Doctoral Seminar

1. بسم الله الرحمن الرحیم Doctoral Seminar Title: The neuroimmunology of Schwann cell Presented by: M. Sh. Mojadadi Advisor: Dr. M. Ebtekar

2. Outline • Introduction to the Schwann cell • Schwann cell development and myelination • Cytokine and chemokine interactions with Schwann cells • Schwann cells as immunomodulatory cells • Guillain-Barre syndrome and the Schwann cell • Schwann cells: The present and the future

3. History Introduction to the Schwann cell • Theodor Schwann: (1810-1882)

4. The Schwann cell Introduction to the Schwann cell • Two major components of nervous system: 1: Central nervous system (CNS) – brain and spinal cord 2: Peripheral nervous system (PNS) a) Axonal processes extending toward or away from CNS (nerves) b) Ganglia (aggregations of nerve cell bodies outside CNS)

5. Introduction to the Schwann cell • Axon may beunmylenated or may bemyelinated by either: • Oligodendroglia in CNS • or • Schwann cells in PNS

6. The Schwann cell Introduction to the Schwann cell • All axons of the peripheral nerves are ensheathed by rows of Schwann cells.

7. The Schwann cell Introduction to the Schwann cell • Nerve cells and Schwann cells are dependent on each other for normal development, function and maintenance. • For example, it is the axon that controls the initiation of myelination, the number of myelin lamellae and the maintenance of the complex Schwann cell organisation. • However, it is the Schwann cell that regulates axonal diameter, neurofilament spacing and phosphorylation, and the clustering of ion channels at the node of Ranvier in myelinated axons. • Furthermore, Schwann cells have the capacity to interact with cells from outside the nervous systems, as evidenced by their well established ability to communicate with cells of the immune system.

8. Types of Schwann cells Introduction to the Schwann cell • Schwann cells in the mature PNS can be categorised for convenience by their morphology, antigenic phenotype, biochemistry and anatomical location. These categories are: 1: Myelinating Schwann cells (MSCs) 2: Non-myelinating Schwann cells (NMSCs) 3: Perisynaptic Schwann cells (PSCs) 4: Satellite cells

9. Satellite cells Nonmyelinating Schwann cells Perisynaptic Schwann cells Myelinating Schwann cells

10. Introduction Schwann cell development and myelination • The glial cells of adult peripheral nerves, myelinating and non-myelinating Schwann cells, are generated during development from neural crest cells. • The protracted embryonic period of gliogenesis involves first the generation of Schwann cell precursors and subsequently the generation of immature Schwann cells. • In the postnatal myelinating or non-myelinating Schwann cells are generated from immature Schwann cells.

11. Schwann cell development Schwann cell development and myelination The generation of Schwann cells from neural crest cells is defined by three major transitions: • 1: From neural crest cells to Schwann cell precursors • 2: From Schwann cell precursors to immature Schwann cells • 3: The divergence of this population into the two mature Schwann cell types (myelinating and non-myelinating) • Most of these events hinge on axonal signals including survival signals, mitogenic signals and differentiation signals.

12. Schwann cell development Schwann cell development and myelination E 14/15 in rat E12/13 in mouse E17/18 in rat

13. Schwann cell development Schwann cell development and myelination

14. Myelination Schwann cell development and myelination • Myelination occurs over an extended period during the first three weeks of postnatal life in rodents, and in humans during a period that begins in embryonic life and ends with puberty. • Considerable evidence now exists for the idea that positive and negative signals are involved. • Just before the onset of myelination at E18, significant changes occur in the relationship between Schwann cells and axons. • They involve radial sorting, a radical change in cellular relationships that allows Schwann cells to start myelinating single large diameter axons.

15. Myelination Schwann cell development and myelination • At the same time Schwann cell numbers are adjusted by controlling survival and proliferation and premature myelination appears to be prevented by the activity of signalling systems that function as ‘myelination brakes’.

16. Myelination Schwann cell development and myelination • The myelin lamellae can be divided into two domains: • 1: compact myelin • 2: non-compact myelin • Compact myelin is highly enriched in lipids, including the glycolipids galactocerebroside and sulphatide. • P0 (the most abundant protein), PMP22 and MBP are proteins of the PNS myelin.

17. Introduction Cytokine and chemokine interactions with Schwann cells • Cytokines, first described as products of the cells of the inflammatory/immune system. • Neuroglial cells, in particular astrocytes, Schwann cells and microglia respond to cytokines by changes in function and phenotype and can themselves produce many of the classically described inflammatory cell cytokines. • Recent studies showing that such cytokines, particularly when produced by cells that are endogenous to the PNS and CNS, are important in PNS and CNS development and perhaps in protection and regeneration of the PNS and CNS in inflammatory, traumatic and even some degenerative diseases.

