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Cold preservation of rat sinusoidal endothelial cells causes actin disassembly, cell rounding, matrix metalloproteinase (MMP) secretion, and platelet adhesiveness. Studies in rats suggest that gelatinases MMP2 and MMP9 are the key mediators of the injury. We created a model of cold preservation injury in mouse sinusoidal endothelial cell (MSEC) to examine the effect of cold on MSEC, specifically on MSEC from genetically deleted mice (MMP9/KO) mice. MSEC were isolated from wild-type and MMP9/KO mice and cold preserved for up to 24 hours. MMP activity was measured in culture supernatants and in effluents from preserved whole mouse livers. Cellular and actin morphology were studied by light and fluorescence microscopy. A platelet-MSEC adhesion assay was performed. Yield, growth, and appearance of MSEC were similar in wild-type and MMP9/KO mice. Cold-preserved wild-type MSEC exhibited actin disassembly and cell rounding as in the rat but at a much slower rate. These morphologic cell changes were attenuated in MSEC from MMP9/KO mice. Both MMP2 and MMP9 were present in liver effluents of wild-type mice, but MMP9 was absent in effluents from MMP9/KO mice. Total MMP activity in culture supernatants was greater after preservation in wild-type than in MMP9/KO mice. There was significantly more platelet adhesion to wild-type MSEC than to MSEC from MMP9/KO mice. In conclusion, MSEC is an excellent model system for the study of cold preservation injury. Injury is similar to rat sinusoidal endothelial cells but delayed. MMP9 is a key mediator of the cold preservation injury.

Leukocyte adhesion on reperfusion is a critical component of cold preservation injury, and involves increased intercellular adhesion molecule 1 (ICAM-1) expression on sinusoidal endothelial cells (SEC). This study determined whether ICAM-1 expression occurs during cold preservation and whether actin disassembly is necessary for ICAM-1 expression and leukocyte adhesion. ICAM-1 expression was measured in isolated rat SEC during 8 hours of cold preservation by immunofluorescence techniques. Leukocyte adhesion to cold-preserved SEC was measured in an assay using fluorescently labeled leukocytes. The calpain inhibitors N-acetyl-leu-leu-norleucinal/N-acetyl-leu-leu-methioninal and the actin stabilizer phalloidin were added in some studies to prevent actin disassembly. Cold-exposed SEC showed a rapid increase of surface ICAM-1 expression, reaching maximum values in 1 hour. Studies in permeabilized cells suggested that ICAM-1 moved from a perinuclear location to the cell surface. Actin stabilization had no effect on the time-dependent increase in ICAM-1 expression, but seemed to affect the distribution of ICAM-1 on the cell surface. Leukocyte adhesion to SEC correlated with ICAM-1 expression and was reduced to control levels by an anti-ICAM-1 antibody. Although actin stabilization did not reduce ICAM-1 expression, it did reduce leukocyte-SEC adhesion to control values. Increased ICAM-1 expression on cold-preserved SEC is a direct effect of cold. It is not related to actin disassembly, although it seems that actin disassembly affects the distribution of ICAM-1. Leukocyte adhesion to cold-preserved SEC requires both increased ICAM-1 and actin disassembly. Agents that inhibit actin disassembly can significantly decrease leukocyte adhesion regardless of increased ICAM-1 expression.

This study was performed to determine the role of intracellular calcium concentration and calpain activity on the cellular events that occur in rat sinusoidal endothelial cells (SEC) in the cold. Intracellular calcium concentrations were measured in isolated cold preserved rat SEC. Dantrolene or 1,2-bis(o-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) was added in some studies. In other studies, calpain activity and m-calpain and mu-calpain expression were measured during cold preservation in the presence or absence of calpain inhibitors. The effect of addition of dantrolene to preservation solutions on function of whole livers after preservation was determined. Cold preservation caused an increase in intracellular calcium concentration first detected at 1 hour of preservation. This was associated with cell rounding and actin disassembly. Dantrolene and BAPTA-AM prevented the increase in intracellular calcium concentration and reduced cell rounding and actin disassembly. Cold preservation also resulted in increased calpain activity and expression on SEC. Calpain expression was reduced by dantrolene. Calpain inhibitors N-acetyl-leu-leu-norleucinal (ALLN) and N-acetyl-leu-leu-methioninal (ALLM) reduced calpain activity and expression and restored SEC cell shape and actin morphology. Dantrolene improved function of livers preserved in Eurocollins (EC) solution when tested on the isolated perfused rat liver (IPRL). In conclusion, exposure of SEC to cold results sequentially in elevated intracellular calcium concentration, increased calpain activity, and actin disassembly.

It has been suggested that the liver traps and deletes activated and potentially harmful T cells, especially of the CD8(+) subset, providing mechanisms to limit systemic immune responses. It is unknown whether this also applies to CD4(+) T cells. In this study, we show that activated stages of CD4(+) T cells were trapped in the liver on intraportal injection. Intravital microscopy showed an immediate adhesion of activated CD4(+) T cells within periportal sinusoids after intraportal injection. Furthermore, we detected high frequencies of interferon gamma (IFN-gamma)-- (Th1) and interleukin 4 (IL-4)-- (Th2) synthesizing effector cells in the liver. Transfer experiments were performed to identify those phenotypes showing specific retention in the liver. Our data show that effector stages and activated cells in general are more efficiently recruited into the liver than resting CD4(+) T cells, similar to what has previously been shown for CD45RB(low) memory cells. In addition, we observed a certain preference for Th1-polarized cells to be trapped by the liver. However, the actual cytokine-producing cells did not specifically enrich among the total population. In conclusion, these data indicate that the liver acts as a filter for activated and memory/effector cells. Cells trapped in the liver might subsequently undergo modulatory influences exerted by the postulated specific microenvironment of the liver.