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The Journal of Immunology, 2001, 167: 5955-5962.
Copyright © 2001 by The American Association of Immunologists

Inability of IL-12 to Down-Regulate IgE Synthesis Due to Defective Production of IFN-{gamma} in Atopic NC/Nga Mice1

Masahiro Matsumoto*,{dagger}, Atsuko Itakura*, Akane Tanaka*, Chie Fujisawa* and Hiroshi Matsuda2,*

* Laboratory of Clinical Immunology, Department of Veterinary Clinic, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan; and {dagger} Department of Pathology, Toxicology Research Laboratories, Fujisawa Pharmaceutical, Osaka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
NC/Nga mice raised in nonsterile circumstances spontaneously suffer from atopic dermatitis-like skin lesions with IgE hyperproduction. We investigated effects of rIL-12 on the IgE production in NC/Nga mice. rIL-12 administration was successful to suppress the increase of IgE levels in BALB/c mice immunized with OVA and aluminum hydroxide, but failed to abrogate that in NC/Nga mice. Both in vivo and in vitro IFN-{gamma} production induced by rIL-12 was less in NC/Nga mice than in BALB/c mice. Addition of rIFN-{gamma} to rIL-4 and LPS completely abrogated IgE production by B cells of BALB/c mice, but was insufficient to suppress it by B cells of NC/Nga mice. In splenic cells pretreated with Con A, STAT4 was phosphorylated at the tyrosine residue by addition of rIL-12, which was more weakly inducible in NC/Nga mice than in BALB/c mice. Finally, we examined the preventive ability of rIL-12 on the clinical aspects of atopic dermatitis in NC/Nga mice. rIL-12 administration resulted in exacerbation of development of the skin lesions and IgE production in NC/Nga mice raised in nonsterile circumstances. These results suggest that defective production of IFN-{gamma} by T cells less sensitive to IL-12 and low responsiveness of B cells to IFN-{gamma} may contribute to IgE hyperproduction in NC/Nga mice, and that IL-12 may have no ability to improve the clinical aspects of NC/Nga mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Strong polarization of Th1 or Th2 is recognized in some pathological conditions: Th1 in organ-specific autoimmunity, contact dermatitis, and chronic inflammation, and Th2 in atopic diseases (1, 2). Especially in atopic subjects, Th2 responses have been reported to be enhanced with reduced Th1 reactions (3, 4, 5, 6, 7, 8, 9). IL-12, a heterodimeric cytokine composed of 40-kDa (p40) and 35-kDa (p35) subunits, is produced by B cells (10, 11), macrophages (12), or dendritic cells (13), and plays a central role in promoting Th1 responses (14). IL-12 has an ability to not only inhibit Th2 responses, but also enhance cytotoxicity (14, 15). Actually, the IL-12 administration is successful to give BALB/c mice, susceptible to Leishmania major infection, the protective immune responses against L. major infection following the differentiation of Th1 (16, 17); and in mice deficient in IL-12 production or IL-12R expression, IFN-{gamma} production is reduced, and the resistance to L. major infection is impaired (18, 19, 20, 21). The fact that the inherited IL-12 and IL-12R deficiencies were found in patients suffering from severe, idiopathic, and disseminated or recurrent mycobacterial or bacterial infection (22, 23, 24, 25) has confirmed an important and indispensable role of IL-12 in acquisition of the resistance to microbial infection through the initiation of the Th1 response. In addition to the induction of Th1, IL-12 is capable of abolishing IgE production in mice immunized with ragweed Ag with aluminum hydroxide (26), infected with intestinal nematode parasites (27), and injected with anti-IgD Ab (28), by inhibition of Th2 responses. On the basis of biological functions of IL-12, preventive or therapeutic trials were performed on several diseases: intracellular microbial infections (16, 18, 29, 30), autoimmune encephalomyelitis (31), allergic airway inflammation (26, 32), tumors (33), and Ag-specific tolerance of contact sensitivity (34).

We recently demonstrated that NC/Nga mice, which were established as an inbred strain, spontaneously developed atopic dermatitis (AD)3-like skin lesions closely associated with severe elevation of IgE levels when they were raised in air-uncontrolled conventional circumstances. Clinical signs and symptoms of the mice begin with scratching behavior and eczema, followed by hemorrhage, superficial erosion, deep excoriation, scaling, and dryness of the skin (35, 36). In the affected skin tissues, epidermal hyperplasia, infiltration of numerous CD4+ T cells and eosinophils, and increased number of mast cells with degranulation are observed (35). Additionally, biophysical and biochemical investigation shows impairment of water retention properties and barrier function with decreased levels of ceramide in the skin (36). These findings resemble those of patients with AD, suggesting that the mice are available as an animal model for human AD. More recently, we have found that a major determinant quantitative-trait locus responsible for dermatitis in NC/Nga mice is located on chromosome 9, which is designated as derm1 (37). Immunization with OVA led to higher production of IgE in NC/Nga mice than that in BALB/c mice, suggesting that NC/Nga mice have an instinctive character to manifest Th2-dominant immune reactions in response to some allergens (38) as well as human subjects with atopic diseases (1, 2, 3, 4, 5, 6, 7, 8, 9). Therefore, we conducted experiments to examine the effects of rIL-12 and rIFN-{gamma} on IgE synthesis in NC/Nga mice to compare with those in BALB/c mice. These cytokines elicit functional activities through the binding to their own receptors, followed by triggering Janus kinase (JAK)-STAT pathways: JAK2, Tyk2, and STAT4 by IL-12; and JAK1, JAK2, and STAT1 by IFN-{gamma} (39).