18. Introduction Cytokine and chemokine interactions with Schwann cells Schwann cell-cytokine interactions can be studied in various ways: 1: Examining tissue obtained at different stages of development. 2: At different phases of experimental and naturally occurring diseases. 3: Employing different in vitro models.

19. In vitro studies of cytokine and Schwann cell interactions Cytokine and chemokine interactions with Schwann cells 1: MHC expression: • IFN-γ induces upregulation of expression of both MHC I and MHC II molecules on the surface of Schwann cells. 2: Expression of adhesion molecules: • Unfractionated cytokines, obtained from mitogen stimulated rat spleen cells (inflammatory cells), as well as IFN-γ, TNF-α and IL-1β upregulated expression of ICAM-1 (CD54). • Incubation of Schwann cells with other cytokines including IL-1β, IL-2 and TGF-β1, all found in the unfractionated cytokines, had no effect on ICAM-1 expression.

20. In vitro studies of cytokine and Schwann cell interactions Cytokine and chemokine interactions with Schwann cells • Expression by TNF-α is predominately via activation of type I TNF-R. • Upregulation of ICAM-1 on Schwann cells can enhance Schwann cell-lymphocyte interactions. • Interestingly, there was no effect of IFN- γ, TNF- α or IL-1β on expression of two integrins, LFA-1α (CD11a) and LFA-1β. • Thus the effect on ICAM-1 expression is not a nonspecific effect on upregulation of all adhesion molecules.

21. In vitro studies of cytokine and Schwann cell interactions Cytokine and chemokine interactions with Schwann cells 3: Effect on Schwann cell proliferation: • It has been shown that unfractionated cytokines stimulate Schwann cell proliferation. • TGF-β1 act as a Schwann cell mitogen. • In one study it has been shown that IL-1α and IL-6 are co-mitogens for Schwann cells, and demonstrated the presence of functional receptor for IL-1 on Schwann cells. • TGF-β, has been shown by several groups to induce Schwann cell proliferation at suboptimal concentrations and can act as a co-mitogen with IL-1. • These findings are relevant to Schwann cell responses to cytokines in vivo, since proliferation is important during development of the PNS as well as during recovery and repair in response to disease.

22. In vitro studies of cytokine and Schwann cell interactions Cytokine and chemokine interactions with Schwann cells 4: Effect on Schwann cell viability: • Studies of TNF-α failed to demonstrate a cytotoxic effect on Schwann cells in myelinating cultures although it has been demonstrated that both the p55 and p75 receptors for TNF are present on rat Schwann cells. • Somewhat unexpectedly, it has been observed that while TGF-β1 alone induced a modest amount of Schwann cell death, when added to TNF-α there was a marked cytotoxic effect.

23. In vitro studies of cytokine and Schwann cell interactions Cytokine and chemokine interactions with Schwann cells 5: Effect on Schwann cell development and maturation: • Upregulation of galactolipids and MAGs are considered indicators of Schwann cell differentiation towards the myelinating phenotype, whereas expression of NGFRp75 is characteristic of promyelinating or non-myelinating phenotype. • High concentrations of intracellular cAMP can induce myelination. Interestingly, low concentrations of cAMP induce Schwann cell proliferation. • It has been shown that supernatants obtained from activated inflammatory cells inhibit cAMP-induced upregulation of surface expression of galactoplipids and also reversed ongoing maturation of Schwann cells. These supernatants induced Schwann cell proliferation.

24. In vitro studies of cytokine and Schwann cell interactions Cytokine and chemokine interactions with Schwann cells • The Schwann cells incubated with IFN-γ, TNF-α, TGF-β1 , IL-1α, IL-1β, IL-2 and IL-6 separately. • It has been shown that IFN-γ, TNF-α and TGF-β1 inhibited cAMP-induced Schwann cell maturation as assessed by upregulation of galactoplipid expression and downregulation of NGFRp75 expression. • TGF-β has been shown to inhibit myelin wrapping of axons by Schwann cells in co-culture experiments. • These findings suggest that increased levels of TGF-β within areas of demyelination may inhibit remyelination.

25. In vitro studies of cytokine and Schwann cell interactions Cytokine and chemokine interactions with Schwann cells 6: Schwann cell synthesis of cytokines: • It has been shown that Schwann cells are able to upregulate genes for several cytokines as well as produce proteins in vitro. • For most of the cytokines this expression of cytokines and specific mRNAs can be shown in vivo. • It has been demonstrated that stimulated Schwann cells upregulate mRNA for IL-1α and β, IL-1RA and IL-1R type I and also synthesis functionally active IL-1. • IL-1, IL-1R and IL-1RA are also expressed in Schwann cells in vivo as well.

