The mechanism of a defective IFN-gamma response to bacterial toxins in an atopic dermatitis model, NC/Nga mice, and the therapeutic effect of IFN-gamma, IL-12, or IL-18 on dermatitis

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-gamma 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-gamma response to staphylococcal enterotoxin B was correlated to the lack of regular Vbeta8(+) T cells and Vbeta8(+) NK T cells, and the low IFN-gamma 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-gamma, 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.     

Deletion polymorphisms in the promoter region of Fcgamma receptor IIB is not associated with antigen-specific IgG2a and IgG2b antibody responses in NC/Nga mice

Fcgamma receptor (R) IIB, a low-affinity FcR for IgG, inhibits B cell Ag R (BCR)-mediated activation when these two receptors are cross-linked by Ag and IgG-containing immune complexs (ICs). We found deletion polymorphisms in the promoter region of fcgr2b in NC/Nga mice, a model for human atopic dermatitis. NC/Nga mice produced significantly higher levels of ovalbumin (OVA)-specific IgG, IgG2a and IgG2b than did BALB/c mice. Analysis of (BALB/c x NC/Nga)F1 x BALB/c or (BALB/c x NC/Nga) F1 x NC/Nga backcross mice revealed that deletion polymorphisms of fcgr2b in NC/Nga mice does not directly regulate hyper OVA-specific IgG2a and IgG2b Ab responses.     

Lack of effect of the abnormal fatty acid metabolism in NC/Nga mice on their atopic dermatitis

Although clinical evidence has suggested that dysregulated fatty acid metabolism is associated with atopic disorders, the molecular basis for such a correlation remains to be demonstrated. In the present study, we analyzed the fatty acid composition in peripheral blood cells of NC/Nga mice, a model for atopic dermatitis (AD). We found that arachidonic acid significantly accumulated in mice with the AD manifestation. In addition, the leucotriene B4-releasing ability upon calcium ionophore A23187 stimulation was potentiated in blood cells. An arachidonic acid accumulation was not apparent in the non-atopic BALB/c strain, but was still observed in healthy NC/Nga mice fed under specific pathogen-free conditions. These results indicate that a disturbed fatty acid metabolism in NC/Nga mice was not a trigger factor for their dermatitis development.     

Orally supplemented Lactobacillus acidophilus strain L‐92 inhibits passive and active cutaneous anaphylaxis as well as 2,4‐dinitroflurobenzene and mite fecal antigen induced atopic dermatitis‐like skin lesions in mice

Oral supplementation of lactic acid bacteria is a potential approach to the prevention and manipulation of allergic diseases such as atopic dermatitis. Our previous report showed that heat‐killed Lactobacillus acidophilus strain L‐92 (L‐92) possessed anti‐allergic properties, although its physiological function in atopic dermatitis has largely remained undefined. To evaluate the anti‐allergic efficacy of L‐92, we used four experimental animal models with the major features of atopic dermatitis and compared the results to those of clinically active drugs. ICR mice were passively sensitized by anti‐dinitrophenyl mouse monoclonal IgE for passive cutaneous anaphylaxis (PCA), and BALB/c mice were actively sensitized by ovalbumin for active cutaneous anaphylaxis (ACA). Allergic reaction was induced by repeated exposure to 2,4‐dinitroflurobenzene (DNFB) and mite (Dermatophagoides farinae) fecal allergen, in BALB/c and NC/Nga mice, respectively. Orally administrated L‐92 significantly inhibited the vascular permeability increase in both PCA and ACA, and the elevation of ovalbumin‐specific IgE titer in ACA. Moreover, repeated applications of DNFB and mite fecal antigen onto the BALB/c and NC/Nga mouse ear, respectively, caused clinical symptoms similar to atopic dermatitis such as ear swelling, scratching behavior and elevation of total serum IgE levels that were also moderately suppressed by L‐92. In addition, L‐92 treated mice exhibited lower levels of mast cells, eosinophil infiltration and Th1/Th2 cytokine expression. Our results, therefore, suggest that oral administration of L‐92 might be useful for alleviating allergic symptoms.     