In the present study, we demonstrated that rIL-12 induced defective production of IFN-{gamma} due to low phosphorylation of STAT4, resulting in failure of rIL-12 to inhibit IgE synthesis in NC/Nga mice immunized with OVA. Furthermore, rIFN-{gamma} was not capable of completely suppressing IgE production by B cells from NC/Nga mice. Thus, the lower responsiveness to rIL-12 associated with reduced phosphorylation of STAT4 may lead to defective production of IFN-{gamma}, thereby resulting in IgE hyperproduction in NC/Nga mice. Additionally, we showed that exogenous administration of rIL-12 exacerbated development of AD-like skin lesions and elevation of IgE levels in NC/Nga mice raised in conventional circumstances.


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

Male specific pathogen-free (SPF) NC/NgaTnd mice and BALB/c mice were obtained from Charles River Japan (Kanagawa, Japan). Mice (4–12 wk old) were maintained in a filter-laminar flow enclosure in a bioclean room, and provided with autoclaved food and water ad libitum for at least 1 wk before use. Several mating pairs of SPF NC/Nga mice were moved to an air-uncontrolled conventional room (conventional NC/Nga mice). Skin lesions very similar to human AD spontaneously appeared from the age of 8 wk in all the progeny (35). Animal experiments complied with the standards in the guidelines of the University Animal Care and Use Committee in Tokyo University of Agriculture and Technology.

Cytokines and Ab

Murine rIL-12 (biologic activity: 4.6 x 106 U/mg) was kindly provided by S. F. Wolf (Genetics Institute, Cambridge, MA). Murine rIL-4 and rIFN-{gamma} were purchased from PharMingen (San Diego, CA). FITC-conjugated rat anti-mouse CD45R/B220 mAb (clone RA3-6B2) were obtained from PharMingen. Rabbit polyclonal Ab against mouse STAT1 that recognize both 91- and 84-kDa proteins and mouse STAT4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-phosphotyrosine mAb (clone 4G10) and rabbit anti-phosphoSTAT1 Ab were provided by Upstate Biotechnology (Lake Placid, NY) and New England Biolabs (Beverly, MA), respectively. Peroxidase-conjugated polyclonal Ab against mouse IgG (H + L) and rabbit IgG (H + L) were obtained from Jackson ImmunoResearch (West Grove, PA) and Sigma (St. Louis, MO), respectively. Goat anti-rat IgG (H + L) F(ab')2 conjugated with magnetic beads were purchased from Miltenyi Biotec (Sunnyvale, CA). For flow cytometric analysis, biotinylated rat anti-mouse IL-12 mAb (p40/p70) (clone C17.8), biotinylated rat anti-mouse IFN-{gamma}R {alpha}-chain mAb (clone GR20), FITC-conjugated rat anti-mouse CD4 mAb (clone RM4-5), FITC-conjugated rat anti-mouse CD8a mAb (clone 53-6.7), and rat anti-mouse CD16/32 (Fc{gamma}RIII/IIa) mAb (clone 2.4G2) were provided by PharMingen. Unless otherwise indicated, all chemicals were purchased from Sigma.

In vivo rIL-12 treatment and immunization with OVA

Three different experiments were conducted to evaluate in vivo effects of rIL-12 on IgE production and development of skin lesions in NC/Nga mice. For administration to mice, rIL-12 was diluted at a concentration of 7000 U/ml in PBS supplemented with 0.01% mouse serum albumin. First, SPF NC/Nga and BALB/c mice were immunized with s.c. injection of 1 µg OVA with 4 mg aluminum hydroxide gel (alum), and 2 wk later, mice were reimmunized with i.p. injection of 2.5 µg OVA with 10 mg alum. rIL-12 (1400 U) was administered i.p. into the mice for consecutive 21 days from the day of the first immunization. The dosage of rIL-12 was selected with reference to the previous reports that showed its curative effect on murine leishmaniasis and its reducing effect on IgE production in mice infected with intestinal nematode parasites (16, 17, 27). Total IgE levels in immunized mice were measured at 7 days after the second immunization. In the second experiments, 1400 U rIL-12 or 200 µg Salmonella typhimurium LPS was applied into SPF NC/Nga and BALB/c mice for 5 days or once, respectively. Six hours after the final dosing of rIL-12 or LPS, plasmas were collected and stored at -20°C until a quantitative analysis of IFN-{gamma} by an ELISA. In the last experiment, 1400 U rIL-12 (daily), 104 U rIFN-{gamma} (twice per week), or vehicle solution alone (twice a week) was injected i.p. into 4-wk-old conventional NC/Nga mice for 4 wk. A total clinical severity score for AD-like skin lesions was defined as the sum of the individual scores graded as 0 (none), 1 (mild), 2 (moderate), and 3 (severe) for each of five signs and symptoms (scratch, erythema/hemorrhage, edema, excoriation/erosion, and scaling/dryness) (35). Plasma total IgE levels were determined by an ELISA.