26. Cytokines and Schwann cells in diseases of the PNS Cytokine and chemokine interactions with Schwann cells • It is important to consider that cytokines are prominent mediators of inflammatory/ immune demyelinating disorders of the PNS. • Many studies support a critical role for cytokines in the pathogenesis of PNS diseases as well as in recovery from many of these disorders. • Acute inflammatory demyelinating polyneuropathy (AIDP) • Diabetic neuropathy • Neurofibromatosis type 1 (NF1) • Leprosy • For example increased levels of several cytokines, including TNFα, have been reported in serum of patients with GBS.

27. Cytokines and Schwann cells in diseases of the PNS Cytokine and chemokine interactions with Schwann cells • In NF1, Schwann cells heterozygous for the NF1 mutation (NF1-/-) secrete five times the normal amount of kit ligand (stem cell factor), which in turn serves as a chemoattractant for mast cells. • Whether the cytokines and growth factors secreted by the activated mast cells act to maintain the Schwann-cellderived neurofibromas in a benign state, or influence progression to malignant nerve sheath tumours is not known. • In leprosy, cytokines are also postulated to influence disease progression. • In experimental autoimmune neuritis (EAN), PNS animal models, genes and gene products for several cytokines, including interleukin-1 (IL-1), IL-6, TNF-α and TGF-β are upregulated in the PNS. • It has been shown that intraneural injection of TNF-α or IL-12, a proinflammatory cytokine produced predominately by cells of the monocyte/macrophage lineage, induces inflammation and demyelination.

28. Cytokines, Schwann cells and PNS regeneration Cytokine and chemokine interactions with Schwann cells • Trauma to the PNS often results in damage or transection of axons, leading to loss of myelination. • Inflammatory cells, primarily macrophages, infiltrate the nerve and phagocytose cellular debris including myelin. • It has been shown that macrophage infiltration is important in regeneration. • Administration of IL-1 receptor antagonist (IL-1RA) also inhibits regeneration, suggesting that IL-1 is an important factor in PNS response to trauma. • It has been shown that IL-1 induces NGF production by Schwann cells and fibroblasts. • It is likely that the production of NGF is important in regeneration.

29. Chemokines and Schwann cells in diseases of the PNS Cytokine and chemokine interactions with Schwann cells • There have been several studies demonstrating several chemokines of both the CXC (alpha chemokines) and CC (beta chemokines) chemokines in the PNS and specifically in Schwann cells. • MCP-1 (CCL2), MIP-1α (CCL3) and RANTES (CCL5) are upregulated in the PNS after peripheral nerve transection. • In one study it was shown that the earliest increase of MCP-1 was seen in Schwann cells, peaking in those cells at 24 h, and observed in inflammatory cells and endothelial cells later in the evolution of the lesion. • MIP-1α-positive cells were seen by 24 h after trauma, but the expression in Schwann cells was seen at 5 days.

30. Chemokines and Schwann cells in diseases of the PNS Cytokine and chemokine interactions with Schwann cells • MIP-1a was also upregulated in Schwann cells present in PNS tumours, suggesting that the chemokine contributes to the recruitment of macrophages into Schwann-cell-derived tumours. • In Guillain-Barre syndrome (GBS) there was upregulation of MCP-1 in Schwann cells as well as inflammatory cells. • CCR2, the receptor for MCP-1, was seen, but at low levels.

31. Chemokines and Schwann cells in diseases of the PNS Cytokine and chemokine interactions with Schwann cells • SDF-1β predominates in embryonic and early postnatal stages of development, and SDF-1γ is more highly expressed in adult PNS. • Neurons and Schwann cells are the predominant SDF-1 expressing cells. • After trauma SDF-1β is transiently increased, suggesting that SDF-1β is important in PNS development and in repair from trauma.

32. Conclusion Cytokine and chemokine interactions with Schwann cells • Cytokine and chemokine interactions with Schwann cells, as well as expression and production of cytokines and chemokines by Schwann cells, are important in the pathogenesis of several different types of disease of the PNS in normal development and PNS repair. • Additional in vitro and in vivo studies are clearly needed to further elucidate the role of these cytokines and chemokines in health and disease.

