Effect of Radiation on Leukocytes Migration and Gene expression of Rat Liver Chemokines: In-vivo and In-vitro Studies

Liver damage is a serious clinical complication of gamma-irradiation. CXC- and CC-chemokines, known to attract inflammatory cells, play a role in transmigration process during inflammation. Chemokines are generated locally at sites of inflammation and orchestrate the recruitment of specific subpopulations of leukocytes from blood into tissues. The aim of this work was to study the recruitment of inflammatory cells in different regions of rat liver tissue by means of immunohistology and to extend the analysis on chemokines known to be involved in recruitment of inflammatory cells. A mild hepatocellular damage was observed after gamma-irradiation by analyzing the liver enzymes in rat serum. Furthermore, CXCL2 serum concentration was also significantly increased up to 3 hours after irradiation as compared to sham-irradiated controls. Immunohistologically, 3-6 hours after irradiation an increased number of neutrophil granulocytes (but not of mononuclear phagocytes) was observed attached to portal vessels between and around the portal (myo)fibroblasts (SMA and Thy-1+ cells) in rat liver. The number of ED-1 (mononuclear phagocytes) positive cells was not increased at any time points after irradiation. Positive staining for monocyte chemoattractant protein -1(MCP-1/CCL2) was also detected in the portal vessel walls. In an attempt to search for differentially expressed genes after gamma-irradiation in rat liver at mRNA level, microarray gene-expression analysis was performed, this showed up-regulation of 31 and down-regulation for 24 genes. A fast and early induction of several leukocytes attracting CC-chemokines (MCP-1/CCL2, MCP-3/CCL7) and CXC- chemokines (CXCL-1/KC, CXCL2 and LIX/CXCL5) genes were observed at RNA level. Furthermore, CXCR-2 receptor gene expression was also upregulated early (1-3h) in irradiated rat liver tissue. Laser capture micro-dissected (LCM) samples were used to localize the gene(s) involved in neutrophils recruitment in the rat liver after irradiation. In the portal area, a strong and early induction in the gene-expression of CCL2 followed by CXCL1, CXCL8 and CXCR2 was detected compared to the parenchymal region of irradiated liver. Therefore, CCL2 can be categorized as a novel gene in periportal region involved in liver inflammation after irradiation. Administration of an antibody against MCP-1/CCL2 30 min before irradiation led to a significant increase in gene expression of IFN-gamma and IP-10/CXCL10 in liver tissue but did not influence recruitment of granulocytes into the portal area. As no major change in studied chemokines was observed in isolated hepatocytes after irradiation, isolated liver (myo)fibroblasts (LMF) (reside in periportal area) were irradiated (8Gy) in order to find the source of these chemokines in the liver. A constitutive expression of these chemokines (CCL2, CXCL1, CXCL2, CXCL5 and CXCL8) and their further up-regulation was found in LMF after irradiation. Single dose (unfractionated) gamma-irradiation administrated percutaneously to the healthy rat liver induces a mild liver damage together with transient accumulation of granulocytes only within the portal area without effecting the recruitment of mono-nuclear phagocytes. This may be due to differently expressed chemokines in the cells of portal vessels; (myo)fibroblasts seem to be one of the major source of the chemokines involved in neutrophils recruitment. Administration of an antibody against MCP-1/CCL2 has not influenced recruitment of granulocytes into the portal area but changed the hepatic gene expression. Inhibition of more than one chemokines (e.g. CXCL1, CXCL8) may be necessary to reduce leukocytes recruitment. Furthermore, normal liver seems to be quite resistant to high single-dose gamma-irradiation. Most results of the present thesis have been published in Malik et al (2010) "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", Am J Pathol; 176 (4): 1801-15, doi:10.2353/ajpath.2010.09050