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The Mechanism of a Defective IFN- Response to Bacterial Toxins in an Atopic Dermatitis Model, NC/Nga Mice, and the Therapeutic Effect of IFN-, IL-12, or IL-18 on Dermatitis¹

Yoshiko Habu^*, Shuhji Seki^2,*, Eiji Takayama^*, Takashi Ohkawa, Yuji Koike, Katsunori Ami^*, Takashi Majima and Hoshio Hiraide^*

^* Division of Basic Traumatology, National Medical Department of Pediatrics, and Department of Surgery I, National Defense Medical College Research Institute, Tokorozawa, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
NC/Nga (NC) mice raised under conventional conditions (Conv. NC mice) spontaneously develop dermatitis similar to human atopic dermatitis, whereas NC mice raised under the specific pathogen-free conditions do not develop dermatitis. In the present study, we show that the representative Th1 cytokine, IFN- levels in the sera of NC mice, injected with either staphylococcal enterotoxin B or endotoxin (LPS), to be severalfold lower than those of normal mice. The low IFN- response to staphylococcal enterotoxin B was correlated to the lack of regular V8⁺ T cells and V8⁺ NK T cells, and the low IFN- response to LPS was correlated to an impaired IL-18 production of macrophages. The CD3-stimulated IL-4 production from liver and spleen T cells from Conv. NC mice in vitro was greatly augmented. The serum IL-4 levels of untreated Conv. NC mice also were higher than those of normal mice and specific pathogen-free NC mice. Treatment of Conv. NC mice either with IFN-, IL-12, or IL-18 twice a week from 4 wk of age substantially inhibited the elevation of the serum IgE levels, serum IL-4 levels, and dermatitis, and IL-12 or IL-18 treatment also reduced the in vitro IL-4 production from CD3-stimulated liver T cells. The systemic deficiency in the Th1 response to bacterial stimulation thus leads to a Th2-dominant state and may induce an abnormal cellular immune response in the skin accompanied with an overproduction of IgE and a susceptibility to dermatitis in NC mice.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Atopic dermatitis (AD)³ in humans is a common disease that frequently occurs in children. AD patients usually have a family history, hypersensitivity to certain Ags and high levels of serum IgE (1, 2). Previous studies suggested that the reduced IFN- production with the concurrent up-regulation of IL-4 induces the hyperproduction of IgE and AD (3, 4, 5), and several clinical trials suggested that IFN- therapy was found to improve AD symptoms (6, 7). However, the mechanism of the cytokine imbalance in atopic dermatitis patients remains to be elucidated. Dermatitis lesions of AD patients are frequently infected with staphylococcal enterotoxin B (SEB)-producing Staphylococcus aureus (8, 9), thus implying that either bacteria or bacterial factors might be involved in the pathogenesis of AD. NC/Nga (NC) mice were established as an inbred strain from Japanese fancy mice in 1957 by Kondo (10, 11, 12). In contrast to NC mice raised under specific pathogen-free conditions (SPF NC mice), NC mice raised under conventional conditions (Conv. NC mice) develop dermatitis with an overproduction of Th2 chemokines (12) and IL-4 associated with elevated serum IgE levels beyond the age of 8 wk (10, 11, 12). Therefore, circumstantial factors including bacterial Ags are suggested to be involved in the onset of dermatitis in these mice. However, the mechanism regarding the polarization to Th2-dominant states of these mice remains unknown.

Regular V8⁺ T cells in mice are main responder T cells to a bacterial superantigen, SEB (13), and produce IFN-. NK Ag 1.1⁺ T (NKT) cells also mainly use V14/V8.2 gene products for their TCR (14) and depend on a MHC class-I like molecule, CD1d, for their development (15, 16, 17). We and others have reported that IL-12 induced NKT cells to acquire a potent antitumor cytotoxicity through their IFN- production (18, 19, 20, 21). We also recently reported that IL-12 produced by SEB-primed Kupffer cells (resident liver macrophages) induced liver NK cells as well as NKT cells to produce IFN- (22). IL-12 produced by Kupffer cells and IFN- produced by these NK-type cells also were both essential for IL-12/LPS-induced generalized Shwartzman reaction (23). IL-18 is a recently identified cytokine that is produced by macrophages and Kupffer cells stimulated with either bacteria or their factors (including LPS; Ref. 24). IL-18 in the presence of IL-12 induces liver NK cells to produce a large amount of IFN- (24, 25). Based on these findings, liver leukocytes are suggested to play a crucial role in the host defense by inducing Th1 immune responses (21).

