PERSPECTIVE
IL-9 and Lung Fibrosis
A Th2 Good Guy?

Gary W. Hoyle and Arnold R. Brody

Program in Lung Biology, Section of Pulmonary Diseases, Environmental and Critical Care Medicine, Department of Medicine, Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, New Orleans, Louisiana

Interleukin (IL)-9 is a cytokine with pleiotropic activities that has been implicated in the pathogenesis of asthma and is shown in the article by Arras and colleagues as having the capacity to modulate the development lung fibrosis. IL-9 was discovered independently as a growth factor for activated T cells (1) and subsequently for mast cells (2). Cloning of the mouse (3) and human (4) IL-9 cDNAs revealed that they encoded polypeptides 144 amino acids in length which were cleaved to form the secreted proteins of 126 amino acid residues. IL-9 binds to a receptor of the cytokine receptor superfamily that is present on mast cells, macrophages, and subsets of T and B lymphocytes. IL-9 is produced primarily by helper T lymphocytes (5) and has diverse activities that are consistent with the fact that its receptor is expressed on multiple cell types. Freshly isolated T cells generally do not respond to IL-9, but activation or continued passaging results in IL-9 receptor expression so that these cells can proliferate in response to IL-9 (6). IL-9 has been implicated in the development of T-cell lymphomas, since transgenic mice overexpressing IL-9 were prone to develop this type of tumor (7) and T cell lines transfected with IL-9 were tumorigenic in wild-type mice (8). IL-9 synergizes with IL-3 to induce mast cell proliferation (9) and also with IL-4 to stimulate the production of IgE and IgG by B lymphocytes (10). IL-9 also stimulates the proliferation of hematopoietic progenitor cells (11).

Further insight into the activities of IL-9 in vivo has been gained from studies in which IL-9 was systemically overexpressed in transgenic mice. These mice, which are designated Tg5, carry an IL-9 transgene under control of a hybrid promoter containing sequences from the pim-1 promoter, Eµ enhancer, and mMLV LTR (7). These promoter sequences were designed to target IL-9 expression preferentially to T lymphocytes, but in Tg5 mice, IL-9 was found to be expressed in all organs examined and accumulated to very high levels (> 1 µg/ml) in serum. IL-9 transgenic mice spontaneously developed thymic lymphomas and exhibited increased susceptibility to chemically induced lymphomas. Tg5 mice displayed increased numbers of mast cells in the gastrointestinal and respiratory tracts and kidneys (12). Tg5 mice also exhibited increased numbers of circulating B cells, particularly of the B-1 class. In addition, serum immunoglobulin levels were elevated, with IgG1 and IgE being most pronounced (13).

Interest in the action of IL-9 in the lung was stimulated by genetic linkage studies that implicated IL-9 as a candidate gene influencing asthma in humans (14) and airway reactivity in inbred strains of mice (15). Examination of the lungs of Tg5 transgenic mice revealed the presence of eosinophils and constitutive expression of CC chemokines such as eotaxin in lung lavage fluid (16). Tg5 mice also exhibited extensive mucus production in airway epithelial cells (17). Following sensitization and pulmonary challenge with allergen, Tg5 mice displayed increases in serum IgE levels, pulmonary eosinophilic inflammation, and airway hyperreactivity compared to nontransgenic mice (18). Targeted expression of an IL-9 transgene specifically to the lungs of mice has been achieved using the airway epithelial-specific CC10 promoter (19). These mice in the absence of allergic sensitization displayed many features characteristic of asthma, including eosinophilic inflammation, airway hyperreactivity, increased numbers of mast cells in the airway epithelium, mucous cell metaplasia, and airway fibrosis. These studies, along with the detection of elevated IL-9 levels in the lungs of asthmatics (20), suggest that IL-9 may be an important mediator in the pathophysiology of asthma.

