Interleukin-12 p40-homodimer production in sensory dorsal root ganglion neurons.

Recently, the reports that sensory nerves contribute to induction and development of peripheral inflammation have been accumulating. Although neuropeptides have been thought to participate in modulation of inflammation, we supposed the involvement of cytokines. Interleukin-12 (IL-12) is a key regulator of cell-mediated immunity. IL-12 is heterodimer cytokine consisting of a p35 and a p40 subunit, but the results that some of immune cell types secrete p40-homodimer have been reported. In this study, we investigated the expression and secretion of IL-12 in mouse sensory neurons in order to evaluate the involvement of sensory neurons in cell-mediated immunity. Expression of IL-12 p40 mRNA was detected and enhanced by interferon-gamma (IFN-gamma), but another subunit of IL-12 p35 mRNA was not detected in sensory dorsal root ganglion (DRG) neurons in culture. IL-12 p40 molecule was detected in DRG neurons by immunocytochemistry. In addition, cultured DRG neurons secreted p40-homodimer that inhibited IL-12-induced STAT4 phosphorylation in T cells. p40 mRNA expression was accumulated in DRG after administration of IFN-gamma into mouse footpad, and this enhancement was eliminated by a cut of sciatic nerve. These results suggest the possibility that p40-homodimer derived from sensory nerves suppresses the excessive peripheral inflammation.

IL-6 up-regulates CNTF mRNA expression and enhances neurite regeneration.

Interleukin-6 (IL-6) is a neurotrophic cytokine, however, its direct effect on nerve regeneration has not been well characterized. We therefore examined the effect of IL-6 on neurite regeneration using the rat dorsal root ganglion. IL-6 significantly enhanced neurite regeneration from transected nerve terminals. We also examined the mRNA expression of IL-6 family cytokines and their receptors during the regeneration. The mRNA expressions of IL-6, IL-6 receptor, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF) receptor alpha, and LIF receptor beta showed no significant differences by the addition of IL-6. In contrast, IL-6 enhanced the mRNA expression of gp130 and CNTF. In addition, CNTF significantly increased neurite regeneration when added exogenously. Our data suggest that IL-6 enhanced regeneration via up-regulating CNTF expression.

Interleukin-12 promotes neurite outgrowth in mouse sympathetic superior cervical ganglion neurons.

To determine the role of cytokines in the nervous system, we examined the effect of interleukin-12(IL-12) on the nerve regeneration of mouse superior cervical ganglion cells (SCG). IL-12 enhanced the neurite outgrowth in a concentration-dependent manner. Immunocytochemical studies demonstrated the expression of IL-12 receptors in neuronal bodies and neurites. The mRNA expression of IL-12 receptors in SCG cells was confirmed by reverse transcription-polymerase chain reaction. Our data demonstrated the presence of IL-12 receptors in sympathetic neurons and suggest that IL-12 plays an important role in neuronal regeneration.

IFN-gamma induces coordinate expression of MHC class I-mediated antigen presentation machinery molecules in adult mouse Schwann cells.

The expression of major histocompatibility complex (MHC) class I molecules on adult mouse Schwann cells (SCs) was examined using immunofluorescence analysis. MHC class I molecules were not expressed on the surface of untreated SCs. Interferon (IFN)-gamma treatment induced expression of the molecules on the SCs. Expression of genes coding for the molecules involved in MHC class I-mediated antigen presentation was also analysed in SCs by reverse transcription-polymerase chain reaction (RT-PCR). Expression of MHC class I heavy chain genes was faintly detected in untreated SCs. IFN-gamma treatment augmented the expression. In addition, IFN-gamma induced expression of the genes for beta2-microglobulin, the peptide transporter TAP-1 and the proteasomal subunit LMP-2, whose expression was not detected in untreated SCs. The expressions of MHC class II molecules and their genes were not detected even after IFN-gamma treatment. These data suggest that MHC class I-mediated antigen presentation machinery functions in adult mouse SCs and that the SCs themselves work as antigen presenting cells and as targets for cytotoxic T cells in some physiological conditions.

