Damaged Neuronal Cells Induce Inflammatory Gene Expression in Schwann Cells: Implication in the Wallerian Degeneration

Lee, Hyun-Kyoung,Choi, Se-Young,Oh, Seog-Bae,Park, Kyung-Pyo,Kim, Joong-Soo,Lee, Sung-Joong The Korean Academy of Oral Biology 2006 International Journal of Oral Biology Vol.31 No.3

Schwann cells play an important role in peripheral nerve regeneration. Upon nerve injury, Schwann cells are activated and produce various proinflammatory mediators including IL-6, LIF and MCP-1, which result in the recruitment of macrophages and phagocytosis of myelin debris. However, it is unclear how the nerve injury induces Schwann cell activation. Recently, it was reported that necrotic cells induce immune cell activation via toll-like receptors (TLRs). This suggests that the TLRs expressed on Schwann cells may recognize nerve damage by binding to the endogenous ligands secreted by the damaged nerve, thereby inducing Schwann cell activation. To explore the possibility, we stimulated iSC, a rat Schwann cell line, with damaged neuronal cell extracts (DNCE). The stimulation of iSC with DNCE induced the expression of various inflammatory mediators including IL-6, LIF, MCP-1 and iNOS. Studies on the signaling pathway indicate that $NF-{\kappa}B$, p38 and JNK activation are required for the DNCE-induced inflammatory gene expression. Furthermore, treatment of either anti-TLR3 neutralizing antibody or ribonuclease inhibited the DNCE-induced proinflammatory gene expression in iSC. In summary, these results suggest that damaged neuronal cells induce inflammatory Schwann cell activation via TLR3, which might be involved in the Wallerian degeneration after a peripheral nerve injury.

Double-stranded RNA Induces Inflammatory Gene Expression in Schwann Cells: Implication in the Wallerian Degeneration

Lee, Hyun-Kyoung,Park, Chan-Hee,Choi, Se-Young,Oh, Seog-Bae,Park, Kyung-Pyo,Kim, Joong-Soo,Lee, Sung-Joong The Korean Society of Pharmacology 2004 The Korean Journal of Physiology & Pharmacology Vol.8 No.5

Schwann cells play an important role in peripheral nerve regeneration. Upon neuronal injury, activated Schwann cells clean up the myelin debris by phagocytosis, and promote neuronal survival and axon outgrowth by secreting various neurotrophic factors. However, it is unclear how the nerve injury induces Schwann cell activation. Recently, it was reported that certain cytoplasmic molecules, which are secreted by cells undergoing necrotic cell death, induce immune cell activation via the toll-like receptors (TLRs). This suggests that the TLRs expressed on Schwann cells may recognize nerve damage by binding to the endogenous ligands secreted by the damaged nerve, thereby inducing Schwann cell activation. Accordingly, this study was undertaken to examine the expression and the function of the TLRs on primary Schwann cells and iSC, a rat Schwann cell line. The transcripts of TLR2, 3, 4, and 9 were detected on the primary Schwann cells as well as on iSC. The stimulation of iSC with poly (I : C), a synthetic ligand for the TLR3, induced the expression of $TNF-{\alpha}$ and RANTES. In addition, poly (I : C) stimulation induced the iNOS expression and nitric oxide secretion in iSC. These results suggest that the TLRs may be involved in the inflammatory activation of Schwann cells, which is observed during Wallerian degeneration after a peripheral nerve injury.

흰쥐 말초신경에서 성체 Schwann 세포의 성공적인 배양

신미영(Mi-Young Shin),노영래(Young-Nae Roh),우지현(Ji Hyun Woo),김영훈(Young-Hoon Kim),황경태(Kyung-Tai Whang),유도성, 조경석(Kyung Suck Cho),김달수(Dal Soo Kim) 대한소아신경학회 2002 대한소아신경학회지 Vol.10 No.1

