NEUTROPHIL-ACTIVATING PEPTIDES NAP-2 AND IL-8 BIND TO THE SAME SITES ON NEUTROPHILS BUT INTERACT IN DIFFERENT WAYS - DISCREPANCIES IN BINDING AFFINITIES, RECEPTOR DENSITIES, AND BIOLOGIC EFFECTS

IL-8 and the neutrophil-activating peptide 2 (NAP-2) are members of th e chemokine family of host defense cytokines. Although IL-8 was shown to interact with two different high affinity receptors on polymorphonu clear neutrophil granulocytes (PMN), direct demonstration of specific binding sites for NAP-2 is difficult, because the NAP-2 molecule lacks iodinable side chains. Here we present a modified labeling procedure for the chemokine that does not affect its biologic activity. The I-12 5-labeled NAP-2 specifically bound to PMN with two different affinitie s (K-D = 0.65 and 22.4 nM). We observed complete cross-competition of unlabeled IL-8 with (125)-I-labeled-2 and of unlabeled NAP-2 with I-12 5-labeled IL-8, indicating the absence of monospecific binding sites f or either chemokine. However, in contrast to former work by others, th e total number of accessible sites was considerably lower for NAP-2 (1 3,000/cell) than for IL-8 (59,000/cell). In addition, PMN prepared fro m heparinized blood expressed significantly more receptors for NAP-2 t han cells prepared from citrated blood, whereas receptor numbers for I L-8 were unchanged. Desensitization experiments suggested a regulatory role for the NAP-2 high affinity site. Short-term priming of PMN with a nonstimulatory dose of NAP-2 (or MGSA) but not with IL-8 led to dra stic down-regulation of the subsequent degranulation response, challen ged by higher dosages of NAP-2, MGSA, or IL-8. Reduced functional resp onsiveness of cells correlated with the rapid down-regulation and inte rnalization of NAP-2 and IL-8 high affinity binding sites. Thus, our d ata indicate that chemokines could mediate by individual modes of inte raction with common receptor's different biologic functions.