Effect of Mouse Strain in a Model of Chemical-induced Respiratory Allergy

The inhalation of many types of chemicals is a leading cause of allergic respiratory diseases, and effective protocols are needed for the detection of environmental chemical–related respiratory allergies. In our previous studies, we developed a method for detecting environmental chemical–related respiratory allergens by using a long-term sensitization–challenge protocol involving BALB/c mice. In the current study, we sought to improve our model by characterizing strain-associated differences in respiratory allergic reactions to the well-known chemical respiratory allergen glutaraldehyde (GA). According to our protocol, BALB/c, NC/Nga, C3H/HeN, C57BL/6N, and CBA/J mice were sensitized dermally with GA for 3 weeks and then challenged with intratracheal or inhaled GA at 2 weeks after the last sensitization. The day after the final challenge, all mice were euthanized, and total serum IgE levels were assayed. In addition, immunocyte counts, cytokine production, and chemokine levels in the hilar lymph nodes (LNs) and bronchoalveolar lavage fluids (BALF) were also assessed. In conclusion, BALB/c and NC/Nga mice demonstrated markedly increased IgE reactions. Inflammatory cell counts in BALF were increased in the treated groups of all strains, especially BALB/c, NC/Nga, and CBA/J strains. Cytokine levels in LNs were increased in all treated groups except for C3H/HeN and were particularly high in BALB/c and NC/Nga mice. According to our results, we suggest that BALB/c and NC/Nga are highly susceptible to respiratory allergic responses and therefore are good candidates for use in our model for detecting environmental chemical respiratory allergens.     

Low susceptibility of NC/Nga mice to the lipopolysaccharide-mediated lethality with D-galactosamine sensitization and the involvement of fewer natural killer T cells

The LPS-mediated lethality of NC/Nga mice, having fewer NKT cells, was examined by using d-galactosamine (d-GalN)-sensitization. The NC/Nga mice were not killed by a simultaneous administration of d-GalN and LPS whereas all C57BL/6 (B6) control mice were killed. The injection of d-GalN and LPS failed to elevate the levels of serum alanine aminotransferase and caspase 3 in the liver tissues of NC/Nga mice. Further, the nitric oxide (NO) level of the d-GalN- and LPS-injected NC/Nga mice was much lower than those of the B6 mice. The expression of an inducible NO synthase (iNOS) was significantly reduced in the livers of NC/Nga mice. However, there was no significant difference in LPS-induced TNF-α production between B6 mice and NC/Nga mice. The NC/Nga mice had an impaired expression of IFN-γ protein and mRNA in response to d-GalN and LPS. The pretreatment with α-galactosylceramide (α-GalCer), which activates Vα14(+) NKT cells and induces the production of IFN-γ, rendered NC/Nga mice more susceptible to the LPS-mediated lethality. The livers of NC/Nga mice had fewer NKT cells compared to B6 mice. Taken together, it is suggested that the resistance of NC/Nga mice to the LPS-mediated lethality with d-GalN sensitization depended on the impaired IFN-γ production caused by fewer NKT cells and reduced NO production that followed.     

Amelioration of the progression of an atopic dermatitis-like skin lesion by silk peptide and identification of functional peptides

The efficacy of silk peptide in treatment of atopic dermatitis was examined in a picryl chloride-induced atopic dermatitis model in NC/Nga mice. Silk peptide ameliorated the development of atopic dermatitis by lowering the serum IgE concentration. Treatment of cultured spleen cells with silk peptide reduced IgE production by enhancing the production of IFN-γ and reducing the level of IL-4. The functional peptides in the silk peptide were identified as mixture of GAGA sequences containing peptides by mass spectrometry and in vitro assay. Our findings indicate that silk peptide exerts an effect on atopic dermatitis by modulating the Th1/Th2 balance.     

B cell sensitivity to IgE-suppressive activity of IFN-gamma is polymorphic and controlled by a non-H-2-linked gene.

It has been suggested that at least two distinct genetic factors are involved in developing atopic diseases. One is the major histocompatibility complex which controls antigen-specific polymorphism of IgE antibody responses and the other is an unidentified factor(s) which controls isotype selection, i.e., class switching to IgE. It is conceivable that both expression of and sensitivity to lymphokines that play central roles in controlling IgE biosynthesis may be involved in the latter polymorphism. To explore this possibility, we have examined the sensitivities of several mouse strains to interleukin (IL)-4 and interferon-gamma (IFN-gamma). The results show that (1) the sensitivity to IgE-suppressive activity of IFN-gamma, but not to the IgE-enhancing activity of IL-4, is polymorphic (e.g., C57BL/6 is 8- to 16-fold more sensitive than BALB/c to IFN-gamma); (2) F1 of these two strains (CByB6F1) are BALB/c type and H-2 congenic mice of d haplotype with B6 background are C57BL/6 type, suggesting that low sensitivity is a non-H-2-linked dominant trait; (3) the polymorphism is determined at B cell levels; and (4) sensitivity to IFN-gamma is not associated with mRNA expression of IFN-gamma receptors (R) by B cells. These data collectively indicate that BALB/c mice have a non-H-2-linked gene which decreases B cell sensitivity to IFN-gamma, but the gene effect is not associated with the expression of IFN-gamma R mRNA on B cells. The possible biological significance of the non-H-2-linked gene is discussed.     

