Augmenter of liver regeneration attenuates inflammatory response in the postischemic mouse liver in vivo.

BACKGROUND: Augmenter of Liver Regeneration (ALR), a protein synthesized in the liver is suggested to be protective against oxidative stress-induced cell death. Hepatic ischemia-reperfusion (I/R) injury is triggered by reactive oxygen species. Here, we tested the hypothesis that ALR attenuates hepatic I/R injury in vivo. METHODS: C57BL6 mice were subjected to warm hepatic ischemia for 90 min. Either recombinant ALR (100 µg/kg) or vehicle were administered to mice prior ischemia. During reperfusion, neutrophil and CD4+ T cell migration and sinusoidal perfusion were analyzed using intravital microscopy. Alanine aminotransferase-aspartate aminotransferase (plasma) and caspase-3 (tissue) activities were determined as markers of hepatocellular necrotic and apoptotic injury. RESULTS: Hepatic I/R led to dramatic enhancement of neutrophil and CD4+ T cell recruitment in hepatic microvessels, sinusoidal perfusion failure, and strong elevation of aspartate aminotransferase-alanine aminotransferase and caspase-3 activities. During early reperfusion (60 min), the pretreatment with ALR improved postischemic perfusion failure (P < 0.05) and attenuated liver enzyme activities. Recruitment of CD4+ T cells, but not of neutrophils was attenuated. After 240 min of reperfusion, the protective effect of ALR was stronger, since the liver enzyme activity, perfusion failure, and leukocyte influx were significantly attenuated. As shown by the measurement of caspase-3 activity, postischemic apoptosis was reduced in the ALR-treated group. CONCLUSIONS: Our in vivo data show that ALR has a therapeutic potential against postischemic liver injury. As a mechanism, we suggest a direct protective effect of ALR on apoptotic and necrotic death of hepatocytes and an attenuation of inflammatory cell influx into the postischemic tissue.

The importance of liver microcirculation in promoting autoimmune hepatitis via maintaining an inflammatory cytokine milieu--a mathematical model study.

In autoimmune diseases, inflammatory cytokine concentrations are important for initiating and maintaining the status of autoimmunity. Autoimmune hepatitis (AIH) is an inflammatory liver disease characterized by a loss of immune tolerance against specific antigens located in hepatocytes. During the progression of the disease, antigen-presenting cells and different classes of T-helper cells secrete specific cytokines important for maintaining the disease. As these cytokines are secreted into the local liver environment, the blood flow in liver sinusoids might influence the local cytokine concentration. Considering the liver tissue as a porous medium, based on Darcy׳s law, the microcirculation within a liver lobule was modelled. Using realistic physiological pressure differences and tissue permeabilities, the blood velocity inside the sinusoids could be calculated and validated with blood velocity data obtained via Orthogonal Polarization Spectral Imaging (OPSI). Furthermore, oxygen consumption is modelled to obtain Rappaport׳s acinus model. Finally, steady state spatial distributions of secreted cytokines within the liver lobule could be estimated for specified realistic production rates of T-helper cells. It could be demonstrated that the characteristics of the liver microcirculation have an important impact on establishing inflammatory cytokine levels within the portal fields and the vascular septa promoting the occurrence of interface hepatitis.

Mechanisms of liver involvement in systemic disease.

The liver may be injured during the course of many systemic diseases. The mechanisms of injury can be broadly divided into four pathways: vascular, toxic, immune, and hormonal. Vascular obstruction may be an early event but is also the late common pathway from all mechanisms. Despite the large number of possible initiating factors, the end results are few, including death of hepatocytes or cholangiocytes, leading to the stereotyped syndromes of acute liver failure, non-cirrhotic portal hypertension, or cirrhosis. This small number of outcomes is a reflection of the few anatomic patterns that can be generated by microvascular obstruction. Vascular obstruction may occur by thrombosis, inflammation, or congestive injury. The innate immunity pathway is activated by endotoxin and other agents, leading to inflammatory infiltration, release of cytokines and reactive oxygen species, and necrosis. The adaptive immune pathway involves the generation of antibodies and antigen-specific cell-mediated attack on hepatic cells. Hormonal effects are principally involved when overnutrition leads to hyperinsulinemia followed by hepatocellular necrosis.

