The role of lipid rafts in LPS-induced signaling in a macrophage cell line

doi:10.1016/j.molimm.2005.04.011

Molecular Immunology

Volume 43, Issue 6, February 2006, Pages 607-612

https://doi.org/10.1016/j.molimm.2005.04.011 Get rights and content

Abstract

The significance of lipid rafts in lipopolysaccharide (LPS) signaling in macrophages was studied through isolation of them by gradient centrifugation and subsequent visualization of signal molecules using antibodies. LPS signaling is initiated by binding to Toll-like receptor-4 (TLR4) and the co-receptor CD-14, leading to activation of downstream targets, such as MAP kinases. In this study, we show that LPS causes translocation of CD-14 and MAP kinases (ERK-2 and p38) to lipid rafts in the macrophage cell line RAW 264.7. The adaptor proteins MyD88 and Gab-2, on the other hand, were not detected in the lipid raft fractions. These results indicate that lipid rafts play a role in LPS-induced signaling in macrophages.

Introduction

Lateral assemblies of lipids, termed lipid rafts, have been postulated to represent a general feature of the plasma membrane of eukaryotic cells (Simons and Ikonen, 1997). Lipid rafts are detergent insoluble/resistant glycolipid-enriched membrane domains which are rich in cholesterol and sphingolipids. Raft associated proteins are enriched in Triton X-100 resistant membrane (DRM or lipid rafts) complexes. Existence of microdomains results in lateral phase separation and compartmentalization of raft associated proteins. The physiological significance of lipid rafts is not yet clear, but one postulated role is in the recruitment and concentration of molecules involved in signaling (Pralle et al., 2000). Recently, it has been postulated that membrane microdomains can be important in lipopolysaccharide (LPS) signaling (Hornef et al., 2003, Triantafilou et al., 2002).

Bacterial LPS activates macrophages by binding to Toll-like receptor-4 (TLR4), leading to production of cytokines (e.g. TNF-α) (Poltorak et al., 1998). Engaged TLR4 activate MyD88-dependent and -independent pathways, leading to activation of downstream targets (Kawai et al., 1999), such as MAP kinases. CD-14 is another receptor able to bind LPS (Muta and Takeshige, 2001), but it is not capable of initiating a transmembrane activation signal on its own. CD-14 is a glycosylphosphatidylinositol (GPI)-anchored protein, and such proteins are often found in membrane microdomains.

To learn more about the putative role of lipid rafts in LPS signaling, sucrose gradient centrifugation was used to isolate lipid rafts, and their content was visualized using antibodies against various signal molecules.

LPS stimulation of the macrophage-like cell line RAW 264.7 induced translocation of CD-14, ERK-2 and p38 to lipid rafts, but not of MyD88, Gab-2, Mnk-1, MEK, JNK-1 or Src. Thus, lipid rafts seem to be involved in TLR signaling, but only some signal components become localized to lipid rafts.

Section snippets

Reagents and antibodies

LPS, lovastatin (Mevinolin) as well as metyl-β-cyclodextrin (MβCD) were from Sigma Chemical Co. (St. Louis, MO, USA). Antibodies against ERK-2, JNK-1, p38, flotillin, CD-14 (mouse), MyD88, Src, Mnk-1, MEK, as well as HRP conjugated secondary antibodies against mouse, rabbit and goat, were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against Gab-2 were from Upstate (Lake Placid, NY, USA) and antibodies against p-ERK were from New England Biolabs (Beverly, MA, USA). Nystatin

Flotillin-1 was used as a marker for lipid rafts

Membrane domains that are highly enriched in lipid raft components can be purified on the basis of Triton X-100 insolubility coupled with buoyant density in sucrose gradients (Harder and Simons, 1997, Harder and Simons, 1999). To elucidate the role of lipid rafts in the innate immune recognition of LPS, lipid rafts were isolated from the macrophage-like cell line RAW 264.7 by sucrose gradient centrifugation as described in Section 2. Flotillin-1 is enriched in detergent-resistant domains in the

Discussion

Very few studies have been conducted addressing the question of a role for lipid rafts in TLR signaling. We show here that the co-receptor CD-14 and the MAP kinases ERK-2 and p38 become translocated to lipid rafts after LPS stimulation, indicating that lipid rafts play a role in LPS-induced signaling in macrophages. The importance of microdomains in LPS signaling in macrophages has also been indicated by the findings of LPS down-regulation of caveolin-1 (Lei and Morrison, 2000) and recruitment

Conclusion

CD-14, ERK and p38 are all involved in LPS-mediated signaling in macrophages and we show here that they become translocated to lipid rafts after stimulation with LPS. Although translocation of some other signaling/adaptor molecules assumed to be involved could not be detected, the results indicate that lipid rafts play a role in LPS-induced signaling.

Acknowledgements

This work was supported by grants from the Swedish fund for research without animal experiments, the Alfred Österlund Foundation, and the Greta and Johan Kock Foundations. The skilful technical assistance by Pia Lundquist is gratefully acknowledged.

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The role of lipid rafts in LPS-induced signaling in a macrophage cell line

Abstract

Introduction

Section snippets

Reagents and antibodies

Flotillin-1 was used as a marker for lipid rafts

Discussion

Conclusion

Acknowledgements

Cytokine

J. Biol. Chem.

J. Biol. Chem.

Curr. Opin. Cell Biol.

Biophys. J.

Immunity

Cytokine

Cell

Blood

Cell

Cell

Clin. Immunol.

J. Biol. Chem.