Daniel Klintman

Important Role of P-Selectin for Leukocyte Recruitment, Hepatocellular Injury, and Apoptosis in Endotoxemic Mice

ABSTRACT Leukocyte recruitment in the liver includes a two-step procedure in which selectin-dependent leukocyte rolling is a prerequisite for subsequent CD18-dependent leukocyte firm adhesion in postsinusoidal venules. However, the roles of the individual selectins in leukocyte rolling and adhesion, hepatocellular injury, and apoptosis remain elusive. Therefore, we examined the pathophysiological role of P-, E-, and L-selectin in male C57BL/6 mice challenged with lipopolysaccharide (LPS) and d-galactosamine (Gal) by use of intravital microscopy of the liver microcirculation. In control animals, administration of LPS-Gal provoked reproducible hepatic damage, including marked increases of leukocyte recruitment, liver enzymes, and hepatocyte apoptosis and reduced sinusoidal perfusion. Interestingly, pretreatment with an anti-P-selectin antibody (RB40.34) markedly reduced leukocyte rolling and firm adhesion by 65 and 71%, respectively. Moreover, interference with P-selectin function significantly improved sinusoidal perfusion and reduced the increase in liver enzymes by 49 to 84% in endotoxemic mice. Moreover, the activity of caspase-3 and the number of apoptotic hepatocytes were significantly reduced by 55 and 54%, respectively, in RB40.34-treated animals. In contrast, administration of an anti-E-selectin antibody (10E9.6) and an anti-L-selectin antibody (Mel-14) did not protect against endotoxin-induced leukocyte responses or hepatic injury. In conclusion, our novel findings document a principal role of P-selectin in mediating leukocyte rolling, a precondition to the subsequent firm adhesion of leukocytes in liver injury. Furthermore, our novel data demonstrate that inhibition of P-selectin function reduces hepatocellular injury and apoptosis, suggesting a causal relationship between leukocyte recruitment on one hand and hepatocellular injury and apoptosis on the other hand. Based on these findings, it is suggested that P-selectin may be an important therapeutic target in endotoxin-induced liver injury.

Leukocyte recruitment in hepatic injury: selectin-mediated leukocyte rolling is a prerequisite for CD18-dependent firm adhesion

BACKGROUND/AIMS This study was designed to examine the role of selectins and CD18 in leukocyte recruitment in hepatic injury induced by tumor necrosis factor-alpha (TNF-alpha) and galactosamine (Gal) in vivo. METHODS Intravital fluorescence microscopy of the hepatic microcirculation was used to quantify leukocyte-endothelium interactions provoked by 24 h of systemic TNF-alpha/Gal challenge in rats. Hepatic injury was evaluated with liver enzymes. RESULTS When administered after 24 h of TNF-alpha/Gal challenge, fucoidan, a selectin-function inhibitor, reduced leukocyte rolling by 69%, whereas firm adhesion was unaltered. In contrast, passive immunization against CD18 decreased leukocyte adhesion by 60%, whereas rolling remained unchanged. Notably, when administered prior to TNF-alpha/Gal, fucoidan attenuated both leukocyte rolling and adhesion, by 57 and 69%, respectively. Pretreatment with an anti-CD18 antibody decreased TNF-alpha/Gal-induced rolling and firm adhesion by 25 and 90%, respectively. Moreover, pretreatment with fucoidan and the anti-CD18 antibody both protected against TNF-alpha/Gal-induced increases in liver enzymes. For example, the pretreatments reduced alanine aminotransferase by 59 and 87%, respectively. CONCLUSIONS Our data suggest that TNF-alpha/Gal-induced leukocyte rolling is selectin-mediated and a precondition for CD18-dependent firm adhesion in hepatic venules. Thus, reducing leukocyte recruitment by inhibition of selectins or CD18 may be useful to control TNF-alpha-induced liver injury.

Lymphocyte function antigen-1 mediates leukocyte adhesion and subsequent liver damage in endotoxemic mice

