Bridget McGarry

Dual Roles of IL-4 in Lung Injury and Fibrosis

Increased lung IL-4 expression in pulmonary fibrosis suggests a potential pathogenetic role for this cytokine. To dissect this role, bleomycin-induced pulmonary inflammation and fibrosis were analyzed and compared in wild type (IL-4+/+) vs IL-4-deficient (IL-4−/−) mice. Lethal pulmonary injury after bleomycin treatment was higher in IL-4−/− vs IL-4+/+ mice. By administration of anti-CD3 Abs, we demonstrated that this early response was linked to the marked T lymphocyte lung infiltration and to the overproduction of the proinflammatory mediators such as TNF-α, IFN-γ, and NO in IL-4−/− mice. In contrast to this early anti-inflammatory/immunosuppressive role, during later stages of fibrosis, IL-4 played a profibrotic role since IL-4−/− mice developed significantly less pulmonary fibrosis relative to IL-4+/+ mice. However, IL-4 failed to directly stimulate proliferation, α-smooth muscle actin, and type I collagen expression in lung fibroblasts isolated from the wild-type mice. Upon appropriate stimulation with other known fibrogenic cytokines, fibroblasts from IL-4−/− mice were relatively deficient in the studied parameters in comparison to fibroblasts isolated from IL-4+/+ mice. Taken together, these data suggest dual effects of IL-4 in this model of lung fibrosis: 1) limiting early recruitment of T lymphocytes, and 2) stimulation of fibrosis chronically.

Eosinophils and T Lymphocytes Possess Distinct Roles in Bleomycin-Induced Lung Injury and Fibrosis

Leukocyte infiltration is characteristic of lung injury and fibrosis, and its role during tissue repair and fibrosis is incompletely understood. We found that overexpression of IL-5 in transgenic mice (IL-5TG) or by adenoviral gene transfer increased bleomycin (blm)-induced lung injury, fibrosis, and eosinophilia. Surprisingly, blm-treated IL-5-deficient (IL-5−/−) mice also developed pronounced pulmonary fibrosis but characterized by marked T lymphocyte infiltration and absence of eosinophilia. In both murine strains however, induction of lung TGF-β expression was evident. Purified lung eosinophils from blm-treated IL-5TG mice stimulated α-smooth muscle actin and collagen expression in mouse lung fibroblasts, without affecting proliferation. Furthermore instillation of purified eosinophils into murine lungs resulted in extension of blm-induced lung fibrosis, thus confirming a role for eosinophils. However, lung T lymphocytes from blm-treated IL-5−/− mice were able to stimulate fibroblast proliferation but not α-smooth muscle actin or collagen expression. Blocking T cell influx by anti-CD3 Abs abrogated lung fibrosis, thus also implicating T lymphocytes as a key participant in fibrosis. Pulmonary fibrosis in IL-5TG mice was preferentially associated with type 2 cytokines (IL-4 and IL-13), whereas fibrotic lesions in IL-5−/− animals were accompanied by proinflammatory cytokine (TNF-α, IL-1β, and IFN-γ) expression. We suggest that eosinophils and T cells contribute distinctly to the development of blm-induced lung fibrosis potentially via their production of different cytokine components, which ultimately induce TGF-β expression that is intimately involved with the fibrosis.

The role of IL‐5 in bleomycin‐induced pulmonary fibrosis

Eosinophils are known to express cytokines capable of promoting fibrosis. Interleukin‐5 (IL‐5) is important in regulating eosinophilopoiesis, eosinophil recruitment and activation. Lung IL‐5 expression is elevated in pulmonary fibrosis, wherein the eosinophil is a primary source of fibrogenic cytokines. To determine the role of IL‐5 in pulmonary fibrosis, the effects of anti‐IL‐5 antibody were investigated in a model of bleomycin‐induced pulmonary fibrosis. Fibrosis was induced in mice by endotracheal bleomycin treatment. Animals were also treated with either anti‐IL‐5 antibody or control IgG. Lungs were then analyzed for fibrosis, eosinophil influx, chemotactic activity, and cytokine expression. The results show that a primary chemotactic activity at the height of eosinophil recruitment is IL‐5. Furthermore, anti‐IL‐5 antibody caused significant reduction in lung eosinophilia, cytokine expression, and fibrosis. These findings taken together suggest an important role for IL‐5 in pulmonary fibrosis via its ability to regulate eosinophilic inflammation, and thus eosinophil‐dependent fibrogenic cytokine production. J. Leukoc. Biol. 64: 657–666; 1998.

Role of Eotaxin-1 (CCL11) and CC chemokine receptor 3 (CCR3) in bleomycin-induced lung injury and fibrosis.

