ProNGF-p75NTR axis plays a proinflammatory role in inflamed joints. A novel pathogenic mechanism in chronic arthritis

To identify the role of mature nerve growth factor (mNGF), its immature form proNGF and their receptors in arthritis inflammatio

Global gene expression analysis in time series following N-acetyl L-cysteine induced epithelial differentiation of human normal and cancer cells in vitro

BACKGROUND: Cancer prevention trials using different types of antioxidant supplements have been carried out at several occasions and one of the investigated compounds has been the antioxidant N-acetyl-L-cysteine (NAC). Studies at the cellular level have previously demonstrated that a single supplementation of NAC induces a ten-fold more rapid differentiation in normal primary human keratinocytes as well as a reversion of a colon carcinoma cell line from neoplastic proliferation to apical-basolateral differentiation [1]. The investigated cells showed an early change in the organization of the cytoskeleton, several newly established adherens junctions with E-cadherin/β-catenin complexes and increased focal adhesions, all features characterizing the differentiation process. METHODS: In order to investigate the molecular mechanisms underlying the proliferation arrest and accelerated differentiation induced by NAC treatment of NHEK and Caco-2 cells in vitro, we performed global gene expression analysis of NAC treated cells in a time series (1, 12 and 24 hours post NAC treatment) using the Affymetrix GeneChip™ Human Genome U95Av2 chip, which contains approximately 12,000 previously characterized sequences. The treated samples were compared to the corresponding untreated culture at the same time point. RESULTS: Microarray data analysis revealed an increasing number of differentially expressed transcripts over time upon NAC treatment. The early response (1 hour) was transient, while a constitutive trend was commonly found among genes differentially regulated at later time points (12 and 24 hours). Connections to the induction of differentiation and inhibition of growth were identified for a majority of up- and down-regulated genes. All of the observed transcriptional changes, except for seven genes, were unique to either cell line. Only one gene, ID-1, was mutually regulated at 1 hour post treatment and might represent a common mediator of early NAC action. The detection of several genes that previously have been identified as stimulated or repressed during the differentiation of NHEK and Caco-2 provided validation of results. In addition, real-time kinetic PCR analysis of selected genes also verified the differential regulation as identified by the microarray platform. CONCLUSION: NAC induces a limited and transient early response followed by a more consistent and extensively different expression at later time points in both the normal and cancer cell lines investigated. The responses are largely related to inhibition of proliferation and stimulation of differentiation in both cell types but are almost completely lineage specific. ID-1 is indicated as an early mediator of NAC action

Nerve growth factor : its contribution to neuroimmunomodulation

Nerve growth factor (NGF) is the best characterised member of the neurotrophin family, a protein group with common structures and functions. Similar to other proteins of neuronal origin NGF, in addition to its well described action on neurons, is involved in the regulation of the immune system. NGF receptors are found on immune cells and autoimmune and inflammatory disorders show significant modifications in the basal concentrations of NGF. The aim of this thesis was to investigate the role of NGF and neuropeptides in autoimmune and inflammatory disorders using in vivo and in vitro models. In the kidneys of lupus prone mice (NZB/W female hybrids) increased concentrations of NGF and neuropeptides were found, whereas in the brain both NGF and neuropeptides were similarly decreased. The spleen of NZB/W is characterised by extensive lymphoproliferation and a decrease in neuropeptides and increase of NGF levels during the disease progression. The increase in NGF concentrations may be explained by the lack of NGF utilisation by neurons or by an increase in the local NGF production. Our results show that NZB/W splenocytes are immunopositive for NGF, and in their conditioned media of high concentrations of NGF were found. Since in lupus, polyclonal B-lymphocyte activation and enhanced IgG production represent the main immune alteration, we elucidated the contribution of B-cells to the modified NGF synthesis found in lupus-prone mice. B-cells express NGF receptors and synthesise NGF and its production increased significantly after stimulation. Neutralisation of endogenous NGF using anti-NGF antibodies decreased the synthesis of bcl2 and induced DNA fragmentation, events that characterise apoptosis. The autocrine synthesis of NGF is essential for the survival of B-cells and it is possible that NGF may be one of the factors influencing the apoptosis and thereby the survival of autoreactive clones. Neuropeptides can also influence lymphocyte development and functions. The synthesis of neuropeptides is a feature not only of neuronal cells but also of immune cells. In unstimulated B-cells there is a very low expression of calcitonin gene-related peptide (CGRP), but a stronger expression is induced after both in vivo and in vitro stimulation. Treatment with anti-NGF antibodies resulted in a marked reduction in CGRP expression in both resting and activated B cells. NGF appears to directly affect the synthesis of CGRP in B-cells in a similar way to that found in sensory neurons. By regulating CGRP synthesis in lymphocytes and neuronal cells, NGF may not only influence the intensity and duration of the immune response, but also exert a protective function. Conclusions: The changes in NGF and neuropeptide basal concentrations observed during the progression of the disease in SLE mice seem to support the hypothesis that a modified communication between the nervous and immune systems contributes to the development of autoimmune diseases. The over-production of NGF found in SLE patients and murine models, by influencing lymphocyte survival, may interfere with the elimination of autoreactive clones

