Mechanism of phagocytosis by Schwann cells

33B rat Schwannoma cell line is known to exhibit phagocytic properties analogous to those of normal Schwann cells. The mechanism of phagocytosis by this cell line was investigated by studying the effect of known modulators of phagocytosis on the uptake of latex particles by these cells. Treatments which block energy production of the host cell, such as incubation at 4 degrees C and treatment with sodium azide, markedly inhibited the phagocytosis of latex particles by these cells. Phagocytosis was dose-dependently, and completely, inhibited by cytochalasin B, demonstrating an important role of microfilaments. Colchicine produced a minor inhibition of phagocytosis only at the highest concentration (10(-3) M) tested, suggesting that intact microtubules are not crucial for latex phagocytosis. Dibutyryl cyclic AMP was without any effect on the phagocytosis. Thus, latex phagocytosis by rat Schwannoma cells is an active, energy-dependent process requiring intact microfilaments with only a minor dependence on microtubules.     

雪旺氏细胞促进共培养大鼠胚胎神经干细胞的分化

目的 探讨诱导神经干细胞分化的因素。方法 新生大鼠雪旺氏细胞与大鼠胚胎神经干细胞在无血清培养液中共培养，观察神经干细胞的形态变化及分别检测特异性标志蛋白tubulin-β、GalC和GAFP的表达。结果 相差显微镜下大部分神经干细胞伸出多个细长突起，且免疫莹光染色tubulin-β高度表达；少数细胞为有多个短小突起的小细胞，且GalC或GFAP阳性表达。结论 雪旺氏细胞与大鼠胚胎神经干细胞共培养可诱导神经干细胞分化。     

The immunocompetence of Schwann cells

Schwann cells are the myelinating glial cells of the peripheral nervous system that support and ensheath axons with myelin to enable rapid saltatory signal propagation in the axon. Immunocompetence, however, has only recently been recognized as an important feature of Schwann cells. An autoimmune response against components of the peripheral nervous system triggers disabling inflammatory neuropathies in patients and corresponding animal models. The immune system participates in nerve damage and disease manifestation even in non-inflammatory hereditary neuropathies. A growing body of evidence suggests that Schwann cells may modulate local immune responses by recognizing and presenting antigens and may also influence and terminate nerve inflammation by secreting cytokines. This review summarizes current knowledge on the interaction of Schwann cells with the immune system, which is involved in diseases of the peripheral nervous system.     

Signals for proinflammatory cytokine secretion by human Schwann cells

Wallerian degeneration is a post-traumatic process of the peripheral nervous system whereby damaged axons and their surrounding myelin sheaths are phagocytosed by infiltrating leukocytes. Our studies indicate that Schwann cells could initiate the process of Wallerian degeneration by releasing proinflammatory cytokines involved in leukocyte recruitment and differentiation including IL-1beta, MCP-1, IL-8 and IL-6. A comparison of the secretory pattern between nerve explants and cultured Schwann cells showed that each cytokine was differentially regulated by growth factor deprivation or axonal membrane fragments. Since Wallerian-like degeneration occurs in a wide variety of peripheral neuropathies, Schwann cell-mediated cytokine production may play an important role in many disease processes.     

GABA uptake by purified rat Schwann cells in culture

Purified rat Schwann cells maintained in culture for up to 6 months retained their ability to take up the neurotransmitter γ-aminobutyric acid (GABA) by a high-affinity mechanism. Although cultured fibroblasts also accumulated GABA, they did so by a low affinity mechanism. These results indicate that Schwann cells continue to express a high affinity GABA transport system in the absence of signals from neurons.     