Eotaxin-1/CCL11 and its receptor CCR3 are involved in recruitment of eosinophils to diverse tissues, but their role in eosinophil recruitment in pulmonary fibrosis is unclear. The present study examined the pulmonary expression of CCL11 and CCR3 during bleomycin (blm)-induced lung injury and determined their importance in the recruitment of inflammatory cells and the development of lung fibrosis. In mice, blm induced a marked pulmonary expression of CCL11 and CCR3. Immunostaining for CCR3 revealed that this receptor was not only expressed by eosinophils but also by neutrophils. CCL11-deficient (CCL11(-/-)) mice developed significantly reduced pulmonary fibrosis. Expression of profibrotic cytokines such as transforming growth factor-beta1 was diminished in the absence of CCL11. Furthermore, increased lung expression of CCL11 significantly enhanced blm-induced lung fibrosis and production of profibrotic cytokines. These effects were also associated with an increase of eosinophil and neutrophil pulmonary infiltration. In contrast, mice treated with neutralizing CCR3 antibodies developed significantly reduced pulmonary fibrosis, eosinophilia, neutrophilia, and expression of profibrotic cytokines. Together, these data suggest that CCL11 and CCR3 are important in the pulmonary recruitment of granulocytes and play significant pathogenic roles in blm-induced lung fibrosis.

Regulation of Macrophage-Derived Fibroblast Growth Factor Release by Arachidonate Metabolites

The macrophage is a source of many mediators with direct and indirect fibrogenic potential. In this study, release of macrophage‐derived fibroblast growth factor (MDGF) activity by murine peritoneal macrophages is examined with regard to its regulation by arachidonate metabolites. Upon stimulation with 10 μg/ml lipopolysaccharide (LPS), resident peritoneal macrophages from CBA/J mice released MDGF activity into media rapidly, reaching maximal levels in approximately 1 h. Lysates of these stimulated cells also revealed significantly increased cell‐associated MDGF activity, composing 45% of the total assayable activity. This activity, as assayed by radioactive thymidine incorporation by primary cultures of rat lung fibroblasts, was separable from interleukin‐1 (IL‐1) activity by reverse phase high performance liquid chromatography (HPLC). Furthermore, purified murine IL‐1 had no MDGF activity in this assay system. This stimulated MDGF release was enhanced by the cylooxygenase inhibitors indomethacin, Ibuprofen, and aspirin at micromolar concentrations, but inhibited in a dose‐dependent manner by prostaglandin E2 (PGE2). On the other hand, nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor was inhibitory at 0.1 and 0.4 μM but not at 2.5 μM. Zymosan‐stimulated macrophages also markedly increased MDGF release, albeit with a different time course which was characterized by a delay of approximately 7 h before peak levels were attained. Such stimulation, which is known to cause increased lipoxygenase activity, was also inhibited by 0.5 μM NDGA. In contrast, the lipoxygenase pathway products leukotrienes B4 (LTB4) and C4 (LTC4) stimulated MDGF release in a dose‐dependent (10‐10‐10‐8 M) manner, with LTC4 being more potent on a per unit dose basis. Stimulation by LTC4 was inhibited by the putative leukotriene receptor antagonist, FPL55712, while LTD4 and LTE4 did not stimulate MDGF release, thus suggesting the mediation of this effect by specific LTC4 receptors. These data suggest also that products of the cyclooxygenase and lipoxygenase pathways are potentially important both as exogenous (ie, derived from cells other than the macrophage itself) and auto‐ or self‐regulators of macrophage MDGF release. This, in turn, implies that cyclooxygenase products are antifibrogenic and important in maintaining or returning to the quiescent or normal state, whereas the lipoxygenase products are profibrogenic and important in induction of fibrosis or wound‐healing and tissue repair. Any alteration in the balance between these two pathways may result in either a desirable or a harmful outcome.

Stimulation of fibroblast proliferation by thrombospondin

Thrombospondin purified from human platelets was examined for its ability to promote proliferation of human dermal fibroblasts. The results show that thrombospondin could stimulate the incorporation of [3H]thymidine by quiescent fibroblasts in a dose-dependent manner without stimulating protein or collagen synthesis. The effect was observed even in the total absence of serum, although the degree of stimulation was substantially lower than that in the presence of 0.4% fetal calf serum, but higher than that in the presence of 4% serum. The effect was specific and not due to contaminants as demonstrated by the ability of antibodies to thrombospondin to specifically inhibit this stimulation. Three monoclonal antibodies directed at different epitopes in the thrombospondin molecule were equally effective in inhibiting this effect. This stimulation of fibroblast proliferation by thrombospondin suggests a potential role for this matrix protein in the mesenchymal cell response in tissue injury and repair.