Ciliary neurotrophic factor inhibits brain and peripheral tumor necrosis factor production and, when coadministered with its soluble receptor, protects mice from lipopolysaccharide toxicity.

BACKGROUND: The receptor of ciliary neurotrophic factor (CNTF) contains the signal transduction protein gp130, which is also a component of the receptors of cytokines such as interleukin (IL)-6, leukemia-inhibitory factor (LIF), IL-11, and oncostatin M. This suggests that these cytokines might share common signaling pathways. We previously reported that CNTF augments the levels of corticosterone (CS) and of IL-6 induced by IL-1 and induces the production of the acute-phase protein serum amyloid A (SAA). Since the elevation of serum CS is an important feedback mechanism to limit the synthesis of proinflammatory cytokines, particularly tumor necrosis factor (TNF), we have investigated the effect of CNTF on both TNF production and lipopolysaccharide (LPS) toxicity. MATERIALS AND METHODS: To induce serum TNF levels, LPS was administered to mice at 30 mg/kg i.p. and CNTF was administered as a single dose of 10 micrograms/mouse i.v., either alone or in combination with its soluble receptor sCNTFR alpha at 20 micrograms/mouse. Serum TNF levels were the measured by cytotoxicity on L929 cells. In order to measure the effects of CNTF on LPS-induced TNF production in the brain, mice were injected intracerebroventricularly (i.c.v.) with 2.5 micrograms/kg LPS. Mouse spleen cells cultured for 4 hr with 1 microgram LPS/ml, with or without 10 micrograms CNTF/ml, were also analyzed for TNF production. RESULTS: CNTF, administered either alone or in combination with its soluble receptor, inhibited the induction of serum TNF levels by LPS. This inhibition was also observed in the brain when CNTF and LPS were administered centrally. In vitro, CNTF only marginally affected TNF production by LPS-stimulated mouse splenocytes, but it acted synergistically with dexamethasone (DEX) in inhibiting TNF production. Most importantly, CNTF administered together with sCNTFR alpha protected mice against LPS-induced mortality. CONCLUSIONS: These data suggest that CNTF might act as a protective cytokine against TNF-mediated pathologies both in the brain and in the periphery.

Selective loss of the 180-kDa form of the neural cell adhesion molecule in hippocampus and cerebellum of the adult mouse following trimethyltin administration.

The neural cell adhesion molecule (NCAM), a membrane glycoprotein which plays critical roles in cell-cell recognition and in the maintenance of cytoarchitecture in the adult brain, may be modulated in response to injury. To determine whether neurotoxic insult alters the profile of NCAM expression, adult female Balb/c mice were given a single injection of trimethyltin chloride (TMT; 3.2 mg/kg ip) and killed 4 hr to 3 weeks later. Hippocampus and cerebellum were isolated and prepared for light microscopy or SDS-PAGE and immunoblot analysis using the monoclonal antibody 5B8, which recognizes the intracellular domains of the 140- and 180-kDa isoforms of NCAM. NCAM140 appeared unaffected in TMT-treated mice at all time points. In contrast, decreased intensity of the 180-kDa band was apparent in both hippocampus and cerebellum 4 hr after TMT administration; maximal loss of NCAM180 was seen in hippocampal immunoblots 8 to 64 hr after treatment. The decrease in NCAM180 followed a similar but less pronounced course in cerebellum. Recovery of the 180-kDa band in hippocampus and cerebellum was apparent around 64 hr and continued until NCAM180 of mice killed 3 weeks after treatment resembled that of controls. No change in glial fibrillary acidic protein was seen by immunoblot analysis, suggesting that the effect is selective for neurons. TMT-induced loss of NCAM180 may be a direct cytotoxic effect, or may represent a reactive neuronal response to injurious stimuli. In either case, loss of NCAM180 accompanies cell injury following toxicant exposure and may be related to cytoskeletal alterations and destabilization of cellular contacts.

N-cadherin in normal and abnormal brain development.

The brain relies upon numerous morphoregulatory molecules to control cell-cell interactions, cell migration and neurite extension. N-cadherin, a calcium-dependent cell adhesion molecule, is essential for normal CNS development. Homophilic binding of N-cadherin depends upon a specific conformation assumed by the molecule when it binds calcium. N-cadherin is a substrate for a specific zinc-dependent protease that may be involved in the regulation of N-cadherin at the cell surface. The reliance of N-cadherin on two cations for proper function makes it a potential target for toxicants which act by replacing or modifying calcium or zinc at ion-binding sites. Exposure of the developing brain to lead, an ubiquitous toxicant known to interact with calcium, disturbs neural tube closure and subsequent maturation of the nervous system. Preliminary data indicates that lead may induce these effects by direct interaction with N-cadherin. Numerous common toxicants, including metals and solvents, also perturb cadherins and cause defective CNS development. These data indicate that changes in the spatio-temporal expression of cadherin can result in profound alterations in neural structure and function, and may underlie CNS malformations caused by numerous toxic agents.