IL-9 belongs to a group of cytokines produced by lymphocytes of the Th2 class. CD4 T cells have been classified into subsets designated Th1 and Th2 based on their distinct cytokine profiles. Th1 cells are induced by most intracellular pathogens, mediate cytotoxicity and delayed-type hypersensitivity reactions, and secrete interferon- (IFN-), IL-12, and IL-18. IFN- and IL-12 are important for the development of the Th1 response, and IFN- inhibits the development of Th2 cells. Th2 cells are typically induced by parasitic infections, are involved in allergic immune responses and asthma, and produce IL-4, IL-5, IL-9, IL-10, and IL-13. IL-4 appears to act early in Th2 cell development to induce the Th2 phenotype, and IL-10 serves to inhibit the development of Th1 cells. Immune responses in which Th1 or Th2 cells and cytokines predominate have been termed type 1 and type 2 responses, respectively. In addition to distinct cytokine profiles, type 1 and type 2 immune responses exhibit production of characteristic immunoglobulin subtypes: IgG2a for Th1, and IgE and IgG1 for Th2.

Th2 cytokines have clearly been implicated as mediators of asthma, and evidence is mounting that type 2 immune responses may also promote the development of pulmonary fibrosis. Increased levels of IL-13 were detected in bronchoalveolar lavage fluid recovered from patients with pulmonary fibrosis compared to control subjects (21). T cells from patients with systemic sclerosis expressed IL-4 and IL-5 messages, whereas cells from normal individuals did not (22). In cryptogenic fibrosing alveolitis, IL-4 and IL-5 expression appears to be increased with a concomitant reduction in expression of IFN- (23). In animal models, IL-5 has been shown to be expressed in mice after bleomycin exposure, and treatment with anti-IL-5 antibodies inhibited bleomycin-induced fibrosis (26, 27). IL-4 production was found to be increased during radiation-induced pulmonary fibrosis in rats (28). T cell depletion studies in this same model demonstrated the importance of IL-4-producing Th2 cells in the development of fibrosis (29). IL-10 expression in the lung was elevated in a murine silicosis model. Exposure of IL-10 knockout mice to silica resulted in increased inflammation but an inhibition of fibrosis, indicating that IL-10 is antiinflammatory but profibrotic in this model (30). In cultured rat lung fibroblasts, IL-4 induced collagen production whereas the Th1 cytokine IFN- inhibited collagen production (31). Similar to IL-9, other Th2 cytokines, when expressed in airway epithelial cells in transgenic mice, have produced phenotypes consistent with the involvement of these cytokines in the pathogenesis of asthma. A common observation in these transgenic models has been the development of airway fibrosis (19, 32, 33). These results taken together provide consistent evidence that Th2 polarization of the immune response is generally profibrotic.

The evidence that Th2 immune responses tend to favor the development of fibrosis would suggest that overexpression of a Th2 cytokine might lead to an exacerbation of the response to a fibrogenic agent. However, Arras and colleagues report that with IL-9 the opposite is the case: IL-9 overexpression results in an inhibition of lung fibrosis with an accompanying reduction in Th2-type immune response. The authors used Tg5 transgenic mice, which systemically overexpress IL-9, to examine the effects of IL-9 on the development of silica-induced pulmonary fibrosis. Tg5 mice exhibited reduced collagen deposition in the lung in response to silica treatment compared with nontransgenic mice as quantitated by hydroxyproline analysis. Histologically, the silicotic nodules in IL-9 overexpressing mice had less collagen staining than those in nontransgenic mice and also displayed within the lesions collections of B lymphocytes which were not present in nontransgenic mice exposed to silica. Silica exposure caused accumulation of B lymphocytes in lavage fluid in both wild-type and Tg5 mice; however, the B cell influx was significantly higher in the IL-9 overexpressing mice. CD4 and CD8 T cell populations in lavage fluid did not differ between the two types of mice. Inhibition of silica-induced fibrosis and stimulation of B cell influx into the lung were also achieved by systemic injection of IL-9 into wild-type mice. This finding was important, since it demonstrated that the observed effects resulted from the actions of IL-9 during silica-induced lung injury as opposed to an impaired fibrogenic response caused by compensatory changes consequent to IL-9 expression during embryonic development of the transgenic animals.

