Pathological Effects

For last 15 years, research efforts in this area have focused on demonstrating the pathophysiological effects of anti-ganglioside antibodies, but it is only recently that the mechanism(s) of anti-ganglioside antibody-mediated axonal injury are beginning to emerge. Clinical, pathological, and experimental findings suggest that there is a spectrum of pathophysiological changes in AMAN that includes recognizable functional and structural axonal injury. Whereas the previous sections have outlined the possible mechanism(s) of generation of specific immune insult(s) directed against the axon, this section outlines evidence derived from clinical and experimental studies providing insights into the mechanisms involved in antibody-mediated axonal injury.

Figure 3 Fresh-frozen cross-sections of rat cauda equina (ventral and dorsal root) triple labeled with anti-ganglioside antibody GD1a-IgG1 (green), neurofilament (red), and IB-4 (blue). Co-localization of three labels is also shown (merged). Neurofilament stains all myelinated fibers, whereas IB-4 stains unmyelinated fibers in dorsal root. This figure shows that monoclonal antibody GD1a-IgG1 preferentially binds to myelinated motor axons in the ventral root but not the sensory myelinated fibers in dorsal roots. (Adapted with permission from Gong et al., 2002.)

Figure 3 Fresh-frozen cross-sections of rat cauda equina (ventral and dorsal root) triple labeled with anti-ganglioside antibody GD1a-IgG1 (green), neurofilament (red), and IB-4 (blue). Co-localization of three labels is also shown (merged). Neurofilament stains all myelinated fibers, whereas IB-4 stains unmyelinated fibers in dorsal root. This figure shows that monoclonal antibody GD1a-IgG1 preferentially binds to myelinated motor axons in the ventral root but not the sensory myelinated fibers in dorsal roots. (Adapted with permission from Gong et al., 2002.)

This discussion is organized to bring out issues relevant to the means by which circulating antibody gains access to myelinated axons in nerves, keeping in view the anatomy of nerves and myelinated nerve fibers (Fig. 3).

In autoimmune diseases, accessibility of target tissues, cells, and antigens to immune effectors is critical in determining the distribution of injury. This is all the more important for the diseases of the nervous system, because the blood-tissue barriers contribute to the immune privileged status of neural tissues. For an immune effector to cause neural injury it has to cross the respective blood tissue barrier (i.e., blood-nerve-barrier for PNS and blood-brain barrier for CNS injury). Peripheral nerve fibers are organized into individual bundles or fascicles. A fibrofatty layer, the epineurium, surrounds and holds individual fascicles in nerve bundles. Each nerve fascicle is surrounded by per-ineurium, which consists of a variable number of layers of specialized perineurial cells attached to each other by tight junctions, with each cell layer covered by basal lamina. The thickness of perineurium varies along the course of peripheral nerves; the thickness diminishes in linear relationship to the diameter of the nerve. The perineurium plays a role in regulating the endoneurial environment, including providing a barrier to diffusion of macromolecules such as antibodies and complement components from the epineurial space. Notably, the perineurium is open-ended near termination of the motor nerve terminal, allowing accessibility to endoneurial space and axon. The endoneurium is a space bounded by the perineurium and includes the nerve fibers, supporting glia, and extracellular matrix containing collagen. It is the endothelial cells of endoneurial blood vessels that form tight junctions with apposing cells, resulting in the formation of blood-nerve barrier. Studies with circulating tracers indicate that the permeability of the blood-nerve barrier to macromolecules, such as horseradish peroxidase, is much higher than that of the blood-brain barrier (Arvidson, 1977). This is also supported by the findings that a low level of IgG is detectable in the normal endoneurium. Further, it appears that along the course of peripheral nerve fibers the blood-nerve barrier permeability varies. This barrier is relatively more lax at the level of dorsal root ganglion and spinal roots and almost nonexistent at motor nerve terminals, making these two sites more vulnerable to attack by circulating immune effectors such as antibodies targeting peripheral nerve antigens.

We have recently examined whether circulating anti-gan-glioside antibodies can access the neural tissues and cross the normal blood-nerve barrier and blood-brain barrier in mice by using tracer studies with a radiolabeled monoclonal anti-ganglioside antibody. Our results indicate a low level accumulation of this antibody in both PNS and CNS, but the accumulation in the CNS was 5- to 10-fold lower than that in the PNS. Further, there was a significant increase in the amount of antibody that accumulated in the neural tissues in the setting of experimental systemic inflammation resulting in an increase in the levels of circulating inflammatory cytokines (Sheikh et al., 2004). In addition, there is experimental evidence suggesting that sera from patients with GBS, particularly those with anti-ganglioside antibodies, perturb the permeability of an in vitro blood-nerve barrier model (Kanda et al., 2000, 2003). These findings suggest that in the absence of peripheral nerve antigen-specific-T-cell inflammation, anti-ganglioside antibodies can access the endoneurium and this accessibility is enhanced in the presence of circulating inflammatory cytokines, which have been reported to be elevated in patients with GBS (Sharief et al., 1993, 1997). Given these experimental findings and the absence of prominent T-cell inflammation in pathological studies of AMAN cases, it is conceivable that, in antibody-mediated neuropathies such as AMAN, T-cell-mediated breakdown of the blood-nerve barrier is not necessary for antibody accessibility to nerve.

