The role of “anti-inflammatory” cytokines in axon regeneration

Cytokine & Growth Factor Reviews 24 (2013) 1–12

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The role of ‘‘anti-inflammatory’’ cytokines in axon regeneration Pı´a M. Vidal, Evi Lemmens, Dearbhaile Dooley, Sven Hendrix * Dept. of Morphology & Biomedical Research Institute, Hasselt University, Belgium

A R T I C L E I N F O

A B S T R A C T

Article history: Available online 15 September 2012

The injured central and peripheral nervous system (CNS and PNS) are difficult to regenerate due to the presence of growth inhibitory molecules which are upregulated around the lesion site. In addition, a strong inflammatory response triggering the production of so-called ‘‘pro’’- and ‘‘anti-inflammatory’’ cytokines, adds to this dilemma. Both pro- and anti-inflammatory cytokines are involved in the regulation of diverse signaling pathways. One of the main aims to induce regeneration is to promote axonal outgrowth and stimulate the formation of new connections. Anti-inflammatory cytokines as modulators of neurite plasticity and outgrowth are of pivotal importance in neuroregeneration with different effects reported. Here we summarize the most relevant information about IL-4, IL-10, IL-13, LIF and TGF-b focusing on their direct and indirect role in axonal outgrowth. ß 2012 Elsevier Ltd. All rights reserved.

Keywords: CNS regeneration PNS regeneration Spinal cord injury IL-4 IL-13 IL-10 TGF-b LIF

Contents 1. 2. 3.

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What are anti-inflammatory cytokines? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Are anti-inflammatory cytokines immunosuppressants? . . . . . . . . . . . . . . . . . . . . . . . . . . Axonal growth and the immune system. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Distinct inflammatory phases after injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of anti-inflammatory cytokines depend on the immune phase, the compartment 3.1. Direct and indirect effects of ‘‘anti-inflammatory’’ cytokines . . . . . . . . . . . . . . . . . . . . . . . 3.2. Effects of anti-inflammatory cytokines in the peripheral and central nervous system . . . . . . . . Interleukin-4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Interleukin-10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Interleukin-13 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Leukemia inhibitory factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Transforming growth factor-b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. What are anti-inflammatory cytokines? The activity of cytokines was first recognized in the mid 1960s, when supernatants derived from in vitro cultures of lymphocytes were found to contain factors that could regulate proliferation, differentiation, and maturation of allogeneic immune cells, induced by activation with antigen or with nonspecific mitogens [1].

* Corresponding author at: Dept. of Morphology & Biomedical Research Institute, Hasselt University, Agoralaan, Building C, B-3590 Diepenbeek, Belgium. Tel.: +32 11 26 9246; fax: +32 11 26 9299. E-mail address: [email protected] (S. Hendrix). 1359-6101/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cytogfr.2012.08.008

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The definitions of cytokines available in the literature tend to be a bit vague and vary among the authors [1–5]. In this review, we base our concept of cytokines on the definitions summarized in Table 1. Cytokines are proteins with pleiotropic, redundant, synergetic and/or antagonistic effects mediated via several signaling cascades, which permit them to regulate cellular activity (such as proliferation, differentiation and maturation) in a coordinated and interactive way over extensive networks. The terms ‘‘cytokine’’ and ‘‘growth factor’’ are sometimes—but not always—used interchangeably. Therefore, in this review we refer to ‘‘Transforming growth factor-b (TGF-b)’’ as a growth factor and cytokine although this may seem pleonastic.

P.M. Vidal et al. / Cytokine & Growth Factor Reviews 24 (2013) 1–12

2 Table 1 Cytokine concepts. Cytokine definition

Reference

Cytokines are small proteins that assist in regulating the development of immune effector cells and/or possess direct effector functions. They are capable of regulating interactions among lymphoid cells, inflammatory cells and hematopoietic cells, thus mediating cell–cell communication. They bind to receptors, thereby triggering signal transduction pathways that ultimately alter gene expression in target cells. Cytokines are multifunctional pleiotropic proteins that play crucial roles in cell–cell communication and cellular activation. Functionally, cytokines have been classified as being merely pro-inflammatory (type 1) or anti-inflammatory (type 2). Cytokines function as intracellular messenger molecules and are defined mainly through their regulatory effects on immune cells. Cytokines are inflammatory mediators important in the host’s response to pathogens and other (foreign) challenges. Cytokines are small cell-signaling molecules secreted by different cell types throughout the body. They can be classified as proteins, glycoproteins or peptides, mainly related to intercellular communication.

[1]

Functionally, cytokines and the cells that secrete them have been classified as either pro-inflammatory (stimulatory, or T helper cell type 1 [Th1] or type 1) or anti-inflammatory (inhibitory or T helper type 2 [Th2] or type 2). In most publications, the terms pro-inflammatory, stimulatory, T helper cell type 1 [Th1] or type 1 are used interchangeably, although it is semantically not correct to use them as synonyms. Similarly, anti-inflammatory, inhibitory, T helper cell type 2 [Th2] or type 2 are also all common terms used. Type 1 cells mainly activate macrophages and control infections; meanwhile type 2 cells activate B cells and eradicate extracellular parasites (reviewed in [6]). However, most of the cytokines have an overlap in function, exerting both pro- and anti-inflammatory effects depending on the tissue milieu, which often makes it difficult to understand the actual effect they induce as mediators of the immune response. For example, it has been suggested that some so-called ‘‘anti-inflammatory’’ cytokines, such as interleukin-4 (IL-4), interleukin-10 (IL-10) and TGF-b may present pro-inflammatory properties under certain experimental conditions [7] (see below). It has been suggested that a T cell subpopulation, called T helper type 2 cells, might be particularly beneficial following CNS and PNS lesions and after neuropathic pain following peripheral nerve injury, especially by producing the anti-inflammatory cytokines IL-4 and IL-10 [8]. To present the message of this review clearly, we consider ‘‘anti-inflammatory’’ cytokines as molecules that control the ‘‘pro-inflammatory cytokine response’’ (see below: ‘‘Are antiinflammatory cytokines immunosuppressants?’’). This limits the effects of sustained or excessive inflammation which can be detrimental for the proper functioning of tissues and organs, such as the regulation of IL-1 and tumor necrosis factor-a (TNF-a) levels [9]. The ‘‘anti-inflammatory’’ cytokines may be secreted by immune cells such as activated lymphocytes, macrophages, microglia and mast cells at or near the site of injury, thus acting mostly locally. We have focused in this review on the role of selected socalled anti-inflammatory cytokines, namely IL-4, IL-10, TGF-b, IL-13 and leukemia inhibitory factor (LIF), in axonal regeneration, in light of a possible new therapeutic application after CNS damage. 1.1. Are anti-inflammatory cytokines immunosuppressants? The term ‘‘anti-inflammatory cytokines’’ seems to suggest that these factors suppress immunity similarly to high doses of corticosteroids, however, this is a wide-spread misunderstanding also shared by many immunologists which is—at least in part—a result of the history of immunology [10]. Historically, the first original immunological experiments were focussed on Th1dominated immune processes such as the tuberkulin reaction. Therefore, immunologists may tend to consider factors that suppress these processes as immunosuppressant. However, quite

