J. Monserrat

Mesenteric Th1 polarization and monocyte TNF-α production: First steps to systemic inflammation in rats with cirrhosis†

A systemic inflammatory state with increased circulating tumor necrosis factor alpha (TNF‐α) has been related to the bacterial infection susceptibility and hemodynamic derangement of patients with cirrhosis. We compared the activation status of immune cell subpopulations defined by 4‐color cytometry in mesenteric and peripheral lymph nodes and blood of rats with CCl4‐cirrhosis to define the immune response initiation site, the T‐cell and monocyte contribution to pro‐inflammatory cytokine production, as well as the pathogenic role of enteric bacteria in the cirrhosis immune response. Th1 cells and monocytes were expanded in the mesenteric nodes (P < .001) and blood (P < .001) of rats with cirrhosis, and activated to produce interferon gamma (P < .0001) and TNF‐α (P < .0001), respectively. The greater numbers of recently activated CD134+ Th cells in mesenteric nodes compared with blood, the correlation between their numbers in mesenteric nodes and blood (r = 0.66, P < .001), and the expansion of activated CD45RC− Th cells, which are unable to re‐enter lymph nodes, in mesenteric nodes but not in blood or axillary nodes points to mesenteric nodes as the origin site of activated Th cells. Abrogation of bacterial translocation by bowel decontamination reduced the number of activated Th cells and monocytes, and normalized interferon gamma production by Th cells and TNF‐α production by monocytes in mesenteric nodes and blood, respectively. In conclusion, in cirrhosis, enteric bacteria start off an orchestrated immune response cascade in mesenteric nodes involving Th1 polarization and monocyte activation to TNF‐α production. Later, the recirculation of these activated effector immune cells into blood promotes systemic inflammation. (HEPATOLOGY 2005;42:411–419.)

Loss of lineage antigens is a common feature of apoptotic lymphocytes

The analysis of apoptosis in cell populations involves the detection of their specific lineage antigen (LAg) expression. This experimental approach relies on their assumed constant expression, but it is unclear whether such expression is actually maintained during cell death. We examined whether the loss of LAgs is a common feature of apoptotic lymphocytes and whether some might completely lose their LAgs. The changes in the expression of CD3, CD5, CD8, CD4, CD28, CD56, and CD19 were monitored in highly purified lymphocyte populations obtained by negative selection in a fluorescence‐activated cell sorter. These were cultured for 24 h with or without phytohemagglutinin or staurosporin. For each LAg‐positive subset studied, apoptosis was consistently more common among cells showing partial or total loss of LAg expression compared with cells maintaining their initial LAg levels. The kinetics of expression loss was rapid for CD8, CD56, and CD28, and more than 80% of initial expression was lost in the early stages of apoptosis but was slower for CD3, CD5, and CD4. For CD3 and CD5, expression was dependent on the apoptotic stimulus used. It is interesting that loss of antigen expression was independent of cell size. This phenomenon was also found in nonmanipulated, highly pure CD19 B lymphocytes of peripheral blood mononuclear cells from B chronic lymphocytic leukemia patients. Loss of LAg expression appeared to be a common feature of apoptotic lymphocytes under all the conditions assayed. The different kinetic patterns of LAg loss suggest apoptotic cells might actively regulate this process.

609 SMALL INTESTINE INFLAMMATION IN RATS WITH CCL4 CIRRHOSIS: ROLE OF ENTERIC BACTERIA

macrophages during conditions of excess iron by blocking the iron exporter ferroportin. In addition, IL-6 mediated upregulation of hepcidin during inflammation is the major mechanism of the socalled ‘anemia of chronic disease’. We here identify H2O2 which is co-released by inflammatory cells as potent transcriptional activator of hepcidin independent of upstream regulators IL-6 and BMP6. Material andmethods: H2O2 release (1–8mM) by immune cells was mimicked using purified enzymes glucose oxidase and catalase as recently described (GOX/CAT system). Huh7 hepatoma cells were treated with either IL-6 or BMP6 alone, or in combination with steady state H2O2 over 24h. Hepcidin regulation was assessed by quantitative real time PCR. Members of the intracellular signaling cascade such as STAT3 were assessed by Western blotting. Promotor studies were performed using hepcidin-promoter constructs with various deletions fused to luciferase as reporter gene with Renilla as control reporter gene. Results: Steady state non-toxic H2O2 concentrations comparable to those by inflammatory cells rapidly and drastically upregulate hepcidin in a dose-dependent way in Huh7 cells by a factor of 10. In addition, H2O2 further potentiates IL6 and BMP6 mediated upregulation of hepcidin 5-fold. Interestingly, induction was also not blunted by confounding hypoxia (2%). The H2O2mediated hepcidin response is an early response reaching the maximum mRNA-concentration already after 6h. Promoter studies identified the STAT3 element as major promoter region of H2O2 mediated hepcidin induction. Indeed, phosphorylation of STAT3 was confirmed under these conditions using Western blotting. Conclusions: Our studies establish H2O2 as as an important regulatory link between systemic iron regulation and oxidative stress. Impaired H2O2-hepcidin signaling in chronic liver diseases could contribute to hepatic accumulation of iron in cirrhotic patients.

528 THE DEFECTIVE FUNCTION OF GUT DENDRITIC CELLS OF RATS WITH CIRRHOSIS AND BACTERIAL TRANSLOCATION IS CORRECTED BY BOWEL DECONTAMINATION WITH ANTIBIOTICS

528 THE DEFECTIVE FUNCTION OF GUT DENDRITIC CELLS OF RATS WITH CIRRHOSIS AND BACTERIAL TRANSLOCATION IS CORRECTED BY BOWEL DECONTAMINATION WITH ANTIBIOTICS L. Munoz, M. Ubeda, M.J. Borrero, M. Lario, R. Frances, D. Diaz, J. Monserrat, L. Lledo, J. Such, M. Alvarez-Mon, A. Albillos. University of Alcala, Ciberehd, Alcala de Henares, Madrid, Hospital General Universitario de Alicante, Ciberhed, Alicante, Hospital Universitario Pŕincipe de Asturias, University of Alcala, Ciberehd, Alcala de Henares, Hospital Universitario Ramon y Cajal, University of Alcala, Ciberehd, Madrid, Spain E-mail: leticia.munoz@uah.es