Autoradiographic and ultrastructural studies of areas of spinal cord occupied by Schwann cells and Schwann cell myelin

Schwann cells, peripheral-type myelin and connective tissue elements develop within the dorsal portion of the X-irradiated spinal cord in immature rats. Factors controlling the distribution of these elements within the irradiated site are not fully understood. In the present study [³H]thymidine autoradiography was used to examine proliferative activities of cells in these areas occupied by peripheral nervous system components, and correlative ultrastructural evaluations were made. At 15 and 20 days post-irradiation (P-I), the Schwann cells occupied the dorsolateral portions of the dorsal funiculi, and heavily labeled cells occurred throughout these areas. By 25 days P-I the Schwann cells extended ventrally into the depths of the dorsal funiculi and into the dorsal gray matter, and labeled cells were concentrated in the deeper portions of these areas. Ultrastructurally, the Schwann cells and peripheral-type myelin were more mature in the superficial portions where proliferative activity was diminished. In contrast, much less mature, peripheral-type myelin occurred in the depths where the labeled cells were concentrated. At 30 and 45 days P-I, labeled cells were much less frequent but usually occurred in the depths when observed. Similarly, a dorsal-ventral gradient in maturity of peripheral-type myelin was evident ultrastructurally. By 60 and 90 days P-I, labeling was rare, and mature Schwann cell myelin was present throughout the areas. Astrocytes and their processes were less numerous in regions invaded by Schwann cells, as compared to controls, and studies are in progress to evaluate the relationships between these glial elements and intraspinal peripheral nervous system components.     

Olfactory ensheathing cells promote migration of Schwann cells by secreted nerve growth factor

Transplantation of Schwann cells (SCs) and olfactory ensheathing cells (OECs) have emerged as very promising therapies for spinal cord repair. The important features of interaction between SCs and OECs are beginning to be appreciated, although the underlying mechanism remains unclear. In the present study, we tested the effects of OECs on SCs migration using a range of in vitro migration assays. We found that SCs migrated abundantly upon OECs monolayer, and the migration-promoting effects were identified to be due to the secreted diffusible factors in OEC-derived conditioned medium (OEC-CM). Furthermore, neutralizing nerve growth factor (NGF) in OEC-CM with NGF antibody could block this effect. Moreover, we found that NGF promotes SCs migration even on astrocyte monolayer. Taken together, these findings provide the first evidence that OECs can promote SCs migration in astrocytic environment by secreted NGF.     

改良的酶消化后植块法体外培养雪旺细胞

目的 观察改良的酶(浓度为2g/L的胶原酶)消化后植块法体外培养雪旺细胞(SCs)效果,探讨高效获取高纯度、高活性SCs的培养纯化方法.方法 取新生SD大鼠坐骨神经,剥离神经外膜,剪块,分别采用单酶消化后植块法与双酶消化法进行培养.以活细胞计数和S-100单抗标记相结合判断SCs增殖和纯化程度,比较2种方法的优劣.结果 改良的单酶消化后植块法可获取3.5x106个SCs,成活率为96%,纯度达94%以上:双酶消化法约获取3.0×106个SCs,成活率为92%,纯度达90%.结论 采用改良的酶消化后植块法培养SCs,可较快得到数量多、纯度高的SCs,符合实验要求,值得推广应用.

Abstract：

Objective To introduce an efficient method for culturing and purifying Schwann cells (SCs) in vitro. Methods Sciatic nerves were harvested from neonatal SD rats and the epineuria were removed. Single enzyme digestion combined with explans and double enzyme digestion were employed, respectively, to digest the nerve tissues following trituration. The proliferation of SCs and degree of purification were evaluated by viable count method and the combination of S-100 labeled monoclonal antibody and SCs. Results Proximally 3.5 × 106 cells were harvested with 96% survival rate and a purity of Schwann cells over 94% by single enzyme digestion combined with explans, however only 3.0×106 cells were gained with the purity being 90% and survival rate being 92% by double enzyme digestion. Conclusion This method yields large amount of viable Schwann cells with high purity and survival rate.     

