Acute necrotizing pancreatitis (ANP) is a common gastrointestinal cause of emergency admissions in dogs and humans and can lead to a systemic inflammatory response syndrome resulting in multiple organ dysfunction syndrome. Among the various complications associated with ANP, acute lung injury (ALI) or its more severe form, acute respiratory distress syndrome (ARDS), are major contributors leading to high mortality rates associated with severe acute pancreatitis (AP) in human patients. The incidence of ALI/ARDS in ANP dogs is not well characterized. However, signs of respiratory complications have been reported clinically in dogs suffering from AP. The pathophysiology of ANP and its systemic complications in dogs and humans are not well understood. Most of the data related to AP comes from rodent models of AP, which may not always represent the true mechanisms occurring in the lungs of dogs or humans with ANP. I decided to undertake evaluation of pancreas and lungs from dogs (N=21) that died of ANP. The cases were selected through the search of the medical records of the Veterinary Medical Center of the Western College of Veterinary Medicine (WCVM). Six healthy SPCA dogs were used as controls. The histology of pancreas was first graded to record the range of ANP severities within dog cases included in this study. Then, characterization of lung inflammation was done with histological grading and qualitative analysis of immunohistochemical staining for von Willebrand Factor (vWF), Toll-Like Receptor-4 (TLR4), interleukin-6 (IL6), and inducible nitric oxide synthase (iNOS). Quantification of the recruitment of septal macrophages in the lungs, designated as pulmonary intravascular macrophages (PIMs), in ANP dogs was achieved by counting the number of positive cells in alveolar septa using a macrophage antibody (MAC387). The results revealed that dogs suffering from ANP have variable lung inflammation, which was characterized by a significant infiltration of mononuclear phagocyte cells in the alveolar septa of all ANP dogs (median, 138; range 31-935) compared to control dogs (median: 1.5; range 0-16; p < 0.001), which suggested that PIMs are induced in ANP. In addition, robust staining for vWF in alveolar septal capillaries in lungs of ANP dogs suggested a strong microvascular inflammatory response. Finally, TLR4, IL6, and iNOS expression was increased in lungs of ANP dogs compared to control dogs. The second study was to investigate whether PIMs are induced in a mouse model of L-arginine-induced ANP. Therefore, lungs of L-arginine treated mice (n=7 per time point) were evaluated at various time points (24 hours, 72 hours and 120 hours) using histology and immunohistochemical staining for CD68 cells and vWF. Nine control mice were used. Counting of CD68-positive cells in the lungs of mice treated with L-arginine showed increased numbers of mononuclear phagocytes in alveolar septa at every time point (p<0.001). Also, the lung’s vasculature from L-arginine-treated mice showed increased vWF staining. Taken together, the data showed that ANP in dogs caused significant recruitment of PIMs, increased expression of vWF, TLR4, IL-6, and iNOS suggesting presence of lung inflammation. The mouse model of L-arginine-induced ANP also showed recruitment of PIMs and increased vascular expression of vWF suggesting that this model may be relevant to study the mechanisms of PIMs recruitment and their functions in lung physiology associated with ANP.

