Schwann Cell-conditioned Medium Research Articles

Stigmasterol alleviates neuropathic pain by reducing Schwann cell-macrophage cascade in DRG by modulating IL-34/CSF1R.

This study aimed to investigate the potential therapeutic applications of stigmasterol for treating neuropathic pain. Related mechanisms were investigated by DRG single-cell sequencing analysis and the use of specific inhibitors in cellular experiments. In animal experiments, 32 male Sprague-Dawley rats were randomly divided into the sham operation group, CCI group, ibuprofen group, and stigmasterol group. We performed behavioral tests, ELISA, H&E staining and immunohistochemistry, and western blotting. Cell communication analysis by single-cell sequencing reveals that after peripheral nerve injury, Schwann cells secrete IL-34 to act on CSF1R in macrophages. After peripheral nerve injury, the mRNA expression levels of CSF1R pathway and NLRP3 inflammasome in macrophages were increased in DRG. Invitro studies demonstrated that stigmasterol can reduce the secretion of IL-34 in LPS-induced RSC96 Schwann cells; stigmasterol treatment of LPS-induced Schwann cell-conditioned medium (L-S-CM) does not induce the proliferation and migration of RAW264.7 macrophages; L-S-CM reduces CSF1R signaling pathway (CSF1R, P38MAPK, and NFκB) activation, NLRP3 inflammasome activation, and ROS production. Invivo experiments have verified that stigmasterol can reduce thermal and cold hyperalgesia in rat chronic compressive nerve injury (CCI) model; stigmasterol can reduce IL-1β, IL-6, TNF-α, CCL2, SP, and PGE2 in serum of CCI rats; immunohistochemistry and western blot confirmed that stigmasterol can reduce the levels of IL-34/CSF1R signaling pathway and NLRP3 inflammasome in DRG of CCI rats. Stigmasterol alleviates neuropathic pain by reducing Schwann cell-macrophage cascade in DRG by modulating IL-34/CSF1R axis.

(+)-Catechin Alleviates CCI-Induced Neuropathic Pain in Rats by Modulating the IL34/CSFIR Axis and Attenuating the Schwann Cell-Macrophage Cascade Response in the DRG.

The aim of this study was to investigate the potential therapeutic applications of (+)-catechin in the treatment of neuropathic pain. In vivo study, 32 SD rats were randomly divided into four groups: sham group, chronic constriction injury (CCI) group, CCI + ibuprofen group and CCI+ (+)-catechin group. They were subjected to behavioural tests, ELISA, immunohistochemistry and Western blotting. The mechanisms involved were investigated using specific inhibitors in cell experiments. Results of in vivo experiments showed that (+)-catechin could reduce the cold sensitivity pain in a rat model of CCI; ELISA and immunohistochemistry results showed that (+)-catechin could decrease the levels of IL-8, IL-6, TNF-α, CCL2 and CCL5 in serum and the expression levels of nNOS, COX2, IL6, TNF-α, IBA-1 and CSF1R in DRG of CCI rats. Finally, western blot confirmed that (+)-catechin could diminish the levels of IL-34/CSF1R/JAK2/STAT3 signalling pathway in DRG of CCI rats. In vitro studies showed that (+)-catechin reduced IL-34 secretion in LPS-induced RSC96 cells. Meanwhile, (+)-catechin administration in LPS-induced Schwann cell-conditioned medium (L-CM) significantly inhibited the proliferation and migration of RAW264.7 cells; in addition, L-CM+(+)-catechin reduced the activation of the CSF1R/JAK2/STAT3 signalling pathway. (+)-Catechin attenuated the Schwann cell-macrophage cascade response in the DRG by modulating the IL34/CSFIR axis and inhibiting activation of the JAK2/STAT3 pathway, thereby attenuating CCI-induced neuropathic pain in rats.

