M. E. Van Der Tol

Complement-mediated phagocytosis of herpes simplex virus by granulocytes. Binding or ingestion.

The role of complement receptors in phagocytosis of herpes simplex virus (HSV) by PMN was examined. Complement components were deposited on the surface of the virus particle in the presence or absence of specific anti-HSV antibodies. Flow cytometry was used to analyze the phagocytosis of fluorescence-labeled viruses and demonstrated that although a virion is able to associate with PMN in the presence of complement alone, the granulocyte is not triggered to mount a metabolic burst. Efficient stimulation of PMN occurs when complexes are formed consisting of virus, specific antibodies, and complement. To address the question whether the viruses were inside or outside the cell, a combined enhancement/quenching method was developed using ammonium chloride as a lysosomotropic agent and trypan blue as a quenching dye. The data indicate that Fc receptor-mediated phagocytosis by PMN results in the ingestion of all cell-associated herpes virions. Interactions of virions through PMN-complement receptors CR1 and CR3 results solely in binding to the PMN but not in internalization. Interactions via both complement and Fc receptors cause synergistic stimulation of the PMN and result in very efficient association of viruses, greater than 80% of which were inside the cell.

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

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 β‐chain of the adhesion molecule family and a combination of antibodies against the α‐chain of LFA‐1 and Mo‐1 also blocked conjugation when target cells were sensitized under suboptimal conditions. The antibody against the β‐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.