Ihtzaz Ahmed Malik

Single-Dose Gamma-Irradiation Induces Up-Regulation of Chemokine Gene Expression and Recruitment of Granulocytes into the Portal Area but Not into Other Regions of Rat Hepatic Tissue

Liver damage is a serious clinical complication of gamma-irradiation. We therefore exposed rats to single-dose gamma-irradiation (25 Gy) that was focused on the liver. Three to six hours after irradiation, an increased number of neutrophils (but not mononuclear phagocytes) was observed by immunohistochemistry to be attached to portal vessels between and around the portal (myo)fibroblasts (smooth muscle actin and Thy-1(+) cells). MCP-1/CCL2 staining was also detected in the portal vessel walls, including some cells of the portal area. CC-chemokine (MCP-1/CCL2 and MCP-3/CCL7) and CXC-chemokine (KC/CXCL1, MIP-2/CXCL2, and LIX/CXCL5) gene expression was significantly induced in total RNA from irradiated livers. In laser capture microdissected samples, an early (1 to 3 hours) up-regulation of CCL2, CXCL1, CXCL8, and CXCR2 gene expression was detected in the portal area but not in the parenchyma; with the exception of CXCL1 gene expression. In addition, treatment with an antibody against MCP-1/CCL2 before irradiation led to an increase in gene expression of interferon-gamma and IP-10/CXCL10 in liver tissue without influencing the recruitment of granulocytes. Indeed, the CCL2, CXCL1, CXCL2, and CXCL5 genes were strongly expressed and further up-regulated in liver (myo)fibroblasts after irradiation (8 Gy). Taken together, these results suggest that gamma-irradiation of the liver induces a transient accumulation of granulocytes within the portal area and that (myo)fibroblasts of the portal vessels may be one of the major sources of the chemokines involved in neutrophil recruitment. Moreover, inhibition of more than one chemokine (eg, CXCL1 and CXCL8) may be necessary to reduce leukocytes recruitment.

Expression of stem cell factor and its receptor c-Kit during the development of intrahepatic cholangiocarcinoma.

Stem cell factor (SCF) and its receptor, c-Kit, constitute an important signal transduction system with proliferative and anti-apoptotic functions. Besides regulating hemopoietic stem cell proliferation and liver regeneration, it has been implicated in the regulation of human malignancies. However, the cellular expression of the SCF–c-Kit gene system in the liver during cholangiocarcinogenesis has not been studied to date. The protein- and mRNA-expression levels of SCF and c-Kit genes were examined in normal rat liver, in isolated normal rat liver cells and in a thioacetamide-induced rat model of intrahepatic cholangiocarcinoma (CC). Immunohistochemical analysis of the normal liver showed that SCF is expressed in the wall of the hepatic artery and in some cells, which were located along the sinusoids, although it was absent from hepatocytes and biliary epithelial cells. The mRNA analysis of isolated normal liver cell populations revealed a co-expression of SCF- and c-Kit-mRNA in sinusoidal endothelial cells and in Kupffer cells, whereas passaged and cultured liver myofibroblasts (MFs) expressed only SCF. Low levels of the SCF- and c-Kit-mRNA expression could be detected in isolated hepatocytes of the normal liver. Immunohistochemical analysis of the CC tissue showed SCF positivity in proliferating biliary cells (CK-19+), in macrophages (ED-1+) and in MFs (α-smooth-muscle-actin, α-SMA+) of the tumoral microenvironment. c-Kit-positivity could be detected on hepatocytes of the regenerating nodules and on the proliferating bile ducts of CC. Compared with the normal liver tissue, SCF-mRNA from the CC tissue was upregulated up to 20-fold, whereas c-Kit-mRNA was upregulated up to fivefold. These data indicate that several cell populations may become able to express SCF and/or c-Kit during cholangiocarcinogenesis. Therefore, the SCF–c-Kit system may contribute to tumor development, for instance, by inducing proliferation of hepatocytes and of biliary cells and by acting as a surviving factor for CC cells.

