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Although it is widely accepted that allergic asthma is driven by T helper type 2 (Th2)-polarized immune responses to innocuous environmental allergens, the mechanisms driving these aberrant immune responses remain elusive. Recent recognition of the importance of innate immune pathways in regulating adaptive immune responses have fueled investigation into the role of innate immune pathways in the pathogenesis of asthma. The phylogenetically ancient innate immune system, the complement system, is no exception. The emerging paradigm is that C3a production at the airway surface serves as a common pathway for the induction of Th2-mediated inflammatory responses to a variety of environmental triggers of asthma (i.e., allergens, pollutants, viral infections, cigarette smoke). In contrast, C5a plays a dual immunoregulatory role by protecting against the initial development of a Th2-polarized adaptive immune response via its ability to induce tolerogenic dendritic cell subsets. On the other hand, C5a drives type 2-mediated inflammatory responses once inflammation ensues. Thus, alterations in the balance of generation of the various components of the complement pathway either due to environmental exposure changes or genetic alterations in genes of the complement cascade may underlie the recent rise in asthma prevalence in westernized countries.

Asthma is a complex inflammatory pulmonary disorder that is on the rise despite intense ongoing research. We aimed to elucidate novel pathways involved in the pathogenesis of asthma. Employing asthma models induced by different allergens (ovalbumin and Aspergillus fumigatus), we uncovered the involvement of two members of the small proline-rich protein (SPRR) family, SPRR2a and SPRR2b, known to be involved in epithelial differentiation but not allergic disease. In situ hybridization revealed induction of SPRR2 signal in a subset of bronchial epithelial cells and mononuclear cells associated with inflammation after allergen challenge. Allergen-induced SPRR2 mRNA accumulation in the lung occurred in a time-dependent manner, with peak expression 10-96 h after a second ovalbumin challenge. Transgenic overexpression of interleukin (IL)-13 in the lungs resulted in a marked increase of SPRR2 expression, and allergen-induced SPRR2 expression was significantly decreased in IL-13-deficient mice. Studies in gene-targeted mice revealed that allergen-induced SPRR2 was dependent upon STAT6. Finally, we aimed to determine if the induction of SPRR2 by allergen was tissue specific. Notably, SPRR2 was markedly increased in the small intestine after induction of allergic gastrointestinal inflammation. Thus, SPRR2 is an allergen- and IL-13-induced gene in experimental allergic responses that may be involved in disease pathophysiology.

Epidemiologic studies have suggested that exposure to airborne particulate matter (PM) can exacerbate allergic airway responses; however, the mechanism(s) are not well understood. We and others have recently shown that development of airway hyperresponsiveness (AHR) may be a complementmediated process. In the present study, we examined the role of complement factor 3 (C3) in the development of PMinduced AHR and airway inflammation by comparing responses between C3-deficient (C3 � / � ) and wild-type mice. Mice were exposed to 0.5 mg of ambient particulate collected in urban Baltimore. Forty-eight hours later, airway responsiveness to intravenous acetylcholine was assessed and bronchoalveolar lavage was conducted. PM exposure of wild-type mice resulted in significant increases in AHR, whereas it did not significantly increase airway reactivity in C3 � / � mice. Interestingly, PM induced similar inflammatory responses in both wildtype and C3 � / � mice. Immunohistochemical staining demonstrated marked C3 deposition in the airway epithelium and connective tissue of wild-type mice after PM exposure. These results suggest that exposure to PM may induce AHR through activation of complement factor 3 in the airways. The morbidity and mortality of asthma has increased dramatically over the last few decades. Although the factors that contribute to this increase are not well understood, epidemiologic studies have suggested that increased exposure to air pollutants such as ozone and particulate matter (PM) are clearly associated with increased hospitalizations, increased asthma medication usage, and decrements in pulmonary function (1, 2). Animal studies have supported a role for PM exposure in development of asthmalike parameters, in that exposure of animals to PM surrogates such as residual oil fly ash (ROFA) and diesel PM induce significant airway inflammation concomitant with increases in airway responsiveness (3‐6). To date, the exact mechanisms by which PM exposure can induce acute bronchoconstriction and airway hyperresponsiveness (AHR) are unknown. Upon activation of the complement system, cleavage of the third and fifth components of the complement system (C3 and C5, respectively) generates the peptides C3a and C5a, both of which are potent anaphylatoxins. C3a and C5a can trigger contraction of smooth muscle, increase the permeability of small blood vessels, and regulate vasodilation (reviewed in Ref. 7). In addition, complement factors are potent chemoattractants for a variety of inflammatory cells including neutrophils, eosinophils, and macrophage/ monocytes. Recent studies suggest that these molecules of the innate immune response are produced by airway epithelial cells and macrophages at the airway surface (8, 9). Receptors for both of these anaphylatoxins (C5aR and C3aR) have been shown to be constitutively expressed on bronchial and alveolar epithelial cells, as well as on vascular endothelial and smooth muscle cells (10). Recent reports suggest that allergen exposure upregulates C3aR expression (11) on bronchial smooth muscle cells. Furthermore, recent studies in C3aR- and C3-deficient mice support a role for C3 in antigen-induced AHR (11, 12). However, the role of complement components in non‐antibody-mediated inflammatory responses has not been explored. We have previously shown that PM collected in urban Baltimore induces AHR concomitant with significant elevations in bronchoalveolar lavage (BAL) cellularity (13). In the present study, we examined the role of C3 in AHR and inflammation induced by ambient Baltimore PM by comparing airway reactivity and inflammatory responses in wild-type and C3 � / � mice. We demonstrate that particulate-induced AHR is C3-dependent, whereas the accompanying inflammatory response is independent of C3 activation.