Cell culture

B220+ B cells were positively collected from spleens of SPF NC/Nga and BALB/c mice by magnetic cell sorting, as described previously (38). Specimens of individual isolated cell populations were analyzed by an EPICS flow cytometer (Coulter, Hialeah, FL); purity of the cells was >90%. Effect of rIL-12 or rIFN-{gamma} on IgE synthesis of B cells was analyzed by an in vitro experimental system (38). Briefly, 105 B220+ B cells isolated from spleens of SPF NC/Nga and BALB/c mice were cultured in 200 µl RPMI 1640 supplemented with 10% FCS, 10-4 M 2-ME, 50 U/ml penicillin, and 50 µg/ml streptomycin in the presence of 200 U/ml rIL-4 and 10 µg/ml LPS in 96-well flat-bottom culture plates (Nunc A/S, Roskilde, Denmark) with various concentrations of rIL-12 or rIFN-{gamma} for 9 days at 37°C in a humidified atmosphere flushed with 5% CO2 in air. The culture supernatants were collected and stored at -20°C until a quantitative analysis for total IgE. To assay a cytokine productivity, splenic cells were incubated with 2 µg/ml Con A at a concentration of 2 x 106 cells/ml in RPMI 1640, FCS, 2-ME, and antibiotics in 24-well culture plates (Nunc A/S) at 37°C in a humidified atmosphere flushed with 5% CO2. Forty-eight hours later, the culture supernatants were collected and stored at -20°C until use. Next, we investigated effect of rIL-12 on IFN-{gamma} synthesis by the method described previously (20). Briefly, Con A-stimulated splenic cells were harvested, resuspended at a concentration of 2.5 x 106 cells/ml in the culture medium, and incubated with various concentrations of rIL-12 for 24 h. The culture supernatants were collected to determine IFN-{gamma} levels by an ELISA.

rIL-12 binding on CD4+ T cells and IFN-{gamma}R expression on B220+ B cells

Flow cytometric analyses for rIL-12 binding and IFN-{gamma}R expression were performed as described previously (40, 41, 42). Briefly, for detection of rIL-12 binding, splenic cells isolated from SPF NC/Nga and BALB/c mice were incubated with 2 µg/ml Con A at a concentration of 2 x 106 cells/ml for 48 h at 37°C. Con A-stimulated and freshly isolated splenic cells were incubated with or without 2300 U/ml rIL-12 in PBS supplemented with 2% FCS and 0.1% sodium azide (washing buffer) for 1 h at 4°C. Cells incubated without rIL-12 were served as a negative control. After treatment with 10 µg/ml anti-mouse CD16/32 mAb for 1 h at 4°C to inhibit nonspecific Ig binding, the cells were incubated with biotinylated anti-mouse IL-12 mAb for 1 h. Then well-washed cells were stained with PE-streptavidin (PharMingen) to detect rIL-12 bound to the cell surface. To specify rIL-12-bearing cell populations, the cells were reincubated with FITC-conjugated anti-CD4 mAb, anti-CD8 mAb, or anti-CD45R/B220 mAb for additional 1 h. The mean fluorescence intensity (MFI) ratio was calculated as: MFI on cells incubated with rIL-12/MFI on cells incubated without rIL-12. For determination of IFN-{gamma}R expression, splenic cells were stimulated with 10 µg/ml LPS for 48 h. LPS-activated and freshly isolated splenic cells were pretreated with 10 µg/ml anti-mouse CD16/32 mAb to block nonspecific binding of Ab, and sequentially incubated with biotinylated anti-mouse IFN-{gamma}R {alpha}-chain mAb for 1 h at 4°C. Then the cells were washed and reacted with PE-streptavidin and FITC-labeled mAb against CD4, CD8, or CD45R/B220. The MFI ratio was calculated as: MFI on cells incubated with anti-mouse IFN-{gamma}R {alpha}-chain mAb/MFI on cells incubated without mAb. Flow cytometric analyses were conducted under gating CD4+ T cells for rIL-12 binding or under gating B220+ B cells for IFN-{gamma}R expression.

Measurement of total IgE

IgE levels were measured by a sandwich ELISA using two kinds of rat anti-mouse IgE mAb (clones 6HD5 and HMK12; Yamasa Shoyu, Chiba, Japan), according to the method described previously (38). The sensitivity of this assay was 2 ng/ml.

Cytokine assay

IFN-{gamma} levels were measured by an ELISA using two different mAb specific for murine IFN-{gamma} according to a slight modification of the method described previously (34). Briefly, immunoplates (Maxi-sorp; Nunc A/S) were coated with 1 µg/ml capture mAb (clone R4-6A2) in 0.1 M NaHCO3 (pH 8.3) overnight at 4°C. After blocking with PBS supplemented with 0.05% Tween 20 and 1% BSA for 1 h at room temperature, collected samples and standard murine rIFN-{gamma} were added and incubated overnight at 4°C, and then reacted with 0.5 µg/ml biotinylated detection mAb (clone XMG1.2) for 1 h. After peroxidase-conjugated avidin (1:2000; Dakopatts, Glostrup, Denmark) was provided, the reaction products were visualized with 0.4 mg/ml orthophenylenediamine and 0.012% H2O2. The absorbance at 490 nm wavelength was measured by an ImmunoMini NJ-2300 (Nalge Nunc International, Tokyo, Japan). The sensitivity of this assay was 78 pg/ml.