Cytoskeleton and cell adhesion molecules: critical targets of toxic agents.

Normal development of the nervous system depends upon complex physical interactions between cells and their local environment. These interactions are mediated by several families of cell adhesion molecules (CAMs). Differential expression and function of CAMs are operative in neural tube formation, neuron migration, in post-migratory differentiation, and maintenance of mature neural structure. CAMs also facilitate contact-dependent cell processes, such as formation of cell junctions. Temporal regulation of these molecules during development may provide "windows of vulnerability" to toxicants. In addition to their extracellular binding activities, some CAMs have membrane-spanning domains by which they communicate directly with the cytoskeleton, permitting extracellular signals to be rapidly translated into cell responses via modifications in cytoskeletal organization. These cytologic changes are particularly critical during migration, neurite formation and synaptogenesis. Toxic perturbation of adhesion molecules can have catastrophic effects on morphogenetic processes both directly and via events which depend upon cytoskeletal rearrangement. Toxicants can also act directly upon the cytoskeleton, resulting secondarily in changes of the membrane distribution and function of CAMs. Toxicant-induced changes in CAMs and cytoskeleton may occur contemporaneously. Interference of cell adhesion-cytoskeleton interactions may be a pivotal molecular event dictating developmental consequences of neurotoxicant exposure.

Immunohistochemical and biochemical analysis of N-cadherin expression during CNS development.

The expression of the calcium-dependent adhesion molecule N-cadherin during chick embryo central nervous system (CNS) development was examined by immunohistochemistry and electrophoresis and immunoblotting. During histogenesis, N-cadherin is expressed at high levels in a uniform fashion in many regions of the CNS. However, during later stages of development, expression becomes restricted to the ependymal cells lining the ventricular system and in the choroid plexus. This down-regulation was confirmed by both immunohistochemical and biochemical techniques. The program of expression lags behind in the cerebellum in concert with the delayed development of this region of the brain. A high level of N-cadherin was found to be expressed in the brainstem and spinal cord floorplate, while a low level was detected at the optic nerve head. The results indicate that while, in general, the program of N-cadherin expression is similar in the retina and the brain, certain structures unique to the eye and brain express locally high or low levels of this adhesion protein.

Evidence for endogenous proteases, mRNA level and insulin as multiple mechanisms of N-cadherin down-regulation during retinal development.

Our previous studies of the role of cell adhesion in retinal development have focused on the expression and function of N-cadherin, the predominant calcium-dependent intercellular adhesion protein of neural tissues. During the course of retinal development, N-cadherin expression undergoes significant qualitative and quantitative changes in its pattern of expression, most prominently a sharp down-regulation of expression throughout most of the retina. The present studies were directed at investigating the epigenetic mechanisms that could mediate this loss of N-cadherin from the retina. Using an in vitro intact retinal organ culture system, results were obtained which suggest that insulin enhances the down-regulation of N-cadherin expression in a protein-synthesis-dependent fashion. Furthermore, the metalloprotease inhibitor 1,10-phenanthroline inhibits the loss of N-cadherin from the retina. While N-cadherin is down-regulated in organ culture, other cell adhesion molecules, which are not down-regulated in vivo, are also not down-regulated in organ culture. The defined organ culture medium conditioned by the retina accumulates both a soluble 90 x 10(3) M(r) N-terminal fragment of N-cadherin as well as a number of secreted proteases. Both of these components are also shown to be present in vivo in the vitreous humor. Northern blot analysis indicates a single mRNA encoding N-cadherin in the retina and no evidence for a second message that could encode the 90 x 10(3) M(r) fragment. However, the amount of N-cadherin mRNA detectable on northern blots decreases during development. The results reported here suggest that the down-regulation of N-cadherin that occurs during retinal development is possibly mediated by multiple mechanisms, which include turnover at the cell surface mediated by endogenous proteolysis, reduced levels of N-cadherin mRNA and modulation by growth factors.

Tissue and age-specificity of post-translational modifications of N-cadherin during chick embryo development.

Our previous studies indicated that regulation of N-cadherin expression differs spatially and temporally among tissues of the eye, possibly reflecting the distinct roles it has in the development and maintenance of eye tissues. To understand this regulation of N-cadherin expression and its function in different tissues during embryonic development, we investigated the post-translational modifications of N-cadherin and its association with the cytoskeleton. We show that N-cadherin is a sulfated and phosphorylated protein. The phosphorylation of N-cadherin occurs in an age- and tissue-specific pattern during development in the neural retina, brain, lens and heart. The extent of sulfation of N-cadherin is also age-dependent, and both sulfated and unsulfated pools of N-cadherin exist in the same tissue as indicated by two-dimensional electrophoresis. The degree of association of N-cadherin with the cytoskeleton differs from one tissue to another, as well as within a single tissue at different stages of development. A positive correlation was found between the extent, developmental timing, and tissue specificity of N-cadherin phosphorylation and the degree of N-cadherin association with the cytoskeleton. Our results suggest the existence of a microheterogeneous population of N-cadherin molecules, within which posttranslational modification of N-cadherin may affect its association with the cytoskeleton and its expression and function during development.

