Antigen-driven bystander effect accelerates epicutaneous sensitization with a new protein allergen
© Wang et al; licensee BioMed Central Ltd. 2009
Received: 22 December 2008
Accepted: 06 March 2009
Published: 06 March 2009
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.
In the past several decades, there has been a progressive increase in the prevalence of atopic disease and an associated increase in the cost of medical management . Atopic diseases are manifested as atopic dermatitis (AD), asthma, and allergic rhinitis. Atopy is associated with the expression of allergen-specific immune response characterized by the production of Th2 cytokines such as IL-4, IL-5, and IL-13; elevated IgE production; and eosinophilia. In contrast, non-atopic individuals display predominant Th1 immune response characterized by the production of IFN-γ. Genetics predisposition and exposure to various environmental allergens, which result in a Th2 immune response, contribute to the pathogenesis of atopic diseases .
AD is often the first clinical manifestation of an atopic triad and is the beginning of the "atopic march" . However, the route of sensitization by an allergen that results in AD is still unclear. There is compelling evidence to show that epicutaneous exposure to a protein allergen is one of the important routes to sensitize the host for AD and other atopic diseases . By the presence of cutaneous lymphocyte-associated antigen (CLA)-positive T cells, it is shown in humans, that T cells are primed or reactivated in the skin or its draining lymph nodes (LNs) [5, 6]. In AD patients, the increased frequency of prior activation and secretion of type 2 cytokines, as well as higher proliferation response to allergen, are largely confined to a CLA-positive subset [7–9]. Moreover, T cell receptor skewing is only detectable within the CLA-positive subset of T cells from those subjects from whom superantigen-secreting, skin-dwelling bacteria could be identified . The recent demonstration of the expression of CLA by CD8 T cells specific for a skin-tropic, but not non-skin-tropic, virus further supports this concept . We and others have demonstrated in an atopic dermatitis animal model, that epicutaneous exposure of protein antigen induces a predominant Th2 response with high IgE production [12, 13]. Furthermore, epicutaneous sensitization with protein antigen induces AD-like skin lesions and development of asthma . The epicutaneously-induced Th2 response requires IL-10 and IL-13 [15, 16], while down-regulation of the response is mediated by C3a, cyclooxygenase-2, and skin scratching [17–19].
It has been demonstrated that through an antigen-driven bystander effect, polarized T cells instruct naïve T cells to differentiate into T cells with a similar phenotype. This bystander effect is observed only when the challenge inoculum contains both the original antigen and a newly introduced antigen. This effect was initially shown to be a mechanism of antigen-driven peripheral tolerance after oral administration of antigens, and it can protect rats from developing experimental autoimmune encephalomyelitis . Adoptive co-transfer of two populations of T-cell receptor transgenic T cells of different specificities demonstrated that polarized Th1 or Th2 effector cells can instruct naïve T cells to differentiate into Th1 or Th2 cells, respectively . Subsequently, study of a murine asthma model revealed that an ongoing Th2 response can induce antigen-specific Th2 response to a new antigen, a process termed "collateral priming" . Further exploration of the underlying mechanisms of the antigen-driven bystander effect has demonstrated that conversion of a naïve T cell occurs only when it interacts with the same APC as the memory T cell, and that the orally immunized memory T cells use IL-4 and IL-10 to "educate" APCs, which in turn induce naïve T cells to produce the same cytokines as those produced by the orally immunized memory T cells . In this present study, we demonstrated that antigen-driven bystander effect, despite its Th1- or Th2-predominant nature, accelerates epicutaneous sensitization with a new protein antigen.
Materials and methods
Mice and reagents
Six to 10-week-old female BALB/c and TCR-OVA-DO11.10 mice were purchased from the animal center of National Taiwan University Collage of Medicine and kept in a specific pathogen-free environment. All animal experiments were approved by the animal care committee of the Medical College of National Taiwan University. OVA (Grade V), BSA, CFA, and 4-nitrophenyl phosphate (pNPP) were purchased from Sigma-Aldrich (St. Louis, MO). Alum adjuvant was purchased from Pierce (Rockford, IL) and carboxyfluorescein succinimidyl ester (CFSE) was obtained from Invitrogen (Carlsbad, CA). Capture and biotin-conjugated detecting antibodies for IFN-γ, IL-4, IL-5 used in the ELISA were from PharMingen (San Diego, CA). Streptavidine-alkaline phosphatase was purchased from Southern Biotechnology (Birmingham, AL). The murine IL-13 ELISA kit purchased from R&D systems (Minneapolis, MN) was used for determination of the IL-13 content of supernatants.
Mice were sensitized as previously described . Briefly, 20 μl of OVA (100 mg/ml or serial dilutions) and 20 μl of BSA (100 mg/ml) were placed on the disc of a Finn chamber (Epitest, Tuusula, Finland). This was then applied to an area of shaved skin on the back of a mouse. For each course of sensitization, freshly prepared patches were applied daily from days 1 to 5. For pretreatment, mice received a subcutaneous (s.c.) injection of 100 μg BSA emulsified in CFA over the bilateral side of tail-base or an intraperitoneal (i.p.) injection of 100 μg BSA in alum adjuvant three weeks before sensitization.
