Transplantation of embryonic spinal cord-derived neurospheres support growth of supraspinal projections and functional recovery after spinal cord injury in the neonatal rat.

Great interest exists in using cell replacement strategies to repair the damaged central nervous system. Previous studies have shown that grafting rat fetal spinal cord into neonate or adult animals after spinal cord injury leads to improved anatomic growth/plasticity and functional recovery. It is clear that fetal tissue transplants serve as a scaffold for host axon growth. In addition, embryonic Day 14 (E14) spinal cord tissue transplants are also a rich source of neural-restricted and glial-restricted progenitors. To evaluate the potential of E14 spinal cord progenitor cells, we used in vitro-expanded neurospheres derived from embryonic rat spinal cord and showed that these cells grafted into lesioned neonatal rat spinal cord can survive, migrate, and differentiate into neurons and oligodendrocytes, but rarely into astrocytes. Synapses and partially myelinated axons were detected within the transplant lesion area. Transplanted progenitor cells resulted in increased plasticity or regeneration of corticospinal and brainstem-spinal fibers as determined by anterograde and retrograde labeling. Furthermore, transplantation of these cells promoted functional recovery of locomotion and reflex responses. These data demonstrate that progenitor cells when transplanted into neonates can function in a similar capacity as transplants of solid fetal spinal cord tissue.

Rudimentary TCR signaling triggers default IL-10 secretion by human Th1 cells.

Understanding the process of inducing T cell activation has been hampered by the complex interactions between APC and inflammatory Th1 cells. To dissociate Ag-specific signaling through the TCR from costimulatory signaling, rTCR ligands (RTL) containing the alpha1 and beta1 domains of HLA-DR2b (DRA*0101:DRB1*1501) covalently linked with either the myelin basic protein peptide 85-99 (RTL303) or CABL-b3a2 (RTL311) peptides were constructed to provide a minimal ligand for peptide-specific TCRs. When incubated with peptide-specific Th1 cell clones in the absence of APC or costimulatory molecules, only the cognate RTL induced partial activation through the TCR. This partial activation included rapid TCR zeta-chain phosphorylation, calcium mobilization, and reduced extracellular signal-related kinase activity, as well as IL-10 production, but not proliferation or other obvious phenotypic changes. On restimulation with APC/peptide, the RTL-pretreated Th1 clones had reduced proliferation and secreted less IFN-gamma; IL-10 production persisted. These findings reveal for the first time the rudimentary signaling pattern delivered by initial engagement of the external TCR interface, which is further supplemented by coactivation molecules. Activation with RTLs provides a novel strategy for generating autoantigen-specific bystander suppression useful for treatment of complex autoimmune diseases.

IL-7 enhances Ag-specific human T cell response by increasing expression of IL-2R alpha and gamma chains.

Interleukin-7 has demonstrated potent enhancing effects on the growth and differentiation of several immature cell types, including thymocytes, and on survival of resting and antigen activated T cells. In this study, we evaluated the effects of IL-7 on post-thymic antigen-specific T cells from human blood. IL-7 was found to enhance proliferation responses and IFN-gamma secretion of myelin or recall Ag-specific Th1 cells through the selective up-regulation of the IL-2Ralpha and gamma but not beta chains in both an Ag-dependent and Ag-independent manner, but did not affect monocytes, B cells, or NK cells. These functions of IL-7 enhanced the detection of Th1 but not Th2 cell frequency by >2.5 fold, and promoted selection of Ag-specific Th1 cells by the limiting dilution method. Moreover, IL-7 pretreatment conferred increased resistance of CD4+ T cells to CD8+ cell lysis. These studies demonstrate that IL-7 promotes the growth and survival of circulating Ag-specific human Th1 cells through a mechanism that probably involves the gammac common receptor for IL-2 family members that includes IL-7.

Overexpression of ciliary neurotrophic factor in vivo rescues chick ciliary ganglion neurons from cell death.

Ciliary ganglion (CG) neurons undergo target-dependent cell death during embryonic development. Although ciliary neurotrophic factor (CNTF) was identified in vitro by its ability to support the survival of chick CG neurons, its function as a target-derived neurotrophic factor has been questioned by those working on mammalian-derived forms of CNTF. We have purified and cloned a chicken CNTF [chCNTF; formerly growth-promoting activity (GPA)] that is expressed in CG targets during the period of cell death and is secreted by cells transfected with chCNTF. In the present study we used a retroviral vector, RCASBP(A), to overexpress chCNTF in CG target tissues. Elevation of chCNTF biological activity three- to fourfold in the embryonic eye rescued an average of 31% of the neurons that would have normally died in vivo. In some individuals, nearly all of the neurons were rescued. ChCNTF had no effect on the number of neurons observed prior to cell death, nor were there any deleterious effects of either viral infection or overexpression of CNTF. These results show that chCNTF is able to function in vivo as a trophic factor for CG neurons, and suggest that limited availability of trophic support is one of the factors regulating CG neuron survival during development.

Identification of functional receptors for ciliary neurotrophic factor on chick ciliary ganglion neurons.

