Gene Knockout Models of Asthma

JOHAN C. KIPS, KURT G. TOURNOY, and ROMAIN A. PAUWELS

Department of Respiratory Diseases, University Hospital Ghent, Ghent, Belgium

	INTRODUCTION

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Airway morphology in asthma is characterized not only by features of an acute allergic inflammation, but also by structural airway changes including subepithelial fibrosis or smooth muscle hypertrophy and/or hyperplasia (1). The key functional abnormality of asthma is the presence of nonspecific bronchial hyperresponsiveness (BHR). This is reflected by an increase both in sensitivity and reactivity of the airways (2, 3). Hypersensitivity is characterized by a leftward shift of the dose-response curve of a bronchoconstrictor agonist, whereas hyperreactivity is reflected in a steeper slope of the dose- response curve and increased maximal airway narrowing. These morphological and functional characteristics are not fully mimicked in the currently developed murine models of allergic airway inflammation. First, no "gold standard" lung function index has been invariably accepted in mice. The indices used include lung resistance, based on the principles described by Amdur and Mead, overflow of insufflation pressure as described by Konzett and Rössler, and sometimes expressed as the airway pressure over time index (APTI), or the enhanced pause (Penh) value (4). These various indices reflect to a variable and ill-defined degree changes in airway sensitivity and reactivity. In the vast majority of murine models, these two components of bronchial hyperresponsiveness are not specifically examined. In addition, the overall increase in airway responsiveness obtained in murine models is smaller than the difference in airway responsiveness that exists between healthy subjects and subjects with asthma. This presumably relates, at least in part, to the differences observed at a morphological level. In most of the murine models of allergen exposure, only acute inflammatory changes are induced, without any concomitant structural change that might affect responsiveness to a larger degree. The distribution of the inflammation is also different from that of human asthma. In animal models, the inflammatory changes not only concentrate in or around the airways, but frequently also include lung parenchymal, mainly perivascular, changes. A serious limitation of the currently available knockout models is that most of these consist of constitutional deletions, precluding the evaluation of the gene of interest in a specific phase of the disease process. As the technology further improves, the possibilities for conditional deletions increase, thus allowing investigators to time the switching off of the gene of interest (8). This will, for example, allow an evaluation in adult life of the role of gene products that are essential during the early stage of development. In addition, conditional deletions will also enable researchers to dissect the role of gene products specifically during the primary antigen sensitization, or during the secondary antigen exposure to memory cells.

Finally, it must be borne in mind that important differences exist between different mouse strains, both in the functional role of components of the immune system and the baseline degree of airway responsiveness (11). Moreover, the sensitization and exposure procedures used in the different models also vary greatly. These different issues must be taken into account, not only when comparing results from different experiments, but especially when trying to extrapolate data from murine models to human asthma.

	INVESTIGATION INTO THE FUNCTIONAL ROLE OF Th2-LIKE CYTOKINES

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We have developed a model of allergen (ovalbumin)-induced airway changes in C57BL/6 mice. In this model, interleukin 4 (IL-4) is essential for the development of ovalbumin (OA)-induced eosinophil influx into the airways, IgE production, and increase in airway responsiveness (15, 16). More recent studies show similar results in mice deficient for signal transducer and activation of transcription 6 (Stat-6), which is considered to be a central signaling pathway for mediating the biological response to IL-4 (17, 18). In our model, mast cell-deficient mice still develop airway eosinophilia in response to OA exposure, whereas MHC class II-deficient mice, lacking effective antigen presentation to CD4⁺ cells and hence functionally active helper T type 2 (Th2) cells, do not. This indicates, as was confirmed by others (19), that the crucial role of IL-4 lies in its effect on Th2 cell development, rather than on the induction of IgE synthesis and subsequent mast cell degranulation. This is in line with a number of other investigations. Takeda and coworkers have shown that mast cell-deficient mice or wild-type littermates develop a comparable increase both in airway eosinophil numbers and airway responsiveness, when sensitized and exposed to OA (20). Similarly, Mehlhop and colleagues reported that in IgE knockout mice sensitization and exposure to Aspergillus extracts induced a comparable degree of airway inflammation and methacholine responsiveness to wild-type animals (21). Of note is that the baseline degree of airway responsiveness is higher in IgE knockout mice than in their wild-type littermates. We made similar observations in IL-4 knockout mice (16). Finally, using B cell-deficient mice, it was shown that the absence of any immunoglobulin production does not prevent development of allergic airway inflammation (22, 23). In these studies, in vivo airway responsiveness was not measured. These observations overall indicate that the IgE-mast cell axis does not play a major role in the induction of allergen-induced airway inflammation and bronchial hyperresponsiveness. These experiments do not allow the exclusion of a possible modulatory role of IgE by enhancing T-B cell interaction (24, 25).