Development of atopic dermatitis-like skin lesions in STAT6-deficient NC/Nga mice

Atopic dermatitis (AD) is a pruritic inflammatory skin disease characterized by elevation of plasma levels of total IgE, infiltration of mast cells and eosinophils, and the expression of cytokines by Th2 T cells. However, the role of Th2 cells in the pathogenesis of AD is not fully understood. In this study we examined the NC/Nga (NC) mouse model of AD and established STAT6-deficient (SATA6(-/-)) NC mice to investigate the relevance of IL-4-mediated immune responses. Surprisingly, these mice elicited AD-like skin lesions at equivalent frequency and time of onset compared with normal NC littermates. Histological features of the lesion in STAT6(-/-) NC mice fulfilled the criteria for the pathogenesis of AD, although these mice fail to produce IgE and Th2 cytokines. The lymph nodes proximal to the regions of skin that developed lesions exhibited massive enlargement elicited by the accumulation of activated IFN-gamma-secreting T cells. Moreover, caspase I, IL-18, IL-12, and IFN-gamma are found to be highly expressed at the skin lesion, occurring simultaneously with elevation of eotaxin 2 and CCR3 expression. Therefore, the Th2-mediated immune response is not necessary for the development of AD-like skin disease in NC mice. The skin microenvironment that favored IFN-gamma production tightly correlates with the skin disease in NC mice through the infiltration of eosinophils.     

Oral administration of a hop water extract ameliorates the development of dermatitis induced by the periodical topical application of a mite antigen in atopic dermatitis model NC/Nga mice

We investigated the inhibitory effect of an oral administration of a hop water extract (HWE) on the development of dermatitis by using NC/Nga atopic dermatitis model mice. The induction of allergic dermatitis was conducted by tape-stripping and topical application of a mite antigen (Dermatophagoides farinae) on to the ear once a week for 10 weeks. HWE was orally administered at a dose of 100 or 500 mg/kg. The total immunoglobulin E (IgE) concentration in serum and the ear thickness were periodically examined. Finally, the antigen-specific IgE level in the serum and the production of interleukin (IL)-4, IL-12 and interferon (IFN)-gamma from splenocytes and cervical lymph node cells were measured. The oral administration of HWE significantly inhibited the increase of total IgE production and ear swelling throughout the experimental period. The production of IL-12 was significantly lower in the HWE administered group than in the control group. The results suggest that the intake of HWE may be effective in preventing and alleviating the development of atopic dermatitis-like skin disease.     

The inhibitory effect of naringenin on atopic dermatitis induced by DNFB in NC/Nga mice

Aims

Atopic dermatitis (AD) is a chronic and relapsing inflammatory dermatitis characterized by pruritic and eczematous skin lesions. Here, we investigated the therapeutic effect of the fruit flavonoid naringenin on DNFB induced atopic dermatitis mice model.

Main methods

AD-like skin lesion was induced by repetitive skin contact with DNFB in NC/Nga mice and the effects of the fruit flavonoid naringenin were evaluated on the basis of histopathological findings of skin, ear swelling and cytokine production of CD4⁺T cells.

Key findings

Intraperitoneal injection of naringenin for one week after DNFB challenge significantly lowered ear swelling and improved back skin lesions. In addition, naringenin significantly suppressed production of interferon-gamma (IFN-γ) by activated CD4⁺ T cells and serum IgE level. Furthermore, naringenin reduced DNFB-induced infiltration of eosinophils, mast cells, CD4⁺ T cells, and CD8⁺ T cells in skin lesions.

Significance

Naringenin may suppress the development of AD-like skin lesions in DNFB-treated NC/Nga mice by reducing IFN-γ production of activated CD4⁺ T cells, serum IgE levels and infiltration of immune cells to skin lesion.     

Reduced expression of STAT4 and IFN-gamma in macrophages from BALB/c mice

BALB/c mice have been shown to easily induce Th2 type responses in several infection models. In this study, to examine the mechanisms of Th2 dominant responses in BALB/c mice, we assessed several macrophage functions using C3H/HeN, C57BL/6, and BALB/c mouse strains. Peritoneal macrophages from three strains of mice equally produced IL-12 by stimulation with LPS plus IFN-gamma. However, IFN-gamma production in response to IL-12 or IL-12 plus IL-18 was much lower in macrophages from BALB/c mice than other strains. IFN-gamma produced by activated macrophages induced IL-12R mRNA expression in T cells and macrophages themselves depending on their amount of IFN-gamma; namely, macrophages from BALB/c mice induced lower expression of IL-12R. Intracellular levels of STAT4 were much lower in macrophages from BALB/c mice. However, other STATs, such as STAT1 or STAT6, were expressed similarly in the three mouse strains. STAT4 and IFN-gamma production by other cell types such as T cells and B cells were equal in C3H/HeN and BALB/c mice. These results indicate that macrophages from Th2-dominant BALB/c mice have different functional characters compared with other mouse strains; that is, STAT4 expression and IFN-gamma production are reduced, which is one of the causes to shift to Th2-type responses.     

