MD-2-dependent pulmonary immune responses to inhaled lipooligosaccharides: effect of acylation state

Abstract

Endotoxins represent one of the most potent classes of microbial immunoactive components that can cause pulmonary inflammation. The aim of this study was to compare the inflammatory potency of two types of Neisseria meningitidis endotoxins (lipooligosaccharides) in lungs: wild type (hexaacylated, LOS(wt)) and mutant type (pentaacylated, LOS(msbB)), and to determine the importance of MD-2 in endotoxin responses in lungs in vivo. Endotoxin-normoresponsive mice (BALB/c) were exposed to selected doses of penta- and hexaacylated lipooligosaccharides (LOS) by nasal aspiration. Cellular and cytokine/chemokine inflammatory responses in bronchoalveolar lavage were measured at 1-, 4-, 8-, 16-, 24-, and 48-hour time points. MD-2-null mice were exposed to one dose of hexaacylated LOS and inflammatory responses were measured after 4 and 24 hours. Inhalation of hexaacylated LOS resulted in strong inflammatory responses, while pentaacylated LOS was much less potent in inducing increases of neutrophils, TNF-alpha, macrophage inflammatory protein-1 alpha, IL-6, granulocyte colony-stimulating factor, and IL-1 beta concentration in bronchoalveolar lavage. Similar kinetics of inflammatory responses in lungs were found in both types of endotoxin exposures. Inhalation of hexaacylated LOS in MD-2-null mice resulted in significantly lower numbers of neutrophils in bronchoalveolar lavage than in normoresponsive mice. Markedly lower inflammatory potency of pentaacylated LOS was observed compared with hexaacylated LOS. Hyporesponsiveness in MD-2-null mice after nasal aspiration of wild-type LOS indicate its essential role in airway responsiveness to endotoxin.

Figure 1.

Dose–response data showing the number of neutrophils in bronchoalveolar lavage (BAL) after nasal aspiration of LOS^wt and LOS^msbB in comparison with controls (***P < 0.001). There was a significantly higher neutrophilic response in LOS^wt- than in LOS^msbB-exposed mice 4 hours after exposure (P < 0.001).

Figure 2.

Cytokine dose–response curves for TNF-α, macrophage inflammatory protein (MIP)-1α, IL-6, granulocyte colony-stimulating factor (G-CSF), and IL-1β in BAL fluid of LOS^wt- in comparison with LOS^msbB-exposed mice. Data show a dose–response relationship where increasing doses of endotoxin produced significant increases of TNF-α, MIP-1α, G-CSF, IL-6, and IL-1β in BAL fluid of LOS^wt-exposed mice in comparison with those exposed to LOS^msbB (P < 0.001 for each cytokine). Each data point compared with respective control: *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3.

Time-course for pulmonary inflammatory responses (total cells and neutrophils) in BAL after the nasal aspiration of LOS^wt in the dose of (top panel) 300 EU/mouse and (middle panel) 30 EU/mouse in comparison with (bottom panel) saline (control). The higher endotoxin dose showed maximal total cells and neutrophils in BAL at 24 hours, with a small elevation of neutrophil counts persisting at 48 hours.

Figure 4.

Pulmonary inflammatory responses after nasal aspiration of LOS^msbB in the dose of 30 EU and 300 EU/mouse at 4- and 24-hour time points shown on the same scale as in Figure 3. Responses at both doses and time points were much lower than observed in LOS^wt-exposed mice.

Figure 5.

Time course for the concentration of TNF-α, MIP-1α, IL-6, G-CSF, and IL-1β in lavage after inhalation of LOS^wt in the dose of 300 and 30 EU/mouse in comparison with controls.

Figure 6.

Representative photomicrographs of lungs stained with hematoxylin and eosin. (A) Hexa- and pentaacylated LOS (LOS^wt and LOS^msbB)-induced accumulation of neutrophils in the peribronchiolar region 4 hours after nasal aspiration of (a) 30 EU, (b) 300, and (c) 3,000 EU/mouse. Magnification: ×100. (B) Neutrophil recruitment in the peribronchiolar region 1, 4, 8, 16, 24, and 48 hours after exposure of 300 EU of LOS^wt. Magnifications: ×40 and ×100.

Figure 6.

Representative photomicrographs of lungs stained with hematoxylin and eosin. (A) Hexa- and pentaacylated LOS (LOS^wt and LOS^msbB)-induced accumulation of neutrophils in the peribronchiolar region 4 hours after nasal aspiration of (a) 30 EU, (b) 300, and (c) 3,000 EU/mouse. Magnification: ×100. (B) Neutrophil recruitment in the peribronchiolar region 1, 4, 8, 16, 24, and 48 hours after exposure of 300 EU of LOS^wt. Magnifications: ×40 and ×100.

Figure 7.

Number of total cells and neutrophils in bronchoalveolar lavage fluid from normoresponsive mice in comparison with MD-2^−/− mice at 4 and 24 hours after exposure (***P < 0.001).