The lung cytokine microenvironment influences molecular events in the lymph nodes during Th1 and Th2 respiratory mucosal sensitization to antigen in vivo.

Originally defined by their patterns of cytokine production, Th1 and Th2 cells have been described more recently to express other genes differentially as well, at least in vitro. In this study we compared the expression of Th1- and Th2-associated genes directly during in vivo sensitization to ovalbumin (OVA) in Th1- and Th2-polarized models of airways inflammation. Th1-polarized airway inflammation was achieved by the intranasal instillation of adenoviral vectors (Ad) encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-12, followed by daily aerosolizations of OVA; instillation of Ad/GM-CSF alone with OVA aerosolization led to Th2-polarized responses. Lymph nodes were obtained at various time-points, RNA extracted, and analysed by real-time quantitative polymerase chain reaction (PCR). Consistent with reports from in vitro and human studies, mice undergoing Th1-polarized inflammation showed preferential expression of the transcription factor t-bet, the chemokines IFN-gamma inducible protein (IP)-10 and macrophage inflammatory protein 1 alpha (MIP-1-alpha), and the chemokine receptor CCR5. In contrast, the transcription factor GATA-3, the chemokines I-309 and thymus and activation regulated chemokine (TARC), and the chemokine receptors CCR3 and CCR4 were preferentially expressed in the Th2 model. Importantly, we also show that Ad/transgene expression remains compartmentalized to the lung after intranasal instillation. Flow cytometric analysis of lung myeloid dendritic cells indicated that B7.1 was expressed more strongly in the Th1 model than in the Th2 model. These studies provide a direct comparison of gene expression in in vivo Th1- and Th2-polarized models, and demonstrate that molecular events in the lymph nodes can be altered fundamentally by cytokine expression at distant mucosal sites.

Inhalation of a harmless antigen (ovalbumin) elicits immune activation but divergent immunoglobulin and cytokine activities in mice.

BACKGROUND: Exposure to aerosolized harmless antigen such as ovalbumin (OVA) has previously been shown to induce inhalation tolerance, a state characterized by inhibition of IgE synthesis and airway inflammation, upon secondary immunogenic antigen encounter. Immune events associated with this phenomenon are still poorly understood. OBJECTIVE: The aim of this study was to investigate cellular and molecular mechanisms underlying this state of 'unresponsiveness'. METHODS: After initial repeated OVA exposure, mice were subjected to a protocol of antigen-induced airway inflammation, encompassing two intraperitoneal injections of OVA adsorbed to aluminium hydroxide followed by airway challenge. We assessed immune events in the draining lymph nodes after sensitization, and in the lungs after challenge. RESULTS: In animals initially exposed to OVA, we observed, at the time of sensitization, considerable expansion of T cells, many of which expressed the activation markers CD69 and CD25, as well as increased numbers of antigen-presenting cells, particularly B cells. While these animals produced low levels of IgE, the observed elevated levels of IgG1 signified isotype switching. Splenocytes and lymph node cells from OVA-exposed mice produced low levels of IL-4, IL-5, IL-13 and IFN-gamma, indicating aborted effector function of both T helper (Th)2- and Th1-associated cytokines. Real time quantitative polymerase chain reaction (PCR) (TaqMan) analysis of costimulatory molecules in the lungs after in vivo challenge showed that B7.1, B7.2, CD28 and CTLA-4 mRNA expression was low in animals initially exposed to OVA. Ultimately, these events were associated with abrogated airway inflammation and attenuated airway hyper-responsiveness. The decreased inflammation was antigen-specific and independent of IL-10 or IFN-gamma. CONCLUSION: Initial exposure to OVA establishes a programme that prevents the generation of intact, fully functional inflammatory responses upon secondary antigen encounter. The absence of inflammation, however, is not associated with categorical immune unresponsiveness.

Temporal-spatial analysis of the immune response in a murine model of ovalbumin-induced airways inflammation.

The objective of this study was to define phenotypic changes of antigen-presenting cells (APCs) and T cells in a murine model of antigen-induced airways inflammation that involves intraperitoneal sensitization with ovalbumin (OVA)/adjuvant followed by antigen aerosolization. We investigated the APC and T-cell compartments both after sensitization (primary immune response) and after challenge (secondary immune response) at the thoracic lymph nodes (initiation site) and the lung (effector site). Our findings document a major cellular expansion in the lymph nodes after both sensitization and challenge. After sensitization, this expansion was comprised mainly of B cells, a considerable proportion of which expressed B7.2. At this time, T cells were markedly expanded and activated as assessed by CD69 expression; further, although GATA-3 and signal transducer and activator of transcription-6 were expressed at this time point, expression of interleukin (IL)-4, IL-5, and IL-13 messenger RNA (mRNA) levels were marginal. However, in vitro stimulation of lymph-node cells with OVA led to cytokine production. In contrast, 24 h after challenge, but not after sensitization, there was a major expansion of dendritic cells and macrophages in the lungs. This expansion was associated with enhanced expression of both B7.1 and B7.2. We also observed expansion of activated CD3(+)/CD4(+) T cells expressing the T helper-2-associated marker T1/ST2 in the lung, most notably 5 d after challenge. Further, IL-4, IL-5, and IL-13, but not interferon-gamma mRNA were expressed at high levels 3 h after challenge. This study helps to elucidate the "geography" of immune responses generated in a conventional murine model of allergic airways inflammation.

Generation of experimental allergic airways inflammation in the absence of draining lymph nodes.

