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Toll-like receptors comprise a family of evolutionary conserved pattern recognition receptors that act as a first defense line in the innate immune system. Upon stimulation with microbial ligands, they orchestrate the induction of a host defense response by activating different signaling cascades. Interestingly, they appear to detect the presence of endogenous signals of danger as well and as such, neurodegeneration is thought to trigger an immune response through ligation of TLRs. Though recent data report the expression of various TLRs in the central nervous system, TLR expression patterns in the peripheral nervous system have not been determined yet. We observed that Schwann cells express relatively high levels of TLRs, with especially TLR3 and TLR4 being prominent. Sensory and motor neurons hardly express TLRs at all. Through the use of NF-κB signaling as read-out, we could show that all TLRs are functional in Schwann cells and that bacterial lipoprotein, a ligand for TLR1/TLR2 receptors yields the strongest response. In sciatic nerve, basal levels of TLRs closely reflect the expression patterns as determined in Schwann cells. TLR3, TLR4, and TLR7 are majorly expressed, pointing to their possible role in immune surveillance. Upon axotomy, TLR1 becomes strongly induced, while most other TLR expression levels remain unaffected. Altogether, our data suggest that similar to microglia in the brain, Schwann cells might act as sentinel cells in the PNS. Furthermore, acute neurodegeneration induces a shift in TLR expression pattern, most likely illustrating specialized functions of TLRs in basal versus activated conditions of the peripheral nerve.

Schwann cells are the myelinating glia cells of the peripheral nervous system (PNS) and can become targets of an autoimmune response in inflammatory neuropathies like the Guillain-Barré syndrome (GBS). Professional antigen presenting cells (APCs) are known to promote autoimmune responses in target tissues by presenting self-antigens. Other cell types could participate in local autoimmune responses by acting as nonprofessional APCs. Using a combined approach of immunocytochemistry, immunohistochemistry, and flow cytometry analysis we demonstrate that human Schwann cells express the antigen processing and presenting machinery (APM) in vitro and in vivo. Moreover, cultured human Schwann cells increase the expression of proteasome subunit delta (Y), antigen peptide transporter TAP2, and HLA Class I and HLA Class II complexes in an inflammatory environment. In correlation with this observation, Schwann cells in sural nerve biopsies from GBS patients show increased expression of antigen processing and presenting molecules. Furthermore, cultured human Schwann cells can proteolytically digest fluorescently-labeled nonmammalian antigen ovalbumin. Taken together, our data suggest antigen processing and presentation as a possible function of Schwann cells that may contribute to (auto)immune responses within peripheral nerves., ((c) 2009 Wiley-Liss, Inc.)

The past 15 years have witnessed the identification of more than 25 genes responsible for inherited neuropathies in humans, many associated with primary alterations of the myelin sheath. A remarkable body of work in patients, as well as animal and cellular models, has defined the clinical and molecular genetics of these illnesses and shed light on how mutations in associated genes produce the heterogeneity of dysmyelinating and demyelinating phenotypes. Here, we review selected recent developments from work on the molecular mechanisms of these disorders and their implications for treatment strategies.

In this article we first discuss the factors that regulate macrophage recruitment, activation, and myelin phagocytosis during Wallerian degeneration and some of the factors involved in the termination of inflammation at the end of the period of Wallerian degeneration after peripheral nerve injuries. In particular, we deal with the early events that trigger chemokine and cytokine expression; the role of phospholipase A(2) in initiating the breakdown of compact myelin, and chemokine, cytokine expression; and the role of MCP-1, MIP-1alpha, and IL-1beta in macrophage recruitment and myelin phagocytosis. We also discuss how inflammation may be switched off and the recently identified role of the Nogo receptor on activated macrophages in the clearance of these cells from the injured nerve. In the second half of the article we focus on the role of certain Schwann cell borne cytokines and chemokines, such as M-CSF and MCP-1 as well as intracellular signaling that regulate their expression in animal models of inherited demyelinating disease. Additionally, we present the preservation of sensory nerves fibers from macrophage attack in these animal models as a challenging paradigm for the development of putative treatment approaches. Finally, we also discuss the similarities and differences in these Schwann cell-macrophage responses in injury-induced Wallerian degeneration and inherited demyelinating diseases. Knowledge of the molecular mechanisms underlying Schwann cell-macrophage interaction under pathological conditions is an important prerequisite to develop effective treatment strategies for various peripheral nerve disorders.

