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Updated: Sep 3, 2026
Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria
Published on: September 16, 2013
Bacterial lipopolysaccharide binds to CD14 in low-density domains of the monocyte-macrophage plasma membrane
P Y Wang1, R L Kitchens, R S Munford
1Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235-9113, USA.
Abstract:
We report that gram-negative bacterial lipopolysaccharide (LPS) binds to CD14 on lipid-enriched, low-density domains of the human monocyte-macrophage (THP-1 cell) plasma membrane. After brief incubation with [3H]LPS under conditions that prevent its internalization, THP-1 cells were disrupted using a detergent-free method and plasma membrane fragments were separated on density gradients. The [3H]LPS-binding fragments had low bouyant densities and were enriched, when compared to high-density membrane fragments, in CD14 (a receptor for LPS and other microbial molecules), p53/56lyn, GTP-binding proteins, ouabain-inhibitable Na+/K+ ATPase, sphingomyelin, and GM1 ganglioside. Monoclonal anti-CD14 antibody 60bca blocked [3H]LPS binding to these membrane fragments. Immunoelectron microscopic analysis identified clusters of CD14 on both large (200-1,000 nm) and small (< or = 200 nm) low-density membrane fragments. GM1 and CD14 were usually found on the same fragments, yet their distributions on those fragments infrequently overlapped. These cells seem to lack arrays of caveolae, the ordered membrane structures that harbor glycosylphosphatidyl-anchored proteins and GM1 in many other cell types. Finding that LPS binds to CD14 predominantly in low-density plasma membrane domains suggests, however, that discrete regions of the monocyte-macrophage plasma membrane may be organized to facilitate rapid responses to, and internalization of, molecules that bind CD14.
Insights
Gram-negative bacterial lipopolysaccharide (LPS) binds to CD14 within lipid-enriched membrane domains in human monocyte-macrophages. This binding occurs in specific plasma membrane regions, suggesting organized structures for rapid cellular responses to microbial molecules.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Gram-negative bacterial lipopolysaccharide (LPS) is a potent immune activator.
- CD14 is a key receptor for LPS on immune cells.
- The precise location and organization of CD14 in the plasma membrane are crucial for its function.
Purpose of the Study:
- To investigate the specific plasma membrane domains where lipopolysaccharide (LPS) binds to CD14 in human monocyte-macrophage cells.
- To characterize the molecular composition of these LPS-binding membrane domains.
- To understand the implications of this localization for cellular response to LPS.
Main Methods:
- Incubation of THP-1 cells with radiolabeled LPS ([3H]LPS).
- Detergent-free disruption of cells and separation of plasma membrane fragments via density gradients.
- Analysis of membrane fragments using immunoelectron microscopy and biochemical assays.
- Blocking of LPS binding using a monoclonal anti-CD14 antibody.
Main Results:
- Lipopolysaccharide (LPS) preferentially binds to CD14 located in lipid-enriched, low-density plasma membrane domains.
- These low-density domains are enriched in CD14, p53/56lyn, GTP-binding proteins, Na+/K+ ATPase, sphingomyelin, and GM1 ganglioside.
- Monoclonal anti-CD14 antibody blocked LPS binding to these membrane fragments.
- CD14 and GM1 ganglioside were found on the same membrane fragments, but their distributions often did not overlap.
- THP-1 cells appear to lack typical caveolae structures.
Conclusions:
- Lipopolysaccharide (LPS) binding to CD14 occurs within specific, organized low-density plasma membrane domains.
- These domains may represent specialized signaling platforms facilitating rapid recognition and internalization of LPS.
- The findings suggest a unique membrane organization in monocyte-macrophages distinct from caveolae-dependent mechanisms seen in other cell types.
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