Experimental pneumococcal meningitis: impaired clearance of bacteria from the blood due to increased apoptosis in the spleen in Bcl-2-deficient mice

Abstract

Necrotic and apoptotic neuronal cell death can be found in pneumococcal meningitis. We investigated the role of Bcl-2 as an antiapoptotic gene product in pneumococcal meningitis using Bcl-2 knockout (Bcl-2(-/-)) mice. By using a model of pneumococcal meningitis induced by intracerebral infection, Bcl-2-deficient mice and control littermates were assessed by clinical score and a tight rope test at 0, 12, 24, 32, and 36 h after infection. Then mice were sacrificed, the bacterial titers in blood, spleen, and cerebellar homogenates were determined, and the brain and spleen were evaluated histologically. The Bcl-2-deficient mice developed more severe clinical illness, and there were significant differences in the clinical score at 24, 32, and 36 h and in the tight rope test at 12 and 32 h. The bacterial titers in the blood were greater in Bcl-2-deficient mice than in the controls (7.46 +/- 1.93 log CFU/ml versus 5.16 +/- 0.96 log CFU/ml [mean +/- standard deviation]; P < 0.01). Neuronal damage was most prominent in the hippocampal formation, but there were no significant differences between groups. In situ tailing revealed only a few apoptotic neurons in the brain. In the spleen, however, there were significantly more apoptotic leukocytes in Bcl-2-deficient mice than in controls (5,148 +/- 3,406 leukocytes/mm2 versus 1,070 +/- 395 leukocytes/mm2; P < 0.005). Bcl-2 appears to counteract sepsis-induced apoptosis of splenic lymphocytes, thereby enhancing clearance of bacteria from the blood.

FIG. 1.

Numbers of apoptotic lymphocytes in spleen follicles in Bcl-2-deficient mice (open bars) and controls (wild type [WT]) (solid bars). (A) Results 36 h after intracerebral injection of 25 μl of saline (n = 3 and n = 4 for the Bcl-2-deficient mice and controls, respectively). (B) Results 36 h after intracerebral injection of 10⁴ CFU of S. pneumoniae in 25 μl (n = 8 and n = 9 for the Bcl-2-deficient mice and controls, respectively). Note the difference in the scales of the ordinate axes. The bars indicate means, and the error bars indicate standard deviations. A number sign indicates that the P value is 0.02, and a plus sign indicates that the P value is <0.005 (as determined by a t test).

FIG. 2.

Histology and immunohistochemistry of the brain and spleen in experimental S. pneumoniae meningitis in Bcl-2-deficient mice and controls. (A) In situ tailing of the brain showing meningeal inflammation in a Bcl-2-deficient mouse with apoptotic PML (arrow). Bar, 20 μm. (B and C) Hematoxylin and eosin staining showing necrotic neuronal damage (arrow) in the CA3 region of the hippocampus of a Bcl-2-deficient mouse (B) and in the dentate gyrus of a Bcl-2-deficient mouse (C). (D) In situ tailing of the brain showing an apoptotic neuron (arrow) in the dentate gyrus of a Bcl-2-deficient mouse. (E and F) In situ tailing of the spleen showing few apoptotic lymphocytes (arrow) in a heterozygous control mouse (E) and in a Bcl-2-deficient mouse (F) into which saline was injected. (G and H) In situ tailing of the spleens of a wild-type mouse (G) with few apoptotic lymphocytes (arrow) and a Bcl-2-deficient mouse (H) showing a markedly increased number of apoptotic lymphocytes (arrow) 36 h after intracerebral infection with S. pneumoniae. Bars in panels B to H, 50 μm. (I) Double staining of spleen slices with in situ tailing (black) and CD3 (red) showing apoptotic lymphocytes (arrow) mainly in the white pulp (wp) within and outside T-cell regions. Note the arteriole (ar) in the center of the T-cell region located in the periarteriolar lymphatic sheath. The red pulp (rp) is visible at the edge and does not contain as many apoptotic cells. Bar, 50 μm. (K) Higher magnification showing that there are no cells that show clear double staining with in situ tailing (black) and CD3 (red) due to the shrunken cytoplasm of apoptotic cells. (L) Double staining with in situ tailing (black) and CD8 (red) showing apoptotic lymphocytes within a cytotoxic T-cell cluster and also outside this area. (M) Bcl-2 staining (arrow) in splenic lymphocytes. Bars in panels K to M, 20 μm.

FIG. 3.

Bacterial titers in blood, cerebellar, and spleen homogenates of Bcl-2-deficient mice (open bars) (n = 8) and controls (solid bars) (n = 9) 36 h after intracerebral infection with 10⁴ CFU of S. pneumoniae in 25 μl. The bars indicate means, and the error bars indicate standard deviations. A number sign indicates that the P value is <0.01 (as determined by a t test).

FIG. 4.

Meningeal inflammation in experimental S. pneumoniae meningitis 36 h after infection. Mice were infected intracerebrally with 10⁴ CFU of S. pneumoniae in 25 μl of saline. ○ and open bars, Bcl-2-deficient mice (n = 8); ▪ and solid bars, controls (wild type [WT]) (n = 9). (A) Meningeal inflammation score (median and 25th and 75th percentiles). (B) Apoptotic PML in the meningeal infiltrate. (C) Apoptotic PML in cerebral infiltrate. The bars indicate means, and the error bars indicate standard deviations. No statistically significant differences were observed.

FIG. 5.

Bcl-2 expression in C57BL/6 wild-type mice 36 h after intracerebral infection with pneumococci (n = 5) or injection of normal saline (n = 5) as determined by Western blotting. ○, mice with pneumococcal meningitis (n = 5); ▪, uninfected controls (n = 5).