At the same time, this illustrates the central role of T cells in these models. Further studies, using different approaches including pretreatment with anti-CD4 antibodies and passive transfer of cells, confirm the functional importance of CD4⁺ cells and more specifically Th2 cells (26). Rankin and coworkers showed that lung-specific expression of IL-4 in transgenic mice causes epithelial hypertrophy and some degree of peribronchial inflammatory changes. However, this was not accompanied by increased airway hyperresponsiveness, suggesting that IL-4 in itself is insufficient to induce all the changes characteristic of asthma, and that other Th2-derived cytokines are also involved (31). An obvious possibility in this respect is IL-5, which by causing eosinophil influx into the airway mucosa would lead to airway hyperresponsiveness (32). Foster and colleagues have indeed shown that ovalbumin sensitization and exposure in IL-5 knockout mice of the C57BL/6 strain does not cause bronchial hyperresponsiveness as opposed to wild-type mice, and that the eosinophil influx can be restored by reconstituting IL-5 production (33). In a subsequent study, they showed that passive transfer of IL-5-secreting CD4⁺ T cells from OA-sensitized wild type animals to nonsensitized IL-5 mice led to airway eosinophilia and an increase in airway responsiveness when these animals were exposed to aerosolized OA (28). They also claimed that in C57BL/6 mice, IL-4 is not essential for the induction of IL-5-producing cells, as OA exposure induced a similar increase in airway responsiveness in wild-type and IL-4 knockout mice with similar eosinophil infiltration around the bronchi. The OA-induced airway changes were inhibited by pretreatment with anti-CD4 or anti IL-5 (34). These data seem somewhat divergent from our own data. This could at least in part be attributed to differences in the methodology used, again illustrating the difficulty in directly comparing results from different groups. In contrast to our model, Foster and coworkers use boosters with alum. It has been shown that IL-4-deficient mice treated with alum, although not producing IgE, continue to produce IL-5 (35), which could explain the apparent discrepancies observed between both models. In addition, experiments using other antigen sources (parasites) or other mouse strains (mainly BALB/c mice) illustrate that although IL-5 seems to be crucial to induce eosinophil infiltration into the airways, modulation of airway responsiveness can occur independently of IL-5 and airway eosinophilia (30, 36). Other Th2 cytokines can indeed influence airway responsiveness. In an elegant study, Cohn and colleagues reported in an OA T cell receptor transgene model that passive transfer of Th2 cells from IL-4 knockout mice can induce airway hyperresponsiveness and pulmonary eosinophil infiltration on OA exposure in the recipient mice. The passively transferred T cells produced high levels not only of IL-5, but also of IL-10 and IL-13 (29). On the basis of observations with exogenously administered IL-10 and of experiments in our model, using IL-10 knockout mice, it would appear that IL-10 actually inhibits allergen-induced eosinophil infiltration and airway hyperresponsiveness (39, 40), thus making it a less likely candidate in the above-mentioned experiment. Conversely, however, IL-13 has been shown to contribute substantially to allergen-induced airway changes, independently of IL-4, through interaction with the  subunit of the IL-4 receptor (41, 42). IL-9 is yet another cytokine that could mediate Th2-induced effects on airway responsiveness (43). Lung-selective expression of IL-9 in transgenic animals has been shown to induce airway hyperresponsiveness, in addition to morphological changes that bear similarities to asthma (44). The overall message that emerges from these various lines of experimentations is that T cells can alter airway responsiveness in their own right, most probably through production of a cytokine cocktail, the composition of which can vary under different experimental conditions. This concept is further illustrated by the studies by De Sanctis and coworkers, showing that transfer of T cells from mice with a different genetically determined baseline airway responsiveness can alter methacholine responsiveness in the recipient strains, without causing notable inflammatory airway changes (45). Whether similar mechanisms apply in humans obviously remains to be proven. It must, however, be noted that, to date, attempts to inhibit the effect of CD4⁺ cells in established severe asthma have not proven successful in improving asthma symptoms (46).

