Table I.

Effect of proteases, high ionic strength and low pH on the binding of PF-4 and rIL-8 to receptors on neutrophils

% Sp. Bound 125I-PF-4a% Sp. Bound 125I-IL-8a
Untreated control100100
Chymotrypsin digestionb109.2 ± 16.142.4 ± 9.2
pH 3.0c98.4 ± 6.80.8 ± 0.8
0.5 M NaClc1.8 ± 0.996.8 ± 5.6
  • a Cells were analyzed for binding of 1 μM 125 I-PF-4 or 1 nM 125I-rIL-8 at 4°C. Data were corrected for unspecific binding, determined in the presence of a 50-fold (PF-4) or 200-fold (rIL-8) molar excess of the unlabeled ligand, respectively. Values were expressed as the percentage of specific binding obtained with the respective ligand in untreated control cells. Unspecific binding was 15.8 ± 4.9% (PF-4) and 3.6 ± 1.4% (IL-8) of total binding by control cells. Data represent mean ± SD of three independent experiments, each performed in duplicate.

  • b PMN were treated with 6 U/mL TPCK-chymotrypsin for 30 min at 37°C or left untreated prior to the binding assay with labeled chemokines.

  • c Neutrophils loaded with the labeled chemokines for 2 h at 4°C were subsequently exposed to PBS buffer containing 0.5 M NaCl or glycine buffer (50 mM glycine 0.1 m NaCl, pH 3.0) for 1 min at 4°C and residual binding was determined. Cells exposed to PBS only served as controls.