Issue Information

Front cover Axonopathy is considered a hallmark of leprosy neuropathy, a chronic infectious disease caused by Mycobacterium leprae infection in Schwann cells. Leprosy neuropathy is associated with irreversible motor and sensory loss seen in infected patients. In the present work, immortalized human Schwann cell line ST8814 were infected with M.leprae. The neurotoxicity of infected Schwann cell-conditioned medium (SCCM) was then evaluated in a human neuroblastoma cell lineage and mouse neurons. ST8814 Schwann cells exposed to M. leprae affected neuronal viability by deviating glial 14C-labeled lactate, important fuel of neuronal central metabolism, to de novo lipid synthesis. We observed that transgenic M. bovis (BCG) cells expressing the M. leprae phenolic glycolipid-1 (PGL-1), a specific cell wall antigen proposed to mediate bacterial-Schwann cell interaction - (BCG-PGL-1) - was able to induce a phenotype similar to M. leprae, unlike the wild type BCG strain. This Schwann cell neurotoxic phenotype, induced by M. leprae PGL-1, occurs through the protein kinase B (Akt) pathway. The pharmacological inhibition of Akt by triciribine significantly reduced free fatty acid content in the SCCM from M. leprae- and BCG-PGL-1-infected Schwann cells and, hence, prevented neuronal death. Overall, these findings provide novel evidence that both M. leprae and PGL-1 induce a toxic Schwann cell phenotype, by modifying the host lipid metabolism, resulting in profound implications for neuronal loss as a basis of leprosy neuropathy. Image content Sensory neurons dissociated from mouse dorsal root ganglion, visualized by immunofluorescence using anti-ß-III tubulin evidencing neurotoxicity of M. leprae-infected Schwann cells conditioned medium over arborization. Read the full article ‘Phenolic glycolipid-1 of Mycobacterium leprae is involved in human Schwann cell line ST8814 neurotoxic phenotype’ by K. do Carmo de Vasconcelos Girardi, B. S. Mietto, K. dos Anjos Lima, G. C. Atella, D. S. da Silva, A. M. R. Pereira, P. S. Rosa, F. A. Lara. 2023, vol.164 (2), pp. 158–171) on doi:10.1111/jnc.15722

Phenolic glycolipid-1 of Mycobacterium leprae is involved in human Schwann cell line ST8814 neurotoxic phenotype.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae infection in Schwann cells. Axonopathy is considered a hallmark of leprosy neuropathy and is associated with the irreversible motor and sensory loss seen in infected patients. Although M. leprae is recognized to provoke Schwann cell dedifferentiation, the mechanisms involved in the contribution of this phenomenon to neural damage remain unclear. In the present work, we used live M. leprae to infect the immortalized human Schwann cell line ST8814. The neurotoxicity of infected Schwann cell-conditioned medium (SCCM) was then evaluated in a human neuroblastoma cell lineage and mouse neurons. ST8814 Schwann cells exposed to M. leprae affected neuronal viability by deviating glial 14 C-labeled lactate, important fuel of neuronal central metabolism, to de novo lipid synthesis. The phenolic glycolipid-1 (PGL-1) is a specific M. leprae cell wall antigen proposed to mediate bacterial-Schwann cell interaction. Therefore, we assessed the role of the PGL-1 on Schwann cell phenotype by using transgenic M. bovis (BCG)-expressing the M. leprae PGL-1. We observed that BCG-PGL-1 was able to induce a phenotype similar to M. leprae, unlike the wild-type BCG strain. We next demonstrated that this Schwann cell neurotoxic phenotype, induced by M. leprae PGL-1, occurs through the protein kinase B (Akt) pathway. Interestingly, the pharmacological inhibition of Akt by triciribine significantly reduced free fatty acid content in the SCCM from M. leprae- and BCG-PGL-1-infected Schwann cells and, hence, preventing neuronal death. Overall, these findings provide novel evidence that both M. leprae and PGL-1, induce a toxic Schwann cell phenotype, by modifying the host lipid metabolism, resulting in profound implications for neuronal loss. We consider this metabolic rewiring a new molecular mechanism to be the basis of leprosy neuropathy.

MicroRNA changes of bone marrow-derived mesenchymal stem cells differentiated into neuronal-like cells by Schwann cell-conditioned medium.

