Intracellular pool of IL-10 receptors in specific granules of human neutrophils: differential mobilization by proinflammatory mediators.

IL-10 has a wide range of effects tending to control inflammatory responses. We used flow cytometry to study IL-10 binding at the polymorphonuclear neutrophil (PMN) surface and its modulation by various proinflammatory agents. Little IL-10 bound to the surface of resting PMN. However, binding was strongly increased after stimulation with LPS and proinflammatory cytokines such as TNF and GM-CSF. IL-1 and IL-8 did not significantly modify IL-10 binding. Cycloheximide had no effect on TNF-induced IL-10 binding, strongly suggesting the release of a pre-existing pool of IL-10R rather than de novo receptor synthesis by PMN. This was confirmed by the inhibitory effect of pentoxifylline, an inhibitor of degranulation. The existence of an intracellular pool of IL-10R was shown by flow cytometry, immunocytochemical staining, and Western blotting with several anti-human IL-10R Abs. In subcellular fractions of resting PMN, IL-10R was mainly located in the specific granule fraction, and was absent from azurophil granules and cytosol. We also tested the mobilization of specific granules by measuring the release of lactoferrin, their reference marker. The differential effects of the proinflammatory agents on IL-10 binding matched their effects on lactoferrin release and may therefore be related to differential mobilization of specific granules by these agents. Furthermore, the kinetics of TNF-induced up-regulation of IL-10 binding to PMN ran parallel to the kinetics of the inhibitory effect of IL-10 on the oxidative burst, suggesting a key role of IL-10R mobilization from specific granules to the membranes in optimal regulation of inflammatory responses.

Lack of IL-10 and IL-13 production by human polymorphonuclear neutrophils.

The ability of polymorphonuclear neutrophils (PMN) to produce and release numerous pro-inflammatory cytokines is now established and play an important role in triggering and maintaining the inflammatory response. We studied the autocrine downregulation of this process by investigating the potential production by human PMN of two major anti-inflammatory cytokines, interleukin 10 (IL-10) and IL-13. The authors' PMN purification method, based in part on immunomagnetic depletion, allows the elimination of all cells known to produce those two cytokines. A wide range of activation conditions were tested and kinetic studies were done. IL-10 and IL-13 proteins were not detected in culture supernatants or cell lysates of PMN from 13 healthy volunteers. This was confirmed by the absence of IL-10 and IL-13 mRNA expression, as shown by RT-PCR. Human PMN thus seem unable to produce or release two of the most potent anti-inflammatory cytokines, IL-10 and IL-13. This could partly account for the persistence of local inflammation, where PMN are the main infiltrating cells.