Involvement of the TNF and FasL Produced by CD11b Kupffer Cells/Macrophages in CCl₄-Induced Acute Hepatic Injury

<div><p>We previously reported that F4/80⁺ Kupffer cells are subclassified into CD68⁺ Kupffer cells with phagocytic and ROS producing capacity, and CD11b⁺ Kupffer cells with cytokine-producing capacity. Carbon tetrachloride (CCl₄)-induced hepatic injury is a well-known chemical-induced hepatocyte injury. In the present study, we investigated the immunological role of Kupffer cells/macrophages in CCl₄-induced hepatitis in mice. The immunohistochemical analysis of the liver and the flow cytometry of the liver mononuclear cells showed that clodronate liposome (c-lipo) treatment greatly decreased the spindle-shaped F4/80⁺ or CD68⁺ cells, while the oval-shaped F4/80⁺ CD11b⁺ cells increased. Notably, severe hepatic injury induced by CCl₄ was further aggravated by c-lipo-pretreatment. The population of CD11b⁺ Kupffer cells/macrophages dramatically increased 24 hour (h) after CCl₄ administration, especially in c-lipo-pretreated mice. The CD11b⁺ Kupffer cells expressed intracellular TNF and surface Fas-ligand (FasL). Furthermore, anti-TNF Ab pretreatment (which decreased the FasL expression of CD11b⁺ Kupffer cells), anti-FasL Ab pretreatment or <i>gld/gld</i> mice attenuated the liver injury induced by CCl₄. CD1d−/− mouse and cell depletion experiments showed that NKT cells and NK cells were not involved in the hepatic injury. The adoptive transfer and cytotoxic assay against primary cultured hepatocytes confirmed the role of CD11b⁺ Kupffer cells in CCl₄-induced hepatitis. Interestingly, the serum MCP-1 level rapidly increased and peaked at six h after c-lipo pretreatment, suggesting that the MCP-1 produced by c-lipo-phagocytized CD68⁺ Kupffer cells may recruit CD11b⁺ macrophages from the periphery and bone marrow. The CD11b⁺ Kupffer cells producing TNF and FasL thus play a pivotal role in CCl₄-induced acute hepatic injury.</p></div

The effect of c-lipo pretreatment on the liver histopathology after CCl₄ injection.

<p>The pathological findings of c-lipo- or PBS-pretreated mice 48 h after CCl₄ challenge. The liver is shown (×100 magnification; H&E staining). (A) The PBS pretreated mice and (B) c-lipo pretreated mice. The data are representative of three mice in each group, with similar results observed for all animals.</p

The serum MCP-1 levels after c-lipo treatment and the MCP-1 production from CD68⁺ Kupffer cells after CCl₄ injection with/without c-lipo pretreatment.

<p>(A) There was an increase in the serum MCP-1 levels in mice early after c-lipo injection. The mice were i.p. injected with c-lipo or PBS, the sera were obtained at the indicated time points, and the MCP-1 levels were measured (n = 3 in each group). (B) The serum MCP-1 levels did not increase after CCl₄ injection in mice pretreated with c-lipo. The mice pretreated with c-lipo or PBS were injected intraperitoneally with CCl₄ or oil, and sera were obtained at the indicated time points to measure the MCP-1 levels (n = 3 in each group).(C) The <i>ex vivo</i> production of MCP-1 from the liver CD68⁺ Kupffer cells from CCl₄-injected mice. One hour after the injection of CCl₄, the liver MNCs were harvested from the liver by collagenase treatment, and CD68⁺ cells were obtained by magnetic beads (MACS system). F4/80⁺ CD11b⁺ cells obtained by F4/80 magnetic beads from the liver MNCs from c-lipo treated mice. Both purified subsets were cultured <i>in vitro</i> for the indicated amounts of time. The data are the means ± SE from three independent experiments. **<i>p</i><0.01, *<i>p</i><0.05 vs. other groups.</p

C-lipo pretreatment aggravates CCl_4-induced hepatic injury.

