Accelerated alveolar bone loss in mice lacking interleukin-10.

Interleukin-10 regulates pro-inflammatory cytokines, including those implicated in alveolar bone resorption. We hypothesized that lack of interleukin-10 leads to increased alveolar bone resorption. Male interleukin-10(-/-) mice, on 129/SvEv and C57BL/6J background, were compared with age-, sex-, and strain-matched interleukin-10(+/+) controls for alveolar bone loss. Immunoblotting was used for analysis of serum reactivity against bacteria associated with colitis and periodontitis. Interleukin-10(-/-) mice had significantly greater alveolar bone loss than interleukin-10(+/+) mice (p = 0.006). The 30-40% greater alveolar bone loss in interleukin-10(-/-) mice was evident in both strains, with C57BL/6J interleukin-10(-/-) mice exhibiting the most bone loss. Immunoblotting revealed distinct interleukin-10(-/-) serum reactivity against Bacteroides vulgatus, B. fragilis, Prevotella intermedia, and, to a lesser extent, against B. forsythus. The results of the present study suggest that lack of interleukin-10 leads to accelerated alveolar bone loss.

Endogenous inhibition of antimycobacterial immunity by IL-10 varies between mycobacterial species.

Interleukin (IL)-10 is an immunoregulatory cytokine that inhibits both Th1-like T cell responses and macrophage activation. Deficiency of IL-10 has been associated with increased Th1-like CD4+ T-cell responses and increased clearance of some intracellular pathogens, however, its role in mycobacterial infections is controversial. In order to examine the effects of mycobacterial virulence on the outcome of infection we compared infection with Mycobacterium avium and virulent Mycobacterium tuberculosis in C57Bl/6 IL-10-/- mice. M. avium infection in IL-10-/- mice resulted in sustained increases in interferon (IFN)-gamma-secreting T-cell responses and was associated with the increased clearance of M. avium from the liver and lung. By contrast, M. tuberculosis infection in IL-10-/- mice led to a transient increase in IFN-gamma T-cell responses at 4 weeks postinfection, with reduced bacterial burden in the lungs. This was not sustained so that by 8 weeks there was no difference to wild-type (WT) mice. In vitro infection of IL-10-/- macrophages with M. avium, but not M. tuberculosis, led to an increased IL-12 production. Therefore, endogenous IL-10 exerts a significant inhibition on specific IFN-gamma T-cell responses to M. avium infection, however, this effect is short lived during the M. tuberculosis infection, and fails to influence the long-term course of infection.

Il-13 and IFN-gamma: interactions in lung inflammation.

Chronic inflammatory diseases of the lungs, such as asthma, are frequently associated with mixed (Th2 and Th1) T cell responses. We examined the impact of critical Th1 and Th2 cytokines, IFN-gamma and IL-13, on the responses in the lungs. In a mouse model of airway inflammation induced by mixed T cell responses, the number of Th1 (IFN-gamma-positive) cells was found to be negatively correlated with airway hyperreactivity. In these mice, blockade of IL-13 partially inhibited airway hyperreactivity and goblet cell hyperplasia but not inflammation. In contrast, in mice that responded with a polarized Th2 response to the same Ag, blockade of IL-13 inhibited airway hyperreactivity, goblet cell hyperplasia, and airway inflammation. These results indicated that the presence of IFN-gamma would modulate the effects of IL-13 in the lungs. To test this hypothesis, wild-type mice were given recombinant cytokines intranasally. IFN-gamma inhibited IL-13-induced goblet cell hyperplasia and airway eosinophilia. At the same time, IFN-gamma and IL-13 potentiated each other's effects. In the airways of mice given IL-13 and IFN-gamma, levels of IL-6 were increased as well as numbers of NK cells and of CD11c-positive cells expressing MHC class II and high levels of CD86. In conclusion, IFN-gamma has double-sided effects (inhibiting some, potentiating others) on IL-13-induced changes in the lungs. This may be the reason for the ambiguous role of Th1 responses on Th2 response-induced lung injury.

Characterization of chemokines and chemokine receptors in two murine models of inflammatory bowel disease: IL-10-/- mice and Rag-2-/- mice reconstituted with CD4+CD45RBhigh T cells.