Approximately 3 billion people-half the worldwide population-are exposed to extremely high concentrations of household air pollution due to the burning of biomass fuels on inefficient cookstoves, accounting for 4 million annual deaths globally. Yet, our understanding of the pulmonary responses to household air pollution exposure and the underlying molecular and cellular events is limited. The two most prevalent biomass fuels in India are wood and cow dung, and typical 24-hour mean particulate matter (PM) concentrations in homes that use these fuels are 300 to 5,000 μg/m(3). We dissected the mechanisms of pulmonary responses in mice after acute or subchronic exposure to wood or cow dung PM collected from rural Indian homes during biomass cooking. Acute exposures resulted in robust proinflammatory cytokine production, neutrophilic inflammation, airway resistance, and hyperresponsiveness, all of which were significantly higher in mice exposed to PM from cow dung. On the contrary, subchronic exposures induced eosinophilic inflammation, PM-specific antibody responses, and alveolar destruction that was highest in wood PM-exposed mice. To understand the molecular pathways that trigger biomass PM-induced inflammation, we exposed Toll-like receptor (TLR)2-, TLR3-, TLR4-, TLR5-, and IL-1R-deficient mice to PM and found that IL-1R, TLR4, and TLR2 are the predominant receptors that elicit inflammatory responses via MyD88 in mice exposed to wood or cow dung PM. In conclusion, this study demonstrates that subchronic exposure to PM collected from households burning biomass fuel elicits a persistent pulmonary inflammation largely through activation of TLR and IL-1R pathways, which could increase the risk for chronic respiratory diseases.

Airborne particulate matter (PM) is hypothesized to play a role in increases in asthma prevalence, although a causal relationship has yet to be established. To investigate the effects of real-world PM exposure on airway reactivity (AHR) and bronchoalveolar lavage (BAL) cellularity, we exposed naive mice to a single dose (0.5 mg/ mouse) of ambient PM, coal fly ash, or diesel PM. We found that ambient PM exposure induced increases in AHR and BAL cellularity, whereas diesel PM induced significant increases in BAL cellularity, but not AHR. On the other hand, coal fly ash exposure did not elicit significant changes in either of these parameters. We further examined ambient PM-induced temporal changes in AHR, BAL cells, and lung cytokine levels over a 2-wk period. Ambient PM-induced AHR was sustained over 7 d. The increase in AHR was preceded by dramatic increases in BAL eosinophils, whereas a decline in AHR was associated with increases in macrophages. A Th2 cytokine pattern (IL-5, IL-13, eotaxin) was observed early on with a shift toward a Th1 pattern (IFN-gamma). In additional studies, we found that the active component(s) of ambient PM are not water-soluble and that ambient PM-induced AHR and inflammation are dose- dependent. We conclude that ambient PM can induce asthma-like parameters in naive mice, suggesting that PM exposure may be an important factor in increases in asthma prevalence.