改良的酶消化后植块法体外培养雪旺细胞

Objective To introduce an efficient method for culturing and purifying Schwann cells (SCs) in vitro. Methods Sciatic nerves were harvested from neonatal SD rats and the epineuria were removed. Single enzyme digestion combined with explans and double enzyme digestion were employed, respectively, to digest the nerve tissues following trituration. The proliferation of SCs and degree of purification were evaluated by viable count method and the combination of S-100 labeled monoclonal antibody and SCs. Results Proximally 3.5 × 106 cells were harvested with 96% survival rate and a purity of Schwann cells over 94% by single enzyme digestion combined with explans, however only 3.0×106 cells were gained with the purity being 90% and survival rate being 92% by double enzyme digestion. Conclusion This method yields large amount of viable Schwann cells with high purity and survival rate.     

改良的酶消化后植块法体外培养雪旺细胞

Objective To introduce an efficient method for culturing and purifying Schwann cells (SCs) in vitro. Methods Sciatic nerves were harvested from neonatal SD rats and the epineuria were removed. Single enzyme digestion combined with explans and double enzyme digestion were employed, respectively, to digest the nerve tissues following trituration. The proliferation of SCs and degree of purification were evaluated by viable count method and the combination of S-100 labeled monoclonal antibody and SCs. Results Proximally 3.5 × 106 cells were harvested with 96% survival rate and a purity of Schwann cells over 94% by single enzyme digestion combined with explans, however only 3.0×106 cells were gained with the purity being 90% and survival rate being 92% by double enzyme digestion. Conclusion This method yields large amount of viable Schwann cells with high purity and survival rate.     

Myelination by Schwann cells in the absence of extracellular matrix assembly

Assembly of extracellular collagen fibrils and Schwann cell basal lamina has previously been identified as a prerequisite for compact myelin formation in the peripheral nervous system. Synthesis of this extracellular matrix (ECM) in vitro required the presence of serum and ascorbic acid. Using rat embryonic dorsal root ganglion neurons and Schwann cells, we have developed a fully defined medium in which myelination occurs. In the absence of ascorbic acid, normal myelin was formed without ECM assembly. This demonstrates that although myelination and ECM assembly are usually closely linked, ECM formation is not a prerequisite for myelination in vitro. GLIA 23:383–388, 1998. © 1998 Wiley-Liss, Inc.     

大鼠雪旺氏细胞支持人胚胎神经干细胞的生长并诱导其分化

目的 探讨大鼠雪旺氏细胞对人胚胎神经干细胞生长和分化的作用。方法 实验分为三组：组一：干细胞生长于培养雪旺氏细胞1天后的培养液中；组二：干细胞与雪旺氏细胞共培养；组三：干细胞生长于DMEM／F12培养液中，观察干细胞的形态学变化和免疫荧光的.组一的神经球分化生长，绝大多数细胞tubulin-β阳性，少数为GalC或GFAP阳性；组二中，在雪旺氏细胞生长密集和稀少的地方，干细胞分别表明为不分化和明显分化两种状态；组三的神经干细胞无法正常生长。结论 大鼠雪旺氏细胞分泌物支持人胚胎神经干细胞的生长并诱导其分化。     

血清和雪旺氏细胞诱导大鼠胚胎神经干细胞分化的比较

目的 比较血清和雪旺氏细胞诱导大鼠胚胎神经干细胞分化的差异。方法 分别采用血清和与雪旺氏细胞共培养的方法诱导大鼠胚胎神经干细胞分化，应用相差显微镜和免疫荧光染色的方法对其进行观察和比较。结果 两种方法都能够诱导绝大多数神经干细胞分化成神经元，少量分化成星形胶质细胞和少突胶质细胞。虽然后一种方法诱导干细胞分化的进程比前一种方法要慢，但细胞形态学上更接近发育成熟的神经元。结论 雪旺氏细胞的分泌物不仅能够诱导共培养的神经干细胞分化，而且使其分化更加成熟。     