목적: 성체포유동물의 말초신경에서 유래한 Schwann 세포는 결체조직이 많고 잘 분화된 세포이기 때문에 성공적인 배양이 어렵고 증식이 잘 되지 않는 것으로 알려져 있다. 그러나 신경손상 등으로 조건화를 하면 성숙말초신경에서도 Schwann 세포의 성공적인 배양이 가능한 것으로 최근에 알려져 있다. 이에 저자들은 국내 최초로 흰쥐의 좌골신경을 이용하여 성숙말초신경에서 조건화된 손상을 줌으로서 Schwann 세포를 배양하는데 성공하였으며 수율도 높일 수 있었다. 방법: 250-300 g의 Sprague-Dawley 수컷 흰쥐를 사용하였으며 배양 2주 전에 좌측 좌골신경을 신경손상을 일으켜 조건화한 뒤 키웠다가 다시 좌골신경에서 20 mm 신경조각을 적출하여 RPMI-1640 배양액에 2주간 배양하였다. Schwann 세포의 확인은 s-100항체를 이용한 면역염색법으로 확인하였다. 결과: 1)조건화된 손상 후 평판에서 배양한지 48시간부터는 양극 및 삼극세포를 발견할 수 있었으며 세포수는 12일까지 지속적으로 증가하였다. 2)3-4시간의 소화시간으로도 분리한 세포의 숫자는 줄지 않고 붙은 세포의 수를 증가시킬 수 있었다. 3)부착된 Schwann 세포는 배양 후 12-14일에 최고로 많이 관찰되었다. 4)부착된 Schwann 세포의 대부분은 전형적인 원형의 세포체를 가지고 있으며 뚜렷한 핵과 양극 혹은 삼극뻗음이 있어서 전체적으로는 방추형의 모양을 하였다. 결론: 이러한 결과로 볼 때 조건화된 손상이 말초신경에서 성체 Schwann 세포를 성공적으로 분리하여 배양할 수 있게 한다는 것을 알 수 있었다. PURPOSE: Schwann cells are difficult to isolate from adult mammalian peripheral nerves because of the abundant connective tissue and the highly differentiated state of the cells, particularly those involved in the formation of myelin. It has been shown that in vivo, both neurons and Schwann cells are conditioned by a nerve lesion, speeding up the Schwann cell proliferation and the neuronal regeneration. In this study with adult rat peripheral nerves, we report that a conditioning lesion increases Schwann cells and of cells which successfully attach to a tissue culture striatum. METHODS: The study was done with Sprague-Dawley male rats(250-300 g). Their left sciatic nerves were exposed, severed at the sciatic notch and deflected. After 14 days, with 20 mm segments the nerves were excised from the distal stump of the conditioned sciatic nerves. Schwann cells were cultured in RPMI-1640 media. The identity of the cells was verified with antibody staining using S-100. RESULTS: The lesion evoked a progressive and significant increase in the number of cells obtained as early as 48 hr of the plating, until day 12. Decreasing the duration of enzyme digestion to 3 and 4 hrs markely increased the number of attached Schwann cells. The peak numbers of attached Schwann cells was observed between day 12 and day 14 of the plating. Most attached Schwann cells had typical oval-shaped cell bodies, with prominent nuclei and bipolar cell extensions, resulting in overall spindle shapes. CONCLUSION: Our findings indicate that a conditioning lesion enables us to isolate and culture adult Schwann cells successfully from the peripheral nerves of rats.

Nidogen is a prosurvival and promigratory factor for adult Schwann cells

Lee, Hyun Kyoung,Seo, In Ae,Park, Hye Kyung,Park, Yoo Mi,Ahn, Kyoung Jin,Yoo, Young Hyun,Park, Hwan Tae Raven Press [etc.] 2007 Journal of neurochemistry Vol.102 No.3

<P>Abstract</P><P>Schwann cells provide a favorable microenvironment for successful regeneration of the injured peripheral nerve. Even though the roles of extracellular matrix proteins in the Schwann cell physiology have long been studied, the precise function of nidogen, a ubiquitous component of the basal lamina, in Schwann cells is unknown. In this study, we show that the protein and mRNA messages for nidogens are up-regulated in the sciatic nerve after sciatic nerve transection. We demonstrate that recombinant nidogen-1 increased the process formation of Schwann cells cultured from adult rat sciatic nerves and that nidogen-1 prevented Schwann cells from serum-deprivation-induced death. In addition, nidogen-1 promoted spontaneous migration of Schwann cells in two-independent migration assays. The Schwann cell responses to the recombinant nidogen-1 were specific because the nidogen-binding ectodomain of tumor endothelial marker 7 inhibited the nidogen responses without affecting Schwann cell response to laminin. Finally, we found that &bgr;1 subunit-containing integrins play a key role in the nidogen-induced process formation, survival, and migration of Schwann cells. Altogether, these results indicate that nidogen has a prosurvival and promigratory activity on Schwann cells in the peripheral nerve.</P>

Double-stranded RNA Induces Inflammatory Gene Expression in Schwann Cells: Implication in the Wallerian Degeneration