Sepsis is associated with leukocyte activation and recruitment in the liver. We investigated the role of lymphocyte function antigen‐1 (LFA‐1) in endotoxin‐induced leukocyte–endothelium interactions, microvascular perfusion failure, hepatocellular injury and apoptosis in the liver by use of gene‐targeted mice, blocking antibodies and a synthetic inhibitor of LFA‐1 (LFA703). For this purpose, mice were challenged with lipopolysaccharide (LPS)+D‐galactosamine (Gal), and intravital microscopy of the liver microcirculation was conducted 6 h later. The number of firmly adherent leukocytes in response to LPS/Gal was reduced by 48% in LFA‐1‐deficient mice. Moreover, endotoxin‐induced increases of apoptosis and enzyme markers of hepatocellular injury were decreased by 64 and 69–90%, respectively, in LFA‐1‐deficient mice. Furthermore, sinusoidal perfusion was improved in endotoxemic mice lacking LFA‐1. A similar protective pattern was observed in endotoxemic mice pretreated with an antibody against LFA‐1. Thus, immunoneutralization of LFA‐1 reduced endotoxin‐induced leukocyte adhesion by 55%, liver enzymes by 64–66% and apoptosis by 42%, in addition to the preservation of microvascular perfusion. Administration of a novel statin‐derived inhibitor of LFA‐1, LFA703, significantly decreased leukocyte adhesion (more than 56%) and the subsequent liver injury in endotoxemic mice. Thus, this study demonstrates a pivotal role of LFA‐1 in supporting leukocyte adhesion in the liver. Moreover, interference with LFA‐1‐mediated leukocyte adhesion protects against endotoxemic liver damage, and may constitute a potential therapeutic strategy in sepsis.

A statin‐based inhibitor of lymphocyte function antigen‐1 protects against ischemia/reperfusion‐induced leukocyte adhesion in the colon

Statins are mainly used to control hypercholesterolemia; however, recent studies have also ascribed anti‐inflammatory effects to the statins. LFA703 is a novel statin‐derived compound, which potently inhibits lymphocyte function antigen‐1 (LFA‐1, CD11a/CD18) but does not affect HMG‐CoA reductase activity. The objective of this study was to examine the anti‐inflammatory mechanisms of LFA703 in ischemia/reperfusion (I/R)‐induced leukocyte–endothelium interactions in the colon. For this purpose, the superior mesenteric artery was occluded for 30 min and leukocyte responses were analyzed in colonic venules after 120 min of reperfusion in mice using inverted intravital fluorescence microscopy. First, the inhibitory mechanisms of LFA703 on leukocyte adhesion were investigated in vitro using a mouse CD4+8+ thymocyte cell line. Immunoneutralization of LFA‐1 and ICAM‐1 abolished leukocyte adhesion, whereas inhibition of VLA‐4 had no effect in this in vitro assay. Indeed, it was found that LFA703 dose‐dependently reduced LFA‐1‐dependent leukocyte adhesion to mouse endothelial cells in vitro with an IC50 of 3.2 μM. I/R caused an increase in leukocyte rolling and adhesion in colonic venules. Immunoneutralization of LFA‐1 significantly reduced I/R‐induced leukocyte adhesion by 89% in colonic venules. In contrast, I/R‐provoked leukocyte rolling was insensitive to inhibition of LFA‐1 function. Administration of 30 mg kg−1 of LFA703 decreased reperfusion‐induced leukocyte adhesion by more than 91%, while the level of leukocyte rolling was unchanged, suggesting that LFA703 effectively blocked LFA‐1‐dependent firm adhesion of leukocyte in the colon. However, LFA703 did not decrease the expression of LFA‐1 on circulating leukocytes. This study demonstrates that LFA‐1 is indeed a critical adhesion molecule in mediating postischemic leukocyte adhesion in the colon. Moreover, this is the first study showing that a statin‐based synthetic compound has the capacity to abolish LFA‐1‐dependent leukocyte adhesion in I/R. These novel findings may have great implications in the clinical treatment of conditions associated with I/R‐induced tissue injury, such as organ transplantation, trauma and major surgery.

p38 Mitogen-Activated Protein Kinase-Dependent Chemokine Production, Leukocyte Recruitment, and Hepatocellular Apoptosis in Endotoxemic Liver Injury

Objective:To determine the role of p38 mitogen-activated protein kinase (MAPK) signaling in endotoxin-induced liver injury. Background:MAPKs have been reported to play a potential role in regulating inflammatory responses, but the role of p38 MAPK signaling in chemokine production, leukocyte recruitment, and hepatocellular apoptosis in the liver of endotoxemic mice is not known. Methods:Endotoxin-induced leukocyte-endothelium interactions were studied by use of intravital fluorescence microscopy in the mouse liver. Tumor necrosis factor-α (TNF-α) and CXC chemokines, liver enzymes, and apoptosis were determined 6 hours after endotoxin challenge. The specific p38 MAPK inhibitor SB 239063 was given immediately prior to endotoxin exposure. Phosphorylation and activity of p38 MAPK were determined by immunoprecipitation and Western blot. Results:Endotoxin increased phosphorylation and activity of p38 MAPK in the liver, which was markedly inhibited by SB 239063. Inhibition of p38 MAPK signaling dose-dependently decreased endotoxin-induced leukocyte rolling, adhesion, and sinusoidal sequestration of leukocytes. SB 239063 markedly reduced endotoxin-induced formation of TNF-α and CXC chemokines in the liver. Indeed, the endotoxin-provoked increase of liver enzymes and hepatocellular apoptosis were abolished and sinusoidal perfusion was restored in endotoxemic mice treated with SB 239063. Conclusions:This study demonstrates that p38 MAPK signaling plays an important role in regulating TNF-α and CXC chemokine production in endotoxemic liver injury and that inhibition of p38 MAPK activity abolishes endotoxin-induced leukocyte infiltration as well as hepatocellular apoptosis. These novel findings suggest that interference with the p38 MAPK pathway may constitute a therapeutic strategy against septic liver damage.