In the present study, we demonstrated that the serum IFN- levels of either SEB- or LPS-injected SPF NC mice or Conv. NC mice were severalfold lower than those of normal mice. This defective IFN- response to these common bacterial toxins, SEB and LPS, was correlated to the absence of V8⁺ T cells and decreased IL-18 production from macrophages, respectively. Consistent with these findings, the in vitro cytokine production from liver and spleen mononuclear cells (MNC) of Conv. NC mice was found to be biased toward IL-4. As a result, the defective Th1 immune response to bacterial stimulations may induce the Th2 response-dominant state in Conv. NC mice and their susceptibility to dermatitis. In addition, we show that not only IFN- and IL-12 but also IL-18 therapy significantly inhibited the elevation of serum IgE, serum IL-4, and dermatitis in Conv. NC mice.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

Eight-week-old SPF or Conv. NC mice and C57BL/6 mice and BALB/c mice raised under the SPF condition were purchased from SLC (Hamamatsu, Japan).

Reagents

SEB and staphylococcal enterotoxin A (SEA) were purchased from Sigma (St. Louis, MO). LPS (Escherichia coli 0111:B4) was purchased from Difco (Detroit, MI). Anti-CD3 Ab (145-2C11) was purchased from BD PharMingen (San Diego, CA). Mouse recombinant IL-18 was purchased from MBL (Nagoya, Japan). Mouse recombinant IL-12 was purchased from R&D System (Minneapolis, MN) and mouse IFN- was purchased from PeproTech (London, U.K.).

Isolation of liver MNC

Under deep ether anesthesia, the mice were euthanized and killed by exsanguination from the subclavian artery and vein, and then the liver was removed. To obtain liver MNC with Kupffer cells, the livers were minced with scissors and then were suspended in 10% FBS RPMI 1640 medium containing 1500 U/ml Dispase II (Godo Shusei, Tokyo, Japan) and finally were incubated in a 37°C water bath for 2 h while shaking. Thereafter, the specimens were washed twice and passed through a 200-gauge stainless steel mesh and liver MNC with Kupffer cells were thus obtained by osmolarity and pH-adjusted 33% Percoll solution containing 100 U/ml heparin and centrifuged at 2000 rpm for 15 min at room temperature (22, 23). The pellet was resuspended in a RBC lysis solution, then was washed twice in 10% FBS RPMI 1640. The proportion of plastic adherent Kupffer cells in all liver MNC was 30%. To obtain liver MNC without Kupffer cells, the liver was passed through a stainless steel mesh and then was suspended in an RPMI 1640 medium. After one washing, the cells were resuspended in 33% Percoll solution and thereafter were centrifuged as described above to obtain liver MNC. The degree of contamination by Kupffer cells or hepatocytes was minimal.

Flowcytometric analysis

Surface phenotypes of the MNC were identified by using mAbs in conjunction with the two-color immunofluorescence test. The mAbs used included FITC-conjugated anti-mouse TCR Ab (hamster IgG), PE-conjugated anti-NK1.1 Ab (mouse IgG2a), FITC-conjugated anti-V8 Ab (F23.1, mouse IgG2a), and FITC-conjugated mouse IgG2a isotype control Ab (BD PharMingen). The presence of fluorescence-positive cells was analyzed by EPICS XL (Coulter, Miami, FL).

PCR analysis of mRNAs for TCR and of genomic DNAs for V gene segments

cDNAs encoding TCR-chains were reverse-transcribed from total RNA isolated from splenic and hepatic MNC from C57BL/6 and NC mice and were amplified by PCR as follows. The portions of cDNAs encoding TCR-chains with V2.1 (350 bp), V8.3 (370 bp), V8.2 and V8.1 (370 bp), and V7.1 (390 bp) were amplified with 5'-TGT GAA CCT ACG CTG CAT CT-3', 5'-TGA CAG TAA CAG GAG GAA AC-3', 5'-TGG CAG TAA CAG GAG GAA AG-3', and 5'-AAC CCA GAT GCC AAG ATA CC-3', respectively as sense primers, and with 5'-GAT GGC TCA AAC AAG GAG AC-3' (correspondent to genes encoding C 1 and C 2) as an antisense primer. The cDNA sequence encoding GAPDH (498 bp) also was amplified by PCR as control experiments with 5'-ATG ACC ACA GTC CAT GCC AT-3' and 5'-GTC CAG GGT TTC TTA CTC CT-3' as a sense primer and an antisense primer, respectively. Each primer was hybridizable with the coding region within the exon 2 of each V gene segment, and each sequence of primer was not found in sequences of other V gene segments. The RNAs isolated by a method with guanidin-isothiocyanate were reverse-transcribed by the Superscript II (Life Technologies, Grand Island, NY) with oligo (dT), and then each specific cDNA was amplified by PCR with an expanded high-fidelity PCR system kit (Boehringer Mannheim, Mannheim, Germany).