Hyunkyoung Lee,Chanhee Park,Se-Young Choi,Seog Bae Oh,Kyungpyo Park,Joong-Soo Kim,Sung Joong Lee 대한생리학회-대한약리학회 2004 The Korean Journal of Physiology & Pharmacology Vol.8 No.5

Schwann cells play an important role in peripheral nerve regeneration. Upon neuronal injury, activated Schwann cells clean up the myelin debris by phagocytosis, and promote neuronal survival and axon outgrowth by secreting various neurotrophic factors. However, it is unclear how the nerve injury induces Schwann cell activation. Recently, it was reported that certain cytoplasmic molecules, which are secreted by cells undergoing necrotic cell death, induce immune cell activation via the toll-like receptors (TLRs). This suggests that the TLRs expressed on Schwann cells may recognize nerve damage by binding to the endogenous ligands secreted by the damaged nerve, thereby inducing Schwann cell activation. Accordingly, this study was undertaken to examine the expression and the function of the TLRs on primary Schwann cells and iSC, a rat Schwann cell line. The transcripts of TLR2, 3, 4, and 9 were detected on the primary Schwann cells as well as on iSC. The stimulation of iSC with poly (I:C), a synthetic ligand for the TLR3, induced the expression of TNF-α and RANTES. In addition, poly (I:C) stimulation induced the iNOS expression and nitric oxide secretion in iSC. These results suggest that the TLRs may be involved in the inflammatory activation of Schwann cells, which is observed during Wallerian degeneration after a peripheral nerve injury.

인회석 박막 피복 도관과 Brain-derived neurotrophic factor(BDNF) 유전자 이입 슈반세포를 이용한 백서 좌골신경 재생에 관한 연구

최원재(Won-Jae Choi),안강민(Kang-Min Ahn),황순정(Soon-Jeong Hwang),정필훈(Pill-Hoon Choung),김명진(Myung-Jin Kim),김남열(Nam-Yeol Kim),유상배(Sang-Bae Yoo),장정원(Jeong-Won Jahng),김현만(Hyun-Man Kim),김중수(Joong-Soo Kim),김윤희(Yun 대한구강악안면외과학회 2005 대한구강악안면외과학회지 Vol.31 No.3

Purpose of Study: Peripheral nerve regeneration depends on neurotrophism of distal nerve stump, recovery potential of neuron, supporting cell like Schwann cell and neurotrophic factors such as BDNF. Peripheral nerve regeneration can be enhanced by the conduit which connects the both sides of transected nerve. The conduit maintains the effects of neurotrophism and BDNF produced by Schwann cells which can be made by gene therapy. In this study, we tried to enhance the peripheral nerve regeneration by using calcium phosphate coated porous conduit and BDNF-Adenovirus infected Schwann cells in sciatic nerve of rats. Materials and Methods: Microporous filter which permits the tissue fluid essential for nerve regeneration and does not permit infiltration of fibroblasts, was made into 2mm diameter and 17mm length conduit. Then it was coated with calcium phosphate to improve the Schwann cell adhesion and survival. The coated filter was evaluated by SEM examination and MTT assay. For effective allogenic Schwann cell culture, dorsal root ganglia of 1-day old rat were extracted and treated with enzyme and antimitotic Ara-C. Human BDNF cDNA was obtained from cDNA library and amplified using PCR. BDNF gene was inserted into adenovirus shuttle vector pAACCMVpARS in which E1 was deleted. We infected the BDNF-Ad into 293 human mammary kidney cell-line and obtained the virus plaque 2 days later. RT-PCR was performed to evaluate the secretion of BDNF in infected Schwann cells. To determine the most optimal m.o.i of BDNF-Ad, we infected the Schwann cells with LacZ adenovirus in 1, 20, 50, 75, 100, 250 m.o.i for 2 hours and stained with 􀝜-galactosidase. Rats(n=24) weighing around 300g were used. Total 14mm sciatic nerve defect was made and connected with calcium phosphate coated conduits. Schwann cells(1x106) or BDNF-Ad infected Schwann cells(1x106) were injected in conduit and only media(MEM) was injected in control group. Twelve weeks after surgery, degree of nerve regeneration was evaluated with gait analysis, electrophysiologic measurements and histomorphometric analysis. Results: 1. Microporous Millipore filter was effective conduit which permitted the adhesion of Schwann cells and inhibited the adhesion of fibroblast. We could enhance the Schwann cell adhesion and survival by coating Millipore filter with calcium phosphate. 2. Schwann cell culture technique using repeated treatment of Ara-C and GDNF was established. The mean number of Schwann cells obtained 1 and 2 weeks after the culture were 1.54±4.0×106 and 9.66±9.6×106. 3. The mRNA of BDNF in BDNF-Ad infected Schwann cells was detected using RT-PCR. In Schwann cell 0.69 μg/μl of DNA was detected and in BDNF-Adenovirus transfected Schwann cell 0.795 μg/μl of DNA was detected. The most effective infection concentration was determined by LacZ Adenovirus and 75 m.o.i was found the most optimal. Conclusion: BDNF-Ad transfected Schwann cells successfully regenerated the 14mm nerve gap which was connected with calcium phosphate coated Millipore filter. The BDNF-Ad group showed better results compared with Schwann cells only group and control group in aspect to sciatic function index, electrophysiologic measurements and histomorphometric analysis.

