Rat neutrophils prevent the development of tuberculosis

Abstract

To understand the role of neutrophils in the development of rat tuberculosis in vivo, we utilized lipopolysaccharide (LPS)-induced neutrophilia in the lungs. LPS (50 micro g/ml) was administered intratracheally to male Fischer rats. Rats were then infected with Mycobacterium tuberculosis by an airborne route. Intratracheal injection of LPS significantly blocked the development of pulmonary granulomas and significantly reduced pulmonary CFU (P < 0.01). LPS treatment with amphotericin B (an LPS inhibitor) or neutralizing anti-rat neutrophil antibody reversed the development of pulmonary lesions. LPS-induced transient neutrophilia prevented early mycobacterial infection. The timing of LPS administration was important. When given intratracheally at least 10 days after aerial infection, LPS did not prevent development of tuberculosis. Neutrophils obtained by bronchoalveolar lavage killed M. tuberculosis cells. These results indicate clearly that neutrophils participate actively in defense against early-phase tuberculosis.

FIG. 1.

(A) Neutrophil-ingesting tubercle bacilli. The results of Ziehl-Neelsen staining are shown at a magnification of ×600. Arrow, neutrophil ingesting tubercle bacilli. (B) Electron micrograph of neutrophil-ingesting tubercle bacilli. Several tubercle bacilli (arrow) are evident in the neutrophil phagosomes. The image is shown at a magnification of ×5,000. (C) Alveoli filled with neutrophils. No exudate was found in the alveoli. The results of hematoxylin and eosin staining are shown at a magnification of ×200.

FIG. 2.

Bacterial load in a time course study starting at day 1 postinfection. The rats were infected by an airborne route at days 1, 3, 5, 7, and 10 after intratracheal injection of LPS (50 μg/0.5 ml). The lung homogenates were grown on Ogawa slant medium at 37°C for 4 weeks, and CFU were counted thereafter.

FIG. 3.

CFU assay of infected lung tissues left untreated (−LPS), treated with LPS (50 μg/0.5 ml) (+LPS), treated with LPS (50 μg/0.5 ml) and amphotericin B (50 μg/0.5 ml) (+LPS +Amphotericin B), treated with LPS (50 μg/0.5 ml) 10 days after aerial infection (+LPS 10 days post infection), and treated with LPS and anti-rat neutralizing neutrophil Ab (500 μg/rat) (+LPS +Ab).

FIG. 4.

Representative histopathology of untreated infected lung tissues (A) and infected lung tissues treated with LPS (50 μg/0.5 ml) 1 day before aerial infection (B), treated with LPS 10 days after aerial infection (C), treated with LPS and amphotericin B (50 μg/0.5 ml) 1 day before aerial infection (D), and treated with LPS and anti-rat neutrophil Ab (500 μg/rat) 1 day before aerial infection (E). The results of hematoxylin and eosin staining are shown at a magnification of ×100.