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LPS and LAM activation of the U373 astrocytoma cell line: differential requirement for CD14
Abstract:
CD14 is a membrane protein (mCD14) found on monocytes and neutrophils that is required for the innate immune response to lipopolysaccharide (LPS) and lipoarabinomannan (LAM). CD14 can also be found in serum as soluble CD14 (sCD14) that when bound to bacterial products, enables many non-CD14 bearing cells to be activated. Lipopolysaccharide binding protein (LBP) is a plasma protein that disaggregates and catalytically transfers LPS to CD14. To examine the role of CD14 and LBP in LAM-dependent activation, we used the U373 astrocyte cell line to stably express membrane-bound CD14 (U373-CD14). In serum-free conditions, U373-CD14 cells could respond to LAM stimulation as measured by expression of intracellular adhesion molecule-1 (ICAM-1). Vector control cells (U373-RSV) could not respond to LAM or LPS; but, upon the addition of serum as a source of soluble CD14, control U373-RSV cells could respond to LPS, but not LAM. Therefore, LAM can activate U373 cells only through membrane CD14 and not soluble CD14. We also demonstrate that this membrane CD14-dependent LAM response is greatly facilitated by the addition of LBP.
Insights
Lipopolysaccharide (LPS) and lipoarabinomannan (LAM) activate innate immunity via CD14. This study shows membrane CD14, not soluble CD14, is essential for LAM-induced responses in astrocytes, with LBP facilitating this activation.
Area of Science:
- Immunology
- Cell Biology
Background:
- CD14, a key innate immune receptor, exists as membrane-bound (mCD14) on immune cells and soluble (sCD14) in serum.
- mCD14 and sCD14 are crucial for recognizing bacterial products like lipopolysaccharide (LPS) and lipoarabinomannan (LAM).
- Lipopolysaccharide binding protein (LBP) facilitates LPS transfer to CD14.
Purpose of the Study:
- To investigate the distinct roles of mCD14 and sCD14 in lipoarabinomannan (LAM)-induced cellular activation.
- To determine the influence of lipopolysaccharide binding protein (LBP) on LAM-mediated responses.
Main Methods:
- U373 astrocyte cell line was engineered to stably express membrane-bound CD14 (U373-CD14).
- Cells were stimulated with LAM and LPS under serum-free and serum-containing conditions.
- Intracellular adhesion molecule-1 (ICAM-1) expression was measured to assess cellular activation.
- The effect of LBP addition was evaluated.
Main Results:
- U373-CD14 cells responded to LAM stimulation in serum-free conditions.
- Control cells (U373-RSV) required serum (sCD14) to respond to LPS but not LAM.
- LAM-induced activation was dependent on membrane CD14 and significantly enhanced by LBP.
Conclusions:
- Membrane CD14 is essential for lipoarabinomannan (LAM) recognition and activation of astrocytes.
- Soluble CD14 does not mediate LAM responses.
- Lipopolysaccharide binding protein (LBP) plays a facilitating role in membrane CD14-dependent LAM activation.