The genomic sequences encoding the V gene segments of the TCR-chains were amplified by PCRs from genomic liver DNAs of NC mice and C57BL/6 mice. The fragments encoding V2.1 (224 bp), V8.3 (214 bp), V8.2 (229 bp), V8.1 (229 bp), and V7.1 (231 bp) were amplified with 5'-TGT GAA CCT ACG CTG CAT CT-3', 5'-TGA CAG TAA CAG GAG GAA AC-3', 5'-TGG CAG TAA CAG GAG GAA AG-3', 5'-TGG CAG TAA CAG GAG GAA AG-3', and 5'-AAC CCA GAT GCC AAG ATA CC-3' as sense primers and with 5'-AGG TGC AGT ACA AGG TTC TG-3', 5'-GGA GAA GCC AAT TCC AGC AG-3', 5'-ACT GAT GTC TGA GAG GGG GT-3', 5'-ACA GCT GTC TGA GAA AGG GA-3', and 5-'GCAGAATCCAGAATCAGGGA-3' as antisense primers, respectively. The portion encoding V14 (261 bp) of TCR-chain also was amplified by PCR as control experiments with 5'-GAA GTG GAG CAG AGT CCT CA-3' and 5'-GAT GTA GGT GGC AGT GTC AT-3' as a sense primer and an antisense primer, respectively. Freshly dissociated livers were incubated in 50 mM Tris-HCl (pH 8.0) containing 100 mM Na₂EDTA and 0.5% sodium dodecylsulfate with 20 µg/ml pancreatic RNase and 100 µg/ml proteinase K (Boehringer Mannheim) at 37°C for 1 h following at 50°C for 3 h. Genomic DNAs were extracted by phenol saturated with 500 mM Tris-HCl (pH 8.0) and precipitated by the addition of sodium acetate and ethanol, and then each specific fragment was amplified by PCRs with an expand high-fidelity PCR system kit (Boehringer Mannheim).

In vivo mouse treatment with SEB, LPS, and anti-CD3 Ab and blood samples

A total of 50 µg of SEB or LPS was i.p. injected into mice, and blood samples were obtained 3 or 6 h after injection by cutting the subclavian artery and vein when the mice were sacrificed. Sera were stocked at -20°C for ELISA. Anti-CD3 Abs (1 µg/200 µl) were i.v. injected into mice, and blood samples were obtained from the retro-orbital plexus at 1.5, 3, 6, and 12 h after Ab injections. Sera were stocked at -20°C for ELISA.

Cell cultures

Liver MNC with Kupffer cells (5 x 10⁵) in 200 µl of 10% FBS RPMI 1640 medium were cultured with SEB (10 µg/ml; Sigma) or SEA (2.5 µg/ml; Sigma) in 96-well flat-bottom plates in 5% CO₂ at 37°C for 48 h and then the culture supernatants were stocked at -20°C. For anti-CD3 Ab stimulation, 96-well flat-bottom plates were coated with 100 µl of anti-CD3 Ab (145-2C11; 10 µg/ml) overnight at 4°C, and 5 x 10⁵ liver MNC without Kupffer cells in 200 µl of 10% FBS RPMI 1640 medium were cultured with immobilized anti-CD3 Ab in 5% CO₂ at 37°C for 48 h and then the culture supernatants were stocked at -20°C.

ELISA of sera and culture supernatants

IFN-, IL-12, and IL-4 levels of sera or culture supernatants were measured by cytokine-specific ELISA kits (Endogen, Woburn, MA). The serum IL-18 levels were assayed with an ELISA kit (MBL). The serum IgE levels were measured with an ELISA kit (Morinaga, Yokohama, Japan). Serum IgG levels were measured by an ELISA kit (Bethyl Laboratory, Montgomery, TX). The sera were usually 20-fold diluted by the assay buffer included in the ELISA kit to measure the IFN-, IL-12, and IL-18 levels, and the sera were usually 5-fold diluted for the IL-4 measurement.

Treatment of Conv. NC mice with IFN-, IL-12 or IL-18

Conv. NC mice were i.p. injected with either recombinant IL-12 (0.1 µg, 1 x 10³U), IL-18 (0.1 µg), IFN- (1 µg, 1 x 10⁴ U), or PBS as a control twice a week from 4 to 12 wk of age.

