Intranasal inoculation of Chlamydia trachomatis mouse pneumonitis agent induces significant neutrophil infiltration which is not efficient in controlling the infection in mice

Abstract

Previous studies have shown that chlamydial infection is accompanied by significant infiltration of neutrophils at the site of infection. However, the role of neutrophils in host defence against chlamydial infection is not clearly understood. Using genetically different inbred mouse strains and CXCR-2 gene knockout (KO) mice, we examined the mechanism for neutrophil recruitment and the role of neutrophils during chlamydial lung infection. Our data showed that C3H mice exhibited significantly higher and more persistent neutrophil infiltration in the lung than did C57BL/6 mice following Chlamydia trachomatis mouse pneumonitis infection. The massive neutrophil infiltration in C3H mice was paralleled by high-level expression of CXCR-2 and its ligands, CXC chemokines (macrophage inflammatory protein 2, cytokine-induced neutrophil attractant (KC) and lipopolysaccharide-induced CXC chemokine), and proinflammatory cytokines (tumour necrosis factor-alpha, interleukin-1 and interleukin-6) in the lung. Although much greater infiltration of neutrophils was observed in C3H mice than in C57BL/6 mice, the former mice had more severe disease and higher in vivo chlamydial growth than the latter. Moreover, CXCR-2 KO mice, which revealed a dramatic reduction in neutrophil activity, showed comparable chlamydial infection to wild-type mice. These results suggest that neutrophils are not efficient for controlling chlamydial lung infection.

Figure 1

C3H mice show higher neutrophil infiltration, CXCR-2 expression and more severe pathological reactions than C57BL/6 mice following MoPn lung infection. Top four panels, mice were intranasally infected with 3 × 10³ IFU of MoPn and killed 1, 2, 7, or 14 days following infection. Lungs were removed and fixed in 10% buffered formalin, embedded in paraffin. Sections were stained with haematoxylin & eosin and the histological changes and cellular infiltration were analysed by light microscopy. Magnification, ×200; inserts, ×400. Bottom panels, mice were killed at day 7 postinfection. Frozen lung sections were stained for CXCR-2 (red) and epithelial cells (green) using methods described in the Materials and methods section; magnification, ×400.

Figure 2

C3H mice show significantly higher neutrophil infiltration than C57BL/6 mice following MoPn lung infection. C3H and C57BL/6 mice (14 mice per group) were infected with MoPn as described in Figure 1 and killed 7 or 14 days following infection. The lungs were digested with collagenase and the recovered cells were stained using Wright–Giemsa staining. The number (a) and percentages (b) of neutrophils in the recovered lung cells are presented as mean ± SEM. *P < 0·05; **P < 0·01, comparison between C3H and C57BL/6 mice.

Figure 3

Higher local myeloperoxidase (MPO) activity in C3H than C57BL/6 mice following MoPn lung infection. C3H and C57BL/6 mice were infected as described in Figure 1. Fresh lung tissues were collected on 7 and 14 days postinfection (five mice per group at each time-point). MPO activity in these tissues was determined as described in the Materials and methods section. The data are presented as mean ± SEM. **P < 0·01, comparison between C3H and C57BL/6 mice.

Figure 4

Higher and persistent chemokine/chemokine receptor expression in the lung of C3H mice following MoPn infection. C3H and C57BL/6 mice were infected as described in Figure 1. Fresh lung tissues were collected on 0, 1, 2, 3, 7 and 14 days (from three to seven mice per group in each time-point) postinfection and snap frozen by liquid nitrogen. Total cellular RNA from frozen lungs was isolated using TRIzol Reagent (Gibco). RT-PCR analysis of chemokine/chemokine receptor mRNA was performed as described in the Materials and methods section. The data are presented as mean ±SEM of the density of the bands of chemokine/chemokine receptor as a percentage of β-actin. *P < 0·05; **P < 0·01, comparison between C3H and C57BL/6 mice.

Figure 5

Higher proinflammatory cytokine production in the lung in C3H mice than C57BL/6 mice following MoPn infection. C3H and C57BL/6 mice were infected as described in Figure 1. Fresh lung tissues were collected 7 and 14 days postinfection (five mice per group at each time-point) and snap frozen in liquid nitrogen. Total cellular RNA from frozen lungs was isolated using TRIzol Reagent (Gibco). RT-PCR analysis of cytokine mRNA was performed as described in the Materials and methods section. The data are presented as mean ± SEM of the density of the bands of cytokines as a percentage of β-actin. *P < 0·05; **P < 0·01, comparison between C3H and C57BL/6 mice.

Figure 6

Slower clearance of chlamydial infection in the lung by C3H mice compared with C57BL/6 mice. Mice (10 mice per group) were infected as described in Figure 1 and were killed on days 7 or 14 postinfection. The levels of Chlamydia in the lung homogenates were analysed as described in the Materials and methods section. The data are presented as mean ± SEM. *P < 0·05; **P < 0·01, comparison between C3H and C57BL/6 mice.

Figure 7

CXCR-2 gene knockout (KO) mice which show dramatic reduction of neutrophils exhibit comparable levels of infection to wild-type mice. CXCR-2 KO mice (BALB/c background) and control wild-type BALB/c mice (four mice per group) were infected intranasally with 1 × 10³ IFUs of MoPn and killed on day 7 postinfection. The levels of MPO (a) and of chlamydial infection (b) in the lungs were analysed as described in the Materials and methods. The data are presented as mean ± SD. *P < 0·01, comparison between CXCR-2 KO and wild-type mice.