We used quantitative PCR to investigate the expression of chemokines and chemokine receptors in two Th1-mediated murine models of inflammatory bowel disease (IBD). First, mRNA levels encoding the chemokines MIG, RANTES, lymphotactin, MIP-3alpha, TCA-3, TARC, MIP-3beta, LIX, MCP-1 and MIP-1beta and the receptors CCR4, CCR6 and CCR2 were significantly increased in chronically inflamed colons of IL-10-/- mice when compared with wildtype mice. Interestingly, reversal of colitis in IL-10-/- mice by anti-IL-12 mAb was accompanied by the inhibition in the expression of LIX, lymphotactin, MCP-1, MIG, MIP-3alpha, MIP-3beta, TCA-3, CCR2 and CCR4, whereas the increased mRNA levels of MIP-1beta, RANTES, TARC and CCR6 were unaffected. Second, to investigate which chemokines and receptors were up-regulated during the inductive phase of colitis, we employed the CD4+CD45RBhigh T cell transfer model. At 4 and 8 weeks after reconstitution of Rag-2-/- mice the mRNA levels of IP-10, MCP-1, MDC, MIG, TARC, RANTES, CCR4 and CCR5 were significantly increased prior to the appearance of macroscopic lesions. Other chemokines and chemokine receptors were clearly associated with the acute phase of the disease when lesions were evident. The sum of our studies with these two models identifies chemokines that are expressed at constant levels, irrespective of inflammatory responses, and those that are specifically associated with acute and/or chronic stages of Th1-driven colitis.

IL-4 exacerbates disease in a Th1 cell transfer model of colitis.

IL-4 is associated with Th2-type immune responses and can either inhibit or, in some cases, promote Th1-type responses. We tested the effect of IL-4 treatment on the development of inflammation in the CD4(+)CD45RB(high) T cell transfer model of colitis, which has been characterized as a Th1-dependent disease. IL-4 treatment significantly accelerated the development of colitis in immunodeficient recipients (recombinase-activating gene-2 (Rag2)(-/-)) of CD4(+)CD45RB(high) T cells. Quantitative analysis of mRNA expression in the colons of IL-4-treated mice showed an up-regulation of both Th1- and Th2-associated molecules, including IFN-gamma, IP-10, MIG, CXCR3, chemokine receptor-8, and IL-4. However, cotreatment with either IL-10 or anti-IL-12 mAb effectively blocked the development of colitis in the presence of exogenous IL-4. These data indicate that IL-4 treatment exacerbates a Th1-mediated disease rather than induces Th2-mediated inflammation. As other cell types besides T cells express the receptor for IL-4, the proinflammatory effects of IL-4 on host cells in Rag2(-/-) recipients were assessed. IL-4 treatment was able to moderately exacerbate colitis in Rag2(-/-) mice that were reconstituted with IL-4Ralpha-deficient (IL-4Ralpha(-/-)) CD4(+)CD45RB(high) T cells, suggesting that the IL-4 has proinflammatory effects on both non-T and T cells in this model. IL-4 did not cause colitis in Rag2(-/-) mice in the absence of T cells, but did induce an increase in MHC class II expression in the lamina propria of the colon, which was blocked by cotreatment with IL-10. Together these results indicate that IL-4 can indirectly promote Th1-type inflammation in the CD4(+)CD45RB(high) T cell transfer model of colitis.

IL-25 induces IL-4, IL-5, and IL-13 and Th2-associated pathologies in vivo.

We have characterized a cytokine produced by Th2 cells, designated as IL-25. Infusion of mice with IL-25 induced IL-4, IL-5, and IL-13 gene expression. The induction of these cytokines resulted in Th2-like responses marked by increased serum IgE, IgG(1), and IgA levels, blood eosinophilia, and pathological changes in the lungs and digestive tract that included eosinophilic infiltrates, increased mucus production, and epithelial cell hyperplasia/hypertrophy. In addition, our studies show that IL-25 induces Th2-type cytokine production by accessory cells that are MHC class II(high), CD11c(dull), and lineage(-). These results suggest that IL-25, derived from Th2 T cells, is capable of amplifying allergic type inflammatory responses by its actions on other cell types.

Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12.

