Resistance to endotoxic shock and reduced neutrophil migration in mice deficient for the Src-family kinases Hck and Fgr

Abstract

Signal transduction through the leukocyte integrins is required for the processes of firm adhesion, activation, and chemotaxis of neutrophils during inflammatory reactions. Neutrophils isolated from knockout mice that are deficient in the expression of p59/61(hck) (Hck) and p58(c-fgr) (Fgr), members of the Src-family of protein tyrosine kinases, have been shown to be defective in adhesion mediated activation. Cells from these animals have impaired induction of respiratory burst and granule secretion following plating on surfaces that crosslink beta2 and beta3 integrins. To determine if the defective function of hck-/-fgr-/- neutrophils observed in vitro also results in impaired inflammatory responses in vivo, we examined responses induced by lipopolysaccharide (LPS) injection in these animals. The hck-/-fgr-/- mice showed marked resistance to the lethal effects of high-dose LPS injection despite the fact that high levels of serum tumor necrosis factor alpha and interleukin 1alpha were detected. Serum chemistry analysis revealed a marked reduction in liver and renal damage in mutant mice treated with LPS, whereas blood counts showed a marked neutrophilia that was not seen in wild-type animals. Direct examination of liver sections from mutant mice revealed reduced neutrophil migration into the tissue. These data demonstrate that defective integrin signaling in neutrophils, caused by loss of Hck and Fgr tyrosine kinase activity, results in impaired inflammation-dependent tissue injury in vivo.

Figure 1

Double mutant hck^−/−fgr^−/− mice are resistant to high-dose LPS but are sensitive to low dose LPS following d-Gal sensitization. (A) Wild-type and hck^−/−fgr^−/− mutant mice were injected i.p. with the indicated doses of LPS, and survival was monitored over the subsequent 8 days. (B) Wild-type and double mutant mice were injected with d-Gal (20 mg) plus the indicated doses of LPS, and survival was monitored. (C) Wild-type, hck^−/−, fgr^−/−, and hck^−/−fgr^−/− mutant mice were injected with 30 mg/kg LPS, and survival was monitored. Data for all graphs are from independent experiments done separately. In A and C, 10 age and weight-matched animals were used per curve (hence A represents a total of 40 hck^−/−fgr^−/− mice and 40 wild-type animals); in B, 10 hck^−/−fgr^−/− and 10 wild-type mice were used for the 0.3 mg/kg doses; other doses used 5 animals per curve.

Figure 2

Serum levels of TNF-α and IL-1 are equivalent in wild-type and mutant mice following LPS injection. Wild-type and hck^−/−fgr^−/− mice were injected i.p. with LPS (30 mg/kg), and serum was collected by retroorbital bleeding at the indicated time points. Serum levels of TNF-α and IL-1α were determined by ELISA. Data shown are averaged values from five mice per genotype ± SD.

Figure 3

Double mutant hck^−/−fgr^−/− mice develop only mild tissue damage during endotoxic shock. Serum chemistry levels assessing liver damage (AST/ALT), renal function (BUN), and muscle/kidney damage (LDH) were determined in wild-type and double mutant mice at the indicated time points following i.p. injection of LPS (30 mg/ml). A total of 15 mice per group were injected, and blood was collected by retroorbital bleeding at the indicated times; however within 24 hr 9 wild-type mice had died and by 48 hr only 2 animals remained; therefore, these two time points reflect data averaged from fewer animals. Over the course of this experiment 13 hck^−/−fgr^−/− mutants survived completely (2 died before 48 hr; hence the final data points reflects results averaged from remaining animals). To facilitate comparison between wild-type and mutant animals, we determined the slopes of curves between the 0 and 24-hr time points for each analyte. For all analytes the slope was greater than 3-fold higher in wild-type (wt) than hck^−/−fgr^−/− animals (AST: wt = 31, mutant = 8; ALT: wt = 22, mutant = 7; BUN: wt = 2.9, mutant 0.6; LDH: wt = 203, mutant = 47). Error bars are SD.

Figure 4

Double mutant mice develop peripheral neutrophilia during endotoxic shock. Total blood neutrophil and lymphocyte counts were determined in the same cohort of mice used in Fig. 3.

Figure 5

Neutrophil migration to the liver during endotoxic shock is reduced in hck^−/−fgr^−/− mice. (A–D) Wild-type and double mutant mice were injected i.p. with LPS (30 mg/kg) and killed 24 hr later. Liver tissues were fixed for 18 hr in 4% paraformaldehyde and mounted in GMA-plastic. Sections were stained with (A and B) hematoxylin/eosin or (C and D) chloroacetylesterase (to detect neutrophil esterases). Arrows indicate neutrophils present either as clusters or as single cells within the liver parenchyma. Photomicrographs are representative of samples from five independent animals. (E and F) Wild-type and double mutant mice were injected with d-Gal (20 mg) and LPS (0.3 mg/kg) and killed 24 hr later. Liver tissues were fixed/mounted as above and stained with hematoxylin/eosin.