Influence of the Th1 Cytokine Environment on CCL5 Production from Langerhans Cells

Abstract

In the chronic skin lesions of atopic dermatitis (AD), T helper type 1 (Th1) cells appear in addition to Th2 cells, but the role played by Th1 cells in skin inflammation during the chronic phase remains unknown. Here we examined CCL5 production from Langerhans cells (LCs) in the Th1 cytokine environment. LCs were generated from mouse bone marrow cells, then stimulated with anti-CD40 antibody and the Th1 cytokine, interferon (IFN)-γ. Their CCL5 production was then measured. In addition, the LCs were incubated with naïve CD4⁺ T cells from a DO 11.10 TCR Tg mouse in the presence of ovalbumin peptide for 5 d, and their IFN-γ, interleukin-4 and CCL5 production was then measured. When LCs were stimulated with the anti-CD40 antibody in the presence of IFN-γ, significant levels of CCL5 production were confirmed. Furthermore, when LCs presented antigen to Th cells in the Th1 cytokine environment, significant levels of CCL5 production were induced. This CCL5 production was associated with IFN-γ activity and CD40L expression by Th cells in the culture. Our present data suggest that LCs augment CCL5 production by responding to IFN-γ while presenting antigen to Th cells, and that this augmentation of CCL5 production would likely contribute to infiltration of eosinophils and other Th1 cells into skin lesions, followed by expansion of chronic inflammation in the skin.

INTRODUCTION

In recent years, allergic diseases such as atopic dermatitis (AD), allergic rhinitis and bronchial asthma have been increasing, and are now a problem for many people in modern society.¹⁾ Most patients with allergies are characterized by an excessive T helper type 2 (Th2) immune response to invading allergens.^2,3) Therefore, it is thought that control of excessive Th2 cell differentiation in vivo would lead to the establishment of effective therapy for allergic disease.^4,5) Allergens from the external environment invade through the skin or mucous membranes, where Langerhans cells (LCs) are localized and act as antigen-presenting cells.^6,7) LCs that have captured allergens then move to secondary lymphoid tissue and trigger Th1/Th2 cell differentiation there.^8,9) They also cause eosinophils, basophils, monocytes, Th1 cells and Th2 cells to migrate into the dermis by producing CCL5/regulated on activation, normal T-cell expressed and secreted (RANTES) and CCL17/thymus and activation-regulated chemokine (TARC) in the epidermis.^10,11) Therefore, any therapeutic method targeting LCs must be designed to control excessive Th1/Th2 cell differentiation, the subsequent excessive Th1/Th2 immune response, and eosinophil-mediated inflammation.

In our previous study, we found that the macrolide antibiotic josamycin inhibited both Th1 and Th2 cell differentiation through LCs, and that the tetracycline antibiotic doxycycline inhibited Th2 cell differentiation without affecting Th1 cell differentiation.^12,13) Furthermore, when josamycin ointment and doxycycline ointment were applied to NC/Nga mice with AD-like skin inflammation (a model mouse of AD), the former effectively suppressed the skin inflammation over a wide range from the acute phase to the chronic phase, whereas the latter suppressed skin inflammation only in the acute phase.^9,13) This suggested that Th1 cells play an important role in the development of skin inflammation in the chronic phase.

It has also been suggested for a long time that, in addition to Th2 cells, Th1 cells play an important role in chronic skin inflammation in patients with AD.^14,15) However, their role in the chronic phase of skin inflammation has remained unclear. In this context, it is thought that LCs may be important agents in the chronicity of skin inflammation because they facilitate the migration of eosinophils, basophils, monocytes and Th1 cells to sites of inflammation through CCL5 production, and —as antigen-presenting cells— determine the directionality of Th1/Th2 cell differentiation.^10,16,17) In the present study, therefore, we examined the effects of CCL5 production from LCs when the latter were placed in a Th1 cytokine environment.

MATERIALS AND METHODS

Mice

Female specific-pathogen-free BALB/c mice (wild type mice) were obtained from Japan SLC (Hamamatsu, Japan) and DO 11.10 TCR Tg mice (ovalbumin (OVA) peptide_323–339-specific I-A^d-restricted T cell receptor (TCR)-transgenic mice) were obtained from Jackson Laboratory (Bar Harbor, ME, U.S.A.), and used at the age of 6–8 weeks. They were housed in plastic cages with sterilized paper bedding in an air-conditioned room at 24 °C and allowed free access to a standard laboratory diet and water. All procedures performed on the mice were approved by the Animal Care and Use Committee of Meiji Pharmaceutical University.