A novel sequence discovered in a computational screen appears distantly related to the p35 subunit of IL-12. This factor, which we term p19, shows no biological activity by itself; instead, it combines with the p40 subunit of IL-12 to form a novel, biologically active, composite cytokine, which we term IL-23. Activated dendritic cells secrete detectable levels of this complex. IL-23 binds to IL-12R beta 1 but fails to engage IL-12R beta 2; nonetheless, IL-23 activates Stat4 in PHA blast T cells. IL-23 induces strong proliferation of mouse memory (CD4(+)CD45Rb(low)) T cells, a unique activity of IL-23 as IL-12 has no effect on this cell population. Similar to IL-12, human IL-23 stimulates IFN-gamma production and proliferation in PHA blast T cells, as well as in CD45RO (memory) T cells.

Role of IL-10 in invasive aspergillosis: increased resistance of IL-10 gene knockout mice to lethal systemic aspergillosis.

IL-10 is associated with a Th2 response, down-regulation of a Th1 response and macrophage activation. We assessed the role of IL-10 during systemic infection with Aspergillus fumigatus. Systemic aspergillosis was established in female C56B1/6 IL-10(-/-) (KO) and wild-type (WT) C57B1/6 mice by i.v. administration of 1 x 10(5)-6 x 10(5) conidia of A. fumigatus. In two experiments, KO survived longer than did WT (P < 0.001). Determination of fungal burdens in the kidneys and brain showed that KO carried significantly lower burdens in both organs than did WT on day 3 (P < 0.001). Semiquantitative histological analyses showed fewer inflammatory foci/mm2 in brain and kidneys of KO than WT (P < 0.03 and < 0.001, respectively) and that extent of infection and associated tissue injury were greater in WT. Although beneficial in some bacterial infections, exogenous IL-10 has been shown deleterious in models of fungal infection. Our data indicate IL-10 is deleterious during systemic aspergillosis infection, increasing the host susceptibility to lethal infection. We speculate this might be related to greater Th2 or lesser Th1 responses, or down-regulation of macrophage responses, in WT compared with KO.

Lessons from genetically engineered animal models. XII. IL-10-deficient (IL-10(-/-) mice and intestinal inflammation.

Interleukin (IL)-10(-/-) mice spontaneously develop intestinal inflammation characterized by discontinuous transmural lesions affecting the small and large intestine and by dysregulated production of proinflammatory cytokines. The uncontrolled generation of IFN-gamma-producing CD4(+) T cells (Th1 type) has been shown to play a causal role in the development of enterocolitis affecting these mutants. This article discusses studies of IL-10(-/-) mice that have investigated the role of enteric organisms in triggering intestinal disease, the mediators responsible for initiating and maintaining intestinal disease, the role IL-10 plays in the generation and/or function of regulatory cells, and the results of IL-10 therapy in experimental animal models of inflammatory bowel disease (IBD) and human patients with IBD.

Iron supplementation affects the production of pro-inflammatory cytokines in IL-10 deficient mice.

BACKGROUND: Iron supplements may increase disease activity in inflammatory bowel disease through the production of the hydroxyl radical because of its catalytic activity in the Fenton reaction. The purpose of this study was to assess the effect of dietary and locally administered iron in the IL-10 knock-out (-/-) mouse, a model of chronic inflammatory bowel disease. MATERIALS AND METHODS: IL10-/- and wild-type mice received a standard or a high-iron diet (35 mg kg(-1) ferrosulphate vs. 500 mg kg(-1) ferrosulphate) after weaning. After 4 weeks the mice were sacrificed. Furthermore, a group of adult IL-10 knock-out mice was given three iron-containing enema's (0.2 mL of 1 mM ferrous-ammonium sulphate) or phosphate buffered saline. These mice were sacrificed after 1 week. Production of pro-inflammatory cytokines by colon tissue cultures, haematological parameters and histology was determined to assess inflammatory activity. RESULTS: Oral as well as rectal administration of iron resulted in increased pro-inflammatory cytokine production in IL-10-/- mice. Neutrophil counts in IL10-/- on a high iron diet increased as well. No enhanced colonic inflammation was noted on histology after iron supplementation. CONCLUSION: We conclude that dietary or topical administered iron increases pro-inflammatory cytokine production in the colon of IL10-/- mice. No significant increase of histological intestinal inflammation was observed.

Blockade of costimulation prevents infection-induced immunopathology in interleukin-10-deficient mice.