Apolipoprotein E (apoE) has immunomodulatory properties and has been implicated in the pathogenic mechanism of autoimmune diseases. Previously, the authors found that apoE deficiency increased the susceptibility to experimental autoimmune neuritis (EAN), an animal model for human Guillain-Barré syndrome. To further elucidate the mechanism behind apoE deficiency exacerbating EAN, the authors investigated the role of major target and important antigen-presenting cells of the peripheral nerve system, Schwann cells (SCs), in apoE knockout mice. Treatment of apoE deficient SCs with recombinant mouse interferon-gamma and lipopolysaccharide resulted in higher MHC-II and CD40 expression as compared with normal SCs derived from wild-type mice. The increased MHC-II and CD40 expression on SCs was accompanied by lower levels of intracellular IL-6 production within SCs of apoE deficiency, which is confirmed by the neutralization with anti IL-6 antibody. The increased antigen-presenting capacity of apoE deficient SCs was further explored by enhancement of T cell proliferation co-cultured with P0 peptide 180-199 specific T cells derived from EAN mice immunized with the P0 peptide. In conclusion, apoE may protect mice from EAN and probably also from chronic inflammatory demyelinating polyneuropathy by affecting the antigen-presenting function of SCs via influence of IL-6 production., ((c) 2007 Wiley-Liss, Inc.)

It was investigated whether retinoic acid (RA) and the proinflammatory cytokines IL-1beta, IL-6, and TNFalpha influence the intracellular distribution of retinoic acid receptors (RAR) and retinoid X receptors (RXR) in Schwann cells. This question arose because nuclear translocation of RARalpha, RXRalpha, and RXRbeta was observed after nerve injury, and because mutual interactions exist between the signal transduction pathways of RA and proinflammatory cytokines. Schwann cell primary cultures from the rat sciatic nerve were incubated with IL-1beta, IL-6, and TNFalpha, with all-trans RA and with a combination of IL-1beta and RA. After incubation periods ranging from 5 min to 5 h, the intracellular distributions of RARalpha, RARbeta, RXRalpha, and RXRbeta were analyzed. All three cytokines caused a shift of RARalpha from the cytosolic compartments into the cell nuclei. This was also observed with RA, and combining RA with IL-1beta produced an additive effect. IL-1beta and IL-6 also affected the distribution of RARbeta, although immunoreactivity of this receptor always remained stronger in the cytosol. No effect of the cytokines on RXRalpha or RXRbeta was observed, whereas RA treatment caused a stronger nuclear signal of both receptors. Effects on the subcellular localization of retinoid receptors may provide a link in a feedback loop between RA/RAR and cytokines.

The human paralytic neuropathy, Miller Fisher syndrome (MFS) is associated with autoantibodies specific for disialosyl epitopes on gangliosides GQ1b, GT1a, and GD3. Since these gangliosides are enriched in synaptic membranes, anti-ganglioside antibodies may target neuromuscular junctions (NMJs), thereby contributing to disease symptoms. We have shown previously that at murine NMJs, anti-disialosyl antibodies induce an alpha-latrotoxin-like effect, electrophysiologically characterized by transient massive increase of spontaneous neurotransmitter release followed by block of evoked release, resulting in paralysis of the muscle preparation. Morphologically, motor nerve terminal damage, as well as perisynaptic Schwann cell (pSC) death is observed. The relative contributions of neuronal and pSC injury to the paralytic effect and subsequent repair are unknown. In this study, we have examined the ability of subsets of anti-disialosyl antibodies to discriminate between the neuronal and glial elements of the NMJ and thereby induce either neuronal injury or pSC death. Most antibodies reactive with GD3 induced pSC death, whereas antibody reactivity with GT1a correlated with the extent of nerve terminal injury. Motor nerve terminal injury resulted in massive uncontrolled exocytosis with paralysis. However, pSC ablation induced no acute (within 1 h) electrophysiological or morphological changes to the underlying nerve terminal. These data suggest that at mammalian NMJs, acute pSC injury or ablation has no major deleterious influence on synapse function. Our studies provide evidence for highly selective targeting of mammalian NMJ membranes, based on ganglioside composition, that can be exploited for examining axonal-glial interactions both in disease states and in normal NMJ homeostasis., ((c) 2005 Wiley-Liss, Inc.)