[2] [3] [4] [5]

often, simply a different type of immune reaction is replacing the original process [10]. Classical immunosuppressants such as corticosteroids only suppress all immune reactions when applied in high doses, while lower doses are only immunomodulatory. Furthermore, the canonical type-2 cytokines IL-4 and IL-13 play key roles during the strong inflammatory responses in allergic diseases such as asthma and atopic dermatitis and can definitely not be labeled ‘‘anti-inflammatory’’ in this context. Following this concept, ‘‘anti-inflammatory’’ cytokines should be considered immunomodulators that inhibit classical type-1 (Th1) and delayed type hypersensivity (DTH) responses [10], however they are not ‘‘immunosuppressants’’. Therefore, we consider the label ‘‘antiinflammatory’’ as partially misleading and prefer to use it only with quotation marks. 2. Axonal growth and the immune system After axotomy, the ends of lesioned axons possess little capability to regrow or elongate; they are able to sprout fibers until they are near to, or in contact with the region of scar tissue formation, where several classes of growth inhibitory molecules are being upregulated [11,12]. These dynamic and constantly moving growth cones contain all the machinery required for membrane remodeling, thereby inducing expression/degradation of surface molecules or receptors in response to repulsive guidance factors. However, unfortunately this does not lead to overt axonal regeneration over large distances [11]. It is believed that the protein synthesis occurring inside the growth cones is used primarily for direction and not for extension [13], and this direction is dependent on the level of cyclic nucleotides (cAMP and cGMP) [13]. On the other hand, direction and length of growth are related, since growth is modulated by the action of attractive and repulsive cues upon growth cones. It is important to note, that axon regeneration mostly takes place in a disturbed immune milieu and that axon growth is substantially modulated by immune factors [6]. Interestingly, many factors that are potent attractive or repulsive factors of axon growth and are influenced by immune factors (Table 2) exert themselves immunological functions (Table 3). For instance, positive and negative regulators are involved in the modulation of cytokine and chemokine levels and activities, angiogenesis, survival processes as well as modulation of macrophages and T cell activation (Table 3). On the other hand, cytokines may influence the expression of axon growth modulators and their receptors as well as their intracellular signaling. Finally, immune factors may not only directly influence axonal outgrowth but may also modulate indirectly attractive and repulsive cues. Therefore, it is no surprise that immunological responses triggered after central or peripheral nervous system injury are very complex and some studies have shown that inflammation has beneficial effects after injury, while others have shown the opposite (reviewed in [6]).

Table 2 ‘‘Anti-inflammatory’’ cytokines modulate positive and negative regulators of axon growth.

Positive regulators Neurotrophic factors (BDNF, NGF, NT-3, NT-4)

Interleukin-10

Interleukin-13

Tumor growth factor-b

Leukemia inhibitory factor

It stimulates immune cells to produce NT-3 mRNA [3] High doses increase NT-4 inducing outgrowth in DRG neurons [47]

BDNF, IL-10 and TGF-b are expressed after transplantation of GAspecific cells in an EAE model [109] IL-10 levels increase after NGF and BDNF treatment in dendritic cells [110]

NGF stimulates IL-13 secretion and modulates IgE-mediated responses in human basophils [111]

TGF-b increases the chemotactic action of NGF in microglia cells [112]. TGF-b3 and NT-3 enhance spiral ganglion neuronal survival [113]; similar to TGF-b5 with NT-3 and TGF-b5 plus BDNF [114] Rho regulates TGF-b1 activation of keratocytes mediating phenotypic [117] and morphological effects of TGF-b1 in stress fiber formation [118] RhoA regulates posttranscriptional regulation of TGF-b in apoptotic cells [119] TGF-b induces an increase of ATP-inducing calcium mobilization in A59 cells (human lung cancer cell line) augmenting cellular migration [123]

LIF acts synergically with NT-3 and BDNF to promote neuronal survival in spiral ganglion cells [114]

Rho-GTPases

IL-4 induces expression of an activator of Rho-GTPase proteins, Dock10, in B cells [115]

Moderate calcium concentrations (100 nM)

IL-4 inhibits calcium transients in airway smooth muscle cells and modulates [Ca2+]i levels [120] IL-4 stimulates increase in calcium activated potassium channels [121] IL-4 induces cAMP and cGMP accumulation in a dose dependent manner in monocytes [125]

Cyclic nucleotide ("cAMP and cGMP)

IL-13 induces upregulation of RhoA in human bronchial smooth muscle cells, which is inhibited by an inhibitor of STAT6 [116]

IL-10 suppresses human osteoclastogenesis by attenuating calcium pathways [122]

IL-13 partly inhibits the calcium activated potassium channels in response to IL-4 [121]

IL-10 mediates inhibitory effects of cAMP elevating agents on bone marrowderived dendritic cells [126]

IL-13 enhances arginase mRNA and protein expression in rat aortic smooth muscle cells, possible by cAMP [127] This cytokine induces a downregulation of NO production through arginase induction via cAMP/PKA in macrophages [128]

Negative regulators High calcium concentrations (>200 nM)

Neurotrophic factors (NGF, NT-3, NT-4)

Fibrotic and glial scar components (tenascin, fibronectin, MBP, MAG, MOG)

Low IL-4 concentrations suppress neurite outgrowth induced by NGF and NT-4 in DRG cells [47] IL-4 increases Tenascin-C mRNA and secretion on cultured keratinocytes [133]

IL-13 induces tenascin-C expression in fibroblasts [134]

In immature airway smooth muscle cells, acetylcholine-induced [Ca2+]i response is enhanced after LIF treatment [124]

TGF-b upregulates CREB levels in advanced breast cancer cells [129] Chronic exposure to TGF-b decreases cAMP-driven Clsecretion (ion transport) in T84 epithelial cells [130]

TGF-b1 increases intracellular Ca2+ concentration, leading to an enhancement of cell adhesion [131] TGF-b1 reduces NT3 mRNA levels in a dose and time dependent manner in Schwann cells [132] TGF-b stimulates astrocytic expression of fibronectin and tenascin [31] and promotes the formation of cell clusters that accumulate extracellular matrix molecules [107]

P.M. Vidal et al. / Cytokine & Growth Factor Reviews 24 (2013) 1–12

Interleukin-4

LIF induces tenascin-C and fibronectin in myoblast cells [135] LIF is required for myelination during development of mouse optic nerve, because a reduction of MPB was seen in LIF / mice [136]