Once the antibodies cross the blood vessels and reach the endoneurial extracellular matrix, how do they access the target antigens on nerve fibers? Does the complex endoneurial interstitium allow antibodies to diffuse through it and access the target antigens on nerve fibers, or is focal disruption of this highly organized structure required? Although uniform low-level endoneurial staining for IgG in normal nerves would favor that antibodies can diffuse through the intersti-tium, a definitive answer to this complex issue is not known and would require the development of sophisticated techniques to study the kinetics of macromolecules in the endoneurium.

The next step in this sequence of events is the antibody binding to axonal antigens on myelinated fibers. In myeli-nated fibers, there are two sites along the axon that are not covered by myelin, but only by Schwann cell basal lamina, the nodes of Ranvier, and motor nerve terminals. Therefore, these sites are relatively more accessible to immune effectors and probably more susceptible to antibody-mediated injury. The susceptibility of these two sites to antibody-mediated injury would be supported by the pathological and immunopathological findings in AMAN and experimental models demonstrating pathogenic effects of anti-ganglioside antibodies on motor nerve terminals (see later). The periax-onal space surrounding the internodal axolemma is relatively inaccessible to ions and macromolecules such as antibody and complement in endoneurial interstitial fluid, because Schwann cell terminal myelin loops attach to para-nodal axolemma by gap junction-like complexes and the periaxonal space in this region only measures 3 to 5 nm compared to 12 to 14 nm in the internodal region. Factors that contribute to the differential susceptibility of different regions of a myelinated motor axon to antibody-mediated injury are summarized in Table 2.

The pathology and immunopathology of AMAN are reviewed, keeping in mind the anatomic organization of myelinated fibers as discussed previously. The pathological examination of both early and late cases has been extremely instructive and has allowed the reconstruction of patho-genetic events over the course of the disease. In AMAN the earliest pathological changes are quite subtle and involve the nodes of Ranvier of motor fibers in the ventral roots (Griffin et al., 1996c). These changes consist of lengthening of the nodal gap at times when the fibers otherwise appear normal. Macrophages are recruited to the nodes of Ranvier early on

Table 2 Properties of Different Regions of a Myelinated Motor Axon Contributing to

Susceptibility to Anti-ganglioside Antibody-Mediated Injury

Table 2 Properties of Different Regions of a Myelinated Motor Axon Contributing to

Susceptibility to Anti-ganglioside Antibody-Mediated Injury

Node/Paranode

Internode

Motor nerve terminal

Ganglioside antigens

++

+

++

Covered by myelin

-

+

-

Ion channels

+ (Na+ and K+)

-

+ (Ca++)

Accessibility to

antibody binding

+

+++

*When periaxonal space is breached or potentially after demyelination.

*When periaxonal space is breached or potentially after demyelination.

(Fig. 4A2), and these macrophages then insert into the nodal gap. The immunopathological changes correlating with motor fiber pathology in ventral roots are also initially observed at nodes of Ranvier. It is observed that early in the pathogenetic process IgG binds at nodes of Ranvier, leading to activation of complement, as suggested by C3d deposition, which causes macrophage recruitment at nodes of Ranvier through complement-derived chemoattractants (Fig. 4B and C) (Hafer-Macko et al., 1996). The currently favored patho-genetic sequence proposes that macrophages at nodes of Ranvier then open the normally impermeable periaxonal space to endoneurial constituents including antibody and complement. This sequence of events is supported by the immunocytochemical findings showing deposition of IgG and complement activation marker C3d in periaxonal space and on internodal axolemma in late cases (Fig. 4D) (Hafer-Macko et al., 1996). Subsequently macrophages are recruited to the periaxonal internodal space after the disruption of paranodal sites of attachment of the Schwann cell myelin sheath to the axon. Insertion of macrophages into the periax-onal space leads to axonal shrinkage and separation away from adaxonal Schwann cell plasmalemma. The shrunken axon surrounded by macrophage survives for some time before undergoing wallerian-like degeneration (Fig. 4E and F) (Griffin et al., 1996a). This sequence of pathological changes in early and late cases would be compatible with the distribution of gangliosides, accessibility of anti-ganglioside antibodies and complement, and anatomic organization of myelinated nerve fibers. In myelinated fibers, the motor nerve terminal is another axonal site susceptible to injury by circulating immune effectors because it is neither covered by myelin nor protected by blood-nerve barrier. A recent case report provides proof of concept for this hypothesis; this description includes a patient with AMAN who recovered rapidly after treatment with IVIg. Pathological examination of the muscle biopsy showed motor fiber degeneration largely limited to intramuscular nerves or motor nerve terminals (Ho et al., 1997a). One explanation for the rapid recovery in this instance is that regeneration over a relatively short distance can be achieved quickly.