Bone marrow-derived mesenchymal stem cells differentiate into neurons under the induction of Schwann cells. However, key microRNAs and related pathways for differentiation remain unclear. This study screened and identified differentially expressed microRNAs in bone marrow-derived mesenchymal stem cells induced by Schwann cell-conditioned medium, and explored targets and related pathways involved in their differentiation into neuronal-like cells. Primary bone marrow-derived mesenchymal stem cells were isolated from femoral and tibial bones, while primary Schwann cells were isolated from bilateral saphenous nerves. Bone marrow-derived mesenchymal stem cells were cultured in unconditioned (control group) and Schwann cell-conditioned medium (bone marrow-derived mesenchymal stem cell + Schwann cell group). Neuronal differentiation of bone marrow-derived mesenchymal stem cells induced by Schwann cell-conditioned medium was observed by time-lapse imaging. Upon induction, the morphology of bone marrow-derived mesenchymal stem cells changed into a neural shape with neurites. Results of quantitative reverse transcription-polymerase chain reaction revealed that nestin mRNA expression was upregulated from 1 to 3 days and downregulated from 3 to 7 days in the bone marrow-derived mesenchymal stem cell + Schwann cell group. Compared with the control group, microtubule-associated protein 2 mRNA expression gradually increased from 1 to 7 days in the bone marrow-derived mesenchymal stem cell + Schwann cell group. After 7 days of induction, microRNA analysis identified 83 significantly differentially expressed microRNAs between the two groups. Gene Ontology analysis indicated enrichment of microRNA target genes for neuronal projection development, regulation of axonogenesis, and positive regulation of cell proliferation. Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that Hippo, Wnt, transforming growth factor-beta, and Hedgehog signaling pathways were potentially associated with neural differentiation of bone marrow-derived mesenchymal stem cells. This study, which carried out successful microRNA analysis of neuronal-like cells differentiated from bone marrow-derived mesenchymal stem cells by Schwann cell induction, revealed key microRNAs and pathways involved in neural differentiation of bone marrow-derived mesenchymal stem cells. All protocols were approved by the Animal Ethics Committee of Institute of Radiation Medicine, Chinese Academy of Medical Sciences on March 12, 2017 (approval number: DWLI-20170311).

Hydrophobic PDMS promotes neural progenitor formation from SHEDs by Schwann cell–cultivated medium induction

ABSTRACTIn this study, the ability of rat Schwann cell-conditioned medium to transdifferentiate stem cells from human exfoliated deciduous teeth (SHEDs) into neural-like cells was evaluated. Furthermore, the potential effects of the hydrophobic polymer polydimethylsiloxane on neurosphere formation by the neural-like transdifferentiated SHED were investigated. The inducing effects of Rat Schwann cell-cultivated medium (RSC-CM) to SHED differentiation were investigated by inverted phase contrast microscopy. Cellular clusters formed fast at 7-day culture. Immune-fluorescent staining and neural gene expression performed by reverse transcription polymerase chain reaction showed the positive expression of nestin, tubulin III, γ-enolase, and glial fibrillary acidic protein of neural marker. Thus, RSC-CM can induce effectively neural differentiation from SHED cells.

PDGF, NT-3 and IGF-2 in Combination Induced Transdifferentiation of Muscle-Derived Stem Cells into Schwann Cell-Like Cells

Muscle-derived stem cells (MDSCs) are multipotent stem cells with a remarkable long-term self-renewal and regeneration capacity. Here, we show that postnatal MDSCs could be transdifferentiated into Schwann cell-like cells upon the combined treatment of three neurotrophic factors (PDGF, NT-3 and IGF-2). The transdifferentiation of MDSCs was initially induced by Schwann cell (SC) conditioned medium. MDSCs adopted a spindle-like morphology similar to SCs after the transdifferentiation. Immunocytochemistry and immunoblot showed clearly that the SC markers S100, GFAP and p75 were expressed highly only after the transdifferentiation. Flow cytometry assay showed that the portion of S100 expressed cells was more than 60 percent and over one fourth of the transdifferentiated cells expressed all the three SC markers, indicating an efficient transdifferentiation. We then tested neurotrophic factors in the conditioned medium and found it was PDGF, NT-3 and IGF-2 in combination that conducted the transdifferentiation. Our findings demonstrate that it is possible to use specific neurotrophic factors to transdifferentiate MDSCs into Schwann cell-like cells, which might be therapeutically useful for clinical applications.

Electrical stimulation of schwann cells promotes sustained increases in neurite outgrowth.