Western blot analysis of tyrosine-phosphorylated STAT1 and STAT4

Splenic cells isolated from SPF NC/Nga and BALB/c mice were incubated with 10 µg/ml LPS or 2 µg/ml Con A for 48 h at a concentration of 2 x 106 cells/ml. LPS- and Con A-stimulated splenic cells (4 x 107) were incubated with or without 100 U/ml rIFN-{gamma} and 100 U/ml rIL-12 for 20 min, respectively. The cells were harvested and lysed in 1 ml ice-cold lysis buffer (50 mM Tris-HCl, pH 8.0, 1% Nonidet P-40, 150 mM NaCl, 100 mM sodium orthovanadate, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 10 µg/ml aprotinin, and 1 mM EDTA) for 30 min. The lysates were centrifuged at 10,000 x g for 30 min at 4°C, and supernatants were immunoprecipitated with anti-STAT1 or anti-STAT4 Ab (10 µg/ml) and protein A-Sepharose CL-4B (50% suspension equilibrated with lysis buffer) (Pharmacia Fine Chemicals, Uppsala, Sweden) for 12 h at 4°C under gentle rotation. The immunoprecipitates were washed four times with 1 ml cold lysis buffer, resuspended in Laemmli’s sample buffer (50 mM Tris-HCl, pH 6.8, 10% glycerol, 1% SDS, 0.1% bromphenol blue, and 1 mM DTT), and boiled for 5 min. The samples were subjected to 7.5% SDS-PAGE, and the resolved samples were transferred electrophoretically to Immobilon-P membranes (Millipore, Bedford, MA). The membranes were immunoblotted with 1 µg/ml anti-phosphoSTAT1 Ab or 1 µg/ml anti-phosphotyrosine mAb for 1 h, washed, and incubated with peroxidase-conjugated Ab for 30 min. Tyrosine-phosphorylated STAT1 and STAT4 were detected with an ECL detection reagent (Amersham, Arlington Heights, IL).

IL-12R mRNA determination by RT-PCR

Total RNA was isolated from 5 x 107 Con A-stimulated splenic cells by using TRIzol reagent (Life Technologies, Gaithersburg, MD), according to the manufacturer’s instruction. The first-strand cDNA was generated by SuperScript Preamplification System (Life Technologies) from 5 µg total RNA. PCR amplification was performed using 1 µl cDNA template and 25 µl of a reaction mixture consisting of 10 mM Tris-HCl, 50 mM KCl, 0.2 mM dNTP, 1.5 mM MgCl2, 2.5 U Taq DNA polymerase (Roche Diagnostics, Mannheim, Germany), and 0.8 µM primer. The thermal cycling conditions were 94°C for 1 min, 60°C for 1 min, and 72°C for 30 s. A negative control was included in each assay to rule out DNA contamination. Primer sequences were designated in our laboratory for IL-12R {beta}1, {beta}2, and {beta}-actin based on the cloned sequences (GenBank accession numbers U23922, U64199, and X03672, respectively). The sequences were as follows: IL-12R {beta}1 sense, 5'-GTC-ACA-ATC-ACA-CGG-GCA-GT-3' and antisense, 5'-AGG-TTC-AGC-TTC-TTG-CCC-AG-3' (product size, 522 bp); IL-12R {beta}2 sense, 5'-CCG-ACG-CTC-TCA-AAA-CTC-AC-3' and antisense, 5'-GCT-GTG-AGA-GTT-CCT-GTA-GC-3' (product size, 534 bp); and {beta}-actin sense, 5'-TGG-TCG-TAC-CAC-AGG-CAT-TG-3' and antisense, 5'-TGA-TGT-CAC-GCA-CGA-TTT-CC-3' (product size, 203 bp). All were amplified 26 cycles that were within the linear range for IL-12R {beta}2. Amplified PCR products were resolved by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining. After 26 cycles amplification, for detection of IL-12R {beta}1 mRNA, secondary PCR was performed 10 cycles under the same thermal cycling condition in the first PCR by using the first PCR product as the DNA template and following primers: sense, 5'-CGA-ATT-GGA-CCT-TGG-GTG-AC-3'; antisense, 5'-ACA-CAG-GCA-TGC-TCC-AAT-CA-3' (product size, 195 bp).

Statistical analysis

A two-tailed Student t test was performed for statistical analysis of the data, and p < 0.05 was taken as the level of significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of rIL-12 on IgE production in mice immunized with OVA and alum

Since administration of IL-12 is capable of inducing Th1, inhibiting Th2 differentiation (2, 14, 15, 16, 17), and reducing IgE synthesis in vivo (26, 27, 28), we attempted to demonstrate whether rIL-12 had an ability to reduce or inhibit IgE synthesis in SPF NC/Nga and BALB/c mice immunized with OVA and alum. Mice were injected with 1400 U rIL-12 daily for 21 days from the day of the first immunization, and plasma IgE levels were measured 7 days after the second immunization. The immunization protocol with OVA and alum induced an increase in plasma levels of total IgE in both strains of mice; rIL-12 administration reduced IgE levels in BALB/c mice, but not in NC/Nga mice (Fig. 1Go). In addition, NC/Nga mice produced higher levels of IgE than control BALB/c mice in vivo. No clinical symptoms, including dermatitis, were observed in both SPF mice treated with rIL-12 (data not shown). Thus, we concluded that NC/Nga mice had a defect in rIL-12 responsiveness to down-regulate IgE production.



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  		FIGURE 1. Effect of rIL-12 on IgE production in BALB/c mice and SPF NC/Nga mice immunized with OVA and alum. One week after the second immunization with OVA and alum, plasma levels of total IgE were determined by an ELISA. Immunized mice were daily injected with 1400 U rIL-12 (hatched) or vehicle alone (open) for 21 days from the day of first immunization. Each value represents the mean ± SE of six to eight mice per group. *, p < 0.01, when compared with control.

