THE MEMBRANE ATTACK COMPLEX OF COMPLEMENT (C5b-9) RESCUES SCHWANN CELLS (SchC) FROM APOPTOSIS IN EXPERIMENTAL ALLERGIC NEURITIS (EAN).
Hafer-Macko C.E., Koski C.L. University of Maryland, Baltimore, Maryland, USA.
C5b-9 is transiently detected on the surface of SchC between days 9 to 13 post-immunization in EAN. C5b-9 initiates macrophage independent myelin sheath disruption. SchC apoptosis is reported to occur in EAN and sublytic C5b-9 prevents SchC apoptosis induced by serum withdrawal in vitro. Therefore, we examined the effects of C5b-9 on SchC apoptosis in inflammatory neuropathy in PVG/C6-rats, a subset of rats which are genetically deficient in C6. The inability to form C5b-9 was confirmed by hemolytic assay. EAN was induced with bovine peripheral nerve myelin in 15 PVG/C6- and 15 PVG/C6+ control rats. Onset and severity of clinical disease was similar in both groups. Lumbosacral roots and sciatic nerves were examined on days 9 through 29 days. Apoptosis was identified by in situ DNA fragment labeling with terminal transferase and digoxigenin 11-dUTP (TUNEL assay). The percentage of apoptotic SchC nuclei per 1000 total SchC during the course of EAN were identified using anti-S100 antibodies and DAPI nuclear staining. The frequency of apoptotic SchC cells/1000 SchC was two to four fold greater in PVG/C6- compared with PVG/C6+ rats on days 9 to 29 in EAN. The percentage of apoptotic SchC was maximal on day 16 (41.3±9.6) in PVG/C6- compared to (12.7± 4.3) in PVG/C6+ rats. In conclusion, our data suggests that C5b-9 rescued SchC from apoptosis during EAN. Preservation of SchC capable of myelin formation could contribute to peripheral nerve repair following immune-mediated demyelination.
MEMBRANE ATTACK COMPLEX OF COMPLEMENT (C5b9) INDUCES PROLIFERATION AND RESCUE FROM APOPTOSIS OF SCHWANN CELLS (SchC)
Dashiell S.M., Rus H., Koski C.L. University of Maryland, Baltimore, Maryland, USA
C5b-9 deposition on SchC of GBS patients or EAN rats does not induce cytolysis secondary to complement regulatory protein expression. To determine if sublytic C5b-9 induces SchC mitogenesis and survival in vitro, rat SchC in serum free medium (N2) were stimulated with C5b-9 assembled from purified proteins (C5,6,7,8,9) or b-neuregulin (NRG). C5b-9 increased 3H-thymidine incorporation 10-fold by 24 hrs over N2 alone or N2 with heat inactivated C5b-9. Cell cycle analysis with propidium iodide and FACS showed SchC in N2 were synchronized in G1/G0 at 24 hrs. C5b-9 shifted 50% of SchC into S phase similar to NRG (100 ng/ml). C5b-9 stimulated SchC doubling as assessed by vital dye exclusion. Pertussis toxin and PD098,059, but not a PKA inhibitor, prevented C5b-9 mediated thymidine incorporation suggesting signal transduction was mediated via Gi proteins and ERK kinase. ERK1 activity, increased 2-fold by 20 min. Serum withdrawal and culture in N2 that caused 40-50% of SchC to undergo apoptosis was prevented by NRG. Likewise, C5b-9 inhibited the number of apoptotic cells and reduced TUNEL-positive SchC. C5b-9, like NRG, is a potent trophic factor stimulating SchC mitogenesis and apoptotic rescue and may contribute to peripheral nerve repair following immune-mediated demyelination.
T CELL RECOGNITION OF NON PROTEIN ANTIGENS IN GUILLAIN BARRÈ SYNDROME.
Cooper J.C., Hughes S., Ben-Smith A., Savage C.O.S., Winer J.B. Divisions of Medical Sciences and Neurosciences, University of Birmingham, UK.
C jejuni is the major antecedent infection associated with acute inflammatory demyelinating poly-neuropathy (AIDP) and other subtypes of the Guillain-Barré Syndrome. Antibody cross-reac-tivity between the lipopolysaccharide of C jejuni and peripheral nerve ganglioside has been demonstrated suggesting a possible mechanism for the pathogenesis of the syndrome. T cell recognition of non protein antigens, however, has so far received little attention as a factor in disease pathogenesis. We have found that two patients with AIDP associated with preceding C jejuni infection had significant T cell proliferation in the peripheral blood to C jejuni antigens (SI 105 and 23). Treatment of the antigen with protease did not prevent substantial T cell proliferation (SI 53 and 7) suggesting that these cells were responding to non-protein antigens. The phenotype of these cells in one of these patients was CD4-CD8- ab TCR+. Such double negative T cells have a T cell receptor capable of recognising non-protein antigens and may be CD1 restricted. gd T cells are similar in having the potential to recognise non protein antigens and we have further shown that we can identify gd T cells in 6 out of 11 (55%) sural nerve biopsy specimens from patients with AIDP by immunocytochemistry. Our results support a role for non-protein recognition by T cells in the pathogenesis of GBS.
EXPOSURE TO NITRIC OXIDE DONORS CAN CAUSE AXONAL DEGENERATION: ELECTRICALLY ACTIVE AXONS ARE ESPECIALLY SUSCEPTIBLE
Smith K.J., Kapoor R., Hall S.M., Davies M. Guy's, King's & St. Thomas' Sch. Med. King's College, London.