Endogenous electric fields are instructive during embryogenesis by acting to direct cell migration, and postnatally, they can promote axonal growth after injury (McCaig 1991, Al-Majed 2000). However, the mechanisms for these changes are not well understood. Application of an appropriate electrical stimulus may increase the rate and success of nerve repair by directly promoting axonal growth. Previously, DC electrical stimulation at 50 mV/mm (1 mA, 8 h duration) was shown to promote neurite outgrowth and a more pronounced effect was observed if both peripheral glia (Schwann cells) and neurons were co-stimulated. If electrical stimulation is delivered to an injury site, both the neurons and all resident non-neuronal cells [e.g., Schwann cells, endothelial cells, fibroblasts] will be treated and this biophysical stimuli can influence axonal growth directly or indirectly via changes to the resident, non-neuronal cells. In this work, non-neuronal cells were electrically stimulated, and changes in morphology and neuro-supportive cells were evaluated. Schwann cell response (morphology and orientation) was examined after an 8 h stimulation over a range of DC fields (0-200 mV/mm, DC 1 mA), and changes in orientation were observed. Electrically prestimulating Schwann cells (50 mV/mm) promoted 30% more neurite outgrowth relative to co-stimulating both Schwann cells with neurons, suggesting that electrical stimulation modifies Schwann cell phenotype. Conditioned medium from the electrically prestimulated Schwann cells promoted a 20% increase in total neurite outgrowth and was sustained for 72 h poststimulation. An 11-fold increase in nerve growth factor but not brain-derived neurotrophic factor or glial-derived growth factor was found in the electrically prestimulated Schwann cell-conditioned medium. No significant changes in fibroblast or endothelial morphology and neuro-supportive behavior were observed poststimulation. Electrical stimulation is widely used in clinical settings; however, the rational application of this cue may directly impact and enhance neuro-supportive behavior, improving nerve repair.

Nf1−/− Schwann Cell-Conditioned Medium Modulates Mast Cell Degranulation by c-Kit-Mediated Hyperactivation of Phosphatidylinositol 3-Kinase

Neurofibromatosis type 1 (NF1) is a common genetic disorder and is characterized by both malignant and nonmalignant neurofibromas, which are composed of Schwann cells, degranulating mast cells, fibroblasts, and extracellular matrix. We and others have previously shown that hyperactivation of the c-Kit pathway in an Nf1 haploinsufficient microenvironment is required for both tumor formation and progression. Mast cells play a key role in both tumorigenesis and neoangiogenesis via the production of matrix metalloproteinases, heparin, and a range of different growth factors. In the present study, we show that tumorigenic Schwann cells derived from Nf1(-/-) embryos promote increased degranulation of Nf1(+/-) mast cells compared with wild-type mast cells via the secretion of the Kit ligand. Furthermore, we used genetic intercrosses as well as pharmacological agents to link the hyperactivation of the p21(Ras)-phosphatidylinositol 3-kinase (PI3K) pathway to the increased degranulation of Nf1(+/-) mast cells both in vitro and in vivo. These studies identify the p21(Ras)-PI3K pathway as a major regulator of the gain in Nf1(+/-) mast cell degranulation in neurofibromas. Collectively, these studies identify both c-Kit and PI3K as molecular targets that modulate mast cell functions in cases of NF1.

Schwann Cells Promote Synaptogenesis at the Neuromuscular Junction via Transforming Growth Factor-β1

Recent studies suggest that glial cells actively participate in the formation, function, maintenance, and repair of the chemical synapse. However, the molecular mechanisms of glia-synapse interactions are largely unknown. We have shown previously that Schwann cell-conditioned medium (SC-CM) promotes synaptogenesis in Xenopus nerve-muscle cocultures. The present study aimed to identify the synaptogenic molecules in SC-CM. Combining biochemical approaches and in vitro bioassays, we found that SC-CM contains transforming growth factor (TGF)-beta1, which is expressed in Schwann cells both in vivo and in vitro. Similar to SC-CM, TGF-beta1 doubled the size of acetylcholine receptor (AChR) clusters at nerve-muscle contacts and significantly increased the percentage of nerve-muscle contacts that show AChR clusters to approximately 60%, compared with approximately 20% seen in control cultures. The synaptogenic effects of SC-CM were abolished if SC-CM was immunodepleted of TGF-beta1 or if the latency-associated protein or a TGF-beta1 receptor kinase inhibitor was added to block the bioactivity of TGF-beta1. Similar to frog SC-CM, mammalian SC-CM also showed synaptogenic effects, which were prevented by immunodepletion of TGF-beta1. TGF-beta1 upregulated agrin expression in spinal neurons, which could explain the increase in AChR clusters in cultures treated with SC-CM. These results suggest that Schwann cells express TGF-beta1, which is both sufficient and necessary for mediating the synapse-promoting effects of Schwann cells at the developing neuromuscular junction. Schwann cell-derived TGF-beta1 thus joins other astrocyte-derived synaptogenic factors in further strengthening the emerging concept that glial cells contribute to synaptogenesis in both the PNS and the CNS.