ABSTRACT Heaves is triggered by exposure to dust and its components, such as endotoxin, and is characterized by clinical signs such as coughing, decreased exercise tolerance, difficulty breathing and abnormal lung sounds which are due to bronchoconstriction and accumulation of neutrophils in the airways. Pulmonary intravascular macrophages (PIMs) are believed to increase horses sensitivity to endotoxemia-induced lung inflammation. The first objective of this study was to investigate a hitherto unknown role of PIMs in equine heaves. I used mouldy hay (MH) to induce heaves and gadolinium chloride (GC) to deplete PIMs in order to compare responses between non-treated and GC-treated heaves horses. A modified randomized crossover study (2X2 factorial) was conducted in which mares (N=9) were exposed to 4 different treatments: alfalfa cubes (Cb), alfalfa cubes + GC (Cb-GC), mouldy hay (MH) and MH + GC (MH-GC). Each treatment was followed by broncholaveolar lavage (BAL). MH was fed for 7 days to induce heaves followed by Cb for 21 days to achieve remission, whereas the treatments in which heaves was not induced (Cb; Cb-GC), the cubes were fed prior to the BAL and for 14 days after the BAL to allow recovery from the BAL procedure. BAL fluids were processed to investigate total cell, neutrophil and alveolar macrophage concentrations. In addition, TNFá protein levels as well as TNFá, IL-8, and TLR4 mRNA expression in BAL cells were assessed in order to infer on their activation state.<p> Data showed higher concentration of dust (3X), endotoxin (20X), and endotoxin per milligram of dust (7X) in MH compared to the Cb environment. Clinical scores and neutrophil concentrations in BAL were higher when mares received MH compared to MH and GC (MH-GC). Real time reverse transcriptase PCR revealed a significant lower expression of IL-8 and TLR4 mRNA in BAL cells from MH-GC mares compared to MH. TNFá mRNA expression as well as protein concentration were not affected by the different treatments. In vitro secondary LPS challenge significantly increased IL-8 mRNA expression in cells from MH treatment compared to without LPS, but not in the MH-GC treatment. TLR4 expression was not affected by the secondary challenge. Although secondary LPS challenge increased expression of TNFá mRNA and protein, the differences among treatment groups were not meaningful. In conclusion, PIM depletion attenuates clinical scores, migration of inflammatory cells into the alveolar space and expression of pro-inflammatory molecules in BAL cells of heaves horses.<p> The observations on the role of PIMs in heaves in horses prompted me to examine the occurrence of PIMs in human lungs. I found a trend for higher numbers of septal macrophages in autopsied lungs from human patients who died of non-pulmonary pathologies compared to those having either COPD or asthma. If these septal macrophages indeed represent the PIMs, this finding is contrary to existing belief that humans, unlike horses, do not have PIMs.

ABSTRACT Heaves is triggered by exposure to dust and its components, such as endotoxin, and is characterized by clinical signs such as coughing, decreased exercise tolerance, difficulty breathing and abnormal lung sounds which are due to bronchoconstriction and accumulation of neutrophils in the airways. Pulmonary intravascular macrophages (PIMs) are believed to increase horses sensitivity to endotoxemia-induced lung inflammation. The first objective of this study was to investigate a hitherto unknown role of PIMs in equine heaves. I used mouldy hay (MH) to induce heaves and gadolinium chloride (GC) to deplete PIMs in order to compare responses between non-treated and GC-treated heaves horses. A modified randomized crossover study (2X2 factorial) was conducted in which mares (N=9) were exposed to 4 different treatments: alfalfa cubes (Cb), alfalfa cubes + GC (Cb-GC), mouldy hay (MH) and MH + GC (MH-GC). Each treatment was followed by broncholaveolar lavage (BAL). MH was fed for 7 days to induce heaves followed by Cb for 21 days to achieve remission, whereas the treatments in which heaves was not induced (Cb; Cb-GC), the cubes were fed prior to the BAL and for 14 days after the BAL to allow recovery from the BAL procedure. BAL fluids were processed to investigate total cell, neutrophil and alveolar macrophage concentrations. In addition, TNFá protein levels as well as TNFá, IL-8, and TLR4 mRNA expression in BAL cells were assessed in order to infer on their activation state.<p> Data showed higher concentration of dust (3X), endotoxin (20X), and endotoxin per milligram of dust (7X) in MH compared to the Cb environment. Clinical scores and neutrophil concentrations in BAL were higher when mares received MH compared to MH and GC (MH-GC). Real time reverse transcriptase PCR revealed a significant lower expression of IL-8 and TLR4 mRNA in BAL cells from MH-GC mares compared to MH. TNFá mRNA expression as well as protein concentration were not affected by the different treatments. In vitro secondary LPS challenge significantly increased IL-8 mRNA expression in cells from MH treatment compared to without LPS, but not in the MH-GC treatment. TLR4 expression was not affected by the secondary challenge. Although secondary LPS challenge increased expression of TNFá mRNA and protein, the differences among treatment groups were not meaningful. In conclusion, PIM depletion attenuates clinical scores, migration of inflammatory cells into the alveolar space and expression of pro-inflammatory molecules in BAL cells of heaves horses.<p> The observations on the role of PIMs in heaves in horses prompted me to examine the occurrence of PIMs in human lungs. I found a trend for higher numbers of septal macrophages in autopsied lungs from human patients who died of non-pulmonary pathologies compared to those having either COPD or asthma. If these septal macrophages indeed represent the PIMs, this finding is contrary to existing belief that humans, unlike horses, do not have PIMs.