The role of CD14 in neutrophil recruitment within the liver microcirculation during endotoxemia.

During Gram-negative sepsis and endotoxemia, CD14 is essential for the recognition of LPS by the TLR4 complex and subsequent generation of systemic inflammation. However, CD14-independent responses to LPS have been reported in vitro and in vivo in selected tissues including the skin. As the liver is a key target organ for neutrophil sequestration and inflammatory pathology during sepsis and endotoxemia, we investigated the role of CD14 in the recruitment of neutrophils into the liver in a mouse model of endotoxemia. Using dynamic in vivo imaging of the liver, we observed that neutrophil recruitment within the sinusoids and post-sinusoidal venules occurred equivalently between LPS-treated wild-type and CD14-knockout mice. Neutrophil recruitment within the liver was completely independent of CD14 regardless of whether it was expressed on cells of hematopoietic or nonhematopoietic origin or in serum as soluble CD14. Whereas CD14 expression was essential for activation of circulating neutrophils and for the development of LPS-induced systemic inflammation (pulmonary neutrophil sequestration, leukopenia, and increased serum proinflammatory cytokine levels), deficiency of CD14 did not limit the adhesion strength of neutrophils in vitro. Furthermore, wild-type and CD14-knockout mice displayed identical deposition of serum-derived hyaluronan-associated protein within liver sinusoids in response to LPS, indicating that the sinusoid-specific CD44/hyaluronan/serum-derived hyaluronan-associated protein-dependent pathway of neutrophil adhesion is activated independently of CD14. Therefore, the liver microcirculation possesses a unique CD14-independent mechanism of LPS detection and activation of neutrophil recruitment.

Hepatic microvascular pressure during anaphylactic shock in anesthetized rats.

OBJECTIVE: Hepatic venoconstriction plays a significant role in anaphylactic hypotension in anesthetized rats. The purpose of this study is to determine whether the primary site of anaphylactic venoconstriction in the liver venous circulation occurs prior to or distal to the sinusoidal capillaries. We also determined whether the hepatic blood volume is increased during anaphylactic hypotension. METHODS: We measured, using a servo-null micropipette pressure-measuring system, the hepatic venular transmural pressure (P micro hv) at the liver surface of anesthetized rats sensitized with the antigen of ovalbumin (1 mg). We also measured the liver lobe thickness, using the ultrasonic crystal dimension measuring system. Anaphylactic hypotension was induced by an intravenous injection of 0.6 mg ovalbumin. RESULTS: When the antigen was injected, the systemic arterial pressure decreased profoundly from 118+/-9 to 45+/-4 mm Hg, which was accompanied by an increase in Ppv and P micro hv: P micro hv only transiently increased from 3.1+/-0.9 to 8.8+/-1.5 cm H(2)O at 1 min and then rapidly returned to the baseline within 2 min, when Ppv continued to increase and reached the peak of 36+/-7 cm H(2)O at 3.5 min after antigen. This greater increase in Ppv-to-P micro hv gradient than that in P micro hv-to-Pcv gradient after antigen indicated that the constriction of the portal veins and the sinusoids much predominates over that of the hepatic veins. Along with this hepatic pre- and sinusoidal constriction, the liver lobe thickness significantly decreased by 4% after antigen. CONCLUSION: Pre-sinusoidal constriction during anaphylactic shock in anaesthetized rats increased the portal venous pressure while the hepatic venular pressure only increased slightly and transiently. This predominant pre-sinusoidal constriction is accompanied by a decrease in liver volume.

Variation in the levels of inflammatory cytokines depending on ischemic time: effects on respiratory variables.

OBJECTIVES: We sought to evaluate the association between ischemic times, cytokines-interleukin (IL)-6, IL-1b, tumor necrosis factor-alpha, sIL-2r, IL-8, and IL-10-and alterations in gaseous exchange. MATERIALS AND METHODS: This prospective study of 42 orthotopic liver transplantation (OLT) recipients examined ischemic times and respiratory variables measured as alterations in intrapulmonary shunt and in the Po(2)/Fio(2) ratio. Centrifuged blood samples were frozen at -80 degrees C for storage. The Inmulite-One system (Euro/Dpc, Gwynedd, UK) was used to determine the concentration of cytokines. For statistical analysis, we used the Pearson correlation coefficient. RESULTS: The average cold ischemic time was 478 minutes (range, 35-929) and warm ischemic time was 69.58 minutes (range, 20-180). The warm ischemic time affected the degree of shunt at the end of the operation (P < .027) and the levels of IL-10 (P < .018) and IL-6 (P < .000). The final degree of shunting and IL-10 (P < .044) showed a correlation. The cold ischemic time affected IL-1 (P < .046) and IL-8 levels (P < .023). The reperfusion syndrome was correlated with the final levels of IL-10 (P < .064) and of IL-8 (P < .066). CONCLUSION: Warm and cold ischemic times affect the final cytokine levels and the degree of intrapulmonary shunt.

