Antigen-driven bystander effect accelerates epicutaneous sensitization with a new protein allergen

Abstract

Exposure to protein allergen epicutaneously, inducing a Th2-dominant immune response, sensitizes the host to the development of atopic disease. Antigen-driven bystander effect demonstrates that polarized T cells could instruct naïve T cells to differentiate into T cells with similar phenotype. In this study, we aimed to determine the contribution of antigen-driven bystander effect on epicutaneous sensitization with a newly introduced protein allergen. BALB/c mice were immunized intraperitoneally with BSA emulsified in alum, known to induce a Th2 response, three weeks before given BSA and OVA epicutaneously. Lymph node cells from these mice restimulated with OVA secreted higher levels IL-4, IL-5 and IL-13 as compared with cells from mice without BSA immunization. In addition, BALB/c mice immunized subcutaneously with BSA emulsified in complete Freund's adjuvant, known to induce a Th1-predominant response, also induced higher Th1 as well as Th2 cytokine response when restimulated with OVA as compared with mice without immunization. We demonstrated that subcutaneous immunization with BSA in CFA induced Th2 as well as Th1 response. The threshold of epicutaneous sensitization to OVA was also reduced, possibly due to increased expressions of IL-4 and IL-10 in the draining lymph nodes during the early phase of sensitization. In conclusion, antigen-driven bystander effect, whether it is of Th1- or Th2-predominant nature, can accelerate epicutaneous sensitization by a newly introduced protein allergen. These results provide a possible explanation for mono- to poly-sensitization spread commonly observed in atopic children.

Figure 1

Antigen-driven bystander effect by Th2 pretreatment enhanced epicutaneously induced Th2 immune response to a new protein antigen. Groups of mice (four per group) were non-pretreated (PBS, no), or intraperitoneally (ip) pretreated with BSA and alum adjuvant on day -21. They were all epicutaneously sensitized with BSA and OVA (no, ip) or PBS (PBS) on day 1–5. On day 11, draining LNs were obtained (A). On day 22, spleens were taken (B). In vitro reactivation culture with OVA was performed. Cytokine contents of 48 h-supernatants were measured by ELISA. Results were shown as mean ± SEM. Four independent experiments were performed with similar results. One representative results are shown.

Figure 2

Antigen-driven bystander effect by Th1 pretreatment also enhanced epicutaneously induced Th2 immune response to a new protein antigen. (A) (B) (C) Group of mice (Four per group) were non-pretreated (PBS, no) or subcutaneously (sc) pretreated with BSA and CFA adjuvant on day -21. They were all epicutaneously sensitized with BSA and OVA (no, sc) or PBS (PBS) on day 1–5. On day 11, draining LNs were obtained (A). On day 22 spleens were taken (B). In vitro reactivation culture with OVA was performed. Cytokines contents of 48 h-supernatants were measured by ELISA. Results were shown as mean ± SEM. (C) On day 22~26, all mice received another course of OVA patch application. On day 29, blood were collected and serum OVA-specific IgG2a and IgE were determined by ELISA. Three independent experiments were performed with similar results. One representative results are shown. (D) Groups of mice (four per group) were immunized by subcutaneous injection with BSA and CFA adjuvant (sc) or BSA and alum adjuvants (ip) three weeks before their spleens were obtained. In vitro reactivation culture of spleen cells with BSA was performed. Cytokine contents of 48 h-supernatants were measured by ELISA. Results were shown as mean ± SEM. Three independent experiments were performed with similar results. One representative results are shown.

Figure 3

Influence of antigen-driven bystander effect on the threshold of epicutaneous sensitization with a new protein allergen. (A) Groups of mice (four per group) were similarly pretreated and immunized as in Fig. 1 (A). On day 11, draining LNs were obtained and OVA-specific proliferation assay was performed. (B) Groups of mice (four per group) were subcutaneously pretreated with BSA (sc) or not (no) on day -21. They received patch co-administration of serial dilutions of OVA (100, 10, 1 mg/ml) and BSA (100 mg/ml) on day 1–5. On day 11, draining LNs were obtained and OVA-specific proliferation assay was performed. (C) Groups of mice (four per group) were non-pretreated (no) or subcutaneously (sc) pretreated on day -21. One day before patch application, all mice received i.v. transfer of CFSE-labeled CD4 T cells prepared from DO11.10 transgenic mice. Patch application with serial dilutions of OVA (100, 10, 1 mg/ml) and BSA (100 mg/ml) were performed on day 1–3. Regional LNs were obtained on day 4 for staining and flow cytometric analysis. All experiments were performed independently for at least three times. One representative results are shown.

Figure 4

The role of cytokine in the epicutaneously induced antigen-driven bystander effect. Groups of mice (three per group) were non-pretreated (no) or pretreated subcutaneously with BSA and CFA adjuvant (sc) three weeks before they received patch sensitization with OVA and BSA on day 1–3. Draining LNs were obtained 24, 48 and 72 hr after start of patch sensitization. Total RNA extraction, cDNA preparation and quantitative real-time PCR were performed individually. The relative cytokine mRNA expression levels of each sample were normalized according to its β-actin expression. Results are shown as mean ± SEM. Three independent experiments were performed with similar results. One representative results are shown.