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Modulation of lipopolysaccharide binding to human granulocytes
A J Weersink1, K P Van Kessel, M E Van der Tol
1Eijkman-Winkler Institute for Medical and Clinical Microbiology, University of Utrecht, The Netherlands.
Abstract:
Using flow cytometry and fluorescein-labelled lipopolysaccharide (LPS) from Salmonella minnesota R595 (FITC-ReLPS), we studied the role of membrane proteins in the recognition of LPS by human polymorphonuclear granulocytes (PMN) in the absence of serum. Treatment of PMN with trypsin, pronase E or proteinase K reduced both the binding of FITC-ReLPS to PMN at 4 degrees and the response of PMN to LPS at 37 degrees, as measured by luminol-enhanced chemiluminescence. Neuraminidase treatment enhanced both activities. Trypsin treatment of PMN after the binding of FITC-ReLPS effectively reduced fluorescence when cells were kept at 4 degrees, while further incubation of FITC-ReLPS-labelled PMN at 37 degrees rendered fluorescence insensible to trypsin. These results indicate a protein structure of the LPS binding site, association of FITC-ReLPS with the cell membrane at 4 degrees and subsequent internalization at 37 degrees. The binding of FITC-ReLPS was not inhibited by the anti-CD14 monoclonal antibody (mAb) 3C10, which recognizes a functional epitope of CD14. Furthermore, binding of FITC-ReLPS was observed to PMN obtained from a patient with paroxysmal nocturnal haemoglobinuria who lacked membrane-bound CD14. Stimulation of PMN with tumour necrosis factor (TNF) or LPS enhanced the binding of FITC-ReLPS at 4 degrees. This was not observed after activation of PMN devoid of granules (cytoplasts), indicating that the binding of LPS at the cell surface is enhanced by mobilization of LPS-binding proteins from intracellular granules. These studies provide evidence that LPS binding and activation of PMN involves protein structures at the cell surface different from CD14, and that granules constitute a pool of LPS-binding proteins that can be translocated to the cell surface upon stimulation.
Insights
Human white blood cells (PMN) recognize lipopolysaccharide (LPS) using membrane proteins distinct from CD14. Intracellular granules also contribute LPS-binding proteins to the cell surface for activation.
Area of Science:
- Immunology
- Cell Biology
Background:
- Lipopolysaccharide (LPS) is a key component of Gram-negative bacteria, triggering immune responses.
- Human polymorphonuclear granulocytes (PMN) are crucial in innate immunity, responding to bacterial components like LPS.
Purpose of the Study:
- To investigate the role of membrane proteins in LPS recognition by human PMN.
- To elucidate the mechanism of LPS binding and activation of PMN in the absence of serum.
Main Methods:
- Flow cytometry using fluorescein-labeled LPS (FITC-ReLPS).
- Enzymatic treatments (trypsin, pronase E, proteinase K, neuraminidase) of PMN.
- Luminol-enhanced chemiluminescence to measure PMN response.
- Use of anti-CD14 monoclonal antibody (mAb) 3C10.
- Analysis of PMN from a patient lacking CD14.
Main Results:
- Protease treatment of PMN reduced FITC-ReLPS binding and activation, indicating protein involvement.
- Neuraminidase treatment enhanced FITC-ReLPS binding and activation.
- FITC-ReLPS binding was not inhibited by anti-CD14 mAb 3C10 and occurred in CD14-deficient PMN.
- PMN stimulation with TNF or LPS enhanced FITC-ReLPS binding, dependent on intracellular granules.
Conclusions:
- LPS recognition and activation of human PMN involve cell surface protein structures distinct from CD14.
- Intracellular granules store LPS-binding proteins that translocate to the cell surface upon stimulation, enhancing LPS binding.