The Role of Glial Cells in the Formation and Maintenance of the Neuromuscular Junction

The vertebrate neuromuscular junction (NMJ) is a "tripartite" synapse, composed of three cellular elements: the presynaptic nerve terminal, the postsynaptic specialization, and synapse-associated glial cells, called perisynaptic Schwann cells (PSCs; also called terminal Schwann cells). During development, PSCs grow beyond nerve terminals and guide nerve terminal extension. Nerve terminals retract or stop extension after PSC ablation by complement-mediated lysis in vivo, suggesting that PSCs can promote synaptic growth and maintenance at developing NMJs. Schwann cell-conditioned medium (SC-CM), which may be mediated by transforming growth factor-beta1, can promote synapse formation in Xenopus nerve-muscle culture. In addition, SC-CM contains small molecules (within 500-5000 Da), which can enhance spontaneous synaptic activities acutely and potently at developing frog NMJs. In adult muscles, PSCs can detect evoked synaptic activities and are capable of modulating transmitter release. Nerve terminals retract and synaptic efficacy is reduced at 1 week, but not within the first few hours, after PSC ablation. Thus, PSCs are essential for the long-term, but not short-term, maintenance of synaptic structure and function at the adult NMJ. During synaptic remodeling in adult muscles, PSC sprouts lead nerve terminal sprouts. After nerve injury, adult PSCs sprout extensive processes, which guide regenerating nerve terminals. Schwann cells express agrin and neuregulins, which may help the postsynaptic differentiation and synaptic repair. Furthermore, neuregulin-ErbB signaling pathways play an essential role in synapse-glial interactions at the NMJ. These recent findings suggest that PSCs play multiple roles and actively participate in synaptic development, modulation, maintenance, and repair of the vertebrate NMJ.

FGF/Heparin Differentially Regulates Schwann Cell and Olfactory Ensheathing Cell Interactions with Astrocytes: A Role in Astrocytosis

After injury, the CNS undergoes an astrocyte stress response characterized by reactive astrocytosis/proliferation, boundary formation, and increased glial fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycan (CSPG) expression. Previously, we showed that in vitro astrocytes exhibit this stress response when in contact with Schwann cells but not olfactory ensheathing cells (OECs). In this study, we confirm this finding in vivo by demonstrating that astrocytes mingle with OECs but not Schwann cells after injection into normal spinal cord. We show that Schwann cell-conditioned media (SCM) induces proliferation in monocultures of astrocytes and increases CSPG expression in a fibroblast growth factor receptor 1 (FGFR1)-independent manner. However, SCM added to OEC/astrocyte cocultures induces reactive astrocytosis and boundary formation, which, although sensitive to FGFR1 inhibition, was not induced by FGF2 alone. Addition of heparin to OEC/astrocyte cultures induces boundary formation, whereas heparinase or chlorate treatment of Schwann cell/astrocyte cultures reduces it, suggesting that heparan sulfate proteoglycans (HSPGs) are modulating this activity. In vivo, FGF2 and FGFR1 immunoreactivity was increased over grafted OECs and Schwann cells compared with the surrounding tissue, and HSPG immunoreactivity is increased over reactive astrocytes bordering the Schwann cell graft. These data suggest that components of the astrocyte stress response, including boundary formation, astrocyte hypertrophy, and GFAP expression, are mediated by an FGF family member, whereas proliferation and CSPG expression are not. Furthermore, after cell transplantation, HSPGs may be important for mediating the stress response in astrocytes via FGF2. Identification of factors secreted by Schwann cells that induce this negative response in astrocytes would further our ability to manipulate the inhibitory environment induced after injury to promote regeneration.

