CD8 alpha-deficient mice are highly susceptible to 5-fluorouracil-induced lethality

Abstract

Intestinal intraepithelial lymphocytes (i-IEL) expressing CD8 alpha are located in the intestine and may confer protection against invasion of intestinal microflora. We found that mice rendered deficient in CD8 alpha molecules by homologous recombination were susceptible to 5-fluorouracil (5-FU)-induced lethality accompanied by translocation of members of the enterobacteria. The number of i-IEL was greatly reduced on day 6 after 5-FU administration in both CD8 alpha(+/-) mice and CD8 alpha(-/-) mice, whereas the recovery of the level of i-IEL thereafter was significantly impaired in CD8 alpha(-/-) mice compared with that in CD8 alpha(+/-) mice. The ability of i-IEL to produce gamma interferon in response to immobilized T-cell receptor (TCR) alpha beta or TCR gamma delta monoclonal antibodies was significantly lower in CD8 alpha(-/-) mice than in CD8 alpha(+/-) mice. Transfer of CD8(+) i-IEL conferred significant protection against 5-FU-induced lethality in CD8 alpha(-/-) mice. The results suggest that CD8(+) i-IEL play an important role in protection against 5-FU-induced lethality with translocation of Enterobacteriaceae.

FIG. 1.

Survival rate of CD8α-deficient mice after 5-FU administration. CD8α^+/− and CD8α^−/− mice were treated i.p. with 600 or 800 mg of 5-FU per kg, and CD4^+/− and CD4^−/− mice were treated i.p. with 600 mg of 5-FU per kg. Twenty (600 mg/kg) or 10 mice (800 mg/kg) were used in each group. ∗, P < 0.05, and ∗∗, P < 0.01 compared with control litermates by the generalized Wilcoxon's test.

FIG. 2.

Growth of endogenous bacteria in CD8α-deficient mice administered 5-FU. Numbers of bacteria in the liver and spleen of CD8α^+/− and CD8α^−/− mice were determined on the indicated days after intraperitoneal administration of 600 mg of 5-FU per kg. Each point and vertical bar represent the mean ± standard deviation (SD) of six animals. ∗, P < 0.05, significant difference compared with CD8α^+/− mice.

FIG. 3.

Kinetics of i-IEL and thymus of CD8α-deficient mice after 5-FU administration. Cell numbers in (A) i-IEL and (B) thymus in CD8α^+/− and CD8α^−/− mice were determined on the days indicated after intraperitoneal administration of 5-FU (600 mg/kg). Each point and vertical bar represent the mean ± SD of six animals. ∗, P < 0.05; ∗∗, P < 0.01, significant difference compared with the value in CD8α^+/− mice.

FIG. 4.

Flow cytometric analysis of i-IEL for expression of CD4, CD8α, αβ TCR, and γδ TCR in CD8α-deficient mice before and after 5-FU administration. The i-IEL CD8α^+/− and CD8α^−/− mice were recovered before (day 0) and 10 days after intraperitoneal administration of 5-FU (600 mg/kg). The i-IEL were stained with PE-anti-CD8α MAb or -anti-TCRγδ MAb and Cy-chrome-anti-CD4 MAb or anti-TCR αβ MAb for FACS analysis. Values represent percentages of subpopulations in selected areas. The FACS analysis results shown are representative of three separate experiments.

FIG. 5.

IFN-γ production by i-IEL in CD8α-deficient mice upon triggering of TCR. i-IEL cells were obtained from CD8α^+/− and CD8α^−/− mice before (day 0) and 10 days after intraperitoneal administration of 5-FU (600 mg/kg). The i-IEL were cultured in the presence or the absence of immobilized anti-TCRαβ or γδ MAb (100 μg/ml). Cytokine levels in the supernatants were determined by ELISA. Data are means ± SD for five mice in each group. ∗, P < 0.05 and ∗∗, P < 0.01 by Student's t test: statistically significant differences from the value for CD8α^+/− mice. Representative data from three independent experiments are shown.

FIG. 6.

Survival rate of CD8α-deficient mice receiving i-IEL transfer following 5-FU administration. i-IEL (10⁷) from Ly5.1 congeneic mice were adoptively transferred into Ly5.2 recipient mice via tail vein inoculation. (A) FACS analysis of i-IEL from CD8α^−/− mice into which i-IEL had been transferred. i-IEL were collected on day 3 after transfer and stained with Cy-chrome-anti-CD4 MAb and PE-anti-CD8α MAb or PE-anti-TCR γδ MAb and anti-Ly5.1 MAb plus Cy-chrome anti-mouse IgG. Dot plot analysis is presented as typical two-dimensional profiles. Numbers represent the percentage of total cells found in each quadrant. (B) At 3 days after the adoptive transfer of these cells, mice were challenged with 5-FU (600 mg/kg) and monitored for survival. Representative data from three independent experiments are shown. ∗, P < 0.01 by the generalized Wilcoxon's test; statistically significant difference from CD8α^−/− mice.

FIG. 6.

Survival rate of CD8α-deficient mice receiving i-IEL transfer following 5-FU administration. i-IEL (10⁷) from Ly5.1 congeneic mice were adoptively transferred into Ly5.2 recipient mice via tail vein inoculation. (A) FACS analysis of i-IEL from CD8α^−/− mice into which i-IEL had been transferred. i-IEL were collected on day 3 after transfer and stained with Cy-chrome-anti-CD4 MAb and PE-anti-CD8α MAb or PE-anti-TCR γδ MAb and anti-Ly5.1 MAb plus Cy-chrome anti-mouse IgG. Dot plot analysis is presented as typical two-dimensional profiles. Numbers represent the percentage of total cells found in each quadrant. (B) At 3 days after the adoptive transfer of these cells, mice were challenged with 5-FU (600 mg/kg) and monitored for survival. Representative data from three independent experiments are shown. ∗, P < 0.01 by the generalized Wilcoxon's test; statistically significant difference from CD8α^−/− mice.