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Multiple sclerosis (MS) is a disabling inflammatory demyelinating disease of the central nervous system (CNS) that primarily affects young adults. Available therapies can inhibit the inflammatory component of MS but do not suppress progressive clinical disability. An alternative approach would be to inhibit mechanisms that drive the neuropathology of MS, which often includes the death of oligodendrocytes, the cells responsible for myelinating the CNS. Identification of molecular mechanisms that mediate the stress response of oligodendrocytes to optimize their survival would serve this need. This study shows that the neurotrophic cytokine leukemia inhibitory factor (LIF) directly prevents oligodendrocyte death in animal models of MS. We also demonstrate that this therapeutic effect complements endogenous LIF receptor signaling, which already serves to limit oligodendrocyte loss during immune attack. Our results provide a novel approach for the treatment of MS.

Cytokines that signal through the leukemia inhibitory factor (LIF) receptor, such as LIF and ciliary neuronotrophic factor, have a wide range of roles within both the developing and mature nervous system. They play a vital role in the differentiation of neural precursor cells into astrocytes and can prevent or promote neuronal differentiation. One of the conundrums regarding signalling through the LIF receptor is how it can have multiple, often conflicting roles in different cell types, such as enhancing the differentiation of astrocytes while inhibiting the differentiation of some neuronal cells. Factors that can modulate signal transduction downstream of cytokine signalling, such as "suppressor of cytokine signalling" proteins, which inhibit the JAK/STAT but not the mitogen-activated protein kinase pathway, may therefore play an important role in determining how a given cell will respond to cytokine signalling. This review discusses the general effects of cytokine signalling within the nervous system. Special emphasis is placed on differentiation of neural precursor cells and the role that regulation of cytokine signalling may play in how a given precursor cell responds to cytokine stimulation.

A number of growth factor families have been implicated in normal inner ear development, auditory neuron survival and protection. Several growth factors, including transforming growth factor-beta5 (TGF-beta5) and TGF-beta3, neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF) and leukemia inhibitory factor (LIF) were tested for their ability, individually or in combination, to promote auditory neuron survival in dissociated cell cultures of early rat post-natal spiral ganglion cells (SGCs). The results indicate that at discrete concentrations all growth factors act in an additive fashion and some in synergy when promoting neuronal survival. These findings support the hypothesis that growth factors from different families may be interdependent when sustaining neuronal integrity.

In the developing forebrain, mounting evidence suggests that neural stem cell proliferation and differentiation is regulated by growth factors. In vitro in the presence of serum, stem cell proliferation is predominantly mediated by fibroblast growth factor-2 (FGF-2) whereas neuronal differentiation can be triggered by FGF-1 in association with a specific heparan sulphate proteoglycan. On the other hand, astrocyte differentiation in vivo and in vitro appears to be dependent on signalling through the leukaemia inhibitory factor receptor (LIFR). The evidence suggests that in the absence of LIFR signalling, the stem cell population is present at approximately the same frequency and can generate neurons but is blocked from producing astrocytes that express glial fibrillary acidic protein (GFAP) or have trophic functions. The block can be overcome by other growth factors such as BMP-2/4 or interferon-gamma, providing further evidence that the inhibition to astrocyte development does not result from loss of a precursor population. Signalling through the LIFR, in addition to stimulating astrocyte differentiation, may also inhibit neuronal differentiation, which may explain why this receptor is expressed at the earliest stages of neurogenesis. Another signalling system which also exerts its influence on neurogenesis through active inhibition is Delta-Notch. We show in vitro that at high cell densities which impede neuronal production by FGF-1, lowering the levels of expression of the receptor Notch by antisense oligonucleotide results in a significant increase in neuronal production. Thus, stem cell differentiation appears to be dependent on the outcome of interactions between a number of signalling pathways, some which promote specific lineages and some which inhibit.

The differentiation of precursor cells into neurons or astrocytes in the developing brain has been thought to be regulated in part by growth factors. We show here that neural precursors isolated from the developing forebrain of mice that are deficient in the gene for the low-affinity leukemia inhibitory factor receptor (LIFR-/-) fail to generate astrocytes expressing glial fibrillary acidic protein (GFAP) when cultured in vitro. Precursors from mice heterozygous for the null allele show normal levels of GFAP expression. These findings support the in vivo findings that show extremely low levels of GFAP mRNA in brains of embryonic day 19 LIFR-/- mice. In addition, monolayers of neural cells from LIFR-/- mice are far less able to support the neuronal differentiation of normal neural precursors than are monolayers from heterozygous or wild-type animals, indicating that endogenous signaling through the LIFR is required for the expression of both functional and phenotypic markers of astrocyte differentiation. LIFR-/- precursors are not irreversibly blocked from differentiating into astrocytes: they express GFAP after long-term passaging or stimulation with bone morphogenetic protein-2. These findings strongly implicate the LIF family of cytokines in the regulation of astrocyte differentiation and indeed the LIF-deficient animals show a significant reduction in the number of GFAP cells in the hippocampus. However, because this reduction is only partial it suggests that LIF may not be the predominant endogenous ligand signaling through the LIFR.

