IL-16 Promotes Leukotriene C4 and IL-4 Release from Human Eosinophils via CD4- and Autocrine CCR3-Chemokine-Mediated Signaling

Abstract

Human eosinophils are potential sources of inflammatory and immunomodulatory mediators, including cysteinyl leukotrienes, chemokines, and cytokines, which are pertinent to allergic inflammation. We evaluated the means by which IL-16, a recognized eosinophil chemoattractant, might act on eosinophils to affect their capacity to release leukotriene C4 (LTC4) or their preformed stores of chemokines (eotaxin, RANTES) or Th1 (IL-12) or Th2 (IL-4) cytokines. IL-16 dose dependently (0.01–100 nM) elicited new lipid body formation, intracellular LTC4 formation at lipid bodies, and priming for enhanced calcium ionophore-activated LTC4 release. IL-16 also elicited brefeldin A-inhibitable, vesicular transport-mediated release of preformed IL-4, but not IL-12, from eosinophils. CD4 is a recognized IL-16R, and accordingly anti-CD4 Fab, soluble CD4, and a CD4 domain 4-based IL-16 blocking peptide inhibited the actions of IL-16 on eosinophils. Although CD4 is not G-protein coupled, pertussis toxin inhibited IL-16-induced eosinophil activation. IL-16 actions were found to be mediated by the autocrine activity, not of platelet-activating factor, but rather of endogenous CCR3-acting chemokines. IL-16 induced the rapid vesicular transport-mediated release of RANTES. The effects of IL-16 were blocked by CCR3 inhibitors (met-RANTES, anti-CCR3 mAb) and by neutralizing anti-eotaxin and anti-RANTES mAbs, but not by platelet-activating factor receptor antagonists (CV6209, BN52021). RANTES and eotaxin each enhanced LTC4 and IL-4 (but not IL-12) release. Therefore, IL-16 activation of eosinophils is CD4-mediated to elicit the extracellular release of preformed RANTES and eotaxin, which then in an autocrine fashion act on plasma membrane CCR3 receptors to stimulate both enhanced LTC4 production and the preferential release of IL-4, but not IL-12, from within eosinophils. Interleukin-16 is a cytokine initially identified as a chemoattractant for CD4+ T cells and subsequently shown to be a chemoattractant for other CD4+ cells, including eosinophils (1, 2). IL-16 can be formed by varied cell types including CD4+ and CD8+ T cells, B cells, mast cells, dendritic cells, epithelial cells, fibroblasts, and eosinophils (1, 3, 4). IL-16 may have roles in diverse immunologic responses (1). In forms of allergic inflammation, including asthma, IL-16 has been found to be prominent. IL-16 was increased in the bronchial mucosa and sputum of patients with atopic asthma (5, 6) and in the bronchial mucosa and bronchoalveolar lavage fluids after segmental allergen challenge (7, 8). Likewise, IL-16 increases in the nasal mucosa of patients with allergic rhinitis after experimental or seasonal allergen exposures (9, 10). In murine models of asthma, administration of anti-IL-16 mAb prevented the production of Ag-specific IgE (11), and administration of an IL-16 blocking peptide inhibited Ag-induced airway hyperresponsiveness (12). IL-16 may participate in other forms of inflammation, including inflammatory bowel disease (13), in which eosinophils may be a source of IL-16 (14). The functions of IL-16 in these varied forms of immunologically mediated diseases may be complex. For instance, IL-16 can inhibit MLR- and anti-CD3-activated lymphocyte responses (15, 16), can participate in dendritic cell-T cell interactions (4, 17), can enhance pro-inflammatory cytokine release from monocytes (18), and can inhibit IL-5 production by Ag-stimulated T cells in atopic subjects (19). We had previously shown that IL-16 was a potent (ED50 ∼ 10−12 M) chemoattractant for human eosinophils and that this activity was dependent on CD4 expressed by eosinophils (2). IL-16, however, did not appear to enhance other responses of eosinophils, such as their capacity for enhanced leukotriene C4 (LTC4)3 formation or their capacity to “degranulate,” as assessed by fluid phase assays of granule-derived arylsulfatase B release (2). Just as there is an increasing recognition of the varied activities of IL-16, there has been an increased understanding of the complexities of eosinophil cell biology and functioning in varied immunologic responses. Eosinophils may have functions in immune responses extending beyond their conventional “degranulation”-based effector responses, and these may include functioning as APCs to promote Th2 CD4+ responses (20, 21). Eosinophils are now recognized to contain preformed stores of diverse cytokines and chemokines within their cytoplasmic granules (22). Thus, in addition to their distinctive cationic proteins, eosinophil granules contain chemokines (e.g., eotaxin (23) and RANTES (24, 25)) and cytokines with disparate and potentially opposing activities, notably including the prototypical Th2 cytokine IL-4 (26, 27, 28) and the Th1 cytokine IL-12 (29, 30, 31). The regulated release of these preformed cytokines occurs not by exocytotic fusion of granules with the plasma membranes to extrude all granule contents, but rather by selective processes based on vesicular mobilization and transport of these granule-derived cytokines (32, 33, 34). We have now applied more sensitive, recently developed investigative methods (32, 33) to evaluate whether IL-16 can promote LTC4 formation by eosinophils and whether IL-16 can participate in the regulated and potentially differential release of chemokines or cytokines that are preformed within human eosinophils. Our data demonstrate that IL-16 can activate specific responses of human eosinophils and in a CD4-dependent manner elicit the release of RANTES and eotaxin from eosinophils, which then function as CCR3-mediated autocrine activators to enhance eosinophil eicosanoid formation, and a selective release of specific cytokines from eosinophil granule stores.