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Production and Pharmacologic Modulation of the Granulocyte-Associated Allergic Responses to Ovalbumin in Murine Skin Models Induced by Injecting Ovalbumin-Specific Th1 or Th2 Cells

2001; Elsevier BV; Volume: 117; Issue: 2 Linguagem: Inglês

10.1046/j.0022-202x.2001.01375.x

1523-1747

Tadashi Terui, Mikiko Okada, Motoko Honda, Maki Ozawa, Hachiro Tagami, Kunio Sano, Hidekazu Shirota, Noriyasu Hirasawa, Gen Tamura,

Dermatology and Skin Diseases

Because interferon-γ, interleukin-4, and interleukin-5 have been identified at the mRNA and protein levels in the lesional skin of patients with atopic dermatitis, we investigated the roles played by granulocytes as effector cells in allergic inflammation by using two unique murine skin models. In vitro generated Th1 and Th2 cells from naïve splenocytes of antiovalbumin T cell receptor transgenic BALB/C mice were adoptively transferred with ovalbumin into the ear pinnae or air-pouches produced in the back skin of naïve, nontransgenic BALB/C mice. The injection of Th1 cells with ovalbumin induced delayed type ear swelling that peaked at 48 h, whereas that of Th2 resulted in ear swelling that peaked at a much earlier time, 24 h. Histologic study of the swollen ear skin and granulocytes recruited into the air-pouch demonstrated that, although the Th1-induced inflammation caused a neutrophil-predominant infiltrate with few eosinophils, larger numbers of eosinophils accumulated in the Th2-induced inflammation. Using these murine models, we further evaluated the effects of drugs used for the treatment of atopic diseases. The results showed that FK506 administration could effectively reduce skin inflammation induced by either Th cells. Interestingly, the neutrophil elastase inhibitor ONO-6818 efficiently inhibited Th1-induced inflammation. In contrast, a leukotriene receptor antagonist, ONO-1078, specifically suppressed Th2-induced inflammation. We also found that each ONO drug exerted direct influence on specified granulocytes, as neither affected in vitro production of relevant Th cytokines. Thus, we succeeded in developing animal skin inflammation models in which we can evaluate the contribution of protein antigen-specific Th1 or Th2 cells through the action of granulocytic effector cells. Because interferon-γ, interleukin-4, and interleukin-5 have been identified at the mRNA and protein levels in the lesional skin of patients with atopic dermatitis, we investigated the roles played by granulocytes as effector cells in allergic inflammation by using two unique murine skin models. In vitro generated Th1 and Th2 cells from naïve splenocytes of antiovalbumin T cell receptor transgenic BALB/C mice were adoptively transferred with ovalbumin into the ear pinnae or air-pouches produced in the back skin of naïve, nontransgenic BALB/C mice. The injection of Th1 cells with ovalbumin induced delayed type ear swelling that peaked at 48 h, whereas that of Th2 resulted in ear swelling that peaked at a much earlier time, 24 h. Histologic study of the swollen ear skin and granulocytes recruited into the air-pouch demonstrated that, although the Th1-induced inflammation caused a neutrophil-predominant infiltrate with few eosinophils, larger numbers of eosinophils accumulated in the Th2-induced inflammation. Using these murine models, we further evaluated the effects of drugs used for the treatment of atopic diseases. The results showed that FK506 administration could effectively reduce skin inflammation induced by either Th cells. Interestingly, the neutrophil elastase inhibitor ONO-6818 efficiently inhibited Th1-induced inflammation. In contrast, a leukotriene receptor antagonist, ONO-1078, specifically suppressed Th2-induced inflammation. We also found that each ONO drug exerted direct influence on specified granulocytes, as neither affected in vitro production of relevant Th cytokines. Thus, we succeeded in developing animal skin inflammation models in which we can evaluate the contribution of protein antigen-specific Th1 or Th2 cells through the action of granulocytic effector cells. ovalbumin transgenic There has been accumulating evidence indicating that both Th1 and Th2 cytokines and their transcripts are present in the lesional skin of patients with atopic dermatitis (AD) (Tsicopoulos et al., 1994Tsicopoulos A. Hamid Q. Haczku A. et al.Kinetics of cell infiltration and cytokine messenger RNA expression after intradermal challenge with allergen and tuberculin in the same atopic individuals.J Allergy Clin Immunol. 1994; 94: 764-772Abstract Full Text PDF PubMed Scopus (96) Google Scholar;Thepen et al., 1996Thepen T. Langeveld-Wildschut E.G. Bihari I.C. van Wichen D.F. van Reijsen F.C. Mudde G.C. Bruijnzeel-Koomen C.A. Biphasic response against aeroallergen in atopic dermatitis showing a switch from an initial TH2 response to a TH1 response in situ: an immunocytochemical study.J Allergy Clin Immunol. 1996; 97: 828-837Abstract Full Text PDF PubMed Scopus (364) Google Scholar;Werfel et al., 1996Werfel T. Morita A. Grewe M. Renz H. Wahn U. Krutmann J. Kapp A. Allergen specificity of skin-infiltrating T cells is not restricted to a type-2 cytokine pattern in chronic skin lesions of atopic dermatitis.J Invest Dermatol. 