Resistance of CD7-deficient mice to lipopolysaccharide-induced shock syndromes

Abstract

CD7 is an immunoglobulin superfamily molecule involved in T and natural killer (NK) cell activation and cytokine production. CD7-deficient animals develop normally but have antigen-specific defects in interferon (IFN)-gamma production and CD8(+) CTL generation. To determine the in vivo role of CD7 in systems dependent on IFN-gamma, the response of CD7-deficient mice to lipopolysaccharide (LPS)-induced shock syndromes was studied. In the high-dose LPS-induced shock model, 67% of CD7-deficient mice survived LPS injection, whereas 19% of control C57BL/6 mice survived LPS challenge (P < 0.001). CD7-deficient or C57BL/6 control mice were next injected with low-dose LPS (1 microgram plus 8 mg D-galactosamine [D-gal] per mouse) and monitored for survival. All CD7-deficient mice were alive 72 h after injection of LPS compared with 20% of C57BL/6 control mice (P < 0.001). After injection of LPS and D-gal, CD7-deficient mice had decreased serum IFN-gamma and tumor necrosis factor (TNF)-alpha levels compared with control C57BL/6 mice (P < 0.001). Steady-state mRNA levels for IFN-gamma and TNF-alpha in liver tissue were also significantly decreased in CD7-deficient mice compared with controls (P < 0.05). In contrast, CD7-deficient animals had normal liver interleukin (IL)-12, IL-18, and interleukin 1 converting enzyme (ICE) mRNA levels, and CD7-deficient splenocytes had normal IFN-gamma responses when stimulated with IL-12 and IL-18 in vitro. NK1.1(+)/ CD3(+) T cells are known to be key effector cells in the pathogenesis of toxic shock. Phenotypic analysis of liver mononuclear cells revealed that CD7-deficient mice had fewer numbers of liver NK1.1(+)/CD3(+) T cells (1.5 +/- 0.3 x 10(5)) versus C57BL/6 control mice (3.7 +/- 0.8 x 10(5); P < 0.05), whereas numbers of liver NK1.1(+)/CD3(-) NK cells were not different from controls. Thus, targeted disruption of CD7 leads to a selective deficiency of liver NK1.1(+)/ CD3(+) T cells, and is associated with resistance to LPS shock. These data suggest that CD7 is a key molecule in the inflammatory response leading to LPS-induced shock.

Figure 1

Survival of CD7-deficient mice treated with LPS. (A) High-dose shock model. C57BL/6 control (▪, n = 16) and CD7-deficient mice (□, n = 30) were injected intraperitoneally with LPS (100 mg/kg). Control C57BL/6 (○, n = 3) and CD7^−/− mice (▴, n = 3) were injected intraperitoneally with saline. Mortality was assessed daily for 7 d. (B) Low-dose LPS plus D-gal shock model. C57BL/6 control mice (▪, n = 10) and CD7-deficient mice (□, n = 10) were injected intraperitoneally with LPS (1 μg) and D-gal (8 mg) in saline. Mortality was assessed for 3 d; *P ≤ 0.001 when comparing survival of C57BL/6 control mice to CD7-deficient mice.

Figure 1

Survival of CD7-deficient mice treated with LPS. (A) High-dose shock model. C57BL/6 control (▪, n = 16) and CD7-deficient mice (□, n = 30) were injected intraperitoneally with LPS (100 mg/kg). Control C57BL/6 (○, n = 3) and CD7^−/− mice (▴, n = 3) were injected intraperitoneally with saline. Mortality was assessed daily for 7 d. (B) Low-dose LPS plus D-gal shock model. C57BL/6 control mice (▪, n = 10) and CD7-deficient mice (□, n = 10) were injected intraperitoneally with LPS (1 μg) and D-gal (8 mg) in saline. Mortality was assessed for 3 d; *P ≤ 0.001 when comparing survival of C57BL/6 control mice to CD7-deficient mice.

Figure 2

CD7-deficient animals have diminished in vivo cytokine serum levels in response to treatment with low-dose LPS and D-gal. (A) Time course of TNF-α serum concentrations after injection of C57BL/6 control mice (▪) or CD7-deficient mice (□) with LPS (1 μg) and D-gal (8 mg). (B) Time course of IFN-γ serum concentration after injection of C57BL/6 control mice (▪) and CD7-deficient mice (□) with LPS and D-gal. Serum cytokine levels, determined by ELISA from one mouse per time point are presented. Data shown are from an experiment that is representative of three separate experiments performed. Statistical significance was determined for each time point by combining data from three experiments; *P ≤ 0.001 when comparing C57BL/6 control mice to CD7-deficient mice.

Figure 3

Steady-state cytokine mRNA levels in the low-dose LPS shock model. Total RNA was extracted from liver (A and C) and spleen (B) from C57BL/6 control (+/+, n = 3) and CD7-deficient (−/−, n = 3) mice 0 and 6 h after injection with low-dose LPS and D-gal. Specific cytokine mRNA levels were quantified by RNase protection assays and reported as percentage of GAPDH signal. Data represent the mean ± SEM of results obtained from three mice at each time point.

Figure 4

In vitro splenocyte IFN-γ response to stimulation with IL-12 and IL-18. Splenocytes from C57BL/6 control mice (n = 3) and CD7-deficient mice (n = 3) were isolated and incubated with a dose-curve of IL-12 (A), IL-18 (B), or IL-12 plus IL-18 (C) for 72 h. Culture supernatants were assayed for IFN-γ by ELISA. Similar results were found for IL-12 and IL-18, and IL-12 plus IL-18 induced proliferation of C57BL/6 and CD7-deficient splenocytes (data not shown). Data are the mean ± SEM for three animals.

Figure 5

CD7-deficient animals have decreased percentages and absolute numbers of liver NK1.1⁺/CD3⁺ T cells. Flow cytometric analysis of isolated liver mononuclear cells from C57BL/6 and CD7-deficient mice was performed as described in Materials and Methods. A minimum of 10⁴ lymphocyte-gated events were analyzed per sample. Panel A shows percentages of CD3⁺/NK1.1⁺ T cells, CD3⁻/NK1.1⁺ NK cells, CD3⁺/ NK1.1⁻ T cells, and B220⁺ B cells. In panel B, the total number of mononuclear cells isolated per liver was determined and used to calculate the absolute number per liver of CD3⁺/NK1.1⁺ T cells, CD3⁻/NK1.1⁺ NK cells, CD3⁺/NK1.1⁻ T cells, and B220⁺ B cells. Data are the mean ± SEM from three experiments with liver mononuclear cells from two to five animals pooled per group, per experiment. *P < 0.05 when comparing C57BL/6 control mice to CD7-deficient mice.