<p>Mice pretreated with c-lipo or PBS were injected intraperitoneally with CCl₄ or oil. (A) The serum ALT levels were evaluated at the indicated times after CCl₄ stimulation. (B) The influence of CCl₄ challenge on the serum TNF levels. The data are the means ± SE from 10 mice in each group. **<i>p</i><0.01 vs. other groups.</p

The intracellular TNF production and FasL expression of Kupffer cells.

<p>(A) The expression of intra-cellular TNF and (B) FasL expression in the F4/80⁺ CD11b⁺ cells or F4/80⁺ CD68⁺ cells was examined 12 h after the CCl₄ injection. (C) The effect of pretreatment with a neutralizing TNF Ab on the FasL expression of F4/80⁺ CD11b⁺ cells. The data are representative of five mice in each experiment, with similar results obtained for each mouse. **<i>p</i><0.01 vs. other groups.</p

The flow cytometric analysis of Kupffer cells/macrophages.

<p>Livers were harvested from mice 36-lipo or PBS, and liver MNCs obtained after collagenase digestion of livers were stained for F4/80, CD11b and CD68. The results of a forward scatter (FS)/side scatter (SS) analysis of the total MNCs are shown (left panels). The mean FS values are also shown (401±2.9 vs. 436±6.8, n = 5, p<005) (left panels). The F4/80/CD11b expression is also presented and the F4/80 positive populations are inside of square gate (middle panels). The CD11b/CD68 expression levels of the gated F4/80-positive cells are demonstrated (right panels). F4/80⁺ CD11b⁺ cells are shown by red dots and the F4/80⁺ CD68⁺ cells are shown by blue dots. **<i>p</i><0.01, *<i>p</i><0.05 vs. other groups. The data are representative of five mice in each group, with similar results for the five mice.</p

The effect of c-lipo pretreatment on the population of F4/80⁺ CD11b⁺ Kupffer cells after CCl₄ injection.

<p>The changes in the population of F4/80-positive Kupffer cells after CCl₄ challenge with c-lipo or PBS pretreatment were examined. Mice were intraperitoneally injected with CCl₄ 36 h after c-lipo or PBS treatment. As an experimental control, the vehicle oil was intraperitoneally injected. The changes in the total amount of liver MNCs, the population of F4/80⁺ cells, and the proportion of each Kupffer cell subset following CCl₄ challenge after c-lipo or PBS pretreatment are shown. The F4/80⁺ CD11b⁺ cells are shown by red dots and the F4/80⁺ CD68⁺ cells are shown by blue dots. The data are representative of five mice in each group, with similar results. **<i>p</i><0.01, *<i>p</i><0.05 vs. other groups.</p

The induction of hepatic injury in mice adoptively transferred mice with CD11b⁺ Kupffer cells from mice injected with CCl₄, and in vitro cytotoxic assay against primary cultured hepatocytes.

<p>Livers were obtained from mice 14₄. Minced liver specimens without collagenase treatment in 1% FBS RPMI 1640 were filtered through a stainless steel mesh. Thereafter, the liver MNCs were obtained using a 33% Percoll solution. The liver MNCs were subjected to MACS sorting to separate the F4/80⁺ cells and F4/80⁻ cells. (A) The purity of the MACS-sorted F4/80⁺ cells and F4/80⁻ cells was confirmed by flow cytometry. The harvested F4/80⁺ cells were mostly CD11b⁺ Kupffer cells. (B) 5×10⁶ F4/80⁺ cells or 5×10⁶ F4/80⁻ cells were adoptively transferred into normal mice or CD68⁺ Kupffer cell-depleted mice (by c-lipo), and the serum ALT levels were evaluated at the indicated time points. As an experimental control, 5×10⁶ liver F4/80⁺ cells from oil-treated mice were transferred into normal mice. (C) Cytotoxic activity of sorted F4/80⁺ cells and F4/80⁻ cells against primary cultured hepatocytes. The data are the means ± SE from six mice in each group. **<i>p</i><0.01, *<i>p</i><0.05 vs. other groups. Cytotoxic activity of F4/80⁺ cells with anti-FasL antibody was also measured. The data are the means ± SE from three independent experiments. *<i>p</i><0.05 vs. other groups.</p