 
Effect of rIL-12 and LPS on IFN-{gamma} production in vivo

IL-12 has its biological properties through induction of IFN-{gamma} synthesis (15, 16, 27, 30, 43). Therefore, we examined plasma levels of IFN-{gamma} in SPF NC/Nga and BALB/c mice daily treated with 1400 U rIL-12 for 5 days. The injection with rIL-12 led to high levels of IFN-{gamma} in BALB/c mice, but very low levels in NC/Nga mice (Fig. 2Go). In control NC/Nga and BALB/c mice injected with vehicle only, IFN-{gamma} was not detected in plasmas. Since LPS is capable of stimulating IFN-{gamma} synthesis in vivo (18, 20, 21), single i.p. injection of 200 µg LPS was performed, and 6 h later plasma samples were collected. Although LPS induced IFN-{gamma} production in both NC/Nga and BALB/c mice, IFN-{gamma} levels in NC/Nga mice were about one-half those in BALB/c mice (Fig. 2Go).



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  		FIGURE 2. rIL-12- and LPS-induced IFN-{gamma} production in SPF NC/Nga mice (hatched) and BALB/c mice (open). Mice were daily injected i.p. with 1400 U rIL-12 or vehicle for 5 days or injected with 200 µg LPS once. At 6 h after the final injection with rIL-12 or single injection with LPS, plasma levels of IFN-{gamma} were determined by an ELISA. No IFN-{gamma} was detected in plasmas of mice treated with vehicle alone. Each value represents the mean ± SE of three to six mice per group. *, p < 0.01, when compared with BALB/c mice.

 
IFN-{gamma} production by splenic cells

The above findings suggested that the low productivity of IFN-{gamma} in NC/Nga mice might be attributable to their low susceptibility to rIL-12. Therefore, we examined a productivity of IFN-{gamma} by splenic cells stimulated with Con A in the presence or absence of rIL-12. First, splenic cells of SPF NC/Nga and BALB/c mice were incubated with 2 µg/ml Con A for 48 h, and IFN-{gamma} levels in the culture supernatants were determined. Con A induced significant production of IFN-{gamma} by splenic cells of NC/Nga and BALB/c mice, but its levels in NC/Nga mice were about half of those in BALB/c mice (Table IGo), which was compatible with the result of the in vivo study. Secondary, to investigate effect of rIL-12 on in vitro IFN-{gamma} production, splenic cells prestimulated with Con A were reincubated with various concentrations of rIL-12 (0.001~10 U/ml) for 24 h. As shown in Fig. 3Go, rIL-12 increased IFN-{gamma} production by Con A-stimulated splenic cells of both NC/Nga and BALB/c mice in a dose-dependent manner. However, the effect of rIL-12 was lower in splenic cells of NC/Nga mice than in those of BALB/c mice. Thus, NC/Nga mice manifested a defective response to rIL-12 to drive IFN-{gamma} production as compared with BALB/c mice.


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  		Table I. IFN-{gamma} production by splenic cells1

 


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  		FIGURE 3. Effect of rIL-12 on IFN-{gamma} production by splenic cells treated with Con A. Splenic cells isolated from SPF NC/Nga mice (•) and BALB/c mice ({circ}) were incubated at a concentration of 2 x 106 cells/ml with 2 µg Con A for 48 h, and reincubated with IL-12 for 24 h. IFN-{gamma} concentrations in culture supernatants were determined by an ELISA. Each point represents the mean ± SE of three separate experiments performed in triplicate.

 
Effect of rIL-12 or rIFN-{gamma} on IgE production in vitro

Since IL-12 and IFN-{gamma} have an ability to reduce in vitro IgE synthesis (44, 45), we examined effect of these cytokines on IgE production by B cells in vitro. B220+ B cells isolated from spleens of NC/Nga and BALB/c mice were incubated with various doses of rIL-12 (10–10,000 U/ml) or rIFN-{gamma} (1–1,000 U/ml) in the presence of 200 U/ml rIL-4 and 10 µg/ml LPS for 9 days. Costimulation with rIL-4 and LPS induced significant IgE production by B cells of both strains of mice, with individual sensitivities to rIL-4 and LPS, which recomfirmed the previous result (38). rIL-12 had no influence on in vitro IgE production by B cells of both NC/Nga and BALB/c mice (Fig. 4GoA). On the other hand, rIFN-{gamma} was capable of decreasing IgE production by B cells of both the strains of mice in a dose-dependent manner (Fig. 4GoB). When >10 U/ml rIFN-{gamma} was added to the culture of B cells isolated from BALB/c mice, IgE levels were under the detection limit of an ELISA. In contrast, IgE production by B cells of NC/Nga mice was not completely suppressed even in the presence of 1,000 U/ml rIFN-{gamma} (Fig. 4GoB). Thus, significant difference in the responsiveness of splenic B cells to rIFN-{gamma} was noted between NC/Nga and BALB/c mice.



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  		FIGURE 4. Effects of rIL-12 (A) and rIFN-{gamma} (B) on IgE production by B220+ B cells. B220+ B cells isolated from spleens of SPF NC/Nga mice (•) and BALB/c mice ({circ}) were incubated with various concentrations of rIL-12 or rIFN-{gamma} in the presence of 200 U/ml rIL-4 and 10 µg/ml LPS for 9 days. IgE levels in the collected culture supernatants were measured by an ELISA. Each point represents the mean ± SE of three separate experiments performed in duplicate.

