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A4487 - Distinctive Actions of Colony-Stimulating Factors on Proliferation and Activation of Two Main Pulmonary Macrophage Populations
Author Block: C. Draijer, R. Penke, M. Peters-Golden; Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, United States.
RATIONALE. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage CSF (M-CSF) were originally defined as factors promoting hematopoietic colony formation. Although both are capable of enhancing macrophage differentiation and their levels may change during inflammatory responses, their effects on resident lung macrophages are incompletely understood. At least two main macrophage populations exist in the healthy lung, distinguished by their localization and unique surface markers. Alveolar macrophages (AMs) are long-lived cells derived from embryonic precursors which replenish by local proliferation, while the origin and maintenance of interstitial macrophages (IMs) is still under debate. Here we compared the proliferative and activation responses of resident AMs and IMs to GM-CSF and M-CSF. METHODS. AMs were obtained by bronchoalveolar lavage and IMs were isolated using CD11b magnetic beads from collagenase-digested lungs of C57BL/6 mice. AMs and CD11b-positive digest cells were cultured overnight to allow attachment. Nonattached cells were washed off. Macrophages were stimulated with either GM-CSF or M-CSF (both 50 ng/mL) for 48 h. Total RNA was isolated, cDNA was synthesized and real-time quantitative PCR was performed. Relative gene expression was analyzed after normalizing for β-actin. A fluorescent DNA-binding dye assay was used to examine proliferation after 5 days of CSF stimulation. RESULTS. As expected, AMs had higher baseline expression of CD11c and YM1 (M2 macrophage marker) and lower CD11b and inducible nitric oxide synthase (INOS; M1 marker) expression than IMs. In addition, the IMs were characterized by high expression of Toll-like receptor 9 (TLR9) and CX3C chemokine receptor 1 (CX3CR1). Stimulation with CSFs increased expression of CD11c and CD11b on both populations while GM-CSF increased YM1 expression on both. GM-CSF down-regulated TLR9, CX3CR1 and INOS expression in IMs while it up regulated INOS expression in AMs. M-CSF did not affect expression of these genes. Increased proliferation was observed after 5 days of stimulation with both CSFs in AMs but not in IMs. This was further confirmed by higher expression of cyclin B1, D1 and survivin in GM-CSF-stimulated AMs. M-CSF stimulation only increased survivin expression and prolonged survival of macrophages in culture as compared to unstimulated control. IMs had higher expression of cyclins at baseline but this was down regulated upon CSF stimulation. CONCLUSIONS. AMs proliferate in response to both CSFs whereas IMs fail to do so with either. GM-CSF promotes an AM-like phenotype in IMs. Individual CSFs can modulate activation and phenotypic markers in a cell-specific manner.