Interleukin-10 (IL-10) is associated with inhibition of cell-mediated immunity and downregulation of the expression of costimulatory molecules required for T-cell activation. When IL-10-deficient (IL-10KO) mice are infected with Toxoplasma gondii, they succumb to a T-cell-mediated shock-like reaction characterized by the overproduction of IL-12 and gamma interferon (IFN-gamma) associated with widespread necrosis of the liver. Since costimulation is critical for T-cell activation, we investigated the role of the CD28-B7 and CD40-CD40 ligand (CD40L) interactions in this infection-induced immunopathology. Our studies show that infection of mice with T. gondii resulted in increased expression of B7 and CD40 that was similar in wild-type and IL-10KO mice. In vivo blockade of the CD28-B7 or CD40-CD40L interactions following infection of IL-10KO mice with T. gondii did not affect serum levels of IFN-gamma or IL-12, nor did it prevent death in these mice. However, when both pathways were blocked, the IL-10KO mice survived the acute phase of infection and had reduced serum levels of IFN-gamma and alanine transaminase as well as decreased expression of inducible nitric oxide synthase in the liver and spleen. Analysis of parasite-specific recall responses from infected IL-10KO mice revealed that blockade of the CD40-CD40L interaction had minimal effects on cytokine production, whereas blockade of the CD28-B7 interaction resulted in decreased production of IFN-gamma but not IL-12. Further reduction of IFN-gamma production was observed when both costimulatory pathways were blocked. Together, these results demonstrate that the CD28-B7 and CD40-CD40L interactions are involved in the development of infection-induced immunopathology in the absence of IL-10.

Chronic colitis in IL-10-/- mice: insufficient counter regulation of a Th1 response.

IL-10-deficient (IL-10-/-) mice, generated by a gene-targeted mutation, develop abnormal immune responses as a result of uncontrolled interactions between antigen presenting cells and lymphocytes. The studies reviewed herein have focused on the enterocolitis that spontaneously develops in IL-10-/- mice. Not unexpectedly, heightened production of proinflammatory mediators accompanied pathologic changes in the gastrointestinal tract of young mutants. In a series of studies, the proinflammatory mediators responsible for initiating the pathogenic response were distinguished from those that were elicited as a consequence of persistent inflammation. We have also investigated the possibility that different mediators are involved in the inductive versus the maintenance phase of disease. The findings of these mechanistic studies as they relate to our understanding of progressive inflammatory disease and the role of IL-10 in controlling the acute and chronic stages are discussed.

The role of IL-10 in inflammatory bowel disease: "of mice and men".

Inflammatory bowel disease (IBD) is a generic term typically used to describe a group of idiopathic inflammatory intestinal conditions in humans that are generally divided into Crohn's disease and ulcerative colitis. Although the etiology of these diseases remains unknown, a number of rodent models of IBD have recently been identified, all sharing the concept that the development of chronic intestinal inflammation occurs as a consequence of alterations in the immune system that lead to a failure of normal immunoregulation in the intestine. On the basis of these models, it has been hypothesized that the development of IBD in humans may be related to a dysregulated immune response to normal flora in the gut. Immunodeficient scid mice injected with CD4+ CD45RB(high) T cells and mice deficient in interleukin (IL)-10 (IL-10-/-) are among the rodent models of IBD. In both models, there is inflammation and evidence of a Th1-like response in the large intestine, characterized by CD4+ T-cell and macrophage infiltrates, and elevated levels of interferon-gamma. Because IL-10 is an immunomodulatory cytokine that is capable of controlling Th1-like responses, the role of IL-10 was investigated in these models. IL-10 was shown to be important in regulating the development of intestinal inflammation in both models. These results provided key data that supported initiation of clinical trials evaluating the efficacy of IL-10 in patients with IBD.

Requirement for IL-13 independently of IL-4 in experimental asthma.

The pathogenesis of asthma reflects, in part, the activity of T cell cytokines. Murine models support participation of interleukin-4 (IL-4) and the IL-4 receptor in asthma. Selective neutralization of IL-13, a cytokine related to IL-4 that also binds to the alpha chain of the IL-4 receptor, ameliorated the asthma phenotype, including airway hyperresponsiveness, eosinophil recruitment, and mucus overproduction. Administration of either IL-13 or IL-4 conferred an asthma-like phenotype to nonimmunized T cell-deficient mice by an IL-4 receptor alpha chain-dependent pathway. This pathway may underlie the genetic associations of asthma with both the human 5q31 locus and the IL-4 receptor.