The next critical question is this: Can anti-ganglioside antibodies with specificity similar to those in patients with AMAN cause axonal injury and reproduce the pathological and immunopathological features of AMAN in experimental animal models? Until recently there were no reproducible or reliable animal models of anti-ganglioside antibody-mediated nerve injury particularly in rodents. Recent studies indicate that repeated immunizations with a mixture of gangliosides, keyhole limpet hemocyanin, and complete Freund's adjuvant in rabbits can produce neuropathic injury. The pattern and distribution of neuropathic injury relate to the specificity of ganglioside used for immunization and the specific anti-ganglioside response. For example, studies with GD1b ganglioside led to the development of sensory ataxic

The Effect The Node Ranvier

Figure 4 Representative pathology and immunopathology of early (A-C) and late (D-F) AMAN cases. (A1) Longitudinal section of a large myelinated fiber showing a normal node of Ranvier (arrow). (A2) Longitudinal section of a motor fiber showing lengthening of the node (arrow) and overlying macrophages (arrowheads). (B) Teased fiber preparations stained for C3d deposition showing deposition of this complement activation product at the nodes of Ranvier. (C) Teased fiber preparations stained with a macrophage marker showing macrophages overlying and inserting processes at the nodes of Ranvier. (D) Cross-section of a ventral root showing deposition of C3d on internodal axolemma. (E) Electron micrograph of a longitudinally sectioned motor nerve fiber showing a macrophage (M) entering the periaxonal space and lying adjacent to axon (A) and separating the overlying myelin sheath. (F) Electron micrograph of cross-sectioned motor nerve fibers at different stages of axonal degeneration; the fiber on the left shows a macrophage (M) in the peri-axonal space surrounding a shrunken but intact axon (A) inside a normal appearing myelin sheath; in the fiber on the right an axon cannot be identified and macrophage (M) has occupied the position normally maintained by axon. (Adapted with permission from Griffin et al., 1995, 1996c; Hafer-Macko et al., 1996.)

Figure 4 Representative pathology and immunopathology of early (A-C) and late (D-F) AMAN cases. (A1) Longitudinal section of a large myelinated fiber showing a normal node of Ranvier (arrow). (A2) Longitudinal section of a motor fiber showing lengthening of the node (arrow) and overlying macrophages (arrowheads). (B) Teased fiber preparations stained for C3d deposition showing deposition of this complement activation product at the nodes of Ranvier. (C) Teased fiber preparations stained with a macrophage marker showing macrophages overlying and inserting processes at the nodes of Ranvier. (D) Cross-section of a ventral root showing deposition of C3d on internodal axolemma. (E) Electron micrograph of a longitudinally sectioned motor nerve fiber showing a macrophage (M) entering the periaxonal space and lying adjacent to axon (A) and separating the overlying myelin sheath. (F) Electron micrograph of cross-sectioned motor nerve fibers at different stages of axonal degeneration; the fiber on the left shows a macrophage (M) in the peri-axonal space surrounding a shrunken but intact axon (A) inside a normal appearing myelin sheath; in the fiber on the right an axon cannot be identified and macrophage (M) has occupied the position normally maintained by axon. (Adapted with permission from Griffin et al., 1995, 1996c; Hafer-Macko et al., 1996.)

neuropathy in rabbits, and pathological analysis showed degeneration of dorsal root ganglion cells and allied axonal projections (Kusunoki et al. 1996b). In contrast, use of whole bovine brain gangliosides or GM1 for immunization resulted in the development of high titer IgG anti-GM1 responses, flaccid paralysis, and degeneration of motor axons in the ventral roots (Yuki et al., 2001). The immunopathology and pathology of these animals showed deposition of IgG on motor axons and the presence of peri-axonal macrophages in motor fibers, features reminiscent of human pathology (Susuki et al., 2003). Moreover, pathological analysis of some animals with severe muscle weakness and death a short time after onset of clinical weakness showed no morphological axonal changes, suggesting that in

AMAN, axonal dysfunction precedes the degeneration (Susuki et al., 2003).

With the availability of a variety of transgenic animals, we have recently taken an alternate approach to generate a mouse model of anti-ganglioside antibody-mediated neuropathy. This paradigm included the generation of monoclonal anti-ganglioside antibodies (Lunn et al., 2000) and implantation of antibody-secreting hybridoma in mice. This led to the development of axonal neuropathy when a GD1a-reactive antibody was implanted in mice (Sheikh et al., 2004). The development of neuropathy in this model was contingent on breakdown of the blood-nerve barrier by circulating cytokines generated by implantation of antibody-secreting tumor. These findings not only provide additional evidence for pathogenicity of anti-ganglioside antibodies, but also provide an example of non-T-cell-dependent breakdown of the blood-nerve barrier. Attempts of passive transfer of neuropathy in animal models with patient-derived or experimental anti-ganglioside antibodies so far have been unsuccessful.

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