Comparison of changes in gene expression of transferrin receptor-1 and other iron-regulatory proteins in rat liver and brain during acute-phase response

The “acute phase” is clinically characterized by homeostatic alterations such as somnolence, adinamia, fever, muscular weakness, and leukocytosis. Dramatic changes in iron metabolism are observed under acute-phase conditions. Rats were administered turpentine oil (TO) intramuscularly to induce a sterile abscess and killed at various time points. Tissue iron content in the liver and brain increased progressively after TO administration. Immunohistology revealed an abundant expression of transferrin receptor-1 (TfR1) in the membrane and cytoplasm of the liver cells, in contrast to almost only nuclear expression of TfR1 in brain tissue. The expression of TfR1 increased at the protein and RNA levels in both organs. Gene expression of hepcidin, ferritin-H, iron-regulatory protein-1, and heme oxygenase-1 was also upregulated, whereas that of hemojuvelin, ferroportin-1, and the hemochromatosis gene was significantly downregulated at the same time points in both the brain and the liver at the RNA level. However, in contrast to observations in the liver, gene expression of the main acute-phase cytokine (interleukin-6) in the brain was significantly upregulated. In vitro experiments revealed TfR1 membranous protein expression in the liver cells, whereas nuclear and cytoplasmic TfR1 protein was detectable in brain cells. During the non-bacterial acute phase, iron content in the liver and brain increased together with the expression of TfR1. The iron metabolism proteins were regulated in a way similar to that observed in the liver, possibly by locally produced acute-phase cytokines. The significance of the presence of TfR1 in the nucleus of the brain cells has to be clarified.

Induction of chemokines and cytokines before neutrophils and macrophage recruitment in different regions of rat liver after TAA administration

Single-dose thioacetamide (TAA) administration induces inflammation and acute liver damage. The mechanism of inflammatory cell recruitment in the liver is still unclear. The aim of this study was to examine the sequence and recruitment of inflammatory cells in different liver regions in relation to CXC- and CC-chemokine and cytokine expression during acute liver injury. Single-dose TAA was administered to rats intraperitoneally, and animals were killed at different time points thereafter. Serum and liver tissue were taken and frozen immediately. Tissue was used for immunostaining cryostat sections, RNA, and protein extraction. RT-PCR and western blotting were performed for RNA and protein analysis, respectively. An early increase (3 h) in CXCL8/IL-8 levels was measured followed by a marked release in MCP1/CCL2 (24 h) serum levels after TAA administration compared with controls. Similarly, an early increase in specific RNA of hepatic chemokines CXCL1/KC and CXCL8/IL-8 was found at 3 h, followed by an upregulation of CXCL5/LIX (6 h), CXCL2/MIP-2 (12 h), and MCP1/CCL2 gene expression at 24–48 h. Further, an induction of pro-inflammatory cytokines IFN-γ and IL-1β followed by IL-6 and TNF-α was observed with a maximum at 12 h. The magnitude of increase in gene expression of TNF-α and MCP1/CCL2 was the highest among all cytokines and chemokines, respectively. By means of immunohistochemistry, an early (12–24 h) increase in the number of only neutrophil granulocytes (NGs) attached to and around portal vessel walls was observed, followed by increased numbers of mononuclear phagocytes (24–48 h) along the sinusoids. Treatment of the human monocytic cell line U-937 with TNF-α increased the gene expression of CXCL1/KC, CXCL8/IL-8, and MCP1/CCL2. Conversely, adding of infliximab (IFX) to the culture medium inhibited this upregulation significantly. In conclusion, single-dose TAA administration induces a sequence of events with a defined upregulation of gene expression of inflammatory chemokines and cytokines and a transient accumulation of NGs within the portal area and macrophages along the sinusoids throughout the liver. Periportal inflammation seems to precede hepatocellular damage.