Because mouse strains susceptible to bleomycin, such as C57BL/ 6J, tend to produce T helper type 1 (Th1) cytokines in response to immune activation, we hypothesized that the inflammatory response to bleomycin is mediated, in part, by local production of the Th1 cytokine interferon-gamma (IFN-gamma). Consistent with this hypothesis, fibrosis-prone C57BL/6J and A/J mice demonstrated significantly elevated expression of IFN-gamma protein (by enzyme-linked immunosorbent assay) in bronchoalveolar lavage fluid at 24 h, and subsequently increased lung inflammation, weight loss, and mortality 10 d after intratracheal bleomycin administration compared with fibrosis-resistant BALB/c mice or saline control mice. To directly determine a role for IFN-gamma in bleomycin toxicity, we exposed C57BL/6J mice with a homozygous null mutation of the IFN-gamma gene (IFN-gamma[-/-]) and wild-type C57BL/6J mice to intratracheal bleomycin. IFN-gamma(-/-) mice demonstrated significantly lower parenchymal inflammation, weight loss, and mortality 10 d after 5 U/kg intratracheal bleomycin administration compared with control mice. At 3 wk after 1.5 U/kg bleomycin exposure, single lung collagen determined by hydroxyproline assay was significantly lower in IFN-gamma(-/-) mice compared with wild-type C57BL/6J mice. Together, these results suggest that IFN-gamma mediates, in part, bleomycin-induced pulmonary inflammation and fibrosis.

Identification of the genetic loci underlying asthma in humans has been hampered by variability in clinical phenotype, uncontrolled environmental influences, and genetic heterogeneity. To circumvent these complications, the genetic regulation of asthma-associated phenotypes was studied in a murine model. We characterized the strain distribution patterns for the asthma-related phenotypes airway hyperresponsiveness (AHR), lung eosinophils, and ovalbumin (OVA)-specific serum immunoglobulin (Ig) E induced by allergen exposure protocols in A/J, AKR/J, BALB/cJ, C3H/HeJ, and C57BL/6J inbred strains and in (C3H/HeJ x A/J)F1 mice. Expression of AHR differed between strains and was sometimes discordant with lung eosinophils or serum IgE. Furthermore, we identified two distinct quantitative trait loci (QTL) for susceptibility to allergen-induced AHR, Abhr1 (allergen-induced bronchial hyperresponsiveness) (lod = 4. 2) and Abhr2 (lod = 3.7), on chromosome 2 in backcross progeny from A/J and C3H/HeJ mice. In addition, a QTL on chromosome 7 was suggestive of linkage to this trait. These QTL differ from those we have previously found to control noninflammatory AHR in the same crosses. Elucidation of the genes underlying these QTL will facilitate the identification of biochemical pathways regulating AHR in animal models of asthma and may provide insights into the pathogenesis of human disease.

Airway hyperresponsiveness (AHR) is a fundamental aspect of asthma that has been shown to be influenced by both environmental and genetic factors. Antigen sensitization and challenge of the A/J inbred mouse strain induced AHR, eosinophilic airway inflammation, and lung goblet cell hyperplasia. We discuss the evidence that supports the role of T helper cells and their subsets in determining the airway inflammatory and contractile responses to antigen in a mouse model. Airway hyperresponsiveness and pulmonary eosinophilic inflammation induced by antigen challenge are associated with a Th2 pattern of cytokine expression in the murine lung. CD4+ T cells mediate the airway reaction to antigen, as depletion of CD4+ T cells attenuates the response. The presence of interleukin (IL)-4 induces the Th2 type of immune response, and this cytokine is required for mice to manifest AHR and inflammation to antigen. The Th1 type of immune response is stimulated by IL-12. Antigen-mediated AHR and inflammation are inhibited by IL-12 administration. Airway hyperresponsiveness in the noninflammatory state (without antigen treatment) is inherited in A/J and C3H/HeJ inbred mouse strains. One quantitative trait locus for AHR in progeny derived from these strains is located on murine chromosome 6. We propose that antigen-inducd AHR and inflammation also have heritable components. Based on the available immunological data, genes that influence the balance between Th1 and Th2 cells are logical candidate genes for antigen-induced AHR and inflammation. Knowledge of the genes that determine this phenotype will help us understand the mechanisms of human asthma.

