5-Lipoxygenase reaction products modulate alveolar macrophage phagocytosis of Klebsiella pneumoniae

Abstract

The leukotrienes are potent lipid mediators of inflammation formed by the 5-lipoxygenase-catalyzed oxidation of arachidonic acid. Although the effects of leukotrienes on neutrophil chemotaxis and activation have been established, their role in modulating innate host defense mechanisms is poorly understood. In a previous study (M. Bailie, T. Standiford, L. Laichalk, M. Coffey, R. Strieter, and M. Peters-Golden, J. Immunol. 157:5221-5224, 1996), we used 5-lipoxygenase knockout mice to establish a critical role for endogenous leukotrienes in pulmonary clearance and alveolar macrophage phagocytosis of Klebsiella pneumoniae. In the present study, we investigated the role of specific endogenous leukotrienes in phagocytosis of K. pneumoniae and explored the possibility that exogenous leukotrienes could restore phagocytosis in alveolar macrophages with endogenous leukotriene synthesis inhibition and enhance this process in leukotriene-competent cells. Rat alveolar macrophages produced leukotriene B4 (LTB4), LTC4, and 5-hydoxyeicosatetraenoic acid (5-HETE) during the process of phagocytosis, and the inhibition of endogenous leukotriene synthesis with zileuton and MK-886 dramatically attenuated phagocytosis. We also observed a reduction in phagocytosis when we treated alveolar macrophages with antagonists to the plasma membrane receptors for either LTB4, cysteinyl-leukotrienes, or both. In leukotriene-competent cells, LTC4 augmented phagocytosis to the greatest extent, followed by 5-HETE and LTB4. These 5-lipoxygenase reaction products demonstrated similar relative abilities to reconstitute phagocytosis in zileuton-treated rat alveolar macrophages and in alveolar macrophages from 5-lipoxygenase knockout mice. We conclude that endogenous synthesis of all major 5-lipoxygenase reaction products plays an essential role in phagocytosis. The restorative and pharmacologic effects of LTC4, LTB4, and 5-HETE may provide a basis for their exogenous administration as an adjunctive treatment for patients with gram-negative bacterial pneumonia.

FIG. 1

Pathways for the oxidative metabolism of AA. The 5-LO pathway is within the box. COX, cyclooxygenase; TX, thromboxane; PG, prostaglandin.

FIG. 2

Representative radioactive HPLC elution profiles from prelabeled AMs incubated in the absence (A) or presence (B) of opsonized K. pneumoniae for 1 h. Eicosanoids were extracted from the medium before separation by HPLC. The radioactivity in each fraction was expressed as a percentage of the total radioactivity incorporated into cells. Peaks are designated on the basis of coelution with authentic standards. TXB₂, thromboxane B₂; HHT, 12-hydroxyheptadecatrienoic acid.

FIG. 3

Effect of endogenous LT synthesis inhibition on AM phagocytosis of K. pneumoniae. AMs were pretreated with medium alone, zileuton (10 μM), or MK-886 (1 μM) for 15 min before the addition of opsonized K. pneumoniae. Data are expressed as the mean phagocytic index (percentage of control) ± SE (n = 4). ∗, P < 0.05 with respect to the control by ANOVA.

FIG. 4

Effect of LT receptor antagonists on AM phagocytosis of K. pneumoniae. AMs were treated with LTB₄ receptor antagonist LY292476) (1 μM), cysteinyl-LT receptor antagonist LY171883) (1 μM), or both for 15 min before the addition of opsonized K. pneumoniae. Data are expressed as the mean phagocytic index (percentage of control) ± SE (n = 4). ∗, P < 0.05 with respect to the control by ANOVA.

FIG. 5

Effects of exogenous lipids on phagocytosis of K. pneumoniae by LT-competent cells. AMs were incubated with lipids for 5 to 10 min before the addition of opsonized K. pneumoniae. Data are expressed as the mean phagocytic index (percentage of control) ± SE (n = 4). ∗, P < 0.05 with respect to the control by ANOVA.

FIG. 6

Effect of LT receptor antagonists on the augmentation of phagocytosis in response to exogenous LTB₄ and LTC₄. AMs were treated with exogenous LTs (LTB₄ or LTC₄) (1 nM), LTB₄ receptor antagonist (LY292476) (1 μM), or cysteinyl-LT receptor antagonist (LY171883) (1 μM) or both before the addition of opsonized K. pneumoniae. Data are expressed as the mean phagocytic index (percentage of control) ± SE (n = 4). ∗, P < 0.05 with respect to the control by ANOVA.

FIG. 7

Effect of exogenous LTB₄, LTC₄, or 5-HETE (1 nM) on phagocytosis of K. pneumoniae by AMs pretreated for 15 min with zileuton (10 μM). Data are expressed as the mean phagocytic index (percentage of control) ± SE of quadruplicate values from one experiment representative of a total of four.

FIG. 8

Ability of exogenous LTs to restore phagocytosis of K. pneumoniae in AMs from 5-LO KO mice. AMs from WT or 5-LO KO mice were treated with medium alone (control) or exogenous LTB₄, LTC₄, or 5-HETE (1 nM) for 5 to 10 min before the addition of opsonized K. pneumoniae. Data are expressed as the mean phagocytic index (percentage of control) ± SE of triplicate values. The phagocytic index for the control was 122 ± 16.