Immunodetection of cyclooxygenase-2 (COX-2) is restricted to tissue macrophages in normal rat liver and to recruited mononuclear phagocytes in liver injury and cholangiocarcinoma

It has been suggested that cyclooxygenase-2 (COX-2)-mediated prostaglandin synthesis is associated with liver inflammation and carcinogenesis. The aim of this study is to identify the cellular source of COX-2 expression in different stages, from acute liver injury through liver fibrosis to cholangiocarcinoma (CC). We induced in rats acute and “chronic” liver injury (thioacetamide (TAA) or carbon tetrachloride (CCl4)) and CC development (TAA) and assessed COX-2 gene expression in normal and damaged liver tissue by RT-PCR of total RNA. The cellular localization of COX-2 protein in liver tissue was analyzed by immunohistochemistry as well as in isolated rat liver cells by Western blotting. The findings were compared with those obtained in human cirrhotic liver tissue. The specificity of the antibodies was tested by 2-DE Western blot and mass spectrometric identification of the positive protein spots. RT-PCR analysis of total RNA revealed an increase of hepatic COX-2 gene expression in acutely as well as “chronically” damaged liver. COX-2-protein was detected in those ED1+/ED2+ cells located in the non-damaged tissue (resident tissue macrophages). In addition COX-2 positivity in inflammatory mononuclear phagocytes (ED1+/ED2−), which were also present within the tumoral tissue was detected. COX-2 protein was clearly detectable in isolated Kupffer cells as well as (at lower level) in isolated “inflammatory” macrophages. Similar results were obtained in human cirrhotic liver. COX-2 protein is constitutively detectable in liver tissue macrophages. Inflammatory mononuclear phagocytes contribute to the increase of COX-2 gene expression in acute and chronic liver damage induced by different toxins and in the CC microenvironment.

Melanocortin receptors in rat liver cells: change of gene expression and intracellular localization during acute-phase response.

MCRs are known to be expressed predominantly in the brain where they mediate metabolic and anti-inflammatory functions. Leptin plays an important role in appetite and energy regulation via signaling through melanocortin receptors (MCRs) in the brain. As serum levels of MCR ligands are elevated in a clinical situation [acute-phase response (APR)] to tissue damage, where the liver is responsible for the metabolic changes, we studied hepatic gene expression of MCRs in a model of muscle tissue damage induced by turpentine oil (TO) injection in rats. A significant increase in gene expression of all five MCRs (MC4R was the highest) in liver at the RNA and protein level was detected after TO injection. A similar pattern of increase was also found in the brain. Immunohistology showed MC4R in the cytoplasm, but also in the nucleus of parenchymal and non-parenchymal liver cells, whereas MC3R-positivity was mainly cytoplasmic. A time-dependent migration of MC4R protein from the cytoplasm into the nucleus was observed during APR, in parallel with an increase in α-MSH and leptin serum levels. An increase of MC4R was detected at the protein level in wild-type mice, while such an increase was not observed in IL-6ko mice during APR. Moreover, treatment of isolated liver cells with melanocortin agonists (α-MSH and THIQ) inhibited the endotoxin-induced upregulation of the acute-phase cytokine (IL-6, IL1β and TNF-α) gene expression in Kupffer cells and of chemokine gene expression in hepatocytes. MCRs are expressed not only in the brain, but also in liver cells and their gene expression in liver and brain tissue is upregulated during APR. Due to the presence of specific ligands in the serum, they may mediate metabolic changes and exert a protective effect on liver cells.

Ferroportin-1 is a 'nuclear'-negative acute-phase protein in rat liver: a comparison with other iron-transport proteins.

Liver is the central organ of iron metabolism. During acute-phase-response (APR), serum iron concentration rapidly decreases. The current study aimed to compare expression and localization of iron transport protein ferroportin-1 (Fpn-1) and of other iron import proteins after experimental tissue damage induced by injecting turpentine oil in the hind limbs of rats and mice. Serum and spleen iron concentration decreased with an increase in total liver, cytoplasmic and nuclear iron concentration. In liver, mRNA amount of Fpn-1, Fpn-1a, Fpn-1b, HFE, hemojuvelin (HJV) and hephaestin (heph) genes showed a rapid decrease. Hepcidin, divalent metal transporter-1 (DMT-1), transferrin (Tf) and Tf-receptor-1 (TfR1), TfR-2 (TfR2) gene expression was increased. Western blot analysis of liver tissue lysate confirmed the changes observed at mRNA level. In spleen, a rapid decrease in gene expression of Fpn-1, Fpn-1a, Fpn-1b, DMT-1, Tf, TfR1 and TfR2, and an increase in hepcidin was observed. Immunohistochemistry of DMT-1 and TfR2 were mainly detected in the nucleus of rat liver and spleen, whereas TfR1 was clearly localized in the plasma membrane. Fpn-1 was mostly found in the nuclei of liver cells, whereas in spleen, the protein was mainly detected in the cell membrane. Western blot analysis of liver fractions confirmed immunohistochemical results. In livers of wild-type mice, gene expression of Fpn-1, Fpn-1a and Fpn-1b was downregulated, whereas hepcidin gene expression was increased. In contrast, these changes were less pronounced in IL-6ko-mice. Cytokine (IL-6, IL-1β and TNF-α) treatment of rat hepatocytes showed a downregulation of Fpn-1, Fpn-1a and Fpn-1b, and upregulation of hepcidin gene expression. Moreover, western blot analysis of cell lysate of IL-6-treated hepatocytes detected, as expected, an increase of α2-macroglobulin (positive acute-phase protein), whereas albumin (negative acute-phase protein) and Fpn-1 were downregulated. Our results demonstrate that liver behaves as a ‘sponge’ for iron under acute-phase conditions, and Fpn-1 behaves as a negative acute-phase protein in rat hepatocytes mainly, but not exclusively, because of the effect of IL-6. These changes could explain iron retention in the cytoplasm and in the nucleus of hepatocytes during APR.