Inhalation of elevated levels of ozone produces a potent inflammatory response in the lung. The magnitude of this response to ozone exposure in mice is inbred strain dependent with the susceptible phenotype being exemplified by the C57BL/6J (B6) strain and the resistant phenotype by the C3H/HeJ (C3) strain. To examine the role of T lymphocytes in the regulation of ozone-induced pulmonary inflammation, mice were pretreated by an intraperitoneal injection of anti-Thy1.2 monoclonal antibody (mAb), anti-CD4+ mAb, or isotype-matched control antibodies (0.5 mg each) and subsequently exposed for 72 h to either filtered air or ozone (0.3 ppm). Immediately after ozone exposure, the cellular profile in the bronchoalveolar lavage fluids (BALF) was assessed. In isotype-treated controls of both strains of mice, ozone exposure induced significant increases in the numbers of macrophages, neutrophils, lymphocytes, and epithelial cells recovered in the BALF; however, the magnitude of each cell type recovered was significantly greater in B6 mice as compared with C3 mice. Both anti-Thy1.2 and anti-CD4+ monoclonal antibody treatments decreased the number of each cell type recovered in the B6 mice and increased the number of cells in the C3 mice. To determine if the CD4+ T-cell-derived cytokine interleukin (IL)-4 was involved in the differential effect of T-cell depletion on the ozone-induced inflammatory responses of C3 and B6 mice, mice were pretreated with either 400 ng of recombinant mouse IL-4 or vehicle, or 5.0 mg anti-IL-4 receptor monoclonal antibody or an isotype-matched antibody before either air or ozone exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

The pathogenesis of asthma remains unclear. An in vivo murine model of antigen-induced airway hyperreactivity and inflammation was developed to investigate the possibility, suggested by a wealth of descriptive human data, that alterations in immunoregulation are important in the genesis of airway hyperreactivity. A/J mice developed airway hyperreactivity and markedly increased numbers of pulmonary inflammatory cells following intraperitoneal sensitization and intratracheal challenge with sheep red blood cells. Notably, eosinophils were a prominent component of the inflammatory infiltrate. The dependence of these phenomena, both pathologic and functional, on CD4+ T lymphocytes was investigated by in vivo depletion of CD4+ cells using the anti-CD4 mAb GK1.5. When administered before antigen challenge, GK1.5 completely prevented both airway hyperreactivity and the infiltration of eosinophils. This model provides the first direct demonstration of the dependence of airway hyperreactivity upon CD4+ T lymphocytes, and the results are consistent with the possibility that eosinophils are effectors of this response.

The challenge of previously sensitized guinea pigs with aerosolized ovalbumin resulted in impairment of the beta-adrenoceptor-mediated relaxation as measured by the in vitro isometric assay of tracheas preconstricted with endothelin-1 or carbamylcholine. Numbers and affinities of beta-adrenoceptors in lung membranes of these animals were not altered under these conditions, although the antigen challenge caused an inflammatory response, as evident from the accumulation of inflammatory cells in the bronchoalveolar lavage fluids. In order to investigate the pathophysiologic role of inflammation in hyperreactive airways, isolated guinea pig tracheas were cultured with proinflammatory cytokines such as human recombinant tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), or interleukin-2 (IL-2). None of these cytokines affected the contractile response of tracheas to carbamylcholine. After preconstriction with carbamylcholine, the TNF-alpha- and IL-1 beta-pretreated tissues produced a significant reduction in the maximal relaxation induced by isoproterenol, whereas the IL-2 pretreatment had no effect. The reduction of the isoproterenol-mediated relaxation by the IL-1 beta treatment was time and dose dependent. Our present observations suggest that in vitro incubation of naive tracheas with proinflammatory cytokines is able to reproduce apparent beta-adrenoceptor impairment as seen in the airways of antigen-challenged guinea pigs of asthma model.