Abbreviations: Brain derived neurotrophic factor (BNDF), calcium (Ca2+), cAMP response element-binding (CREB), cyclic adenosine monophosphate (cAMP), cyclic guanoside monophosphate (cGMP), dorsal root ganglion (DRG), experimental autoimmune encephalomyelitis (EAE), immunoglobulin E (IgE), glatiramer acetate (GA), interleukin 4 (IL-4), interleukin 10 (IL-10), interleukin 13 (IL-13), leukemia inhibitory factor (LIF), nerve growth factor (NGF), neurotrophin factor 3 (NT-3), neurotrophin factor 4 (NT-4), myelin binding protein (MBP), myelin associated glycoprotein (MAG), myelin oligodendrocyte glycoprotein (MOG), nitric oxide (NO), protein kinase A (PKA), Ras homolog gene family, member A (RhoA), signal transducer and activator of transcription (STAT6), transforming growth factor-b (TGF-b). 3

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Table 3 Axon growth-modulating factors with immunological functions. Molecules

Examples of functions/effects in the immune system

Reference

Netrin-1

Its administration before or after ischemia-reperfusion injury protects kidneys by suppressing leukocyte infiltration. The authors suggest that netrin-1 suppresses cytokine and chemokine production in these cells Netrin-1 administration reduces the levels of inflammatory cytokines within the alveolar space, reducing the intra-alveolar inflammation during acute lung injury (ALI) in a porcine model Macrophage migration mediated by TGF-b is regulated by activation/inactivation of RhoA RhoA and its downstream effector ROK are activated in synovial tissue of rheumatoid arthritis (RA) patients. The blockage of ROK inhibits pro-inflammatory cytokine production via inhibition of NF-kB activation Ca2+ dependent signaling pathways mediate gene induction and repression of activated T cells from patients with severe-combined immunodeficiency LPS induces HMGB1, a chromatin binding factor that acts as a late mediator of mortality in murine endotoxemia and sepsis, released by Ca2+-dependent signals T cell proliferation is inhibited in human T cells expressing cGK1, stimulated with NO and cGMP analogs Neutrophil retraction is regulated by an increase in intracellular cAMP levels in response to chemoattractants NGF interacts directly with endothelial cells in vitro and induces an angiogenic response in the chick embryo in vivo BDNF, NGF, NT-3 or NT-4 increase viability of eosinophils from bronchoalveolar lavage fluid Tenascin–C expressed in glioblastoma cells inhibits the migration of T cells Fibronectin induces a more immature phenotype on dendritic cells, leading an increase in their endocytic ability

[137]

Rho-GTPases

Calcium

cAMP/cGMP Neurotrophins Glial scar compounds

[138] [139] [140] [141] [142] [143] [144] [145] [146] [147] [148]

Abbreviations: Brain derived neurotrophic factor (BNDF), calcium (Ca2+), cyclic adenosine monophosphate (cAMP), cyclic guanoside monophosphate (cGMP), cGMP-activated kinase 1 (cGK1), high-mobility group protein B1 (HMGB1), lipopolysaccharide (LPS), nerve growth factor (NGF), neurotrophin factor 3 (NT-3), neurotrophin factor 4 (NT-4), nuclear factor kappa B (NF-kB), nitric oxide (NO), Ras homolog gene family A (RhoA), Rho-associated protein kinase (ROK), transforming growth factor–b (TGF-b).

3. Distinct inflammatory phases after injury Inflammation is part of the initial response injury and is characterized in the acute phase by increased blood flow and vascular permeability, with accumulation of fluid (edema formation), leukocytes and inflammatory mediators, such as cytokines. The immune response also varies according to location, with differences for instance between the CNS and PNS. It has been suggested that inflammation in the nervous system might be sitespecific with characteristic immunological molecules involved. For example, the response of T cells to axonal injury is more limited in the CNS than in the PNS and T cell apoptosis occurs extensively in the injured CNS when compared to the PNS [14]. In addition, the time-course of the injury and its repair is of pivotal importance. After CNS injury, phase-specific immune responses are starting to be recognized (Kramer & Hendrix unpublished observations; [15]). After spinal cord injury (SCI), at least four main stages can be distinguished: acute, sub-acute, early chronic and chronic phase.

The acute phase, which typically lasts for a few hours, is characterized by an upregulation of pro-inflammatory cytokines, such as IL-1b and TNF-a [16,17]. This phase has been defined by an augmentation in damage, i.e. neuronal and axonal destruction, as well as demyelination close to the injury site [18]. There is also an infiltration of neutrophils, reaching the highest level 1 day after injury [19], and activated B and T cells increase in the spleen and bone marrow [20]. In the sub-acute phase, between 2 and 7 days after injury, the levels of some pro-inflammatory cytokines start to decrease [15,17]. Meanwhile, there is an increase in the number of monocytes and lymphocytes and the levels of anti-inflammatory factors (Fig. 1). Examples of molecules involved in the acute inflammatory phase are the cytokines IL-1b, IL-6, IL-8, IL-11 and TNF-a as well as the chemokines granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Together these factors activate macrophages, neutrophils as well as natural killer cells. During the acute and subacute phases, recovery of locomotor skills in rodents is observed.

CNS

A

Relative mRNA levels

LIF TGF- β IL-4 IL-10

0

24 Hours

Time frame

7 Days

6 Weeks

PNS Relative mRNA levels

B

IL-4

IL-10

24

0

Time frame

Hours

7 Days

6 Weeks

Fig. 1. Temporal expression patterns of ‘‘anti-inflammatory’’ cytokines in SCI and PNI of rodents. (A) Curves represent mRNA levels after SCI for IL-4, IL-10, LIF, and TGF-b. (B). Curves represent mRNA levels after PNI for IL-4 and IL-10. Curves were adapted and generated using data from the following references: [15,17,46,47,53,106–108] and own unpublished data.