Acquisition of CD24 expression by Lin-CD43+B220(low)ckit(hi) cells coincides with commitment to the B cell lineage.

The present study describes three subsets of bone marrow-derived Lin CD43+B220(low) (B220(low)) progenitor cells which represent distinct stages in hematopoietic development. These populations differ in their expression of CD24 and ckit and the occurrence of IgH gene rearrangement. B220(low) CD24-ckit(hi) progenitors have their IgH loci in germ-line configuration and are multipotent since they can give rise to B cells, T cells and myeloid cells. B220lowckit(hi) cells which have acquired CD24 expression have retained IgH loci in germ-line configuration and the ability to generate B cells, however, they have lost the ability to generate T cells and myeloid cells. Thus acquisition of CD24 by B220(low) cells occurs concurrently with the transition from a multipotent to a lineage-restricted progenitor population. B220(low)CD24+ cells expressing low levels of ckit are also lineage restricted, giving rise to only B cells and have begun D-J(H) rearrangement, implying that initiation of IgH rearrangement coincides with the down-regulation of ckit expression.

A role for NK cells as regulators of CD4+ T cells in a transfer model of colitis.

Previous studies have shown that the chronic inflammation observed in the colon of IL-10-deficient (IL-10(-/-)) mice is mediated by CD4+ Th1 T cells and is dependent on the presence of IFN-gamma for its initial development. As CD4+ T cells from IL-10(-/-) mice will cause colitis when transferred into recombinase-activating gene (Rag)-deficient recipients, we considered the possibility that the recipients' NK cells could be an important source of IFN-gamma for the development of colitis. Therefore, the ability of IL-10(-/-) CD4+ T cells to cause colitis in Rag-deficient recipients that had been depleted of NK cells was tested. Contrary to our expectations, NK cell-depleted recipients of IL-10(-/-) CD4+ T cells developed accelerated disease compared with nondepleted recipients. Furthermore, CD4+ T cells from normal mice (IL-10(+/+)) also caused colitis in NK cell-depleted recipient mice, but not in nondepleted recipients. NK cells inhibited effector CD4+CD45RBhigh T cells, and subsequent experiments showed that this effect was dependent on perforin. Thus NK cells can play an important role in down-regulating Thl-mediated colitis by controlling the responses of effector T cells to gut bacteria.

Resident enteric bacteria are necessary for development of spontaneous colitis and immune system activation in interleukin-10-deficient mice.

Mice with targeted deletion of the gene for interleukin-10 (IL-10) spontaneously develop enterocolitis when maintained in conventional conditions but develop only colitis when kept in specific-pathogen-free (SPF) environments. This study tested the hypothesis that enteric bacteria are necessary for the development of spontaneous colitis and immune system activation in IL-10-deficient mice. IL-10-deficient mice were maintained in either SPF conditions or germfree conditions or were populated with bacteria known to cause colitis in other rodent models. IL-10-deficient mice kept in SPF conditions developed colitis in all segments of the colon (cecum and proximal and distal colon). These mice exhibited immune system activation as evidenced by increased expression of CD44 on CD4(+) T cells; increased mesenteric lymph node cell numbers; and increased production of immunoglobulin A (IgA), IgG1, and IL-12 p40 from colon fragment cultures. Mice populated with bacterial strains, including Bacteroides vulgatus, known to induce colitis in other rodent models had minimal colitis. Germfree IL-10-deficient mice had no evidence of colitis or immune system activation. We conclude therefore that resident enteric bacteria are necessary for the development of spontaneous colitis and immune system activation in IL-10-deficient mice.

IL-12, but not IFN-gamma, plays a major role in sustaining the chronic phase of colitis in IL-10-deficient mice.

