Immuno-phenotypic and functional characterization of rabbit pulmonary intravascular macrophages.

Pulmonary intravascular macrophages (PIMs) are present in species such as cattle, sheep and horse and promote acute lung inflammation (ALI). Rabbits are often used as a model of ALI but there is controversy about the presence of PIMs in these species. Rabbits were treated with 10 mg/kg of gadolinium chloride intravenously (GC; n = 6) or saline (n = 6) followed by euthanasia at 48 h post-treatment to determine the presence of PIMs. In a subsequent study, rabbits were pre-treated with GC or 0.9 % saline followed by 100 µg/kg of E. coli lipopolysaccharide intravenously 48 h later. Rabbits were euthanized 24 h post-LPS treatment. Light and electron microscopy showed that PIMs attached to the capillary endothelium and were positive for RAM-11 anti-macrophage antibody. While GC treatment induced apoptotic PIMs, there was no difference in the PIM number between control and GC-treated rabbits. Rabbits administered with LPS were 3.5 times more likely to die before the end of the 24-h period than those pre-treated with GC. Lung heterophil accumulation and IL-1ß, TNFα and IL-6 mRNA expression were significantly higher in rabbits administered with LPS compared to those administered with GC before the LPS injection. PIMs from the LPS-treated rabbits were positive for TNFα. Lung, BAL and serum IL-8 and MCP-1 expression was not different between LPS rabbits with or without pre-treatment with GC. We conclude that rabbit lungs contain PIMs and that their depletion reduces endotoxin-induced lung inflammation. The presence of PIMs in rabbit lungs may need to be considered while using rabbit to model acute lung injury.

Expression and activities of N-myristoyltransferase and calcineurin in normal and inflamed lungs.

The role of N-myristoyltransferase and calcineurin is well established in signaling pathways. However, there are no data on their expression and activities in normal and inflamed lungs. The mechanisms of lung inflammation induced following administration of lipopolysaccharides (LPS) or exposure to swine barn air remain unclear. Therefore, we examined expression and activities of N-myristoyltransferase and calcineurin in normal and inflamed lungs of rats. Histopathology showed acute inflammation in the lungs of rats exposed to barn air or LPS but not of control rats. There was no difference in the activities of N-myristoyltransferase and calcineurin among the control, barn-exposed, and LPS-treated rat lungs. Although N-myristoyltransferase and calcineurin were localized in airway epithelium, blood vessel walls, alveolar macrophages, and septa in the lungs of rats from all the groups, the staining intensity was increased in the lungs from rats exposed to intravenous LPS or barn air. Densitometric analyses of Western blots of 55- and 60-kDa polypeptide bands corresponding to N-myristoyltransferase and calcineurin, respectively, in the lung homogenates revealed no differences among the groups. These results show that expression of myristoyltransferase and calcineurin in lung epithelium and endothelium and a cell-specific increase in immunohistochemical expression.

Expression of toll-like receptor 4 and 2 in horse lungs.

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.