Ciliary neurotrophic factor and an avian homolog, growth promoting activity, are members of the cytokine/neurokine family of trophic factors and have been proposed to function as survival and developmental factors for ciliary ganglion neurons in vivo. Here we identify for the first time functional receptors for ciliary neurotrophic factor and growth promoting activity on cultured ciliary ganglion neurons. [(125)I]Rat ciliary neurotrophic factor binding studies indicate that rat ciliary neurotrophic factor and growth promoting activity bind to these receptors with a single affinity, while human ciliary neurotrophic factor recognizes both a high- and low-affinity site. Comparison of the relative potency of human ciliary neurotrophic factor and avian growth promoting activity in biological assays indicates that growth promoting activity is three to five times more active in promoting survival and in regulating acetylcholine receptors. The binding of ciliary neurotrophic factor is specific, sensitive to phosphatidylinositol-specific phospholipase C and partially inhibited by leukemia inhibitory factor, but not inhibited by other members of the human neurokine family, including interleukin-6, interleukin-22 and oncostatin M. Cross-linking of [(125)I]rat ciliary neurotrophic factor to ciliary neurons results in the specific labeling of three proteins with estimated molecular masses of 153,000, 81,000 and 72,000. Only the 81,000 molecular weight component is released from the cells after treatment with phosphatidylinositol-specific phospholipase C, suggesting a membrane attachment via a glycosylphosphatidylinositol linkage. Stimulation with ciliary neurotrophic factor or growth promoting activity, but not by other neurokines, results in the rapid tyrosine phosphorylation of a 90,000 molecular weight protein that is inhibited by pretreatment with phosphatidylinositol-specific phospholipase C. In conclusion, we report here the pharmacological and functional properties of ciliary neurotrophic factor receptors on embryonic ciliary ganglion neurons. These results provide the means for elaborating the molecular mechanisms of ciliary neurotrophic factor action and understanding its physiological role in a defined neuronal population.

Expression of a chicken ciliary neurotrophic factor in targets of ciliary ganglion neurons during and after the cell-death phase.

Ciliary ganglion (CG) neurons, like other neuronal populations, become dependent on their targets for survival during development. We have previously purified and cloned a secreted ciliary neurotrophic factor that was called growth-promoting activity (GPA). We report here the expression and purification of a highly active form of recombinant GPA, the preparation of GPA-specific polyclonal and monoclonal antibodies, and the use of these antibodies to investigate the cellular location and timing of GPA expression in tissues innervated by CG neurons. Virtually all of the trophic activity in extracts of embryonic eyes could be depleted by GPA-specific antibodies. GPA-like immunoreactivity was found in both targets of the CG: the arterial vasculature of the choroid layer and the ciliary body of the eye. In the choroid layer, GPA was localized to smooth muscle cells surrounding the choroid arteries. Staining in the choroid layer was first detectable at embryonic day (E) 10, or about 2 days after cell death has begun in the ganglion, then increased in intensity through E19. Quantification of trophic activity from whole eye extracts at various ages showed a small increase in activity observed between E9 and E12 and at least a 10-fold increase between E12 and E18. The presence of GPA protein in target cells of CG neurons during the specific developmental period when these neurons undergo cell death is consistent with its proposed function as a target-derived ciliary neurotrophic factor.

Analysis of function and expression of the chick GPA receptor (GPAR alpha) suggests multiple roles in neuronal development.

Growth promoting activity (GPA) is a chick growth factor with low homology to mammalian ciliary neurotrophic factor (CNTF) (47% sequence identity with rat CNTF) but displays similar biological effects on neuronal development. We have isolated a chick cDNA coding for GPA receptor (GPAR alpha), a GPI-anchored protein that is 70% identical to hCNTFR alpha. Functional analysis revealed that GPAR alpha mediates several biological effects of both GPA and CNTF. Soluble GPAR alpha supports GPA- and CNTF-dependent survival of human TF-1 cells. In sympathetic neurons, GPAR alpha mediates effects of both GPA and CNTF on the expression of vasoactive intestinal peptide (VIP) as shown by the inhibition of GPA- and CNTF-mediated VIP induction upon GPAR alpha antisense RNA expression. These results demonstrate that GPAR alpha is able to mediate effects of two neurokines that are only distantly related. GPAR alpha mRNA expression is largely restricted to the nervous system and was detected in all neurons that have been shown to respond to GPA or CNTF by increased survival or differentiation, i.e. ciliary, sympathetic, sensory dorsal root, motoneurons, retinal ganglion cells and amacrine cells. Interestingly, GPAR alpha mRNA was additionally found in neuronal populations and at developmental periods not known to be influenced by GPA or CNTF, suggesting novel functions for GPAR alpha and its ligands during neurogenesis and neuron differentiation.

GPA and CNTF produce similar effects in sympathetic neurones but differ in receptor binding.

The effects of growth promoting activity (GPA) on sympathetic neurone development were investigated in vitro and compared with the effects of ciliary neurotrophic factor (CNTF). GPA interfered with sympathetic neurone proliferation and induced the expression of vasoactive intestinal peptide (VIP) in neurones from 7-day-old (E7) chick embryos. The biological effects observed with saturating levels of GPA are indistinguishable from the effects of CNTF. The effects on VIP expression suggest that GPA may be involved in the specification of sympathetic neurone phenotypes. Whereas half maximal effects are achieved at lower concentrations of GPA than CNTF, GPA competes less efficiently than CNTF for the binding of 125I-labelled CNTF. This suggests similar, but not identical interactions of CNTF and GPA with receptors on chick sympathetic neurones.