5-Lipoxygenase activating protein (FLAP) dependent leukotriene biosynthesis inhibition (MK591) attenuates Lipid A endotoxin-induced inflammation

Abstract

The Lipid A moiety of endotoxin potently activates TLR-4 dependent host innate immune responses. We demonstrate that Lipid-A mediated leukotriene biosynthesis regulates pathogen-associated molecular patterns (PAMP)-dependent macrophage activation. Stimulation of murine macrophages (RAW264.7) with E. coli 0111:B4 endotoxin (LPS) or Kdo2-lipid A (Lipid A) induced inflammation and Lipid A was sufficient to induce TLR-4 mediated macrophage inflammation and rapid ERK activation. The contribution of leukotriene biosynthesis was evaluated with a 5-lipoxygenase activating protein (FLAP) inhibitor, MK591. MK591 pre-treatment not only enhanced but also sustained ERK activation for up to 4 hours after LPS and Lipid A stimulation while inhibiting cell proliferation and enhancing cellular apoptosis. Leukotriene biosynthesis inhibition attenuated inflammation induced by either whole LPS or the Lipid A fraction. These responses were regulated by inhibition of the key biosynthesis enzymes for the proinflammatory eicosanoids, 5-lipoxygenase (5-LO), and cyclooxygenase-2 (COX-2) quantified by immunoblotting. Inhibition of leukotriene biosynthesis differentially regulated TLR-2 and TLR-4 cell surface expression assessed by flow cytometry, suggesting a close mechanistic association between TLR expression and 5-LO associated eicosanoid activity in activated macrophages. Furthermore, MK591 pre-treatment enhanced ERK activation and inhibited cell proliferation after LPS or Lipid A stimulation. These effects were regulated in part by increased apoptosis and modulation of cell surface TLR expression. Together, these data clarify the mechanistic association between 5-lipoxygenase activating protein-mediated leukotriene biosynthesis and 5-LO dependent eicosanoid metabolites in mediating the TLR-dependent inflammatory response after endotoxin exposure typical of bacterial sepsis.

Figure 1. MK591 pre-treatment of RAW264.7 cells reduced LPS and Lipid A induced TNF-α secretion.

RAW264.7 cells were pre-treated for 30 minutes with MK591 or vehicle following by LPS or Lipid A stimulation for 0.5 hr (A) and 4 hr (B). Conditioned media were collected and TNF-α levels analyzed by ELISA. Quantitative data are presented as means ± SEM from 6–7 independent experiments. *p<0.01 and ^†p<0.001 compared to LPS-treated cells and ^‡p<0.01 and ^§p<0.001 compared to Lipid A-treated cells.

Figure 2. MK591 pre-treatment of RAW264.7 cells reduced LPS and Lipid A induced MIP-2 secretion.

RAW264.7 cells were pre-treated for 30 minutes with MK591 or vehicle followed by LPS or Lipid A stimulation for 0.5 hr (A) and 4 hr (B). Quantitative data are presented as means ± SEM from 6–7 independent experiments. *p<0.05 and ^†p<0.001 compared to LPS-treated cells and ^‡p<0.01 compared to Lipid A-treated cells.

Figure 3. MK591 pre-treatment enhanced ERK activation in RAW264.7 cells stimulated by LPS and Lipid A.

(A) RAW264.7 cells were pre-treated for 30 minutes with MK591 or vehicle followed by LPS or Lipid A stimulation for 0.5 hr or 4 hr. Activation of ERK was evaluated by immunoblotting of RAW264.7 cell lysates. Representative immunoblots of 5 independent experiments are presented. Total ERK was quantified as an internal control. (B) Quantitative data are presented as means ± SEM from 5 independent experiments. *p<0.05 compared to LPS-treated cells and ^†p<0.05 compared to Lipid A-treated cells.

Figure 4. MK591 pre-treatment inhibits 5-LO expression in RAW264.7 cells stimulated by LPS and Lipid A.

(A) RAW264.7 cells were pre-treated for 30 minutes with MK591 or vehicle followed by LPS or Lipid A stimulation for 0.5 hr and 4 hr. Expression of 5-LO were evaluated by immunoblotting of RAW264.7 cell lysates. Actin density was quantified as internal control. Representative immunoblots of 5 independent experiments are presented. (B) Quantitative expression is the mean ± S.E.M. of 5 independent experiments. At 0.5 hr, *p<0.05 and ^†p<0.01 compared to LPS-treated cells, ^‡p<0.05 compared to Lipid A-treated cells; as at 4 hr, ^§p<0.05, ^¶p<0.01 and ^||p<0.001 compared to LPS-treated cells, ^@p<0.01 compared to Lipid A-treated cells.

Figure 5. MK591 pre-treatment inhibits COX-2 expression in RAW264.7 cells stimulated by LPS and Lipid A.

(A) RAW264.7 cells were pre-treated for 30 minutes with MK591 or vehicle followed by LPS or Lipid A stimulation for 0.5 hr and 4 hr. Expression of COX-2 were examined by immunoblotting using RAW264.7 cell lysates. Actin density was quantified as internal control. Quantitative expression is the mean ± S.E.M. of 5 independent experiments. (B) Quantitative expression is the mean ± S.E.M. of 5 independent experiments. *p<0.05 compared to LPS-treated cells and ^†p<0.05 compared to Lipid A-treated cells.

Figure 6. MK591 pre-treatment inhibits RAW264.7 cell proliferation using WST-1 proliferation assay.

RAW264.7 cells were pre-treated for 30 minutes with MK591 or vehicle followed by LPS or Lipid A stimulation for 0.5 hr and 4 hr. Cell viability was analyzed by WST-1 and quantitative assessment of proliferation is presented as the mean ± S.E.M. *p<0.001 compared to non-treated control cells, ^†p<0.001 compared to LPS-treated cells and ^‡p<0.001 compared to Lipid A-treated cells.

Figure 7. MK591 pretreatment mediates dose-dependent increases in RAW264.7 apoptosis assessed by TUNEL staining.

RAW264.7 cells were pre-treated for 30 minutes with MK591 or vehicle followed by LPS or Lipid A stimulation for 0.5 hr and 4 hr. Apoptotic nuclei were detected with FITC (green fluorescence), and all nuclei were counterstained with DAPI (blue fluorescence). Apoptotic cell number increased in a MK591 dose-dependent fashion. Original magnification, ×400.