Neutrophil-activating peptide 2 and gro/melanoma growth-stimulatory activity interact with neutrophil-activating peptide 1/interleukin 8 receptors on human neutrophils.
Neutrophil-activating peptide 1/interleukin 8 (NAP-1/IL-8), neutrophil-activating peptide 2 (NAP-2), and gro/melanoma growth-stimulatory activity (gro/MGSA) are potent inflammatory cytokines with homologous structure and similar neutrophil-activating properties. Receptors on human neutrophils that interact with these peptides were studied. Analysis of 125I-NAP-1/IL-8 binding at 0-4 degrees C revealed 64,500 +/- 14,000 receptors/cell with an apparent Kd of 0.18 +/- 0.07 nM (mean +/- S.D. of six independent experiments). Unlabeled NAP-1/IL-8, NAP-2, and gro/MGSA competed with 125I-NAP-1/IL-8 for binding to human neutrophils. Competition with increasing concentrations of unlabeled NAP-2 and gro/MGSA resolved two classes of NAP-1/IL-8 binding sites: about 70% of them bound NAP-2 and gro/MGSA with high affinity (Kd: 0.34 +/- 0.2 and 0.14 +/- 0.02), while 30% were of low affinity (Kd: 100 +/- 20 and 130 +/- 10 nM). Different binding sites, however, were not apparent upon competition with unlabeled NAP-1/IL-8, suggesting that both classes of receptors have similar affinities for NAP-1/IL-8. The existence of two receptors was also suggested by ligand cross-linking and cross-desensitization experiments. Two neutrophil membrane proteins with apparent Mr of 66,000-74,000 and 42,000-46,000 became cross-linked to 125I-NAP-1/IL-8, and the labeling was decreased when excess NAP-1/IL-8, NAP-2, or gro/MGSA was present. Stimulation of neutrophils with NAP-1/IL-8 resulted in desensitization toward a subsequent challenge with NAP-2 or gro/MGSA as shown by the rise in cytosolic free calcium. By contrast, following primary stimulation with NAP-2 or gro/MGSA, responses to NAP-1/IL-8 were only moderately attenuated, supporting the existence of NAP-1/IL-8 receptors which bind NAP-2 or gro/MGSA with low affinity. In conclusion, our results demonstrate that NAP-2 and gro/MGSA act upon human neutrophils by directly interacting with two classes of receptors for NAP-1/IL-8.
Moser
B
B
Theodor-Kocher Institute, University of Bern, Switzerland.
Schumacher
C
C
von Tscharner
V
V
Clark-Lewis
I
I
Baggiolini
M
M
D000595:Amino Acid Sequence; D001345:Autoradiography; D001667:Binding, Competitive; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006133:Growth Substances / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000378:metabolism*; D023062:Receptors, Interleukin-8A
C475829:CXCL1 protein, human; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D003432:Cross-Linking Reagents; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D016209:Interleukin-8; D008024:Ligands; D009363:Neoplasm Proteins; D011971:Receptors, Immunologic; D023062:Receptors, Interleukin-8A