Oral Administration of a Hop Water Extract Ameliorates the Development of Dermatitis Induced by the Periodical Topical Application of a Mite Antigen in Atopic Dermatitis Model NC/Nga Mice

We investigated the inhibitory effect of an oral administration of a hop water extract (HWE) on the development of dermatitis by using NC/Nga atopic dermatitis model mice. The induction of allergic dermatitis was conducted by tape-stripping and topical application of a mite antigen (Dermatophagoides farinae) on to the ear once a week for 10 weeks. HWE was orally administered at a dose of 100 or 500 mg/kg. The total immunoglobulin E (IgE) concentration in serum and the ear thickness were periodically examined. Finally, the antigen-specific IgE level in the serum and the production of interleukin (IL)-4, IL-12 and interferon (IFN)-γ from splenocytes and cervical lymph node cells were measured. The oral administration of HWE significantly inhibited the increase of total IgE production and ear swelling throughout the experimental period. The production of IL-12 was significantly lower in the HWE administered group than in the control group. The results suggest that the intake of HWE may be effective in preventing and alleviating the development of atopic dermatitis-like skin disease.     

Suppression of atopic dermatitis and tumor metastasis in mice by small amounts of radon

We examined the effect of radon in two experimental disease models in mice by administering radon dissolved in water at 68-203 Bq/liter. Administration of radon in drinking water to NC/Nga mice significantly delayed the progression of atopic dermatitis-like skin lesions induced by picrylchloride when administered prior to the induction of disease signs. The number of pulmonary metastatic foci in C57BL/6 mice inoculated with B16 melanoma cells was also reduced significantly by administration of radon in drinking water when the number of tumor cells was small and the radon treatment was started prior to tumor inoculation. The ratio of Ifng to Il4 produced by splenocytes from BALB/c mice immunized with DNP-Ascaris was significantly increased by administration of radon in drinking water. From these results, a modulation of immunity by radon was suggested.     

Sensitivity difference to the suppressive effect of prostaglandin E2 among mouse strains: a possible mechanism to polarize Th2 type response in BALB/c mice

PGE2 has been shown to play a prominent role in regulating Th1 and Th2 type responses. We studied the role of PGE2 in IFN-gamma production by Staphylococcus aureus Cowan I-stimulated spleen cells from several mouse strains such as BALB/c, C3H/HeN, and C57BL/6. When spleen cells were pretreated with indomethacin (cyclooxygenase (COX)-1 and COX-2 inhibitor) or NS-398 (COX-2-specific inhibitor), S. aureus Cowan I -induced IFN-gamma production was increased more markedly in spleen cells from BALB/c mice than from C3H/HeN and C57BL/6 mouse. However, PGE2 production was not significantly different among spleen cells from three mouse strains. When various concentrations of PGE2 were exogeneously added to spleen cells, PGE2 showed a stronger suppressive effect on IFN-gamma production in spleen cells from BALB/c mice than from other strains of mice. This suppressive effect of PGE2 in BALB/c mice mainly depended on IL-12p70 production by APCs. More PGE2 binding sites were found in BALB/c spleen cells than in C3H/HeN spleen cells, indicating that the sensitivity difference to the suppressive effect of PGE2 was due to the difference of the number of PGE2 receptors. The administration of NS-398 into BALB/c mice enhanced Ag-specific IFN-gamma production, but not IL-4 production. This effect is the same as IL-12 administration in vivo. From these results, we propose that the modulation of PGE2 is important for Th1 activation via IFN-gamma and IL-12p70 production in vitro and in vivo and that PGE2 is one of the pivotal factors in the Th2-dominant immune response in BALB/c mice.     

