The Harry M. Zweig Memorial Fund for Equine Research

The Role of the Pulmonary Macrophages in Initiating the Inflammatory Response of Recurrent Airway Obstruction

Dr. Dorothy M. Ainsworth

The problem. Recurrent airway obstruction (RAO) or “heaves” is an age-old respiratory disorder that develops in certain mature horses that are fed hay. The chronic inflammatory process is characterized by constriction of the small airways, excess production of mucus and accumulation of neutrophils in the airways. The severity of the disease can be reduced by eliminating hay exposure and by keeping the horse outside all of the time. Resolution of the pulmonary inflammation is best achieved by glucocorticoid administration (Ainsworth et al., 2003a). Unfortunately, for performance horses traveling the show circuit, neither husbandry alterations nor steroid administration are feasible or permissible. Ideally, a physiological approach to the treatment and/or prevention of RAO—that is natural and permissible for show animals—is needed. In order to develop such a therapeutic approach, the events underlying the development of the pulmonary inflammation must be understood. This has been and continues to be the goal of our laboratory investigations.

Challenging the dogma. For years, the inflammation of RAO was assumed to reflect an allergic reaction to inhaled dusts or molds found in poorly-cured hay. Advocates of the “allergic theory” likened the equine condition to asthma in humans. Although RAO shares many clinical features of asthma—bronchoconstriction and excessive mucus production—the inflammatory cells plugging the airways in horses with RAO consist of neutrophils and not eosinophils, as found in human asthmatics. In a series of studies recently completed in our laboratory (Ainsworth et al., 2003b), we tested the hypothesis that RAO is an allergic airway disorder and found evidence to the contrary! Molecular biology techniques were used to measure the gene expression of inflammatory mediators in cells isolated from the airways. The mediators, or cytokines, typically associated with allergies and asthma, are interleukin-4 (IL-4) and IL-13. In contrast to what occurs in human asthmatics, IL-4 and IL-13 concentrations were not elevated in the chronically-diseased horses . Instead, another cytokine called interferon-gamma (IFN-?), as well as a neutrophil signaling cytokine called interleukin-8, were increased. Interestingly, we also found that the neutrophil counts in the airways of RAO-prone horses that were studied when asymptomatic or after they had been exposed to dusty hay for only 24 hours, always exceeded those of the controls. This increase in airway neutrophils occurred in the absence of increases in the IFN-? or IL-8!

The significance of this study is that it provides strong evidence that allergen skin testing and desensitization techniques have absolutely no scientific basis in the diagnosis and treatment of RAO. The 2 nd significant finding is that other sources of neutrophil signaling proteins must exist. The most likely candidate is the airway epithelial cell.

The airway epithelium—not an innocent by-stander! During the past 10 months, studies in our lab have specifically examined the role of the airway lining cell, the epithelium, in initiating and in maintaining the inflammation of RAO. Traditionally the airway epithelium has been considered to be a passive tissue that merely provides a physical barrier between the environment (inhaled particulates) and the internal milieu of the body. However, studies conducted in humans with asthma or COPD have demonstrated that the epithelium is capable of synthesizing a multitude of signaling proteins and cytokines. These proteins contribute to migration of cells into the airways and to remodeling of the lung tissue.

Data generated in our laboratory (Zweig funded studies) have shown that in horses chronically-affected with RAO (weeks in duration), the gene expression of IL-8 in the airway epithelium is 3-fold higher than that found in healthy horses. This suggests that the epithelium is an important source for signaling of inflammatory cells into the airways of horses. The question of what is signaling the airway epithelium to produce IL-8 in RAO-affected horses is the focus of this year's proposal. As demonstrated in the figure shown below, we hypothesize that the pulmonary macrophage, a cell whose function is to “process” inhaled molds, viruses, bacteria, parasites and particulates, is the pivotal player in establishing this over-exuberant inflammatory response. In its encounter with inhaled dusts and molds in the hay, the macrophage produces and secretes interleukin-23. This cytokine targets lymphocytes (immune cells) to increase production of interleukin-17. This cytokine serves as the link between the airway lymphocytes and the epithelial lining cell and induces the epithelium to produce IL-8. [Note in the schematic that although the macrophage can produce IL-8, as discussed earlier, this cell does not increase its synthesis in RAO-susceptible horses that are asymptomatic or that are exposed to hay dust for 24 hours].

In studies proposed for year 2004, our laboratory would measure the gene expression of IL-17, IL-23, IL-8 and IFN-? in airway cells isolated from healthy horses and from RAO-susceptible horses during the asymptomatic period and following acute and chronic exposure to hay dust. In the second year of the study, we would isolate macrophages from those same horses, establish cell cultures and expose the cells ( in vitro ) to increasing concentrations of a hay dust solution. The hay dust solution would be prepared from hay used to induce the disorder. (The supernatants from the cell cultures, containing IL-23, would be saved for later analysis). The gene expression of IL-23 would be measured in the macrophage cultures. This set of experiments would test the hypothesis that increasing concentrations of hay dust increase the gene expression and production of IL-23 in the macrophages and that the macrophages isolated from RAO-susceptible horses are more sensitive (produce more IL-23) to hay dust exposure than macrophages isolated from healthy horses that never develop RAO.

These studies will improve our understanding of the mechanisms inducing the inflammation of RAO and provide sites by which physiological interventions may be designed and implemented.

Inhaled hay dust

Figure 1: Overview of major cells and cytokines involved in the development of RAO.

Ainsworth DM, Grunig G, Matychak MB, Young J, Erb HN, Antczak, DF. The effect of feeding alterations and dexamethasone administration on cytokine mRNA profiles in bronchoalveolar lavage cells of horses with recurrent airway obstruction. (Submitted Eq. vet J, 2003a).

Ainsworth D.M. , Grunig G, Matychak MB, Young J, Erb NH, Antczak DF. Recurrent airway obstruction in horses is characterized by IFN-? and IL-8 production. Vet Immunol Immunopath 96: 83-91, 2003b).