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Later on, the early chronic and later chronic phases are characterized by the development of specific humoral and cellular immune responses directed to facilitate cleaning of the injury site [15]. This time frame can last weeks or even months. During these two phases, the levels of T cells, macrophages and neutrophils start to increase again showing a second peak of these immune factors. It is not clear whether this is related to changes in functional recovery. It has been suggested that inflammation may support regeneration in the chronic phase because a reduction of macrophage/microglial infiltration lead to a decrease in functional recovery following SCI [19,20]. 3.1. Effects of anti-inflammatory cytokines depend on the immune phase, the compartment and the cell type Axonal growth is actively promoted during the development of the CNS and PNS in mammalian vertebrates. Conversely, in the adult CNS, axon extension is limited even after axotomy. This is in part due to a limited expression of proteins that can promote rapid growth during development and adult peripheral nerve regeneration [21]. The presence of structural barriers (e.g. scar tissue) and molecules (e.g. myelin inhibitory molecules such as NogoA) [22], as well as the complex inflammatory response generated after injury, are also responsible for limiting axonal regeneration. Cellular types such as microglia, astrocytes and oligodendrocytes play a central role during CNS inflammation, as they can produce and modulate the secretion of cytokines and growth factors. Microglia are among the first cells to be activated after CNS injury, thus initiating the response to brain injury, while the other cellular types are mainly involved in intermediate or later responses [2,15,23]. Although the immune response is one of the first reactions to occur following injury to the nervous system, there is still controversy in the literature about the role of the immune system, especially T cells and macrophages, in nervous system repair. The first inflammatory response in rodents is characterized by infiltrating neutrophils, macrophages/microglia and T cells; meanwhile in a later phase, the levels of macrophages and neutrophils increase again peaking at around day 60 after injury [19]. They can have either beneficial or detrimental effects after damage (reviewed in [6]). For instance, after mechanical damage, such as SCI, traumatic brain injury or crush injury of the optic nerve, injections or vaccines of active autoimmune T cells specific to myelin-associated proteins (MBP) or ovalbumin, lead to improvement in some models and exacerbation in others [24]. This suggests that a specific molecular crosstalk between the immune and nervous systems, determines the outcome of T cell actions after damage. For instance, some studies provide convincing evidence that specific inflammatory cascades play a direct role in axonal outgrowth [25], either by improving the integrity of neurites [26] or by promoting cellular survival [27,28]. The environment within which a cytokine is produced may also be responsible for its beneficial or detrimental effect; this means that in a Th1-dominated immune milieu, a cytokine may have ‘‘suppressive’’ effects while the same cytokine may lead to a dramatic exacerbation in a Th2-dominated environment [6]. For example, TGF-b promotes neurite outgrowth from dorsal root ganglia (DRG) explants, however, it neutralizes outgrowth promoted by IL-1a or IL-1b [29]. Furthermore, cytokine effects may dramatically be altered due to a change in the location; thus, immune cells and their secreted factors may exert distinct effects in one compartment (e.g. blood vessels or perivascular space) whilst displaying other effects after invading a certain tissue (e.g. brain parenchyma) [30]. In addition, there is extensive literature outlining that cytokines exert cell-type-specific effects. For example, IL-4 seems to promote

5

the proliferation of fibroblasts [31] and endothelial cells [32], while having anti-proliferative effects on other cellular types such as carcinoma cells [33]. Striking examples of phase-specific effects of anti-inflammatory cytokines have been described in other disease contexts such as asthma, experimental autoimmune encephalomyelitis (EAE) as well as wound healing and pregnancy [34–38]. Studies using anti-IL-13 monoclonal antibodies to treat asthma, have suggested that IL-13 may play a protective role in acute inflammatory settings, while having a detrimental effect in chronic inflammatory settings [34]. Similarly, blocking IL-4/IL-13 receptor prevents allergic airway inflammation in asthma, but has no effect in the established disease, suggesting that both cytokines play a predominant role in the acute onset of disease [35]. IL-10 may also have different effects depending on the time of application in EAE, an animal model of multiple sclerosis; there is a delay in the onset of the disease in IL-10 transgenic mice treated with anti-IL10 antibodies the day of immunization, while in mice treated 8 days before immunization, this delay is absent [36]. Additionally, anti-TGF-b1, 2 and 3 monoclonal antibodies also display different effects on wound healing and hypertrophic scar formation. Early treatment impairs wound healing and has no effect on scar formation, while middle and later treatments reduce scar formation [37]. Finally, it is known that LIF enhances embryonic implantation, but it has no effect on early embryonic development [38]. Many researchers have achieved a substantial understanding of the cellular mechanism behind these findings; however, much still remains unknown due to the enormous complexities in both the nervous and immune systems. It is also of pivotal importance that the effects of cytokines may differ depending on the immunological milieu. However, it is safe to assume that the ability to maximize specific immune elements, while suppressing others that are aspects of the immune response responsible for further damage, is critical for repair. 3.2. Direct and indirect effects of ‘‘anti-inflammatory’’ cytokines In order to understand the contradictory effects reported about cytokines, it is not only necessary to characterize the phasespecific and compartment-specific effects of the cytokines, but also the direct and indirect effects on neurite growth. Direct effects of cytokines are mediated via cytokine receptors on a neuron which is directly stimulated or inhibited by a factor to regrow an axon. In addition, cytokines may have indirect effects on axon growth by influencing other cells and their secreted factors, for example by stimulating astrocytes to produce neurotrophins [24]. It is known that IL-13 is capable of modulating the inflammatory response by suppressing the production of inflammatory mediators such as IL1b, TNF-a and IL-6 from microglia in vitro and in vivo [39,40]. In Table 2 we have summarized selected examples of positive and negative neurite growth modulators which are influenced by antiinflammatory cytokines. Another example is seen in EAE, where IL13 elicits an inhibitory function on MBP-directed T cell or B cell immunoreactivity in vitro and in vivo [41]. The treatment of EAE animals with RTL401, a peptide construct used to prevent relapses and reverse EAE switching cytokines profiles, induces a reduction of infiltrating mononuclear cells into the CNS and inflammatory lesions in the spinal cord. It also preserves injured axons through a strong Th2 response and is accompanied by upregulated IL-13 levels in the spleen, blood, spinal cord and brain [42]. These data suggest that IL-13 is involved in the preservation of injured axons in the spinal cord and the reversal of clinical signs of EAE via suppression of the induction of type 1-mediated autoimmune responses. Studies using vaccination models for the treatment of CNS injury have shown that type 2-inducing adjuvants prevent the development of EAE and promote axon regeneration [43]. In addition, type 2 cells promote neuronal survival better than type 1