Effect of irradiation on gene expression of rat liver adhesion molecules: in vivo and in vitro studies.

Background and Purpose:Migration of leukocytes into tissue is a key element of innate and adaptive immunity. An animal study showed that liver irradiation, in spite of induction of chemokine gene expression, does not lead to recruitment of leukocytes into the parenchyma. The aim of this study was to analyze gene expression of adhesion molecules, which mediate leukocyte recruitment into organs, in irradiated rat liver in vivo and rat hepatocytes in vitro.Material and Methods:Rat livers in vivo were irradiated selectively at 25 Gy. Isolated hepatocytes in vitro were irradiated at 8 Gy. RNA extracted within 48 h after irradiation in vivo and in vitro was analyzed by real-time PCR (polymerase chain reaction) and Northern blot. Adhesion molecule concentration in serum was measured by ELISA (enzyme-linked immunosorbent assay). Cryostat sections of livers were used for immunohistology.Results:Significant radiation-induced increase of ICAM-1 (intercellular adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule-1), JAM-1 (junctional adhesion molecule-1), β1-integrin, β2-integrin, E-cadherin, and P-selectin gene expression could be detected in vivo, while PECAM-1 (platelet-endothelial cell adhesion molecule-1) gene expression remained unchanged. In vitro, β1-integrin, JAM-1, and ICAM-2 showed a radiation-induced increased expression, whereas the levels of P-selectin, ICAM-1, PECAM-1, VCAM-1, Madcam-1 (mucosal addressin cell adhesion molecule-1), β2-integrin, and E-cadherin were downregulated. However, incubation of irradiated hepatocytes with either tumor necrosis factor-(TNF-)α, interleukin-(IL-)1β, or IL-6 plus TNF-α led to an upregulation of P-selectin, ICAM-1 and VCAM-1.Conclusion:The findings suggest that liver irradiation modulates gene expression of the main adhesion molecules in vivo and in cytokine-activated hepatocytes, with the exception of PECAM-1. This may be one reason for the lack of inflammation in the irradiated rat liver.Hintergrund und Ziel:Tierexperimentelle Studien haben gezeigt, dass es in der Akutphase nach Einzeitbestrahlung der Leber mit 25 Gy im Gegensatz zu anderen toxischen Leberschädigungen trotz strahleninduzierter Expression von Chemokinen nicht zu einer inflammatorischen Reaktion mit Einwanderung von Entzündungszellen kommt. Ziel der vorliegenden experimentellen Studie war die Messung der Auswirkungen ionisierender Strahlung auf die Expression der wichtigsten Adhäsionsmoleküle nach Leberbestrahlung in vivo und Bestrahlung von Hepatozyten in vitro im etablierten Modell.Material und Methodik:Innerhalb von 48 h nach selektiver Leberbestrahlung in vivo (25 Gy) sowie Bestrahlung von Hepatozyten in vitro (8 Gy) wurde RNA extrahiert und mittels Real-Time-PCR (Polymerase-Kettenreaktion) und Northern-Blot analysiert. Neben alleinig bestrahlten Hepatozyten wurden dabei in vitro auch Zellen untersucht, die zusätzlich zur Bestrahlung mit Tumor-Nekrose-Faktor-(TNF-)α, Interleukin-(IL-)1β oder einer Kombination aus IL-6/TNF-α inkubiert wurden. Adhäsionsmolekülkonzentrationen im Serum wurden mittels ELISA („enzyme-linked immunosorbent assay“) gemessen, Lebergewebe auch mittels Immunhistochemie untersucht.Ergebnisse:ICAM-1 („intercellular adhesion molecule-1“), VCAM-1 („vascular cell adhesion molecule-1“), JAM-1, („junctional adhesion molecule-1“), β1-Integrin, β2-Integrin, E-Cadherin und P-Selectin waren in vivo nach Bestrahlung vermehrt exprimiert, die PECAM-1-Expression („platelet-endothelial cell adhesion molecule-1“) blieb jedoch unverändert. In vitro kam es zu einer vermehrten Expression von β1-Integrin, JAM-1 und ICAM-2 und einer verminderten Expression von P-Selectin, ICAM-1, PECAM-1, VCAM-1, Madcam-1 („mucosal addressin cell adhesion molecule 1“), β2-Integrin und E-Cadherin. Nach zusätzlicher Inkubation der Hepatozyten mit TNF-α, IL-1β oder IL-6 und TNF-α kam es auch in vitro zu einer vermehrten Expression von P-Selectin, ICAM-1 und VCAM-1.Schlussfolgerung:Leberbestrahlung führt zu einer vermehrten Expression der wichtigsten Adhäsionsmoleküle in vivo und in durch Zytokine aktivierten Hepatozyten. Die PECAM-1-Expression wird allerdings nicht beeinflusst. Dies könnte einer der Gründe für die fehlende Inflammation in diesem Modell sein.

