Pathogenesis of pneumococcal pneumonia in cyclophosphamide-induced leukopenia in mice

Abstract

Streptococcus pneumoniae pneumonia frequently occurs in leukopenic hosts, and most patients subsequently develop lung injury and septicemia. However, few correlations have been made so far between microbial growth, inflammation, and histopathology of pneumonia in specific leukopenic states. In the present study, the pathogenesis of pneumococcal pneumonia was investigated in mice rendered leukopenic by the immunosuppressor antineoplastic drug cyclophosphamide. Compared to the immunocompetent state, cyclophosphamide-induced leukopenia did not hamper interleukin-1 (IL-1), IL-6, macrophage inflammatory protein-1 (MIP-1), MIP-2, and monocyte chemotactic protein-1 secretion in infected lungs. Leukopenia did not facilitate bacterial dissemination into the bloodstream despite enhanced bacterial proliferation into lung tissues. Pulmonary capillary permeability and edema as well as lung injury were enhanced in leukopenic mice despite the absence of neutrophilic and monocytic infiltration into their lungs, suggesting an important role for bacterial virulence factors and making obvious the fact that neutrophils are ultimately not required for lung injury in this model. Scanning and transmission electron microscopy revealed extensive disruption of alveolar epithelium and a defect in surfactant production, which were associated with alveolar collapse, hemorrhage, and fibrin deposits in alveoli. These results contrast with those observed in immunocompetent animals and indicate that leukopenic hosts suffering from pneumococcal pneumonia are at a higher risk of developing diffuse alveolar damage.

FIG. 1.

Bacterial counts in lungs (A) and bloodstream (B) of immunocompetent and leukopenic mice infected with S. pneumoniae (means + SD [error bars] for five mice). Values that were significantly greater than those observed for infected immunocompetent mice are indicated by symbols as follows: ∗, P < 0.05; •, P < 0.01; §, P < 0.001.

FIG. 2.

WBCs in peripheral bloodstream of immunocompetent and leukopenic mice infected with S. pneumoniae (means ± SD [error bars] for five mice). Values that were significantly greater than those observed for infected leukopenic mice are indicated by symbols as follows: ∗, P < 0.05; §, P < 0.01. Cyclophosphamide injections resulted in blood leukocyte depletion from 7.2 × 10⁹cells/liter (pretreatment, straight line) to 1.0 × 10⁹cells/liter (throughout the experiment, dotted line) in uninfected control mice.

FIG. 3.

MPO in lungs of immunocompetent and leukopenic mice infected with S. pneumoniae (means ± SD [error bars] for 5 mice). Levels in uninfected immunocompetent and leukopenic mice were, respectively, 6 ± 4 (straight line) and 4 ± 1 U/lung (dotted line). ∗, P < 0.001 (value was significantly greater than that observed for infected leukopenic mice).

FIG. 4.

Leukocyte counts in BAL fluids of immunocompetent and leukopenic mice infected with S. pneumoniae (means + SD [error bars] for five mice). Values that were significantly greater than those observed for infected leukopenic mice are indicated by symbols as follows: ∗, P < 0.01; •, P < 0.05; §, P < 0.001. Values of total leukocytes, macrophages, and neutrophils in BAL fluids of uninfected mice were, respectively, 1.3 × 10⁴ ± 0.71 × 10⁴, 1.3 × 10⁴ ± 0.71 × 10⁴, and 0 cells/ml for both immunocompetent and leukopenic mice.

FIG. 5.

Levels of proinflammatory cytokines and chemokines in lungs of immunocompetent and leukopenic mice infected with S. pneumoniae (means + SD [error bars] for five mice). Values of tumor necrosis factor (TNF), IL-1, IL-6, MIP-2, and MIP-1 for uninfected immunocompetent versus leukopenic mice were, respectively, 200 ± 10 and 160 ± 15, 1,600 ± 100 and 1,500 ± 200, 700 ± 50 and 450 ± 250, 700 ± 200 and 450 ± 400, and 100 ± 100 and 100 ± 100. MCP-1 values for both uninfected groups were below the limit of detection of the assay, which was between 20 and 500 pg/ml.

FIG. 6.

Levels of nitric oxide (NO) in BAL fluids of immunocompetent and leukopenic mice infected with S. pneumoniae (means + SD [error bars] for five mice). Values for uninfected immunocompetent versus leukopenic animals were, respectively, 10 ± 2 and 9 ± 2 μm/ml. ∗, value was significantly greater than that for infected leukopenic mice (P < 0.05).

FIG. 7.

Pulmonary vascular permeability of immunocompetent and leukopenic mice infected with S. pneumoniae (means + SD [error bars] for five mice). Values for uninfected immunocompetent versus leukopenic mice were, respectively, of 1.64 ± 0.10 versus 1.60 ± 0.14. Symbols: •, P < 0.05 (value was significantly lower than that for infected immunocompetent mice); §, P < 0.05 (value was significantly greater than that for infected immunocompetent mice).

FIG. 8.

Wet/dry lung weight ratio for immunocompetent and leukopenic mice infected with S. pneumoniae (means + SD [error bars] for five mice). Values that were significantly greater than those observed for infected immunocompetent mice are indicated by symbols as follows: ∗, P < 0.01; §, P < 0.001. Values for uninfected immunocompetent versus leukopenic mice were, respectively, 4.4 ± 0.2 and 4.2 ± 0.2.

FIG. 9.

Morphological analysis of lung tissues by light microscopy. Studies of infected immunocompetent (A) and leukopenic (C) mice were conducted on day 2 postinfection. Healthy lungs were observed in noninfected immunocompetent (B) and leukopenic (D) mice. Abbreviations: A, alveolar space; B, bronchiolus; V, blood vessel. Scale bar = 20 μm.

FIG. 10.

Morphological analysis of lung tissues by scanning electron microscopy. Healthy lungs were observed in noninfected immunocompetent (A) and leukopenic (D) mice. Micrographs were taken of lungs from infected immunocompetent mice on days 2 (B) and 3 (C) postinfection and of lungs from infected leukopenic mice on days 2 (E) and 3 (F) postinfection. Abbreviations: B, bacteria; F, fibrosis; M, macrophage; P, polymorphonuclear cell; R, red blood cell. Scale bar = 10 μm.

FIG. 11.

Morphological analysis of lung tissues by transmission electron microscopy. Studies were conducted in infected immunocompetent (A and B) and leukopenic (D and E) mice on day 3 postinfection. Normal ultrastructure of endothelia and epithelia was observed in noninfected immunocompetent (C) and leukopenic (F) mice. Abbreviations: A, alveolar space; B, bacteria; BL, basal lamina; E, edema; En, endothelia; Ep, epithelia; F, fibrosis; N, necrotized tissue; P, polymorphonuclear cell; S, surfactant; V, blood vessel. Scale bar = 1 μm.