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cells in vitro [24], while in brain slices, type 2 cells suppress type 1induced inflammatory signals [44]. Thus, the induction of a Th2 milieu may be another important indirect effect of the antiinflammatory cytokines described here. During CNS and PNS trauma, we consider the cytokine/ neurotrophin axis [45] to be of particular interest. After trauma, nerve growth factors (such as neurotrophins) and cytokines are mostly present at the same time. From an evolutionary point of view, it seems plausible that these two systems are closely intermingled and display synergistic effects. For example, using DRG explants we demonstrated that neurotrophin-induced neurite outgrowth is dose-dependently modulated by pro- and anti-inflammatory cytokines such as TNF-a, IFN-g, IL-4 and IL-6. These data suggest a significant relation between neurotrophins and cytokines in the response after peripheral nerve injury that may help to modulate outgrowth and axonal regeneration [45]. 4. Effects of anti-inflammatory cytokines in the peripheral and central nervous system In this part of the review we will focus on the effects of selected ‘‘anti-inflammatory’’ factors on neurite outgrowth and regeneration, namely IL-4, IL-10, IL-13, LIF and TGF-b. 4.1. Interleukin-4 IL-4 is a 30 kDa protein that is produced mainly by mature Th2 cells, mast cells, B cells and stromal cells. It plays a key role in inducing CD4+ T cells to differentiate into type 2 cells, while suppressing the development of type 1 cells. It has been shown that after sciatic nerve injury, there is a downregulation of IL-4 mRNA levels in the ipsilateral DRG within hours after injury, while on the other hand, pro-inflammatory levels are upregulated [46,47], suggesting a positive role of IL-4 after peripheral nerve injury (PNI). Similarly to IL-10, it also has positive effects on axonal regeneration. In a model of PNI, IL-4 promoted facial motor neuron survival after axotomy though STAT6 signaling [25], while in retinal cell cultures, IL-4 enhanced the survival, by regulating the cholinergic uptake in a dose- and time-dependent manner [48]. It seems clear that IL-4 is capable of modulating cellular survival via different signaling pathways depending on the environment. In addition, IL-4 displays clear cell-type-specific effects. For example, it promotes the proliferation of fibroblasts [31,33] and endothelial cells [49], while it exerts anti-proliferative effects on tumor cells such as renal cell carcinoma cells (RCC) [33] and breast and colon cancer cell lines [32], possibly by the inhibition of angiogenesis [50]. IL-4 also has biphasic effects; in vivo, low doses weakly induce neovascularization, while high doses inhibit angiogenesis by acting directly on endothelial cells. These biphasic or dual effects of IL-4 are also seen in the case of migration, where low doses of the cytokine (between 1 and 100 pg/mL) stimulate migration of endothelial cells and high doses inhibit migration [50]. In cultured DRG cells, IL-4 has also been seen to modulate peripheral axon regeneration (Table 4), suggesting even more that T cells may affect neuroregeneration via local IL-4 secretion, which in turn stimulates local neurotrophin secretion in a dose dependent manner [45]. It has been suggested that IL-4 and NT3 have a synergistic relationship since this cytokine is able to induce an enhancement of NT-3 mRNA expression in human immune cells [3], while NT-3 enhances IL-4 production by stimulated type 2 cells [51]. Since NT-3 is known to enhance the release of neurotransmitters [52], these interactions may well have an effect on synaptic efficacy and neuronal plasticity. IL-4 has also been associated with the control of brain inflammation and neuroprotective effects in the CNS. In this respect, after SCI, IL-4 and IL-4Ra mRNA levels increased within 24 h after

injury while declining thereafter. IL-4 neutralizing antibodies do not change the levels of pro-inflammatory cytokines, but lead to an increase of ED1 immunoreactivity around the lesion site and augmentation of the cavity 4 weeks after injury. This finding suggests that IL-4 may endogenously exert protective effects regulating the acute and chronic macrophage responses [53]. In rod photoreceptors, IL-4 protects against thapsigargin, a potent inducer of apoptosis, by blocking cell death through the cAMP/PKA pathway [54]; and, IL-4 also plays an important role in controlling GABA-ergic and cholinergic phenotypes, where it can stimulate the uptake of neurotransmitters in rat retina cultures [48,55]. At the same time, IL-4 is able to enhance the survival of hippocampal neurons in vitro, in a dose and time dependent manner, since low concentrations (nM-mM) for less than 48 hours have protective effects and high concentrations (mM) for a longer incubation period, seem to be neurotoxic [27,28]. On the other hand, treatment of hippocampal neuronal cultures with IL-4, leads to increased proliferation of astrocytes and microglia [27]. In microglial cells, IL-4 promotes growth, phagocytic functions as well as proliferation [56], and inhibits the production of nitric oxide (NO) and of pro-inflammatory cytokines such as TNF-a [57] thus exerting a neuroprotective effect in the CNS. In astrocytes, IL-4 also has diverse functions, such as the inhibition of LPS-induced NO synthesis due to blocking of inducible nitric oxide synthase (iNOS) expression. A similar effect was seen in microglia, and additionally, the induction of adhesion molecules such as ICAM-1 and the induction of NGF secretion by cortical and cerebellar astrocytes have also been observed [58]. Oligodendrocytes, the cells responsible for myelin synthesis in the CNS, are also a source of cytokines and NO under inflammatory conditions [59]. Both IL-4 and IL-10 protect against cell death, also by modulating iNOS expression and NO production following LPS/IFN-g stimulation [23]. In addition, IL-4 has been shown to increase oligodendrocyte branching and maturation through microglia interaction [60]. However, analysis of neurites from newly formed neurons in cocultures of neuronal progenitor cells (NPCs) and microglia previously treated with IL-4, did not significantly differ from the culturing of NPCs alone [60]. In summary, IL-4 regulates cellular survival, proliferation and branching in the PNS as well as in the CNS, and promotes peripheral axon regeneration. 4.2. Interleukin-10 IL-10 is one of the most vastly studied ‘‘anti-inflammatory’’ cytokines and is mainly produced by monocytes/macrophages, Th2 cells and B cells as an 18kDa protein. It inhibits monocytes/ macrophages and modulates lymphocyte and neutrophil responses as well as cytokine production. More detailed information about its structure and function has been widely reviewed previously [9]. Treatment with IL-10 increases both cell survival and axonal regeneration after PNS injury. For example, after facial nerve axotomy, flow cytometry analyses showed that the levels of IL-10, amongst other cytokines, were increased [61], providing a protective effect from cell death following injury to facial motor neurons (FMN). It was shown that IL-10 works cooperatively with CD4+ T cells, with T cells being involved in maintaining glialderived IL-10 levels in the vicinity of injured cell bodies [62]. In a model of sciatic nerve injury, enhanced axon regeneration and reduced glial scarring after administration of IL-10 was found [63], while in retinal ganglion cells (RGCs), active glial cells express iNOS, synthesizing high levels of NO which is toxic to neuronal cells. IL-10 was able to increase the survival of axotomized RGCs and the integrity of the axons of the nerve fiber layer (NFL) in vivo, via the inhibition of NO synthesis, leading to a decrease in free

P.M. Vidal et al. / Cytokine & Growth Factor Reviews 24 (2013) 1–12

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Table 4 Selected effects of IL-4, IL-10, IL-13, LIF and TGF-b on neurite outgrowth in PNS and CNS. Cytokine

Effect

Experimental model

Specie

Reference

IL-4

Promotion of facial motoneuron survival after axotomy In the presence of neurotrophins, IL-4 modulates peripheral axon regeneration in a dose dependent manner Enhanced survival of axotomized RGCs, reduction of monocytes in the NFL and inhibition of NO production Significantly increased number and length of cell - bearing processes and increased cellular survival No effects on neurite length of newly formed neurons from NPCs, but increases oligodendrocyte branching IL-10 enhances survival of axotomized RGCs and improves axonal integrity of the nerve fiber layers by inhibition of NO production Increases of axonal regeneration and long term (up to 8 weeks) improvement of locomotor skills after a single dose of IL-10 (30 min after injury) Reduction of inflammation and neuronal damage after SCI by systemic administration of IL-10 Reduction of spinal tissue damage at 12 weeks post injury by IL-10 (30 min before injury), MP, or a combination of both agents, without improvement of hindlimb locomotor function Increase of neuronal survival and improvement of motor function after IL-10 vector injections (30 min after injury) up to 6 weeks after injury LIF induces an increase of phosphorylated Akt473 but no effect on dendrite growth in hippocampal neurons LIF increases diameter, number and conduction velocity of the regenerated sciatic nerve LIF augments corticospinal axon growth and expression of NT-3 after spinal cord injury LIF is unable to promote neurite arborization and growth by itself; however, the addition of LIF to cells treated with NGF increases neurite length and induces neurite restructuration in vitro. Furthermore LIF is required for regeneration of injured sensory neurons in vivo TGF-b1 reduces neurite extension of cerebellar neurons TGF-b has a weak positive effect on neurite outgrowth in astrocytes TGF-b1 inhibits the neurite outgrowth of cortical neurons in vitro via activation of RhoA/ Rho-kinase pathway; while in vivo anti-TGF-b1 treatment promotes growth and preservation of raphespinal axons caudally to the lesion site and improves locomotor skills TGF-b alone does not increase neurite outgrowth in DRG neurons, but it is involved in the stimulation of neurite growth by SPARC

