This study focused on reducing the chronic rather than acute phase of allergic asthma. We show that targeting an alternative family of chemoattractants, namely, extracellular cyclophilins, can lead to a reduced persistence of leukocytes during the chronic phase, and also reduces the severity of subsequent acute asthma reactivation. These findings provide a potential novel target for reducing chronic inflammation in allergic asthma.
Allergic asthma is a complex disease of airway inflammation that results from a variety of genetic and environmental factors, and is characterized by acute airway constriction, the hypersecretion of mucus, and lung inflammation (1
). Although their exact immunopathophysiology is not clearly understood, acute asthmatic responses are known to be driven by specific subsets of leukocytes, namely, antigen-specific T cells and IgE-switched B-cells, mast cells, and eosinophils, as well as neutrophils in some cases (2
). Samples from the airways of patients with asthma show an increased presence of CD4+
T cells expressing the activation markers CD25 and major histocompatibility complex Class II (3
). These activated CD4+
T cells create a cytokine environment that favors the initiation and perpetuation of the asthmatic response, making them important regulators of disease pathogenesis (2
). The Th2-associated cytokines IL-4, IL-5, and IL-13 are found at increased concentrations in asthmatic airway cells and fluid, and they colocalize with T-cell markers in biopsies (7
). These Th2 cytokines promote the generation of IgE-secreting B-cells, and the mobilization and activation of eosinophils (9
). After they are recruited and activated, these leukocytes exacerbate inflammation by secreting a myriad of cytokines, chemokines, and enzymes, resulting in the additional recruitment of proinflammatory leukocytes, tissue damage, and airway constriction.
Allergic asthma is a chronic disease insofar as airway inflammation is never completely resolved, even in the absence of acute allergen challenge. One of the downstream effects of this unresolved inflammation involves the increased deposition of collagen, resulting in airway remodeling that can drastically reduce airway diameter (10
). Another hallmark of chronic asthma involves the sustained presence of proinflammatory leukocytes within airways and submucosal tissue during the quiescent (or chronic) phases of the disease, despite an absence of allergen. Indeed, elevated numbers of eosinophils, activated lymphocytes, and mast cells, as well as increased concentrations of IL-5, were found in biopsies of patients with asthma in clinical remission (11
). Along with promoting tissue pathology, the sustained presence of activated, antigen-specific T cells and other effector leukocytes within asthmatic airways and tissues creates an environment that is primed for rapid re-initiation upon allergen challenge. The factors regulating the observed persistence of leukocytes in the absence of allergen stimulation are unknown. However, insofar as most effector leukocytes have a limited lifespan (12
) and do not proliferate in situ
), the persistent airway inflammation seen during chronic asthma must involve recruitment stimuli to maintain an elevated numbers of leukocytes.
Obvious candidates that could regulate this recruitment comprise the chemokines known to be associated with asthma, including eotaxins 1–3, regulated upon activation, normal T-cell expressed and presumably secreted (RANTES), macrophage inflammatory protein (MIP)-1a, and monocyte chemotactic protein (MCP)-1, all of which were shown to increase after exposure to allergens. Although an acute burst of production of these classic chemokines occurs within 2–4 hours of exposure, they return to baseline concentrations within 24 hours (16
). In addition, studies in which patients with asthma were sampled during remission phases of their disease showed concentrations of chemokines similar to those in healthy control subjects, despite elevated numbers of eosinophils and T cells in their lung airways (11
). Similar findings were reported for eotaxin in a guinea pig model of asthma (18
), and for eotaxin, RANTES, MIP-1α, and MCP-1 in a murine model (19
). These observations demonstrate a timeline whereby the majority of chemokines associated with the recruitment of asthma-associated leukocytes, including T cells and eosinophils, are produced acutely after allergen challenge, but return to low, or even baseline, concentrations within 24 hours. This finding begs the question of how the recruitment of leukocytes may be regulated during the chronic phases of asthma, when acute allergen challenge is absent. Although low, residual concentrations of chemokines may be sufficient to mediate this recruitment, alternative types of chemoattractants may take over as regulatory factors.
Cyclophilins are present in high abundance in all eukaryotic cells (20
). Although cyclophilins exhibit many different functions (20
), they are probably best known as receptors for the immunosuppressive drug cyclosporine A (CsA) (21
). However, cyclophilins can also be secreted in response to inflammatory stimuli (22
), and high concentrations of extracellular cyclophilins were reported in several inflammatory diseases (24
). Interestingly, extracellular cyclophilins demonstrate potent chemoattractant properties both in vitro
) and in vivo
), suggesting a capacity to contribute to the recruitment of leukocytes during inflammatory responses. In support of this idea, we previously showed that blocking cyclophilin function in vivo
, using various nonimmunosuppressive analogues of CsA, reduced the recruitment of leukocytes into inflamed tissues by 40–80% in two different murine models of acute lung inflammation (27
). In one of these, an ovalbumin (OVA)–induced model of acute allergic asthma, the inhibition of cyclophilins resulted not only in a reduced influx of leukocytes into the airways and lung tissue, but also led to reductions in Th2-associated cytokines, mucus hypersecretion, and airway resistance (31
Although these studies demonstrated a significant role for cyclophilins in mediating the recruitment of leukocytes during the acute phase of an asthmatic response, their potential contribution to the chronic recruitment of leukocytes was not examined. Thus, in the present study, we investigated whether inhibiting the function of cyclophilins during the chronic phase of asthma would lead to a reduction in the persistence of pulmonary leukocytes, and whether this reduction would in turn exert an impact on the severity of subsequent acute responses upon allergen challenge (i.e., acute reactivation).
Several murine models of chronic allergic lung inflammation that mimic most of the hallmark features of chronic human asthma, including the persistence of leukocytes, were previously described (32
). Using a modified version of one of these models, we show here that: (1
) extracellular cyclophilins (but not classic chemokines) are present during the chronic phase of disease; (2
) inhibiting the function of cyclophilins during the chronic phase leads to a 45–80% reduction in the persistence of leukocytes; and (3
) inhibiting the function of cyclophilins during the chronic phase also results in a significant decrease in the severity of acute disease reactivation upon allergen challenge, including reduced influx of leukocytes, tissue pathology, and airway hyperreactivity.