 
Binding of rIL-12 on T cells and expression of IFN-{gamma}R on B cells

Since IL-12 and IFN-{gamma} exert its biological activities through the binding of these cytokines to their own receptors, we next examined the binding of rIL-12 on CD4+ T cells and the expression of IFN-{gamma}R on B220+ B cells by a flow cytometric analysis. Binding of rIL-12 was detected on Con A-stimulated CD4+ T cells of NC/Nga and BALB/c mice (Fig. 5GoA), but not on freshly isolated CD4+ T cells of both strains of mice. The MFI ratio was increased by stimulation with Con A, whereas the ratio on CD4+ T cells was not different between NC/Nga and BALB/c mice (Fig. 5GoC), indicating that the IL-12R was expressed on CD4+ T cells of NC/Nga as well as on those of BALB/c mice in stimulation with Con A. These results suggested that lower responsiveness to rIL-12 for IFN-{gamma} production in NC/Nga mice than in BALB/c mice was not due to lower expression of IL-12R. The IFN-{gamma}R was expressed constitutively on freshly isolated splenic B cells of NC/Nga and BALB/c mice; there was no significant difference in its expression (Fig. 5Go, B and D). Even when B cells were stimulated with LPS for 48 h, IFN-{gamma}R expression was detected in the same intensity on B cells of NC/Nga and BALB/c mice (Fig. 5Go, B and D).



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  		FIGURE 5. Flow cytometric analyses for rIL-12 binding on Con A-stimulated CD4+ T cells and for expression of the IFN-{gamma}R on LPS-stimulated B220+ B cells. Splenic cells isolated from SPF NC/Nga and BALB/c mice were incubated with or without Con A (2 µg/ml) or LPS (10 µg/ml) for 48 h. A, Con A-stimulated cells were incubated with 2300 U/ml rIL-12, and were stained with biotinylated anti-IL-12 mAb and PE-conjugated streptavidin, and followed by incubation with FITC-conjugated anti-CD4 mAb. B, LPS-stimulated cells were stained with biotinylated anti-IFN-{gamma}R {alpha}-chain mAb and PE-conjugated streptavidin, and followed by incubation with FITC-conjugated anti-B220 mAb. C, rIL-12 binding: the MFI ratio on freshly isolated and Con A-stimulated CD4+ T cells. D, Expression of the IFN-{gamma}R: the MFI ratio on freshly isolated and LPS-stimulated B220+ cells.

 
Tyrosine phosphorylation of STAT1 and STAT4

We hypothesized that hyporesponsiveness to IFN-{gamma} and IL-12 in NC/Nga mice may be caused by a defect in signal transduction of those cytokines. IFN-{gamma} and IL-12 trigger JAK-STAT signaling pathways: JAK1, JAK2, and STAT1 by IFN-{gamma}, and JAK2, Tyk2, and STAT4 by IL-12 following the binding to their own receptors (39). Therefore, we investigated tyrosine phosphorylation of STAT1 and STAT4 of splenic cells stimulated with rIFN-{gamma} and rIL-12. The addition of rIFN-{gamma} or rIL-12 led to tyrosine phosphorylation of STAT1 or STAT4 in splenic cells of NC/Nga and BALB/c mice, respectively, whereas little or no tyrosine phosphorylation was detectable in splenic cells pretreated with LPS or Con A without the cytokines (Fig. 6Go). The degree of STAT1 phosphorylation in splenic cells of NC/Nga mice induced by rIFN-{gamma} was comparable with that in splenic cells of BALB/c mice (Fig. 6GoA). Since the anti-STAT1 Ab used for this analysis are capable of recognizing both 84- and 91-kDa proteins differentially spliced by a single gene (39), two phosphorylated bands were detected. On the other hand, although there was no significant difference in the loaded amount of STAT4 in splenic cells between NC/Nga and BALB/c mice, STAT4 phosphorylation was remarkably decreased in splenic cells of NC/Nga mice as compared with that of BALB/c mice (Fig. 6GoB).



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  		FIGURE 6. Western blot analysis for phosphorylation of STAT1 (A) and STAT4 (B) in splenic cells stimulated with rIFN-{gamma} and rIL-12. Splenic cells isolated from SPF NC/Nga and BALB/c mice were pretreated with 10 µg/ml LPS or 2 µg/ml Con A for 48 h. A, LPS-stimulated splenic cells were incubated with or without 100 U/ml rIFN-{gamma} for 20 min. Whole cell lysates were immunoprecipitated with anti-STAT1 Ab. Following separation by SDS-PAGE and transfer to a nitrocellulose membrane, the blot was probed with anti-phosphoSTAT1 Ab, and followed by stripping and reprobing with anti-STAT1 Ab. B, Con A-stimulated splenic cells were incubated with or without 100 U/ml rIL-12 for 20 min. Whole cell lysates were immunoprecipitated with anti-STAT4 Ab. The blot was probed with anti-phosphotyrosine mAb, and followed by stripping and reprobing with anti-STAT4 Ab.

 
IL-12R {beta}1 and IL-12R {beta}2 mRNA levels in splenic cells

Since expression of the IL-12R {beta}2 subunit is closely related to phosphorylation of STAT4 induced by IL-12 (22, 46, 47), we next examined expression of IL-12R {beta}2 mRNA in Con A-stimulated splenic cells by RT-PCR. Con A stimulation induced expression of IL-12R {beta}1 mRNA in NC/Nga mice, which was roughly comparable with that in BALB/c mice (Fig. 7Go). On the other hand, expression of IL-12R {beta}2 mRNA was markedly decreased in splenic cells of NC/Nga mice as compared with that in BALB/c mice (Fig. 7Go).