Following axotomy most medial septal neurons in the adult rat brain have dramatically reduced numbers of choline acetyltransferase (ChAT) positive neurons. Since leukemia inhibitory factor (LIF) promotes cholinergic expression in several neuronal populations, the aim of this study was to determine if LIF would continue to support cholinergic expression in axotomized medial septal neurons. Mini-osmotic pumps were used to infuse saline or LIF into the lateral cerebral ventricle. Counts of ChAT and low-affinity nerve growth factor (p75NGFR) immunostained neurons indicated that LIF-treated animals retained ChAT expression in > 90% of axotomized neurons whereas in saline-infused animals this was < 30%. Also, LIF was equally effective in maintaining p75NGFR expression levels in axotomized medial septal neurons.

The survival of auditory neurones depends on the continued supply of trophic factors. Early postnatal spiral ganglion cells (SGC) in a dissociated cell culture were used as a model of auditory innervation to test the trophic factors leukaemia inhibitory factor (LIF) and neurotrophin-3 (NT-3) for their ability, individually or in combination, to promote neuronal survival. The findings suggest that LIF supports neuronal survival in a concentration-dependent manner. Moreover LIF potentiated NT-3-mediated spiral ganglion neuronal survival in a synergistic fashion.

The cytokine leukemia inhibitory factor (LIF) favors the survival and growth of axons in vitro and in vivo. Fibronectin has been shown to enhance nerve regeneration when added in combination with various growth factors including LIF. The goal of this study was to evaluate the effect of LIF plus fibronectin on the regeneration of transected nerve and functional recovery of reinnervated skeletal muscle, in one experimental model of peripheral nerve repair, at two recovery times. The rat sciatic nerve was cut at mid-thigh level and a silicone cuff containing either saline (control), LIF, or LIF plus fibronectin (L+F) was used to bridge the proximal and distal nerve stumps leaving a 1 cm gap between them. Rats were then explored at 6 or 12 weeks following the initial surgery. Regenerating nerves were assessed by measuring the diameter of myelinated axons, conduction velocity, and number of myelinated fibers. Muscle reinnervation was assessed by measuring muscle mass, force of contraction, and histologically for changes in muscle fiber type (type I and type II). In this report we demonstrate that at 6 weeks there were significant increases in 1) nerve conduction velocity, 2) myelinated axon diameter, and 3) number of myelinated axons over that of control (saline-treated) animals. Both LIF groups demonstrated a shift in type II muscle fiber area compared to saline-treated controls, with the L+F group having a significant increase in muscle mass. At 12 weeks there was an improved recovery over and above that demonstrated at 6 weeks. Muscle mass was 65% and 42% greater than control for LIF and L+F, respectively. Force of contraction, conduction velocity, myelinated fiber number, and diameter were also significantly greater for both LIF- and L+F-treated rats than saline-treated rats. These results demonstrate that LIF significantly improves the regeneration of damaged peripheral nerves and the preservation of muscle viability, resulting in greatly enhanced recovery of skeletal muscle function.

The effects of leukemia inhibitory factor (LIF) on muscle atrophy and the reinnervation of muscle were investigated. The rat medial gastrocnemius (MG) nerve was either cut (denervation groups), crushed (reinnervation group) or left intact (normal group). Muscles were injected with LIF in phosphate buffered saline (PBS) containing pluronic gel, the contralateral control muscles were injected with the vehicle alone. The muscles from the LIF-injected denervation groups were analysed for muscle fibre area; this was found to be significantly larger than controls. The greatest change was observed in the reinnervation group, where the muscle fibre area following LIF treatment was 53% (Type 1) and 84% (Type 2) greater. In addition, nerve fibre diameters were analysed in the reinnervation-treated group and these were also significantly larger. However, LIF injected into normally innervated muscle resulted in a decrease in muscle fibre area. These results show that LIF ameliorates denervation-induced muscle atrophy and improves regeneration of muscle and nerve.

