Apical splenic nerve electrical stimulation discloses an anti-inflammatory pathway relying on adrenergic and nicotinic receptors in myeloid cells.

The autonomic nervous system innervates all lymphoid tissues including the spleen therefore providing a link between the central nervous system and the immune system. The only known mechanism of neural inhibition of inflammation in the spleen relies on the production of norepinephrine by splenic catecholaminergic fibers which binds to ß2-adrenergic receptors (ß 2-ARs) of CD4+ T cells. These CD4+ T cells trigger the release of acetylcholine that inhibits the secretion of inflammatory cytokines by macrophages through α7 nicotinic acetylcholine receptor (α7nAchRs) signaling. While the vagal anti-inflammatory pathway has been extensively studied in rodents, it remains to be determined whether it coexists with other neural pathways. Here, we have found that three nerve branches project to the spleen in mice. While two of these nerves are associated with an artery and contain catecholaminergic fibers, the third is located at the apex of the spleen and contain both catecholaminergic and cholinergic fibers. We found that electrical stimulation of the apical nerve, but not the arterial nerves, inhibited inflammation independently of lymphocytes. In striking contrast to the anti-inflammatory pathway mechanism described so far, we also found that the inhibition of inflammation by apical nerve electrical stimulation relied on signaling by both ß 2-ARs and α7nAchRs in myeloid cells, with these two signaling pathways acting in parallel. Most importantly, apical splenic nerve electrical stimulation mitigated clinical symptoms in a mouse model of rheumatoid arthritis further providing the proof-of-concept that such an approach could be beneficial in patients with Immune-mediated inflammatory diseases.

Detection of alpha7 nicotinic acetylcholine receptors with the aid of antibodies and toxins.

Neuronal nicotinic acetylcholine receptors (nAChRs) containing alpha7 subunit are well represented in the brain and some non-neuronal tissues, and their malfunctioning is associated with diverse pathologies. Therefore, detection and quantification of alpha7 nAChR are important tasks. The affinity-purified antibodies were prepared against the 1-23 and 179-190 fragments of the human and rat alpha7 nAChR extracellular domain. The specificity and selectivity of these alpha7 (1-23) and alpha7 (179-190) antibodies was tested by ELISA in model systems: the E. coli-expressed alpha7 subunit extracellular domain and the pituitary cell line GH(4)C(1) stably expressing human alpha7 nAChR. On the rat brain slices two antibodies and biotinylated alpha-cobratoxin specifically stained the hippocampus region known to be rich in alpha7 nAChR. Western blot analysis revealed that in the human thalamus membranes and in rat brain membranes, antibodies alpha7 (1-23) stained a single band of 62 kDa, while the alpha7 (179-190) antibodies stained a doublet of 53-54 kDa. The results obtained show that utilization of model systems and a combination of several antibodies with appropriately labeled toxins may provide better ways for detection of alpha7 nAChR.

Increased expression of the nicotinic acetylcholine receptor in stimulated muscle.

Chronic low-frequency stimulation has been used as a model for investigating responses of skeletal muscle fibres to enhanced neuromuscular activity under conditions of maximum activation. Fast-to-slow isoform shifting of markers of the sarcoplasmic reticulum and the contractile apparatus demonstrated successful fibre transitions prior to studying the effect of chronic electro-stimulation on the expression of the nicotinic acetylcholine receptor. Comparative immunoblotting revealed that the alpha- and delta-subunits of the receptor were increased in 10-78 day stimulated specimens, while an associated component of the surface utrophin-glycoprotein complex, beta-dystroglycan, was not drastically changed in stimulated fast skeletal muscle. Previous studies have shown that electro-stimulation induces degeneration of fast glycolytic fibres, trans-differentiation leading to fast-to-slow fibre transitions and activation of muscle precursor cells. In analogy, our results indicate a molecular modification of the central functional unit of the post-synaptic muscle surface within existing neuromuscular junctions and/or during remodelling of nerve-muscle contacts.

Neuronal nicotinic receptors in human epilepsy.

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a rare monogenic idiopathic partial epilepsy characterized by clusters of frontal lobe motor seizures during sleep. Recently, it has been shown that mutations of the chromosome-20q-located neuronal nicotinic acetylcholine receptor alpha4-subunit (CHRNA4) are associated with ADNFLE in some families, but that other families are not linked to this locus. Both CHRNA4 mutations (Ser248Phe and 776ins3) identified so far are found in the pore-forming second transmembrane region of the gene. Electrophysiological studies showed that mutations in this functional important part of the receptor subunit have a profound effect on the permeability for calcium ions. Interestingly, the Ser248Phe mutation was found again in a second ADNFLE family. Haplotype analysis excluded a founder effect and showed that Ser248Phe occurred independently twice. This provides the possibility to study the effect of the same mutation on different genetic backgrounds. Several attempts have been made to identify additional genes responsible for ADNFLE. But despite some positive linkage results including the CHRNA3-CHRNA5-CHRNB2 cluster on chromosome 15q24, no further mutations have been found so far. The mutation screening of functionally important parts of CHRNA5 in 12 ADNFLE patients did not support a causative role of this nicotinic acetylcholine receptor subunit.