Peripheral nerve injury (facial nerve axotomy)

Adult mice

[25]

DRG outgrowth model

E13 mouse

[47]

Transection of the optic nerve

Adult rats

[26]

Retinal cell culture

Postnatal rats (1 or 2 days)

[50]

Co-culture of IL-4 treated microglia with NPCs or oligodendrocyte

Adult mice

[62]

Transection of the optic nerve

Adult rats and rat retinal ganglion cell line

[26,66]

Moderate SCI at T9 and T10 level in

Adult rats

[16]

Quisqualic acid (QUIS), a model of SCI, between spinal segments T12-L2

Adult rats

[149]

Contusion spinal cord injury at T8 level

Adult rats

[78]

Laminectomy at T11-12 vertebral level and also lateral hemisection of the spinal cord at T13 level Mouse hippocampal neurons from E16 embryos

Adult rats

[76]

E16 mouse

[99]

Transected sciatic nerve model

Adult rats

[93]

Hemisection at T7 level

Adult rats

[97]

Cultures of adult DRG neurons Crush injury of the sciatic nerve in LIF +/+ and LIF -/-mice of both sexes

Adult rats Adult rats

[96] [96]

Co-culture of cerebral astrocytes and meningeal fibroblasts with cerebellar neurons P1 rat DRG explants in 3-dimensional astrocytes cultures For the in vitro assay cortical neurons from E18 were used and for the in vivo experiments contusion spinal cord injury at vertebral level T9-T10 Mice lumbar DRG from E13.5 embryos

Postnatal rats (1 or 2 days) Postnatal rats (1 day) E18 rats and adult rats E13.5 mouse

[107] [31] [102] [100]

IL-10

LIF

TGF-b

Abbreviations: Dorsal root ganglia (DRG), embryonic day (E), interleukin-4 (IL-4), interleukin-10 (IL-10), leukemia inhibitory factor (LIF), nerve growth factor (NGF), neurotrophin factor 3 (NT-3), methylprednisolone (MP), nerve fiber layer (NFL), neuronal progenitor cells (NPCs), nitric oxide (NO), retinal ganglion cells (RGCs), secreted protein acidic rich in cystein (SPARC), spinal cord injury (SCI), thoracic (T), transforming growth factor-b (TGF-b). Remark: no published information is available that directly relates IL-13 with axonal/neurite outgrowth; therefore no data on IL-13 are included.

radicals [26]. Finally, IL-10 exerts a dose and time dependent increase on RGCs survival by inhibiting apoptotic cell death by a mechanism that involves activation of the STAT3 pathway, without any effect on cellular proliferation [64]. It also induces a rapid decrease in caspase-3 activity, but has no effect on intracellular Ca2+ levels, whose levels are able to modulate caspase-3 induction, suggesting that IL-10 has an intrinsic ability to inhibit directly or indirectly, caspase-3 activity. It has also been suggested that cell death occurs in neurons when NF-kB is permanently activated, for

instance after trauma [7] or toxic concentrations of glutamate [65], and the inhibition of NF-kB activity results in inactivation of caspases [66]. IL-10 blocks the glutamate mediated NF-kB DNA binding activity in neuronal [67] and nonneuronal cells [68], suggesting that IL-10 reduces or prevents the activity of caspase-3 and NF-kB activity. In neurons of the CA1 hippocampal region, it has been shown that combined treatment with hypothermia and IL-10 may induce neuronal survival [69], as well as in astrocytes, in which IL-10 and IL-13 inhibit apoptosis through the stimulation of

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phosphatidylinositol 3-kinase (PI3K) [70]. In microglial cells, IL-10 also serves as a survival factor by inducing STAT3 phosphorylation while not enhancing proliferation [71]. IL-10 can significantly reduce vulnerability of neurons to CNS ischemia and trauma [72,73]. For example, in vitro, in spinal cord neurons, IL-10 induces a number of signaling cascades through binding to its receptor thereby inducing NF-kB and transcription of the anti-apoptotic Bcl-2 and Bcl-xL genes, while after exposure to glutamate, it blocks cytochrome c release and caspase cleavage [74,75]. Furthermore, these direct neuroprotective effects of IL-10 have been demonstrated in a retinal ganglion cell line [64], oligodendroglial cells [23] and cerebellar granule cells [67]. Similarly, in vivo, IL-10 overexpression in the spinal cord using a herpes simplex virus based vector resulted in increased neuronal survival and improved motor function for up to 6 weeks after injury [74]. Others showed that also a single dose of IL-10 given intraperitoneally during the first period after SCI (considered as the early inflammatory response) may be neuroprotective, by attenuating TNF-a protein levels between 1 h and 1 day after SCI in rats. This was also seen to induce an improvement in the hind limb motor function for up to two months after SCI [16]. IL-10 is also able to reduce TNF-a and IL-1 levels and improve the outcome after traumatic brain injury [72]. However, while most studies show neuroprotective effects of IL-10 after SCI, improved locomotor recovery was found in some, but not all SCI rodents [16,74,76]. In summary, IL-10 is implicated in cell survival (CNS and PNS) and may promote recovery after SCI. 4.3. Interleukin-13 IL-13 is a 10 kDa protein which mediates its effects via the IL-13 receptor, expressed on human B cells, basophils, eosinophils, mast cells, endothelial cells, fibroblasts, monocytes, macrophages, respiratory epithelial cells, and smooth muscle cells. Two types of receptors exist, one being a heterodimer of IL-13Ra1 and IL-4Ra which can bind to IL-4 as well, and a second type consisting of an IL-13Ra2 chain [77]. Signal transduction via IL-4Ra is known to be responsible for most of the functional characteristics of IL-4 and IL-13. Both cytokines activate JAK/STAT signaling, and it is already known that IL-4 and STAT6 are involved in promotion of axonal regeneration [25]. It is therefore probable that IL-13 and STAT6 may also contribute to axonal regeneration. In addition, administration of recombinant IL-13 leads to the enhancement of macrophage development and function [41] which in turn can modulate production of pro-inflammatory cytokines such as IL-6, TNFa, IL12 and the ‘‘anti-inflammatory’’ cytokine IL-10 [78]. This cytokine is also capable of modulating the inflammatory response by suppressing the production of inflammatory mediators such as IL-1b, TNF-a and IL-6 from microglia in vitro and in vivo [39,40]. Among these cytokines, IL-10 and TNF-a have been implicated in axonal regeneration in both a beneficial and detrimental way [16,76]. In the CNS, IL-13 mRNA levels are upregulated within hours after CNS trauma (our own unpublished data). The effect of IL-13 on neuronal survival is not clear and both positive [79–81] and negative effects [82] have been reported. In the brain, neurons and microglia act cooperatively to downregulate brain inflammation by inducing IL-13 expression in microglia and enhancing COX-2 expression [80] which has been previously associated with cytotoxicity in brain diseases [83], thereby causing death of activated microglia and leading to an increase in neuronal survival [79,81]. Using thrombin to activate microglia leads to an upregulation of IL-13 and ROS levels, resulting in a decrease in neuronal survival. On the other hand, the blockade of IL-13 reduces inflammatory cytokine expression, thus increasing neuronal survival in the hippocampus in vivo [82]. One possible reason

