Effects of exogenous MIP-2 and KC treatment on hepatocyte proliferation and liver regeneration after partial hepatectomy.

<p>Wild-type mice were injected intravenously with high doses or low doses of MIP-2 and KC, starting 24 hours after hepatectomy and continued daily. An identical volume of sterile phosphate-buffered saline (PBS) was used as a vehicle control. (A) Hepatocyte proliferation was determined by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and quantitative analysis of PCNA labeling. Data are mean ± SEM with n = 4–8 per group. *<i>P</i><0.05 compared to vehicle group. Original magnification was 400X. (B) Liver regeneration was determined by increases in liver mass. Data are mean ± SEM with n = 4–8 per group. *<i>P</i><0.05 compared to vehicle group.</p

Effects of exogenous MIP-2 and KC treatment on hepatocyte proliferation in the normal liver.

<p>Wild-type mice were injected intravenously with low doses of MIP-2 and KC every 24 hours. An identical volume of sterile phosphate-buffered saline (PBS) was used as a vehicle control. Hepatocyte proliferation was determined by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and quantitative analysis of PCNA labeling. Data are mean ± SEM with n = 4–8 per group. Original magnification was 400X.</p

Effect of CXCR2 on hepatocyte proliferation and liver regeneration after I/R injury (A) and partial hepatectomy (B) was determined by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and quantitative analysis of PCNA labeling.

<p>(A) Hepatocyte proliferation in post-ischemic liver (upper panel) after I/R injury showed a significant increase in CXCR2−/− mice. Data are mean ± SEM with n = 3–14 per group. *<i>P</i><0.05 compared to wild-type mice. Hepatocyte proliferation and liver regeneration in non-ischemic liver (lower panel) after I/R injury was significantly reduced in CXCR2−/− mice. Data are mean ± SEM with n = 3–6 per group. *<i>P</i><0.05 compared to wild-type mice. (B) Hepatocyte proliferation and liver regeneration after partial hepatectomy showed a significant decrease in CXCR2−/− mice. Data are mean ± SEM with n = 3–4 per group. *<i>P</i><0.05 compared to wild-type mice.</p

Hepatocyte proliferation and liver regeneration in ischemic and non-ischemic liver lobes after I/R injury.

<p>(A) Hepatocyte proliferation was determined by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and quantitative analysis of PCNA labeling. Data are mean ± SEM with n = 3–5 per group. *<i>P</i><0.05 compared to ischemic liver. (B) Liver mass of ischemic and non-ischemic lobes after I/R injury. Data are mean ± SEM with n = 3–5 per group. *<i>P</i><0.05 compared to sham.</p

CXC Chemokines Function as a Rheostat for Hepatocyte Proliferation and Liver Regeneration

<div><p>Background</p><p>Our previous in vitro studies have demonstrated dose-dependent effects of CXCR2 ligands on hepatocyte cell death and proliferation. In the current study, we sought to determine if CXCR2 ligand concentration is responsible for the divergent effects of these mediators on liver regeneration after ischemia/reperfusion injury and partial hepatectomy.</p><p>Methods</p><p>Murine models of partial ischemia/reperfusion injury and hepatectomy were used to study the effect of CXCR2 ligands on liver regeneration.</p><p>Results</p><p>We found that hepatic expression of the CXCR2 ligands, macrophage inflammatory protein-2 (MIP-2) and keratinocyte-derived chemokine (KC), was significantly increased after both I/R injury and partial hepatectomy. However, expression of these ligands after I/R injury was 30-100-fold greater than after hepatectomy. Interestingly, the same pattern of expression was found in ischemic versus non-ischemic liver lobes following I/R injury with expression significantly greater in the ischemic liver lobes. In both systems, lower ligand expression was associated with increased hepatocyte proliferation and liver regeneration in a CXCR2-dependent fashion. To confirm that these effects were related to ligand concentration, we administered exogenous MIP-2 and KC to mice undergoing partial hepatectomy. Mice received a “high” dose that replicated serum levels found after I/R injury and a “low” dose that was similar to that found after hepatectomy. Mice receiving the “high” dose had reduced levels of hepatocyte proliferation and regeneration whereas the “low” dose promoted hepatocyte proliferation and regeneration.</p><p>Conclusions</p><p>Together, these data demonstrate that concentrations of CXC chemokines regulate the hepatic proliferative response and subsequent liver regeneration.</p></div

Differential hepatic expression of CXC chemokines in I/R injury and hepatectomy.

<p>Expression of MIP-2 and KC in the remnant liver after partial hepatectomy or ischemic and non-ischemic lobes after I/R injury. Data are mean ± SEM with n = 3–6 per group. *<i>P</i><0.05 compared to sham group. **<i>P</i><0.05 compared to ischemic liver.</p

Increased survival and decreased bacterial sepsis-associated tissue damage of mice with T-cell targeted deletion of HIF-1α.

