IL-1{alpha} induced an increase in GM-CSF release much higher in cultures of AD KC compared with those of control KC. Consistent with these observations, we have shown in our studies that KC are stimulated to produce GM-CSF by IL-1{alpha}, and this KC-derived GM-CSF also strongly inhibited IL-12 p40 production by LC (% inhibition = 89.4 ± 1.4%). It has been proposed that Th2-type immune responses play a key pathogenetic role in AD, and this is supported by the presence of blood eosinophilia and enhanced serum IgE levels in the majority of AD patients (15). However, the immuneregulatory mechanism that induces Th2 development in AD is presently unknown. Our results suggest that in the skin, GM-CSF may play a critical role in Th2 development via interference with LC, especially in the inflammatory state of AD in which KC are stimulated to overproduce GM-CSF.

Although our finding that GM-CSF down-regulates IL-12 production and promotes maturation of LC at the same time is surprising, similar observations were very recently made for human DC by Kalinski et al. (39, 40). They generated immature CD1a+ CD83- DC from human peripheral monocytes and allowed them to mature by incubation with IL-1ß and TNF-{alpha} (39). Although maturing DC was responsive to stimulation with anti-CD40 and IFN-{gamma}, fully matured DC became unresponsive to the same stimuli, and eventually IL-12 secretion was strongly down-regulated. In another study (40), both IL-10 and PGE2 inhibited IL-12 production by maturing human DC, but these reagents failed to do so on fully matured DC. From these studies, they concluded that mature DC are resistant to the reagents that modulates IL-12 production. The present study shows that mouse LC become unresponsive to IL-12-triggering factors as a result of exposure to GM-CSF, which has not been thus far described.

Rissoan et al. (41) have recently shown that distinct human DC subsets, DC1 and DC2, produce different cytokines and directly induce Th1 and Th2 differentiation, respectively. Maldonado-Lopez et al. (35) have also shown that mouse spleen-derived DC subclasses, CD8{alpha}+ and CD8{alpha}-, induce Th1 and Th2 differentiation, respectively. We speculate that LC have capacity to induce both Th1 and Th2 in the same way, and GM-CSF is able to turn LC from "type 1 (LC1)" to "type 2 (LC2)." As we mentioned above, KC in AD overproduce GM-CSF both spontaneously and following stimulation with IL-1{alpha} (36). It is intriguing to hypothesize that sustained overproduction of GM-CSF by KC of AD results in down-regulation of LC IL-12 production in the skin, thus favoring the development of Th2-type cytokine profiles at a certain stage of dermatitis. This study is now under investigation in our laboratory.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Yayoi Tada, Department of Dermatology, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8655 Japan. Back

2 Abbreviations used in this paper: LC, Langerhans cells; AD, atopic dermatitis; DC, dendritic cells; KC, keratinocytes. Back

Received for publication August 16, 1999. Accepted for publication March 2, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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