Feasibility of Quantification of Intracranial Aneurysm Pulsation with 4D CTA with Manual and Computer-Aided Post-Processing

<div><p>Background and Purpose</p><p>The analysis of the pulsation of unruptured intracranial aneurysms might improve the assessment of their stability and risk of rupture. Pulsations can easily be concealed due to the small movements of the aneurysm wall, making post-processing highly demanding. We hypothesized that the quantification of aneurysm pulsation is technically feasible and can be improved by computer-aided post-processing.</p><p>Materials and Methods</p><p>Images of 14 cerebral aneurysms were acquired with an ECG-triggered 4D CTA. Aneurysms were post-processed manually and computer-aided on a 3D model. Volume curves and random noise-curves were compared with the arterial pulse wave and volume curves were compared between both post-processing modalities.</p><p>Results</p><p>The aneurysm volume curves showed higher similarity with the pulse wave than the random curves (Hausdorff-distances 0.12 vs 0.25, p<0.01). Both post-processing methods did not differ in intra- (r = 0.45 vs r = 0.54, p>0.05) and inter-observer (r = 0.45 vs r = 0.54, p>0.05) reliability. Time needed for segmentation was significantly reduced in the computer-aided group (3.9 ± 1.8 min vs 20.8 ± 7.8 min, p<0.01).</p><p>Conclusion</p><p>Our results show pulsatile changes in a subset of the studied aneurysms with the final prove of underlying volume changes remaining unsettled. Semi-automatic post-processing significantly reduces post-processing time but cannot yet replace manual segmentation.</p></div

Mouse CD11b⁺Kupffer Cells Recruited from Bone Marrow Accelerate Liver Regeneration after Partial Hepatectomy

<div><p>TNF and Fas/FasL are vital components, not only in hepatocyte injury, but are also required for hepatocyte regeneration. Liver F4/80⁺Kupffer cells are classified into two subsets; resident radio-resistant CD68⁺cells with phagocytic and bactericidal activity, and recruited radio-sensitive CD11b⁺cells with cytokine-producing capacity. The aim of this study was to investigate the role of these Kupffer cells in the liver regeneration after partial hepatectomy (PHx) in mice. The proportion of Kupffer cell subsets in the remnant liver was examined in C57BL/6 mice by flow cytometry after PHx. To examine the role of CD11b⁺Kupffer cells/Mφ, mice were depleted of these cells before PHx by non-lethal 5 Gy irradiation with or without bone marrow transplantation (BMT) or the injection of a CCR2 (MCP-1 receptor) antagonist, and liver regeneration was evaluated. Although the proportion of CD68⁺Kupffer cells did not significantly change after PHx, the proportion of CD11b⁺Kupffer cells/Mφ and their FasL expression was greatly increased at three days after PHx, when the hepatocytes vigorously proliferate. Serum TNF and MCP-1 levels peaked one day after PHx. Irradiation eliminated the CD11b⁺Kupffer cells/Mφ for approximately two weeks in the liver, while CD68⁺Kupffer cells, NK cells and NKT cells remained, and hepatocyte regeneration was retarded. However, BMT partially restored CD11b⁺Kupffer cells/Mφ and recovered the liver regeneration. Furthermore, CCR2 antagonist treatment decreased the CD11b⁺Kupffer cells/Mφ and significantly inhibited liver regeneration. The CD11b⁺Kupffer cells/Mφ recruited from bone marrow by the MCP-1 produced by CD68⁺Kupffer cells play a pivotal role in liver regeneration via the TNF/FasL/Fas pathway after PHx.</p></div