Method for production of neuronal hybridoma using emetine and actinomycin D.

Neuronal hybrid cells established by somatic cell fusion are useful for studies of neuronal properties at the molecular level (Hammond, D.N., Lee, H.J., Tonsgard, J.H. and Wainer, B.H., Development and characterization of clonal cell lines derived from septal cholinergic neurons, Brain Res., 512 (1990) 190-200; Wainwright, M.S., Perry, B.D., Won, L.A., O'Malley, K.L., Wang, W.Y., Ehrlich, M.E. and Heller, A., Immortalized murine strial neuronal cell lines expressing dopamine receptors and cholinergic properties, J. Neurosci., 15 (1995) 676-688). The somatic cell fusion method requires a fusion partner which is unable to survive in the selection medium if it does not fuse with primary cells to isolate the hybrid cells. Hypoxanthine guanine phosphoribosyltransferase (HPRT)-deficient partner cells and hypoxanthine, aminopterin and thymidine (HAT) selection medium are commonly used for this procedure (Harlow, E. and Lane, D. (Eds.), Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory Publications, New York, 1988, pp. 139-243). The present method requires neither HPRT-deficient cells nor HAT medium. Primary neurons are fused with the C1300 neuroblastoma cells pretreated with emetine (Grollman, A.P., Inhibitors of protein biosynthesis, J. Biol. Chem., 243 (1968) 4089-4094), an inhibitor of ribosomes and actinomycin D (Perry, R.P., Selective effects of actinomycin D on the intracellular distribution of RNA synthesis in tissue culture cells, Exp. Cell Res., 29 (1963) 400-406), an inhibitor of ribosomal RNA (rRNA) synthesis, before fusion. By this treatment, we are able to isolate hybrid cells after fusion because non-fused C1300 cells die due to the loss of active ribosomes and protein synthesis, whereas C1300 cells fusing with primary cells survive due to the supply of intact ribosomes and rRNA from primary cells. This method produces neuronal hybrids at high efficiency.

Delayed neurite regeneration and its improvement by nerve growth factor (NGF) in dorsal root ganglia from MRL-lpr/lpr mice in vitro.

We studied neurite regeneration in MRL-lpr/lpr mice, a murine model of systemic lupus erythematosus, using a culture system to investigate the influences of immunological abnormalities on neurons. The regeneration of cultured dorsal root ganglion (DRG) neurons from MRL-lpr/lpr mice was delayed compared with control MRL-+/+ mice. This modification of regeneration was age-dependent. MRL-lpr/lpr mice older than 16 weeks of age exhibited less neurite regeneration than controls but those younger than 6 weeks of age showed equal regeneration. Regeneration was improved by adding nerve growth factor (NGF) to culture medium. Following immunocytochemical staining, we counted the low affinity NGF receptor p75-positive DRG neurons in MRL mice. The percentage of p75-positive neurons in MRL-lpr/lpr mice older than 16 weeks of age was higher than that in MRL-+/+ mice. These neuronal abnormalities were thought not to be directly dependent on the genetic defect of Fas antigen, which is related to apoptosis in MRL-lpr/lpr mice, but to be the result of immunological abnormalities. The present study is the first to demonstrate a modification of neurite regeneration by immunological dysfunction in autoimmune mice.

Mature sensory neuron-derived hybrid cell line expressing NGF-dependent neurite extension.

We established a hybrid cell line which extended neurites in the presence of nerve growth factor by fusion between adult mouse sensory neurons and neuroblastoma C1300 cells using emetine and actinomycin D. The serine-phosphorylated 200 and 160 kDa neurofilament proteins were detected in the hybrid cells. In comparison, C1300 cells expressed both subunit of non-phosphorylated neurofilaments at serine residues.

Sensory neurons regulate immunoglobulin secretion of spleen cells: cellular analysis of bidirectional communications between neurons and immune cells.