The liver as a lymphoid organ.

The liver receives blood from both the systemic circulation and the intestine, and in distinctive, thin-walled sinusoids this mixture passes over a large macrophage population, termed Kupffer cells. The exposure of liver cells to antigens, and to microbial products derived from the intestinal bacteria, has resulted in a distinctive local immune environment. Innate lymphocytes, including both natural killer cells and natural killer T cells, are unusually abundant in the liver. Multiple populations of nonhematopoietic liver cells, including sinusoidal endothelial cells, stellate cells located in the subendothelial space, and liver parenchymal cells, take on the roles of antigen-presenting cells. These cells present antigen in the context of immunosuppressive cytokines and inhibitory cell surface ligands, and immune responses to liver antigens often result in tolerance. Important human pathogens, including hepatitis C virus and the malaria parasite, exploit the liver's environment, subvert immunity, and establish persistent infection.

Reciprocal activation between CD4+ T cells and Kupffer cells during hepatic ischemia-reperfusion.

BACKGROUND: Mechanisms mediating CD4+ T-cell recruitment during alloantigen-independent hepatic ischemia-reperfusion (I/R) remain not fully understood. We hypothesized that Kupffer cells activate CD4+ T-cells in the postischemic liver, by the release of free oxygen radicals and cytokines. METHODS: Recruitment of freshly isolated and fluorescence-labeled CD4+ T-cell was analyzed after hepatic I/R (90/30-120 min) using intravital microscopy in sham-operated mice, in mice after hepatic I/R and in postischemic groups after Kupffer cell depletion, after treatment with antioxidant glutathione, in interleukin (IL)-6-/- mice; and in wild-type mice after infusion of tumor necrosis factor (TNF) receptor-1-/-CD4+ T-cells. Using flow cytometry and immunohistochemistry, we assessed whether Kupffer cell-derived mediators activate CD4+ T-cells and sinusoidal endothelial cells. The clearance kinetics of fluorescence-labeled latex beads was determined as a marker of Kupffer cell activity in vivo. RESULTS: I/R-induced accumulation of CD4+ T-cells in hepatic sinusoids was significantly attenuated on Kupffer cell depletion, after scavenging of free radicals and after interruption of the IL-6- and TNF-alpha-dependent pathways. These mediators directly activate CD4+ T-cells and up-regulated the expression of T cell-relevant adhesion molecules on sinusoidal endothelial cells. Postischemic activity of Kupffer cells was significantly impaired in wild-type mice, and was even more depressed in CD4-/- animals. CONCLUSION: Kupffer cells trigger recruitment of CD4+ T-cells in the postischemic liver by the release of reactive oxygen species, IL-6, and TNF-alpha. These mediators are capable of activating CD4+ T-cells and sinusoidal endothelial cells. CD4+ T-cells, in turn, influence the activation of Kupffer cells.

Liver: An organ with predominant innate immunity.

UNLABELLED: Blood circulating from the intestines to the liver is rich in bacterial products, environmental toxins, and food antigens. To effectively and quickly defend against potentially toxic agents without launching harmful immune responses, the liver relies on its strong innate immune system. This comprises enrichment of innate immune cells (such as macrophages, natural killer, natural killer T, and gammadelta T cells) and removal of waste molecules and immunologic elimination of microorganisms by liver endothelial cells and Kupffer cells. In addition, the liver also plays an important role in controlling systemic innate immunity through the biosynthesis of numerous soluble pathogen-recognition receptors and complement components. CONCLUSION: The liver is an organ with predominant innate immunity, playing an important role not only in host defenses against invading microorganisms and tumor transformation but also in liver injury and repair. Recent evidence suggests that innate immunity is also involved in the pathogenesis of liver fibrosis, providing novel therapeutic targets to treat such a liver disorder.