1996; 107: 871-876Crossref PubMed Scopus (215) Google Scholar) as well as in an animal model of AD (Spergel et al., 1999Spergel J.M. Mizoguchi E. Oettgen H. Bhan A.K. Geha R.S. Roles of Th1 and Th2 cytokines in a murine model of allergic dermatitis.J Clin Invest. 1999; 103: 1103-1111Crossref PubMed Scopus (305) Google Scholar). Although the etiology of AD is not fully understood, it is speculated that hypersensitivity to environmental macromolecular protein antigens, e.g., food allergens and Dermatophagoides, rather than to small molecular haptens plays an important role in its pathomechanism. To investigate the mechanisms underlying the development of AD, we need an animal model in which cutaneous inflammation is induced by Th1 or Th2 cells reactive against these protein antigens. In antigen-primed individuals, the initiation stage of delayed type hypersensitivity (DTH) includes the activation of vascular endothelial cells and the recruitment and activation of antigen-specific T cells. The next effector stage of DTH involves the synthesis of chemotactic and activating cytokines from antigen-specific T cells. Subsequently, there occur the recruitment, activation, and cytokine and reactive-oxygen-intermediate secretion of effector cells such as granulocytes and macrophages and an increase in vascular permeability (Askenase, 1992Askenase P.W. Delayed-type hypersensitivity recruitment of T cell subsets via antigen-specific non-IgE factors or IgE antibodies: relevance to asthma, autoimmunity and immune responses to tumors and parasites.Chem Immunol. 1992; 54: 166-211Crossref PubMed Google Scholar). Adoptively transferred DTH that is induced by injecting primed T cells and antigen into naïve mouse ear lobes represents the latter effector stage of the DTH process (Bianchi et al., 1981Bianchi A.T.J. Hooijkaas H. Benner R. Clones of helper T cells mediate antigen-specific, H-2-restricted DTH.Nature. 1981; 290: 62-63Crossref PubMed Scopus (89) Google Scholar;Scovern and Kantor, 1982Scovern H. Kantor F.S. Local passive transfer of delayed-type hypersensitivity in the mouse.J Immunol. 1982; 129: 25-29PubMed Google Scholar). It has been revealed that Th1 clones can induce footpad DTH (Cher and Mosmann, 1987Cher D.J. Mosmann T.R. Two types of murine helper T cell clone. II. Delayed type hypersensitivity is mediated by Th1 clones.J Immunol. 1987; 138: 3688-3694PubMed Google Scholar;Fong and Mosmann, 1989Fong T.A. Mosmann T.R. The role of IFN-γ in delayed-type hypersensitivity mediated by Th1 clones.J Immunol. 1989; 143: 2887-2893PubMed Google Scholar;Müller et al., 1993Müller K.M. Jaunin F. Masouye I. Saurat J.-H. Hauser C. Th2 cells mediate IL-4-dependent local tissue inflammation.J Immunol. 1993; 150: 5576-5584PubMed Google Scholar). On the other hand, preactivated bulk-cultured and alloreactive Th2 populations can induce interleukin 4 (IL-4) dependent footpad DTH (Müller et al., 1993Müller K.M. Jaunin F. Masouye I. Saurat J.-H. Hauser C. Th2 cells mediate IL-4-dependent local tissue inflammation.J Immunol. 1993; 150: 5576-5584PubMed Google Scholar), although resting Th2 clones do not induce DTH (Fong and Mosmann, 1989Fong T.A. Mosmann T.R. The role of IFN-γ in delayed-type hypersensitivity mediated by Th1 clones.J Immunol. 1989; 143: 2887-2893PubMed Google Scholar). With hematoxylin and eosin (H&E) stained histologic sections, it has been shown that neutrophils, but not eosinophils, accumulate in Th2 clone-mediated inflammatory responses (Müller et al., 1993Müller K.M. Jaunin F. Masouye I. Saurat J.-H. Hauser C. Th2 cells mediate IL-4-dependent local tissue inflammation.J Immunol. 1993; 150: 5576-5584PubMed Google Scholar). These results suggest that Th2 cells also have the potential to induce DTH-like inflammation, partly through the action of granulocytic effector cells. CD4+ T cells can develop into Th1 and Th2 cells, which are characterized by the production of specific cytokines, i.e., Th1 cells produce interferon-γ (IFN-γ), IL-2, and low levels of IL-10, whereas Th2 cells produce IL-4, IL-5, IL-13, and high levels of IL-10 (Mosmann and Coffman, 1989Mosmann T.R. Coffman R.L. TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties.Ann Rev Immunol. 1989; 7: 145-173Crossref PubMed Scopus (6652) Google Scholar;Abbas et al., 1996Abbas A.K. Murphy K.M. Sher A. Functional diversity of helper T lymphocytes.Nature. 1996; 383: 787-793Crossref PubMed Scopus (3759) Google Scholar). Naive T cells can be induced to differentiate either into Th1 effector cells when stimulated in the presence of IL-12 or into Th2 effector cells when stimulated in the presence of IL-4 (Seder et al., 1992Seder R.A. Paul W.E. Davis M.M. de St Groth B.F. The presence of interleukin 4 during in vitro priming determines the lymphokines-producing potential of CD4+ T cells from T cell receptor transgenic mice.J Exp Med. 1992; 176: 1091-1098Crossref PubMed Scopus (922) Google Scholar;Hsieh et al., 1993Hsieh C.-S. Macatonia S.E. Tripp C.S. Wolf S.F. O'Garra A. Murphy K.M. Development of Th1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages.Science. 