Schwann Cell-Derived Factors Modulate Synaptic Activities at Developing Neuromuscular Synapses

Glial cells are active participants in the function, formation, and maintenance of the chemical synapse. To investigate the molecular basis of neuron-glia interactions at the peripheral synapse, we examined whether and how Schwann cell-derived factors modulate synaptic function at developing neuromuscular junctions (NMJs). Schwann cell-conditioned medium (SC-CM) from Xenopus Schwann cell cultures was collected and applied to Xenopus nerve-muscle cocultures. We found that SC-CM increased the frequency of spontaneous synaptic currents (SSCs) within 3-15 min by an average of approximately 150-fold at developing neuromuscular synapses. The increase in SSC frequency by SC-CM is a presynaptic effect independent of neuronal excitability and requires the influx of Ca2+. In contrast to its potentiating effect on spontaneous transmitter release, SC-CM suppressed the evoked transmitter release. The SC-CM effect required the presence of motoneuron soma but not protein synthesis. Using molecular weight cutoff filters and dialysis membranes, we found that the molecular weight of functional factor(s) in SC-CM was within 500 and 5000 Da. The SC-CM effect was not attributable to currently known factors that modulate synaptic efficacy, including neurotrophins, glutamate, and ATP. SC-CM also enhanced spontaneous synaptic release at developing NMJs in Xenopus tadpoles in situ. Our results suggest that Schwann cells release small molecules that enhance spontaneous synaptic activities acutely and potently at developing neuromuscular synapses, and the glial cell-enhanced spontaneous neurotransmission may contribute to synaptogenesis.

Neuregulin isoforms in dorsal root ganglion neurons: Effects of the cytoplasmic domain on localization and membrane shedding of Nrg‐1 type I

Embryonic sensory neurons express membrane-anchored growth factors that stimulate proliferation and differentiation of Schwann cells. The most important of these are members of the neuregulin-1 (Nrg-1) family that activate the erbB2/erbB3 receptor kinase on Schwann cells. Nrg-1 growth factors display a complex pattern of alternative mRNA splicing. We investigated the expression of the Nrg-1 type I in rat embryo dorsal root ganglion (DRG) neurons. Nrg-1 type I mRNA was abundantly expressed in DRG neurons; molecular cloning identified three distinct isoforms. The most prominent structural difference produced by alternative splicing was truncation of the C-terminal cytoplasmic domain. In sensory neurons and other cells, Nrg-1 type I proteins with the full-length 374-amino-acid cytoplasmic domain were expressed on the cell surface. In contrast, an isoform with a partially truncated cytoplasmic domain was retained in an intracellular compartment. Deletion studies demonstrated the presence of a cryptic intracellular retention signal that was exposed in the truncated cytoplasmic domain. Cell surface Nrg-1 type I molecules were subject to protease-dependent release of the biologically active ectodomain. As a consequence of their intracellular localization, the Nrg-1 type I isoform with a truncated cytoplasmic domain was not subject to membrane shedding. Nrg-1 type I ectodomain release was accelerated by factors present in Schwann cell-conditioned medium. In cells with active Nrg-1 type I ectodomain, shedding products corresponding to the cytoplasmic domain were not detected, because of rapid gamma-secretase- and proteasome-dependent degradation. These results demonstrate that sensory neurons express alternatively spliced neuregulin polypeptides with distinct subcellular localizations and processing.

