2015 Volume 38 Issue 2 Pages 317-320
Patients with atopic dermatitis (AD) show increased numbers of Th2 cells in their acute skin lesions and superficial skin colonization by Staphylococcus aureus. The purpose of this study was to clarify the effect of S. aureus cell wall components on Th2 chemokine production by murine Langerhans cells (LCs). Murine LCs were stimulated with peptidoglycan (PEG) and/or muramyldipeptide (MDP) for 24 or 48 h, and Th1 and Th2 chemokine production was investigated by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). PEG-stimulation of LCs induced production of the Th2 chemokine CCL17 and this was enhanced in the presence of interleukin (IL)-4. A low-molecular weight PEG fragment, MDP, did not induce CCL17 production by LCs. However, when LCs were stimulated with PEG in combination with MDP, PEG-induced CCL17 production was synergistically enhanced by MDP. Furthermore, PEG- and MDP-induced CCL17 production was enhanced to a greater extent in the presence of IL-4. These results suggest that S. aureus colonization in AD patients may enhance the Th2-prone immune response through upregulation of CCL17 production by LCs, which would occur as a result of simultaneous stimulation with PEG and MDP from S. aureus in a Th2 environment.
The majority of patients with atopic dermatitis (AD) show increased expression of Th2 cytokines such as interleukin (IL)-4 and IL-5 in their acute skin lesions and superficial skin colonization by Staphylococcus aureus.1) S. aureus can be isolated from 96–100% of AD skin lesions, whereas skin colonization by this organism is evident in only 0–10% of healthy individuals.2,3) We have also found previously that the rate of detection of S. aureus in the lesioned skin of AD patients is higher than in non-lesioned skin.3) Furthermore, the S. aureus bacterial cell count in the lesioned skin of AD patients is also significantly higher than that in non-lesioned skin. Many attempts have been made to characterize the role of S. aureus in the skin of AD patients, and most studies have focused on the role of staphylococcal exotoxins.4) However, half of the S. aureus strains isolated from AD patients are not capable of producing superantigens,3,5,6) and also there is no significant difference in the rate of detection of S. aureus producing superantigenic exotoxin between lesioned and non-lesioned skin in AD patients.3) Therefore, staphylococcal exotoxins do not fully explain the role of S. aureus in AD.
The cell walls of Gram-positive bacteria are composed of highly cross-linked peptidoglycan (PEG), which is thought to be the major inflammatory product. Therefore, we hypothesized that PEG from S. aureus may play a more important pathogenic role than superantigenic exotoxins in AD. In previous studies, we found that PEG was able to induce IL-5 production by peripheral blood mononuclear cells (PBMCs) from patients with AD,7) and that percutaneous invasion of PEG induced Th2 cell infiltration in the dermis through production of the Th2 chemokine, CCL17, by Langerhans cells (LCs).8) However, the role of muramyldipeptide (MDP; N-acetylmuramyl-L-alanyl-D-isoglutamine), which represents the minimal bioactive structure of PEG, on Th2 chemokine production by LCs is not clear. In the present study, therefore, we investigated the influence of MDP on PEG-induced Th2 chemokine production by LCs and the influence of the Th2 cytokine, IL-4, on this process.
PEG derived from S. aureus was obtained from Fluka (Buchs SG, Switzerland), reconstituted in phosphate-buffered saline (PBS), pH 7.4, at a concentration of 1 mg/mL, and sonicated for 1 h before use. MDP was purchased from EMD Biosciences (La Jolla, CA, U.S.A.) and reconstituted in PBS, pH 7.4, at a concentration of 1 mg/mL.
MiceFemale specific-pathogen-free BALB/c mice were obtained from Japan SLC (Hamamatsu, Japan) and used at the age of 6 to 8 weeks. They were housed in plastic cages with sterilized paper bedding in a clean, air-conditioned room at 24°C and allowed free access to a standard laboratory diet and water. All procedures performed on the mice were in accordance with the Guidelines of the Animal Care and Use Committee of Meiji Pharmaceutical University, Tokyo.