Schwann cells are best known as myelinating glial cells of the peripheral nervous system, but they also participate actively in the sphere of immunity by producing pro-inflammatory cytokines, such as interleukin-1beta (IL-1beta). In a previous study, we demonstrated that posttranslational processing of IL-1beta by immune-challenged Schwann cells required the P2X7 receptor. Remarkably, the release of IL-1beta was not associated with cell death, indicating the involvement of an active mechanism. ATP binding cassette (ABC) transporters are known to transport leaderless secretory proteins, such as IL-1beta; therefore, we investigated whether such transporters were at work in Schwann cells. Mouse Schwann cells expressed ABC1 transporter mRNA and displayed the functional protein. Glybenclamide and diisothiocyanato-stilbene-disulfonic acid (DIDS), two blockers of chloride fluxes that drive the export activity of ABC1 transporters, inhibited IL-1beta release without altering its intracellular processing. Enhancing chloride efflux potentiated the release of IL-1beta, while decreasing it led to a strong reduction in its release. Because the stimulation of the P2X7 receptor also activates a chloride conductance, we investigated the possibility of a sole anionic pathway mobilized by the P2X7 receptor and ABC1. Glybenclamide and DIDS had no significant effects on the P2X7-activated chloride current suggesting therefore the existence of two different pathways. In summary, ABC1 transporters are required for the release of IL-1beta by mouse Schwann cells. Being associated together with chloride conductance, P2X7 receptors and ABC1 transporters delineate a subtle and complex regulation of IL-1beta production in mammalian Schwann cells. Furthermore, ABC1 transporters could be a target of therapeutic interest for regulating IL-1beta activity in neuroinflammation disorders.

The monoclonal antibody 4C5 recognizes a cell surface antigen of the developing central nervous system (CNS) and peripheral nervous system (PNS). In vitro antibody perturbation experiments have shown that the 4C5 antigen is involved in horizontal and vertical migration processes of granule cells during development of the rodent cerebellum. Moreover, results concerning the cellular localization and temporal expression of the 4C5 antigen during development and after injury of the rat sciatic nerve suggested that it may participate in Schwann cell migrations that occur during the above processes. To test this possibility, we examined the effects of our function-blocking antibody on Schwann cell migration in three in vitro bioassays: in tissue cultures from developing sciatic nerve, in dorsal root ganglion cultures on cryostat sections of normal or denervated adult sciatic nerve, and in pure Schwann cell cultures. The results showed that the presence of monoclonal antibody 4C5 in all the above culture systems strongly inhibited Schwann cell migration, indicating that the 4C5 antigen participates in migration processes that take place during development and regeneration of the peripheral nervous system. Moreover, staining of migrating Schwann cells in the presence of monoclonal antibody 4C5 with rhodamine-phalloidin showed that 4C5 antigen activity is associated with actin cytoskeletal organization of these cells, and more specifically with lamellipodia formation., (Copyright 2003 Wiley-Liss, Inc.)

The ability of resident cells to induce apoptosis of invading immune cells is a major regulatory factor operating in immune-privileged tissues, including the nervous system. We investigated the cellular and molecular factors participating in modulation of immune response in peripheral nerves, focusing on two cytotoxic pathways: fas ligand (fasL) and perforin. fasL and perforin expression was found by immunochemistry on Schwann cells (Sc) in nerve biopsies from patients with chronic inflammatory demyelinating polyneuritis and on human Sc cultures. Treatment of Sc with tumor necrosis factor (TNF) alpha and interferon (IFN) gamma upregulated the expression of both molecules. In a coculture model, Sc exposed to TNFalpha or IFN gamma were able to induce both apoptotic and lytic injury of T-lymphocytes. Inactivation of fasL with the neutralizing antibody NOK-2 abolished T-cell apoptosis induced by Sc treated with TNFalpha, but not by Sc treated with IFN gamma. Conversely, T-cell lysis was significantly decreased when IFN gamma-activated Sc were treated with concanamycin A, which inhibited perforin release. At variance with T-lymphocytes, B-cells were less sensitive to cytokine-treated Sc toxicity. Thus, Sc exposed to inflammatory cytokines have the capacity of inducing selective damage of T-lymphocytes and have the potential of regulating the immune response in the peripheral nervous system., (Copyright 2003 Wiley-Liss, Inc.)

