Defining counterstructures for L-selectin and beta2-integrins on human neutrophils

Neutrophil adhesion is crucial in host defense and inflammation. The aggregation of neutrophils in the circulation can also have important pathophysiological consequences when leukocyte plugs form and occlude the microvasculature. Molecules involved in neutrophil adhesion are from four distinct families: integrins, selectins, intercellular adhesion molecules (ICAMS), and mucins. Previous work by us and others indicated that beta2-integrins and L-selectin molecules found on the neutrophil surface were essential to neutrophil aggregation. In this project, we used flow-cytometry to study neutrophil aggregation in suspension in order to define potential counter-structures for neutrophil integrins and selectins. By using function blocking antibodies against L-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) the high affinity ligand for P-selectin on neutrophils, we were able to show that PSGL-1 could serve as a ligand for L-selectin as well. ICAM-1, -2, and -3 are all found on the neutrophil surface and are potential counter-structures for the neutrophil integrins. Chinese Hamster Ovary (CHO) cell lines expressing human ICAM-1 and -3 were used in neutrophil aggregation assays to determine if either molecule could serve as a counter-structure for beta2-integrins. We found that under shear, when stimulated with fMLF in suspension, neutrophils aggregated with ICAM-1 and ICAM-3 CHO cells. Blocking antibodies to the alpha-subunits on neutrophils, variably inhibited the interactions between ICAM-1 or ICAM-3 with Mac-1 and LFA-1. Neutrophil-CHO-ICAM-3 interactions could be blocked to some extent with an antibody against Mac-1 as well as a peptide, NIF (Neutrophil Inhibitory Factor) which is known to inhibit the function of Mac-1. The antibody against LFA-1 could also block neutrophil-CHO-ICAM-3 interactions to some extent. Taken together, the data provide conflicting evidence about the role of Mac-1 and ICAM-3 as potential counter-structures. Lastly, we studied the potential of neutrophil activation due to P-selectin engagement with PSGL-1. We made double transfected CHO cells with P-selectin and ICAM-3 (PIC-3 cells) and demonstrated that under shear neutrophils aggregated with PIC-3 cells without additional stimulation in a P-selectin-dependent manner.