given by the authors may be that thrombin and LPS activate distinct signaling pathways when inducing microglia activation and IL-13 may influence differentially these distinct pathways. It has also been shown that IL-13 (in a dose dependent manner) can influence the morphology of macrophage colonies, B cells and monocytes; cells growing in the absence of IL-13 first showed a large round morphology whereas cells cultured in its presence, were more flattened and formed extensive processes and cellular aggregates [84], acting as cells in the late stage of macrophage colony formation. Similarly, it has been shown that IL-13 can modulate 3 stages of the B cell maturation process: early activation phase, proliferation and differentiation [85] while others showed that in human monocytes, IL-13 can modulate the development of long processes and alter survival [86]. In the CNS, activated microglia or macrophages seem to create a favorable environment for regeneration by degrading inhibitory molecules which prevent axonal growth and reactive neurite sprouting [87]. It is possible that modulating this microglial response after CNS injury by factors such as IL-13, could be helpful to either prevent or degrade depositions of these regeneration-inhibiting factors. In summary, IL-13 has been implicated in cellular survival, with both detrimental and beneficial effects reported. However, no studies have been performed yet to investigate any direct effect of this cytokine on axonal regeneration. 4.4. Leukemia inhibitory factor LIF belongs to the IL-6 cytokine family, which transduce their signals through the gp130 subunit and the low affinity LIF receptor (LIFR). It has hematopoietic, neuronal and endocrine functions [88]. LIF is transported retrogradely by sensory and sympathetic neurons; this transport is increased after nerve lesions [89,90]. LIF is absent from the adult mammalian nervous system but it is upregulated after injury to the sciatic nerve [90]. This upregulation seems to enhance the regeneration of the transected sciatic nerve by improving the conduction velocity of the regenerated nerve and the number of myelinated fibers [91]. In addition, LIF also inhibits the transport of ciliary neurotrophic factor (CNTF) in lesioned sciatic nerve [92]. LIF is also involved in cell viability and supporting survival of sensory neurons [93], possibly via direct mechanisms, since LIF binds specifically to DRG neurons in vitro [89]. Moreover it can also promote survival of sensory and motor neurons after axotomy in vivo [91]. However in vitro studies have shown that LIF has no effect on neurite outgrowth in DRG cells, either alone or in combination with NGF; moreover, LIF initiates arborization of sensory neurons [94]. After CNS injury, such as SCI and cortical lesioning, there is also an increase of LIF mRNA levels (Fig. 1). In contrast, mRNA levels of the LIFR decrease slightly after cortical injury. Using in situ hybridization, it was possible to identity that LIF is mainly expressed by astrocytes and to some extent also by microglia cells after cortical injury and SCI [17]. This upregulation seems to have a positive effect after SCI. Fibroblasts genetically modified to produce LIF were embedded in a collagen matrix and grafted to the lesioned spinal cord, promoting corticospinal axon growth and resulting in an increase of NT-3 [95]. These findings support the hypothesis that ‘‘anti-inflammatory’’ factors not only directly mediate regeneration, but can also indirectly regulate the nervous system response to injury by increasing the production of trophic factors via the cytokine/neurotrophin axis [45]. Using LIF knockout mice, it was shown that LIF plays an important role in the initial infiltration of inflammatory cells after cortical and sciatic nerve injury, acting as a chemotactic factor for macrophages and activation of microglia and astrocytes [96]. In hippocampal neurons, LIF induces activation of signaling pathways associated

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with neuroprotection and regeneration, increasing STAT3 levels and phosphorylation of Akt473. However, it has no effect on dendrite morphology and outgrowth, compared to other hematopoietic cytokines, such as erythropoietin [97]. In summary, LIF increases regeneration after PNS and CNS injury (directly or indirectly) and modulates cell survival. 4.5. Transforming growth factor-b TGF-b is synthesized as an inactive precursor with three isoforms: TGF-b1, b2 and b3. The active molecule is a 25 kDa protein which is constitutively expressed in many cell types, such as platelets, monocytes and T cells. It is mainly involved in inhibition of monocytes/macrophages and pro-inflammatory cytokine synthesis [9]. We will focus more on the effects of TGF-b1, since this ‘‘anti-inflammatory’’ factor has been studied in a greater depth than the other isoforms. In the PNS, TGF-b1 has different effects depending of the cellular target. Using DRG explants in three-dimensional cultures of astrocytes, TGF-b was found to promote neurite outgrowth. However, when these explants were treated with IL-1a or b, TGF-b neutralized the outgrowth promoted by IL-1a or IL-1b [29]. In Schwann cells, secreted protein acidic and rich in cysteine (SPARC), a matricellular protein, mediates outgrowth via TGF-b and laminin-1 mechanisms [98]. The addition of TGF-b1 to pre-treated astrocyte cultures has no effect on cellular migration, but suppresses the migrationpromoting action of IL-1 and basic fibroblast growth factor (bFGF) on primary oligodendrocyte precursors and astrocytes [29]. TGFb1 also suppresses the proliferation of astrocytes [99] and inhibits neurite outgrowth via activation of RhoA/Rho kinase signaling in cortical neurons in vitro [100]. As for the other ‘‘anti-inflammatory’’ factors mentioned previously, TGF-b levels are increased after SCI, but later than the other factors (Fig. 1), and also after brain injury, specifically TGF-b1 [99]. TGF-b was also seen to be involved in glial scar formation. For example, after brain injury TGF-b, via TGF-b receptor/Smad signaling, induces an increase in the expression of neurocan, one of the inhibitory molecules which mediates glial scar formation in astrocytes [101]. Similarly, fibrinogen, a growth inhibitory factor known to be involved in the glial scar formation, acts as a carrier of latent TGF-b to the injury sites, facilitating the interaction of TGF-b with astrocytes and the subsequent activation of TGF-b [102]. Thus, TGF-b is known to enhance the production of abundant factors after CNS injury, which may limit spontaneous axon regeneration and mediate neuronal survival after axonal injury [103]. Nevertheless, treatment with TGF-b1 and b2 antibodies in a unilateral nigrostriatal transection model, leads to a reduction of astrocyte response and reduction of gliosis. However, this combination did not enhance the regeneration of dopaminergic axons [104]. Similar results were obtained after SCI, where treatment with TGF-b1 neutralizing antibodies after injury lead to an enhancement of locomotor skills and reduction of glial scar formation. However, this improvement did not lead to an increase in outgrowth of the corticospinal tract fibers, but presumably may be responsible for restoring the injured serotonergic fibers [100]. Finally, in vitro, TGF-b1 enhances proliferation and the formation of clusters using a meningeal fibroblast and astrocyte in vitro model of the scar. The cellular clusters of extracellular matrix molecules and axonal growth inhibitory factors accumulate similarly to that in the glial scar following CNS injury. In clusters of cerebellar neurons, neurite extension is inhibited in the presence of TGF-b1 on flat meningeal fibroblasts [105]. In summary, TGF-b may either promote or inhibit neurite outgrowth and may modulate cellular proliferation and migration.