Generation of LCs

Murine bone marrow cells for generation of LCs were prepared and cultured as described previously.¹⁸⁾ Briefly, bone marrow cells from BALB/c mice were cultured in RPMI 10 (RPMI 1640 medium with L-glutamine (Sigma-Aldrich, St. Louis, MO, U.S.A.) containing 10% fetal bovine serum (Sigma-Aldrich), 25 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (Hepes) (Sigma-Aldrich), 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco RBL, Grand Island, NY, U.S.A.) supplemented with recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) (20 ng/mL; PeproTech, Rocky Hill, NJ, U.S.A.), recombinant murine interleukin (IL)-4 (100 ng/mL; PeproTech) and recombinant human transforming growth factor (TGF)-β1 (10 ng/mL; PeproTech) at 37 °C in a humidified atmosphere with 5% CO₂. Half of the total volume of the culture medium was changed every 48 h, and 7 d after the start of culture the grown cells were collected and treated with mouse anti-mouse I-A^d monoclonal antibody (mAb) (clone 34-5-3s, mouse immunoglobulin G (IgG)2a) (1 : 200; Cedarlane Laboratories Ltd., ON, Canada) in RPMI 10 for 1 h on ice. The cells that had reacted with the anti-I-A^d antibody were then purified using a CELLection Pan Mouse IgG Kit (Invitrogen Dynal AS, Oslo, Norway), and used as LCs. LCs positive for I-A^d were purified to around 95% as determined by flow cytometry.

Quantification of CCL5

LCs were adjusted to 1 × 10⁵ cells/mL in RPMI 10. The cultures (0.2 mL/well) were incubated in a 96-well culture plate (Nunc, Roskilde, Denmark) in the presence of 0.5–2 µg/mL interferon (IFN)-γ (PeproTech) and/or 1–4 µg/mL anti-CD40 mAb (clone 1C10; rat IgG2a) (BioLegend, San Diego, CA, U.S.A.) at 37 °C in a humidified atmosphere with 5% CO₂. The culture supernatants were collected after incubation for 48 h, and the CCL5 concentrations were measured using enzyme-linked immunosorbent assay (ELISA) kits for quantification of murine CCL5 (R & D Systems, Minneapolis, MN, U.S.A.).

Induction of Th1 Cell Differentiation in Vitro and CCL5 Production

Naïve CD4⁺ T cells were separated from spleen cells of a DO 11.10 TCR Tg mouse using a Mouse Naïve CD4⁺ T Cell Isolation Kit (StemCell Technologies, Inc., Vancouver, BC, Canada). The naïve CD4⁺ T cells (5 × 10⁵ cells/mL) in a 96-well culture plate (Nunc) were cultured with LCs (5 × 10⁴ cells/mL) in the presence of 30 nM OVA peptide (323-ISQAVHAAHAEINEAGR-339; obtained from Operon Biotechnologies, Tokyo, Japan) for 5 d at 37 °C in a humidified atmosphere with 5% CO₂. The grown Th cells in the cultures were stained with phycoerythrin-conjugated control mAb (clone HTK888; American hamster IgG) (BioLegend) or phycoerythrin-conjugated anti-mouse CD40L mAb (clone MR1; American hamster IgG) (BioLegend), and analyzed by flow cytometry. The linear plot obtained, showing granularity (side scatter) versus cell size (forward-angle light scatter), revealed mainly a single-cell population. Analytic gates were set around this population to exclude cellular debris. The number of positive cells was then obtained from a histogram representing mean fluorescence intensities on a 5-decade logarithmic amplifier.

In some experiments, coculture of LCs and naïve CD4⁺ T cells was carried out in the presence or absence of 10 µg/mL control mAb (clone RTK2071; rat IgG1) (BioLegend), 10 µg/mL anti-mouse IFN-γ neutralizing mAb (clone XMG1.2; rat IgG1) (BioLegend) or 10 µg/mL anti-mouse CD40L (CD154) neutralizing mAb (clone #208109; rat IgG2a) (R & D Systems). The culture supernatants were finally removed and tested for production of IFN-γ, IL-4 and CCL5 using ELISA kits (R & D Systems).