Inhibitory Effects of Heartwood Extracts of Broussonetia kazinoki Sieb on the Development of Atopic Dermatitis in NC/Nga Mice

We investigated the effects of a topically applied extract of the heartwood of Broussonetia kazinoki Sieb (B. kazinoki) on atopic dermatitis (AD)-like skin lesions induced by an extract of the house-dust mite Dermatophagoides farina in NC/Nga mice. We found that topically applied B. kazinoki extract suppressed the histological manifestations of AD-like skin lesions, and decreased the levels of plasma immunoglobulin E (IgE) and interleukin-4 (IL-4) in the mice. Moreover, B. kazinoki inhibited the induction of thymus-and-activation-regulated chemokine (TARC/CCL17), macrophage-derived chemokine (MDC/CCL22), and regulated-on-activation-normal T cell-expressed-and-secreted chemokine (RANTES/CCL5) in HaCaT cells activated by tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). In conclusion, our results suggest that B. kazinoki extract has therapeutic advantages in the treatment of AD.     

The involvement of neutrophils in the resistance to Leishmania major infection in susceptible but not in resistant mice

To understand the immunomodulatory roles of neutrophils in Leishmania major infection, we examined the expression of cytokine and chemokine mRNAs from neutrophils of the infected resistant C3H/HeJ and susceptible BALB/c mice. We also examined the effects of neutrophil depletion on the expression of cytokine by peritoneal macrophages and draining lymph node cells and on the footpad lesions and parasite burdens in these mice. Neutrophils from resistant C3H/HeJ but not from susceptible BALB/c mice expressed mRNAs for IL-12p40, IFN-gamma,TNF-alpha and monokine induced by IFN-gamma(MIG). Neutrophil depletion of the resistant mice reduced the expression of IFN-gammaandTNF-alpha in peritoneal macrophages but did not affect the expression of IL-12p40 and IFN-gamma in draining lymph node cells and the growth of footpad lesions. On the other hand, neutrophil depletion of susceptible BALB/c mice did not affect the expression of TNF-alpha and monocyte-derived chemokine (MDC) in peritoneal macrophages but induced the early stage expression of IL-4 in draining lymph node cells and exacerbated the footpad lesions and increased the parasite burden. The exacerbation of footpad lesions induced by neutrophil depletion was abolished by rIL-12 treatment. Our results suggest that even in susceptible BALB/c but not in C3H/HeJ mice there is a certain resistance requiring neutrophils at the early stage of infection.     

Intranasal mite allergen induces allergic asthma-like responses in NC/Nga mice

Airway responses induced by intranasal administration of mite allergen without adjuvant were studied in NC/Nga mice. A crude extract of Dermatophagoides farinae (Df) was administered for 5 consecutive days and a single intranasal challenge booster dose was given 1 week after the last sensitization. 24 h after the single challenge, the airway hyperresponsiveness (AHR) was measured and the bronchoalveolar lavage fluid (BALF) was analyzed for numbers of eosinophils and neutrophils, and both cytokine and chemokine levels. There were marked increases in number of eosinophils in the BALF, AHR, Th2 cytokines (IL-5 and IL-13), and chemokine (eotaxin-1 and eotaxin-2) levels in the BALF following Df exposure. C57BL/6N, A/J, BALB/c, and CBA/JN mouse strains were also exposed to Df crude extract, but all of the measured responses were strongest in NC/Nga mice. Furthermore, Df-exposed NC/Nga mice showed the goblet cell hyperplasia, pulmonary eosinophilic inflammation, and increases in both total serum IgE and Df-specific IgG1. After intranasal exposure of NC/Nga mice to crude extract of Dermatophagoides pteronyssinus, the BALF eosinophilia and AHR were similar to responses induced by Df. None of the study parameters were increased in response to intranasal exposure to ovalbumin. These data demonstrated that NC/Nga mice developed allergic asthma-like responses after intranasal exposure to mite allergens.     

An immunostimulatory DNA sequence from a probiotic strain of Bifidobacterium longum inhibits IgE production in vitro

The immunostimulatory oligodeoxynucleotide (ODN) BL07 (5'-GCGTCGGTTTCGGTGCTCAC-3') was identified from the genomic DNA of the probiotic strain Bifidobacterium longum BB536. ODN BL07 stimulated B-lymphocyte proliferation and induced interleukin-12 (IL-12) production in macrophage-like J774.1 cells. ODNs BL07 and BL07S (modified with phosphorothioate backbone) significantly inhibited immunoglobulin E (IgE) production and stimulated interferon-gamma (IFN-gamma) and IL-12 production, but did not affect IL-4 secretion in murine splenic cells of ovalbumin-primed BALB/c mice. These ODNs also significantly inhibited production of IgE in purified murine B cells in the presence of IL-4 and anti-CD40. The results suggest the potential of ODNs BL07 and BL07S in preventing IgE-related immune responses and the possible involvement of ODN BL07 in the antiallergic efficacy of B. longum BB536.