The cytokine/neurotrophin axis in peripheral axon outgrowth

Inflammation is part of the physiological wound healing response following mechanical lesioning of the peripheral nervous system. However, cytokine effects on axonal regeneration are still poorly understood. Because cytokines influence the expression of neurotrophins and their receptors, which play a major role in axonal outgrowth after lesioning, we investigated the hypothesis that cytokines influence specifically neurotrophin-dependent axon elongation. Therefore, we have characterized neurotrophin-dependent neurite outgrowth of murine dorsal root ganglia (DRG) in vitro and investigated the influence of pro- and anti-inflammatory cytokines on these outgrowth patterns. Embryonic day 13 (E13) DRG were cultured in Matrigel for 2 days and axonal morphology, density and elongation were determined using an image analysis system. Nerve growth factor (NGF), neurotrophin-3 (NT-3) and -4 (NT-4) were applied alone (50 ng/mL), in double or in triple combinations. NT-3, NT-4 and NT-3 + NT-4 combined induced a moderate increase in axonal outgrowth (P < 0.001) compared with controls, while NGF and all combinations including NGF induced an even more pronounced increase in axonal outgrowth (P < 0.001). After characterizing these outgrowth patterns, interleukin (IL)-1 beta, IL-4, IL-6, interferon-gamma (IFN gamma) and tumour necrosis factor-alpha (TNF alpha) (50 or 500 ng/mL) were added to the different neurotrophin combinations. Low doses of TNF alpha and IL-6 influenced neurite extension induced by endogenous neurotrophins. IL-4 increased NT-4-induced outgrowth. IL-6 stimulated NT-3 + NT-4-induced outgrowth. IFN gamma stimulated neurite extension in the presence of NT-3 + NT-4 and NT-3 + NGF. TNF alpha inhibited NT-3-, NT-3 + NGF-, NT-4 + NGF- and NT-3 + NT-4 + NGF-induced outgrowth. These data suggest that inflammation following nerve injury modulates re-innervation via a cytokine/neurotrophin axis.