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Background: Bile-duct ligated (BDL) rats recruit pulmonary intravascular macrophages (PIMs) and are highly susceptible to endotoxin-induced mortality. The mechanisms of this enhanced susceptibility and mortality in BDL rats, which are used as a model of hepato-pulmonary syndrome, remain unknown. We tested a hypothesis that recruited PIMs promote endotoxin-induced mortality in a rat model. Methods: Rats were subjected to BDL to induce PIM recruitment followed by treatment with gadolinium chloride (GC) to deplete PIMs. Normal and BDL rats were treated intravenously with E. coli lipopolysaccharide (LPS) with or without GC pre-treatment followed by collection and analyses of lungs for histopathology, electron microscopy and cytokine quantification. Results: BDL rats recruited PIMs without any change in the expression of IL-1β, TNF-α and IL-10. GC caused reduction in PIMs at 48 hours post-treatment (P < 0.05). BDL rats treated intravenously with E. coli LPS died within 3 hours of the challenge while the normal LPS-treated rats were euthanized at 6 hours after the LPS treatment. GC treatment of rats 6 hours or 48 hours before LPS challenge resulted in 80% (1/5) and 100% (0/5) survival, respectively, at 6 hours post-LPS treatment. Lungs from BDL+LPS rats showed large areas of perivascular hemorrhages compared to those pre-treated with GC. Concentrations of IL-1β, TNF-α and IL-10 were increased in lungs of BDL+LPS rats compared to BDL rats treated with GC 48 hours but not 6 hours before LPS (P < 0.05). Conclusion: We conclude that PIMs increase susceptibility for LPS-induced lung injury and mortality in this model, which is blocked by a reduction in their numbers or their inactivation. [ABSTRACT FROM AUTHOR]

N-myristoyltransferase (NMT) attaches a 14 carbon fatty acid, myristic acid, to the N-terminal glycine residue of proteins. NMT exists in two isoforms NMT1 and NMT2. Myristoylated proteins play critical roles in protein-protein interactions, cell signaling and oncogenesis. Although elevated expression of NMT1 has been described in colorectal carcinoma, its expression and roles in normal and inflamed lungs of the cattle are unknown. Therefore, we investigated the expression and activity of NMT in a bovine model of lung inflammation induced with Mannheimia hemolytica and in vitro in neutrophils and macrophages. Western blots revealed increased expression of NMT1 in lungs from infected animals compared to control animals. Total NMT activity was reduced in inflamed lungs compared to control animals (p < 0.05) along with increased expression of enolase, a putative inhibitor of NMT. NMT1 staining was observed in the septum, vascular endothelium and the epithelium in the lungs from control as well as infected calves. NMT1 expression was intense in neutrophils in the necrotic areas in the inflamed lungs. Immuno-electron microscopy localized NMT1 in cytoplasm and nuclei of endothelium, pulmonary intravascular macrophages and airway epithelium. Total NMT activity and NMT1 expression were increased in neutrophils and macrophages exposed to Escherichia coli LPS in vitro. NMT knockdown increased apoptosis in activated neutrophils. This is the first report demonstrating expression of NMT in normal and inflamed lungs and a novel role for NMT in regulation of neutrophil lifespan.

Background: Lipopolysaccharide (LPS) contained in tobacco smoke and a variety of environmental and occupational dusts is a toxic agent causing lung inflammation characterized by migration of neutrophils and monocytes into alveoli. Although migration of inflammatory cells into alveoli of LPS-treated rats is well characterized, the dynamics of their accumulation in the perivascular space (PVS) leading to a perivascular inflammation (PVI) of pulmonary arteries is not well described., Methods: Therefore, we investigated migration of neutrophils and monocytes into PVS in lungs of male Sprague-Dawley rats treated intratracheally with E. coli LPS and euthanized after 1, 6, 12, 24 and 36 hours. Control rats were treated with endotoxin-free saline. H&E stained slides were made and immunohistochemistry was performed using a monocyte marker and the chemokine Monocyte-Chemoattractant-Protein-1 (MCP-1). Computer-assisted microscopy was performed to count infiltrating cells., Results: Surprisingly, the periarterial infiltration was not a constant finding in each animal although LPS-induced alveolitis was present. A clear tendency was observed that neutrophils were appearing in the PVS first within 6 hours after LPS application and were decreasing at later time points. In contrast, mononuclear cell infiltration was observed after 24 hours. In addition, MCP-1 expression was present in perivascular capillaries, arteries and the epithelium., Conclusion: PVI might be a certain lung reaction pattern in the defense to infectious attacks.

Toll-like receptor (TLR) is a key component in launching innate immune response to microbial challenge. TLR4 and TLR2 are recognized as specific receptors for components of Gram-negative and Gram-positive bacteria, respectively. Horses are extremely sensitive to endotoxin-induced cardiopulmonary distress and mortality which causes significant economic losses. To date, there are no data on the expression of TLR4 and TLR2 in horse lungs. Therefore, we examined the expression of TLR4 and TLR2 in lungs from normal or Escherichia coli lipopolysaccharide (E. coli LPS; 50 ng/kg; iv) treated horses. We also studied the impact of the depletion of pulmonary intravascular macrophages (PIM) on TLR4 and TLR2 expression in normal or LPS-treated horses. RT-PCR showed TLR4 mRNA but not TLR2, in normal horse lungs. PIM depletion reduced TLR4 mRNA expression without affecting TLR2. The LPS treatment increased the expression of TLR4 and TLR2 mRNA in normal and PIM-depleted horses compared to normal saline-treated horses. Light and electron microscopic immunocytochemistry showed TLR4 protein in PIM, alveolar macrophages and septal endothelium in lungs from normal or LPS-treated horses. Immuno-gold electron microscopy showed TLR4 in PIM and dual-label immuno-electron microscopy co-localized TLR4 and LPS in the cytoplasm and nucleus of PIM of LPS-treated horses. The present manuscript is the first report on the expression of TLR4 and TLR2 in normal and LPS-treated horses and direct co-localization of TLR4 with LPS molecules in PIM. These data provide evidence that PIM are equipped with TLR4 to handle and rapidly respond to circulating endotoxins.