The effects of primary sensory neurons in the dorsal root ganglia (DRG) on immunoglobulin (Ig)-secreting activity of spleen cells were investigated in culture. The conditioned medium (CM) of normal spleen cells stimulated DRG neurons to release neurokinin A which increased the number of Ig-secreting spleen cells. In contrast, the CM of concanavalin A-induced suppressor spleen cells induced the release of vasoactive intestinal peptide, an inhibitor of Ig secretion of spleen, from DRG neurons. These findings indicate that sensory neurons can help and suppress Ig secretion, and these bidirectional activities are controlled by the factors released from immune cells.

IFN-gamma induces expression of MHC class I molecules in adult mouse dorsal root ganglion neurones.

The expression of major histocompatibility complex (MHC) class I molecules on adult mouse DRG neurones was analysed using immunofluorescence and reverse transcription-polymerase chain reaction (RT-PCR). MHC class I molecules were not expressed in untreated dorsal root ganglion (DRG) neurones. The molecules, however, were detected on the surface of neurones following IFN-gamma treatment. In addition, expression of the MHC class I beta 2-microglobulin and genes was induced in neurones following IFN-gamma treatment. Expression of Tap-1 and Lmp-2 genes, which are involved in antigen processing and presentation, was also induced by IFN-gamma treatment. These results indicated that DRG neurones are able to express MHC class I molecules following IFN-gamma treatment and suggest that DRG neurones themselves are susceptible to recognition by cytotoxic T cells.

Improved method for producing neuronal hybrids using emetine and actinomycin D.

We modified the method of somatic cell fusion for neurons to improve the efficiency of hybrid production. C1300 neuroblastoma cells were incubated with emetine and actinomycin D before fusion. After fusion between C1300 cells and adult mouse dorsal root ganglia neurons with polyethyleneglycol, we were able to select the hybrids from non-fused cells. We obtained hybrid clones at high efficiency (7.2 clones/10(4) neurons).

Myelin-stimulated macrophages release neurotrophic factors for adult dorsal root ganglion neurons in culture.

1. Our previous study demonstrated that cultured macrophages release neurotrophic factors spontaneously. In a histological study of Wallerian degeneration, macrophages phagocytosed myelin debris and expressed activated markers. 2. To investigate the role of myelin-stimulated macrophages on neurite regeneration, we prepared conditioned media from cultured mouse peritoneal macrophages which had phagocytosed a myelin fraction. This conditioned media enhanced both neurone survival and neurite regeneration of adult dorsal root ganglia (DRG) neurons compare to conditioned media from macrophage cultures without myelin. 3. The production of the neurotrophic supernatant was dose-dependent on myelin fraction and specific for myelin because supernatants from macrophages incubated with LPS (lipoplysaccharide), MDP (N-acetylmuramyl-L-alanyl-D-isoglutamine) or latex beads were not neurotrophic. 4. The neurotrophic factors from myelin-stimulated macrophages were different from spontaneously released macrophage factors as they differed in heat-sensitivity. 5. These results suggest that myelin-stimulated macrophages contribute to axon regeneration after Wallerian degeneration.

Mitogen induced proliferation of isolated adult mouse Schwann cells.

The proliferation of neonatal Schwann cells (SCs) in response to mitogenic agents has been well analyzed in vitro, but a limited range of mitogens have been defined. We investigated whether three identified neonatal SC mitogens [glial growth factor (GGF), platelet-derived growth factor BB (PDGF-BB), and basic fibroblast growth factor (bFGF)] are required to stimulate mitosis of adult SCs. Adult SCs were isolated from mouse sciatic nerves by mechanical and chemical dissociation, following three experimental steps: 1) culturing the dissociated cells for 24 hr in 10% FCS-F12 medium, 2) culturing these cells in serum-free medium for the next 48 hr, and 3) purifying adult SCs by differential adhesion. We describe a new method for preparation of SCs from peripheral nerves of adult mouse that provides 99.5% pure SCs populations at cell yields of greater than 3 x 10(3) cells/mg of starting nerve wet weight within 5 culture days. Although mitosis of SCs in culture in response to mitogens requires the presence of serum, the complex nature of serum renders difficult a complete analysis of mitogens required for SCs DNA synthesis, so we examined the proliferating response of adult SCs to GGF, PDGF-BB, and bFGF in serum-free medium. GGF alone had mitogenicity for adult SCs in a dose-dependent manner, and synergistic activation coupling with forskolin was not observed. Neither PDGF-BB nor bFGF was mitogenic for adult SCs when used alone or with forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)