The most appropriate antimitotic treatment of Ara-C in Schwann cell-enriched culture from dorsal root ganglia of new born rat

Soung-Min Kim,Jeongwon Jahng,Jong-Ho Lee 대한구강악안면외과학회 2006 대한구강악안면외과학회지 Vol.32 No.1

Schwann cell, one of important components of peripheral nervous system, interact with neurons to mutually support the growth and replication of embryonal nerves and to maintain the different functions of adult nerves. The Ara-C, known as an antimitotic agent, have been used to have high effectiveness in eliminating fibroblasts during Schwann cell culture period. This enrichment effect is also known to be cummulative with each successive pulse of Ara-C applied and is due to a progressive loss of fibroblasts. But the cytotoxicity by Ara-C is also cummulative and noticeable over the period. To determine the most effective application time and interval of Ara-C in the Schwann cell culture, we observed the Schwann cell purity and density with the Ara-C treatment in plain and three-dimensional culture from dorsal root ganglion of new born rat. By culturing dispersed dorsal root ganglia, we can repeatedly generate homogenous Schwann cells, and cellular morphology and cell count with mean percentages were evaluated in the plain culture dishes and in the immunostainings of S-100 and GFAP in the three-dimensional culture. The Ara-C treated cultures showed a higher Schwann cell percentage (31.0%±8.09% in P4 group to 65.5%±24.08% in P2 group), compared with that obtained in the abscence of Ara-C (17.6%±6.03%) in the plain culture after 2 weeks. And in the three-dimensional culture, S-100 positive cells increased to 56.22%±0.67% and GFAP positive cells to 66.46%±1.83% in G2 group (p<0.05), higher yield than other groups with Ara-C application. Therefore, we concluded that the Ara-C treatment is effective for the proliferation of Schwann cells contrast to the fibroblasts in vitro culture, and the first application after 24 hours from cell harvesting and subsequent 2 pulse treatment (P2 group in plain culture and G2 group in three-dimensional culture) was more effective than other application protocols.

Transient lysosomal activation is essential for p75 nerve growth factor receptor expression in myelinated Schwann cells during Wallerian degeneration

Junyang Jung,Wenting Cai,So Young Jang,Yoon Kyoung Shin,Duk Joon Suh,Jong Kuk Kim,Hwan Tae Park 대한해부학회 2011 Anatomy & Cell Biology Vol.44 No.1

Myelinated Schwann cells in the peripheral nervous system express the p75 nerve growth factor receptor (p75NGFR) as a consequence of Schwann cell dedifferentiation during Wallerian degeneration. p75NGFR has been implicated in the remyelination of regenerating nerves. Although many studies have shown various mechanisms underlying Schwann cell dedifferentiation, the molecular mechanism contributing to the re-expression of p75NGFR in differentiated Schwann cells is largely unknown. In the present study, we found that lysosomes were transiently activated in Schwann cells after nerve injury and that the inhibition of lysosomal activation by chloroquine or lysosomal acidification inhibitors prevented p75NGFR expression at the mRNA transcriptional level in an ex vivo Wallerian degeneration model. Lysosomal acidification inhibitors suppressed demyelination, but not axonal degeneration, thereby suggesting that demyelination mediated by lysosomes may be an important signal for inducing p75NGFR expression. Tumor necrosis factor-α (TNF-α) has been suggested to be involved in regulating p75NGFR expression in Schwann cells. In this study, we found that removing TNF-α in vivo did not significantly suppress the induction of both lysosomes and p75NGFR. Thus, these findings suggest that lysosomal activation is tightly correlated with the induction of p75NGFR in demyelinating Schwann cells during Wallerian degeneration.