1993; 260: 547-549Crossref PubMed Scopus (2791) Google Scholar), although a number of other factors have been shown to influence the differentiation pathway of naïve CD4+ T cells (Abbas et al., 1996Abbas A.K. Murphy K.M. Sher A. Functional diversity of helper T lymphocytes.Nature. 1996; 383: 787-793Crossref PubMed Scopus (3759) Google Scholar). In this study, we freshly prepared ovalbumin (OVA) specific Th1 and Th2 cells by culturing naïve splenocytes obtained from anti-OVA T cell receptor (TCR) transgenic (tg) mice in the presence of appropriate cytokines and anticytokine antibody. Either Th1 or Th2 cells were injected into murine ear lobes and air-pouches developed on the mid-back of naïve mice to re-evaluate the roles of effector cells played by granulocytes. Using these unique murine skin models, we further investigated how therapeutic drugs used for the treatment of AD and other atopic diseases would influence the ear swelling or granulocyte accumulation that is induced by these CD4+ T cells. Male BALB/C mice bred in our animal facility were used at 8–12 wk of age. BALB/C mice tg for TCR specific for OVA323-339 (anti-OVA TCR tg mice) were established as described previously (Sato et al., 1994Sato T. Sasahara T. Nakamura Y. et al.Naive T cells can mediate delayed-type hypersensitivity response in T cell receptor transgenic mice.Eur J Immunol. 1994; 24: 1512-1516Crossref PubMed Scopus (90) Google Scholar). FK506 solution and ointment (0.1%) were gifts from Fujisawa Pharmaceutical (Osaka, Japan). Drugs capable of inhibiting granulocyte infiltration, ONO-1078 and ONO-6818, were gifts from Ono Pharmaceutical (Kyoto, Japan). FK506, ONO-1078, and ONO-6818 were administered once intraperitoneally or orally just before the adoptive transfer of Th cells. FK506 ointment was also applied topically once on the ear lobes 1 h after injection of Th cells. Spleen cells (3 × 107) from unimmunized anti-OVA TCR tg mice were cultured in 12 ml of RPMI 1640 medium with OVA (100 μg per ml) together with IL-12 (1 ng per ml; Genzyme, Cambridge, MA) for the induction Th1 cells or IL-4 (10 ng per ml; Genzyme) plus anti-IL-12 monoclonal antibody (0.1 μg per ml; Genzyme) for that of Th2 cells for 3 d. The cells were cultured in fresh medium without any cytokine or anticytokine antibody for another 3 d. Graded doses of these cells were adoptively transferred subcutaneously into the ear lobe without fractionation. From the in vitro culture made without any cytokines or anticytokine antibody for another 3 d, viable lymphocytes were further enriched for CD4+ T cells by a panning method as described previously (Sano et al., 1999Sano K. Haneda K. Tamura G. Shirato K. Ovalbumin (OVA) and Mycobacterium tuberculosis bacilli cooperatively polarize anti-OVA T-helper (Th) cells toward a Th1-dominant phenotype and ameliorate murine tracheal eosinophils.Am J Respir Cell Mol Biol. 1999; 20: 1260-1267Crossref PubMed Scopus (54) Google Scholar). The CD4+ fraction was cultured with mitomycin C (Wako Chemical Industries, Osaka, Japan) treated spleen cells of BALB/C mice as antigen-presenting cells in the presence of OVA (100 μg per ml) in quadruplicate for 2 d in 96-well plates (Falcon, Lincoln Park, NJ). Culture supernatants were assayed for IFN-γ and IL-4. Where indicated, ONO-6817 or ONO-1078 was added to Th stimulation cultures at a final concentration of 10 μg per ml [0.02% dimethylsulfoxide (DMSO)] or 30 μg per ml (0.067% DMSO). DMSO was included in the control cultures at the same concentrations. Supernatants obtained from 48 h cultures of Th1 or Th2 cells developed from anti-OVA TCR tg mice were assayed for cytokines by sandwich enzyme-linked immunosorbent assay (ELISA), as described previously (Shirota et al., 2000Shirota H. Sano K. Kikuchi T. Tamura G. Shirato K. Regulation of murine airway eosinophilia and Th2 cells by antigen-conjugated CpG oligodeoxynucleotides as a novel antigen-specific immunomodulator.J Immunol. 2000; 164: 5575-5582Crossref PubMed Scopus (157) Google Scholar). Purified and biotinylated monoclonal antibodies to IFN-γ and IL-4 were purchased from Pharmingen (San Diego, CA). Standard recombinant mouse IFN-γ and IL-4 were purchased from Genzyme. Unless otherwise stated, 1 × 106 cells were injected subcutaneously into the right ear lobes of naïve, non-tg male BALB/C mice. A total of 30 or 50 μl of the cell suspension was injected with a 1 ml syringe equipped with a 27 gauge × 1/2 needle. The left ear lobes were left untreated. The thickness of each ear lobe was measured with a precision caliper (Mitsutoyo, Tokyo, Japan), immediately before and at various time periods after injection. Ear swelling was expressed as (thickness of the right ear after injection - thickness of the left ear) - (thickness of the right ear before the injection - thickness of the left ear) × 0.01 mm. Results are shown as the mean swelling ± SD. Each experimental group comprised four to five mice, unless otherwise stated. In several experiments, determination of ear thickness was performed in a blinded fashion. Student's t test was used for statistical analysis. For histologic examination, 1 × 106 cells of Th1 or Th2 cells were adoptively transferred subcutaneously into the ears of naïve BALB/C. Animals were sacrificed 24 h or 48 