We report a novel method for measuring forward and reverse kinetic rate constants, kf0 and kr0, for the binding of individual receptors and ligands anchored to apposing surfaces in cell adhesion. Not only does the method examine adhesion between a single pair of cells; it also probes predominantly a single receptor-ligand bond. The idea is to quantify the dependence of adhesion probability on contact duration and densities of the receptors and ligands. The experiment was an extension of existing micropipette protocols. The analysis was based on analytical solutions to the probabilistic formulation of kinetics for small systems. This method was applied to examine the interaction between Fc gamma receptor IIIA (CD16A) expressed on Chinese hamster ovary cell transfectants and immunoglobulin G (IgG) of either human or rabbit origin coated on human erythrocytes, which were found to follow a monovalent biomolecular binding mechanism. The measured rate constants are Ackf0 = (2.6 +/- 0.32) x 10(-7) micron 4 s-1 and kr0 = (0.37 +/- 0.055) s-1 for the CD16A-hIgG interaction and Ackf0 = (5.7 +/- 0.31) X 10(-7) micron 4 s-1 and kr0 = (0.20 +/- 0.042) s-1 for the CD16A-rIgG interaction, respectively, where Ac is the contact area, estimated to be a few percent of 3 micron 2.

Surface presentation of adhesion receptors influences cell adhesion, although the mechanisms underlying these effects are not well understood. We used a micropipette adhesion frequency assay to quantify how the molecular orientation and length of adhesion receptors on the cell membrane affected two-dimensional kinetic rates of interactions with surface ligands. Interactions of P-selectin, E-selectin, and CD16A with their respective ligands or antibody were used to demonstrate such effects. Randomizing the orientation of the adhesion receptor or lowering its ligand-and antibody-binding domain above the cell membrane lowered two-dimensional affinities of the molecular interactions by reducing the forward rates but not the reverse rates. In contrast, the soluble antibody bound with similar three-dimensional affinities to cell-bound P-selectin constructs regardless of their orientation and length. These results demonstrate that the orientation and length of an adhesion receptor influences its rate of encountering and binding a surface ligand but does not subsequently affect the stability of binding.

Abstract-There is increased interest in measuring kinetic rates, lifetimes, and rupture forces of single receptor/ligand bonds. Valuable insights have been obtained from previous experiments attempting such measurements. However, it remains difficult to know with sufficient certainty that single bonds were indeed measured. Using exemplifying data, evidence supporting single-bond observation is examined and caveats in the experimental design and data interpretation are identified. Critical issues preventing definitive proof and disproof of single-bond observation include complex binding schemes, multimeric interactions, clustering, and heterogeneous surfaces. It is concluded that no single criterion is sufficient to ensure that single bonds are actually observed. However, a cumulative body of evidence may provide reasonable confidence.

Kinetic rates and affinity are essential determinants for biological processes that involve receptor-ligand binding. By using a micropipette method, we measured the kinetics of human Fc␥ receptor III (CD16) interacting with IgG when the two molecules were bound to apposing cellular membranes. CD16 is one of only four eukaryotic receptors known to exist natively in both the transmembrane (TM, CD16a) and glycosylphosphatidylinositol (GPI, CD16b) isoforms. The biological significance of this anchor isoform coexistence is not clear. Here we showed that the anchor influenced kinetic rates; compared with CD16a-TM, CD16a-GPI bound faster and with higher affinities to human and rabbit IgGs but slower and with lower affinity to murine IgG2a. The same differential affinity patterns were observed using soluble IgG ligands. A monoclonal antibody bound CD16a-GPI with higher affinity than CD16a-TM, whereas another monoclonal antibody reacted strongly with CD16a-TM but weakly with CD16a-GPI. No major differential glycosylation between the two CD16a isoforms was detected by SDS-polyacrylamide gel electrophoresis analysis. We suggest a conformational difference as the mechanism underlying the observed anchor effect, as it cannot be explained by the differing diffusivity, flexibility, orientation, height, distribution, or clustering of the two molecules on the cell membrane. These data demonstrate that a covalent modification of an Ig superfamily receptor at the carboxyl terminus of the ectodomain can have an impact on ligand binding kinetics.

