INTRODUCTION

TOP ABSTRACT INTRODUCTION CONCLUSION REFERENCES

Although asthma is apparently restricted to the human species, animal models can be used to investigate particular aspects of this human disease. Chronic airway inflammation is thought to play a major role in the pathogenesis of asthma. Bronchial biopsies and bronchoalveolar lavage (BAL) fluid recovered from patients with asthma have been reported to contain an increased number of activated T lymphocytes, eosinophils, and mast cells (1). Many patients with asthma are sensitized and have specific immunoglobulin E (IgE) antibodies to one or more inhalant allergens. Atopy is, indeed, considered to be a major risk factor for the development of asthma. However, the pathological and immunopathological findings in biopsies and BAL from asthmatic patients with or without atopy are very similar and demonstrate the presence of a T helper 2 (Th2)-lymphocyte-dominated chronic airway inflammation with the presence of an increased number of cells producing interleukin (IL)-4 and IL-5 (2, 3). The chronic airway inflammation in asthma is thought to be responsible for the increased airway responsiveness that is so characteristic of the disease. This hypothesis is based on the finding of a significant relationship between airway inflammation and airway hyperresponsiveness, as well as the observation that treatment with inhaled glucocorticosteroids decreases both airway inflammation and airway hyperresponsiveness.

We have developed a murine in vivo model of allergic airway inflammation characterized by the presence of IgE antibodies to an inhaled antigen, peribronchial infiltrates with an increased number of eosinophils, and increased airway responsiveness to nonantigenic bronchoconstrictor stimuli (4). In this animal model we have investigated the role of different cytokines in the development of IgE antibodies to inhaled antigen, eosinophilic airway inflammation, and airway hyperresponsiveness. The studies were performed by using knockout mice or by exogenous administration of cytokines or cytokine antagonists.

Mouse Model of Asthma

The C57 Black 6 (C57Bl/6) mice were actively sensitized on Day 0 by intraperitoneal injection of 10 µg of ovalbumin adsorbed to 1 mg of alum and from Day 14 to 21 exposed daily to aerosolized ovalbumin over a 30-min period. On Day 22, airway inflammation, characterized by the presence of peribronchial and peribronchiolar mixed cellular infiltrates and consisting mainly of mononuclear cells and eosinophils, could be demonstrated. This was reflected by an increase in the number of eosinophils in BAL fluid recovered from these animals. Furthermore, this inflammatory response was accompanied by an increase in airway responsiveness to carbachol. Ovalbumin-specific IgE antibodies could be demonstrated in the serum of the sensitized and exposed animals.

Role of Interleukin-4

Interleukin-4 has a variety of effects that suggest that this cytokine plays a major role in the pathogenesis of asthma. It promotes isotype switching of B cells toward IgE synthesis, stimulates proliferation of Th2 cells, and suppresses interferon-gamma (IFN-) production by Th1 cells. The potential importance of IL-4 in inducing allergic airway inflammation has been addressed in IL-4 knockout mice. In these mice, the sensitization and exposure procedure outlined above did not induce bronchopulmonary eosinophilia as it did in their wild-type littermates (4). Similarly, whereas active sensitization induced synthesis of ovalbumin-specific IgE that was enhanced by repeated exposure to aerosolized ovalbumin in wild-type animals, no specific IgE was found in similarly treated IL-4 knockout mice. Finally, repeated exposure to ovalbumin did not induce airway hyperresponsiveness in actively sensitized IL-4 knockout mice when compared to a saline-exposed control group (5). Additional experiments showed that major histocompatibility complex (MHC) class II-deficient mice lacking functionally active T cells developed neither allergic airway inflammation nor IgE antibodies, suggesting that the crucial role of IL-4 lies in its effect on Th2 cell development (4). This hypothesis was confirmed by Coyle and coworkers (6), again in a murine model of allergen-induced airway inflammation. They demonstrated that the development of airway inflammation in this model was accompanied by the presence of Th2 cells in the airways (6). In IL-4 knockout mice, T cells recovered from the airways did not synthesize a Th2 cytokine pattern, which correlated with the absence of inflammatory airway changes. They also showed that when wild-type animals were treated with anti-IL-4 during the exposure to aerosolized ovalbumin but not during the sensitization process, the influx of eosinophils to the airways could not be inhibited. This finding again points toward a role of IL-4 in the initial Th2 cell development. Additional evidence of the role of IL-4 in mediating the eosinophilic response to antigen sensitization and challenge comes from studies of Stat6 knockout mice. A major signaling pathway involved in many cytokine-mediated responses is based on tyrosine phosphorylation of signal transducers and activators of transcription (Stat). Stat6 is essential for mediating the biological responses to IL-4 (7, 8).

Role of Interleukin-5

In vitro data indicate that several cytokines, including IL-3 granulocyte/macrophage colony-stimulating factor (GMCSF), and IL-5 can influence the production, maturation, and activation of eosinophils. Interleukin-5 is thought to act predominantly at the later stages of eosinophil maturation and activation (9). Despite this apparent redundancy, it appears that IL-5 is the main cytokine involved in the development of eosinophilia in vivo. The administration of exogenous IL-5 has been shown to cause eosinophilia in a variety of in vivo models (10). Another line of evidence is offered by observations in transgenic animals. In the Il-5 transgenic mice developed by Tominaga and colleagues, a marked eosinophilia in blood and various organs was observed (11). Dent and colleagues developed IL-5 transgenic mice in which transcription of IL-5 is coupled to the dominant control region of the gene encoding for the constitutive T-cell marker CD2 (12). A T-cell specific expression of IL-5 was thus obtained, resulting in a lifelong eosinophilia, distributed mainly in organs with predicted T-cell expression such as bone marrow, spleen, and peritoneum, with fewer cells in the airway mucosa. These animals did not show increased expression of IL-3 or granulocyte/macrophage colony-stimulating factor (GMCSF), thus illustrating that IL-5 can induce the full pathway of eosinophil differentiation and production in vivo. Strikingly, these IL-5 transgenic animals behaved normally and did not die prematurely, suggesting that eosinophils need other factors for degranulation and subsequent tissue damage. It has to be pointed out that even in IL-5 knockout and in IL-5Ra knockout mice, a small number of morphologically normal eosinophils remain detectable in blood (13, 14). Therefore, a minor contribution of minimal amounts of constitutively expressed IL-3 and GMCSF to the production of eosinophils in these transgenic animals cannot be excluded.