h after injection. Ears were fixed in 10% formalin for 2–3 d, and processed for paraffin embedding. Tissue sections (4 μm) were stained with H&E, mounted, and observed in a blinded fashion. For staining of eosinophils, tissue sections were stained with May-Grünwald-Giemsa. Air-pouches were produced in the mid-back of naïve, non-tg BALB/C mice by injecting 4 ml of air subcutaneously 6 d before the adoptive transfer. One day before their injection, 2 ml of air were re-injected to maintain the pouches. Two milliliters of Th cell suspensions were adoptively transferred at a cell density of 1 × 106 cells per ml phosphate-buffered saline (PBS) in the presence or absence of OVA. At indicated time points after the adoptive transfer of Th cells, the back skin was incised and opened without rupturing the air-pouches encapsulated in a fibrous membrane. After an injection of 2 ml of PBS and shaking, the contents of the cystic pouches were recovered on a Petri dish by cutting the pouch membrane. Their total cell number was calculated and differential lymphocyte cell count was carried out with smeared samples stained with May-Grünwald-Giemsa. Th1 and Th2 cells were prepared by a short-term culture of splenic cells obtained from naïve anti-OVA TCR tg mice in the presence of cytokines and anticytokine antibody as described in Materials and Methods. Partially purified CD4+ cells were incubated with OVA (100 μg per ml) and mitomycin-C-treated spleen cells at a density of 2 × 105 per well that were used as antigen-presenting cells. Culture supernatants were assayed for IFN-γ and IL-4. The results showed that Th1 cells secreted IFN-γ but little IL-4, whereas Th2 cells secreted IL-4 but little IFN-γ, as shown in Figure 1. Cytokine secretions from Th1 or Th2 cells without OVA stimulation or with irrelevant antigens (bovine serum albumin or keyhole limpet lemocyani) were less than 0.05 ng per ml. As the levels of cytokine production by purified CD4+ T cells were not much different from those obtained by bulk-cultured splenocytes (data not shown), in later experiments we used the bulk splenic cells cultured under the above-mentioned conditions as Th1 or Th2 cells. These cells, suspended in 30 or 50 μl of PBS with OVA (5 μg per site), were injected into the right ear lobes of naïve, non-tg BALB/C mice to induce Th1- or Th2-induced inflammation. The time course of ear swelling measured at several time points after injection was different between Th1- and Th2-induced inflammation as shown in Figure 2. The ear thickness in the Th1-injected mice started to increase at 12 h and reached a peak at 48 h after injection, whereas that in the Th2-injected mice was detectable at 6 h and became maximal at 24 h in every experiment Figure 2. We also carried out a dose-response study to determine the optimal number of T cells to induce the ear swelling. The intensity of the response with either Th1 or Th2 was dependent on the number of cells injected, whereas the overall time course remained unchanged (data not shown). As the ear swelling induced by either type of T cells reached a plateau approximately when 1 × 106 cells per site were injected, we chose 1 × 106 cells for subcutaneous injection in the later experiments. In the next experiment, we investigated the histologic differences between Th1- and Th2-induced swollen ears. In H&E specimens, untreated ear pinnae showed little inflammatory cell infiltration Figure 3a. Only mild cell infiltration was detected in the specimen obtained with injection of OVA alone (data not shown). With injected Th1 or Th2 cells in the presence of OVA, marked cell infiltration and edematous changes were found at either injected site Figure 3b, c. The inflammatory cells were composed of mononuclear cells and polymorphonuclear leukocytes. It was hard to differentiate eosinophils from neutrophils in these H&E specimens, however. We also used May-Grünwald-Giemsa staining, which enabled us to easily discriminate eosinophils from neutrophils. The May-Grünwald-Giemsa specimens showed that a vast majority of the polymorphonuclear leukocytes were neutrophils and that eosinophils were absent in the Th1-injected swelling Figure 3d. In sharp contrast, Th2 cells induced inflammation characterized by a considerable number of eosinophils in addition to neutrophils Figure 3e. As mentioned above, we could histologically demonstrate a distinct pattern of granulocyte infiltration induced by protein-antigen-activated Th1 or Th2 cells. In the next experiment, to more quantitatively analyze the granulocyte accumulation, we produced air-pouches in the mid-back of naïve, non-tg BALB/C mice as described in Materials and Methods, and then adoptively transferred Th1 or Th2 cells together with OVA into the pouches. At indicated time points after injection, the infiltrated cells were recovered and counted for the total cell numbers. With smear samples of these cells on slide glasses stained with May-Grünwald-Giemsa, we further counted the differential leukocyte numbers to analyze infiltrating cells. A kinetic study showed that the numbers of total infiltrating cells reached a peak at time points corresponding to those when ear thickness after injection with each type