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5. Concluding remarks Many ‘‘anti-inflammatory’’ factors are down- or upregulated after CNS and PNS injury, either immediately or at a later stage. Their expression at one particular time point after injury appears to be crucial in defining whether or not regeneration is going to take place. Most of the ‘‘anti-inflammatory’’ factors reviewed here participate in the regulation of cell survival, proliferation and migration (IL-4, IL-10, IL-13, LIF and TGF-b), promoting in this way regeneration after injury. Others have a more direct effect on neurite regeneration (IL-4, LIF and TGF-b). The understanding in vitro and also in vivo of how cytokines interact with other cytokines as well as with the injured environment could bring to light their role after injury. Such information would also help in determining the most effective ways to utilize their properties in order to stimulate and enhance regeneration. Today the number of new therapies involving a combination of different molecules, all aiming to promote axonal regeneration, has increased considerably. Thus, the modulation of the inflammatory phases, possibly through cytokine modulation, could play a key role in regenerative therapies. In conclusion, the therapeutic use of the cytokines discussed here is still extremely limited due to their phase-specific and compartment-specific effects. Instructive ‘‘interactomics’’ models are still a major necessity to develop complex neuro-immunomodulatory therapies. References [1] Goldsby R, Kindt R, Psborne B. Kuby Immunology 2000. [2] Sholl-Franco A, da Silva AG, Adao-Novaes J. Interleukin-4 as a neuromodulatory cytokine: roles and signaling in the nervous system. Annals of the New York Academy of Sciences 2009;1153:65–75. [3] Besser M, Wank R. Cutting edge: clonally restricted production of the neurotrophins brain-derived neurotrophic factor and neurotrophin-3 mRNA by human immune cells and Th1/Th2-polarized expression of their receptors. Journal of Immunology 1999;162:6303–6. [4] Kelly-Welch AE, Hanson EM, Boothby MR, Keegan AD. Interleukin-4 and interleukin-13 signaling connections maps. Science 2003;300:1527–8. [5] Gilman A, Goodman L, Hardman J, Limbird L. Goodman & Gilman’s the Pharmacological Basis of Therapeutics 2001. [6] Hendrix S, Nitsch R. The role of T helper cells in neuroprotection and regeneration. Journal of Neuroimmunology 2007;184:100–12. [7] Bethea JR, Castro M, Keane RW, Lee TT, Dietrich WD, Yezierski RP. Traumatic spinal cord injury induces nuclear factor-kappaB activation. Journal of Neuroscience 1998;18:3251–60. [8] Maini RN, Taylor PC. Anti-cytokine therapy for rheumatoid arthritis. Annual Review of Medicine 2000;51:207–29. [9] Opal SM, DePalo VA. Anti-inflammatory cytokines. Chest 2000;117:1162–72. [10] Matzinger P. Friendly and dangerous signals: is the tissue in control? Nature Immunology 2007;8:11–3. [11] Yiu G, He Z. Glial inhibition of CNS axon regeneration. Nature Reviews Neuroscience 2006;7:617–27. [12] Tom VJ, Steinmetz MP, Miller JH, Doller CM, Silver J. Studies on the development and behavior of the dystrophic growth cone, the hallmark of regeneration failure, in an in vitro model of the glial scar and after spinal cord injury. Journal of Neuroscience 2004;24:6531–9. [13] Ming GL, Wong ST, Henley J, Yuan XV, Song HJ, Spitzer NC, Poo MM. Adaptation in the chemotactic guidance of nerve growth cones. Nature 2002;417:411–8. [14] Moalem G, Monsonego A, Shani Y, Cohen IR, Schwartz M. Differential T cell response in central and peripheral nerve injury: connection with immune privilege. FASEB Journal 1999;13:1207–17. [15] Donnelly DJ, Popovich PG. Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury. Experimental Neurology 2008;209:378–88. [16] Bethea JR, Nagasima H, Acosta MC, Briceno C, Gomez F, Marcillo AE, Loor K, Green J, Dietrich WD. Systemically administered interleukin-10 reduces tumor necrosis factor-alpha production and significantly improves functional recovery following traumatic spinal cord injury in rats. Journal of Neurotrauma 1999;16:851–63. [17] Pineau I, Lacroix S. Proinflammatory cytokine synthesis in the injured mouse spinal cord: multiphasic expression pattern and identification of the cell types involved. Journal of Comparative Neurology 2007;500:267–85. [18] Schwab JM, Brechtel K, Mueller CA, Failli V, Kraps HP, Tuli SK, Schluesener HJ. Experimental strategies to promote spinal cord regeneration—an integrative perspective. Progress in Neurobiology 2006;78:91–116.

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Evi Lemmens studied Biomedical Sciences at Hasselt University, Belgium (1999–2003), and obtained her Ph.D. at Maastricht University, the Netherlands, in 2008. During her doctoral studies, she investigated the long-term effects of early-life febrile seizures on brain development and behavioral outcome. She currently works at Hasselt University as a postdoctoral fellow of the Flanders Research Foundation (FWO). Her research focuses on neuro-immune interactions to promote regeneration after nervous system trauma such as spinal cord injury and traumatic brain injury.

Dearbhaile Dooley obtained her B.Sc. (Hons) from the Discipline of Anatomy at the National University of Ireland, Galway in 2010. She is currently a Ph.D. student in the Dept. of Morphology at the Biomedical Research Institute at Hasselt University, Belgium. Her research focuses on the immunomodulatory role of stem cells in spinal cord injury and how the combination of stem cells with pro- and anti-inflammatory cytokines, alters the surrounding microenvironment.

Sven Hendrix M.D. is a full professor for anatomy and director of the Doctoral School for Medicine & Life Sciences at Hasselt University, Belgium. He obtained his M.D. degree at the Charite´ Berlin, Germany. His research group studies neuroinflammation with a special focus on CNS trauma and axonal regeneration.