Schwann cells differentiate in vivo in response to contact with axons, and cAMP simulates some of these aspects of differentiation in vitro, particularly morphologic changes and expression of certain phenotypic molecules. Unfractionated inflammatory cytokines inhibit cAMP-induced Schwann cell expression of galactolipids (Gal). We sought to identify which cytokines were responsible for this inhibition and to determine whether other phenotypic indicators of Schwann cell differentiation were also affected. Neonatal rat Schwann cells were incubated in vitro with 1 mM 8 Bromo cAMP (8 Br cAMP) with or without the addition of interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-2, IL-6, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), or transforming growth factor-beta (TGF-beta). Cells were then examined for morphologic changes and for expression of surface Gal and low-affinity nerve growth factor receptor (NGFRp75), employing indirect immunofluorescence. 8 Br cAMP induced Schwann cell upregulation of Gal, downregulation of NGFRp75, and the cells became enlarged and somewhat amorphous and irregular in appearance. Cells treated with IFN-gamma or TNF-alpha alone were more bipolar and more evenly distributed on coverslips than were control cells, whereas TGF-beta alone induced elongated cells often in a swirling pattern. None of the cytokines alone induced upregulation of Gal or downregulation of NGFRp75. TNF-alpha, IFN-gamma, and TGF-beta inhibited the 8 Br cAMP-induced morphologic changes, as well as the upregulation of Gal and downregulation of NGFRp75. The other cytokines had no effects on Gal or NGFRp75 expression. Thus, these three cytokines, which are present in inflammatory lesions in the peripheral nervous system, are capable of inhibiting Schwann cell differentiation., (Copyright 2001 Wiley-Liss, Inc.)

Oligodendrocytes and Schwann cells are the glia principally responsible for the synthesis and maintenance of myelin. Damage may occur to these cells in a number of conditions, but perhaps the most studied are the idiopathic inflammatory demyelinating diseases, multiple sclerosis in the CNS, and Guillain-Barré syndrome and its variants in the peripheral nervous system (PNS). This article explores the effects on these cells of cytotoxic immunological and inflammatory mediators: similarities are revealed, of which perhaps the most important is the sensitivity of both Schwann cells and oligodendrocytes to many such agents. This area of research is, however, characterised and complicated by numerous and often very substantial inter-observer discrepancies. Marked variability in cell culture techniques, and in assays of cell damage and death, provide artifactual explanations for some of this variability; true inter-species differences also contribute. Not the least important conclusion centres on the limited capacity of in vitro studies to reveal disease mechanisms: cell culture findings merely illustrate possibilities which must then be tested ex vivo using human tissue samples affected by the relevant disease., (Copyright 2001 Wiley-Liss, Inc.)

An association between macrophages and remyelination efficiency has been observed in a variety of different models of CNS demyelination. In order to test whether this association is causal or coincidental, we have examined the effects of macrophage depletion on the rate of remyelination of lysolecithin-induced demyelination in the spinal cord of young adult female rats. Macrophage depletion was achieved by reducing the monocyte contribution to the macrophages within the lesion using the clodronate-liposome technique. This technique not only resulted in a decrease in Ox-42-positive cells in the spleen of treated animals but also in the levels of macrophage scavenger receptor type B mRNA expression within the demyelinating lesion. In animals treated with clodronate-liposomes throughout the remyelination process, there was a significant decrease in the extent of oligodendrocyte remyelination at 3 weeks after lesion induction, but no effect on Schwann cell remyelination. If macrophage depletion was delayed until the second half of the remyelination phase, then there was no effect on the repair outcome, implying that macrophages are required for the early stages of CNS remyelination. The results of this study indicate that the macrophage response is an important component of successful CNS remyelination and that approaches to the treatment of demyelinating disease based on inhibition of the inflammatory response may also impair regenerative events that follow demyelination., (Copyright 2001 Wiley-Liss, Inc.)

A duplication of the gene for myelin protein PMP22 is by far the most common cause of the hereditary demyelinating neuropathy CMT1A. A role for PMP22 in cell growth in addition to its function as a myelin protein has been suggested because PMP22 is homologous to a gene specifically upregulated during growth arrest. Furthermore, transfected rat Schwann cells overexpressing PMP22 show reduced growth. In addition, abnormal Schwann cell differentiation has been described in nerve biopsies from CMT1A patients. To analyse whether the duplication of the PMP22 gene in CMT1A neuropathy primarily alters Schwann cell differentiation and to exclude nonspecific secondary responses, we improved human Schwann cell culturing. This allowed us long-term passaging of human Schwann cells with unchanged phenotype, assessed by expression of different Schwann cell markers. Subsequently we established Schwann cell cultures from CMT1A nerve biopsies. We find decreased proliferation of Schwann cells from different CMT1A patients in all passages. We also demonstrate PMP22 mRNA overexpression in cultured CMT1A Schwann cells. We conclude that decreased proliferation in cultured Schwann cells that carry the CMT1A duplication indicates abnormal differentiation of CMT1A Schwann cells. The identification of an abnormal phenotype of CMT1A Schwann cells in culture could possibly lead to an in vitro disease model.