A third line of evidence pointing toward the importance of IL-5 for eosinophilia in vivo is the observation that in mice, guinea pigs, and monkeys, pretreatment with anti-IL-5 monoclonal antibodies can suppress allergen-induced airway eosinophilia (15).

Whether this eosinophil influx or IL-5 production is the direct cause of increased airway responsiveness remains a matter of debate. In some experiments, anti-IL-5 pretreatment has been reported not only to inhibit allergen-induced eosinophil influx, but also the increased airway responsiveness. This finding is not invariably confirmed in other studies (18). For example, Corry and colleagues reported that pretreatment of Balb/C mice with anti-IL-5 in a dose sufficient to block antigen-induced eosinophil influx did not affect the increase in airway responsiveness (19). In a similar experiment, we pretreated C57Bl/6 mice with the combination of anti-IL-5 and anti-IL-5 receptor antibodies in an attempt to fully inhibit the effect of endogenously released IL-5. Again, despite a pronounced inhibition of the antigen-induced airway eosinophilia, the increase in airway responsiveness was not prevented (20).

The use of transgenic animals could clarify these seemingly controversial results. To date, only limited data are available. Foster and colleagues convincingly demonstrated that both the allergen-induced eosinophilia and airway hyperreactivity are abolished in IL-5 knockout mice (21). Additional experiments in this area are eagerly awaited.

The role of cytokines in the sequence of events leading to allergen-induced airway inflammation and airway hyperresponsiveness can be studied by administering anticytokine antibodies at specific times. For example, treating mice with anti-IL-4 or anti-IL-5 during exposure to aerosolized ovalbumin effectively inhibited antigen-induced airway eosinophilia (6). These experiments therefore suggest a sequential involvement of IL-4 and IL-5, with IL-4 directing T cells to a Th2 phenotype, which in turn release IL-5 upon stimulation by an inhaled allergen, leading to airway eosinophilia, and possibly to airway hyperresponsiveness. This is in line with recent data from Rankin and colleagues showing that lung-specific expression of IL-4 in transgenic mice causes epithelial hypertrophy and some degree of peribronchial inflammatory changes consisting of a mixed cellular infiltrate (22). However, this was not accompanied by increased airway hyperresponsiveness, suggesting that IL-4 itself is insufficient to induce all the changes characteristic of asthma.

Role of Interleukin-12 and Interferon-

In the mouse model of allergic airway inflammation, we have shown that the intraperitoneal administration of recombinant murine IL-12 during active immunization prevented the production of specific IgE-antibodies as well as the airway eosinophilia and increase in airway responsiveness provoked by aerosol challenge. In contrast, administration of IL-12 to actively immunized mice during aerosol exposure abolished airway eosinophilia and airway hyperresponsiveness without affecting the production of specific IgE. Interleukin-12 stimulates the differentiation of naive Th cells into Th1 cells, at the same time suppressing the development of Th2 cells (23). This mechanism explains how IL-12 administered during active sensitization inhibits allergen-induced airway eosinophilia and increased responsiveness in mice (24, 25). It has been reported that once Th cells have been committed to the Th2 phenotype, the potentially suppressive IL-12-mediated signaling is rapidly lost (26). However, IL-12 retains the capacity to inhibit allergen-induced eosinophilia and airway hyperresponsiveness, even when only administered during secondary antigen exposure (24, 27). This effect could be mediated through the secondary release of immunomodulatory cytokines such as IL-10 and IFN-. Administration of exogenous IFN- prevents the airway eosinophilia and hyperresponsiveness following allergen exposure in mice (28, 29). Li and colleagues recently reported that liposome-mediated IFN- gene transfer to the pulmonary epithelium in sensitized mice before secondary antigen exposure also inhibited the pulmonary allergic response (30). Others have shown that IFN--receptor knockout mice develop a prolonged airway eosinophilia in response to allergen (31). These and other studies suggest the potential modulating effect of IFN- on allergen-induced airway changes. We have therefore investigated the role of IFN- in the inhibitory effects of IL-12 on allergic airway inflammation by comparing the effect of IL-12 on the primary and secondary responses in wild-type mice and in IFN- receptor-deficient mice. Administration of IL-12 during aerosol exposure inhibited both the airway eosinophilia and the production of IgE antibodies in both wild-type mice and IFN- receptor-deficient mice. However, treatment with IL-12 during the active immunization increased the airway eosinophilia and the production of IgE antibodies in the IFN- receptor-deficient mice, probably by stimulating Th2 lymphocytes (32).

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION CONCLUSION REFERENCES

Animal models suggest that cytokines play an important role in the pathogenesis of allergic airway inflammation and airway hyperresponsiveness. The validity of these concepts can be really tested in human asthma only when tools like specific cytokine antagonists and agonists become available for therapeutic studies. In view of the potential role of these cytokines, it also seems attractive to investigate the genetic factors that control their synthesis and to study the relationship between these genetic factors and asthma.