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  		FIGURE 7. IL-12R mRNA expression in splenic cells stimulated with Con A. Splenic cells isolated from NC/Nga and BALB/c mice were stimulated with Con A (2 µg/ml) for 6 and 12 h. mRNA expression of IL-12R {beta}1 and {beta}2 chains and {beta}-actin was assayed by RT-PCR, followed by electrophoresis and ethidium bromide staining. This is representative of two separate experiments.

 
Effect of rIL-12 and rIFN-{gamma} on severity of skin lesions and plasma IgE levels

We finally examined effect of rIL-12 and rIFN-{gamma} on the onset or progression of dermatitis and IgE hyperproduction in conventional NC/Nga mice. NC/Nga mice that were raised in nonsterile air-uncontrolled conventional circumstances started to scratch their faces, necks, ears, and dorsal skins from the age of 7 wk, resulting in various grades of dermatitis; and clinical condition of the dermatitis got more severe, correlating with elevation of plasma total IgE levels (Fig. 8Go), as described previously (35). When treated with 1400 U rIL-12 or 104 U rIFN-{gamma} from the age of 4 wk, all NC/Nga mice revealed more severe clinical symptoms at the ages of 7 and 8 wk as compared with those of age-matched control mice treated with vehicle alone; the exacerbating effect of rIL-12 was stronger than that of rIFN-{gamma} (Fig. 8GoA). Simultaneously, we measured total IgE levels in plasma of mice treated with rIL-12, rIFN-{gamma}, or vehicle alone. Although rIL-12 treatment led to elevation in levels of total IgE as early as the age of 6 wk, there was no difference in IgE levels between rIFN-{gamma}-treated and control mice (Fig. 8GoB). These findings clearly demonstrated, unexpectedly, that administration with rIL-12 and rIFN-{gamma} exacerbated the onset and progression of AD-like skin lesions; and the former, unlike the latter, promoted the elevation of plasma total IgE levels, suggesting that rIL-12 had no ability to improve the dermatitis.



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  		FIGURE 8. Clinical skin conditions (A) and plasma levels of total IgE (B) in conventional NC/Nga mice treated with rIL-12. Four-week-old littermates raised in conventional circumstances were given 1400 U rIL-12 (•), 104 U rIFN-{gamma} ({blacktriangleup}), or vehicle alone ({circ}) until the age of 8 wk. A total clinical severity score for skin lesions and IgE levels was quantitated according to the criteria described (35 ). Each value represents the mean ± SE of three to four mice per group. *, p < 0.05, when compared with age-matched vehicle control. **, p < 0.01, when compared with age-matched vehicle control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In addition to the generation of mice having disrupted the genes for IL-12 itself (18, 19) and its receptors (20, 21), immunological findings reported in IL-12 (22, 23)- or IL-12R {beta}1 chain-deficient patients (24, 25) demonstrate that IL-12 has an indispensable role for initiation of Th1 responses. Administration of IL-12 induced Th1 and inhibits Th2 differentiation in murine models for infection with parasites or for allergy (16, 17, 27, 32, 48). The i.p. injection with IL-12, >100 ng/day, is successful to reduce the increase of IgE levels in mice inoculated with intestinal nematode parasites, Nippostrongylus brasiliensis, and in mice injected with anti-IgD Ab (27, 28). Therefore, first, we conducted the present experiments to investigate how rIL-12 affected IgE synthesis in NC/Nga mice available as a model for human AD. Administration of rIL-12 (1400 U/day), unexpectedly, failed to suppress IgE synthesis in SPF NC/Nga mice immunized with OVA and alum.

IL-12 exerts its biological functions through production of IFN-{gamma} (15, 16, 27, 28, 43), which is a key cytokine to down-regulate IgE both in vivo and in vitro (27, 45). In fact, rIL-12 induced IFN-{gamma} synthesis in BALB/c mice in vivo and rIFN-{gamma} abolished in vitro IgE production by B cells isolated from BALB/c mice. However, NC/Nga mice showed a low IFN-{gamma} productivity by stimulation with rIL-12 in vivo; and the inhibitory ability of rIFN-{gamma} on in vitro IgE production was insufficient at even high concentrations to B cells of NC/Nga mice. These results suggested that NC/Nga mice were low responsive not only to IL-12 for induction of IFN-{gamma} synthesis, but also to IFN-{gamma} for inhibition of IgE synthesis by B cells as compared with BALB/c mice. Thus, we speculated that hyporesponsiveness to these cytokines that down-regulate IgE synthesis may contribute to IgE hyperproduction in NC/Nga mice. Human allergen-specific Th2 cells are unresponsive to IL-12 for IFN-{gamma} production due to the lack of both the DNA-binding activity of STAT4 and the IL-12-induced phosphorylation of STAT4 (49). Moreover, STAT4 phosphorylation in response to IL-12 was depleted in the process of development of murine Th2 cells despite no change in expression of the IL-12R (50). In deed, STAT4 phosphorylation in splenic cells induced by rIL-12 was less in NC/Nga mice than in BALB/c mice despite equivalent binding of rIL-12 to activated CD4+ T cells of the both mice. In the additional experiments with RT-PCR, equivalent expression of IL-12R {beta}1 mRNA and weak expression of IL-12R {beta}2 mRNA were detected in Con A-stimulated splenic cells of NC/Nga mice as compared with those of BALB/c mice, suggesting that defective expression of the IL-12R {beta}2 subunit necessary for the signal transduction may result in low phosphorylation of STAT4 in CD4+ T cells of NC/Ng mice. On the other hand, there was no significant difference in the expression of the IFN-{gamma}R on B cells or the STAT1 phosphorylation in LPS-pretreated splenic cells following the stimulation with rIFN-{gamma} between NC/Nga and BALB/c mice, suggesting that B cells of NC/Nga mice had no defect in IFN-{gamma} signaling. IFN-{gamma} leads to a loss of IL-4-induced STAT6 tyrosine phosphorylation, nuclear translocation, and DNA binding, at least in part, through induction of silencer of cytokine signaling 1 (51). However, STAT1 activated by IFN-{gamma} is not capable of recognizing the STAT6-specific IL-4 response element in the {epsilon} promoter (51). Previously, we showed that JAK3 phosphorylation in stimulation with IL-4 and CD40 ligand was more inducible in B cells of NC/Nga mice than those of BALB/c mice (38). Therefore, we speculated that IFN-{gamma}-induced silencer of cytokine signaling 1 might be insufficient to suppress enhanced JAK3 phosphorylation, followed by STAT6 activation in B cells of NC/Nga mice, resulting in the incomplete inhibition of IgE production by IFN-{gamma} in B cells of NC/Nga mice.

