-Horses are unique in their extreme sensitivity to endotoxin-induced cardio-pulmonary shock and mortality. The mechanisms behind increased sensitivity of the horse to endotoxin remain unknown. Pulmonary intravascular macrophages (PIMs) are pro-inflammatory cells occurring in horses. Because the functions of equine PIMs in endotoxemia remain unknown, we studied the role played by equine PIMs in endotoxin-induced pulmonary pathophysiology. We achieved this by using a recently developed protocol to deplete PIMs in order to compare lipopolysaccharide (LPS)-induced pulmonary responses in horses with or without PIMs. Horses treated with gadolinium chloride (GC; 10 mg/kg intravenous) to deplete PIMs or endotoxin-free saline (n = 4) were injected with Escherichia coli LPS (E. coli LPS; 50 ng/kg intravenously) 48 h after GC or saline. Control horses (n = 5) received two injections of endotoxin-free saline at 48 h intervals. All the horses were euthanized 2 h after LPS or saline challenge. Immunohistology for the PIMs showed their reduced numbers in GC-treated horses. The LPS treatment of normal and GC-treated horses increased diastolic and systolic pulmonary arterial pressures at 30 min compared to the saline-treated horses (P < 0.05). However, horses pre-treated with GC did not have an LPS-induced increase in mean pulmonary arterial pressure compared to the LPS-treated horses (P < 0.05). Light and electron microscopic immunocytochemistry detected extensive labeling for LPS in PIMs of LPS-treated horses. Both the LPS-treated groups had more alveolar septal cells positive for TNF-α and IL-1β compared to control horses, which did not receive LPS (P < 0.05). However, GC-treated horses challenged with the LPS showed less IL-1β-positive cells (P < 0.05). Immuno-electron microscopy localized TNF-α and IL-1β in PIMs. These new data show that PIMs endocytose LPS and contain TNF-α and IL-1β and their depletion partially inhibits LPS-induced pulmonary inflammatory responses. PIMs

-Pulmonary intravascular macrophages (PIMs) are present in horses and are believed to increase their sensitivity to endotoxin-induced cardio-pulmonary shock. However, owing to a lack of a marker for PIMs and the inability to isolate them, their precise contributions in the horse remain unknown. We designed this study to identify an immuno-phenotypic marker for PIMs and to develop a protocol for their transient depletion with gadolinium chloride (GC). GC is a lanthanide that has been used to deplete liver and lung macrophages. The horses (N = 15) were divided into control (n = 5) and GC-treated (n = 10) groups and the lung samples were examined by routine and immunocytochemical light and electron microscopy. GC-treated horses were euthanized at 48 h (n = 6) and 72 h (n = 4) post-treatment. The PIMs reacted with MAC-387 but not with ED-1 and CD-68 anti-macrophage antibodies. GC reduced the number of PIMs in horses at 48 and 72 h compared with the control (p < 0.05). There were increased intravascular TUNEL-positive cells in GC-treated horses and electron microscopy showed apoptotic PIMs in these horses. These data show that MAC-387 is a reliable marker for PIMs and GC is a safe tool to reduce the number of PIMs.PIMs / gadolinium / MAC-387 / apoptosis / immunohistology / immuno-electron microscopy / TUNEL

-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 Gramnegative and Gram-positive bacteria, respectively. Horses are extremely sensitive to endotoxininduced 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 colocalized 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. immunohistochemistry / lungs / immuno-gold EM / pulmonary intravascular macrophages / LPS