CD16, the low affinity Fc ␥ receptor III for IgG (Fc␥RIII), exists as a polypeptide-anchored form (Fc␥RIIIA or CD16A) in human natural killer cells and macrophages and as a glycosylphosphatidylinositol-anchored form (Fc␥RIIIB or CD16B) in neutrophils. CD16A requires association of the ␥ subunit of Fc⑀RI or the subunit of the TCR-CD3 complex for cell surface expression. The CD16B is polymorphic and the two alleles are termed NA1 and NA2. In this study, CD16A and the two alleles of CD16B have been expressed in Chinese hamster ovary (CHO) cells and their ligand binding and phagocytic properties analyzed. The two allelic forms of CD16B showed a similar affinity toward human IgG1. However, the NA1 allele showed approximately 2-fold higher affinity for the IgG3 than the NA2 allele. Although all three forms of CD16 efficiently bound rabbit IgG-coated erythrocytes (EA), only CD16A coexpressed with the ␥ subunit phagocytosed EA. The phagocytosis mediated by CD16A expressed on CHO cells was independent of divalent cations but dependent on intact microfilaments. CHO cells expressing CD16A-␥ and CD16A-chimeras also phagocytosed EA. The phagocytosis was specifically inhibited by tyrphostin-23, a tyrosine kinase inhibitor. In summary, our results demonstrate that glycosylphosphatidylinositol-anchored CD16B alleles differ from CD16A in their ability to mediate phagocytosis. Furthermore, since studies with other Fc␥Rs have shown that CHO cells lack the phagocytic pathway mediated by the cytoplasmic domain of Fc␥Rs, the phagocytosis of EA by CHO cells stably transfected with CD16A and CD16A-subunit chimera provides an ideal system to dissect the phagocytic signaling pathways mediated by these Fc␥R-associated subunits. In humans, CD16 is expressed as two distinct (CD16A and CD16B) forms (9 -15) which are products of two different highly homologous genes. CD16B is expressed on neutrophils in a glycosylphosphatidylinositol (GPI)-anchored form, whereas CD16A is expressed on NK cells, macrophages, and placental trophoblasts as a polypeptide-anchored transmembrane protein (8, 16 -18). The GPI-anchored CD16B exists as two allelic forms termed NA1 (CD16B NA1) and NA2 (CD16B NA2 ) (19). The polypeptide-anchored CD16A expressed on NK cells and macrophages is associated with subunits such as the chain of the 21) or the ␥ chain of . The NA1 and NA2 alleles of CD16B are 95% homologous to each other and are 95-97% homologous to CD16A in their extracellular domain (12). The functional significance of the existence of membrane isoforms and the polymorphism of CD16 are not clear. However, some studies have shown that CD16A differs from CD16B by triggering killing of tumor targets and signaling for IL-2 production (16,25,26).We established CHO cell lines expressing isoforms of CD16 and determined their ligand binding and phagocytic properties. The results show that the polypeptide-anchored CD16A is able to mediate phagocytosis of antibody-coated target cells, whereas under similar conditions the NA1 and NA2 alleles of GPI-anchored CD16B are not. ...

Although kodecytes are created artificially, they can be designed to mimic the serologic and flow cytometric profiles of native ABO subgroup RBCs.

Recently, there has been an increasing interest in measuring the interaction forces between cell adhesion receptors and their ligands [1–3]. These molecules are either anchored on the membrane of a cell or coated on the surface of a substratum. The two surfaces are joined together as a result of the formation of non-covalent bonds between the receptors and ligands. The forces are measured when the two surfaces are separated. In a theoretical paper published nineteen years ago, George Bell estimated the force required to break a receptor-ligand bond and that required to uproot the receptor from the cell membrane to be of the same order of magnitude [4]. The interpretation of the force data therefore requires the knowledge of detachment mode, i.e., via adhesive mechanism if the receptor-ligand bond is dissociated or via cohesive mechanism if the receptor-membrane anchor is disrupted.

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