Allopurinol inhibits CXC chemokine expression and leukocyte adhesion in endotoxemic liver injury

AbstractObjective: Leukocyte activation and recruitment are rate limiting steps in endotoxemic liver injury. We investigated the effect of allopurinol on the expression of CXC chemokines and leukocyte-endothelium interactions, microvascular perfusion failure, and cellular injury and apoptosis in endotoxemic liver injury. Materials and methods: Mice were treated with allopurinol prior to challenge with lipopolysaccharide (LPS) + D-Galactosamine (Gal). Intravital microscopy of the liver microcirculation and analysis of liver enzymes were conducted 6 h later. Results: We observed that allopurinol pre-treatment reduced the number of firmly adherent leukocytes by more than 57% in postsinusoidal venules of endotoxemic mice. Indeed, endotoxin-induced liver injury enzymes were decreased by 76% and sinusoidal perfusion failure was reversed in mice pre-treated with allopurinol. Moreover, administration of allopurinol reduced LPS-induced hepatocyte apoptosis by 56%. Notably, it was found that allopurinol significantly decreased the levels of CXC chemokines (more than 83% reduction) in livers of endotoxemic mice. Conclusions: This study shows that allopurinol markedly protects against endotoxemic liver injury and indicates that reactive oxygen species (ROS) mediate synthesis of CXC chemokines and leukocyte adhesion in the liver in vivo.

Staphylococcal enterotoxin A-induced hepatotoxicity is predominantly mediated by Fas ligand (CD95L).

Objective:To determine the role of tumor necrosis factor α (TNF-α) and Fas ligand (FasL, CD95L) in superantigen-induced and endotoxin-induced liver injury. Summary Background Data:Gram-positive bacteria are increasingly common causes of sepsis and multiorgan failure, but the pathophysiologic mechanisms of superantigen-provoked hepatotoxicity remain elusive. Methods:Intravital fluorescence microscopy was used to study the liver microcirculation in mice challenged with superantigen (staphylococcal enterotoxin A, SEA) or endotoxin (lipopolysaccharide, LPS) combined with d-galactosamine. Results:Administration of 10 &mgr;g LPS and 50 &mgr;g SEA caused similar hepatocellular damage as determined by liver enzymes and apoptosis. Notably, TNF-α–deficient mice were completely protected against hepatic injury provoked by LPS, whereas no protection was observed in response to SEA. On the other hand, FasL-deficient mice were protected against liver injury induced by SEA, but no protection was found when challenged with LPS. LPS increased clear-cut leukocyte recruitment, whereas SEA had no significant effect on leukocyte responses in the liver microcirculation. Leukocyte responses to LPS were decreased by >56% in TNF-α gene-targeted animals. Moreover, antiadhesive therapy, ie, immunoneutralization of P-selectin, which is an effective inhibitor of leukocyte recruitment, protected against LPS-induced but not against SEA-induced hepatic damage. Conclusions:These novel findings demonstrate that the mechanisms of hepatic injury in endotoxin-induced and superantigen-induced sepsis are principally different. On one hand, SEA-provoked hepatotoxicity is mediated by FasL and is not associated with leukocyte recruitment. On the other hand, liver damage provoked by LPS is mediated by TNF-α and characterized by prominent leukocyte responses. These data may facilitate development of more specific therapies against sepsis of different origins.

Protective effect of Linomide on TNF-α-induced hepatic injury

Abstract Background / Aims : Linomide is an immunomodulator that ameliorates several autoimmune and inflammatory diseases. We assessed the effect of Linomide on microvascular perfusion failure, leukocyte recruitment and hepatocellular injury induced by tumor necrosis factor alpha (TNF-α) and d-Galactosamine (Gal). Methods : After 3 days of Linomide pretreatment (1, 10 and 100mg/kg/day), rats were challenged with TNF-α/Gal for 24h. Microvascular perfusion, leukocyte–endothelium interactions in hepatic postsinusoidal venules and leukocyte sequestration in sinusoids were evaluated using intravital microscopy. Liver enzymes were measured spectrophotometrically. Results : Challenge with TNF-α/Gal significantly reduced sinusoidal perfusion, and increased leukocyte rolling, adhesion and liver enzymes. Interestingly, pretreatment with Linomide (10 and 100mg/kg/day) significantly reduced TNF-α/Gal-induced leukocyte rolling by 65 and 63%, and leukocyte adhesion by 87 and 84%, respectively. Moreover, Linomide (10 and 100mg/kg/day) decreased sinusoidal sequestration of leukocytes by 71 and 51%, and markedly improved sinusoidal perfusion. Moreover, Linomide reduced aspartate aminotransferase by 87–97%, and alanine aminotransferase by 79–96%. However, Linomide had no protective effect when administered concomitantly with TNF-α/Gal. Conclusions : These data demonstrate a dose-dependent inhibitory effect of Linomide on perfusion failure, leukocyte recruitment and hepatocellular injury provoked by TNF-α. Indeed, these findings suggest that Linomide may be an effective substance for protection of the liver in sepsis.

