Concentration limits and localization of hydrogen peroxide in the extracellular space of solid tissues

H₂O₂ released to the extracellular space (ECS) regulates diverse physiological processes, yet its concentrations and spatial distribution in tissues remain poorly defined. This uncertainty hampers mechanistic understanding of redox signaling. Here, we used reaction–diffusion modeling to estimate extracellular H₂O₂ concentrations and transport ranges in various scenarios. Idealized analytical models were combined with numerical models incorporating localized NADPH oxidase (NOX) clusters, ECS microstructure, membrane permeability, and the thioredoxin- and GSH-dependent clearance systems. Using maximal neutrophil and NOX O ₂ ^•− /H₂O₂ release rates, we obtained upper bounds for extracellular H₂O₂. Adjacent to isolated average-sized, fully active NOX2 clusters H ₂ O ₂ peaked at ∼540 nM at adhesion cell–cell separations, and decreased radially over ∼50– 100 nm. At the receptor cell surface, peak concentration decreased inversely with intercellular separation, to <5 nM at 1 µm separation. Radial decrease here, for this wide separation, was over ∼2.5 µm. Even the former maximal extracellular concentrations induce just a minimal, highly localized oxidation of the intracellular Prdx, Trx and GSH pools. In turn, maximally activated neutrophils carry ∼2000 such NOX2 clusters, inducing 10’s of µM peak ECS H ₂ O ₂ concentrations. These cause extensive Prdx and Trx oxidation near the exposed membranes. However, the GSH-dependent system still sustains a strong transmembrane gradient if the permeation barrier remains intact, and ECS H ₂ O ₂ concentrations decay to sub-µM within a few µm of the source cell. Extracellular H ₂ O ₂ concentrations scaled linearly with source flux in all the examined conditions. These results establish stringent constraints on autocrine, juxtacrine and next-cell paracrine H₂O₂ signaling.