of Th cells became maximal, as shown in Figure 2. The infiltrating cells induced 48 h after injection of Th1 cells were mainly composed of neutrophils with a small number of eosinophils Figure 4a. 60%-80% of infiltrating cells noted at 48 h were found at 24 h, although differential leukocyte numbers were similar (data not shown). On the other hand, Th2 induced an infiltration consisting of a considerable number of eosinophils at 24 h after injection Figure 4b. The cell numbers were slightly reduced at 48 h. Using our ear swelling and air-pouch models, we evaluated the effects of systemic administration and topical application of a well-known immunosuppressant, FK506, which inhibits T cell function through its action on calcineurin (Schreiber and Crabtree, 1992Schreiber S.L. Crabtree G.R. The mechanism of action of cyclosporin A and FK506.Immunol Today. 1992; 13: 136-142Abstract Full Text PDF PubMed Scopus (1880) Google Scholar), on the ear swelling and granulocyte accumulation induced by Th cells. Single intraperitoneal administration of FK506 reduced the ear swelling induced by Th1 or Th2 cells in a dose-dependent manner Figure 5a, b. Systemic FK506 also reduced the infiltration of both neutrophils and eosinophils into the air-pouch induced by Th1 or Th2 cells Figure 5c, d. We further noted that a single topical application of 0.1% FK506 ointment resulted in a marked reduction in the ear swelling induced by these T cells Figure 6.Figure 6Topical application of FK506 induced a significant reduction in ear swelling. Th1 or Th2 cells (1 × 106 cells per site) were adoptively transferred into the right ear lobes of naïve BALB/C mice with OVA to produce Th1- or Th2-induced inflammation. 0.1% FK506 ointment was applied once topically on the right ear lobes immediately after injection. Topical application of FK506 resulted in a marked reduction of ear swelling induced by either type of Th cells. The ear thickness was measured with a precision caliper, immediately before and at various time points after injections (see Materials and Methods). Ear swelling was expressed as (thickness of the right ear after injection - thickness of the left ear) - (thickness of the right ear before the injection - thickness of the left ear) × 0.01 mm. Results are shown as the mean swelling ± SD. Statistical significance was calculated using Student's t test: *p < 0.05, **p < 0.001.View Large Image Figure ViewerDownload (PPT) Next, we evaluated the effects of two drugs that have a specific influence on granulocyte infiltration: ONO-6818 specifically inhibits neutrophil elastase, whereas ONO-1078 is a leukotriene receptor antagonist that inhibits allergen-induced airway eosinophilic inflammation. Single intraperitoneal administration of ONO-6818 caused a dose-dependent reduction in the ear swelling induced by Th1 cells, whereas it exerted little effect on that induced by Th2 cells Figure 7a. In contrast, ONO-1078 inhibited ear swelling induced by Th2 cells, but not that by Th1 cells Figure 7b. Similar observations were made in experiments with the air-pouch: the Th1-induced accumulation of neutrophils in the air-pouch was inhibited by ONO-6818 but not by ONO-1078 Figure 7c, whereas Th2-induced eosinophilia was inhibited significantly by ONO-1078 but not significantly by ONO-6818 Figure 7d. We further carried out in vitro experiments to examine the effects of these two drugs on the lymphokine production by OVA-stimulated Th cells. Neither drug affected the production of respective lymphokines by the antigen-stimulated Th1 or Th2 cells Figure 8. Taken together, ONO-6818, a neutrophil-specific elastase inhibitor, and ONO-1078, a leukotriene receptor antagonist, specifically inhibited Th1-induced neutrophilic and Th2-induced eosinophilic inflammation, respectively, through their direct effects on granulocytes, but not by their indirect effects via Th cells.Figure 8Lack of inhibitory effects of ONO-6818 or ONO-1078 on in vitro cytokine production by Th1 or Th2 cells. Th1 and Th2 cells were stimulated with 100 μg per ml OVA and antigen-presenting cells in 96-well plates. ONO-6818 or ONO-1078 was added to the cultures at a final concentration of 10 μg per ml containing 0.02% DMSO or 30 μg per ml containing 0.067% DMSO. DMSO was included in the control cultures at the same concentrations. The levels of IFN-γ from Th1 cells and IL-4 from Th2 cells were determined by ELISA. These two drugs did not affect the IFN-γ or IL-4 production.View Large Image Figure ViewerDownload (PPT) In this study, we succeeded in producing allergic skin inflammation by subcutaneous injection of Th1 and Th2 cells prepared from anti-OVA TCR tg mice with OVA into the skin of naïve mice. The results showed that Th2-mediated ear swelling reached a peak at 24 h after the injection of Th2 cells with OVA, whereas Th1-mediated ear swelling peaked at 48 h. Histologically, a massive neutrophil infiltration was found in the Th1-induced inflammation. By contrast we could observe a significantly higher number of eosinophils mixed with neutrophils in the Th2-induced inflammation. For quantitative analysis, we produced air-pouches in the mid-back of naïve BALB/C mice. Then, Th cells were adoptively