Sham-operated rats with anesthesia and opening of celiac cavity but without blocking of hepatic or renal blood flow (= 10) were used as controls

Sham-operated rats with anesthesia and opening of celiac cavity but without blocking of hepatic or renal blood flow (= 10) were used as controls. Collection and measurement methods of specimens Blood, hepatic and renal tissues were collected at different time points. sinusoidal endothelial cells and renal tubular epithelial cells 1 h after ischemia-reperfusion, and the expression of ICAM-1 was up-regulated in hepatic sinusoid and renal vessels after 6 h. CD1a+CD80+DCs gradually increased in hepatic sinusoidal endothelium and renal tubules and interstitium 1 h after ischemia-reperfusion, and there was the most quantity of DCs in 24-h group. The localization of DCs was associated with rat hepatic/renal function. These changes became less significant in NE 10790 rats treated with anti-PsL-EGFmAb. CONCLUSION: DCs play an important role in immune pathogenesis of hepatic/renal ischemia-reperfusion injury. Anti-PsL-EGFmAb may regulate and inhibit local DC immigration and accumulation in liver/kidney. Keywords: Adhesion molecules, Dendritic cells, Hepatic/renal ischemia-reperfusion injury, Anti-P-selectin lectin-EGF domain name monoclonal antibody INTRODUCTION Hepatic/renal ischemia-reperfusion injury is very common clinically, but the exact mechanism is unknown[1-6]. Recently, it has been reported that cell adhesion molecules play NE 10790 a crucial role in ischemia-reperfusion injury by mediating the conversation of polymorphonuclear neutrophils with endothelium[7-12]. You will find evidences that inhibition of the activities of adhesion molecules especially selectins prevented leukocyte adhesion, migration and recruitment[12-15]. In addition, dendritic cells (DCs) and their biological functions have also been implicated in inflammatory diseases, autoimmune diseases, graft rejection and tumors[16-21]. Thus, the functions of DCs in adhesion and migration have attracted great interest because of the observations that their migration into inflamed tissue is usually mediated by P- or E-selectins and that they Rabbit polyclonal to AKAP5 play an important initiating and modulating role in immune responses within inflamed tissues[15,17-21]. However, the functions of DCs involved in leukocyte infiltration and immune pathogenesis of hepatic/renal ischemia-reperfusion injury are largely unknown. In view of the previous studies[22,23], we further investigated the functions of P-selectin, intercellular adhesion molecule-1 (ICAM-1) and DCs in rat hepatic/renal ischemia-reperfusion injury and the preventive effect of anti-P-selectin lectin-EGF domain name monoclonal antibody (anti-PsL-EGFmAb) around the injury. MATERIALS AND METHODS Reagents The antibodies and reagents used in the experiments included goat anti-mouse CD1a polyclonal antibody (Santa Cruz Biotech Co., California, USA), rabbit anti-mouse CD80 polyclonal antibody (BD Biotech Co., San Diego, CA), anti-rabbit IgG-FITC and anti-goat IgG-RPE (Jackson Co., Baltimore, USA), P-selectin and ICAM-1 immunohistochemistry LSAB packages (Mei Hua Biotech Development Co., Beijing, China), serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), serum creatinine (SCr) packages (Shanghai Institute of Medical and Chemistry, Shanghai, China) and anti-PsL-EGFmAb was produced in-house[24]. Animal models Ninety male Wistar rats (Shanghai Experimental Animal Centers of Chinese Academy of Sciences), weighing 20010 g were given free access to food and water for three days before the experiments. The rats were anesthetized with 2.5% sodium pentobarbital intraperitoneally and randomly divided into two groups. In group one (= 40), the ligament linking liver, diaphragm and abdominal wall were separated, then portal vein and liver artery that drain blood to left hepatic lobe were blocked with a microvascular clamp for 60 min. After that the clamp was removed, and reperfusion occurred. In group two (= 40), the left renal artery was blocked with a microvascular clamp for 60 min, then the clamp was removed and reperfusion started. The right kidney was cut off before the process. The two groups were then randomly divided into two subgroups, one treated with anti-PsL-EGFmAb (anti-PsL-EGFmAb-treated group, = 20) and one treated with normal saline only (saline-treated group, = 20). Anti-PsL-EGFmAb (2 mg/kg) or normal saline was injected intravenously 5 min before reperfusion. Five rats in each group were sacrificed respectively at 1, 3, 6 and 24 h after reperfusion. Sham-operated rats with anesthesia and opening of celiac cavity but without blocking of hepatic or renal blood flow (= 10) were used as controls. Collection and measurement methods of specimens Blood, hepatic and renal tissues were collected at different time points. Levels of serum AST, ALT, BUN and SCr were measured with a 747 automatic analyzer (Hitachi Boehringer Mannheim, Mannheim, Germany). Hepatic and renal tissue samples were fixed in 10% formalin and embedded in paraffin. Sections were slice 5 m solid and stained with hematoxylin and eosin for light microscopic examination. Expression of P-selectin and ICAM-1 NE 10790 in hepatic/renal tissue was detected by an immunohistochemistry method with an LSAB kit. Distribution of DCs in hepatic and renal tissues Dual-label immunofluorescence staining for microscopic image analysis was used[25]. Hepatic and renal tissue sections were washed with PBS, and then blocked with 0.3% BSA for 20 min. The sections were incubated with the appropriate concentration of goat anti-mouse CD1a polyclonal antibody and rabbit anti-mouse CD80 polyclonal antibody overnight at 4 C. Subsequently, the sections.