Neuron-glia signaling and the protection of axon function by Schwann cells

The interaction between neurons and glial cells is a feature of all higher nervous systems. In the vertebrate peripheral nervous system, Schwann cells ensheath and myelinate axons thereby allowing rapid saltatory conduction and ensuring axonal integrity. Recently, some of the key molecules in neuron–Schwann cell signaling have been identified. Neuregulin-1 (NRG1) type III presented on the axonal surface determines the myelination fate of axons and controls myelin sheath thickness. Recent observations suggest that NRG1 regulates myelination via the control of Schwann cell cholesterol biosynthesis. This concept is supported by the finding that high cholesterol levels in Schwann cells are a rate-limiting factor for myelin protein production and transport of the major myelin protein P0 from the endoplasmic reticulum into the growing myelin sheath. NRG1 type III activates ErbB receptors on the Schwann cell, which leads to an increase in intracellular PIP3 levels via the PI3-kinase pathway. Surprisingly, enforced elevation of PIP3 levels by inactivation of the phosphatase PTEN in developing and mature Schwann cells does not entirely mimic NRG1 type III stimulated myelin growth, but predominantly causes focal hypermyelination starting at Schmidt–Lanterman incisures and nodes of Ranvier. This indicates that the glial transduction of pro-myelinating signals has to be under tight and life-long control to preserve integrity of the myelinated axon. Understanding the cross talk between neurons and Schwann cells will help to further define the role of glia in preserving axonal integrity and to develop therapeutic strategies for peripheral neuropathies such as CMT1A.     

Conduction properties of central nerve fibers remyelinated by Schwann cells.

Demyelination of central axons arises from a number of conditions, including multiple sclerosis and spinal cord compression. The demyelination disrupts conduction and leads directly to the production of symptoms. Repair of the demyelination by peripheral myelinating cells could potentially relieve the symptoms, but the conduction properties of central axons remyelinated by Schwann cells have yet to be studied in detail. This paper examined the conduction properties of such axons. Large focal demyelinating and remyelinating lesions were induced in the dorsal columns of rats by the intraspinal injection of ethidium bromide. Recordings of compound action potentials conducted through these lesions were then made at various recovery times. Thus the changing conduction properties of the affected fibers could be correlated with the different stages of lesion development. During the early stages of demyelination there was widespread conduction block, with no evidence of appreciable conduction occurring with prolonged latency or refractory period of transmission (RPT). However, with the onset of remyelination by Schwann cells, conduction was restored in many axons, and most, if not all, of the affected axons eventually showed successful conduction through the lesion. Initially the conduction was characterized by very prolonged latency, long RPT, and an inability to conduct fast trains of impulses. These deficits became less prominent as remyelination progressed. In chronically remyelinated axons the RPT was restored to within normal limits, although some deficit in both conduction velocity and the ability to conduct trains of impulses persisted. Since these deficits were not severe we conclude that remyelination of central demyelinated axons by Schwann cells should be effective in promoting the restoration of normal function.     

Synthesis of progesterone in Schwann cells: regulation by sensory neurons

In peripheral nerves, progesterone synthesized by Schwann cells has been implicated in myelination. In spite of such an important function, little is known of the regulation of progesterone biosynthesis in the nervous system. We show here that in rat Schwann cells, expression of the 3 beta-hydroxysteroid dehydrogenase and formation of progesterone are dependent on neuronal signal. Levels of 3 beta-hydroxysteroid dehydrogenase mRNA and synthesis of [3H]progesterone from [3H]pregnenolone were low in purified Schwann cells prepared from neonatal rat sciatic nerves. However, when Schwann cells were cultured in contact with sensory neurons, both expression and activity of the 3 beta-hydroxysteroid dehydrogenase were induced. Regulation of 3 beta-hydroxysteroid dehydrogenase expression by neurons was also demonstrated in vivo in the rat sciatic nerve. 3 beta-hydroxysteroid dehydrogenase mRNA was present in the intact nerve, but could no longer be detected 3 or 6 days after cryolesion, when axons had degenerated. After 15 days, when Schwann cells made new contact with the regenerating axons, the enzyme was re-expressed. After nerve transection, which does not allow axonal regeneration, 3 beta-hydroxysteroid dehydrogenase mRNA remained undetectable. The regulation of 3 beta-hydroxysteroid dehydrogenase mRNA after lesion was similar to the regulation of myelin protein zero (P0) and peripheral myelin protein 22 (PMP22) mRNAs, supporting an important role of locally formed progesterone in myelination.     

