CINCINNATI—A study using a broad molecular approach has highlighted a specific biochemical pathway’s previously unrecognized potential importance in asthma etiology: Gene microarray technology has revealed up-regulation of the enzyme arginase in two distinct models of experimental asthma, as well as in the lungs of asthma patients.[1] Up-regulation of arginase occurs in response not only to allergens but also to interleukin 4 (IL-4) and IL-13, cytokines thought to be central to development of the asthma phenotype. The findings point to new targets for asthma therapy.

In an effort to determine which genes are active in the asthma disease process, principal author Marc E. Rothenberg, MD, PhD, and coworkers used DNA microarray analysis to detect sets of genes up-regulated in the lungs of mice in two experimental models of asthma. “The asthma genome is different depending upon which asthma model you examine,” observed Dr. Rothenberg, Professor of Pediatrics at the University of Cincinnati and Director of the Division of Allergy and Immunology at Children’s Hospital Medical Center in Cincinnati. This suggests that “patients with very different causes for asthma may have significant differences in the genes and pathways involved,” he said.

Nevertheless, reported Dr. Rothenberg, “We have identified a set of asthma-associated genes—291 so-called signature genes” common to both models. He added, “We also identified a potentially critical role for arginase” in asthma etiology. Because this enzyme is dramatically up-regulated in both types of experimental asthma, the data suggest that “arginase may be involved in asthma … independent of the underlying cause,” noted Dr. Rothenberg.

In one model of asthma, mice were sensitized with intraperitoneal injections of ovalbumin combined with an alum adjuvant. They subsequently received two separate intranasal ovalbumin challenges. In the second, mice were sensitized to Aspergillus antigen through repeated intranasal exposure. Both experimental models yielded eosinophilic inflammation, mucus production, and airway hyperresponsiveness—hallmarks of asthma.

Microarray analysis demonstrated induction of 496 and 527 genes in the ovalbumin and Aspergillus models, respectively. Although some of these genes were specific to each sensitization protocol, the majority of the induced transcripts overlapped. This core group of 291 genes induced in both experimental asthma models, the authors argue, probably reflects pathways of asthma pathogenesis, rather than allergen-specific responses.

Among the transcripts up-regulated most dramatically were three genes involved in arginine metabolism: arginase I, arginase II, and the cationic amino acid transporter 2 (CAT2). Of particular note was the fact that arginase I was up-regulated in perivascular and peribronchial pockets of inflammation within the lungs of asthmatic mice; specifically, arginase I was expressed in alveolar macrophages.

Figure 1 How Arginine Metabolism May Be Involved in Asthma Etiology

Genes for both the cationic amino acid transporter 2 (CAT2) and arginase can be induced in experimental asthma. Elevated CAT2 activity increases availability of arginine (depicted at left), the substrate for both arginase and nitric oxide synthase (NOS). Arginase competes with NOS for arginine, thereby limiting NO production and possibly affecting bronchoconstriction. In addition, products of arginase may affect cell growth and differentiation, as well as collagen synthesis. (Adapted from Zimmerman et al. J Clin Invest. 2003.[1])

TH2 CYTOKINES REGULATE ARGINASE

Because experimental expression of IL-4 can trigger features of asthma, the researchers examined arginase induction in transgenic mice overexpressing this cytokine in the lung. These mice showed increased expression of arginase I. However, if IL-4–overexpressing mice lacked STAT6, a downstream signaling protein known to mediate responses to IL-4 and IL-13, arginase I was not induced. In contrast, arginase II expression seemed largely STAT6 independent. When sensitized to ovalbumin, STAT6-deficient mice showed 90% lower arginase I induction than did wild-type asthmatic mice, demonstrating that asthma-associated increases are predominantly mediated by the arginase I gene.

ARGINASE EXPRESSION MIRRORS HYPERRESPONSIVENESS

Because experimental intranasal application of IL-13 can produce features of asthma such as eosinophilic inflammation, chemokine induction, mucus production, and airway hyperresponsiveness, the researchers tested its effects on arginase expression. IL-13 administration induced arginase I and, to a lesser extent, arginase II within 12 hours—timing paralleling the development of IL-13–induced airway hyperresponsiveness but preceding IL-13–induced leukocyte recruitment.

ARGINASE INDUCED IN HUMAN ASTHMA

To test the clinical relevance of these experimental findings, the researchers examined bronchoalveolar lavage and bronchial biopsy samples from patients with or without asthma. As compared with nonasthmatic samples, lavage samples from asthma patients contained a higher proportion of cells expressing arginase I. These arginase-expressing cells appeared to be mononuclear macrophages. In situ hybridization revealed strong arginase I expression in biopsy samples from asthma patients but barely detectable expression in nonasthmatic samples. Specifically, expression was observed in submucosal inflammatory cell infiltrates as well as in patches of epithelial cells within the lung.

WHAT DOES ARGINASE DO?

Increased arginase activity could enhance production of polyamines, small signaling molecules that are important in cell growth and differentiation, Dr. Rothenberg pointed out. As Figure 1 demonstrates, the product of arginase, L-ornithine, is a precursor of polyamines involved in cell growth and differentiation. Effects on cell growth could influence smooth muscle, fibroblasts, and lymphocytes, while effects on cell differentiation could increase mucus production associated with asthma, Dr. Rothenberg explained.

Polyamines are also linked with increased smooth muscle contractility, which could worsen bronchoconstriction. He added, “Arginase also regulates proline generation, which affects collagen production, … important for the fibrosis associated with asthma.”

Another potential effect of increased arginase activity stems from the fact that the enzyme shares a substrate with nitric oxide synthase (NOS) (shown in the illustration above). “Arginase and NOS compete for arginine, and therefore, induction of one indirectly affects the other,” Dr. Rothenberg pointed out. “Thus, elevated arginase likely lowers NO production—and its bronchodilatory action.” Reduced NOS activity is a phenomenon implicated in enhanced airway hyperresponsiveness in experimental asthma.

FUTURE THERAPEUTIC TARGETS?

Although specific inhibitors of arginase I activity exist, the enzyme’s importance in the liver and other organs might be problematic. Theoretically, said Dr. Rothenberg, an arginase inhibitor “would be helpful, especially if you deliver the drug directly to the lung and [do] not affect liver arginase.”

As an alternative, however, agents that block elements upstream of arginase I induction might be more fruitful targets for therapy. For example, “IL-13 appears to be important, but it synergizes with IL-4,” he noted. “Both require STAT6 for arginase induction.”

—Mimi Zucker, PhD

Reference
1. Zimmerman N, King NE, Laporte J, et al. Dissection of experimental asthma with DNA microarray analysis identifies arginase in asthma pathogenesis. J Clin Invest. 2003;111:1863-1874.

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