Publications

Loss of function mutations in the autoimmune regulator (Aire) gene in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy patients and mutant mice lead to autoimmune manifestations that segregate as a monogenic trait, but with wide variation in the spectrum of organs targeted. To investigate the cause of this variability, the Aire knockout mutation was backcrossed to mice of diverse genetic backgrounds. The background loci strongly influenced the pattern of organs that were targeted (stomach, eye, pancreas, liver, ovary, thyroid, and salivary gland) and the severity of the targeting (particularly strong on the nonobese diabetic background, but very mild on the C57BL/6 background). Autoantibodies mimicked the disease pattern, with oligoclonal reactivity to a few antigens that varied between Aire-deficient strains. Congenic analysis and a whole genome scan showed that autoimmunity to each organ had a distinctive pattern of genetic control and identified several regions that controlled the pattern of targeting, including the major histocompatibility complex and regions of Chr1 and Chr3 previously identified in controlling type 1 diabetes.

The extracellular Ca(2+)-sensing receptor (CaR) plays an essential role in mineral homeostasis. Studies to generate CaR-knockout (CaR(-/-)) mice indicate that insertion of a neomycin cassette into exon 5 of the mouse CaR gene blocks the expression of full-length CaRs. This strategy, however, allows for the expression of alternatively spliced CaRs missing exon 5 [(Exon5(-))CaRs]. These experiments addressed whether growth plate chondrocytes (GPCs) from CaR(-/-) mice express (Exon5(-))CaRs and whether these receptors activate signaling. RT-PCR and immunocytochemistry confirmed the expression of (Exon5(-))CaR in growth plates from CaR(-/-) mice. In Chinese hamster ovary or human embryonic kidney-293 cells, recombinant human (Exon5(-))CaRs failed to activate phospholipase C likely due to their inability to reach the cell surface as assessed by intact-cell ELISA and immunocytochemistry. Human (Exon5(-))CaRs, however, trafficked normally to the cell surface when overexpressed in wild-type or CaR(-/-) GPCs. Immunocytochemistry of growth plate sections and cultured GPCs from CaR(-/-) mice showed easily detectable cell-membrane expression of endogenous CaRs (presumably (Exon5(-))CaRs), suggesting that trafficking of this receptor form to the membrane can occur in GPCs. In GPCs from CaR(-/-) mice, high extracellular [Ca(2+)] ([Ca(2+)](e)) increased inositol phosphate production with a potency comparable with that of wild-type GPCs. Raising [Ca(2+)](e) also promoted the differentiation of CaR(-/-) GPCs as indicated by changes in proteoglycan accumulation, mineral deposition, and matrix gene expression. Taken together, our data support the idea that expression of (Exon5(-))CaRs may compensate for the loss of full-length CaRs and be responsible for sensing changes in [Ca(2+)](e) in GPCs in CaR(-/-) mice.

Mucus hypersecretion is a prominent manifestation in patients with chronic inflammatory airway diseases and contributes to their morbidity and mortality by plugging airways and causing recurrent infections. Human neutrophil elastase (HNE) exists in high concentrations (1-20 microM) in airway secretions of these patients and induces overproduction of MUC5AC mucin, a major component of airway mucus. Previous studies showed that HNE induces MUC5AC mucin production involving reactive oxygen species (ROS) generation and TGF-alpha-dependent epidermal growth factor receptor (EGFR) activation in human airway epithelial cells. However, the molecular mechanisms involved in these responses are not defined. TNF-alpha-converting enzyme (TACE) cleaves pro-TGF-alpha into soluble TGF-alpha and can be activated by ROS. We hypothesize that HNE activates TACE via ROS generation, resulting in cleavage of pro-TGF-alpha, EGFR activation, and MUC5AC mucin expression in airway epithelial cells. Here we show that in human airway epithelial cells HNE increases TGF-alpha release, EGFR phosphorylation, and MUC5AC mucin expression, effects that were attenuated by TACE inhibitor TAPI-1 and by specific knockdown of TACE expression with small interfering RNA, implicating TACE in HNE-induced responses. These responses to HNE were also reduced by pretreatment with ROS scavengers, implicating ROS. Furthermore, we show that HNE causes protein kinase C (PKC) activation and translocation from cytosol to plasma membrane; blockade of this effect by PKC inhibitors reduced HNE-induced ROS generation and other responses, implicating PKC. We conclude that HNE induces MUC5AC mucin expression via a cascade involving PKC-ROS-TACE in human airway epithelial cells.

Excessive mucus production is an important pathological feature of asthma. The Th2 cytokines IL-4 and IL-13 have both been implicated in allergen-induced mucus production, inflammation, and airway hyperreactivity. Both of these cytokines use receptors that contain the IL-4Ralpha subunit, and these receptors are expressed on many cell types in the lung. It has been difficult to determine whether allergen-induced mucus production is strictly dependent on direct effects of IL-4 and IL-13 on epithelial cells or whether other independent mechanisms exist. To address this question, we used a cell type-specific inducible gene-targeting strategy to selectively disrupt the IL-4Ralpha gene in Clara cells, an airway epithelial cell population that gives rise to mucus-producing goblet cells. Clara cell-specific IL-4Ralpha-deficient mice and control mice developed similar elevations in serum IgE levels, airway inflammatory cell numbers, Th2 cytokine production, and airway reactivity following OVA sensitization and challenge. However, compared with control mice, Clara cell-specific IL-4Ralpha-deficient mice were nearly completely protected from allergen-induced mucus production. Because only IL-13 and IL-4 are thought to signal via IL-4Ralpha, we conclude that direct effects of IL-4 and/or IL-13 on Clara cells are required for allergen-induced mucus production in the airway epithelium.

RATIONALE

Macrophages are believed to play a central role in emphysema based largely on data from mouse models. However, the relevance of these models to smoking-related lung disease in humans is uncertain.

OBJECTIVES

We sought to comprehensively characterize the effects of smoking on gene expression in human alveolar macrophages and to compare these with effects seen in transgenic mouse models of emphysema.

METHODS

We used DNA microarrays with genomewide coverage to analyze alveolar macrophages from 15 smokers, 15 nonsmokers, and 15 subjects with asthma (disease control). Selected gene expression changes were validated by polymerase chain reaction and ELISA. Expression changes were compared with those identified by microarray analysis of interleukin-13-overexpressing and integrin-beta6-deficient mice, which both develop emphysema.

MEASUREMENTS AND MAIN RESULTS

All 15 smokers shared a common pattern of macrophage gene expression that distinguished them from nonsmokers, a finding not observed in subjects with asthma. We identified 110 genes as differentially expressed in smokers despite using conservative statistical methods. Matrix metalloproteinase 12, a proteinase that plays a critical role in mouse models, was the third most highly induced gene in smokers (ninefold, p < 0.0001). However, most changes in smokers were not reflected in mouse models. One such finding was increased osteopontin expression in smokers (fourfold, p = 0.006), which was confirmed at the protein level and correlated with the degree of airway obstruction.

CONCLUSIONS

Smoking induces a remarkably consistent and distinctive pattern of alveolar macrophage activation. These studies identify aspects of mouse models that are directly relevant to human smokers and also reveal novel potential mediators of smoking-related diseases.