Abstract
IntroductionWe have previously established a link between impaired phagocytic capacity and deregulated S1P signaling in alveolar macrophages from COPD subjects. We hypothesize that this defect may include a disruption of epithelial-macrophage crosstalk via Spns2-mediated intercellular S1P signaling.MethodsPrimary alveolar macrophages and bronchial epithelial cells from COPD subjects and controls, cell lines, and a mouse model of chronic cigarette smoke exposure were studied. Cells were exposed to 10% cigarette smoke extract, or vehicle control. Spns2 expression and subcellular localization was studied by immunofluorescence, confocal microscopy and RT-PCR. Phagocytosis was assessed by flow-cytometry. Levels of intra- and extracellular S1P were measured by S1P [3H]-labeling.ResultsSpns2 expression was significantly increased (p<0.05) in alveolar macrophages from current-smokers/COPD patients (n = 5) compared to healthy nonsmokers (n = 8) and non-smoker lung transplant patients (n = 4). Consistent with this finding, cigarette smoke induced a significant increase in Spns2 expression in both human alveolar and THP-1 macrophages. In contrast, a remarkable Spns2 down-regulation was noted in response to cigarette smoke in 16HBE14o- cell line (p<0.001 in 3 experiments), primary nasal epithelial cells (p<0.01 in 2 experiments), and in smoke-exposed mice (p<0.001, n = 6 animals per group). Spns2 was localized to cilia in primary bronchial epithelial cells. In both macrophage and epithelial cell types, Spns2 was also found localized to cytoplasm and the nucleus, in line with a predicted bipartile Nuclear Localization Signal at the position aa282 of the human Spns2 sequence. In smoke-exposed mice, alveolar macrophage phagocytic function positively correlated with Spns2 protein expression in bronchial epithelial cells.ConclusionOur data suggest that the epithelium may be the major source for extracellular S1P in the airway and that there is a possible disruption of epithelial/macrophage cross talk via Spns2-mediated S1P signaling in COPD and in response to cigarette smoke exposure.
Highlights
We have previously established a link between impaired phagocytic capacity and deregulated S1P signaling in alveolar macrophages from Chronic obstructive pulmonary disease (COPD) subjects
Our data suggest that the epithelium may be the major source for extracellular S1P in the airway and that there is a possible disruption of epithelial/macrophage cross talk via Spns2mediated S1P signaling in COPD and in response to cigarette smoke exposure
Defective phagocytosis of apoptotic bodies in the airway leads to their accumulation and potential secondary necrosis with ensuing inflammation that cannot be resolved even after smoking cessation [6]
Summary
Primary alveolar macrophages and bronchial epithelial cells from COPD subjects and controls, cell lines, and a mouse model of chronic cigarette smoke exposure were studied. Spns expression and subcellular localization was studied by immunofluorescence, confocal microscopy and RTPCR. The primary antibody to Spns was a goat polyclonal antibody directed against an intracellular domain (G-14, Santa Cruz, Dallas, TX, USA). Rabbit polyclonal antibodies directed against the aa120 domain (LifeSpan BioSciences, Seattle, WA, USA) or the N-terminus (GeneTex, Irvin, CA, USA) were used to confirm similar patterns of immunofluorescence. Rabbit SPHK1 and SPHK2 polyclonal antibodies were from Bioss (Woburn, MA, USA). Secondary donkey IgG F(ab’) fragment antibodies conjugated to AF488, AF594 or AF647, specific to rabbit, goat, and mouse IgG (respectively) were all from Jackson ImmunoResearch (West Grove, PA, USA)
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