Neutrophils contribute to liver damage during endotoxin shock. The objective of this investigation was to document where neutrophils localize in the hepatic vasculature and whether they migrate out of sinusoids or postsinusoidal venules. A well-characterized model of galactosamine and endotoxin shock and immunostaining for neutrophil-associated migration inhibition factor-related protein complex 8/14 S100 calcium-binding proteins were used. Treatment of C3Heb/FeJ mice with 100 micrograms/kg Salmonella abortus equi endotoxin alone or in combination with 700 mg/kg galactosamine induced a time-dependent increase of neutrophil margination in sinusoids and postsinusoidal venules at 4 h. The number of venular neutrophils decreased in both groups at later time points without evidence for transmigration. Extravasation of neutrophils was only observed from sinusoids in galactosamine plus endotoxin-treated animals between 4 and 7 h, which correlated with parenchymal cell injury. After endotoxin alone, large numbers of neutrophils remained sequestered in sinusoids without injury. These data suggest that neutrophils cause hepatocellular injury during endotoxemia after extravasation and are less likely to cause damage when sequestered in the vasculature. In the liver, neutrophils migrate out of sinusoids and not out of postsinusoidal venules.

We studied the role of P-selectin, an adhesion molecule known to be important for neutrophil localization to sites of inflammation, in a model of inflammatory liver injury. Male C3Heb/ FeJ (ET-sensitive) mice treated with 700 mg/kg galactosamine and 100 g/kg Salmonella abortus equi endotoxin (Gal/ET), murine tumor necrosis factor ␣ (TNF-␣, 15 g/kg), or interleukin-1 (IL-1, 13-23 g/kg), showed increased P-selectin mRNA levels in the liver. In contrast, C3H/HeJ (ET-resistant) mice responded only to cytokines with P-selectin mRNA formation. Whereas no P-selectin expression was detectable in control livers, there was temporary staining of endothelium in large blood vessels but not in sinusoids between 3 and 5 h after ET, TNF-␣, or IL-1 treatment. Severe liver injury induced by Gal/ET at 7 h was not inhibited by an anti-P-selectin antibody in C3Heb/FeJ mice or in P-selectin-deficient animals. Sequestration of neutrophils in sinusoids, i.e. those neutrophils that have been identified as critical for the injury, was not affected by the antibody or in P-selectindeficient mice. However, the temporary margination in portal and post-sinusoidal venules was reduced by 75% in anti-P-selectin antibody-treated animals and by 51% in P-selectin-deficient mice. We conclude that hepatic P-selectin gene transcription in vivo involves cytokines. However, blocking P-selectin neither attenuated sinusoidal neutrophil sequestration nor prevented neutrophil-induced liver injury during endotoxin shock but attenuated neutrophil margination in larger vessels. Thus, our data demonstrate similarities and fundamental differences in the requirement for adhesion molecules to localize neutrophils in the liver vasculature compared to other organs during an inflammatory response.

Neutrophil-induced liver injury during endotoxemia is dependent on the adhesion molecule Mac-1 (CD11b/CD18) on neutrophils. The potential involvement of its counterreceptor, intercellular adhesion molecule-1 (ICAM-1), in the pathogenesis was investigated after administration of 100 micrograms/kg Salmonella abortus equi endotoxin (ET) in galactosamine-sensitized mice (Gal). In ET-sensitive mice (C3Heb/FeJ), which generated large amounts of tumor necrosis factor-alpha (TNF-alpha), massive neutrophil infiltration and severe liver injury were observed. In an ET-resistant strain (C3H/HeJ), which did not generate TNF-alpha Gal/ET failed to cause neutrophil accumulation or injury. ICAM-1 messenger RNA (mRNA), negligible in control livers, was selectively induced by Gal/ET in ET-sensitive mice. Intravenous injection of murine TNF-alpha, interleukin-1 alpha (IL-1 alpha) or IL-I beta (13 to 23 micrograms/kg) strongly induced the ICAM-1 message in both strains, showing a comparable capacity for ICAM-1 mRNA synthesis. All cytokines caused similar neutrophil accumulation in the liver; however, only Gal/TNF-alpha also caused upregulation of Mac-1 on circulating neutrophils and liver injury. The anti-murine ICAM-1 monoclonal antibody YN.1 (3 mg/kg) attenuated liver injury in ET-sensitive mice by 67% to 90% compared with isotype-matched control antibody-treated animals but did not reduce neutrophil accumulation in hepatic sinusoids. Our data suggest that the cytokines TNF-alpha and IL-1 are the main mediators responsible for upregulation of ICAM-1 mRNA in the liver during endotoxemia. The upregulation of both adhesion molecules, ICAM-1 and Mac-1, is necessary for a neutrophil-induced liver injury to occur. (ABSTRACT TRUNCATED AT 250 WORDS)

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