Endotoxin induced peritonitis elicits monocyte immigration into the lung: implications on alveolar space inflammatory responsiveness

Abstract

Background: Acute peritonitis developing in response to gram-negative bacterial infection is known to act as a trigger for the development of acute lung injury which is often complicated by the development of nosocomial pneumonia. We hypothesized that endotoxin-induced peritonitis provokes recruitment of monocytes into the lungs, which amplifies lung inflammatory responses to a second hit intra-alveolar challenge with endotoxin.

Methods: Serum and lavage cytokines as well as bronchoalveolar lavage fluid cells were analyzed at different time points after intraperitoneal or intratracheal application of LPS.

Results: We observed that mice challenged with intraperitoneal endotoxin developed rapidly increasing serum and bronchoalveolar lavage fluid (BALF) cytokine and chemokine levels (TNFalpha, MIP-2, CCL2) and a nearly two-fold expansion of the alveolar macrophage population by 96 h, but this was not associated with the development of neutrophilic alveolitis. In contrast, expansion of the alveolar macrophage pool was not observed in CCR2-deficient mice and in wild-type mice systemically pretreated with the anti-CD18 antibody GAME-46. An intentional two-fold expansion of alveolar macrophage numbers by intratracheal CCL2 following intraperitoneal endotoxin did not exacerbate the development of acute lung inflammation in response to intratracheal endotoxin compared to mice challenged only with intratracheal endotoxin.

Conclusion: These data, taken together, show that intraperitoneal endotoxin triggers a CCR2-dependent de novo recruitment of monocytes into the lungs of mice but this does not result in an accentuation of neutrophilic lung inflammation. This finding represents a previously unrecognized novel inflammatory component of lung inflammation that results from endotoxin-induced peritonitis.

Figure 1

Increased serum and bronchoalveolar lavage fluid TNFα, MIP-2 and CCL2 levels in mice in response to intraperitoneal endotoxin application. Mice were either left untreated (0 h time points) or received a single intraperitoneal endotoxin application (50 μg/mouse). At various time points thereafter, mice were sacrificed and serum (A) and BAL fluid (B) TNFα (black bars), MIP-2 (grey bars) and CCL2 (white bars) levels were quantified by ELISA. The values are shown as mean ± SEM of 4–6 independent experiments. * indicates p < 0.01 versus respective control (0 h).

Figure 2

Intraperitoneal endotoxin application elicits alveolar macrophage expansion in mice. Mice were either left untreated (0 h time point) or were challenged intraperitoneally with a single dose of endotoxin (50 μg/mouse). At various time points post treatment, mice were sacrificed and subjected to bronchoalveolar lavage for determination of alveolar macrophage (black bars) and neutrophil (white bars) numbers, as indicated. Values are given as mean ± SEM of six mice per treatment group (96 h time point, n = 15). * indicates p < 0.01 versus control (0 h), + indicates p < 0.01 versus 96 h time point.

Figure 3

Alveolar macrophage expansion elicited in endotoxin-induced peritonitis is CCR2-dependent. Wild-type mice or CCR2-deficient mice were either left untreated or received a single intraperitoneal LPS application (50 μg/mouse) or wild-type mice were pretreated with function-blocking anti-CD18 antibodies prior to and every 24 h post intraperitoneal LPS application, as indicated. At 96 h post-treatment, mice were sacrificed and alveolar macrophages (black bars) and neutrophils (white bars) contained in bronchoalveolar lavage were quantified. Values are given as mean ± SEM of five mice per treatment group. * indicates p < 0.01 compared to all other treatment groups.

Figure 4

Alveolar macrophage expansion elicited by intraperitoneal LPS application does not enhance neutrophilic alveolitis and cytokine release in response to subsequent intratracheal LPS application. Mice were either left untreated or received an intraperitoneal LPS application (50 μg/mouse) for either 96 h or 144 h or intratracheal LPS alone (1 μg/mouse) for 24 h or combinations of intraperitoneal LPS followed by intratracheal LPS application, as indicated. Subsequently, bronchoalveolar lavage fluid neutrophil numbers (A) and BAL fluid (B) TNFα (black bars), MIP-2 (grey bars) and CCL2 (white bars) levels were quantified by ELISA. Values are given as mean ± SEM of at least five mice per treatment group. * indicates p < 0.01 versus control.

Figure 5

Monocytes recruited into the lungs in response to intraperitoneal LPS do not enhance but rather decrease neutrophilic alveolitis in mice challenged with CCL2 plus LPS. (A) Treatment groups of experiments shown in (B). Mice were either left untreated (bar 1) or received LPS intraperitoneally for 96 h (bar 2) or received intratracheal LPS applications alone for 24 h (bar 3) (1 μg/mouse). In addition, groups of mice received either combined intraperitoneal LPS applications (50 μg/mouse) and 96 h later, an intratracheal LPS application for 24 h (1 μg/mouse) (bar 4) or combined intratracheal applications of CCL2 for 48 h (50 μg/mouse) plus LPS for 24 h (1 μg/mouse) (bar 6) or were pretreated with intraperitoneal LPS for 48 h, followed by instillation of CCL2 (50 μg/mouse) for another 48 h followed by an intratracheal LPS application for 24 h (bar 5). Subsequently, mice were sacrificed and total numbers of alveolar recruited neutrophils were calculated. Values are given as mean ± SEM of at least five mice per treatment group. * indicates p < 0.01 versus control, + indicates p < 0.01 compared to all other treatment groups.