Analysis of Chemokine Production by LCsLCs in the epidermis were separated from mouse skin as described previously,8) and adjusted to 5×105 cells/mL in RPMI 1640 medium with L-glutamine (Sigma, St. Louis, MO, U.S.A.) containing 10% fetal bovine serum (Sigma), 25 mM Hepes (Sigma), 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco RBL, Grand Island, NY, U.S.A.) (RPMI 10). They were then stimulated with 10 µg/mL PEG and/or MDP, which approximates the likely concentration in vivo and was used as the optimal concentration for in vitro experiments, in the presence or absence of 200 ng/mL IL-4 (Peprotech, Rocky Hill, NJ, U.S.A.) at 37°C in a humidified atmosphere with 5% CO2. In order to determine the levels of expression of mRNAs for Th1- and Th2-type chemokines in LCs, mRNA was extracted from LCs after incubation for 24 h, and then the corresponding cDNA was synthesized as described previously.8) Polymerase chain reaction (PCR) was performed using a GeneAmp PCR System 9700 (Perkin-Elmer, Norwalk, CT, U.S.A.) in a 25-µL reaction volume containing 1.5 µL of cDNA (corresponding to 16 ng mRNA as a starting material) and the PCR products were separated on a 2% agarose gel containing ethidium bromide. The primers used for amplification of β-actin,8) CXCL9,9) CXCL10,10) CCL178) and CCL228) have been described elsewhere. The culture supernatants were collected after incubation for 48 h, and the chemokine concentrations were measured using enzyme-linked immunosorbent assay (ELISA) kits for quantification of murine CXCL10 and CCL17 (R & D Systems, Minneapolis, MN, U.S.A.).
Statistical AnalysisThe data were expressed as means (±S.D.), and differences between means were analyzed using Student’s t-test with a two-tailed test of significance. Differences at p<0.05 were considered to be statistically significant.
CXCL9 and CXCL10 are Th1-type chemokines that are structurally related and share the common receptor CXCR3.11) On the other hand, CCL17 and CCL22 are Th2-type chemokines that share the receptor CCR4. Initial experiments designed to examine the influence of IL-4 on PEG-induced Th1-type and Th2-type chemokine production, respectively, used murine epidermal LCs. The LCs were treated with 10 µg/mL PEG, which signals through TLR-2,12,13) in the presence or absence of IL-4, and subsequently, reverse transcription (RT)-PCR analysis was performed to investigate whether a Th2-environment influences the expression of mRNAs for Th2 chemokines in LCs. After 24 h of culture in the presence of PEG, LCs began to express mRNA for CXCL10 and CCL17, but not that for CXCL9 and CCL22 (Fig. 1A). Furthermore, the levels of expression of CCL17 mRNA, but not CXCL10 mRNA, were augmented by the presence of IL-4 in the culture. To determine whether CXCL10 and CCL17 were produced at the protein level by cultured LCs and secreted into the culture medium, ELISA was carried out using medium cultured for 48 h. As shown in Figs. 1B, C, PEG-stimulated LCs produced significant levels of CXCL10 and CCL17, and IL-4 augmented the level of CCL17 production but not CXCL10 production. These results indicate that the effects of IL-4 are more specific to Th2-type chemokine production.
LCs (5×105/mL) from mouse epidermis were incubated with 10 µg/mL PEG in the presence or absence of 200 ng/mL IL-4. (A) After 24 h of incubation, cytoplasmic mRNA was extracted from LCs, reverse-transcribed and amplified by PCR using primer sets for β-actin, CXCL9, CXCL10, CCL17 and CCL22. The data shown are representative results of four independent experiments. (B) After 48 h of incubation, culture supernatants were assayed for CXCL10 production using ELISA. The results are expressed as means±S.D. (n=5). (C) After 48 h of incubation, culture supernatants were assayed for CCL17 production using ELISA. The results are expressed as means±S.D. (n=5).
