Immunophenotypic characterization and depletion of pulmonary intravascular macrophages of horses

- 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

Depletion of pulmonary intravascular macrophages partially inhibits lipopolysaccharide-induced lung inflammation in horses

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-$\alpha$ and IL-1$\beta$ compared to control horses, which did not receive LPS (P < 0.05). However, GC-treated horses challenged with the LPS showed less IL-1$\beta$-positive cells (P < 0.05). Immuno-electron microscopy localized TNF-$\alpha$ and IL-1$\beta$ in PIMs. These new data show that PIMs endocytose LPS and contain TNF-$\alpha$ and IL-1$\beta$ and their depletion partially inhibits LPS-induced pulmonary inflammatory responses