Hemostatic analysis of a 13 year old with antiphospholipid syndrome and restrictive pericarditis.

Antiphospholipid syndrome is an autoimmune thrombophilic disorder that is uncommon in adults and remarkably rare in children. Thrombotic etiological factors are variable in antiphospholipid syndrome, including antibody-antigen complex-mediated platelet activation, inhibition of anticoagulants, or attenuation of fibrinolysis. We present the case of a child with antiphospholipid syndrome presenting with syncope, constrictive pericarditis and hepatic enlargement that was found to have platelet-mediated hypercoagulability and marked clot lysis via thrombelastography in the preoperative period. Restoration of circulation following pericardectomy and inotropic support was associated with attenuation of hypercoagulability and fibrinolysis. It is concluded that the etiological factors responsible for antiphospholipid syndrome-mediated hemostatic abnormalities and the probable effects of hepatic hypoperfusion on clot lysis in this patient were detected with thrombelastography, and similar thrombelastographic analyses are recommended to compliment standard coagulation assessments of patients with antiphospholipid syndrome.

The CD154-CD40 T-cell co-stimulation pathway in liver ischemia and reperfusion inflammatory responses.

BACKGROUND: Ischemia-reperfusion (I/R) injury is a prime antigen-independent inflammatory factor in the dysfunction of liver transplants. The precise contribution of T cells in the mechanism of I/R injury remains to be elucidated. As the CD154-CD40 co-stimulation pathway provides essential second signal in the initiation and maintenance of T-cell-dependent immune responses, this study was designed to assess the role of CD154 signaling in the pathophysiology of liver I/R injury. METHODS: A mouse model of partial 90-min warm hepatic ischemia followed by 6 hr of reperfusion was used. Three animal groups were studied: (1) wild-type (WT) mice treated with Ad-(-gal versus Ad-CD40 immunoglobulin; (2) untreated WT versus CD154 (MR1) monoclonal antibody-treated WT mice; and (3) untreated WT versus CD154 knockout mice. RESULTS: The disruption of CD154 signaling in all three animal groups ameliorated otherwise fulminant liver injury, as evidenced by depressed serum glutamic oxaloacetic transaminase levels, compared with controls. These beneficial effects were accompanied by depressed hepatic T-cell sequestration, local decrease of vascular endothelial growth factor expression, inhibition of tumor necrosis factor-(and T-helper type 1 cytokine production, and induction of antiapoptotic (Bcl-2/Bcl-xl) but depression of proapoptotic (caspase-3) proteins. CONCLUSIONS: By using in parallel a gene therapy approach, pharmacologic blockade, and genetically targeted mice, these findings document the benefits of disrupting CD154 to selectively modulate inflammatory responses in liver I/R injury. This study reinforces the key role of CD154-CD40 T-cell co-stimulation in the pathophysiology of liver I/R injury.

The portal immunosuppressive storm: relevance to islet transplantation?

Outcomes in clinical islet transplantation improved substantially with the introduction of combined sirolimus and tacrolimus immunosuppression. However, multiple islet preparations are often required to achieve insulin independence, suggesting that islet engraftment may not be optimal when these agents are absorbed via the portal vein. The current study was designed to assess the differential concentrations of immunosuppressive drugs within the portal and systemic circulations of a large animal model, to assess the local concentrations of drugs to which islets are exposed early after implantation. Chronic catheters were placed in the portal vein and carotid artery of 6 mongrel dogs, and immunosuppressants were administered orally. Blood samples were drawn simultaneously from portal and systemic catheters, and drug concentrations were analyzed. Peak immunosuppressant levels as well as area under the curve were dramatically elevated in portal blood relative to systemic levels for all drugs tested. This "portal storm" of immunosuppression may be relevant to intrahepatic islet transplantation.

Hepatic T cells and liver tolerance.

The T-cell biology of the liver is unlike that of any other organ. The local lymphocyte population is enriched in natural killer (NK) and NKT cells, which might have crucial roles in the recruitment of circulating T cells. A large macrophage population and the efficient trafficking of dendritic cells from sinusoidal blood to lymph promote antigen trapping and T-cell priming, but the local presentation of antigen causes T-cell inactivation, tolerance and apoptosis. These local mechanisms might result from the need to maintain immunological silence to harmless antigenic material in food. The overall bias of intrahepatic T-cell responses towards tolerance might account for the survival of liver allografts and for the persistence of some liver pathogens.