transferred into the pouch with OVA. Similar to the results of the histology of swollen ears, we found that Th1 caused a massive infiltration of neutrophils and a few eosinophils into the air-pouch, whereas Th2 induced the accumulation of a much larger number of eosinophils mixed with neutrophils. Since the discovery of the Th1 and Th2 cytokine pattern among CD4+ T cells, many studies have focused on the elucidation of the significance of these cytokine patterns in immunologic responses. Th1 and Th2 cytokines normally regulate different types of immune responses. DTH or contact hypersensitivity is often associated with Th1 rather than Th2 responses (Cher and Mosmann, 1987Cher D.J. Mosmann T.R. Two types of murine helper T cell clone. II. Delayed type hypersensitivity is mediated by Th1 clones.J Immunol. 1987; 138: 3688-3694PubMed Google Scholar;Fong and Mosmann, 1989Fong T.A. Mosmann T.R. The role of IFN-γ in delayed-type hypersensitivity mediated by Th1 clones.J Immunol. 1989; 143: 2887-2893PubMed Google Scholar;Hauser, 1990Hauser C. Cultured epidermal Langerhans cells activate effector T cells for contact sensitivity.J Invest Dermatol. 1990; 95: 436-440Abstract Full Text PDF PubMed Google Scholar;Müller et al., 1993Müller K.M. Jaunin F. Masouye I. Saurat J.-H. Hauser C. Th2 cells mediate IL-4-dependent local tissue inflammation.J Immunol. 1993; 150: 5576-5584PubMed Google Scholar). In fact the cytokines, e.g., IL-2 and IFN-γ, can be detected locally in DTH sites (Murray et al., 1992Murray J.S. Pfeiffer C. Madri J. Bottomly K. Major histocompatibility complex (MHC) control of CD4 T cell subset activation. II. A single peptide induces either humoral or cell-mediated responses in mice of distinct MHC genotype.Eur J Immunol. 1992; 22: 559-565Crossref PubMed Scopus (120) Google Scholar;Tsicopoulos et al., 1992Tsicopoulos A. Hamid Q. Varney V. Ying S. Moqbel R. Durham S.R. Kay A.B. Preferential messenger RNA expression of Th1-type cells (IFN-γ+, IL-2+) in classical delayed-type (tuberculin) hypersensitivity reactions in human skin.J Immunol. 1992; 148: 2058-2061PubMed Google Scholar) or in lymph nodes draining the DTH site (Heinzel et al., 1989Heinzel F.P. Sadick M.D. Holaday B.J. Coffman R.L. Locksley R.M. Reciprocal expression of interferon γ or interleukin 4 during the resolution or progression of murine leishmaniasis.J Exp Med. 1989; 169: 59-72Crossref PubMed Scopus (1239) Google Scholar). Furthermore, resting Th1 clones can induce footpad DTH in naïve mice when locally transferred with corresponding antigens, and the DTH reaction is partially regulated by IFN-γ (Cher and Mosmann, 1987Cher D.J. Mosmann T.R. Two types of murine helper T cell clone. II. Delayed type hypersensitivity is mediated by Th1 clones.J Immunol. 1987; 138: 3688-3694PubMed Google Scholar;Fong and Mosmann, 1989Fong T.A. Mosmann T.R. The role of IFN-γ in delayed-type hypersensitivity mediated by Th1 clones.J Immunol. 1989; 143: 2887-2893PubMed Google Scholar). Thus, in animal studies, Th1-like cells have been demonstrated to be responsible for inflammatory reactions such as DTH and contact sensitivity. By contrast, it has been controversial whether Th2 cells can really induce such immune-mediated inflammatory changes. Whereas Cher and Mosmann described that Th2 clones were inactive in a DTH assay (Cher and Mosmann, 1987Cher D.J. Mosmann T.R. Two types of murine helper T cell clone. II. Delayed type hypersensitivity is mediated by Th1 clones.J Immunol. 1987; 138: 3688-3694PubMed Google Scholar), there are certain conditions in which Th2 cells or their cytokines are involved in the inflammatory responses. For example, Müller et al demonstrated that preactivated bulk-cultured Th2 cells and alloreactive Th2 clones induced footpad inflammatory responses (Müller et al., 1993Müller K.M. Jaunin F. Masouye I. Saurat J.-H. Hauser C. Th2 cells mediate IL-4-dependent local tissue inflammation.J Immunol. 1993; 150: 5576-5584PubMed Google Scholar). In addition, the notion that IL-4 is involved in the development of specific inflammation is supported by the observation that IL-4-tg mice developed allergic-like blepharitis (inflammation of the eyelid) (Tepper et al., 1990Tepper R.I. Levinson D.A. Stanger B.Z. Campos-Torres J. Abbas A.K. Leder P. IL-4 induces allergic-like inflammatory disease and alters T cell development in transgenic mice.Cell. 1990; 62: 457-467Abstract Full Text PDF PubMed Scopus (342) Google Scholar). These results suggest that Th2 cells also have the potential to induce DTH-like inflammatory reactions. In humans, antigen-specific Th1 cells can readily be isolated from the sites of DTH responses observed in diseases (Del Prete et al., 1994Del Prete G. Maggi E. Romagnani S. Human Th1 and Th2 cells: functional properties, mechanisms of regulation, and role in disease.Lab Invest. 1994; 70: 299-306PubMed Google Scholar) and skin test sites induced by purified protein derivative (Del Prete et al., 1991Del Prete G.F. De Carli M. Mastromauro C. et al.Purified protein derivative of Mycobacterium tuberculosis and excretory-secretory antigen (s) of Toxocara canis expand in vitro human T cells with stable and opposite (type 1 T helper or type 2 T helper) profile of cytokine production.J Clin Invest. 