Nerve growth factor (NGF) restores depletions of calcitonin gene-related peptide and substance P in sensory neurons from diabetic mice in vitro.

Dorsal root ganglion neurons from streptozotocin (STZ)-induced diabetic, genetic diabetic and normal mice were cultured in serum-containing media with or without nerve growth factor (NGF). The immunocytochemical analysis carried out after 1 week in culture revealed that the ratios of neurons immunoreactive to calcitonin gene-related peptide (CGRP) in NGF-free medium in the STZ-diabetic mice (average 23.2%) were significantly lower than those in the normal mice (45.1%). The ratios of neurons immunoreactive to CGRP and substance P (SP) in the NGF-free medium were also lower in the genetic diabetic mice (23.6% and 21.8%) than those in the normal ones (40.7% and 34.2%). However, treatment with NGF restored these reduced immunoreactivities in the diabetic groups in a dose-dependent manner. These results show that NGF can be effective for the diabetes-induced depletion of CGRP and SP in sensory neurons, and suggest its possible role in the prevention and improvement of diabetic sensory neuropathy.

Bradykinin-responsive cells of dorsal root ganglia in culture: cell size, firing, cytosolic calcium, and substance P.

1. We analyze bradykinin-sensitive cells of the mouse dorsal root ganglion in culture from the viewpoints of cell size, electrical responses, and Ca2+ concentration change due to bradykinin and immunocytochemistry of substance P. 2. Sixteen percent of cells in the cell group 26-30 microns in diameter fired in response to 10 microM bradykinin. None of other cell groups showed a firing response to bradykinin. 3. We measured a cytosolic Ca2+ change due to bradykinin using a Ca(2+)-sensitive fluorescent dye, Fura 2. The rapid rise (peak time, 20 sec) in the Ca2+ concentration was ascribed to Ca2+ release from intracellular Ca2+ stores. The profound change in the Ca2+ concentration was observed again in the cell group 26-30 microns in diameter. Seventeen percent of cells in this group increased the Ca2+ concentration by approximately seven times that at resting level. 4. Among cells which increase Ca2+ concentration responding to bradykinin, 83% of them contain substance P (an immunocytochemical study). 5. We conclude that 16-17% of the cell group 26-30 microns in diameter of the dorsal root ganglia in culture are polymodal nociceptors and respond to bradykinin.

Macrophage-enhanced neurite regeneration of adult dorsal root ganglia neurones in culture.

To study the effects of macrophages on nerve regeneration, purified low population dorsal root ganglia (DRG) neurones from adult mice were cultured in protein-free medium (PFM) or in conditioned media (CM) from peritoneal macrophages cultured in PFM. In PFM, only 20% of the neurones survived at 5 days in culture, but the macrophage CM more than doubled the proportion of neurone survival and neurite extension of controls. These factors did not act on superior cervical ganglia (SCG) neurones, and were considered as being proteins because of their trypsin-sensitive property. Additionally, they may be different from the previously known neurotrophic factors derived from macrophages, because the target cells and effective concentration of these factors were different from those of known ones. The factors investigated in this study may play an important role in nerve regeneration.

Isolation and age-related characterization of mouse Schwann cells from dorsal root ganglion explants in type I collagen gels.

