Modulation of lipopolysaccharide binding to human granulocytes.

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.

• Authors: A J Weersink, K P Van Kessel, M E Van der Tol, J Verhoef
• Journal: Immunology (Immunology) Vol. 83 Issue 4 Pg. 617-23 (Dec 1994) ISSN: 0019-2805 [Print] England
• PMID: 7533136 (Publication Type: Journal Article)

Chemical References

• Antibodies, Monoclonal
• Antigens, CD
• Antigens, Differentiation, Myelomonocytic
• Lipopolysaccharide Receptors
• Lipopolysaccharides
• Tumor Necrosis Factor-alpha
• Peptide Hydrolases

Topics

• Antibodies, Monoclonal (immunology)
• Antigens, CD (immunology)
• Antigens, Differentiation, Myelomonocytic (immunology)
• Cell Membrane (metabolism)
• Cell Separation
• Flow Cytometry
• Humans
• Lipopolysaccharide Receptors
• Lipopolysaccharides (immunology, metabolism)
• Neutrophils (drug effects, metabolism)
• Peptide Hydrolases (pharmacology)
• Staphylococcus
• Tumor Necrosis Factor-alpha (immunology)