33. Introduction Schwann cells as immunomodulatory cells ? • The nervous system is an immunologically privileged site. • The blood-nerve barrier (BNB) does restrict access of immune cells and soluble mediators to a certain degree; however, this restriction is not complete, either anatomically (e.g. the BNB is absent or relatively deficient at the roots, in the ganglia and the motor terminals) or functionally. • Activated T lymphocytes can penetrate intact barriers irrespective of their antigen specificity, and, under certain circumstances, release cytokines that upregulate the expression of major histocompatibility complex MHC II molecules, key molecules required for antigen presentation. • In the central nervous system (CNS) tissue-resident neuroglial cells are present that actively participate in the regulation of immune responses within the tissue.

34. Schwann cells as immunomodulatory cells Schwann cells as immunomodulatory cells • In recent years, several lines of evidence have pointed to Schwann cells as immunocompetent cells within the peripheral nervous system (PNS), which, in addition to their physiological roles, exhibit a broad spectrum of immune-related functions and might be involved in the local immune response in the PNS. • Spectrum of an immune response can be displayed by Schwann cells; recognition of antigens, presentation of antigens, mounting an immune response, and, finally, terminating an immune response within the inflamed peripheral nerve.

35. Antigen recognition by Schwann cells Schwann cells as immunomodulatory cells • The family of toll-like receptors (TLRs) belong to the group of pattern-recognition receptors that are key to recognising specific conserved components of microbes such as LPS. • TLRs are usually found on antigen presenting cells, such as dendritic cells. • TLR-2 has been shown to be constitutively expressed on primary human and rat Schwann cells, and has been invoked as a target receptor for M. leprae. • Under inflammatory conditions, expression of various TLRs, especially TLR-4, is inducible on rat Schwann cells in vitro.

36. Schwann cells as antigen-presenting cells Schwann cells as immunomodulatory cells • It has been shown that human and rat Schwann cells in vitro constitutively express low levels of MHC class I but not MHC class II. • In human nerve biopsies from patients with GBS and its chronic variant, CIDP Schwann cells stained positive for MHC class II. • This suggests that these cells may indeed act as APC in immune-mediated disorders of the PNS. • Schwann cells in vitro have been shown to present foreign and exogenous autoantigen, such as MBP, to antigen-specific syngeneic T line cells.

37. Schwann cells as antigen-presenting cells Schwann cells as immunomodulatory cells • Recent data suggest that BB-1, a member of the family of co-stimulatory molecules, can be detected on unmyelinated Schwann cells and appears upregulated on myelinating Schwann cells in nerve biopsies from CIDP patients. • These findings suggest that Schwann cells possess the necessary markers enabling them to act as an APC in the inflamed PNS.

38. Schwann cells as regulators of immune response Schwann cells as immunomodulatory cells • For a long time cytokines as mediators of an immune response within the peripheral nerve were considered to be the exclusive product of inflammatory cells. • There is now a large body of evidence implying that Schwann cells are capable of producing and secreting a large variety of cytokines, which could act as immunomodulators. • IL-1 (initiator of immune response), in addition, other proinflammatory cytokines, such as IL-6, TNF-α, and TGF-β are generated and released by Schwann cells under certain conditions, some in vitro, others also in vivo. • Other proinflammatory and immunoregulatory mediators, such as PGE2, Tx A2 and LTC4, are synthesised in large amounts by Schwann cells, and may regulate the immune cascade within the inflamed PNS.

39. Schwann cells as regulators of immune response Schwann cells as immunomodulatory cells • NFkB plays a pivotal role in the regulation of the host innate antimicrobial response. • It governs the expression of many immunological mediators, including cytokines, their receptors and components of their signal transduction. • Recent studies suggest that two NF-kB complexes, p65/p50 and p50/p50, can be activated and regulated in human Schwann cells under certain conditions. • Interestingly, the natural inhibitor of NF-kB, IkB, can also be detected in large amounts in Schwann cells.

40. Schwann cells as terminators of the immune response Schwann cells as immunomodulatory cells • In order to control the massive expansion of cellular and soluble immune mediators within the target tissue, certain mechanisms must operate with high fidelity to regulate the immune response. • Schwann cells reveal surface expression of FasL after stimulation with proinflammatory cytokines in vitro. • Functional analysis indicates that the interaction between Fas on T cells and FasL on Schwann cells promotes apoptosis of T lymphocytes. • This raises the possibility that Schwann cells are important in terminating the immune response in the inflamed PNS.