Astrocytoma and Schwann cells in coculture

Glial fibrillary acidic protein (GFAP) is the principal intermediate filament protein found in mature astrocytes. Although the exact function of GFAP is poorly understood, it is presumed to stabilize the astrocyte’s cytoskeleton and help in maintaining cell shape. Previous studies from our laboratory have shown that when astrocytes were cocultured with primary Schwann cells (pSCs), astrocytes became hypertrophied and fibrous with intensely positive GFAP staining and segregated Schwann cells (SCs) into pockets. In order to understand the functional role of GFAP in this already established astrocyte-SC coculture model, we generated GFAP-negative cell lines from a GFAP-positive astrocytoma cell line and cocultured both the cell lines with pSCs. Our studies demonstrate that the GFAP-positive cell line put out processes toward the SCs, whereas the GFAP-negative cells did not form processes and the majority of the cells remained round. The most significant and interesting finding of this study, however, is the formation of elaborate processes by SCs when grown in coculture with the astrocytoma cells, unlike SCs cultured alone, which showed their typical bipolar spindle-shaped morphology. The extent of processes did not seem to be dependent on GFAP, since SCs cultured with both the cell lines formed similar processes. This coculture model may be useful in elucidating the factor(s) responsible for the formation of processes by SCs and can be further help in our understanding of the mechanism of morphological transformation of SCs.     

Schwann cells transplanted into the CNS

A small volume of purified Schwann cells, cultured from early postnatal rat sciatic nerve, was injected into the hippocampus or fimbria of syngeneic adult hosts. The procedure caused minimal structural disturbance at the transplantation site, with close graft-host contact and maximal opportunity for integration.The donor Schwann cells were identified by a combination of light and electron microscopic features (which include characteristic deep and complex infoldings of a well marked nuclear envelope), antigenic profile (especially low affinity nerve growth factor receptor immunoreactivity), uptake of fluorescent latex microspheres and autoradiography of [³H]thymidine-labelled dividing cells.The donor Schwann cells adopted a distinctive elongated form, with a central, ovoid nucleus flanked by processes which were up to 300 μm long, and which ranged from swollen segments with a diameter as large as 12 μm down to thread-like fibres of 1 μm or less with growth cone-like expansions. Transplanted cells migrated from the graft, particularly along blood vessels, and could permeate all cytoarchitectonic regions of the adjacent host hippocampal neuropil. Donor Schwann cells also migrated along the longitudinal axis of the fimbria, where they were interspersed in parallel with the interfascicular glial rows and axons.The grafted cells induced a transient but marked host astrocytic hypertrophy, which did not appear to impede the migration of the donor Schwann cells.The transplanted Schwann cells did not form peripheral myelin (as detected by P_o immunoreactivity), and it is not clear whether they survive beyond the period at which we detect them.     

Inhibition of the interaction of mycobacteria with Schwann cells by antimycobacterial antibodies

The effect of antimycobacterial serum on the uptake of Mycobacterium w by 33B rat Schwannoma cell line and rat peritoneal macrophages was studied. Incubation of Mycobacterium w with antimycobacterial serum caused a marked inhibition of its uptake by 33B cells but did not diminish its uptake by macrophages. These results suggest that the mechanism of the interaction of mycobacteria with Schwann cells differs from that with macrophages.