Immunomodulatory Effects Of Pulmonary Surfactant Protein A Induced By Endotoxin Activation Of Alveolar Macrophages And Its Mechanism

The host defense in the lung is the most vulnerable interface for pathogens clearance. Innate defenses of the lung consist of first, structural defenses, second, antimicrobial molecules generated in the airway, and third, phagocytic defenses provided by the resident alveolar macrophages and the polymorphonuclear leukocytes (PMNs) that are recruited into the lung in response to pulmonary infection. The primary antimicrobial defenses in the lung are the pulmonary epithelial, the resident alveolar macrophages, and protein components of the innate immune system. An impressive number of studies carried out over the past 10 years provided compelling evidence that components of the surfactant lining layer function as primary immunomodulators in pulmonary host defence. Lining at the air-liquid interface, pulmonary surfactant is a complex mixture of 90% lipids and 10% proteins. The major phospholipid (75%) is dipalmitoylphosphatidylcholine (DPPC) . The protein components consist of hydrophobic protein SP-B, SP-C and hydrophilic protein SP-A and SP-D. The properties, localization and structure of SP-A suggest its role in surface film formation and surface tension reduction in lung homeostasis. However, studies utilising SP-A-deficient mice have failed to confirm the major role of SP-A in surfactant metabolism in vivo. These SP-A-/- mice have no major abnormalities respiratory function but exhibit a markedly delayed microbial clearance of clinically relevant pathogens compared with SP-A +/+ mice. These data demonstrate the main role of SP-A is in regulating immune response of the lung.Lipopolysaccharide (LPS) is among the first specific ligands identified for SP-A. In vitro data showed that (LPS-free) SP-A not only reduces LPS-elicited pro-inflammatory cellresponses but also stimulates the deacylation and uptake of LPS by isolated alveolar macrophages, a process thought to contribute to the detoxification of LPS by immune cells. Importantly, SP-A -/- mice produced more bronchoalveolar TNF and nitric oxide than do wild type mice after LPS treatment. Intratracheal instillation of exogenous SP-A into the lungs of SP-A-deficient mice restored pro-inflammatory mediator release upon LPS challenge. These in vivo and in vitro data highlight the idea that SP-A plays an important role in modulating LPS-induced immune cell activation via multiple mechanism. In the current study, we focused on the relationship between the anti-inflammatory effect of SP-A and the LPS induced NF-KB/lKB-a signalling pathway.First, in the present investigation SP-A has no stimulatory effect on resting cell in terms of the TNF-a release and NF-KB activity. As measured by Western blot, SP-A increased the basal level of IKB in a dose-dependent manner. Since even a modest increase in IKB is sufficient to redistribute p65 from the nucleus to the cytoplasm, the observed inhibition of NF-KB activation by SP-A might be a consequence of this basal enhancement of IKB-a. The effect of SP-A on NF-KB activity was confirmed by CHO cell lines stably transfected with CD14 and Toll like receptor. The flow cytometry analysis of CD25 membrane expression demonstrated that neither TLR4 nor TLR2 in these cell lines conferred responsiveness to SP-A. These data strongly suggest that the SP-A used in the present study was free of contaminating LPS and lipopeptide.Next finding of the current study is that SP-A inhibited both R- and S-LPS-induced cell activation of alveolar macrophages. Upon stimulation with LPS, control cells showed a rapid degradation of cytosolic IKB within 30-40 min and reappearance at 1 h.In contrast, in SP-A pretreated cells, both of the cytosolic and nuclear IKB was strongly increased and the reappearance was accelerated. The inhibitory role of IKB is not only the cytosolic sequestration of NF-KB, but also the inhibition of NF-KB binding to specific DNA sequences and shifting its steady state back to the cytosol. Therefore, the data presented here imply the possibility that SP-A down regulates NF-KB-dependent gene by enhancing IKB protei...