Statistical Analysis

The data were expressed as means (± standard error of the mean (S.E.M.)), and differences between means were analyzed by the Tukey–Kramer multiple comparison test. Differences at p < 0.05 were considered to be statistically significant.

RESULTS

CCL5 Production from LCs in the Presence of Anti-CD40 mAb or IFN-γ

LCs were cultured in the presence of three different concentrations of anti-CD40 mAb, and a suboptimal concentration of anti-CD40 mAb for examination of CCL5 production. As shown in Fig. 1a, stimulation with anti-CD40 mAb (1–4 µg/mL) induced CCL5 production by LCs in a dose-dependent manner, and 1 µg/mL anti-CD40 mAb was selected as the suboptimal concentration to investigate the role of a Th1 cytokine environment in CCL5 production. However, stimulation with IFN-γ alone (0.5–2 µg/mL) did not induce CCL5 production from LCs at any concentration (Fig. 1b).

Fig. 1. Effects of Anti-CD40 mAb and IFN-γ on CCL5 Production from LCs

LCs were incubated in the presence of anti-CD40 mAb (1–4 µg/mL) (a) or IFN-γ (0.5–2 µg/mL) (b) for 48 h, and the concentrations of CCL5 in the culture supernatants were determined by ELISA. The results are expressed as means ± S.E.M. (n = 6). * p < 0.05, ** p < 0.01 versus non-treatment.

Effects of IFN-γ on CCL5 Production from Anti-CD40 mAb-Stimulated LCs

We next studied the production of CCL5 from LCs in the presence of both IFN-γ and anti-CD40 mAb. As shown in Fig. 2, although production of CCL5 by LCs stimulated with the suboptimal concentration (1 µg/mL) of anti-CD40 mAb did not surpass that from non-treated LCs, the LCs responded to IFN-γ in a dose-dependent manner when simultaneously exposed to anti-CD40 mAb (1 µg/mL) and IFN-γ (0.5–2 µg/mL), and significant levels of CCL5 production were induced.

Fig 2. Synergistic Effects of Anti-CD40 mAb and IFN-γ on CCL5 Production from LCs

LCs were incubated in the presence of anti-CD40 mAb (1 µg/mL) and IFN-γ (0.5–2 µg/mL) for 48 h, and the concentrations of CCL5 in the culture supernatants were determined by ELISA. The results are expressed as means ± S.E.M. (n = 6). * p < 0.05, ** p < 0.01 versus anti-CD40 mAb (1 µg/mL).

Induction of Th1/Th2 Cell Differentiation Mediated by LCs and CCL5 Production

In order to clarify the influence of a Th1 cytokine environment on CCL5 production from LCs, LCs and naïve CD4⁺ T cells were cocultured in the presence of OVA peptide for 5 d. As shown in Fig. 3a, the cytokine released by Th cells in the culture supernatant was predominantly IFN-γ rather than IL-4, suggesting that the cytokine environment of the culture was Th1 cytokine-dominant. Furthermore, significant levels of CCL5 production were confirmed upon coculture of LCs and naïve CD4⁺ T cells in the presence of OVA peptide (Fig. 3b).

Fig. 3. Th1/Th2 Cell Differentiation by Coculture of LCs with Naïve CD4⁺ T Cells, and Its Effect on CCL5 Production

LCs were incubated with naïve CD4⁺ T cells from DO11.10 TCR Tg mouse in the presence of OVA peptide for 5 d. The culture supernatants were assayed for IFN-γ and IL-4 production (a), and CCL5 production (b) using ELISA. The results are expressed as means ± S.E.M. (n = 6). * p < 0.01 versus LCs + Th cells.

Influences of Anti-IFN-γ mAb on CCL5 Production

To clarify whether CCL5 production from LCs in the Th1 cytokine environment is associated with IFN-γ production from Th1 cells, the influence of anti-IFN-γ neutralizing mAb (10 µg/mL) on CCL5 production was investigated. As shown in Fig. 4, CCL5 production induced by coculture of LCs and naïve CD4⁺ T cells in the presence of OVA peptide was significantly inhibited by addition of anti-IFN-γ neutralizing mAb, but was not inhibited by control mAb.

