Influence of lipopolysaccharide-binding protein on pulmonary inflammation in gram-negative pneumonia

Abstract

Lipopolysaccharide-binding protein (LBP) is upregulated as part of the acute-phase response. Lipopolysaccharide-binding protein has a known multifunctional role in potentiating the recognition, clearance, and killing of gram-negative bacteria. In a Klebsiella pneumonia model, we previously demonstrated that LBP gene-deficient mice (LBP-/-) mice experience increased mortality when compared with wild-type (Wt) mice (98% vs. 59%). We hypothesize that LBP is essential to bacterial clearance from the lung, and its absence leads to alteration of the pulmonary inflammatory response to pneumonia. Twelve- to 16-week-old female C57Bl/6 Wt mice and age-matched LBP-/- mice were administered 1 × 10(3) colony-forming units of Klebsiella pneumoniae by intratracheal injection. Animals were euthanized at 6, 12, 24, or 36 h after inoculation. Lung tissue and bronchoalveolar lavage samples were obtained. Lung homogenate samples were assayed to determine quantitative bacterial load per whole lung, proinflammatory cytokine concentrations, myeloperoxidase activity, and assessment of pulmonary leukocyte populations. In vitro production of inflammatory mediators were also assayed after LPS stimulation of peritoneal macrophages isolated from Wt, Toll-like receptor 4 (TLR4)-deficient, and LBP-/- mice. The LBP-/- mice demonstrated significantly elevated levels of bacteria in the lung at 24 and 36 h when compared with Wt controls. The average lung levels of proinflammatory cytokines interleukin-1β (IL-1β), IL-6, keratinocyte-derived chemokine, and macrophage-inflammatory protein-2 were greater in the LBP mice and remained elevated longer when compared with those in the Wt mice. Myeloperoxidase activity, an indicator of neutrophil content, was significantly increased at time 36 h in the LBP mice. After in vitro stimulation of peritoneal macrophages with LPS, production of IL-1β, IL-6, IL-10, keratinocyte-derived chemokine, and macrophage-inflammatory protein-1α were suppressed in LBP and TLR4-deficient mice compared with that in Wt. Absence of a functional LBP-/- gene results in diminished clearance of gram-negative bacteria from the pulmonary system. Failure to recognize and clear gram-negative bacteria via the LBP/TLR4 axis results in an initial delayed inflammatory response. This delay in LBP-/- mice is followed by excessive amplification and prolonged elevation of proinflammatory mediators and neutrophil sequestration within the lungs.

Figure 1

Quantification of lung bacteria levels. All values are Wt vs. LBP-/- and represent median group values of CFU's in100μL of the whole lung homogenate. (A) CFU's at 6, 12, 24, and 36 hours post inoculation of Klebsiella pneumonia. At 6 hours N=5, at 12 hours N=5, at 24 hours N=22, and at 36 hours N=10 for both Wt and LBP -/- species. (*p < 0.05). (B) BAL macrophages from Klebsiella infected lungs. Upper frame, high power magnification (x100) of BAL macrophages (stained with toluidine blue) obtained from Klebsiella sp. infected Wt mouse lungs 12 hr after lung challenge. Lower frame, similarly obtained BAL macrophages from Klebsiella sp. infected LBP-/- mouse lungs. Arrows show the inclusions within vesicles of the cytosol.

Figure 1

Quantification of lung bacteria levels. All values are Wt vs. LBP-/- and represent median group values of CFU's in100μL of the whole lung homogenate. (A) CFU's at 6, 12, 24, and 36 hours post inoculation of Klebsiella pneumonia. At 6 hours N=5, at 12 hours N=5, at 24 hours N=22, and at 36 hours N=10 for both Wt and LBP -/- species. (*p < 0.05). (B) BAL macrophages from Klebsiella infected lungs. Upper frame, high power magnification (x100) of BAL macrophages (stained with toluidine blue) obtained from Klebsiella sp. infected Wt mouse lungs 12 hr after lung challenge. Lower frame, similarly obtained BAL macrophages from Klebsiella sp. infected LBP-/- mouse lungs. Arrows show the inclusions within vesicles of the cytosol.

Figure 2

In vivo cytokine and chemokine analysis. (A) Wt vs. LBP-/- mice lung homogenates at 6, 12, 24, and 36 hours. (B) Wt vs. LBP-/- BAL fluid at 6, 12, 24, and 36 hours. (*p < 0.05). Normal group represents baseline values from mice at 0 hours that did not undergo bacterial challenge and were undetectable for all measured cytokines. At 6 hours N=12, at 12 hours N=11, at 24 hours N=24, and at 36 hours N=10 for both Wt and LBP-/- species.

Figure 3

Flow cytometric analysis of inflammatory cell populations in lung parenchyma at 24 hours post-inoculation. (A) Significantly reduced recruitment of macrophages into lung parenchyma was found in LBP-/- animals compared to the Wt. (B) F4/80+ CD11b+ CD11c+ macrophages represented a greater percentage of the total population of lung parenchymal cells in Wt mice (19% vs 9%). (*p < 0.05). N=4 for both Wt and LBP-/- species.

Figure 4

In vitro cytokine and chemokine analysis. Thioglycollate-elicited peritoneal macrophages were stimulated with 50ng/mL of LPS for 8 hours in serum-free medium. Supernatants were assayed for inflammatory mediators. N=4 for Wt, LBP-/-, and TLR4-/- species.

Figure 5

Lung myeloperoxidase assay. Wt vs. LBP-/- mice MPO activity in lung homogenates at 6, 12, 24, and 36 hours. MPO activity was significantly higher in Wt mice vs. LBP-/- mice at 6 hours (0.094 μg/mL ± 0.012 vs. 0.037 μg/mL ± 0.03, p < 0.05). At 36 hours, MPO activity was significantly higher in LBP-/- mice compared to Wt (0.352 μg/mL ± 0.03 vs.0.123 μg/mL ± 0.012, p < 0.05). At 6 hours N=5, at 12 hours N=5, at 24 hours N=22, and at 36 hours N=10 for both Wt and LBP-/- species.

Figure 6

Flow cytometric analysis cells in bronchial lavage fluid (BAL) at 24 hours post bacteria inoculation. BAL macrophages were identified as forward light scatter high, F4/80+,CD11c+, CD11b+ cells. BAL neutrophils were identified as FSC low, Gr-1+, CD11b++, F4/80- cells. Apoptotic cells were identified as Annexin V+ Live/Dead-. Necrotic cells were identified as Annexin V+ Live/Dead+. (*p < 0.05). N=4 for both Wt and LBP-/- species.

Figure 7

Summary of changes in Wt lungs (left) and LBP-/- lungs (right) after challenge with Klebsiella sp. Wt lungs showed fewer CFUs (arrows indicating Klebsiella organisms). Macrophages were more numerous and PMNs eventually declined to lower levels. In LBP-/- lungs (right), many more Klebsiella prevail, the number of PMNs is considerably greater, and intrapulmonary cytokine levels are considerably higher, possibly due to a heightened load of bacteria.