Evaluation on the severity of dermatitis

The evaluation of the severity of dermatitis was conducted essentially according to the same scoring method as that was recently reported by Hiroi et al. (26): no symptoms, 0; mild inflammation with scratching, 1; moderate inflammation and/or mild hemorrhage, 2; severe inflammation and/or hemorrhage or ulcer, 3.

Statistical analysis

Differences between the groups were analyzed by the Mann-Whitney U test or an ANOVA analysis with Fisher’s protected least-significant difference or with Scheffe’ F test by using the Stat View program (SAS Institute, Cary, NC) on an Apple computer (Apple Computer, Cupertino, CA). The differences in the severity of dermatitis in the NC mice were analyzed by the Mann-Whitney U test or the Kruskal-Wallis test. Differences were considered to be significant at p < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Defective IFN- response to SEB of NC mice both in vivo and in vitro

Six hours after SEB injection through the tail veins into SPF NC mice, Conv. NC mice, SPF C57BL/6 mice, and SPF BALB/c mice, sera were obtained and the IFN- levels were examined. The results showed the IFN- levels of NC mice to be severalfold lower than those of BALB/c mice (Fig. 1A). We also examined the SEB-stimulated IFN- production from the liver, spleen, and lymph node MNC. The results showed that the production of IFN- in vitro also was severely impaired (Fig. 1, B–D) in NC mice as compared with BALB/c mice. The low IFN- response to SEB of C57BL/6 mice (Fig. 1, A–D) is probably related to the absence of MHC class-II molecule I-E in C57BL/6 mice because SEB predominantly requires I-E molecules over I-A molecules to evoke a full immune response (27). However, the liver and spleen MNC of SPF NC mice responded normally to SEA and also produced large amounts of IFN- in vitro comparable to those of BALB/c mice and C57BL/6 mice. In addition, the spleen MNC from Conv. NC mice stimulated with SEA in vitro produced a significantly lower amount of IFN- than the spleen MNC from other mice groups (Fig. 1, E and F).

View larger version (27K): [in this window] [in a new window]   FIGURE 1. An impaired IFN- production in response to SEB in NC mice. Serum IFN- levels in C57BL/6 mice, BALB/c mice, and NC mice injected with SEB (A). Six hours after an i.p. injection of SEB (50 µg) into either SPF NC mice, Conv. NC mice, or SPF control mice, sera were obtained for ELISA. The serum IFN- levels were the means ± SE from 10 mice of each strain. The in vitro IFN- production in response to SEB in the liver MNC (B), spleen MNC (C) and lymph node (LN) MNC (D) from SPF C57BL/6 mice, SPF BALB/c mice, SPF NC mice, and Conv. NC mice. In vitro IFN- production in response to SEA in the liver and spleen MNC from control mice and NC mice (E and F). A total of 5 x 105 liver MNC with Kupffer cells, spleen, and LN MNC in 200 µl of 10% FBS RPMI 1640 medium were cultured with SEB (10 µg/ml) or SEA (2.5 µg/ml) in 96-well flat-bottom plates in 5% CO2 at 37°C for 48 h and then the culture supernatants were stocked at -20°C and thereafter subjected to ELISA. The data represent the means ± SE from five independent experiments.

Absence of V8⁺ T cells in NC mice

Because V8⁺ T cells are a major responder to SEB, we examined the V8⁺ T cells in the SPF NC mice. Interestingly, the NC mice had a much smaller population of NKT cells in the liver MNC than C57BL/6 mice (4.8% vs 19.1%, n = 6) and lacked both regular V8⁺ T cells and V8⁺ NKT cells (Fig. 2, A and B), whereas the other T cells were present (Fig. 2B; n = 6). The staining of liver MNC with isotype control Ab for anti-V8 Ab (F23.1; mouse IgG2a) and anti-NK1.1 Ab indicated that few V8⁺ cells in the liver MNC of NC mice demonstrated a nonspecific background (Fig. 2A). Essentially similar results were obtained in Conv. NC mice (not shown). We next examined the expression of V gene products (mRNA) and genomic DNA for V genes of SPF NC mice and SPF C57BL/6 mice by PCR. A simplified diagram of the location of V, D, and C genes in genomic DNA is shown (Fig. 3A). NC mice lacked any expression of the TCR-chains using V8 gene products in the liver and spleen MNC, whereas mRNAs of the TCR-chains using V2.1 and V7.1 gene products were expressed in the liver and spleen MNC of NC mice in a manner similar to those in C57BL/6 mice (Fig. 3B). Furthermore, we could not detect all of the V8.3-, V 8.2-, and V 8.1-encoding segments in the genomic DNA from NC mice, whereas V2.1-, V7.1-, and V14-encoding segments were detected (Fig. 3C). These results strongly suggest that NC mice lack V8⁺ T cells and V8⁺ NKT cells because the locus involving the V8-encoding gene cluster is deleted whereas the V14 gene is present.