	INVESTIGATION INTO THE MECHANISMS UNDERLYING PREFERENTIAL Th2-LIKE DEVELOPMENT

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Dendritic cells (DCs) in the airway epithelium are the major antigen-presenting cells (APCs) involved in the primary immune reponse to inhaled allergen. After migrating to the local lymph nodes, DCs present the processed antigen to naive T cells. The activation of T cells requires in addition the expression of cofactors, of which CD40/CD40 ligand and CD28/B7 have been most extensively investigated (47). Again, the exact importance of each of these cofactors in the immune response in vivo remains to be fully assessed. From the currently available data, it appears that blockade of either CD40 or B7-1 diminishes allergen-induced airway eosinophilia, without, however, affecting IL-5 production or the number of circulating eosinophils, indicating that the development of Th2 cells is not abolished (48, 49). Using the technique of conditional deletion, we showed that DCs also play a major role in the secondary immune response to inhaled allergens (50). Others have illustrated the importance of B7-2 as cofactor in the immune response during allergen presentation to memory T cells (51- 53). Again, this area is in full development.

The development of Th2 cells as opposed to Th1 cells during the primary immune response is also known to be influenced, among other factors, by the surrounding cytokine milieu. Initial studies have shown that exogenous administration of IL-12 during the primary antigen presentation suppresses allergen-induced Th2 cell development (54, 55), thus inhibiting IgE production, airway eosinophilia, and increased responsiveness. Concordantly, airway inflammation has been shown to be enhanced in IL-12 receptor (p40)-deficient mice (56). Interestingly, IL-12 also retains the capacity to inhibit allergen-induced eosinophilia and airway hyperresponsiveness, even when administered only during secondary antigen exposure (54, 57). It has been hypothesized that this effect could be mediated through the secondary release of immunomodulatory cytokines such as IL-10 and interferon  (IFN-). Administration of exogenous IFN- prevents the development of airway eosinophilia and hyperresponsiveness after allergen exposure (58, 59). Others have shown that IFN- receptor knockout mice develop a prolonged airway eosinophilia in response to allergen (60). However, we have shown that IL-12 retains its immunomodulatory effect when given during secondary antigen exposure in IFN- receptor-deficient mice, indicating that these effects are not due to the endogenous release of IFN- (61). Whether IL-12 mediates these effects through the secondary release of IL-10 remains to be further evaluated.

	CONCLUSION

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From the currently available gene knockout models of asthma, it emerges that T cells themselves are the main determining factor establishing the degree of airway responsiveness, and that altering the Th1/Th2 cell balance, irrespective of exogenous factors, does have an effect on airway behavior. It would appear that neither the influx of eosinophils nor production of IgE is the cause of bronchial hyperresponsiveness. It remains to be fully established to what extent they might modulate the T cell-determined degree of airway responsiveness in these models.

	DISCUSSION

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Holt: One of the problems in these models is the lack of a late phase.

Kips: The currently used techniques for lung function measurement in mice are mainly aimed at measuring airway responsiveness, not the early- or late-phase reaction. It can be argued that airway responsiveness is a relevant outcome measure in asthma and that the absence of a documented late-phase reaction does not reduce the value of murine models. The allergen-induced late-phase reaction also merely represents a laboratory model, albeit applicable to man, but not fully representing day-to-day asthma.

Platts-Mills: In the human the only epidemiology we have relates to IgE antibodies. So, my question to you is: If a T cell really is essential to the pathology of the process, how would you search for a model in the human where we could put antigen in the lung and show that the T cells are responsible for the process that follows?

Kips: The gene knockout models of asthma all point towards the central role of allergen-driven T cells, irrespective of the presence of IgE. One way of addressing the role of human T cells in airway behavior is the use of models based on SCID mice. This offers the opportunity to study specific human T cell function without intervention by other inflammatory cell types in airway responsiveness.

Platts-Mills: But that is a circular answer, because the central question from our point of view is whether this bronchial reactivity which you see in the mouse is really a model for human bronchial reactivity.

Kips: IgE could be merely an epiphenomenon of other things that are actually going on in the airways.

Platts-Mills: But that is my third point. If IgE was really an epiphenomenon, somewhere in the human epidemiology someone would have found a break, where we would have found a population of people who would have a T cell response alone to something without the IgE.

Kips: How about occupational asthma?

Platts-Mills: That is very strikingly different.

Holgate: As you move away from childhood (and much of the epidemiology we are going to hear about today relates to childhood) to what you and I would call real asthma, the influence of atopy seems to diminish quite markedly. And yet, when we look at the lung tissue, Th2 cells are there, as are the IL-5 and other cytokines; you cannot see any differences within the tissue itself, and if they are there, they are very subtle. And a second thing we have got to explain, is why not all allergic people are asthmatic, because they are sensitized and the antigen has to get into the system via the inhaled route. So, they are systemically sensitized, they are locally tolerant. In order to understand the dynamics of what is going on in the tissue, we have got to start asking what pulls away a small population of animals that may be very responsive locally and not so responsive systemically.

Kips: The sensitization schemes we use are potent and overrule any differences between animals. What additional factors are needed to express the phenotype cannot be deduced from these models.

