title: Quantitation of conjugate formation between human polymorphonuclear leukocytes and antibody-coated target cells by flow cytometry: the role of Fc receptor and LFA-1 antigen.
abstract: The specific binding of human polymorphonuclear leukocytes (PMN) to antibody-coated target cells was characterized by flow cytometry. PMN were labeled with phycoerythrin-E (PE) via a granulocyte-specific monoclonal antibody (leu-M1) and mixed with fluorescein isothiocyanate-labeled K562 tumor cells sensitized with rabbit antiserum. Specific conjugates were formed as analyzed by two-color fluorescence in a flow cytometer. The formation of stable conjugates was dependent on initiation of contact, temperature, time, and antiserum concentration. Studies with inhibitors implicate that microfilaments, but not microtubules, Ca2+, Mg2+, or energy-dependent processes were a prerequisite for binding of PMN to the antibody-coated target cells. No conjugates were formed when uncoated target cells were used or when the experiment was performed in the presence of protein A, indicating that binding was specifically mediated through Fc receptors (FcR). Monoclonal antibodies against the FcRII and FcRIII were used to address the role of these receptors in conjugation. One of the two anti-FcRIII antibodies and an anti-FcRII antibody effectively prevented conjugation. A monoclonal antibody directed against the common beta-chain of the adhesion molecule family and a combination of antibodies against the alpha-chain of LFA-1 and Mo-1 also blocked conjugation when target cells were sensitized under suboptimal conditions. The antibody against the beta-chain also diminished killing of antibody-coated K562, as measured by chromium release when included in the cytotoxicity assay. These results indicate that flow cytometry permits accurate quantitation and characterization of the binding between PMN and antibody-coated target cells, which in principle, can be prevented by monoclonal antibodies against surface receptors. Binding is primarily established by both the FcRII and FcRIII. Adhesion-associated molecules on the PMN surface contribute to optimal binding.
authors.affiliation: Eijkman-Winkler Laboratory of Medical Microbiology, University of Utrecht, The Netherlands.
authors.forename: K P
authors.initials: KP
authors.lastname: van Kessel
authors.forename: J A
authors.initials: JA
authors.lastname: van Strijp
authors.forename: M E
authors.initials: ME
authors.lastname: van der Tol
authors.forename: H J
authors.initials: HJ
authors.lastname: van Kats-Renaud
authors.forename: R M
authors.initials: RM
authors.lastname: Thijssen
authors.forename: A C
authors.initials: AC
authors.lastname: Fluit
authors.forename: J
authors.initials: J
authors.lastname: Verhoef
mesh_terms: D000911:Antibodies, Monoclonal; D000920:Antibody-Dependent Cell Cytotoxicity; D000943:Antigens, Differentiation / Q000276:immunology / Q000502:physiology*; D002450:Cell Communication; D005434:Flow Cytometry; D006801:Humans; D016169:Lymphocyte Function-Associated Antigen-1; D008562:Membrane Glycoproteins / Q000502:physiology*; D009504:Neutrophils / Q000502:physiology*; D011961:Receptors, Fc / Q000502:physiology*; D016028:Receptors, Leukocyte-Adhesion / Q000276:immunology / Q000502:physiology*; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D000943:Antigens, Differentiation; D016169:Lymphocyte Function-Associated Antigen-1; D008562:Membrane Glycoproteins; D011961:Receptors, Fc; D016028:Receptors, Leukocyte-Adhesion