View larger version (28K): [in this window] [in a new window]   FIGURE 2. Absence of V8+ T cells in the NC mice. A, Reduction in the number of NK1.1+ T cells and the absence of NK1.1+V8+ T cells as well as NK1.1- V8+ T cells in the liver MNC of NC mice. The numbers in each right quadrant represent the percentage (means ± SE, n = 6) of respective T cells in the whole liver MNC. B, Absence of V8+ T cells not only in the liver but also in the spleen and thymus from NC mice.

View larger version (28K): [in this window] [in a new window]   FIGURE 3. PCR analysis of mRNA and genomic DNA of TCR-chains in C57BL/6 and NC mice. A, Simplified diagram of the genes encoding TCR -chains. Boxes indicate V-region-encoding segments or C genes. Vertical lines indicate D or J gene segments. B, Expressions of mRNAs for TCR-chains were analyzed by RT-PCR in spleens and livers from C57BL/6 and NC mice. Expressions of mRNAs for GAPDH also were analyzed as a control. C, Detection of gene segments encoding V-regions of TCR-chains by PCR in genomic DNAs from C57BL/6 and NC mice. The gene segment encoding V14 of TCR-chain also was analyzed.

The IL-4 production from the liver and spleen MNC in either C57BL/6 mice or NC mice in response to SEB also was significantly lower than that in BALB/c mice (Fig. 4, A and B). The defective IL-4 production in NC mice also may be related to the absence of V8⁺ T cells, whereas that in C57BL/6 mice may be related to the absence of the I-E molecule. However, the IL-4 production from the liver and spleen MNC of the Conv. NC mice in response to SEA was larger than that from the liver and spleen MNC in either SPF C57BL/6, BALB/c, or SPF NC mice (Fig. 4, C and D).

View larger version (28K): [in this window] [in a new window]   FIGURE 4. In vitro IL-4 production from liver and spleen MNC of SPF control mice, SPF NC mice, and Conv. NC mice in response to SEB or SEA. A total of 5 x 105 liver MNC with Kupffer cells and spleen MNC in 200 µl of 10% FBS RPMI 1640 medium were cultured with SEB (10 µg/ml) or SEA (2.5 µg/ml) in 96-well flat-bottom plates in 5% CO2 at 37°C for 48 h and then the culture supernatants were stocked at -20°C and thereafter subjected to ELISA. The data represent the means ± SE from five independent experiments.

Defective IFN- response of NC mice in response to LPS by impaired IL-18 production

SPF NC mice also produced much lower amounts of IFN- and IL-18 after the in vivo LPS stimulation despite the fact that they produced a substantial amount of IL-12 (Fig. 5, A–C) and the low IFN- response to LPS returned to normal levels when IL-18 (0.2 µg) was simultaneously injected with LPS (Fig. 5D). Essentially similar results also were obtained from Conv. NC mice (data not shown).

View larger version (25K): [in this window] [in a new window]   FIGURE 5. An impaired IFN- and IL-18 production in NC mice injected with LPS. A, Comparison of the serum IFN- levels among the mice injected with LPS. B, Comparison of the serum IL-12 levels among the mice injected with LPS. C, Comparison of the serum IL-18 levels among the mice injected with LPS. D, Effect of IL-18 on serum IFN- levels of the NC mice injected with LPS. IFN- and IL-18 were measured in the sera from SPF NC mice or control mice at 6 h after the injection of LPS (50 µg, i.p.) and the total IL-12 levels were measured in the sera from mice at 3 h after LPS injection. In the case of IL-18 injection, 0.2 µg of IL-18 was i.p. injected into NC mice simultaneously with LPS. A–C, Data represent the means ± SE from eight mice of each strain. D, Data represent the means ± SE from five NC mice of each group.

NC mice lack anti-CD3-stimulated early IFN- and IL-4 production in vivo

Because NKT cells were reportedly the source of early IFN- and IL-4 on in vivo anti-CD3 stimulation (15, 16, 17) and NC mice lacked V8⁺ NKT cells (a largest population of NKT cells), we examined the effect of the deletion of V8⁺ NKT cells on response to in vivo anti-CD3 stimulation in NC mice. When either SPF NC mice or Conv. NC mice (8 wk of age) were injected with anti-CD3 Ab in vivo, the serum IL-4 and IFN- levels at 3 h after Ab injection were much lower than those in the control mice (Fig. 6, A and B), substantiating the absence of V8⁺ NKT cells in NC mice.