Previous studies have shown that Schwann cell implantation offers a potential therapeutic approach to a variety of neurodegenerative disorders and traumatic injuries. In a clinically relevant paradigm, however, the implantation of autologous Schwann cells is problematic and the use of heterogenetic Schwann cells will be required. In the present study we addressed this important issue and analysed the immunogenicity and survival of allogeneic and xenogeneic Schwann cell suspension grafts in a prelesioned CNS fiber tract, the transected postcommissural fornix of the adult Wistar rat. Cultured Schwann cells from Wistar rat or human peripheral nerve were injected either immediately or after a delay into the transection site and the spatio-temporal pattern of leukocyte infiltration and of major histocompatibility antigen expression was characterized and semiquantified with immunocytochemical methods. Our main findings are that (1) invasive cerebral lesions induce the expression of MHC class I and II antigens, but only sparse infiltration of T-lymphocytes, (2) both allogeneic and xenogeneic discordant Schwann cell suspension grafts, from either neonatal or adult peripheral nerve, survive without any overt signs of rejection for up to 10 weeks after implantation; and (3) delayed implantation procedures have no effect on immune responses to allogeneic Schwann cell grafts. These results demonstrate that there is no marked ongoing immune reactions to heterogenetic Schwann cell suspension grafts and that long-term survival of cross-species Schwann cell grafts can be achieved in the absence of any immunsuppressive treatment. Thus the conditions for functional transplantation of Schwann cells across immunological barriers seem to be favourable and will have implications for future cross-species studies, and possibly also for clinical application.

In an attempt to identify and characterize novel Schwann cell surface molecules with putative functions during development, maintenance, and regeneration of the peripheral nervous system (PNS), we have produced monoclonal antibodies against viable neonatal rat Schwann cells. Using a sensitive live cell ELISA protocol, three monoclonal antibodies reactive with cultured Schwann cells, designated 27B10, 26F2, and 27C7 were isolated. The 27B10 and 26F2 antibodies specifically labelled forskolin-stimulated secondary Schwann cells in vitro as determined by live cell ELISA implying that the expression of the antigens in situ is regulated by axonal contact. The observation that the antigens seemed to be associated with both Schwann cell phenotypes clearly discriminated them from the well characterized myelin proteins as well as from molecules known to be confined to the non-myelin-forming phenotype. Interestingly, both antigens were found to be concentrated at the nodes of Ranvier. Further studies therefore have to show whether the identified antigens share structural or functional homology with adhesion or channel molecules, which display a similar distribution. Following transection of the adult sciatic nerve, the 26F2 antigen was rapidly down-regulated in the distal nerve stump. The 27C7 antibody reacted with an 80 kDa cell surface molecule common to non-myelin-forming Schwann cells. No differences in expression of the antigen between forskolin-treated and untreated Schwann cells in vitro were found, suggesting that the antigen is expressed independently from axonal contact. Two weeks after nerve transection in the absence of myelinating Schwann cells, the antigen was associated with S-100-positive Schwann cells of the distal nerve stump. The antigen was found to be expressed also by non-neuronal tissues, the level of the protein declined towards the adult stage. Comparison of the 27C7 antigen with previously described marker molecules suggests that we have identified a novel Schwann cell surface antigen of the non-myelin-forming phenotype.

We have examined both how the molecular phenotype of Schwann cells in vitro is regulated by transforming growth factor beta (TGF-beta), using immunohistochemistry and immunoblotting, and the distribution of TGF-beta 2 and 3 in embryonic and mature nerves and ganglia, using immunohistochemistry and in situ hybridisation. We find that TGF-beta 2 and -3 upregulate expression of the neural cell adhesion molecules NCAM and L1. In TGF-beta-treated cultures, in addition to the 140 and 120 kD isoforms known to be present in Schwann cells, small amounts of the 180 kD isoform can be detected. TGF-beta s also block cAMP-induced expression of the lipid antigens galactocerebroside (GalC) and O4, in addition to blocking expression of protein zero (P0), the major peripheral myelin glycoprotein, as previously shown. Using antibodies specific to TGF-beta 2 and -3, respectively, we confirm the presence of these proteins in myelin-forming Schwann cells and show also that TGF-beta 2 and -3 are clearly expressed by peripheral glia that are not involved in myelination. This includes Schwann cell precursors, embryonic Schwann cells, non-myelin-forming Schwann cells and satellite cells from adult nerves and ganglia, and neonatal Schwann cells in purified cultures without neurones. In situ hybridisation with a digoxygenin-labelled riboprobe reveals a strong TGF-beta 3 mRNA signal in Schwann cells, satellite cells, and some neurones. Schwann cells in culture also secrete TGF-beta in a latent form, whereas purified cultures of dorsal root ganglion neurones from 1-day-old rats secrete active TGF beta during the first 48 h in culture.