Neuron-Like Differentiation of PC12 Cells Treated With Media Conditioned by Either Sciatic Nerves, Optic Nerves, or Schwann Cells

In previous works we reported the finding of neurotrophic activity in a serum-free Dulbecco's modified Eagle's medium conditioned by rat sciatic nerves, previously maintained in culture for 11 days. This medium produces rapid neuron-like differentiation of cultured PC12 cells, as revealed by an increase in the size of the cell body and by the extension of short and/or long neurites by most of the cells. Neuregulin present in the conditioned medium was demonstrated to play a key role in the observed differentiation. In the present work, taking into consideration those latter results, the neurotrophic activity of conditioned media prepared with sciatic and optic nerves cultured during days 1-4 and 9-12 were studied. Evaluation of the trophic activities of those media revealed an opposite timing in the activities of sciatic and optic nerves conditioned media. The activity of the sciatic nerve was not observed in the 1-4-day period, increasing then up to the 9-12-day period. On the contrary, the optic nerve conditioned medium was active in the 1-4-day period, decreasing down to the 9-12-day period. These results led us to explore the contribution of the different cellular constituents of those nerves to their neurotrophic properties. As a first step in that direction we also investigated the neurotrophic activity of media conditioned during 12 days by cultured Schwann cells isolated from rat sciatic nerves. The Schwann cell conditioned media did produce a rapid differentiation of the PC12 cells similar to that caused by the sciatic nerve conditioned medium, though of a lower magnitude. Variations in the trophic activities of the conditioned media used in the present work is discussed taking into consideration the production of trophic and inhibitory factors by the peripheral and central glial cells. The role played by the optic nerve glia and myelin is being investigated at present.

Schwann cells exhibit excitotoxicity consistent with release of NMDA receptor agonists

Neurodegenerative effects of Schwann cells transplanted into the central nervous system have been observed previously. We report here that conditioned medium from Schwann cell cultures exhibit degenerative influences on hippocampal neurons. Aliquots of Schwann cell-conditioned medium compromised the morphologic integrity of the neurons, markedly elevated their intracellular calcium concentrations, and decreased their viability. The degenerative effects of Schwann cell medium on neuronal morphology and viability were blocked by N-methyl-D-aspartate (NMDA) receptor antagonists D-(-)-2-amino-5-phosphonopentanoic acid (D-APV) and 5,7-dicholorokynurenic acid (DCKA). Glutamate was detected in Schwann cell-conditioned medium at a concentration on the order of 10(-5) M. D-Amino acid oxidase (DAAOx) also attenuated the neurotoxicity exhibited by Schwann cells. These data suggest that Schwann cells release biologically relevant concentrations of excitotoxins that include glutamate and D-serine.

Effects of Schwann cell secreted factors on PC12 cell neuritogenesis and survival

We have used PC12 cells to examine the effects of factors secreted by Schwann cells that promote cell survival and neurite outgrowth, and hence are likely candidates for promoting neuronal regeneration. RT-PCR showed that primary Schwann cells produced a range of neurotrophins, excluding NT3, but this profile was different from either of two cell lines SCTM41 or PVGSCSV40T, or forskolin-expanded Schwann cells. The effects of Schwann cell conditioned media on neurite outgrowth was tested against a range of factors, and showed clear neuritogenic effects. Of the factors tested, only NGF had a significant response on neuritogenesis. Western blotting for neurofilaments showed that primary Schwann cells induced a strong response close to that of NGF. The Trk tyrosine kinase inhibitor K252a did not block the neuritogenic effects of primary Schwann cells. In contrast, K252a blocked both NGF and the SCTM41 cell effects. Schwann cell conditioned media also enhanced PC12 cell survival. Again, in contrast with NGF or SCTM41 cells, the primary Schwann cell effect was Trk tyrosine kinase independent. The Schwann cell conditioned medium contains a protein factor (greater than 12 kDa and broken down by trypsin treatment) with remarkable thermal stability (unaffected at 95 degrees C for 15 min) and the ability to bind heparin. Our results provide clear evidence that Schwann cells produce factors other than those already known to stimulate a neural phenotype in PC12 cells, and which thus have potential regeneration enhancing effects.