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.

Influence of humoral immunoreaction on hepatic nonparenchymal cells in ex situ xenoperfused rat livers.

BACKGROUND: The influence of xenogeneic humoral immunoreaction on hepatic nonparenchymal cells (NPCs) was evaluated ex situ in xenoperfused rat livers. METHODS: Isolated rat livers were perfused via the portal vein (PV) for 240 min. The perfusates consisted of fresh rat blood (group 1), fresh human blood (group 2), and fresh human blood containing 5 microg/mL soluble complement receptor type 1 (Group 3). RESULTS: Deposition of human IgM and C(5b-9) complement was observed in group 2, although only human IgM deposition was detected in group 3. Portal vein pressure in group 2 rose drastically during the first 10 min. Creatine kinase BB component gradually increased in all groups, followed by an elevation in alanine aminotransferase and both parameters were significantly higher in group 2 than in groups 1 and 3. In group 2, platelet thrombi in the peripheral PVs and periportal hemorrhage were observed after 10 min, and massive necrosis around the central veins after 240 min; these changes were not observed in group 1 or 3. Production of tumor necrosis factor alpha and alpha interferon and expression of intercellular adhesion molecule 1 (ICAM-1) were lower in group 2 than in groups 1 and 3. In group 2, there were negative areas for ICAM-1 and tumor necrosis factor alpha staining around the central veins after 240 min, which were consistent with necrotic areas. CONCLUSIONS: In xenoperfused rat livers, humoral mediators initially caused the disturbance of microcirculation, which would induce long ischemia in the pericentral areas, resulting in massive necrosis. NPC necrosis may be responsible for less production of cytokines and adhesion molecules in the xenoperfused livers.

Liver perfusion and hepatocellular inflammatory response in sepsis.

Sepsis is characterized by disturbances in liver perfusion and alterations in intrahepatic cellular functions and interactions. This provokes structural and functional liver damage as well as hepatocellular activation that is believed to perpetuate the immuno-inflammatory response. Changes in hepatic perfusion during sepsis are still poorly understood due to the heterogeneity of septic animal models and the difficult accessibility of the hepatic circulation in humans. Sinusoidal blood flow is severely compromised during sepsis due to a decline in perfused sinusoidal area in association with a decrease in sinusoidal flow velocity. Imbalances in the production of nitric oxide may account for these (micro) circulatory disorders. Interactions between liver macrophages, activated endothelial cells and hepatocytes determine the intensity of inflammation and contribute to initial liver damage. Hepatocellular injury is then enhanced by attracted and invading neutrophils. The management of hepatic dysfunction during sepsis is largely supportive and based on prevention and vigorous resuscitation including early nutritional support and adequate oxygenation. Interestingly, experimental studies suggest that pharmacological interventions with significant hemodynamic effects, such as dobutamine and nitric oxide synthase inhibitors, may adversely affect the liver during the septic process.

Enhanced CD34 expression of sinusoid-like vascular endothelial cells in hepatocellular carcinoma.

The immunohistochemical expression of CD34 (human hematopoietic stem cell and endothelial cell marker) and laminin were studied in chronic liver diseases and hepatocellular carcinoma (HCC) to elucidate whether their expression reflected phenotypic differences between non-cancerous sinusoids and sinusoid-like tumor vessels. In normal liver, hepatic sinusoids were always negative for CD34 and laminin. In chronic hepatitis and cirrhosis, the two antigens were sparsely expressed in capillarized sinusoids at periportal and perinodular area. In advanced HCC, CD34 was strongly and diffusely expressed by the endothelial lining of sinusoid-like tumor vessels. However, early-stage HCC showed a wide spectrum of CD34 expression from negative to focal and diffuse, strongly positive staining in sinusoid-like vessels. Laminin was strongly expressed in advanced HCC but not in early-stage HCC. The results indicate that the enhanced expression of CD34 by sinusoidal endothelial cells may reflect the phenotypic change of endothelial cells in chronic liver diseases and HCC, and that the expression may correlate with the processes of angiogenesis induced by hepatocarcinogenesis.