Regulation of interleukin-8 binding and function by heparin and α₂- macroglobulin

Background: Increased expression of interleukin-8 (IL-8), a potent neutrophil chemoattractant, is associated with a number of inflammatory diseases. Interleukin-8 binds to the glycosaminoglycan (GAG) heparin and the protease inhibitor α₂-macroglobulin, molecules which regulate the function of a number of cytokines. Heparan sulphate was previously shown to enhance neutrophil chemotactic responses to IL-8. Objective: The purpose of this study was to investigate the effect of heparin, heparan sulphate and α₂- macroglobulin on IL-8 binding to neutrophils and subsequent functional effects in vitro. Methods: The binding of ¹²⁵I-IL-8 to normal neutrophils at 4 °C was studied and the IL-8 induced neutrophil chemotactic response was investigated using micro-Boyden chambers. Complexation of IL-8 with α₂- macroglobulin was confirmed using gel filtration chromatography. Results: Heparin, but not heparan sulphate, inhibited the binding of ¹²⁵I-IL-8 to neutrophils (IC₅₀ = 26 μg/mL) and IL-8 induced neutrophil chemotactic responses (IC₅₀ = 4 μg/mL). The specific inhibitory effect of heparin was apparently due to an interaction with IL-8 which was charge-dependent, since dextran sulphate had a greater inhibitory effect on chemotactic responses (IC₅₀ = 2 μg/mL) and FITC-heparin did not bind to neutrophils. The heparin-induced inhibition of IL-8 binding and chemotactic responses was reversed in a dose-dependent manner in the presence of α₂-macroglobulin. The binding of ¹²⁵I-IL-8 to neutrophils in the presence of α₂- macroglobulin appears to be, in part, through the specific IL-8 receptor. Conclusion: These results point to an anti-inflammatory role for heparin and a novel, potentially, pro-inflammatory role for α₂-macroglobulin which together indicate the importance of cytokine-binding macromolecules in determining net cytokine function.