<p><u>A: </u><i>Use of the hypoxic marker EF5 reveals that CD4+ and CD8+ T cells have been exposed to low oxygen tension (hypoxic) conditions in the peritoneum during sepsis in mice.</i> Single cell suspensions from peritoneal lavage fluid and spleens were analyzed by flow cytometry using anti-EF5 mAb (Elk3-52 Cy5). <u>B: </u><i>High Efficiency of Cre recombinase-mediated deletion of HIF-1</i> α <i>in T-Cells.</i> Efficiency of deletion was calculated by quantitative real-time PCR as described. Constitutively synthesized HIF-1 α mRNA was detected in control (lck-Cre negative) but not in (lck-Cre positive) HIF1 α gene targeted mice. N = 3 per group. <u>C: </u><i>T cell lineage specific HIF-1</i> α <i>deficient mice are more resistant to lethal sepsis after cecal ligation and puncture procedure.</i> Mice underwent CLP and were observed for mortality. N = 13 per group. p = 0.0326, Logrank (Mantel-Cox). <u>D: </u><i>T cell lineage specific HIF-1</i> α <i>deficient mice have less sepsis-associated liver damage as evaluated by levels of ALT transaminase activity in serum.</i> Serum samples obtained from mice 72 hrs after CLP. *:p<0.05 vs. WT, N = 5–6 per group.</p

HIF-1α is a negative regulator of TCR-triggered pro-inflammatory cytokine secretion <i>in vitro</i> and <i>in vivo</i>.

<p><u>A: </u><i>T lymphocytes deficient in HIF-1α undergo more cell divisions as compared to wild type T cells.</i> Splenic T cells were purified, stained using CFSE, activated for 72 hours and analyzed by flow cytometry as described. A representative dot plot of wild type CD4 T cells (A) and HIF-1α deficient T cells (B) showing both activation by CD25 expression and cell divisions. (C) Comparison of number of divisions by CD4 T cells.*: p<0.05 vs. WT, N = 3 per group.<u> B: </u><i>T cell specific disruption of HIF-1 α gene substantially increases pro-inflammatory cytokine secretion by ex vivo TCR-activated T cells.</i> Spleen T cells were derived from T cell lineage specific HIF-1 α deficient mice. Cells were activated for 24 h under hypoxic conditions (1% O₂). Extracellularly secreted cytokines were determined by ELISA. *:p<0.05 vs. WT, N = 5.<u> C: </u><i>Higher intracellular levels of IFN-γ production by inflamed peritoneum-located hypoxic HIF-1 α deficient CD8+ T cells as compared with similarly located in vivo hypoxic HIF-1 α expressing T cells after CLP</i>. Peritoneal lavage was performed 72 hrs after CLP and 1,5×10⁶ T cells were restimulated and stained with anti-IFN-<i>γ</i> mAb. <u>D: </u><i>Levels of proinflammatory cytokines TNF-α and IL-6 are significantly higher as compared to mice with selective disruption of HIF-1α gene in T-cells.</i> Peritoneal fluid (TNF-α, left panel) and serum (IL-6, right panel) were withdrawn at the indicated times after CLP and cytokines were determined by ELISA. Closed circles: HIF-1α KO, open circles: WT. *:p<0.05 vs. WT, N = 4</p

Increased NF-κB m-RNA expression and activity of <i>ex vivo</i> activated T cells of mice with T-cell targeted deletion of HIF-1 α.

<p>For all three panels, T-cells from spleens were isolated from age and sex matched lck Cre (−) and lck Cre (+) HIF-1α loxP mice and stimulated as described (WT-S, κΟ−S). Unstimulated cells served as controls (WT, κΟ).<u> A: </u><i>T cell specific disruption of HIF-1 α gene substantially increases NF-κB binding activity in ex vivo TCR-activated T cells.</i> Nuclear extracts were prepared from harvested cells and EMSA was conducted. The experiment was repeated and representative data of two experiments are shown. All lanes contain hot binding probe for NF-κB. Specificity of EMSA was tested in the presence of 50 fold excess of either unlabeled probe (Con 1) or CRE specific probe (Con 2), respectively.<u> B: </u><i>T cell specific disruption of HIF-1 α gene increases NF-κB p50 and p65 binding activity in ex vivo TCR-activated T cells.</i> NF-κB-ELISA was conducted with nuclear extracts. *:p<0.05 vs. WT, N = 4.<u> C: </u><i>T cell specific disruption of HIF-1 α gene increases NF-κB p50 mRNA expression in ex vivo TCR-activated T cells.</i> RNA was prepared and subsided to quantitative RT-PCR. *:p<0.01 vs. WT. N = 4.</p

Decreased bacterial burden in mice with T-cell targeted deletion of HIF-1 α.

<p><u>A: </u><i>T cell lineage specific HIF-1</i> α <i>deficient mice have much less bacterial burden in liver and spleen 72 hrs after CLP</i> *:p<0.05 vs. WT, means±SEM, N = 3 per group. <u>B: </u><i>Growth of gas-forming bacteria and tissue destruction during CLP-induced sepsis in mice with HIF-1</i> α<i>-expressing T cells, but not in mice with HIF-1</i> α <i>gene–deleted T cells.</i> Masses of bacteria form rings around gas bubbles in spleens of mice with HIF-1 α expressing T cells. Much less bacteria could be seen in the spleen taken 72 h after CLP from mice with HIF-1 α deficiency in T cells.<u> C: </u><i>T-cell specific deficiency in HIF-1 α enhances effector functions of bactericidal granulocytes. </i><u>Left Panel:</u> Much stronger upregulation of activation marker CD11b on tissue granulocytes (CD11b⁺/Gr-1^bright cells) isolated from HIF-1 α-deleted mice compared to HIF-1 α -expressing control mice. *:p<0.05 vs. WT, N = 3. <u>Right Panel:</u> Higher spontaneous production of hydrogen peroxide by tissue granulocytes (CD11b⁺/Gr-1^bright cells) in HIF-1 α-deleted mice than in HIF-1 α-expressing control mice. *:p<0.05 vs. WT, N = 3.</p