1991; 88: 346-350Crossref PubMed Scopus (601) Google Scholar;Haanen et al., 1991Haanen J.B.A.G. Malefijt R.W. Res P.C.M. Kraakman E.M. Ottenhoff T.H.M. de Vries R.R.P. Spits H. Selection of a human T helper type 1-like T cell subset by mycobacteria.J Exp Med. 1991; 174: 583-592Crossref PubMed Scopus (261) Google Scholar). Moreover, there is also accumulating evidence that Th2 cells can induce inflammatory responses. Th2-like cells were cloned from inflammatory skin lesions of AD (Wierenga et al., 1990Wierenga E.A. Snoek M. de Groot C. Chretien I. Bos J.D. Jansen H.M. Kapsenberg M.L. Evidence for compartmentalization of functional subsets of CD4+ T lymphocytes in atopic patients.J Immunol. 1990; 144: 4651-4656PubMed Google Scholar;Van der Heijden et al., 1991Van der Heijden F.L. Wierenga E.A. Bos J.D. Kapsenberg M.L. High frequency of IL-4 producing CD4+ allergen-specific T lymphocytes in atopic dermatitis lesional skin.J Invest Dermatol. 1991; 97: 389-394Abstract Full Text PDF PubMed Google Scholar) and also identified in late phase skin reactions or in bronchial lavage specimens recovered from patients with allergic asthma (Kay et al., 1991Kay A.B. Ying S. Varney V. et al.Messenger RNA expression of the cytokine gene cluster, interleukin 3 (IL-3), IL-4, IL-5, and granulocyte/macrophage colony-stimulating factor, in allergen-induced late-phase cutaneous reactions in atopic subjects.J Exp Med. 1991; 173: 775-778Crossref PubMed Scopus (567) Google Scholar;Robinson et al., 1992Robinson D.S. Hamid Q. Ying S. et al.Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma.N Eng J Med. 1992; 326: 298-304Crossref PubMed Scopus (2494) Google Scholar). Th1 cells and their cytokines are believed to play an important role in DTH reactions. Among these cytokines, IFN-γ is a potent activator for both macrophages and neutrophils, which are the major effector cells in DTH reactions. It has been controversial, however, whether eosinophils are effector cells in Th2-mediated inflammatory responses. In an IgE-independent animal asthma model, antigen-specific Th2 cells can induce eosinophil infiltration (Li et al., 1998Li X.-M. Schofield B.H. Wang Q.-F. Kim K.-H. Huang S.-K. Induction of pulmonary allergic responses by antigen-specific Th2 cells.J Immunol. 1998; 160: 1378-1384PubMed Google Scholar), although nonspecific neutrophil influx was observed during the early time period in the same model. In contrast, neutrophil infiltration was found to predominate in Th2-induced DTH-like footpad inflammatory responses (Müller et al., 1995aMüller K.M. Jaunin F. Masouye I. Piguet P.-F. Saurat J.-H. Hauser C. Involvement of granulocytes and the adhesion receptor intercellular adhesion molecule-1 and lymphocyte function-associated antigen-1 in tissue inflammation induced by Th2-type helper cells.J Invest Dermatol. 1995; 104: 350-354Crossref PubMed Scopus (8) Google Scholar) when evaluated with H&E-stained histologic sections. As it was hard to differentiate eosinophils from neutrophils in the H&E-stained histologic sections, in this study the histologic materials were stained with May-Grünwald-Giemsa. The results indicated that there were a larger number of eosinophils in the Th2-induced swollen ears and in the inflammatory infiltrates of the air-pouch model. Although the mechanism for Th2-mediated inflammation is not fully understood, it is known that IL-4 exerts dual effects on the inflammatory responses. Its negative effects include inhibition of the upregulation of ICAM-1 and ELAM-1 on endothelial cells (Thornhill et al., 1991Thornhill M.H. Wellicome S.M. Mahiouz D.L. Lanchbury J.S.S. Kyan-Aung U. Haskard D.O. Tumor necrosis factor combines with IL-4 or IFN-γ to selectively enhance endothelial cell adhesiveness for T cells: the contribution of vascular cell adhesion molecule-1-dependent and -independent mechanisms.J Immunol. 1991; 146: 592-598PubMed Google Scholar) and suppression of the thrombomodulin anticoagulation pathway (Kapiotis et al., 1991Kapiotis S. Besemer J. Bevec D. Valent P. Bettelheim P. Lechner K. Speiser W. Interleukin-4 counteracts pyrogen-induced downregulation of thrombomodulin in cultured human vascular endothelial cells.Blood. 1991; 78: 410-415Crossref PubMed Google Scholar). In synergy with IL-1, tumor necrosis factor (TNF), or IFN-γ, however, IL-4 enhances the expression of VCAM-1 on endothelial cells both in vitro (Masinovsky et al., 1990Masinovsky B. Urdal D. Gallatin W.M. IL-4 acts synergistically with IL-1β to promote lymphocyte adhesion to microvascular endothelium by induction of vascular cell adhesion molecule-1.J Immunol. 1990; 145: 2886-2895PubMed Google Scholar) and in vivo (Briscoe et al., 1992Briscoe D.M. Cotran R.S. Pober J.S. Effects of tumor necrosis factor, lipopolysaccharide, and IL-4 on the expression of vascular cell adhesion molecule-1 in vivo. Correlation with CD3+ T cell infiltration.J Immunol. 1992; 149: 2954-2960PubMed Google Scholar), and this was correlated with the increased adhesion and infiltration of T cells and eosinophils (Schleimer et al., 1992Schleimer R.P. Sterbinsky S.A. Kaiser J. et al.IL-4 induces adherence of human eosinophils and basophils but not neutrophils to endothelium. Association with expression of VCAM-1.J Immunol. 