NGF in early embryogenesis, differentiation, and pathology in the nervous and immune systems

The physiology of NGF is extremely complex, and although the study of this neurotrophin began more than 60 years ago, it is far from being concluded. NGF, its precursor molecule pro-NGF, and their different receptor systems (i.e., TrkA, p75NTR, and sortilin) have key roles in the development and adult physiology of both the nervous and immune systems. Although the NGF receptor system and the pathways activated are similar for all types of cells sensitive to NGF, the effects exerted during embryonic differentiation and in committed mature cells are strikingly different and sometimes opposite. Bearing in mind the pleiotropic effects of NGF, alterations in its expression and synthesis, as well as variations in the types of receptor available and in their respective levels of expression, may have profound effects and play multiple roles in the development and progression of several diseases. In recent years, the use of NGF or of inhibitors of its receptors has been prospected as a therapeutic tool in a variety of neurological diseases and injuries. In this review, we outline the different roles played by the NGF system in various moments of nervous and immune system differentiation and physiology, from embryonic development to aging. The data collected over the past decades indicate that NGF activities are highly integrated among systems and are necessary for the maintenance of homeostasis. Further, more integrated and multidisciplinary studies should take into consideration these multiple and interactive aspects of NGF physiology in order to design new therapeutic strategies based on the manipulation of NGF and its intracellular pathways

NGF and Its Receptors in the Regulation of Inflammatory Response

There is growing interest in the complex relationship between the nervous and immune systems and how its alteration can affect homeostasis and result in the development of inflammatory diseases. A key mediator in cross-talk between the two systems is nerve growth factor (NGF), which can influence both neuronal cell function and immune cell activity. The up-regulation of NGF described in inflamed tissues of many diseases can regulate innervation and neuronal activity of peripheral neurons, inducing the release of immune-active neuropeptides and neurotransmitters, but can also directly influence innate and adaptive immune responses. Expression of the NGF receptors tropomyosin receptor kinase A (TrkA) and p75 neurotrophin receptor (p75NTR) is dynamically regulated in immune cells, suggesting a varying requirement for NGF depending on their state of differentiation and functional activity. NGF has a variety of effects that can be either pro-inflammatory or anti-inflammatory. This apparent contradiction can be explained by considering NGF as part of an endogenous mechanism that, while activating immune responses, also activates pathways necessary to dampen the inflammatory response and limit tissue damage. Decreases in TrkA expression, such as that recently demonstrated in immune cells of arthritis patients, might prevent the activation by NGF of regulatory feed-back mechanisms, thus contributing to the development and maintenance of chronic inflammation

Effects of intranasally-delivered pro-nerve growth factors on the septo-hippocampal system in healthy and diabetic rats

Pro-nerve growth factor (proNGF) is the predominant form of NGF in the brain and its levels increase in neurodegenerative diseases. The balance between NGF receptors may explain the contradictory biological activities of proNGF. However, the specific role of the two main proNGF variants is mostly unexplored. proNGF-A is prevalently expressed in healthy brain, while proNGF-B content increases in the neuro-degenerating brain. Recently we have investigated in vitro the biological action of native mouse proNGF variants. To gain further insights into the specific functions of the two proNGFs, here we intranasally delivered mouse-derived proNGF-A and proNGF-B to the brain parenchyma of healthy and diabetic rats, the latter characterized by dysfunction in spatial learning and memory, in the septo-hippocampal circuitry and by relative increase in proNGF-B hippocampal levels. Exogenous proNGF-B induces depression of hippocampal DG-LTP and impairment of hippocampal neurogenesis in healthy animals, with concomitant decrease in basal forebrain cholinergic neurons and cholinergic fibers projecting to the hippocampus. proNGF-A, while ineffective in healthy animals, rescues the diabetes- induced impairment in DG-LTP and hippocampal neurogenesis, promoting the concomitant recovery of the basal forebrain cholinergic phenotype. Our experimental evidences suggest that the balance between different proNGFs may influence the development and progression of neurodegenerative diseases