Linomide and antibody-targeted superantigen therapy abolishes formation of liver metastases in mice.

Hematogenous spread of tumor cells and metastasis formation in the liver are insidious aspects of cancer progression and are not frequently amenable to curative treatment. We examined the effect of Linomide and antibody-targeted therapy against the formation of hepatic metastases in vivo. For this purpose, syngenic B16 melanoma cells transfected with GA733-2 (a human colon cancer cell surface antigen) were injected into a mesenteric vein of C57/Bl6 mice. To test bacterial superantigen (Sag) targeting for immunotherapy of liver metastases, we used genetically fused proteins consisting of SEA and a Fab moiety of a GA733-2 tumor-reactive antibody (C215Fab-SEA). Linomide dose-dependently reduced hepatic metastases, and at 300 mg/kg this reduction was more than 80%. Treatment with C215Fab-SEA decreased metastases formation by 49% and the combination of Linomide and C215Fab-SEA was found to completely abolish liver metastases (>99% reduction). Taken together, our novel data suggest that Linomide and antibody-targeted superantigen therapy individually markedly reduce and together abolish liver metastases. Considering that current therapy of hepatic metastases is mainly limited to surgical resection in a subgroup of patients, these findings indicate that Linomide alone or in combination with antibody-targeted superantigen may provide a novel approach against liver metastases.

Mo1247 Neutrophil and Monocyte Activity in Stable Cirrhosis: Relation With Cirrhosis Etiology and Severity As Well As Cytokine Profile and 25(OH) Vitamin D Levels

Background: Chronic liver disease and cirrhosis are a major cause of morbidity and mortality worldwide. Hospitalizations due to complications of cirrhosis are associated with substantial economic burden. We hypothesized that the hospitalizations related to complications of cirrhosis varies by season based on individuals various indoor/outdoor activities, diet and seasonal infection; and we aim to study seasonal variations in hospitalizations due to complications of cirrhosis in the USA from a large national inpatient database. Methods: The National Inpatient Sample database of year 2011 which was collected as part of Healthcare Cost and Utilization Project by Agency for Healthcare Research and Quality were utilized for this study. The NIS is the largest all payer inpatient care database containing around 8 million hospitalizations from around 1000 hospitals in the United States. The hospitalizations related to cirrhosis and its complications including hepatic encephalopathy, esophageal variceal bleeding, and ascites requiring paracentesis were captured using various ICD-9 diagnosis codes. Four seasons were categorized based on the months of hospitalization (spring: March, April, May; summer: June, July, August; fall: September, October, November and winter: December, January, February). Analyses were performed separately different regions of United States. SAS 9.3 was used for analyses. Results: Our study included 36,684 hospitalizations due to complications of cirrhosis which include hepatic encephalopathy, gastrointestinal bleeding, and ascites requiring paracentesis. The hospitalizations related to these complications were comparable across all regions with 24.64% in Spring, 24.70% in Summer, 25.62% in Fall, and 25.00% in Winter. Though wide variation was seen while analyzing data by different regions with highest rate of hospitalizations due to variceal bleeding in the Northeast during spring (34.8%), in the Midwest during fall (31.43%), in the South during winter (27.96%), and in the West highest during summer (27.08%) months. Similarly, hospitalizations due to hepatic encephalopathy in the Northeast were highest in spring (26.22%), in the Midwest during fall (26.25%), in the South during winter (26.13%), and in theWest during summer (25.52%). Hospitalizations due to ascites requiring paracentesis were highest during fall for Northeast (26.98%) and South (27.17%) regions while Midwest and West regions had higher rates during spring (25.60%), and summer (25.87%) respectively.Conclusion: Seasonal variations in hospitalizations due to the complications of cirrhosis are present when hospitalizations due to hepatic encephalopathy, variceal bleeding, and ascites requiring paracentesis are analyzed by various regions. Understanding the factors behind such variations may help guide preventing hospitalizations secondary to these complications.