The functional hierarchy between IL-8, GCP-2, and NAP-2, as manifested by their abilities to induce CXCR1 or CXCR2 internalization.

(A) IL-8 functionally competes for and displaces GCP-2–induced internalization of CXCR1 expression. CXCR1-transfected HEK 293 cells were incubated with IL-8 (1000 ng/mL) or GCP-2 (3000 ng/mL) for the indicated time at 37°C. GCP-2 + IL-8 (1 hour) indicates a concomitant exposure to both chemokines for 1 hour. GCP-2 (2 hours) + IL-8 (1 hour) indicates an exposure to GCP-2 for 2 hours, to which IL-8 was added during the second hour of incubation with GCP-2. The cells were washed and stained with anti-CXCR1–specific antibodies, and subjected to fluorescence-activated cell sorting (FACS) analysis as described in “Materials and methods.” Each value represents the mean ± SD of 3 independent experiments. *P = .001 for GCP-2 exposure for 2 hours vs the GCP-2 (2 hours) + IL-8 (1 hour) treatment. **P < .001 for GCP-2 exposure for 1 hour vs the GCP-2 + IL-8 (1 hour) treatment. (B) GCP-2 functionally competes for and displaces NAP-2–induced downmodulation of CXCR2 expression. CXCR2-transfected HEK 293 cells were incubated with GCP-2 (1000 ng/mL) or NAP-2 (2000 ng/mL) for the indicated time at 37°C. NAP-2 + GCP-2 (1 hour) indicates a concomitant exposure to both chemokines for 1 hour. NAP-2 (2 hours) + GCP-2 (1 hour) indicates an exposure to NAP-2 for 2 hours, to which GCP-2 was added during the second hour of incubation with NAP-2. The cells were washed and stained with anti-CXCR2–specific antibodies, and subjected to FACS analysis as described in “Materials and methods.” Each value represents the mean ± SD of 3 independent experiments. * P = .01 for GCP-2 exposure for 1 hour vs the NAP-2 + GCP-2 (1 hour) treatment. **P = .006 for NAP-2 exposure for 2 hours vs the NAP-2 (2 hours) + GCP-2 (1 hour) treatment.