Anionic Pulmonary Surfactant Phospholipids Inhibit Inflammatory Responses from Alveolar Macrophages and U937 Cells by Binding the Lipopolysaccharide-interacting Proteins CD14 and MD-2

Koji Kuronuma,Hiroaki Mitsuzawa,Katsuyuki Takeda,Chiaki Nishitani,Edward D Chan,Yoshio Kuroki,Mari Nakamura,Dennis R Voelker

doi:10.1074/jbc.m109.040832

Abstract

Lipopolysaccharide (LPS), derived from Gram-negative bacteria, is a major cause of acute lung injury and respiratory distress syndrome. Pulmonary surfactant is secreted as a complex mixture of lipids and proteins onto the alveolar surface of the lung. Surfactant phospholipids are essential in reducing surface tension at the air-liquid interface and preventing alveolar collapse at the end of the respiratory cycle. In the present study, we determined that palmitoyl-oleoyl-phosphatidylglycerol and phosphatidylinositol, which are minor components of pulmonary surfactant, and synthetic dimyristoylphosphatidylglycerol regulated the inflammatory response of alveolar macrophages. The anionic lipids significantly inhibited LPS-induced nitric oxide and tumor necrosis factor-alpha production from rat and human alveolar macrophages and a U937 cell line by reducing the LPS-elicited phosphorylation of multiple intracellular protein kinases. The anionic lipids were also effective at attenuating inflammation when administered intratracheally to mice challenged with LPS. Binding studies revealed high affinity interactions between the palmitoyl-oleoyl-phosphatidylglycerol and the Toll-like receptor 4-interacting proteins CD14 and MD-2. Our data clearly identify important anti-inflammatory properties of the minor surfactant phospholipids at the environmental interface of the lung.

Highlights

Grants PHL 073907 and GCRC M01-RR00051. ࡗ This article was selected as a Paper of the Week. 1 Both authors contributed to this work. 2 To whom correspondence should be addressed: Dept. of Medicine, National molecules are engaged by the plasma LPS-binding protein (LBP) [3] and transferred to CD14, a glycosylphosphatidylinositol-anchored protein, abundantly expressed on macrophages
POPG and PI Inhibit LPS-induced Production of Proinflammatory Cytokines—In our initial studies, we investigated the ability of purified lipids normally present as minor components of pulmonary surfactant to modulate LPS-induced cytokine secretion
Administered POPG, but Anionic Phospholipids Antagonize LPS Activation of Human not DPPC, significantly inhibited the TNF-␣ secretion in bronchoalveolar lavage fluid (BALF), Alveolar Macrophages—We examined the lipid antago- indicating that this anionic lipid has an anti-inflammatory effect nism of primary human alveolar macrophages, isolated by for sepsis originating outside the lung

Summary

EXPERIMENTAL PROCEDURES

Cells and Reagents—LPS (0111:B4) purified from Escherichia coli was purchased from Sigma-Aldrich. The macrophages were plated at 5 ϫ 104 cells/well in 96-well plates (Falcon) in RPMI 1640 medium containing 10% bovine growth serum and allowed to adhere for 48 h prior to the addition of agonists and antagonists. The blots were washed in Tris-Tween-buffered saline (20 mM Tris-HCl buffer, pH 7.6, containing 137 mM NaCl and 0.05% (v/v) Tween 20), blocked with 5% (w/v) nonfat dry milk for 1 h, and probed according to the method described by Towbin et al [35] with phospho-specific antibodies to p46p54 JNK, p42/p44 ERK, and p38 MAPK, or I␬B␣ or with a polyclonal MKP-1 or I␬B␣ antibodies in 5% (w/v) bovine serum albumin dissolved in Tris-Tween-buffered saline. The p value for significance was set at 0.05

RESULTS

These latter findings are consistent with earlier findings about

DISCUSSION