Fig. 4. Effects of Anti-IFN-γ Neutralizing mAb on CCL5 Production Induced by Coculture of LCs with Naïve CD4⁺ T Cells

LCs were incubated with OVA peptide and naïve CD4⁺ T cells from DO11.10 TCR Tg mouse in the presence or absence of anti-IFN-γ neutralizing mAb (10 µg/mL) or control mAb (10 µg/mL) for 5 d. The culture supernatants were assayed for CCL5 production using ELISA. The results are expressed as means ± S.E.M. (n = 6). * p < 0.01 versus LCs + Th cells, ** p < 0.01 versus LCs + Th cells + OVA.

Induction of CD40L Expression on Th Cells Activated by Antigen Presentation by LCs, and Influence of Anti-CD40L mAb on CCL5 Production from LCs

To clarify whether Th cells activated by LC antigen presentation show increased surface expression of CD40L, the level of CD40L expression on Th cells grown in culture was analyzed by flow cytometry. As shown in Fig. 5, it was confirmed that CD40L expression on Th cells was enhanced by coculture with LCs in the presence of OVA peptide (94% of Th cells positive). However, CD40L expression was not detected on most fresh naïve CD4⁺ T cells. Furthermore, CCL5 production induced by coculture of LCs and naïve CD4⁺ T cells in the presence of OVA peptide was significantly inhibited by addition of anti-CD40L neutralizing mAb, but was not inhibited by control mAb (Fig. 6).

Fig. 5. CD40L Expression on Activated Th Cells Developed by Coculture of LCs with Naïve CD4⁺ T Cells

The activated Th cells developed after 5 d of coculture of LCs with naïve CD4⁺ T cells from DO11.10 TCR Tg mouse in the presence of OVA peptide, and were stained with phycoerythrin-conjugated control mAb (open histogram, dashed line) or phycoerythrin-conjugated anti-CD40L mAb (solid histogram), then analyzed by flow cytometry. The naïve CD4⁺ T cells were also stained with phycoerythrin-conjugated anti-CD40L mAb (open histogram, solid line), and were compared with the CD40L expression on the activated Th cells. The data shown are representative of four independent experiments.

Fig. 6. Effects of Anti-CD40L Neutralizing mAb on CCL5 Production Induced by Coculture of LCs with Naïve CD4⁺ T Cells

LCs were incubated with OVA peptide and naïve CD4⁺ T cells from DO11.10 TCR Tg mouse in the presence or absence of anti-CD40L neutralizing mAb (10 µg/mL) or control mAb (10 µg/mL) for 5 d. The culture supernatants were assayed for CCL5 production using ELISA. The results are expressed as means ± S.E.M. (n = 6). * p < 0.01 versus LCs + Th cells, ** p < 0.01 versus LCs + Th cells + OVA.

DISCUSSION

It has long been known that the chronic skin lesions of AD patients show increased amounts of Th1 cells in addition to Th2 cells.^14,15) However, it has remained unclear whether these Th1 cells actually participate in the exacerbation of skin inflammation. In our recent studies, we investigated the treatment of AD-like skin inflammation induced in the NC/Nga mouse with josamycin, which inhibits both Th1 and Th2 cell differentiation, and doxycycline, which inhibits only Th2 cell differentiation.^9,13) This revealed that doxycycline, which does not inhibit Th1 cell differentiation, was unable to fully control skin inflammation in the chronic phase.¹³⁾ This suggested that Th1 cells play an important role in the formation of chronic skin lesions, although the specific mechanisms responsible for the increase of Th1 cells in the lesional skin remained unidentified.

We previously demonstrated that peptidoglycan derived from Staphylococcus aureus, which can be isolated at high frequency (>96%) from the lesional skin of AD patients,¹⁹⁾ stimulated LCs in the epidermis and promoted the production of CCL5 and CCL17.^10,11) CCL5 production promotes the migration of eosinophils, basophils, monocytes and Th1 cells to sites of inflammation,^20,21) and CCL17 production promotes the migration of Th2 cells.^22–24) However, expression of other chemokines such as CCL11 (involved in eosinophil migration) or CCL22 (involved in Th2 cell migration) was not detected in the skin.^10,11) Therefore, the production of CCL5 and CCL17 induced by S. aureus colonization of AD skin lesions might play an important role in the induction of chronic inflammation characterized by a Th1-prone immune response with eosinophil migration and acute inflammation characterized by a Th2-prone immune response, respectively. Furthermore, we found that CCL17 production was increased when LCs were placed in a Th2 cytokine environment.²⁵⁾ This means that a Th2 cytokine environment increases the Th2 immune response still further through augmentation of CCL17 production from LCs.