View larger version (14K): [in this window] [in a new window]   FIGURE 6. In vivo IFN- and IL-4 production by the anti-CD3 Ab stimulation. Either Conv. NC mice, SPF NC mice, or SPF control mice of 8 wk of age were injected with 1 µg of anti-CD3 Ab through the tail vein and then sera were obtained at the indicated time points after Ab injection from the retro-orbital plexus. The data represent the means ± SE from five mice of each group.

Decreased in vitro IFN- production and increased IL-4 production from theCD3-stimulated liver and spleen MNC of Conv. NC mice, and increased serum IL-4 levels of untreated Conv. NC mice

When liver, spleen, and lymph node MNC from NC mice (10 wk of age) were stimulated in vitro with anti-CD3 Ab for 48 h, liver and spleen MNC from Conv. NC mice produced a smaller amount of IFN- and a greater amount of IL-4 than those of the control mice (Fig. 7, A–D). In contrast, liver and spleen MNC from SPF NC mice stimulated with anti-CD3 Ab in vitro produced only a small amount of IL-4 while also producing a substantial amount of IFN- (Fig. 7, A–D). Productions of cytokines from lymph node MNC showed a similar tendency, but differences among mouse groups were not statistically significant (Fig. 7, E and F). Consistent with these results, the serum IL-4 levels of the untreated Conv. NC mice (10 wk of age) were higher than those of the control mice and SPF NC mice (Fig. 7G).

View larger version (28K): [in this window] [in a new window]   FIGURE 7. In vitro IFN- and IL-4 production from liver, spleen and lymph node MNC and the serum IL-4 levels of mice. Liver MNC without Kupffer cells, spleen, and lymph node (LN) MNC were obtained from mice of each group of 10 wk of age and 5 x 105 MNC were cultured with immobilized anti-CD3 mAb in 200 µl of 10% FBS RPMI 1640 medium in 96-well flat-bottom plates in 5% CO2 at 37°C for 48 h and culture supernatants were subjected to ELISA for IFN- (A, C, and E) and IL-4 (B, D, and F). Sera also were obtained from five 10-wk-old mice and were subjected to ELISA for IL-4 (G). The data represent the means ± SE from five mice of each group.

Treatment of Conv. NC mice either with IFN-, IL-12, or IL-18 retarded the onset of dermatitis and the elevation of serum IgE

Because the experiments so far revealed that NC mice have defects in IFN- production, we examined the therapeutic effect on NC mice of recombinant IFN- as well as IL-12 and IL-18, both of which are potent IFN- inducers. Conv. NC mice were i.p. injected with either recombinant IL-12 (0.1 µg, 1 x 10³U), IL-18 (0.1 µg), IFN- (1 µg, 1 x 10⁴ U), or PBS as a control twice a week from 4 to 12 wk of age. In PBS-injected Conv. NC mice, seven of eight mice developed severe dermatitis, and one of eight mice developed moderate dermatitis at 12 wk of age, whereas only two of eight mice developed mild dermatitis in mice treated with either IL-12 or IL-18, respectively (p < 0.01). Although IFN- also significantly inhibited dermatitis (p < 0.01), the effect was less than that of IL-12 or IL-18 (p < 0.05) because in IFN--treated mice, five mice and one of eight mice at 12 wk of age showed mild and moderate dermatitis, respectively. The severity of dermatitis was scored as described in Materials and Methods, and dermatitis scores of each mouse group at the indicated ages were presented (Fig. 8A).

View larger version (21K): [in this window] [in a new window]   FIGURE 8. A, Therapeutic effect of IFN-, IL-12, and IL-18 on dermatitis of Conv. NC mice. Conv. NC mice were injected with either cytokine or PBS as a control twice a week from 4 wk of age, and the severity of dermatitis was scored at 9, 10 and 12 wk of age. No symptoms, 0; mild inflammation with scratching, 1; moderate inflammation and/or mild hemorrhage, 2; severe inflammation and/or hemorrhage or ulcer, 3. The data are the means ± SE from eight mice of each group at indicated ages. B, Inhibition of the elevation of serum IgE by cytokine therapy. Conv. NC mice were injected with either cytokine or PBS as a control twice a week from 4 wk age and blood samples were obtained from retro-orbital plexus at the indicated mouse ages. The sera were subjected to ELISA. The data represented were the means ± SE from five Conv. NC mice of each group at the indicated mouse ages.