The association of B lymphocytes with reduced collagen deposition in silicotic nodules raises the possibility that these cells may mediate the antifibrotic effect in this model. In mice and rats exposed to silica, B cells have been detected in granulomas and in increased numbers in thoracic lymph nodes (34). However, little mechanistic information is available regarding a possible role of B lymphocytes in regulating fibrogenesis. In murine schistosomiasis models, B cell-deficient mice exhibited increased hepatic fibrosis, suggesting that B cells can serve to inhibit liver fibrosis (37, 38). It will be interesting to determine whether these B cell-deficient mice are resistant to the development of lung fibrosis.

Arras and coworkers also obtained evidence for a reduced shift toward a type 2 immune response in silica- exposed Tg5 mice. In wild-type mice silica induced increases in lung IL-4 and lavage fluid IgG1 levels and a decrease in lung IFN- levels, all of which are indicators of a type 2 immune response. In IL-9 overexpressing mice, IL-4 was not increased, and IFN- was not decreased by silica. Silica exposure slightly increased the concentration of IgG1 in lavage fluid, but at the highest dose of silica was significantly lower than in wild-type mice. The levels of IgG2a, an indicator of a type 1 response, were increased by silica in lavage fluid from both wild-type and Tg5 mice, but the increase was significantly larger in the transgenic mice. The correlation between reduced type 2 polarization and inhibition of silica-induced collagen deposition is consistent with the concept that a type 2 immune response will promote fibrogenesis.

How does overexpression of a Th2 cytokine such as IL-9 result in a shift away from a type 2 immune response? The answer to this question may lie with the fact that the network of cytokine expression induced during a natural Th2 response will necessarily be different from that induced by forced overexpression of a single cytokine. The natural controls that maintain the spectrum of cytokine expression observed during a Th2 response and that inhibit a Th1 response will not be in place when IL-9 is overexpressed by itself. IL-9 may normally inhibit type 2 immune polarization, but these properties are not obvious when other Th2 cytokines such as IL-4 and IL-10 are present at the levels observed during a type 2 immune response. Recently developed IL-9 knockout mice displayed normal Th2 cytokine responses to ovalbumin allergen challenge and schistosome egg granuloma formation but exhibited enhanced T cell expression of IL-4, IL-5, and IL-10 after nematode infection (39). These results provide further evidence that IL-9 in certain situations can serve to limit type 2 immune responses.

The results of Arras and coworkers highlight the importance of examining the expression of other cytokine genes in addition to the gene being overexpressed. In cytokine overexpression studies the gene of interest can have indirect effects via altered production of other cytokines in addition to its direct effects on responding cells. To date, researchers have had to make educated guesses regarding the nature of the molecules whose expression may be altered by manipulating expression of a given cytokine. With the advent of microarray technology, it is now possible to systematically examine cytokine networks as they occur naturally and in genetically manipulated animals. This type of experimental approach is likely to significantly accelerate the progress in our understanding of the immune system. The complexity of the cytokine networks involved in pulmonary fibrosis suggests that it will be a significant undertaking to understand how all the molecules involved interrelate during development of the disease. However, the bright side of this complexity is that once a thorough understanding of the mechanisms involved is achieved, it may allow identification of multiple pathways for manipulating the immune system to inhibit profibrotic gene expression and promote the production of antifibrotic molecules.

	Footnotes

Address correspondence to: Gary W. Hoyle, Ph.D., Tulane University Medical Center, 1430 Tulane Avenue, New Orleans, LA 70112. E-mail: ghoyle{at}tulane.edu

(Received in original form February 23, 2001).

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