Impact of T-cell receptor Vbeta haplotypes on the development of dermatitis in DS-Nh mice: synergistic production of interleukin-13 caused by staphylococcal enterotoxin C and peptide glycans from Staphylococcus aureus

Abstract

Although the pathogenic role of interleukin-13 (IL-13) is a key for atopic dermatitis (AD), the mechanism of IL-13 production in AD remains unclear. To investigate the role of the T-cell receptor Vbeta (TCR Vbeta) haplotype in the development of dermatitis and the production of IL-13 in the naturally occurring dermatitis model by staphylococcal enterotoxin C (SEC)-producing Staphylococcus aureus, we raised DS-Nh mice harbouring the TCR Vbeta(a) haplotype with a central deletion in the TCRBV gene segments, including TCR Vbeta8S2. Observation and histopathological analysis of the two mouse substrains with spontaneous dermatitis indicated that later onset and weaker severity of AD-like dermatitis were identified in mice with TCR Vbeta(a) compared to those with TCR Vbeta(b). Immunohistochemical examination revealed the infiltration of a large number of CD4-bearing T cells in the skin lesions in mice with TCR Vbeta(b) but not in those with TCR Vbeta(a). Interestingly, much lower levels of serum IL-13 were detected in mice with the TCR Vbeta(a) than in those with the TCR Vbeta(b) haplotype. In vitro, synthetic ligands (Pam(2)CSK4) of toll-like receptor 2 (TLR2) synergistically produced IL-13 with SEC in splenocytes of mice with TCR Vbeta(b) but not of those with TCR Vbeta(a), and natural killer T cells were essential for this synergism. Our findings suggested that this TCR Vbeta-haplotype-dependent synergism with TLR2 plays an important role in the development of AD-like dermatitis in DS-Nh mice.

Figure 3

Serological analysis in both substrains. (a) Cytokine profile in serum from each substrain at 5 and 20 weeks of age. Mice were housed under conventional conditions for 0 and 15 weeks, and mice from 15 weeks onwards developed dermatitis (see Fig. 1). Relative cytokine expression was calculated using the following formula: (level of cytokine in each group of mice) / (level of cytokine for TCR Vβ^b mice at 5 weeks of age). (b, c) Serological conditions with aging, and kinetic levels of IL-13 and IgE. Sera collected from mice kept under conventional conditions at 5, 15 and 20 weeks of age were used for measurement of IL-13 (b) and IgE (c) levels. Results are means ± SD of 10 mice. *P < 0·05 and **P < 0·01 for comparison between the age-matched substrains. ND, not detected so the amount of IgE was estimated as 0.

Figure 1

TCR Vβ haplotypes and clinical features of DS-Nh mice with each haplotype.(a)Detection by PCR of gene segments encoding the TCR Vβ regions of TCR chains from genomic DNA from mouse substrains with either TCR Vβ haplotype. (b) Predicted diagram of genes encoding TCR Vβ segments. Boxes indicate TCR Vβ region-encoding segments or C genes. (c) Clinical features of both substrains kept under specific pathogen-free conditions. Mice did not develop spontaneous dermatitis.

Figure 7

Accumulation of collagen in skin lesions of mice. The skin lesion specimens were sampled from mice at 0, 5, 10, 15 and 20 weeks of age and the concentration of collagen was measured by the specific affinity of dye for collagen. Each value represents the mean ± SD of eight mice. *P < 0·05 and **P < 0·01 for comparison between the age-matched substrains.

Figure 2

Effects of TCR Vβ haplotypes on development of AD-like dermatitis. (a) Incidence of erythema and changes in clinical skin conditions with aging inconventionally housed TCR Vβ^a and TCR Vβ^b mice. Incidence of erythema (% frequency) was calculated using the following formula: [number of mice with erythema/total number of mice (n = 10)] × 100 (upper graph). Total clinical scores for skin conditions were determined from severity of erythema, oedema, dry skin and erosion (lower graph). Results are means ± SD of 10 conventionally housed mice. *P < 0·05 and **P < 0·01 for comparison between age-matched mouse strains. (b)Clinical features of both substrains kept under conventional conditions at 5 and 20 weeks.

Figure 4

Selectivity of TCR Vβ and reactivity of splenocytes to SA. (a) TCRBV repertoires in spleen cells from TCR Vβ^a and TCR Vβ^b mice at 5 weeks of age under specific pathogen-free conditions, which were stimulated in vitro with SAs (SEA, SEB, SEC, and TSST). Blocks show means +2 SD of TCRBV repertoires in PBMC from five mice without in vitro stimulation. Dots show the percentage frequency of TCRBV-bearing T cells in spleen cells stimulated in vitro with each SA. In this study, we assessed the selectivity of TCR Vβ to SA using the following definition: (i) percentage was greater than the mean percentage +2 SD of five control experiments, without stimulation, and (ii) actual percentage obtained in the assay was >10%. (b) Proliferative responses of spleen cells to SEC from TCR Vβ^a and Vβ^b mice at 5 weeks of age under specific pathogen-free conditions. Blocks show means ± SD of five mice.

Figure 5

Cytokine response to synergistic stimulation by SEC and Pam₂CSK4. (a) Sixty-two cytokine responses to synergistic stimulation by SEC and Pam₂CSK4. Using the RayBiotech system (Raybiotech Inc. GA), we identified expression profiles for 62 cytokines using spleen cells from TCR Vβ^a and TCR Vβ^b mice at 5 weeks of age raised under specific pathogen-free conditions. (b) IL-13 production in cultured spleen cells from TCR Vβ^a and TCR Vβ^b mice stimulated with SEC, Pam₂CSK4, or both. Splenocytes from mice with TCR Vβ^b were treated with MACS to eliminate NKT cells. Results are means ± SD of five mice. **P < 0·01 for comparison between age-matched substrains. (c) IL-13 produced in cultured spleen cells from TCR Vβ^b mice stimulated with SAs, Pam₂CSK4, or both. Results are means ± SD of five experiments. **P < 0·01 compared with no-stimulation.

Figure 6

Histological and immunohistological features of facial skin tissues. Frozen sections from conventionally housed mice at 20 weeks of age were stained with anti-CD4 antibodies. Paraffin sections from conventionally housed mice at 5, 15 and 20 weeks were stained with Masson Trichrome (MTC).