Cytokine therapies inhibited the in vitro IL-4 production from CD3-stimulated liver T cells and inhibited the elevation of serum IL-4 levels in Conv. NC mice

Next, we examined how cytokine therapies affect the T cell function from Conv. NC mice. Four days after the last injection of cytokines into 12-wk-old mice, liver MNC were obtained and stimulated with immobilized anti-CD3 Ab in vitro for 48 h and culture supernatants were subjected to ELISA. The results showed that IL-12 and IL-18 therapy but not IFN- therapy inhibited IL-4 production (Fig. 9A). Interestingly, the IL-18 treatment inhibited IL-4 more profoundly than did IL-12 (Fig. 9A). Although the effect of IL-18 treatment was less dramatic than in the case of IL-4 production, the IL-18 treatment also significantly inhibited CD3-stimulated IFN- production from liver T cells (Fig. 9B). In addition, the serum IL-4 levels of the Conv. NC mice treated with all cytokines, especially in those treated with IL-18, significantly decreased (Fig. 9C).

View larger version (16K): [in this window] [in a new window]   FIGURE 9. The effect of cytokine therapy on in vitro IL-4 and IFN- production from CD3-stimulated liver T cells and serum IL-4 levels in Conv. NC mice. The Conv. NC mice were sacrificed 4 days after the last injection of each cytokine (PBS as a control) into mice (12 wk of age), and liver MNC were stimulated with anti-CD3 Ab for 48 h in vitro. Culture supernatants were subjected to ELISA. The data represented were the means ± SE from four experiments using four individual mice of each group. Sera also were obtained from five mice of each group, and the IL-4 levels were expressed by the means ± SE

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The Th1 response plays an important role in the host defense against bacterial infections (28, 29), whereas the Th2 response plays an important role in Ab production against various Ags. It is generally accepted that the Th1 and Th2 immune responses cross-regulate each other (30, 31, 32). The Th1 cytokine, IFN-, decreased the proliferation of Th2 cells and, conversely, IL-4 and IL-10 (33) down-regulated the IFN--mediated Th1 response (30, 31). SEB is one of the known staphylococcal superantigens and induces V7⁺, V8⁺, and V17⁺ T cells to proliferate and also produce IFN-, whereas SEA activates V15⁺ T cells (13). Because V8⁺ T cells make up the largest population among all mouse VT cells (34), it is reasonable to assume that V8⁺ T cell-deficient NC mice responded poorly to SEB. We also recently reported that liver NK cells as well as NKT cells produce IFN- in response to IL-12 produced by SEB-primed Kupffer cells (22). Therefore, the absence of regular V8⁺ T cells and V8⁺NKT cells may indeed be responsible for the IFN- hyporesponsiveness of NC mice to SEB. PBMC of AD patients, when stimulated with SEB in vitro, also have been reported to produce less IFN- and more IL-4 than those of healthy persons (35, 36).

In contrast, both IL-12 and IL-18 produced by macrophage lineage cells (including liver Kupffer cells) have been reported to be essential for LPS-stimulated IFN- production (24, 25). NC mice could not effectively produce IL-18 in response to LPS and therefore produced a low amount of IFN-. B cells stimulated with LPS and IL-4 have been demonstrated to produce IgE (37), whereas IFN- inhibited IgE production (38). Spleen B cells from NC mice also have been reported to produce more IgE than B cells of BALB/c mice after the stimulation by LPS and IL-4 in vitro (11), and IL-4-producing T cells were present in skin lesions of NC mice (12). These findings, together with the present results, suggest that Conv. NC mice may be polarized to a Th2-dominant state by a defective IFN- responsiveness to bacterial stimulations. As a result, regular T cells from Conv. NC mice stimulated with anti-CD3 Ab in vitro are thus suggested to produce a greater amount of IL-4 than those of normal mice and SPF NC mice.