IL-10-deficient (IL-10(-/-)) mice develop chronic enterocolitis mediated by CD4+ Th1 cells producing IFN-gamma. Because IL-12 can promote Th1 development and IFN-gamma production, the ability of neutralizing anti-IL-12 mAb to modulate colitis in IL-10(-/-) mice was investigated. Anti-IL-12 mAb treatment completely prevented disease development in young IL-10(-/-) mice. Treatment of adult mice resulted in significant amelioration of established disease accompanied by reduced numbers of mesenteric lymph node and colonic CD4+ T cells and of mesenteric lymph node T cells spontaneously producing IFN-gamma. In contrast, anti-IFN-gamma mAb had minimal effect on disease reversal, despite a significant preventative effect in young mice. These findings suggested that IL-12 sustains colitis by supporting the expansion of differentiated Th1 cells that mediate disease independently of their IFN-gamma production. This conclusion was supported by the finding that anti-IL-12 mAb greatly diminished the ability of a limited number of CD4+ T cells expressing high levels of CD45RB from diseased IL-10(-/-) mice to expand and cause colitis in recombination-activating gene-2(-/-) recipients, while anti-IFN-gamma mAb had no effect. Furthermore, IL-12 could support pathogenic IL-10(-/-) T cells stimulated in vitro in the absence of IL-2. While these studies show that IL-12 plays an important role in sustaining activated Th1 cells during the chronic phase of disease, the inability of anti-IL-12 mAb to abolish established colitis or completely prevent disease transfer by Thl cells suggests that additional factors contribute to disease maintenance.

Radioprotective effects of flk2/flt3 ligand.

The radioprotective properties of flk2/flt3 ligand (FL) were evaluated in lethally irradiated mice. Optimum survival rates (70-80%) were observed when 5 to 20 microg of FL was administered at both 20 and 2 hours before LD100/30 radiation. Administration of FL well in advance of irradiation was essential for conferring most of the radioprotection, since a single dose given at -20 hours still resulted in a significant survival rate (65%), whereas a single dose given at -2 hours was relatively nonprotective. Histopathologic examination at 7 and 9 days postirradiation revealed significant myelopoietic activity in the bone marrow (BM) of FL-treated mice, suggesting that their survival might be due to sparing of radiosensitive hematopoietic cells. By comparison, the BM of mice treated with phosphate-buffered saline was extremely hypocellular and remained that way until they died of bacterial infection. Hematopoietic assays confirmed a marked stimulation of early white blood cell (WBC) recovery in the BM and blood of FL-protected mice relative to PBS-treated controls. By day 21, FL-protected mice showed circulating WBC numbers that were higher than preirradiation values; however, their BM colony-forming units in culture were still depressed. Moreover, these mice experienced a prolonged anemia and thrombocytopenia. These findings are discussed in light of the restricted subset of hematopoietic progenitors shown to be responsive to FL in vitro.

Requirements for allergen-induced airway hyperreactivity in T and B cell-deficient mice.

BACKGROUND: The pathogenesis of asthma is believed to reflect antigen-induced airway inflammation leading to the recruitment of eosinophils and activation of mast cells through cell-associated IgE. Controversies persist however, regarding the relative importance of different pathogenic cells and effector molecules. MATERIALS AND METHODS: A variety of gene-targeted mice were examined for the induction of cholinergic airway hyperresponsiveness (AH), allergic airway inflammation, mucus production, and serum IgE reactivity following intratracheal challenge with a potent allergen. AH was determined using whole-body plethysmography following acetylcholine challenge. Where possible, results were confirmed using neutralizing antibodies and cell-specific reconstitution of immune deficient mice. RESULTS: T and B cell-deficient, recombinase-activating-gene-deficient mice (RAG -/-) failed to develop significant allergic inflammation and AH following allergen challenge. Reconstitution of RAG -/- mice with CD4+ T cells alone was sufficient to restore allergen-induced AH, allergic inflammation, and goblet cell hyperplasia, but not IgE reactivity. Sensitized B cell-deficient mice also developed airway hyperreactivity and lung inflammation comparable to that of wild-type animals, confirming that antibodies were dispensable. Treatment with neutralizing anti-IL-4 antibody or sensitization of IL-4-deficient mice resulted in loss of airway hyperreactivity, whereas treatment with anti-IL-5 antibody or sensitization of IL-5-deficient mice had no effect. CONCLUSIONS: In mice, CD4+ T cells are alone sufficient to mediate many of the pathognomonic changes that occur in human asthma by a mechanism dependent upon IL-4, but independent of IL-5, IgE, or both. Clarification of the role played by CD4+ T cells is likely to stimulate important therapeutic advances in treatment of asthma.