Leukaemia inhibitory factor (LIF) and Interleukin-6 (IL-6) are multifunctional cytokines that are related on the basis of their predicted structural similarities and shared signal transducing receptor components. Both these factors stimulate myoblast proliferation, and whereas LIF is neurotrophic for sensory neurons, and for the motor neurons which innervate muscle, IL-6 has only been reported to act on a population of septal neurons in the brain. We have looked at the effect of peripheral nerve trauma on the expression of these factors. We show here that whereas LIF and IL-6 mRNAs are expressed in low levels in normal sciatic nerve and skeletal muscle, there is significant up-regulation in the nerve segments after injury, with proximally and distally. There is also an increase in LIF and IL-6 expression in the denervated muscle located largely in the muscle cells. In addition, while there is retrograde axonal transport of LIF by the sciatic nerve, IL-6 is not retrogradely transported, and as a result, IL-6 does not stimulate the survival of sensory neurons in vitro. Both growth factors are produced by Schwann cells. These results show a rapid response in the expression of these genes after injury and suggest that LIF and IL-6 act as trauma factors but with different roles in injured peripheral nerve.

Previously we have shown that leukaemia inhibitory factor (LIF) potentiates the development of murine spinal cord neurons in vitro, suggesting that it, or related factors, may play an important regulatory role in neuronal development. We have further investigated this role and show here that the generation of neurons in cultures of embryonic day 10 spinal cord cells is inhibited by antibodies to the beta subunit of the LIF receptor. Since there are more undifferentiated precursors in antibody-treated cultures than in control and LIF-treated cultures, it is concluded that the primary action of LIF, or related molecules, is to promote neuronal differentiation, not precursor survival. In addition, the failure of LIF to support neuronal survival in the period immediately following differentiation suggests that the increased numbers of neurons generated with LIF are not attributable to its neurotrophic action. By selecting neuronal precursors on the basis of their inability to express class 1 major histocompatibility complex molecules, it was shown that LIF acted directly upon these cells and not via an intermediary cell. LIF also appears to be involved in regulating the differentiation of astrocytes, since it increases the number of glial fibrillary protein (GFAP)-positive cells present in the cultures and since the spontaneous production of GFAP-positive cells is blocked by antibodies to the LIF beta receptor. These findings suggest that LIF or related factors promote the differentiation of neural precursors in the spinal cord, but that they are not involved in preferentially promoting precursors down a specific differentiation pathway.

Two of the key early events in the development of the peripheral nervous system are the proliferation of neural crest precursor cells and their subsequent differentiation into different neural cell types. We present evidence that members of the fibroblast growth factor family, (FGF1 or FGF2) act directly on the neural crest cells in vitro to stimulate proliferation in the presence of serum. These findings correlate with in situ hybridisation analysis, which shows FGF2 mRNA is expressed in cells both in the neural tube and within newly formed sensory ganglia (dorsal root ganglia, DRG) at embryonic day 10 in the mouse, when neural crest precursors are proliferating within the DRG. This data infers an autocrine/paracrine loop for FGF regulation of proliferation. Evidence supporting this notion is provided by the finding that part of the endogenous proliferative activity in the NC cultures is related to FGF. It was also found, in early neural crest cultures, that exogenous FGF completely inhibited neuronal differentiation, probably as a direct consequence of its mitogenic activity. In order to stimulate neuronal differentiation significantly, it was necessary to remove the FGF and replace it with leukemia inhibitory factor (LIF) or related factors. Under these conditions, 50% of the cells differentiated into neurons, which developed a sensory neuron morphology and were immunoreactive for the sensory markers CGRP and substance P. These data support a model of neural crest development, whereby multipotential neural crest precursor cells are stimulated to divide by FGF and subsequent development into sensory neurons is regulated by LIF or other cytokines with a similar signalling mechanism.

The death of spinal motoneurones after axotomy provides a useful model for studying novel factors which prevent motoneurone loss in vivo. Peripheral nerves of newborn rats were unilaterally transected and treated with either a vehicle solution or leukaemia inhibitory factor (LIF). Compared with the vehicle controls, treatment with a gelfoam containing LIF significantly reduced motoneurone loss: from 38% to 22% after 3 days and from 55% to 38% after 7 days. The loss of motoneurones was further reduced by placing the LIF-containing gelfoam inside a silicone chamber: from 39% to 15% after 7 days, which represented a 62% rescue. Thus, LIF is a potential therapeutic agent for preventing the loss of injured or diseased motoneurones.