For adoptive transfer of OVA-TCR CD4 T cells, spleen cells from DO.11.10 mice were positively selected for CD4 T cells using CD4 microbeads. Then, 107 CD4 T cells/ml were incubated with 1 μM CFSE for 10 min at 37°C. Prewarmed FCS-containing PBS was added and washed by cold PBS. Labeled OVA-TCR CD4 T cells (5 × 106) were intravenously injected into BALB/c recipients 24 h before sensitization.
LN and spleen cell cytokine production and proliferation assay
Ten days after the start of a sensitization course, mice were sacrificed to obtain axillary, subscapular, and inguinal LNs. Pooled LN cells (1 × 106) were cultured in the presence or absence of 100 μg/ml OVA. Supernatants were harvested 48 h later and stored at -80°C. IFN-γ, IL-4, IL-5, and IL-13 content of supernatants was each measured by a standard sandwich ELISA. The limit of detection for IL-4, IL-5, and IL-13 were all 10 pg/ml, whereas that for IFN-γ was 50 pg/ml. For spleen cells, spleens were harvested 3 weeks after sensitization, and pooled spleen cells were stimulated with 100 μg/ml OVA. For the proliferation assay, graded doses of OVA were added. 48 h after the initiation of culture, [3H] thymidine was added and the cells were harvested 18 h later.
Total RNA extraction, cDNA preparation and quantitative real-time PCR
The patched skin and draining lymph node samples were obtained 24, 48, and 72 h after patch co-administration of BSA and OVA. They were frozen with liquid nitrogen and soaked in 1 ml TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). After homogenization, the total RNA was extracted, cDNA was synthesized, and quantitative real-time PCR was performed according to the manufacturer's instructions. Each sample was analyzed in triplicate. The relative cytokine mRNA expression level of each sample was normalized according to its β-actin expression.
Antigen-driven bystander effect enhances epicutaneously-induced Th2 immune response to a co-administered new protein allergen
Antigen-driven bystander effect decreases the threshold of epicutaneous sensitization with a new protein allergen
The role of cytokines in epicutaneously induced antigen-driven bystander effect
The data presented here clearly demonstrate that the antigen-driven bystander effect, despite its Th1- or Th2-predominant nature, accelerates epicutaneous sensitization with a new protein allergen by enhancing the Th2 immune response and by lowering the sensitization threshold. To our knowledge, this is the first report to address the contribution of antigen-driven bystander effect to the epicutaneous sensitization of protein allergens in atopic disease.
The continuation of fetal allergen-specific Th2 responses associated with decreased capacity to produce Th1 cytokine IFN-γ is a defining feature of atopic disease in infancy . After infancy, the major sensitized allergens shift from oral food allergens to aeroallergens . Several observations are consistent with the view that some factors might modulate the susceptibility to allergen sensitization. Firstly, the dose-response relationship between allergen exposure and sensitization differs depending on allergens and locales . Secondly, once sensitized to one allergen, atopic patients are more likely to become sensitized to other allergens . Moreover, there are strong associations of multiple sensitizations both within and between different allergen classes . Skin is continuously exposed to many kinds of allergens and microorganisms in the environment. Microorganisms can gain access to the human body through different routes and sensitize the immune system before contact with the skin. Therefore, it is very likely that the antigen-driven bystander effect affects our skin constantly, although its effect may vary. Thus, the present study provides a possible mechanistic explanation to the clinical observation of the spread of Th2 responses from mono-allergen to multiple allergens in atopic individuals. Another clinical implication of our data is that our results do not conflict with "hygiene hypothesis". The "hygiene hypothesis" predicts that nonspecific suppression of Th2 immune response by a predominant systemic Th1 milieu at the time of sensitization prevents the development of allergy. However, our present study demonstrated specific, but not non-specific, promotion of Th2 immune responses by antigen-driven bystander effect. Collectively, our results support the wisdom of recognizing allergen sensitization early in life and taking precautions to prevent exposure of skins to these allergens.
The mechanism underlying the antigen-driven bystander effect is still obscure. Alpan et al. suggests that orally induced memory T cells utilize IL-4 and IL-10 they produced, but not CD40 ligand, to educate DCs which in turn induce conversion of naïve T cells . However, since co-culturing DCs with naïve T cells plus various concentrations of IL-4 and IL-10 does not simulate the situation in which DC was educated by orally induced memory T cells in their studies, the authors suggested that there must be other factors involved . Our current study provides more information about the underlying mechanism of antigen-driven bystander effect. As we demonstrated in this report, systemic immunization plus skin sensitization to protein antigens induce increased cytokine expression in the draining LNs, but not in the patched skin. This observation supports the notion that the draining LNs are the site where antigen-driven bystander effect takes place .
In summary, in this study we demonstrated that antigen-driven bystander effect contributes to accelerated epicutaneous sensitization with a new protein allergen. These results provide a possible explanation for a mono- to poly-sensitization spread commonly observed in atopic children.
cutaneous lymphocyte-associated antigen.
We thank Dr. Betty A. Wu-Hsieh for helpful advice and careful review of the manuscript. This work was supported by grants from National Taiwan University Hospital (94N049, 94A18-4 and 95A21-4) and from National Health Research Institutes (NHRI-EX95-9516SC).
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