The epithelium of the respiratory mucosa provides a barrier against injurious luminal agents, including bacteria, enzymes, and toxins. The normal respiratory epithelium is coated with mucus, which provides a variety of protective functions, including protection of the lower airways from dehydration and from damaging airborne irritants, particles, and microorganisms. The adhesive and viscoelastic properties of mucus glycoproteins (mucins), the major protein components of airway mucus, allow the trapping of foreign substances and their transport and removal on the tips of beating cilia toward the throat, a process termed mucociliary clearance. However, overzealous production of mucus may significantly contribute to the morbidity and mortality associated with certain respiratory diseases. In particular, mucus hypersecretion and plugging of the airways are characteristic features of patients who die from asthma (1, 2), chronic bronchitis, and cystic fibrosis (2). In human airways, mucins are produced and secreted by specialized cells in the epithelium, including the goblet cells in the surface airway epithelium and the secretory (mucous and serous) cells in the submucosal glands. Because of their greater prominence in histologic sections, submucosal glands, rather than goblet cells, have been thought to contribute the greater quantity of mucus to airway surface fluid (3). However, recent studies suggest that goblet cells may contribute more to the overall quantity of mucus produced than do the submucosal glands. Mucins constitute a heterogeneous group of high molecular weight, richly glycosylated molecules. To date, nine human mucin genes ( MUC1 , MUC2 , MUC3 , MUC4 , MUC5/5AC , MUC5B , MUC6 , MUC7 , and MUC8 ) encoding the protein core of mucin have been identified (reviewed in Reference 4). The biologic importance of these diverse mucin proteins is not currently known. Nonetheless, there appears to be some degree of specificity in the tissue expression of the various MUC genes. In the respiratory tract, seven of the nine MUC genes are expressed ( MUC1 , MUC5/5AC , MUC2 , MUC4 , MUC5B , MUC7 , and MUC8 ) (reviewed in Reference 4). The mechanisms governing mucin glycoprotein synthesis and secretion are not well understood in either health or disease. Control of mucus secretion is a complex process involving regulation at many different levels, including ( 1 ) cell proliferation and differentiation, ( 2 ) mucin gene expression, and ( 3 ) release of mature mucin molecules from storage granules (3). Under normal conditions, there are relatively few goblet cells in the human airway and virtually none in the airways of animals kept in clean environments. In response to a wide variety of stimuli, including proteinases, irritant gases, inflammatory mediators, reactive oxygen species, and cholinergic and nonadrenergic, noncholinergic nerve activation, a rapid increase in the number of airway goblet cells is observed via both hyperplastic (cell division) and metaplastic (cell differentiation) mechanisms. In the airways of rodents, these increases are primarily due to metaplasia, since goblet cells are not normally seen in the airways of clean animals. Secondly, MUC gene transcription has been shown to be induced upon exposure of the airways to a number of substances that induce mucus secretion, such as endotoxin, SO 2 , and allergens (5, 6). Thus, it is hypothesized that an important point of control of mucus secretion is the synthesis of these protein backbones of the mature mucin glycoproteins. Although the exact molecular mechanisms regulating mucin-gene expression are virtually unknown, there is evidence that the individual genes may be differentially regulated. For example, Muc-2 expression is induced in the lungs of rats exposed to SO 2 and Sendai virus, but not by allergen exposure (5, 6). Conversely, allergen challenge has been shown to induce MUC5 gene expression (6). Lastly, secretion of mature mucin molecules requires their release from the intracellular granules in which they are stored. To date, there are a number of inflammatory mediators implicated in the allergic diathesis that are known to influence goblet-cell secretion, including the prostaglandins E2 and F2a, leukotrienes, 15-HETES, platelet-activating factor, mast-cell and neutrophil proteases, eosinophil cationic protein, and the cytokines interleukin (IL)-1 and tumor necrosis factor (TNF) (3). As stated above, mucus hypersecretion is a key feature of allergic asthma and is associated with the clinical symptoms, airway obstruction, and mortality of the disease. Because of the difficulty of studying molecular processes in the human lung, much of our current knowledge of mucus regulation has come from the study of murine models of allergic disease. Recently, several groups of investigators have shown that respiratory challenge with allergens causes physiologic and pathologic changes similar to those seen in human allergic asthma, including airway hyperresponsiveness, airway inflammation, and airway goblet-cell metaplasia (GCM), as evidenced by periodic acid-Schiff (PAS) ( Received in original form May 3, 2000 )