A technique for isolation of adult Schwann cells (ScC) from dorsal root ganglia (DRG) is described. Decapsulated DRG explants embedded into type I collagen gels were cultured for 3 days in serum-free medium during which ScC migrated from the explant. These explants were then grown in serum-supplemented medium to allow ScC proliferation. On day 10 the number of ScC isolated from DRG explants per mouse was about 2.5 x 10(5), and the purity was greater than 95%. This culture system provided sufficient numbers of highly purified adult ScC in a shorter culture period (2-3 times) than other methods. We used ScC from this method to determine the age-related changes in attachment, growth, and survival of ScC cultured in serum-free medium. The attachment capacity of adult ScC on type I collagen or polylysine was similar to that of newborn ScC. However, the collagen promoted growth and survival of adult ScC but not that of neonatal ScC, indicating age-related differences of ScC properties in vitro.

Mechanism of inhibitory action of capsaicin on particulate axoplasmic transport in sensory neurons in culture.

The inhibitory effect of capsaicin on axoplasmic transport in cultured dorsal root ganglion cells was analyzed by video-enhanced contrast microscopy. Capsaicin inhibited particle transports in a dose-dependent manner, irrespective of the diameter of axons. The effect of capsaicin was reversible at low concentrations. Capsaicin affected both the anterograde and retrograde transport. Large organelles were more sensitive to capsaicin than small ones in the retrograde transport. An experiment using calcium-sensitive dye, Fura 2, indicated that capsaicin raised the intraneuronal free calcium concentration preceding the inhibition of the transport. Electron microscopy revealed that microtubules and neurofilaments are disorganized and disoriented by capsaicin. We reached a conclusion that capsaicin inhibits fast axoplasmic transport of both anterograde and retrograde directions in all types of somatosensory neurons in culture by disorganizing intraaxonal cytoskeletal structures, through the elevated intracellular Ca2+ concentration.

How neurotransmitters affect axoplasmic transport?

The effects of acetylcholine (ACh) and adrenaline on fast axoplasmic transport of cultured superior cervical ganglion cells were analyzed with a computer-assisted video-enhanced differential interference contrast microscopic system. ACh suppressed the transport reversibly in both anterograde and retrograde directions, and adrenaline increased the transport reversibly. These effects are related to the amount of c-AMP. This amount of c-AMP in connection with neurotransmitters controls the axoplasmic transport, which in turn is related to the activity of the neuron.

Effect of neurotransmitters on axoplasmic transport: acetylcholine effect on superior cervical ganglion cells.

The effect of acetylcholine (ACh) on particle movements along axons of cultured superior cervical ganglion cells was analyzed with a computer-assisted video-enhanced differential interference contrast microscope system. ACh suppressed the axoplasmic transport reversibly in both anterograde and retrograde directions. A muscarinic agonist, arecoline, mimicked the ACh effect, but nicotine did not. An experiment with the Ca(2+)-indicator dye, fura-2, revealed that ACh suppressed the transport without any change of intracellular Ca2+ concentration. ACh also suppressed the axoplasmic transport in Ca(2+)-free medium. Islet-activating protein (IAP), pertussis toxin, blocked the ACh effect. These results indicate that ACh activates muscarinic receptors and suppresses fast axoplasmic transport through the activation of IAP-sensitive GTP-binding protein, irrespective of Ca2+ ions.

Increase in intracellular calcium induced by stimulating histamine H1 receptors in macrophage-like P388D1 cells.

The addition of histamine to macrophage-like P388D1 cells resulted in a dose-dependent increase in intracellular calcium [Ca2+]i measured by fura-2 in single cells. The maximum level of [Ca2+]i was obtained by addition of 1 x 10(-4) M histamine. The increase was primarily due to release from the intracellular store. The addition of an H1 specific antagonist pyrilamine before histamine treatment inhibited the increase reversibly, while an H2 specific antagonist cimetidine had no inhibitory effect. Histamine also resulted in a dose-dependent increase in cGMP but not in cAMP. These data suggest the existence of histamine H1 receptors in these cells and histamine may have some biological effect on the function of macrophages via [Ca2+]i and cGMP as the second messengers.