On the other hand, the effects of CCL5 production from LCs when the latter are placed in a Th1 cytokine environment have remained unclear. Our data showed that LCs stimulated with an anti-CD40 mAb, which crosslinks CD40 molecules on the surface of LCs, increased their production of CCL5 in the presence of the Th1 cytokine IFN-γ, relative to that when stimulated with anti-CD40 mAb alone. However, Fujita et al. have shown that LCs separated from mouse epidermis responded to IFN-γ and increased CCL5 production in the absence of anti-CD40 mAb.²⁶⁾ In their study, the mouse epidermal cells including LCs received strong stimuli such as various enzyme treatments or vigorous pipetting in the process of LC separation. Therefore, the purified LCs would have already been primed to a state that did not require stimulation by anti-CD40 mAb. Since CCL5 acts as a chemokine for Th1 cells, it seems likely that a Th1 cytokine environment increased the Th1 immune response through an increase of CCL5 production from LCs.

It has been clarified previously that CD40L on activated Th cells is responsible for CD40 signaling on LCs during antigen presentation.²⁷⁾ We confirmed in vitro that naïve CD4⁺ T cells receiving antigen from LCs proliferated over time and expressed CD40L on their surfaces, leading to gradual induction of IFN-γ production. Furthermore, since production of the Th2 cytokine IL-4 was negligible in comparison with IFN-γ production, the environment in this culture would have been Th1 cytokine-dominant. Accordingly, it is thought that Th1 cells themselves in the culture enhanced the production of CCL5 by stimulation of LCs through CD40L-CD40 binding and IFN-γ exposure. As another possible mechanism for the increase of CCL5 production from LCs, it is thought that IFN-γ may act on Th1 cells themselves to enhance CD40L expression on the Th1 cells. This augmentation of CD40L expression on Th1 cells may also play an important role in CCL5 production through CD40L-CD40 binding. This excessive production of CCL5 would also occur in AD patients, thus further augmenting the Th1 immune response in their skin lesions and leading to the formation of chronic skin lesions accompanied by eosinophilic infiltration. Once these conditions promoting Th1 cell differentiation become established in the lesional skin, the concentration of CCL5 would rise in the dermis through this mechanism, and as a result the number of Th1 cells in the dermis would increase steadily. Although the cells producing CCL5 in the lesional skin of AD patients are unclear, immunohistochemical analysis by Park et al. using anti-CCL5 mAb showed that the whole epidermis and part of the dermis adjacent to it were stained positively for CCL5.²⁸⁾ Therefore, even in the lesional skin of AD patients, epidermal LCs can be a source of CCL5, and these LCs may interact with Th1 cells in the epidermis, as observed by Kabashima and Egawa,²⁹⁾ or in the dermis during passage to lymph vessels.

We next considered the type of trigger that would be important for initiation of Th1-prone cell differentiation in the dermis. One possible mechanism would be antigen presentation by mast cells and subsequent Th1/Th2 cell differentiation in the dermis.³⁰⁾ We previously demonstrated that mast cells stimulated with peptidoglycan from S. aureus during antigen presentation promoted Th1 cell differentiation.³⁰⁾ As the chronic skin lesions of AD show sustained colonization by S. aureus and an increased number of mast cells in the dermis,^19,31–35) it is possible that peptidoglycan stimulation of, and antigen presentation by these mast cells would be involved in triggering excessive Th1 cell differentiation in the dermis. In addition, it would be expected that CCL5 production from LCs would increase synergistically in the co-presence of peptidoglycan and IFN-γ, considering our previous findings and the present results.¹⁰⁾ Since mast cells do not induce CCL5 production even if they are cultured under the same conditions as LCs (data not shown), CCL5 production from LCs would eventually be required for migration of Th1 cells to the lesional skin. In any event, for the above reason, it would seem advisable to avoid the use of Th1 adjuvant for treatment of AD, aimed at down-regulation of Th2 immune response.