The result that IFN- treatment of Conv. NC mice did not decrease in vitro IL-4 production from CD3-stimulated liver MNC was somewhat unexpected. However, IFN- was reported to not inhibit the IL-4 production itself from Th2 cells stimulated with anti-CD3 Ab in vitro while nevertheless suppressing the proliferation of Th2 cells (31). In addition, the half-life time of IFN- is very short (19–32 min; Ref. 39), and the liver MNC were obtained from mice 4 days after the last injection of cytokines in the present study. Therefore, the effect of the previous IFN- treatment on in vitro culture may be minimal. Because IL-12 injection into NC mice significantly elevated the serum IFN- levels at least for 12 h after injection whereas IL-18 injection did not (data not shown), the therapeutic effect of IL-12 on Conv. NC mice was at least partly mediated by IFN-, whereas IL-18 may induce a Th1-dominant state in Conv. NC mice without any direct induction of IFN- production. In fact, the IL-4 production from CD3-stimulated liver T cells greatly decreased after the IL-18 treatment. Although it is unknown at present why the IL-18 treatment also decreased the amount of IFN- produced by CD3-stimulated liver T cells, IL-18 more profoundly decreased IL-4 production than IFN- production. However, it should be noted that although IL-18 was originally thought to be a representative Th1 cytokine (by its capacity to induce IFN- production and an antitumor immunity; Ref. 25), IL-18 also appears to possess one aspect similar to that of Th2 cytokine, especially in the absence of IL-12. Surprisingly, it was reported very recently that daily injections of IL-18 (1 or 5 µg/mouse) into SPF BALB/c mice for 13 days increased the serum IgE levels (40, 41) presumably by inducing IL-4 production from CD4⁺ T cells, whereas daily injections of 0.1 µg of IL-18 did not elevate the serum IgE levels (41). These findings conflict with the present results in Conv. NC mice. However, in our experiments, 0.1 µg of IL-18 was injected only twice a week for 8 wk. In addition, when Conv. NC mice were treated with IL-18 (0.1 µg) once a week, the therapeutic effect on dermatitis was more limited (our unpublished observation). Although the reason for this discrepancy is unclear at present, it has recently been reported that IL-18 induced IL-4 production more predominantly than IFN- production from the CD3-stimulated spleen MNC of BALB/c mice (known as a Th2-dominant mouse strain) whereas IL-18 enhanced the IFN- production but not the IL-4 production from the CD3-stimulated spleen MNC of the C57BL/6 mice and CBA mice (known as Th1-dominant strains) (42). Thus, these findings suggest that IL-18 is a more complex cytokine than previously thought and may thus be an important coordinator for either the Th1 or Th2 immune response depending on the conditions of the hosts.

However, it should be noted that IL-18 production alone without IL-12 production does not likely occur in many bacterial infections. Because NC mice have a deficiency in both IL-18 and IFN- productions in response to bacterial components, it is possible that NC mice under Conv. conditions indeed need IL-18 as a Th1 cytokine to adjust their Th1 and Th2 imbalance. Nevertheless, the responses of Conv. normal mice of various strains and either Conv. or SPF NC mice to various doses of IL-18 or different injection schedules should be examined in a future study.

The development of dermatitis in NC mice recently has been suggested to be controlled by an autosomal recessive gene because none of the F₁ progeny between NC mice and BALB/c mice develop dermatitis, whereas one-quarter of F₂ mice developed dermatitis (43). However, it might not be so simple because approximately one-half of all F₁ mice between NC mice and C57BL/6 mice developed late onset dermatitis, which was less severe, and showed a moderate IgE elevation (Y.H. and S.S., unpublished observation). BALB/c mice may have a certain inhibitory gene for dermatitis that is not present in C57BL/6 mice. Because SPF NC mice do not demonstrate dermatitis, it is apparent that the dermatitis gene alone does not induce dermatitis. The immune response of NC mice to environmental Ags is essential for dermatitis, which is quite different from many other gene-mutated mice with certain diseases. Although we cannot conclude that the absence of V8⁺ T cells and the decreased IL-18 production in NC mice in response to LPS is directly associated with the dermatitis gene, it can nevertheless be concluded that these defects are important factors regarding the induction of dermatitis in NC mice.

Taken together, systemic IFN- hyporesponsiveness to bacterial stimulations may lead to a Th2-dominant state accompanied by IgE hyperproduction, thus resulting in a susceptibility to dermatitis in NC mice. In addition to IFN- and IL-12, IL-18 is therefore also considered to be a potentially effective therapy for some AD patients.

	Footnotes

 
¹ This work was supported in part by the Kawano Memorial Foundation.

² Address correspondence and reprint requests to Dr. Shuhji Seki, Division of Basic Traumatology, National Defense Medical College Research Institute, Tokorozawa 359-8513, Japan.

³ Abbreviations used in this paper: AD, atopic dermatitis; SEB, staphylococcal enterotoxin B; NC, NC/Nga; SPF, specific pathogen free; Conv., conventional; NKT, NK1.1⁺ T; MNC, mononuclear cells; SEA, staphylococcal enterotoxin A.

Received for publication July 17, 2000. Accepted for publication February 22, 2001.

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