1992; 148: 1086-1092PubMed Google Scholar). These findings suggest that IL-4 can play either a positive or a negative role during inflammation, depending on other environmental factors.Müller et al., 1995bMüller K.M. Rocken M. Carlberg C. Hauser C. The induction and functions of murine T-helper cell subsets.J Invest Dermatol. 1995; 105: 8S-13SCrossref PubMed Scopus (14) Google Scholar reported that IL-4 and TNF-α are important regulators for Th2-mediated footpad DTH. In this study we also assessed the effects of therapeutic drugs used for atopic diseases with both the ear swelling and air-pouch models. We first showed that systemic and topical FK506 reduced the ear swelling and infiltrations of both neutrophils and eosinophils into air-pouches induced by either Th1 or Th2 cells. In the literature, FK506 is known to exert its effect through the inhibition of cytokine or chemokine production by Th cells (Schreiber and Crabtree, 1992Schreiber S.L. Crabtree G.R. The mechanism of action of cyclosporin A and FK506.Immunol Today. 1992; 13: 136-142Abstract Full Text PDF PubMed Scopus (1880) Google Scholar;Sasakawa et al., 2000Sasakawa Y. Sakuma S. Higashi Y. Sasakawa T. Amaya T. Goto T. FK506 suppresses neutrophil chemoattractant production by peripheral blood mononuclear cells.Eur J Pharmacol. 2000; 403: 281-288Crossref PubMed Scopus (29) Google Scholar). It still remains to be elucidated how FK506 influences granulocyte function and accumulation. As for eosinophils, although there is a report indicating that FK506 reduces IL-8 expression in human eosinophils (Kohyama et al., 1999Kohyama T. Takizawa H. Kawasaki S. Akiyama N. Sato M. Ito K. Yamamoto K. A potent immunosuppressant FK506 inhibits IL-8 expression in human eosinophils.Mol Cell Biol Res Commun. 1999; 1: 72-77Crossref PubMed Scopus (22) Google Scholar), it was not observed to affect recruitment of eosinophils or delay worm expulsion in a murine parasite-infected model (Fujino et al., 1998Fujino T. Ichikawa H. Fried B. The immunosuppressive compound FK506 does not affect expulsion of Echinostoma trivolvis in C3H mice.Parasitol Res. 1998; 84: 519-521Crossref PubMed Scopus (12) Google Scholar). As for neutrophils, it has been reported that FK506 inhibits motility of neutrophils (Hendey et al., 1992Hendey B. Klee C.B. Maxfield F.R. Inhibition of neutrophil chemokinesis on vitronectin by inhibitors of calcineurin.Science. 1992; 258: 296-299Crossref PubMed Scopus (170) Google Scholar;Burnett et al., 1994Burnett D. Adams D.H. Martin T.J. Liu Q. Grant R.A. Stockley R.A. Lord J.M. Inhibition by FK506 of formyl peptide-induced neutrophil activation and associated protein synthesis.Biochem Pharmacol. 1994; 48: 1081-1088Crossref PubMed Scopus (12) Google Scholar) and their degranulation and production of destructive superoxide radicals (Forrest et al., 1991Forrest M.J. Jewell M.E. Koo G.C. Sigal N.H. FK-506 and cyclosporin A: selective inhibition of calcium ionophore-induced polymorphonuclear leukocyte degranulation.Biochem Pharmacol. 1991; 42: 1221-1228Crossref PubMed Scopus (30) Google Scholar;Burnett et al., 1994Burnett D. Adams D.H. Martin T.J. Liu Q. Grant R.A. Stockley R.A. Lord J.M. Inhibition by FK506 of formyl peptide-induced neutrophil activation and associated protein synthesis.Biochem Pharmacol. 1994; 48: 1081-1088Crossref PubMed Scopus (12) Google Scholar); a recent report indicates that FK506 has no direct suppressive effect on neutrophil chemotaxis induced by several chemotactic factors (Sasakawa et al., 2000Sasakawa Y. Sakuma S. Higashi Y. Sasakawa T. Amaya T. Goto T. FK506 suppresses neutrophil chemoattractant production by peripheral blood mononuclear cells.Eur J Pharmacol. 2000; 403: 281-288Crossref PubMed Scopus (29) Google Scholar). Taken together, we assume that suppression of Th-induced inflammatory skin responses by FK506 is mediated via its effects on granulocytes as well as on Th cells, although it was not conclusive from the results of this study. Interestingly, administration of a neutrophil-specific elastase inhibitor, ONO-6818, caused a reduction in the Th1-induced skin inflammation, but less effectively reduced the Th2-induced one. On the other hand, a leukotriene receptor antagonist, ONO-1078, specifically inhibited the Th2-mediated ear swelling and eosinophil infiltration, whereas it had little effect on the Th1-mediated ones. We also confirmed that granulocytes are the therapeutic target cells of these two drugs, as neither drug affected in vitro production of IFN-γ or IL-4 by antigen-stimulated Th cells. These results suggest that neutrophils or eosinophils are the major effector cells in Th1- or Th2-mediated cutaneous inflammation. In conclusion, we demonstrated the usefulness of two unique murine models to evaluate the contribution of Th1 and Th2 cells to the development of skin allergic inflammation. Using these models we confirmed the usefulness of not only well-known but also potential therapeutic drugs for the treatment of AD and other atopic diseases. The drugs studied could reduce the relevant cutaneous inflammation and granulocyte infiltration induced by protein-antigen-stimulated Th cells.