CONCLUSION

Our present findings suggest that a Th1 cytokine environment in the lesional skin of AD patients might promote the chronicity of skin inflammation through augmentation of CCL5 production from LCs. Therefore, any factor, such as S. aureus colonization, promoting an Th1 immune response would need to be excluded from the lesions. For example, topical application of josamycin makes it possible to exclude S. aureus from the lesional skin, and at the same time inhibits the induction of a Th1 immune response via LCs and mast cells. This approach could potentially achieve complete cure of AD and shows promise as a new therapeutic strategy.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Dierick BJH, van der Molen T, Flokstra-de Blok BMJ, Muraro A, Postma MJ, Kocks JWH, van Boven JFM. Burden and socioeconomics of asthma, allergic rhinitis, atopic dermatitis and food allergy. Expert. Rev. Pharmacoecon. Outcomes Res., 20, 437–453 (2020).
• 2) Lloyd CM, Snelgrove RJ. Type 2 immunity: expanding our view. Sci. Immunol., 3, eaat1604 (2018).
• 3) Hassoun D, Malard O, Barbarot S, Magnan A, Colas L. Type 2 immunity-driven diseases: towards a multidisciplinary approach. Clin. Exp. Allergy, 51, 1538–1552 (2021).
• 4) van de Veen W, Akdis M. The use of biologics for immune modulation in allergic disease. J. Clin. Invest., 129, 1452–1462 (2019).
• 5) Manka LA, Wechsler ME. New biologics for allergic diseases. Expert Rev. Clin. Immunol., 14, 285–296 (2018).
• 6) Katz SI, Tamaki K, Sachs DH. Epidermal Langerhans cells are derived from cells originating in bone marrow. Nature, 282, 324–326 (1979).
• 7) MacPherson GG, Liu LM. Dendritic cells and Langerhans cells in the uptake of mucosal antigens. Curr. Top. Microbiol. Immunol., 236, 33–53 (1999).
• 8) Igyártó BZ, Kaplan DH. Antigen presentation by Langerhans cells. Curr. Opin. Immunol., 25, 115–119 (2013).
• 9) Matsui K, Tachioka K, Onodera K, Ikeda R. Topical application of josamycin inhibits development of atopic dermatitis-like skin lesions in NC/Nga mice. J. Pharm. Pharm. Sci., 20, 38–47 (2017).
• 10) Matsui K, Wirotesangthong M, Nishikawa A. Percutaneous application of peptidoglycan from Staphylococcus aureus induces eosinophil infiltration in mouse skin. Clin. Exp. Allergy, 37, 615–622 (2007).
• 11) Matsui K, Nishikawa A. Percutaneous application of peptidoglycan from Staphylococcus aureus induces infiltration of CCR4⁺ cells into mouse skin. J. Investig. Allergol. Clin. Immunol., 21, 354–362 (2011).
• 12) Matsui K, Tamai S, Ikeda R. Effects of macrolide antibiotics on Th1 cell and Th2 cell development mediated by Langerhans cells. J. Pharm. Pharm. Sci., 19, 357–366 (2016).
• 13) Matsui K, Nojima Y, Kajiwara Y, Busujima K, Mori Y. Topical application of doxycycline inhibits Th2 cell development mediated by Langerhans cells and exerts a therapeutic effect on atopic dermatitis. J. Pharm. Pharm. Sci., 23, 86–99 (2020).
• 14) Herz U, Bunikowski R, Renz H. Role of T cells in atopic dermatitis. New aspects on the dynamics of cytokine production and the contribution of bacterial superantigens. Int. Arch. Allergy Immunol., 115, 179–190 (1998).
• 15) Grewe M, Bruijnzeel-Koomen CAFM, Schöpf E, Thepen T, Langeveld-Wildschut AG, Ruzicka T, Krutmann J. A role for Th1 and Th2 cells in the immunopathogenesis of atopic dermatitis. Immunol. Today, 19, 359–361 (1998).
• 16) de Nadaï P, Chenivesse C, Gilet J, Porte H, Vorng H, Chang Y, Walls AF, Wallaert B, Tonnel AB, Tsicopoulos A, Zerwes HG. CCR5 usage by CCL5 induces a selective leukocyte recruitment in human skin xenografts in vivo. J. Invest. Dermatol., 126, 2057–2064 (2006).
