P-selectin glycoprotein ligand-1 (PSGL-1) is a ligand for L-selectin in neutrophil aggregation.

In inflammation, activated neutrophils adhere to endothelial cells and aggregate with one another. While beta 2-integrin and L-selectin are essential for aggregation, their ligands remain to be identified. We have previously shown that L-selectin mediates a carbohydrate-dependent interaction in aggregation (Simon et al: J Immunol 149:2765, 1992; Rochon et al: J Immunol 152:1385, 1994). We have suggested that the L-selectin counter-structure is a mucinlike protein and proposed that aggregation occurs through a two-step process involving L-selectin, beta 2-integrin, and their distinct counter-structures (Bennett et al: J Leuk Biol 58:510, 1995). A candidate ligand for L-selectin is P-selectin glycoprotein ligand-1 (PSGL-1), a mucinlike protein on neutrophils that binds P-and E-selectin. Using flow cytometry we show that the number and size of neutrophil aggregates is reduced with Fab fragments of PL1, an anti-PSGL-1 monoclonal antibody that blocks the interaction between P-selectin and PSGL-1 (Moore et al: J Cell Biol 128:661, 1995). In addition, monoclonal antibodies to L-selectin and PSGL-1 were used simultaneously to modulate the availability of these adhesion molecules on individual cell populations. The inhibition of aggregation by these antibodies is consistent with L-selectin and PSGL-1 being counter-structures. We suggest that L-selectin and PSGL-1 support a collisional cell-cell interaction that represents the first step in neutrophil aggregation.

Fixation traps formyl peptide receptors in high and low affinity forms that can be regulated by GTP[S] in the absence of ligand.

The formyl peptide receptor on human neutrophils recognizes bacterial, N-formylated peptides and initiates a cascade of intracellular signals via a pertussis toxin sensitive Gi protein. We used fluorescence techniques to investigate the interactions of ligand (L), receptor (R), and G proteins (G), the ternary complex, in both live and fixed human neutrophils. By lightly fixing permeabilized neutrophils with a procedure that retained ligand binding, we were able to "capture' R and G in different configurations in the absence of ligand. Fixed receptors were trapped in a high affinity form (attributed to LRG) that could not be rapidly converted to low affinity by the addition of GTP[S]. Adding saturating nucleotide prior to fixation trapped receptors in a low affinity form (attributed to LR). The low affinity receptors retained the sensitivity of the native receptors to the presence of NA+. The distribution between high and low affinity receptors was modulated by GTP[S] in a dose dependent manner. The ability to redistribute low and high affinity receptor forms prior to fixation was unique to GTP[S], as compared to other non-activating nucleotides, suggesting that GTP[S] can regulate the distribution between R and RG. We suggest that precoupled receptors that give rise to high affinity ligand binding are likely to exist in native membranes in human neutrophils.

Evidence for a third component in neutrophil aggregation: potential roles of O-linked glycoproteins as L-selectin counter-structures.

The homotypic aggregation of neutrophils requires the participation of L-selectin and the beta 2-integrins, but it has not been clear whether the two receptors recognize one another as counter-structures or whether other adhesion molecules are involved. We have examined aggregation of live neutrophils with target populations, manipulated to alter expression of adhesive epitopes, using flow cytometry. A target population depleted of both L-selectin and activatable beta 2-integrin displayed an ability to aggregate with live neutrophils, suggesting that these two molecules are not counter-structures. We also found that an O-sialoglycoprotease (GCP) from Pasteurella haemolytica is capable of inhibiting homotypic aggregation. Neutrophils treated with GCP lose O-glycosylated proteins but retain L-selectin and activatable beta 2-integrin. One or more of the GCP substrates appears to function in L-selectin-dependent binding but not in beta 2-integrin-dependent binding. Together the data suggest a mechanism of aggregation that is analogous to leukocyte-endothelial cell adhesion in which a low-affinity carbohydrate-dependent interaction precedes a high-affinity integrin-dependent adhesion.