Double-stranded RNA induces iNOS gene expression in Schwann cells, sensory neuronal death, and peripheral nerve demyelination

Lee, Hyunkyoung,Park, Chanhee,Cho, Ik-Hyun,Kim, Hyun Yeong,Jo, Eun-Kyeong,Lee, Soojin,Kho, Hong-Seop,Choi, Se-Young,Bae Oh, Seog,Park, Kyungpyo,Kim, Joong Soo,Lee, Sung Joong Wiley Subscription Services, Inc., A Wiley Company 2007 Glia Vol.55 No.7

<P>Inflammation in the peripheral nervous system (PNS) is one of the characteristics of virus-induced peripheral neuropathy. In this inflammatory response, Schwann cells are actively involved. Previously, toll-like receptor 3 (TLR3) was reported as a receptor for double-stranded RNA (dsRNA) that induces antiviral and inflammatory responses in cells of the innate immune system. In this study, we investigated the expression and putative role of TLR3 in Schwann cells. TLR3 was constitutively expressed in Schwann cells. Stimulation with polyinosinic–polycytidylic acid, a synthetic dsRNA analogue, induced the expression of inducible nitric oxide synthase (iNOS) gene in Schwann cells. Studies on the intracellular signal transduction pathways using iSC, an immortalized Schwann cell line, revealed that dsRNA induces the activation of NF-κB, p38, and c-Jun N-terminal kinase (JNK). The activation of NF-κB, p38, JNK, and dsRNA-dependent protein kinase is required for dsRNA-mediated iNOS gene expression. However, the activation of PI3 kinase and GSK-3β inhibited iNOS gene induction, a process mediated by their inhibitory effects on NF-κB and p38 activation. dsRNA-induced NO production caused neuronal cell death in cultured dorsal root ganglion. Finally, the introduction of dsRNA into the rat sciatic nerve induced iNOS gene expression and peripheral nerve demyelination in vivo. Taken together, these data suggest that viral RNA may induce inflammatory Schwann cell activation via TLR3 and peripheral nerve damage in the PNS. © 2007 Wiley-Liss, Inc.</P>

신생 백서 척수후근절의 슈반세포 배양을 위한 Ara-C 분열억제제의 최적 효과에 대한 연구

김성민(Soung-Min Kim),이종호(Jong-Ho Lee),안강민(Kang-Min Ahn),김남열(Nam-Yeol Kim),성미애(Mi-Ae Sung),황순정(Soon-Jeong Hwang),김지혁(Ji-Hyuck Kim),장정원(Jeong-Won Jahng) 대한구강악안면외과학회 2004 대한구강악안면외과학회지 Vol.30 No.2

Schwann cell, one of important components of peripheral nervous system, interact with neurons to mutually support the growth and replication of embryonal nerves and to maintain the different functions of adult nerves. The Ara-C, known as an antimitotic agent, have been used to have high effectiveness in eliminating fibroblasts during Schwann cell culture period. This enrichment effect is also known to be cummulative with each successive pulse of Ara-C applied and is due to a progressive loss of fibroblasts. But the cytotoxicity by Ara-C is also cummulative and noticeable over the period. To determine the most effective application time and interval of Ara-C in the Schwann cell culture, we observed the Schwann cell purity and density with the Ara-C treatment in plain and three-dimensional culture from dorsal root ganglion of new born rat. By culturing dispersed dorsal root ganglia, we can repeatedly generate homogenous Schwann cells, and cellular morphology and cell count with mean percentages were evaluated in the plain culture dishes and in the immunostainings of S-100 and GFAP in the three-dimensional culture. The Ara-C treated cultures showed a higher Schwann cell percentage (31.0%±8.09% in P4 group to 65.5%±24.08% in P2 group), compared with that obtained in the abscence of Ara-C (17.6%±6.03%) in the plain culture after 2 weeks. And in the three-dimensional culture, S-100 positive cells increased to 56.22%±0.67% and GFAP positive cells to 66.46%±1.83% in G2 group (p<0.05), higher yield than other groups with Ara-C application. Therefore, we concluded that the Ara-C treatment is effective for the proliferation of Schwann cells contrast to the fibroblasts in vitro culture, and the first application after 24 hours from cell harvesting and subsequent 2 pulse treatment (P2 group in plain culture and G2 group in three-dimensional culture) was more effective than other application protocols.