Cytokine kinetics and other host factors in response to pneumococcal pulmonary infection in mice

Abstract

There is a need for more insight into the pathogenesis of Streptococcus pneumoniae pneumonia, as the fatality rate associated with this disease remains high despite appropriate antibiotherapy. The host response to pneumococci was investigated after intranasal inoculation of CD1 mice with 10(7) log-phase CFU of bacteria. We identified five major pathogenesis steps from initial infection to death. In step 1 (0 to 4 h), there was ineffective phagocytosis by alveolar macrophages, with concurrent release of tumor necrosis factor alpha (TNF), interleukin-6 (IL-6), and nitric oxide (NO) in bronchoalveolar lavage (BAL) fluid, TNF, IL-6, and interleukin-1 alpha (IL-1) in lung tissues, and IL-6 in serum, which were associated with tachypnea and hemoconcentration. In step 2 (4 to 24 h), bacterial growth in alveoli and polymorphonuclear cell recruitment from bloodstream to lung tissue (high myeloperoxidase levels) to alveoli were associated with high release of all three cytokines and leukotriene B4 (LTB4) in tissue and BAL fluid, as well as transient spillover of IL-1 in serum. In step 3 (24 to 48 h), despite downregulation of TNF and IL-1 in BAL fluid and lungs, there was appearance of injury to alveolar ultrastructure, edema to interstitium, and increase in lung weight as well as regeneration of type II pneumocytes and increased secretion of surfactant; bacteria progressed from alveoli to tissue to blood, and body weight loss occurred. In step 4 (48 to 72 h), strong monocyte recruitment from blood to alveoli was associated with high NO release in tissue and BAL fluid, but there was also noticeable lymphocyte recruitment and leukopenia; bacteremia was associated with TNF and IL-6 release in blood and thrombocytopenia. In step 5 (72 to 96 h), severe airspace disorganization, lipid peroxidation (high malondialdehyde release in BAL fluid), and diffuse tissue damage coincided with high NO levels; there was further increase in lung weight and bacterial growth, loss in body weight, and high mortality rate. Delineation of the sequential steps that contribute to the pathogenesis of pneumococcal pneumonia may generate markers of evolution of disease and lead to better targeted intervention.

FIG. 1

Light and electron microscopy of lung architecture of normal mice (A to C) and mice infected with 10⁷ S. pneumoniae cells and sacrificed 72 h later (D to F). Perivascular areas (arrowheads) close to bronchioles (B) (A and D; magnification, ×400) were greatly enlarged after infection, due to edema and phagocyte recruitment. Interstitial tissues (I) in alveolar areas (B and E; ×400) were also enlarged, and leukocytes (L) could be seen in alveoli. Tissue injury characterized the infectious and inflammatory processes (C and F; ×5,000). Macrophages (M) containing ingested bacteria (arrowhead) were seen, as well as recruited neutrophils (N) and lymphocytes (LY). T₁, type I pneumocyte; T₂, type II pneumocyte; E, endothelial cell.

FIG. 2

Electron microscopy of lungs of mice infected with 10⁷ S. pneumoniae cells and sacrificed 72 h later. Type II pneumocytes (T₂) proliferated after infection (A; magnification, ×9,600) and secreted abnormal amounts of surfactant (S) in alveoli (C; ×6,000). Although S. pneumoniae cells (arrows) were partly eradicated through phagocytosis (M, macrophage) (B; ×18,000), extracellular killing also seemed to occur, as the polysaccharide capsule (C) of bacteria localized outside phagocytes in areas of intense inflammation appeared more disaggregated (thinner and more diffuse) (B [arrows] and inset; ×44,000) than the capsule of bacteria localized in less severely inflamed areas (D [×18,000] and inset [×44,000]). LY, lymphocyte.

FIG. 3

Recruitment of inflammatory cells from blood vessels (C) to lung tissue (B) to BAL (A) as a function of time after intranasal infection with 10⁷ S. pneumoniae cells. Cell populations (mean ± SEM) in blood and BAL fluid were counted, and neutrophils in lung homogenate were measured by quantifying MPO. ∗, P < 0.05 compared with preinfection values; +, P < 0.01 compared with preinfection values.

FIG. 4

Mean (SEM) cytokine levels in cell-free BAL, lung homogenates previously cleared from blood, and serum of mice infected with 10⁷ S. pneumoniae cells. TNF in BAL (A), lung (B), and serum (C), IL-1 in BAL (D), lung (E), and serum (F), and IL-6 in BAL (G), lung (H), and serum (I) are reported. ∗, P < 0.05 compared with preinfection values; +, P < 0.01 compared with preinfection values.

FIG. 5

Mean (SEM) LTB₄ levels in cell-free BAL (A), in lung homogenates previously cleared from blood (B), and in serum (C) of mice infected with 10⁷ S. pneumoniae cells. ∗, P < 0.05 compared with preinfection values; +, P < 0.01 compared with preinfection values.

FIG. 6

Mean (SEM) NO levels in cell-free BAL (A), in lung homogenates previously cleared from blood (B), and in serum (C) of mice infected with 10⁷ S. pneumoniae cells. ∗, P < 0.05 compared with preinfection values; +, P < 0.01 compared with preinfection values.

FIG. 7

Mean (SEM) MDA levels in BAL 12 and 96 h after infection with 10⁷ S. pneumoniae cells. ∗, P < 0.05 compared with preinfection values; +, P < 0.01 compared with preinfection values.