The objective of this study was to investigate the contribution of secondary lymphoid organs in the generation and maintenance of experimental allergic airway inflammation. We employed a previously reported murine model of respiratory mucosal allergic sensitization, induced by repeated aerosolizations of ovalbumin in the context of a GM-CSF airway environment. We executed this protocol in wild-type (WT) and lymphotoxin-alpha-deficient mice (LTalpha-KO) mice, which are devoid of lymph nodes (LNs) and possess rudimentary spleen structures. Despite the lack of pulmonary LNs draining the airway compartment, LTalpha-KO mice were fully capable of mounting a robust inflammatory response in the airways, consisting of Th2 polarized CD4+ T cells and eosinophils. This was accompanied by IL-5, IL-13, and IFN-gamma production by splenocytes and generation of ovalbumin-specific serum IgE. Exposure to the same antigen 7 weeks after complete resolution of airway inflammation once again induced a Th2 polarized infiltrate, demonstrating intact immunological memory. To investigate inherent plasticity in establishing antigen-specific immunity, mice were splenectomized before sensitization. Allergic sensitization was completely abrogated in splenectomized LTalpha-KO mice, compared with eusplenic LTalpha-KO controls. These data demonstrate that secondary lymphoid organs, either LN or spleen, are essential for the generation of allergic airway responses.

Determinants of the immune-inflammatory response in allergic airway inflammation: overview of antigen presentation and cellular activation.

Under normal circumstances the lung is in a state of immunologic homeostasis, a condition in which exposure to innocuous antigens does not lead to immune-inflammatory responses. This is the only reasonable solution to the dilemma faced by the lung: The need to interact with the external environment and the need to avoid responding to most of the environmental antigens to which it is exposed. In allergic diseases, such as asthma, this homeostasis is undermined, and immuneinflammatory responses to harmless aeroallergens are activated. We describe the changes in antigen presentation and cellular activation observed in a model of allergic airway inflammation. Further, we present a summary of our work that investigated the impact of the airway cytokine microenvironment on the development of immune responses in the respiratory tract.

Interleukin-10 gene transfer to the airway regulates allergic mucosal sensitization in mice.

The objective of this study was to investigate the effect of airway gene transfer of interleukin (IL)-10, a cytokine with potent anti-inflammatory and immunoregulatory activities, on allergic mucosal sensitization. We used a recently described murine model that involves repeated exposures to aerosolized ovalbumin (OVA), daily for 10 d, in the context of granulocyte macrophage colony-stimulating factor (GM-CSF) expression in the airway environment achieved by intranasal delivery of a replication-deficient adenovirus carrying the GM-CSF transgene. The resulting inflammatory response was characterized by a T-helper 2 cytokine profile and marked airway eosinophilia. After complete resolution of the inflammatory response (Day 28), a single exposure to OVA reconstituted airway eosinophilia and induced airway hyperresponsiveness. We show that concurrent expression of IL-10 inhibited GM-CSF-driven OVA-specific inflammation in a dose-dependent manner. Specifically, IL-10 decreased the number of mononuclear cells, neutrophils, and eosinophils in the bronchoalveolar lavage fluid (BALF). Histologic evaluation of the tissue corroborated the findings in the BALF. Concurrent expression of IL-10 at the time of mucosal sensitization abrogated both the cellular and physiologic recall responses in vivo. Studies in interferon (IFN)-gamma knockout mice demonstrated that prevention of airway eosinophilia by IL-10 was IFN-gamma-independent and that expression of IL-10 was associated with decreased levels of IL-4, IL-5, and tumor necrosis factor-alpha in the BALF. Flow cytometric analysis of dispersed lung cells showed that expression of IL-10 in the airway reduced the absolute number of Class II major histocompatibility complex (MHC)(+)/CD11c(+) (dendritic cells) and Class II MHC(+)/Mac-1(bright) (macrophages) cells expressing the costimulatory molecules B7.1 and B7.2 by 30%. However, IL-10 coexpression did not prevent expansion of CD4 and CD8 T cells or expression of the early activation marker CD69 on T cells. Thus, airway gene transfer of IL-10 altered the immune response to OVA in a way that resulted in inhibition of airway inflammation. These findings suggest that development of an immunoregulatory strategy based on IL-10, alone or in combination with GM-CSF, warrants further consideration.

GM-CSF transgene expression in the airway allows aerosolized ovalbumin to induce allergic sensitization in mice.

The purpose of this study was to explore whether repeated exposure to aerosolized ovalbumin (OVA) in the context of local expression of GM-CSF can initiate a Th2-driven, eosinophilic inflammation in the airways. On day -1, Balb/c mice were infected intranasally with an adenovirus construct expressing GM-CSF (Ad/GM-CSF). From day 0 to day 9 mice were exposed daily to an OVA aerosol. Mice exposed to OVA alone did not show any evidence of airway inflammation. Mice receiving both Ad/GM-CSF and aerosolized OVA exhibited marked airway inflammation characterized by eosinophilia and goblet cell hyperplasia. Migration of eosinophils into the airway was preceded by a rise in IL-5 and IL-4. Both IL-5 and class II MHC were critically required to generate airway eosinophilia. After resolution, airway eosinophilia was reconstituted after a single OVA exposure. Flow cytometric analysis of dispersed lung cells revealed an increase in macrophages and dendritic cells expressing B7.1 and B7.2, and expansion of activated (CD69-expressing) CD4 and CD8 T cells in mice exposed to OVA and Ad/GM-CSF. Our data indicate that expression of GM-CSF in the airway compartment increases local antigen presentation capacity, and concomitantly facilitates the development of an antigen-specific, eosinophilic inflammatory response to an otherwise innocuous antigen.