41. Summary Schwann cells as immunomodulatory cells

42. Introduction Guillain-Barre syndrome and the Schwann cell • Guillain-Barre´ syndrome (GBS) was introduced by Guillain, Barre´ and Strohl in 1916. • GBS is an immunologically PNS disease that characterized by progressive weakness of the limbs reaching its worst typically within four weeks and loss of the tendon reflexes. • About two thirds of patients with GBS develop symptoms of peripheral neuropathy between one and six weeks after symptoms of an antecedent infection, most commonly an upper respiratory infection but also after gastrointestinal infection. • Campylobacter jejuni, EBV and Mycoplasma pneumoniae

43. Types of GBS Guillain-Barre syndrome and the Schwann cell • GBS consists of three subtypes: 1: Acute inflammatory demyelinating polyradiculoneuropathy (AIDP) (in Europe and North America) 2: Acute motor axonal neuropathy (AMAN) (in china and east Asia) 3: Acute motor and sensory axonal neuropathy (AMSAN)

44. GBS and the Schwann cell Guillain-Barre syndrome and the Schwann cell • GBS is characterized by the infiltration of macrophages and T cells into the endoneurium and myelin sheath. • In moderate disease, axons are not attacked and hence they remyelinate after few weeks. • Schwann cells synthesize neurotropic factors, growth factors, neurite promoting factors and cell adhesion molecules that help in regenerating the peripheral nerve axons.

45. Schwann cells: The present and the future Introduction • Cell transplant therapies are currently under active consideration for a number of degenerative diseases. • In the immune-mediated demyelinating-neurodegenerative disease multiple sclerosis (MS), only the myelin sheaths of the CNS are lost, while Schwann cell myelin of the PNS remains normal. • Schwann cells play a crucial role in endogenous repair of peripheral nerves due to their ability to dedifferentiate, migrate, proliferate, express growth promoting factors, and myelinate regenerating axons. • On the other hand following spinal cord injury (SCI), Schwann cells migrate from periphery into the injury site, where they apparently participate in endogenous repair processes. • These, and the finding that Schwann cells can myelinate CNS axons, has focused interest on Schwann cell transplants to repair myelin in MS and SCI.

46. Schwann cells: The present and the future Schwann cells transplant for CNS repair • Therapeutic strategies for MS: 1: Attempts to block the disease process 2: Attempts to repair the damage, notably the demyelination, caused by the disease Stimulation of the inherent remyelination capacity of the CNS • Myelin repair Transplantation of exogenous cells that can populate the MS lesions, remyelinate the axons and prevent further axon loss.

47. Schwann cells: The present and the future Schwann cells transplant for CNS repair • Choice of cell type for transplantation: 1: Cells from various stages of the oligodendrocyte lineage 2: Olfactory bulb ensheathing cells 3: Schwann cells from the post-natal PNS • It has been shown that transplanted Schwann cells myelinate CNS axons in vivo in a variety of experimental models, and can restore function to demyelinated axons. • In one study it has been shown that transplantation of Schwann cells to subarachnoid space induces repair in contused rat spinal cord (Masoumeh Firouzi et al. 2008). • Indeed Schwann cells were chosen for the first clinical trial of transplanted cells in MS patients.

48. Schwann cells: The present and the future Potential drawbacks to the use of Schwann cells • Potential drawbacks to the use of Schwann cells within the CNS: 1: Migration • Unfortunately according to most studies, implanted Schwann cells do not migrate significantly through the normal CNS. 2: Survival • Schwann cells appear to survive very poorly in normal CNS, although in lesions where the cells can access naked axons and myelinate, they thrive. 3: Complex interplay between astrocytes and Schwann cells • Studies show that co-existence or close interaction between astrocytes and Schwann cells, in vivo or in vitro, is rarely obtained, and that under most conditions astrocytes inhibit Schwann cell myelination in the CNS.

49. Schwann cells: The present and the future Potential drawbacks to the use of Schwann cells 4: Ethical and immunological compatibility issues • Use of fetal tissue as a source for Schwann cells and MHC incompatibility 5: Preparation of enough cells for effective transplantation • It is necessary to generate enough cells as soon as possible because of progressive nature of spinal injuries and limited time window for effective therapy

50. Schwann cells: The present and the future Strategies for the future 1: Use of Schwann cell precursors (SCPs) instead of mature Schwann cells • It has been shown that implanted SCPs from embryo day 14 (E14) rat nerves into CNS survive well, migrate through normal CNS tissue, interface smoothly and intimately with host glial cells and myelinate. 2: The use of host-derived, autologous peripheral nerves as a source for Schwann cells 3: Improve methods to generate enough cells for transplantation 4: Combinatorial therapy • It has been shown that co-transplantation of olfactory ensheathing glia (OEG) with Schwann cells promotes efficacy of therapy in SCI.