Since, in human allergic disorders including AD (3, 4, 5, 6, 7, 8) and atopic asthma (9), Th2-dominant responses are strongly involved in their pathogenesis, there is a possibility that a resolution of the imbalance of Th1/Th2 responses may be effective as a treatment for such diseases. Actually, IL-12 evoked Th1 responses in BALB/c mice, which instinctively manifested Th2-dominant immune response and protected the mice from L. major infection (16, 17). Therefore, we attempted to investigate the therapeutic or prophylaxis potential of rIL-12 to develop or progress AD-like skin lesions and IgE hyperproduction in conventional NC/Nga mice. Unexpectedly, rIL-12 exacerbated the development of dermatitis and elevation of IgE levels. Finkelman et al. (27) showed that IL-12 needed to be administrated by day 4 of primary infection with parasites to inhibit IgE production through IFN-{gamma} synthesis. Furthermore, the potent immunoregulatory effect of IL-12 on allergic lung inflammation is dependent on timing of IL-12 administration relative to the sensitization and challenge (26). These findings demonstrated that IL-12 was ineffective on suppression of IgE synthesis committed in vivo, suggesting a possibility that IL-12 is not capable of reducing IgE levels in conventional NC/Nga mice due to the existence of B cells committed to synthesize IgE. Additionally, IL-12 potentiates not only IFN-{gamma}, but also IL-4 production by an established human Th2-like clone (52) and to exacerbate Th2-dependent responses in IFN-{gamma} knockout mice: granuloma formation, eosinophil infiltration, and IgE synthesis induced by injection of Schistosoma mansoni eggs (53). These reports suggested another possibility that IL-12 directly enhances Th2 differentiation and IgE synthesis in NC/Nga mice.

Much is known regarding how IFN-{gamma} produced in the process of Th1 responses is strongly involved in the elicitation of delayed-type hypersensitivity, including contact sensitivity in human subjects and in rodents (18, 54, 55). Injection with rIFN-{gamma} increased clinical skin severity scores in conventional NC/Nga mice, whereas it did not affect plasma IgE levels, suggesting that IFN-{gamma} may participate in manifestation of dermatitis in NC/Nga mice. Although rIL-12 administration led to lower production of IFN-{gamma} in NC/Nga mice as compared with BALB/c mice, subsequently produced IFN-{gamma} may enhance Th1 responses, resulting in exacerbation of the dermatitis in NC/Nga mice. It is now emphasized that both Th1 and Th2 cells play important roles in pathogenesis in AD (56, 57). Therefore, we speculated that the consecutive treatment of conventional NC/Nga mice with rIL-12 may drive both Th1 and Th2 responses, thereby resulting in the aggravation of the skin lesions and the enhancement of IgE synthesis. Therefore, to identify the immunological characteristics of NC/Nga mice and patients with AD, further investigations including not only Th2 responses, but also Th1 responses should be done.


   Acknowledgments
 
We thank Dr. S. F. Wolf (Genetics Institute) for providing rIL-12. We are also grateful to Dr. K. Tsubota (Fujisawa Pharmaceutical) for his technical help with RT-PCR.


   Footnotes
 
1 This work was supported by grants from the Ministry of Education, Science, Sports, and Culture; from the Pioneering Research Project in Biotechnology, the Ministry of Agriculture, Forestry, and Fisheries, Japan; and for Specially Promoted Research on Atopic Disorders from the Tokyo Metropolitan Government. Back

2 Address correspondence and reprint requests to Dr. Hiroshi Matsuda, Laboratory of Clinical Immunology, Department of Veterinary Clinic, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan. E-mail address: hiro{at}cc.tuat.ac.jp Back

3 Abbreviations used in this paper: AD, atopic dermatitis; JAK, Janus kinase; MFI, mean fluorescence intensity; SPF, specific pathogen-free. Back

Received for publication March 15, 2001. Accepted for publication September 10, 2001.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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