Renz: I agree with your key point that there is no mouse model for asthma yet. We agree that T cells modulate airway responsiveness and that they are required to get airway hyperresponsiveness. However, there are different pathways, which we now need to sort out.

Kips: I could not agree more. Ovalbumin might not be the right choice. House dust mite might be a better choice as it behaves differently from ovalbumin. In addition to exposing animals to small doses of allergen we need to expose them to confounding factors, to look at the interrelationship between allergens and viruses, LPS, and so on. Some investigators are doing that. One of the disadvantages of the gene knockout technology as we have it at the moment is that it is black and white. We first of all knockout a gene throughout the entire development of the animal. Then we usually expose the animals to quite high doses under various experimental conditions. We need some fine-tuning, so that we can bring down the exposure to match what is encountered in the daily environment. More importantly, the switching off and on of the genes should become far better controllable.

Martinez: Timing of sensitization is very important. Very few children age 3 years or less have IgE to aeroallergens, but out of the few that do, 80 or 90% have chronic asthma by the age of 13. If we go to age 6 still there is a very high risk for having asthma among the children who are sensitized against our culprit, which is Alternaria. But by the age of 11, the risk has decreased significantly, in the sense that those who have become sensitized between the ages 6 and 11 have a much lower risk, that is not significantly higher than that of children that never become sensitized. Dr. Peat has shown similar things in Australia. So, there is sensitization and sensitization: Timing is important. This is an answer to what Dr. Holgate was saying before, in the sense that of course allergic sensitization is not asthma. There is a second set of epidemiological data and that is the asthma epidemic in Barcelona. The asthma epidemic data has been interpreted as showing that exposure to the soy bean antigen induced or provoked asthma in these people, but when you ask in a little more detail you find out that 90 or 95% of the people who developed these severe asthmatic attacks were already asthmatics. But it does not mean that only asthmatics became sensitized, because when you ask even further, a lot of people became sensitized who were not asthmatics before and these did not develop significant asthma. So again: The timing of sensitization is important. I wonder if you think that in the future we will have an animal model that will take these issues into account.

Kips: Dr. Renz presented some of the data at this meeting that points towards the animal models that are emerging now, looking even before birth and through the development of the systems.

Vercelli: Talking about the role of IL-13 and the IL-13 KO, it should be mentioned that the results from these models should be interpreted with caution, because mice do not have IL-13 receptors on T cells. Thus, the effects of IL-13 on lungs and airways cannot be directly T cell mediated.

Kips: I agree that the effects of certain cytokines (e.g., IL-13, but also IL-5) can be different in man and in mice. This has to be taken into account when extrapolating the observations from mouse models to human disease. However, even if IL-13 does not directly affect T cells in mice, it does have indirect effects (e.g., effects via IL-12 and IL-10) that can influence T cell function.

Oosterhout: One of the reasons for discrepancies between different murine models of allergic asthma may be the genetic background. In fact, this was nicely illustrated by the data of Foster and coworkers that you mentioned: IL-5 KO black-6 mice do not develop AHR, whereas IL-5 KO BALB/c mice still develop AHR.

Kips: Your approach, neutralization with antibodies, or with the CTL4 fusion protein, is often probably far better, in that it allows a better timing of the intervention. There have been a few animal models using, for example, Dr. Heusser's nonanaphylactogenic anti-IgE, showing that you can reduce the allergen-induced airway inflammation. But in all these studies, where IgE proved to be effective, anti-IgE was given during the secondary antigen exposure, to memory T cells. When Dr. Coyle gives the antibody during his booster phase, there is no effect whatsoever on IL-4 or IL-5 production. So anti-IgE given during sensitization does not appear to do the trick. If you give it afterwards, during the secondary antigen presentation, it does seem to have an effect.

Aalberse: T cells can do it, but do they do it in the human, and particularly, do they do it in the young asthmatic patients. Your argument is in fact: IgE is a readout system, if you have IgE, then you have Th2. Wouldn't you then expect that high IgG₄ levels could also be used as a readout?

Kips: The point is taken.

Aalberse: Would you accept that in the human, particularly in young patients, Th2 alone is not sufficient?

Kips: Yes, but there is no proof either that IgE is the missing link. It might well be that structural alterations in the airways are the missing link. Dr. Holgate in the collaborative study with Chech investigators have found alterations at the airway level in very young children prior to clinical overt disease.

	Footnotes

Correspondence and requests for reprints should be addressed to J. C. Kips, M.D., University Hospital Ghent, Department of Respiratory Diseases, De Pintelaan 185, B 9000 Ghent, Belgium. E-mail: johan.kips{at}rug.ac.be

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