Background Neutralization of the neurotrophin Nerve Growth Factor (NGF) has recently emerged as a potential treatment of pain in humans. In normal condition NGF is ubiquitously present in the organism. However, NGF levels are markedly up-regulated in inflamed tissues, including joints of patients with chronic arthritis. Although immune cells are known to express NGF receptors, TrkA and p75-NTR, it is not clear how NGF levels affect immune cell activity during the inflammatory response. The aim of our study was to investigate NGF effects on cytokine production induced by pathogen-associated molecular patterns in human monocytes. Methods Using peripheral blood monocytes, purified by Percoll gradient, we investigated the NGF effects on human monocytes after Toll-like receptor (TLR) activation. To mimic the physiological condition in which cells are constantly exposed to NGF, monocytes were pre-incubated overnight with NGF before adding TLR ligands. To mimic the inflammatory condition in which there is an increase in NGF concentration, monocytes were stimulated with TLR ligands and NGF was added either at same or at later times. The expression of NGF receptors and TLRs, the activation of signalling pathways and the synthesis of IL-6 and IL-1? were evaluated using real-time PCR, western blot, flow cytometry and ELISA. Summary of the results Pre-incubation with NGF caused an enhanced response to TLR ligands (LPS, LTA, PAM) with a two-fold NGF dose-dependent increase in IL-6 production. NGF alone did not have any effect. The NGF pre-treated cells showed an up-regulation of TLR-2 expression after TLR2 ligand addition. The addition of NGF at the time of or after TLR stimulation induced a two-fold reduction in IL-6 and IL-1? synthesis and a down-regulation of TLR expression. These dual, apparently opposite, effects of NGF on monocytes appear to be related to a modified ratio of p75-NTR/TrkA receptor expression. After overnight incubation with NGF a marked increase in p75-NTR/TrkA ratio is present. It is known that p75-NTR signals through the NF-kB pathway, and we found a marked increase in nuclear NF-kB translocation in monocytes preincubated with NGF and stimulated with TLR ligands. On the contrary a marked up-regulation of TrkA expression was found following TLR stimulation suggesting that the inhibitory effect of NGF on cytokine production may depend on TrkA signalling. Conclusions NGF is part of a physiological mechanism regulating inflammatory response. NGF can amplify or down-regulate monocyte activation via TLRs depending on the differential expression of NGF receptors with subsequent changes in the p75-NTR/TrkA ratio. A better knowledge of the NGF effects, via interaction with the two NGF receptors, on inflammatory response may help to identify proper targets of novel anti-inflammatory analgesic approaches.
Nerve Growth Factor Differential Effect On Toll-Like Receptor-Mediated Cytokine Production in Human Monocytes Is Due to Changes in p75-NTR/Trk A Receptor Ratio
BracciLaudiero L;
2009
Abstract
Background Neutralization of the neurotrophin Nerve Growth Factor (NGF) has recently emerged as a potential treatment of pain in humans. In normal condition NGF is ubiquitously present in the organism. However, NGF levels are markedly up-regulated in inflamed tissues, including joints of patients with chronic arthritis. Although immune cells are known to express NGF receptors, TrkA and p75-NTR, it is not clear how NGF levels affect immune cell activity during the inflammatory response. The aim of our study was to investigate NGF effects on cytokine production induced by pathogen-associated molecular patterns in human monocytes. Methods Using peripheral blood monocytes, purified by Percoll gradient, we investigated the NGF effects on human monocytes after Toll-like receptor (TLR) activation. To mimic the physiological condition in which cells are constantly exposed to NGF, monocytes were pre-incubated overnight with NGF before adding TLR ligands. To mimic the inflammatory condition in which there is an increase in NGF concentration, monocytes were stimulated with TLR ligands and NGF was added either at same or at later times. The expression of NGF receptors and TLRs, the activation of signalling pathways and the synthesis of IL-6 and IL-1? were evaluated using real-time PCR, western blot, flow cytometry and ELISA. Summary of the results Pre-incubation with NGF caused an enhanced response to TLR ligands (LPS, LTA, PAM) with a two-fold NGF dose-dependent increase in IL-6 production. NGF alone did not have any effect. The NGF pre-treated cells showed an up-regulation of TLR-2 expression after TLR2 ligand addition. The addition of NGF at the time of or after TLR stimulation induced a two-fold reduction in IL-6 and IL-1? synthesis and a down-regulation of TLR expression. These dual, apparently opposite, effects of NGF on monocytes appear to be related to a modified ratio of p75-NTR/TrkA receptor expression. After overnight incubation with NGF a marked increase in p75-NTR/TrkA ratio is present. It is known that p75-NTR signals through the NF-kB pathway, and we found a marked increase in nuclear NF-kB translocation in monocytes preincubated with NGF and stimulated with TLR ligands. On the contrary a marked up-regulation of TrkA expression was found following TLR stimulation suggesting that the inhibitory effect of NGF on cytokine production may depend on TrkA signalling. Conclusions NGF is part of a physiological mechanism regulating inflammatory response. NGF can amplify or down-regulate monocyte activation via TLRs depending on the differential expression of NGF receptors with subsequent changes in the p75-NTR/TrkA ratio. A better knowledge of the NGF effects, via interaction with the two NGF receptors, on inflammatory response may help to identify proper targets of novel anti-inflammatory analgesic approaches.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