Effect of Irradiation on Gene Expression of Rat Liver Adhesion Molecules

Background and Purpose:Migration of leukocytes into tissue is a key element of innate and adaptive immunity. An animal study showed that liver irradiation, in spite of induction of chemokine gene expression, does not lead to recruitment of leukocytes into the parenchyma. The aim of this study was to analyze gene expression of adhesion molecules, which mediate leukocyte recruitment into organs, in irradiated rat liver in vivo and rat hepatocytes in vitro.Material and Methods:Rat livers in vivo were irradiated selectively at 25 Gy. Isolated hepatocytes in vitro were irradiated at 8 Gy. RNA extracted within 48 h after irradiation in vivo and in vitro was analyzed by real-time PCR (polymerase chain reaction) and Northern blot. Adhesion molecule concentration in serum was measured by ELISA (enzyme-linked immunosorbent assay). Cryostat sections of livers were used for immunohistology.Results:Significant radiation-induced increase of ICAM-1 (intercellular adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule-1), JAM-1 (junctional adhesion molecule-1), β1-integrin, β2-integrin, E-cadherin, and P-selectin gene expression could be detected in vivo, while PECAM-1 (platelet-endothelial cell adhesion molecule-1) gene expression remained unchanged. In vitro, β1-integrin, JAM-1, and ICAM-2 showed a radiation-induced increased expression, whereas the levels of P-selectin, ICAM-1, PECAM-1, VCAM-1, Madcam-1 (mucosal addressin cell adhesion molecule-1), β2-integrin, and E-cadherin were downregulated. However, incubation of irradiated hepatocytes with either tumor necrosis factor-(TNF-)α, interleukin-(IL-)1β, or IL-6 plus TNF-α led to an upregulation of P-selectin, ICAM-1 and VCAM-1.Conclusion:The findings suggest that liver irradiation modulates gene expression of the main adhesion molecules in vivo and in cytokine-activated hepatocytes, with the exception of PECAM-1. This may be one reason for the lack of inflammation in the irradiated rat liver.Hintergrund und Ziel:Tierexperimentelle Studien haben gezeigt, dass es in der Akutphase nach Einzeitbestrahlung der Leber mit 25 Gy im Gegensatz zu anderen toxischen Leberschädigungen trotz strahleninduzierter Expression von Chemokinen nicht zu einer inflammatorischen Reaktion mit Einwanderung von Entzündungszellen kommt. Ziel der vorliegenden experimentellen Studie war die Messung der Auswirkungen ionisierender Strahlung auf die Expression der wichtigsten Adhäsionsmoleküle nach Leberbestrahlung in vivo und Bestrahlung von Hepatozyten in vitro im etablierten Modell.Material und Methodik:Innerhalb von 48 h nach selektiver Leberbestrahlung in vivo (25 Gy) sowie Bestrahlung von Hepatozyten in vitro (8 Gy) wurde RNA extrahiert und mittels Real-Time-PCR (Polymerase-Kettenreaktion) und Northern-Blot analysiert. Neben alleinig bestrahlten Hepatozyten wurden dabei in vitro auch Zellen untersucht, die zusätzlich zur Bestrahlung mit Tumor-Nekrose-Faktor-(TNF-)α, Interleukin-(IL-)1β oder einer Kombination aus IL-6/TNF-α inkubiert