To examine whether CXCL10 production and CCL17 production induced by PEG-stimulated LCs are influenced by coexisting MDP, LCs were stimulated with 10 µg/mL PEG and 10 µg/mL MDP. As shown in Fig. 2, MDP-stimulated LCs did not induce significant levels of CXCL10 production and CCL17 production like those seen in PEG-stimulated LCs. However, production of both CXCL10 and CCL17 by PEG-stimulated LCs was significantly enhanced in combination with MDP. In addition, CCL17 production, but not CXCL10 production, by LCs simultaneously stimulated with PEG and MDP was significantly enhanced by addition of IL-4 to the culture medium (Fig. 3). Furthermore, the level of IL-4-dependent enhancement of CCL17 production by LCs treated with PEG and MDP was superior to that for LCs treated with PEG and IL-4, although the presence of IL-4 in the culture medium did not induce CCL17 production by MDP-stimulated LCs.
LCs (5×105/mL) from mouse epidermis were incubated with 10 µg/mL PEG and/or 10 µg/mL MDP. After 48 h of incubation, culture supernatants were assayed for CXCL10 production and CCL17 production using ELISA. The results are expressed as means±S.D. (n=5).
LCs (5×105/mL) from mouse epidermis were incubated with 10 µg/mL PEG and 10 µg/mL MDP in the presence or absence of 200 ng/mL IL-4. After 48 h of incubation, culture supernatants were assayed for CXCL10 production and CCL17 production using ELISA. The results are expressed as means±S.D. (n=5).
Chemokines are involved in recruitment of inflammatory cells to the site of an immune reaction. Our results demonstrated that PEG augmented production of CXCL10 and CCL17 by LCs and that this effect was further enhanced in combination with MDP. Furthermore, we found that the Th2 cytokine, IL-4, augmented production of the Th2-type chemokine, CCL17, but not that of the Th1-type chemokine, CXCL10, by LCs stimulated with PEG alone or in combination with MDP. A Th2-type response is observed in the acute-phase of AD and is associated with induction of the early allergic reaction.1) Therefore, topical improvement of the Th2-environment might be effective for relief of the acute-phase inflammatory reaction in AD patients with S. aureus colonization. Although it is well known that MDP is a nucleotide-binding oligomerization domain-2 (Nod-2) agonist,14) MDP itself induced neither CXCL10 production nor CCL17 production. This would probably have been due to the unresponsiveness of MDP to TNF-α production.15) However, since PEG itself induces TNF-α production, which in turn enhances Nod-2 expression, TLR-2 stimulation by PEG and Nod-2 stimulation by MDP would exert a synergistic effect to induce production of CXCL10 and CCL17 from LCs.8,15,16) We must prove that this hypothesis is right in future. Furthermore, it has been reported that simultaneous stimulation with TNF-α and IL-4 enhances the production of CCL17, whereas IL-4 alone does not induce CCL17 production.17) These observations would explain the synergistic effect of PEG and IL-4 on CCL17 production. However, the mechanisms underlying the absence of an IL-4 effect on PEG-induced CXCL10 production remain largely elusive.
Since S. epidermidis-derived PEG also had similar effects to CCL17 production (data not shown), it was thought that effects of PEG observed in this study was not unique to S. aureus-derived PEG. However, the number of Gram-positive bacteria except S. aureus in the lesioned skin of AD patients is very few and therefore its influence would be negligible. As the density of S. aureus in the skin lesions of AD patients exceeds 1×107 organisms/cm2, it seems that 10 µg/mL PEG and MDP used for in vitro stimulation would roughly correspond to the concentration in vivo.2) Therefore, sustained S. aureus colonization in AD patients might cause epidermal LCs to elicit a Th2-prone immune response to PEG and MDP, which are common cell components of superantigenic exotoxin-producing and -non-producing S. aureus strains. As the skin of most AD patients shows superficial S. aureus colonization and barrier disruption due to reduced levels of filaggrin,18) it is likely that PEG and MDP would penetrate continuously into the skin and might play a critical role in perpetuating skin tissue inflammation through Th2 cells infiltration.8) Therefore, in at least a subgroup of AD patients, irrespective of whether they show clinical signs of superinfection, a combination of antimicrobial treatment and anti-Th2 immune response medication might provide a new therapeutic strategy for AD.
The authors declare no conflict of interest.