• 17) Matsui K, Nishikawa A. Peptidoglycan from Staphylococcus aureus induces Th2 immune response in mice. J. Investig. Allergol. Clin. Immunol., 22, 80–86 (2012).
• 18) Matsui K, Mori A, Ikeda R. Langerhans cell-like dendritic cells stimulated with an adjuvant direct the development of Th1 and Th2 cells in vivo. Clin. Exp. Immunol., 182, 101–107 (2015).
• 19) Matsui K, Nishikawa A, Suto H, Tsuboi R, Ogawa H. Comparative study of Staphylococcus aureus isolated from lesional and non-lesional skin of atopic dermatitis. Microbiol. Immunol., 44, 945–947 (2000).
• 20) Crane IJ, Xu H, Wallace C, Manivannan A, Mack M, Liversidge J, Marquez G, Sharp PF, Forrester JV. Involvement of CCR5 in the passage of Th1-type cells across the blood-retina barrier in experimental autoimmune uveitis. J. Leukoc. Biol., 79, 435–443 (2006).
• 21) Rot A, Krieger M, Brunner T, Bischoff SC, Schall TJ, Dahinden CA. RANTES and macrophage inflammatory protein 1 alpha induce the migration and activation of normal human eosinophil granulocytes. J. Exp. Med., 176, 1489–1495 (1992).
• 22) Imai T, Baba M, Nishimura M, Kakizaki M, Takagi S, Yoshie O. The T cell-directed CC chemokine TARC is a highly specific biological ligand for CC chemokine receptor 4. J. Biol. Chem., 272, 15036–15042 (1997).
• 23) Imai T, Chantry D, Raport C, Wood C, Nishimura M, Godiska R, Yoshie O, Gray P. Macrophage-derived chemokine is a functional ligand for the CC chemokine receptor 4. J. Biol. Chem., 273, 1764–1768 (1998).
• 24) Imai T, Nagira M, Takagi S, Kakizaki M, Nishimura M, Wang J, Gray PW, Matsushima K, Yoshie O. Selective recruitment of CCR-4 bearing Th2 cells toward antigen-presenting cells by the CC chemokines thymus and activation-regulated chemokine and macrophage-derived chemokine. Int. Immunol., 11, 81–88 (1999).
• 25) Matsui K, Tofukuji S, Ikeda R. CCL17 production by mouse Langerhans cells stimulated with Staphylococcus aureus cell wall components. Biol. Pharm. Bull., 38, 317–320 (2015).
• 26) Fujita H, Asahina A, Gao P, Fujiwara H, Tamaki K. Expression and regulation of RANTES/CCL5, MIP-1α/CCL3, and MIP-1β/CCL4 in mouse Langerhans cells. J. Invest. Dermatol., 122, 1331–1333 (2004).
• 27) van Kooten C, Banchereau J. CD40-CD40 ligand. J. Leukoc. Biol., 67, 2–17 (2000).
• 28) Park CW, Lee BH, Han HJ, Lee CH, Ahn HK. Tacrolimus decreases the expression of eotaxin, CCR3, RANTES and interleukin-5 in atopic dermatitis. Br. J. Dermatol., 152, 1173–1181 (2005).
• 29) Kabashima K, Egawa G. Intravital multiphoton imaging of cutaneous immune response. J. Invest. Dermatol., 134, 2680–2684 (2014).
• 30) Matsui K, Kanai S, Ikuta M, Horikawa S. Mast cells stimulated with peptidoglycan from Staphylococcus aureus augment the development of Th1 cells. J. Pharm. Pharm. Sci., 21, 296–304 (2018).
• 31) Leung DYM. Atopic dermatitis: the skin as a window into the pathogenesis of chronic allergic diseases. J. Allergy Clin. Immunol., 96, 302–319 (1995).
• 32) Gloor M, Peters G, Stoika D. On the resident aerobic bacterial skin flora in unaffected skin of patients with atopic dermatitis and in normal controls. Dermatologica, 164, 258–265 (1982).
• 33) Mikhail GR, Miller-Milinska A. Mast cell population in human skin. J. Invest. Dermatol., 43, 249–254 (1964).
• 34) Mihm MCJ Jr, Soter NA, Dvorak HF, Austen KF. The structure of normal skin and the morphology of atopic eczema. J. Invest. Dermatol., 67, 305–312 (1976).
• 35) Damsgaard TE, Olesen AB, Sørensen FB, Thestrup-Pedersen K, Schiøtz PO. Mast cells and atopic dermatitis. Stereological quantification of mast cells in atopic dermatitis and normal human skin. Arch. Dermatol. Res., 289, 256–260 (1997).