Effects of antibody binding on structural transitions of the nicotinic acetylcholine receptor.

Patch-clamping and photoaffinity-labeling techniques were used to study the effects of binding of monoclonal antibodies (mAbs) on the function of Torpedo californica nicotinic acetylcholine receptor (nAChR). The rat anti-Torpedo nAChR mAbs examined here are known to inhibit ligand binding to either the high-affinity (mAb 247) or both the high- and low-affinity binding sites (mAb 370 and mAb 387) [Mihovilovic, M. & Richman, D. P. (1984) J. Biol. Chem. 259, 15051-15059; Mihovilovic, M., & Richman, D. P. (1987) J. Biol. Chem. 262, 4978-4986]. Single-channel analysis shows that mAb 247 and the Fab fragment of mAb 247 inhibit the opening of the nAChR ion channel, although they have no effects on the structural transition from the resting to desensitized state as monitored by the extent of decreased labeling by the photoreactive probe 3-(trifluoromethyl)-3-(m- [125I]iodophenyl)diazirine ([125I]-TID). In the presence of mAb 387, the nAChR single-channel amplitude was decreased by 20%, whereas Fab 387 completely inhibited channel opening. [125I-TID]-labeling studies suggest that the mAb 387-nAChR and Fab 387-nAChR complexes are able to undergo the transition between resting and desensitized states. This result confirms that the nAChR can assume a desensitized state without prior channel opening. In addition, mAb 35 and mAb 132, which recognize the main immunogenic region (MIR) of the nAChR, and mAb 370 do not alter either single-channel behavior or labeling patterns. Combining the results from characterization with respect to their epitopes and their effects on agonist (carbamylcholine) and antagonist [alpha-bungarotoxin (alpha-BTX) and curare] binding, these results indicate that mAbs could be used to map functional and structural domains.

Regulatory action of Astragalus saponins and buzhong yiqi compound on synthesis of nicotinic acetylcholine receptor antibody in vitro for myasthenia gravis.

With overall analysis of symptoms and signs of myasthenia gravis (MG) basing on "Pi (Spleen) Deficiency" [symbol: see text] theory and with years of our clinical experience in treating MG, we performed a pharmacological study of Astragalus saponins and Buzhong Yiqi Compound (Tonic granulae invigorating vital energy) in 14 peripheral blood mononuclear cell (PBMNC) cultures from 10 MG patients. PBMNG from two groups of patients given dexamethasone (Dxm) and cobalt 60 (60Co) treatment were used as controls. The results showed that water soluble Astragalus saponins significantly reduced the titer of nicotinic acetylcholine receptor antibodies (nAchR-Ab) in the cell culture supernatants, from 418.8-2328 to nil in 6 cases, and from 1143-1235 to 43-157 fmol/ml in 2 cases, and that Buzhong Yiqi compound also had inhibitory immunoregulatory action.

T cells reactive with a small synthetic peptide of the acetylcholine receptor can provide help for a clonotypically heterogeneous antibody response and subsequently impaired muscle function.

The influence of T cell specificity was evaluated with regard to its role in the antibody response against the acetylcholine receptor (AChR) and resulting AChR-dependent muscle dysfunction. The reactivity of immune Th cells was restricted to a small region of the AChR alpha-subunit (amino acid residues 100-116) reported to be highly immunogenic. T cells primed to this peptide were found to demonstrate significant proliferation when challenged in vitro with either the homologous peptide or the intact AChR. Adoptive transfer of the peptide-immune T cells into immunologically naive recipient rats followed by AChR challenge resulted in the production of anti-AChR antibodies very similar to those produced under the regulation of T cells immune to the entire intact AChR with regard to overall clonotypic heterogeneity (measured by IEF) and their ability to interfere with AChR-dependent muscle contraction. Interestingly, when the threonine at position 106 was substituted with a proline, the resulting peptide continued to be equally, if not exceedingly, capable of stimulating T cell-proliferative responses, but was found to be ineffective at stimulating the levels of anti-AChR antibodies necessary for producing neuromuscular dysfunction.

Immunologic similarities between the hypothalamic alpha-bungarotoxin receptor and the Torpedo californica nicotinic cholinergic receptor.

The solubilized rat central nervous system (hypothalamic) nicotinic cholinergic receptor and the Torpedo nicotinic cholinergic receptors are immunologically similar and show greater than 60% immunologic cross-reactivity using a double-antibody technique. Antibodies to the Electrophorus and Torpedo receptors also decrease the rate of alpha-bungarotoxin binding to these membraneous receptors. It is concluded that the Torpedo and hypothalamic nicotinic receptors are immunologically similar and that receptor binding sites for alpha-bungarotoxin and antibodies are physically close. These studies indicate that alpha-bungarotoxin can be used to study the nicotinic cholinergic receptor of the rat hypothalamus.