wurden. Adhäsionsmolekülkonzentrationen im Serum wurden mittels ELISA („enzyme-linked immunosorbent assay“) gemessen, Lebergewebe auch mittels Immunhistochemie untersucht.Ergebnisse:ICAM-1 („intercellular adhesion molecule-1“), VCAM-1 („vascular cell adhesion molecule-1“), JAM-1, („junctional adhesion molecule-1“), β1-Integrin, β2-Integrin, E-Cadherin und P-Selectin waren in vivo nach Bestrahlung vermehrt exprimiert, die PECAM-1-Expression („platelet-endothelial cell adhesion molecule-1“) blieb jedoch unverändert. In vitro kam es zu einer vermehrten Expression von β1-Integrin, JAM-1 und ICAM-2 und einer verminderten Expression von P-Selectin, ICAM-1, PECAM-1, VCAM-1, Madcam-1 („mucosal addressin cell adhesion molecule 1“), β2-Integrin und E-Cadherin. Nach zusätzlicher Inkubation der Hepatozyten mit TNF-α, IL-1β oder IL-6 und TNF-α kam es auch in vitro zu einer vermehrten Expression von P-Selectin, ICAM-1 und VCAM-1.Schlussfolgerung:Leberbestrahlung führt zu einer vermehrten Expression der wichtigsten Adhäsionsmoleküle in vivo und in durch Zytokine aktivierten Hepatozyten. Die PECAM-1-Expression wird allerdings nicht beeinflusst. Dies könnte einer der Gründe für die fehlende Inflammation in diesem Modell sein.

Ferritin L is the sole serum ferritin constituent and a positive hepatic acute phase protein

ABSTRACT Ferritin L (FTL) and ferritin H (FTH) subunits are responsible for intracellular iron storage. Serum ferritin levels are not only dependant on body iron stores. Aims of the present study are to demonstrate nature, source, and major regulatory mediators of serum ferritin in an animal model of acute-phase (AP) response. Animals (rats, wild-type [WT] mice, and interleukin [IL]-6ko mice) were injected with turpentine oil (TO) intra-muscularity to induce a sterile abscess and sacrificed at different time points afterward. Rat hepatocytes were isolated for cell culture and, after reaching confluence, stimulated with major AP cytokines to induce AP conditions. We found a significantly increased expression of both ferritin subunits in liver at mRNA and protein levels during AP response. In the serum of both control and TO-injected rats, only FTL was detectable by Western blotting, whereas no increase in serum FTL was measured by Western blot or enzyme-linked immunosorbent assay. An increase in protein expression of FTL and FTH was observed in lysates of rat hepatocytes after treatment with IL-6, IL-1&bgr;, and tumor necrosis factor-&agr;; however, only FTL was increasingly released into supernatant. In both TO-injected rats and WT mice, a dramatic increase in serum IL-6 levels was observed, along with an increased amount of hepatic ferritin subunits. However, an increase of hepatic FTL but not of FTH protein expression was observed in IL-6ko mice after TO injection. Our data demonstrate that FTL is the only rat serum ferritin whose release into circulation from the hepatocytes is increased by the effect of AP cytokines (e.g., IL-6). In contrast, FTH expression is intracellular in both under physiological and AP conditions.