Flow Cytometric Analysis of Peripheral Blood Mononuclear Cells Induced by Experimental Endotoxemia in Horse

Abstract

ABSTRACT. Cellular activation and functional cell surface markers were evaluated during experimentally-induced endotoxemia in healthy horses. Eight healthy adult horses were infused a low dose of endotoxin (lipopolysaccharide from Escherichia coli O26: B6, 30 ng/kg of body weight, IV) and five control horses were given an equivalent volume of sterile saline solution. Venous blood samples were collected for flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) and to measure plasma endotoxin concentrations. Clinical signs of endotoxemia were recorded at 10, 20, 30, 40, 50 min, 1, 2, 3, 4, 8, 16, 24 and 48 hr after endotoxin or saline solution administration. Clinical findings characteristic of endotoxemia (tachycardia, tachypnea, increased rectal temperature, and leukopenia) occurred transiently in all horses administered endotoxin; however, plasma endotoxin concentrations were detectable in only 50% (4/8) of the endotoxin-infused horses. Endotoxin (lipopolysaccharide; LPS) has been shown to play a central role in the pathogenesis of severe sepsis and septic shock accompanied with systemic inflammatory response syndrome (SIRS) caused by gram-negative bacteria [2,9]. In horses with gastrointestinal disorders, endotoxemia is major cause of morbidity and mortality associated with septic shock [18]. Inflammatory mediators such interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α are increased in horses with endotoxemia [5,28,36]. Many important equine diseases are associated with endotoxemia, thus elucidating important mechanisms in the pathogenesis of this condition in horse could lead to improved treatment.The monocyte is a pivotal cell in mediating the body's response to endotoxemia. Monocytes express CD14, a cell surface receptor that binds complexes formed between LPS and a plasma LPS-binding protein (LBP) [38]. CD14 is a glycosyl-phosphatidyl-inositol (GPI) anchored membrane protein that is expressed on mature monocytes [15]. Moreover, CD14 was identified as a monocyte differentiation marker expressed on the surface of macrophages, neutrophils, and other cells of myeloid linkage [33]. It functions as a co-receptor for bacterial LPS and triggers the induction of inflammatory responses. Recently it has been demonstrated that Toll-like receptor (TLR) 4 may be the predominant cell surface molecule mediating the response to LPS [1,7,20] and the binding of LPS to the CD14-TLR4 complex is effective in generating a functional cellular response [7,20]. CD14 initiates the activation of macrophages for the synthesis of proinflammatory cytokines [13] that stimulate both proliferation and differentiation of CD4 + cell for helper T cell, CD5 + cell specific for peripheral B1-a cell, and CD8 + cell for suppressor and cytotoxic T cell and IgM + cell for B cell. The LPS, an outer-membrane component of gram-negative bacteria, is recognized as one of major causes of septic shock [8]. Downregulation of CD14 has been reported in circulating monocytes of human septic patient [8,14,21,22]. A continuous decrea...

Effects of an intravenous endotoxin challenge on glucose and insulin dynamics in horses

“…[13][14][15][16][17] In 1 study, 17 IV administration of E coli O55:B5 LPS at a dose of 30 ng/kg decreased the WBC count, compared with baseline values, and the nadir occurred 2 hours after infusion. [13][14][15][16][17] In 1 study, 17 IV administration of E coli O55:B5 LPS at a dose of 30 ng/kg decreased the WBC count, compared with baseline values, and the nadir occurred 2 hours after infusion.…”

“…Administration of a high dose of LPS (125 µg/kg) to ponies induces hyperglycemia within the first hour, followed by significant hypoglycemia, compared with blood glucose concentrations before endotoxin administration; however, plasma insulin concentrations remained unchanged. [13][14][15][16][17] However, endotoxemia may still play a role in the development of laminitis, and this method allows physiologic responses to LPS administration to be isolated and studied. 12 If IR is an important risk factor for laminitis, then endotoxemia may exacerbate this disturbance in glucose metabolism and further increase the risk of disease.…”

Induction and characterization of endotoxin tolerance in equine peripheral blood mononuclear cells in vitro

“…The administration of CD14-antibodies was effective in preventing septic shock even when administered after LPS exposure. In horses infused with endotoxin, the expression of CD14 on PBMCs increases and a correlation between this enhanced expression and the clinical signs of endotoxic shock was reported (Kiku et al 2003). CD14 is involved in the clearance of gram negative pathogens during infection and improves the sensitivity of the immune system to infection (Haziot et al 1994).…”

Endotoxaemia: a review with implications for the horse

“…Moreover, CD14 mAbs that block LPS-CD14 binding protected against organ injury and death in an in vivo rabbit model of endotoxic shock (Schimke et al 1998). In horses infused with endotoxin, the expression of CD14 on peripheral blood mononuclear cells (PBMCs) increased and authors reported a correlation between this enhanced expression and the clinical signs of endotoxic shock (Kiku et al 2003). Additionally, different bacterial products utilise CD14 as a binding protein, which subsequently transports these products to their respective receptors on the cell membrane (Delude et al 1995;Muroi et al 2002).…”