Axonal loss is a major feature of some inflammatory demyelinating peripheral neuropathies, but its underlying mechanisms are frequently obscure. In the absence of a direct immune attack on the axons it is often assumed that they succumb to the mêlée of potentially deleterious factors found at inflammatory sites, such as degradative enzymes and cytokines. The inducible form of nitric oxide synthase (iNOS) is also expressed at sites of inflammation, and this enzyme is capable of producing nitric oxide (NO) at low micromolar concentrations. We have previously shown that such concentrations can cause temporary axonal conduction block (Redford et al., 1997, Brain;120: 2149-2157), and now show that they can also cause axonal degeneration, especially if the axons are electrically active during NO exposure. The dorsal roots of anaesthetised rats were exposed in vivo to NO (using the donor DETA NONOate, [NO]=l.5-25mM) while serially monitoring conduction. At lower [NO], conduction was blocked reversibly (as previously described), but the block was rendered persistent at higher [NO] (>20mM). Notably, the block was also rendered persistent when exposure to the lowest NO concentrations examined was combined with sustained impulse activity within the physiological range (50 or 100Hz for 2 hours). Morphological examination of such roots demonstrated axonal degeneration in some axons, particularly smaller axons. We conclude that relatively brief (2-4 hrs) exposure to NO can cause axonal degeneration. Moreover, the degeneration occurs at pathophysiological concentrations if the axons are firing at physio-logical frequencies. We propose that the combination of natural impulse traffic and NO at sites of inflammation may contribute to axonal degeneration.
EVIDENCE FOR A COMBINED PRE- AND POSTSYNAPTIC BLOCKADE OF NEUROMUSCULAR TRANSMISSION BY ANTI-GQ1B ANTIBODIES FROM PATIENTS WITH MILLER FISHER SYNDROME (MFS)
Buchwald B., Bufler J.(1), Carpo M.(2), Nobile-Orazio E.(2), Toyka K.V. Departments of Neurology, Universities Würzburg, (1)Munich, Germany, (2)Milano, Italy.
BACKGROUND: Neuromuscular blocking factors have been demonstrated in sera of patients with Miller Fisher syndrome (MFS), but the crucial antigen remains uncertain. MATERIALS AND METHODS: We have investigated serum, purified IgG and purified anti-GQlb antibodies from patients with typical MFS (n=ll) and after recovery from acute disease (n=3). For combined pre- and postsynaptic analysis sera were applied to mouse motor nerve terminals by means of the perfused macro-patch clamp electrode. For a more direct analysis of the effects on acetylcholine-induced postsynaptic currents, we performed outside-out patch clamp measurements on mouse myotubes. RESULTS: All MFS-sera induced combined pre- and postsynaptic blockade with a decrease in quantal content and reduction in amplitude of postsynaptic currents. Purified anti-GQlb antibodies were as effective as whole serum. No blockade could be induced by sera obtained after recovery from MFS. Five sera examined by outside-out patch clamp analysis caused a concentration dependent and reversible decrease in acetylcholine induced currents. IgG and sera from healthy subjects or from patients with other inflammatory, non-neuropathic diseases had no effect. CONCLUSION: In MFS, circulating antibodies depress the presynaptic transmitter release and prevent the activation of postsynaptic channels and thus seem to be of pathogenetic relevance in causing muscle weakness during acute stage of disease. In MFS, GQlb may be the principal but not exclusive antigen to which the humoral immune response is directed.
ANTI-GQ1B ANTIBODIES CLONED FROM MICE IMMUNISED WITH MILLER FISHER SYNDROME STRAINS OF C. JEJUNI LPS INDUCE NMJ BLOCK
Goodyear C.S.(1,2), Plomp J.J.(3,4), O'Hanlon G.M.(1), Morrison I.(1), Veitch J.(1), Conner J.(2), Molenaar P.C.(3) and Willison H.J.(1) (1)Department of Neurology, University of Glasgow, (2)Department of Biological Sciences, Glasgow Caledonian University, UK, Departments of (3)Physiology, (4)Neurology, Leiden University Medical Centre, The Netherlands
Miller Fisher syndrome (MFS) is associated with anti-GT1a/GQlb
antibodies in >90% of cases. MFS follows C. jejuni enteritis
and structural studies on C. jejuni lipopolysaccharide (LPS) have
revealed they bear GD3/GT1a ganglioside-like, GQ1b cross-reactive
carbohydrate moieties. Our electro-physiological studies using
MFS sera have demonstrated latrotoxin-like effects on the neuro-muscular
junction (NMJ), i.e. massive increase in spontaneous acetylcholine
release followed by transmission block. We have now proven that
LPS-induced antibodies which react with GQ1b can mediate this
effect as follows: C3H/HeN mice were immunised with 50 micro-grams
of LPS from C. jejuni strains OH4382 (GD3-like) and OH4384 (GT1a-
and GM1-like) and 15 anti-GD3/GT1a/GQ1b IgM/IgG monoclonal anti-bodies
(mabs) were derived by spleen cell fusion. Mabs bound to the NMJ.
Electrophysiological recordings at NMJs from normal mice hemi-diaphragms
exposed in vitro to the IgM mabs in the presence of complement
showed a-latrotoxin-like effects. Immuno-fluorescence
studies revealed deposits of IgM and complement products at these
NMJs. These studies demonstrate that anti-GQ1b antibodies arising
as part of the immune response to C. jejuni LPS can produce NMJ
block.