Neutrophils are the predominant infected phagocytic cells in the airways of patients with active pulmonary TB - PubMed (original) (raw)

Comparative Study

Neutrophils are the predominant infected phagocytic cells in the airways of patients with active pulmonary TB

Seok-Yong Eum et al. Chest. 2010 Jan.

Abstract

Background: The exact role of neutrophils in the pathogenesis of TB is poorly understood. Recent evidence suggests that neutrophils are not simply scavenging phagocytes in Mycobacterium tuberculosis (Mtb) infection.

Methods: Three different types of clinical specimens from patients with active pulmonary TB who underwent lung surgery were examined: sputum, BAL fluid, and cavity contents. Differential cell separation and quantification were performed for intracellular and extracellular bacteria, and bacterial length was measured using microscopy.

Results: Neutrophils were more abundant than macrophages in sputum (86.6% +/- 2.2% vs 8.4% +/- 1.3%) and in BAL fluid (78.8% +/- 5.8% vs 11.8% +/- 4.1%). Inside the cavity, lymphocytes (41.3% +/- 11.2%) were the most abundant cell type, followed by neutrophils (38.8% +/- 9.4%) and macrophages (19.5% +/- 7.5%). More intracellular bacilli were found in neutrophils than macrophages in sputum (67.6% +/- 5.6% vs 25.2% +/- 6.5%), in BAL fluid (65.1% +/- 14.4% vs 28.3% +/- 11.6%), and in cavities (61.8% +/- 13.3% vs 23.9% +/- 9.3%). The lengths of Mtb were shortest in cavities (1.9+/- 0.1 microm), followed by in sputum (2.9 +/- 0.1 microm) and in BAL fluid (3.6 +/- 0.2 microm).

Conclusions: Our results show that neutrophils are the predominant cell types infected with Mtb in patients with TB and that these intracellular bacteria appear to replicate rapidly. These results are consistent with a role for neutrophils in providing a permissive site for a final burst of active replication of the bacilli prior to transmission.

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Figures

Figure 1.

Figure 1.

Localization of Mtb bacilli in sputum (A), in BAL (B), and in cavity caseum (C). Cytospin slides of each sample were prepared and stained for acid-fast bacilli (Ziehl-Neelsen, original magnification ×200) and human cell morphology (Hemacolor, original magnification ×1,000). The bacilli (arrows) collocate with macrophages as well as neutrophils and the extracellular matrix. M ɸ = macrophages; Mtb = Mycobacterium tuberculosis; PMN = neutrophils.

Figure 2.

Figure 2.

Differential cell counts in sputum, in BAL, and in cavity caseum. The percentage of each cell type was calculated when the counted total cell numbers were more than 200 and represented as neutrophil, macrophage, lymphocyte, and epithelial cells. Horizontal bars indicate mean value. The data among groups were analyzed using one-way analysis of variance, and the differences between two groups were tested using the unpaired t test; P ≤.05 was considered significant.

Figure 3.

Figure 3.

Localization of Mtb bacilli in sputum, in BAL, and in cavity caseum. Cytospin slides were prepared and stained by the Ziehl-Neelsen method followed by Hemacolor staining. The number of bacilli was counted both inside and outside of the cells in more than 100 fields (original magnification ×1,000) under a light microscope, and the total number ranged from 5 to 855. The percentage of extracellular and intracellular bacilli was calculated and represented in the extracellular (bacilli in extracellular space) and intracellular (bacilli in intracellular space) columns. Horizontal bars indicate mean value. The data among groups were analyzed using one-way analysis of variance, and the differences between two groups were tested using the unpaired t test; P ≤.05 was significant. See Figure 1 legend for expansion of abbreviation.

Figure 4.

Figure 4.

Differential counts of the cells ingesting Mtb bacilli in sputum, in BAL, and in cavity caseum. Cytospin slides were prepared and stained by the Ziehl-Neelsen method followed by Hemacolor staining. The number of cells engulfing bacilli was counted in more than 100 fields (original magnification ×1,000) under a light microscope and ranged from 4 to 47. The percentage of each cell type was calculated and represented as Neutrophil, Macrophage, and Epithelial Cell. Horizontal bars indicate mean value. The data among groups were analyzed using one-way analysis of variance, the differences between two groups were tested using the unpaired t test, and no significance was found among groups. See Figure 1 legend for expansion of the abbreviation.

Figure 5.

Figure 5.

Frequency of Mtb bacilli lengths measured in sputum, in BAL, and in cavity caseum. More than 200 bacilli in each specimen were measured, grouped (as described in the “Materials and Methods ” section), and plotted. Individual measurements were used for the calculation of average length and other statistical analyses. Extensive heterogeneity was observed in cell length in different specimens. The average values of Mtb cell lengths were 2.9 ± 0.1 μm, 3.6 ± 0.2 μm, and 1.9 ± 0.1 μm for sputum, BAL and cavity caseum, respectively. A statistically significant difference between cavity caseum and both BAL and sputum was observed (P ≤.001). See Figure 1 legend for expansion of the abbreviation.

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