Differential Effects of Neurotrophins and Schwann Cell-Derived Signals on Neuronal Survival/Growth and Synaptogenesis

Recent studies have shown that the survival of mammalian motoneurons in vitro is promoted by neurotrophins (NTs) and cAMP. There is also evidence that neurotrophins enhance transmitter release. We thus investigated whether these agents also promote synaptogenesis. Cultured Xenopus spinal cord neurons were treated with a mixture of BDNF, glia-derived neurotrophic factor, NT-3, and NT-4, in addition to forskolin and IBMX or the cell-permeant form of cAMP, to elevate the cAMP level. The outgrowth and survival of neurons were dramatically increased by this trophic stimulation. However, when these neurons were cocultured with muscle cells, the trophic agents resulted in a failure of synaptogenesis. Specifically, the induction of ACh receptor (AChR) clustering in cultured muscle cells was inhibited at nerve-muscle contacts, in sharp contrast to control, untreated cocultures. Because AChR clustering induced by agrin or growth factor-coated beads in muscle cells was unaffected by trophic stimulation, its effect on synaptogenesis is presynaptic in origin. In the control, agrin was deposited along the neurite and at nerve-muscle contacts. This was significantly downregulated in cultures treated with trophic stimuli. Reverse transcriptase-PCR analyses showed that this decrease in agrin deposition was caused by an inhibition of agrin synthesis by trophic stimuli. Both agrin synthesis and induction of AChR clustering were restored under trophic stimulation when Schwann cell-conditioned medium was introduced. These results suggest that trophic stimulation maintains spinal neurons in the growth state, and Schwann cell-derived factors allow them to switch to the synaptogenic state.

Denervated Schwann cells attract macrophages by secretion of leukemia inhibitory factor (LIF) and monocyte chemoattractant protein-1 in a process regulated by interleukin-6 and LIF.

Injury to peripheral nerves results in the infiltration of immune cells, which remove axonal- and myelin-derived material. Schwann cells could play a key role in this process by regulating macrophage infiltration. We show here that medium conditioned by primary denervated Schwann cells or the Schwannoma cell line RN22 produces chemotactic activity for macrophages. The presence of blocking antibodies to macrophage chemoattractant protein-1 (MCP-1) or leukemia inhibitory factor (LIF) reduced this activity to approximately 35 and 65% of control levels, respectively, and only 15% remained in the presence of both antibodies. The presence of chemotactic LIF in Schwann cell-conditioned medium was confirmed by using cells from lif-/- mice. Although interleukin-6 (IL-6) is not itself a chemotactic factor, we found that medium from il-6-/- nerves showed only 40% of the activity secreted by wild-type nerves. Furthermore, IL-6 rapidly induced LIF mRNA in primary Schwann cells, and LIF rapidly induced MCP-1 mRNA expression. Treatment of RN22 Schwannoma cells with IL-6 or LIF enhanced the secretion of the chemotactic activity of these cells. These observations show that Schwann cells attract macrophages by secreting MCP-1 and LIF. They also provide evidence for an autocrine-signaling cascade involving IL-6, LIF, and MCP-1, which amplifies the Schwann cell-derived chemotactic signals gradually, in agreement with the delayed entry of macrophages to injured nerves.

Denervated Schwann Cells Attract Macrophages by Secretion of Leukemia Inhibitory Factor (LIF) and Monocyte Chemoattractant Protein-1 in a Process Regulated by Interleukin-6 and LIF

Injury to peripheral nerves results in the infiltration of immune cells, which remove axonal- and myelin-derived material. Schwann cells could play a key role in this process by regulating macrophage infiltration. We show here that medium conditioned by primary denervated Schwann cells or the Schwannoma cell line RN22 produces chemotactic activity for macrophages. The presence of blocking antibodies to macrophage chemoattractant protein-1 (MCP-1) or leukemia inhibitory factor (LIF) reduced this activity to ∼35 and 65% of control levels, respectively, and only 15% remained in the presence of both antibodies. The presence of chemotactic LIF in Schwann cell-conditioned medium was confirmed by using cells from<i>lif−/−</i> mice. Although interleukin-6 (IL-6) is not itself a chemotactic factor, we found that medium from<i>il-6−/−</i> nerves showed only 40% of the activity secreted by wild-type nerves. Furthermore, IL-6 rapidly induced LIF mRNA in primary Schwann cells, and LIF rapidly induced MCP-1 mRNA expression. Treatment of RN22 Schwannoma cells with IL-6 or LIF enhanced the secretion of the chemotactic activity of these cells. These observations show that Schwann cells attract macrophages by secreting MCP-1 and LIF. They also provide evidence for an autocrine-signaling cascade involving IL-6, LIF, and MCP-1, which amplifies the Schwann cell-derived chemotactic signals gradually, in agreement with the delayed entry of macrophages to injured nerves.