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 the chemokine family of host defense cytokines. Although IL-8 was shown to interact with two different high affinity receptors on polymorphonuclear 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 125I-labeled NAP-2 specifically bound to PMN with two different affinities (KD = 0.65 and 22.4 nM). We observed complete cross-competition of unlabeled IL-8 with 125I-labeled-NAP-2 and of unlabeled NAP-2 with 125I-labeled IL-8, indicating the absence of monospecific binding sites for either chemokine. However, in contrast to former work by others, the total number of accessible sites was considerably lower for NAP-2 (13,000/cell) than for IL-8 (59,000/cell). In addition, PMN prepared from heparinized blood expressed significantly more receptors for NAP-2 than cells prepared from citrated blood, whereas receptor numbers for IL-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 drastic down-regulation of the subsequent degranulation response, challenged by higher dosages of NAP-2, MGSA, or IL-8. Reduced functional responsiveness of cells correlated with the rapid down-regulation and internalization of NAP-2 and IL-8 high affinity binding sites. Thus, our data indicate that chemokines could mediate by individual modes of interaction with common receptor's different biologic functions.