Leukemia inhibitory factor (LIF) has several characteristics of a neurotrophic factor for sensory neurons. Here we have investigated whether LIF also supports the survival of axotomised sensory neurons in vivo. Newborn rat pups received a unilateral sciatic nerve transection and the injury site was treated with gelfoam soaked in phosphate buffered saline (PBS), nerve growth factor (NGF), or LIF. Neuronal nucleoli in the L5 dorsal root ganglia were counted, appropriate corrections applied, and the resultant neuronal loss expressed as a percentage of the contralateral intact side. In animals where LIF was administered neuronal loss was significantly reduced: 2 days after LIF treatment neuronal loss was 19.5% compared to 43% in PBS-treated animals; 3 days after LIF treatment neuronal loss was 20.4% compared to 40.2% in PBS-treated animals; however, 7 days after LIF treatment there was no significant reduction in the number of neurons lost. The degree of rescue of sensory neurons in vivo by LIF was found to be similar to NGF, which was not surprising as both factors supported the survival of a similar population of sensory neurons in vitro. Rescue was not observed when LIF-containing gelfoam was placed away from the axotomised nerve, suggesting that LIF's action may be associated with its retrograde transport or direct signalling at the site of nerve injury.

Leukemia inhibitory factor (LIF) was recently shown to stimulate the generation of sensory neurons from the murine neural crest in vitro. Here, we examine the respective activities of LIF and nerve growth factor (NGF) throughout the embryonic development of sensory neurons in dorsal root ganglia (DRG) and neural crest. In cultures of embryonic day 12 (E12) DRG, which contain sensory neuron precursor cells, a combination of both LIF and NGF are required for the differentiation of mature sensory neurons from their neurofilament negative (NF-) precursors. The primary differentiation step from NF- cell to NF+ immature neuron is promoted by LIF, whereas the survival and further maturation of the newly differentiated neurons depends on NGF. In cultures of sensory neurons isolated at the time of target innervation (E14 and E15 DRG), the survival of the majority of the neurons is dependent on NGF. However, LIF acts as a survival agent for a discrete population of NGF non-responsive neurons. From E16, the number of neurons maintained by LIF increases to > 90% by birth. Consistent with the in vitro observations, LIF mRNA could be detected at early developmental stages (E12-E13), within the spinal column and DRG as well as the limbs and, later (after E15), in areas of sensory innervation (skin, limbs, feet and gut). This supports the idea that LIF, as well as NGF, may regulate sensory development in vivo.

Recent evidence from our laboratory has shown that leukemia inhibitory factor (LIF) can act early in peripheral nervous system development. We have investigated a potential role of LIF in the developing spinal cord. In explants and dissociated cultures of spinal cord primordium, LIF stimulated a profuse neurite outgrowth. To determine if these effects were related to neuronal differentiation, cells were plated at low cell density and stained for neurofilament. LIF stimulated an increase in the number of newly differentiated neurons, without inducing proliferation of the precursors. Given that LIF has previously reported effects as a cholinergic switching factor for sympathetic neurons, we investigated whether LIF had similar effects in these spinal cord cultures. LIF increased the number of cholinergic neurons in proportion to its overall effect on the stimulation of all neurofilament positive neurons in the culture. These data show that LIF stimulates the generation of spinal cord neurons from their precursors and further implicates a role for LIF in nervous system development.

Leukemia inhibitory factor (LIF), a peptide growth factor with multiple activities, has recently been shown to support the generation and survival of sensory neurons in cultures of mouse neural crest and dorsal root ganglia (DRG). We have conducted binding experiments with 125I-LIF on cultures of DRG to determine the receptor distribution for LIF on these cells and found that at least 60% of the sensory neurons in the cultures bound 125I-LIF, all of which could be eliminated by the addition of unlabeled LIF. The other cells in the culture, which morphologically appeared to be Schwann cells, did not bind appreciable quantities of 125I-LIF. In order to investigate whether LIF is retrogradely transported to sensory neurons in vivo, 125I-LIF was injected into the footpads and gastrocnemius muscles of newborn and adult mice, following sciatic nerve ligation. Radioactivity accumulated in the distal portion of the sciatic nerve, indicating retrograde transport of LIF. Subsequent experiments on mice with unligated sciatic nerves showed that 125I-LIF is specifically transported into the sensory neurons of the DRG. There was no apparent transport of 125I-LIF into motor neurons in the spinal cord. These experiments demonstrate that LIF can specifically bind to and be transported by sensory neurons and further support the idea that LIF acts as a target-derived neurotrophic factor, analogous to NGF.

The processes that regulate the development of peripheral neurons from their precursors in the embryonic neural crest are essentially unknown. In this report, we show that leukemia inhibitory factor stimulates the generation of neurons in cultures of mouse neural crest. These neurons have the morphology of sensory neurons and contain neuropeptides found in mammalian sensory neurons. Consistent with these neurons being of the sensory lineage is the finding that they arise from nondividing precursors within the neural crest. In addition, we show that leukemia inhibitory factor supports the generation and/or maturation of sensory neurons in cultures of cells obtained from embryonic dorsal root ganglia. In cultures of postnatal dorsal root ganglia, which contain mature sensory neurons, leukemia inhibitory factor acts directly as a survival molecule on the majority of neurons.