Identification of mammalian and invertebrate analogues of the avian calcium-dependent cell adhesion protein N-cadherin with synthetic-peptide directed antibodies against a conserved cytoplasmic domain.

N-cadherin, a 130kD transmembrane adhesive glycoprotein, is a mediator of specific cellular interactions during development. Analysis of N-cadherin at the protein level, to date, has been largely dependent upon monoclonal antibody NCD-2 which recognizes only avian N-cadherin. We produced a monospecific polyclonal antiserum, C-NCAD(838-856), to a synthetic peptide corresponding to a portion of the highly conserved c-terminal cytoplasmic domain of chick N-cadherin. Using polyacrylamide gel electrophoresis and immunoblotting to map tissue distribution we show that the antiserum detects chick N-cadherin with a similar tissue distribution as NCD-2. Unlike NCD-2, however, anti-C-NCAD(838-856) recognizes N-cadherin analogues in a wide variety of species, including mouse, human, fish and drosophila. The results of comparative immunoblot studies demonstrate similar tissue-specific patterns and apparent molecular weight variation in the chick, mouse and human. This indicates that N-cadherin structure and expression, and most likely function as well, have been highly conserved in evolution. The antiserum recognizes an epitope unique to N-cadherin which is conserved among N-cadherins from a variety of species but is absent from other members of the cadherin gene family, as no immunoreactivity was detected with tissues bearing these other cadherins. The antiserum is thus a useful tool for the phylogenetic and biochemical investigation of N-cadherin from a variety of tissue sources.

Expression of calcium-dependent cell adhesion during ocular development: a biochemical, histochemical and functional analysis.

Previous studies of the adhesive properties of embryonic chick neural retina cells indicate a gradual decrease in the expression of calcium-dependent adhesions during retinal histogenesis, a function which has been attributed in part to gp130/4.8, a retinal calcium-dependent adhesion-associated cell surface membrane glycoprotein with a molecular weight of approximately 130 kDa and an isoelectric point of 4.8 (G. B. Grunwald, R. Pratt, and J. Lilien, 1982, J. Cell Sci. 55, 69-83). The experiments described here were done to define the relationship of gp130/4.8 to N-cadherin, another calcium-dependent adhesion molecule found in chick retina, which has a reported molecular weight of 127 kDa and which is recognized by monoclonal antibody NCD-2 (K. Hatta and M. Takeichi, 1986, Nature (London) 320, 447-449). Using two-dimensional gel electrophoresis followed by Western blotting as well as quantitative solid-phase immunoassays, polyspecific antisera recognizing gp130/4.8 were compared with monoclonal antibody NCD-2 for reactivity with proteins of retina and other tissues. The data lead us to conclude that retinal calcium-dependent adhesion proteins gp130/4.8 and N-cadherin are likely to be the same molecule. In order to obtain evidence for a direct correlation of changes in expression of these adhesion proteins with changes in retinal cell adhesivity and related morphogenetic events, parallel studies were carried out with cells from various ocular tissues to examine the functional, biochemical, and immunohistochemical expression of N-cadherin during ocular development. Immunohistochemical mapping of N-cadherin in the developing chick eye reveals three modes of N-cadherin expression which occur simultaneously in different ocular tissues: (1) down-regulation, (2) up-regulation, and (3) steady-state expression. These patterns of expression correlate with changes in the adhesive behavior of cells as well as with discrete stages in the morphogenesis of several ocular tissues. The results suggest that N-cadherin is a versatile cell adhesion protein with a role in both the development of several ocular tissues and the maintenance of specialized structures in the mature eye.

Heterogeneity of tubulin epitopes in mouse fetal tissues.

A panel of six monoclonal antibodies against alpha (TU-01, TU-03, TU-04, TU-05, TU-09) or beta (TU-13) subunits of tubulin was used to study expression of tubulin epitopes in 14-day-old mouse embryos. Specificity of antibodies was confirmed by immunoblotting experiments. Monoclonal antibodies TU-01, TU-09 and TU-13, like the polyclonal antibody reacted essentially with all tissues, whereas other antibodies displayed differential reactivity. Most notably, TU-03 reacted very strongly with simple epithelia and basal layer of stratified epithelial layers. TU-04 recognized maturation related changes in spinal cord. Reactivity of TU-05 was restricted to central nervous system and peripheral nerves. Present results document immunohistochemical heterogeneity of tubulin in fetal tissues and suggest the existence of maturation and tissue specific epitopes of tubulin in developing organs.