Protection of Axons via Facilitation of Remyelination

The existence of pathogenic autoantibodies is well established for several peripheral neurological syndromes including myasthenia gravis, Lambert-Eaton syndrome, Guillain-Barre syndrome, and acquired neuromyotonia (Vincent et al., 1999). Involvement of pathogenic autoantibodies in a particular dis ease has been defined by several lines of experimental and clinical evidence. For example, antibodies to a defined target should be present in the majority of patients that present with the disease. The presence of these antibodies is often demonstrated by using the purified antibodies to immuno-stain tissues that express the target antigen. Moreover, immunization with the target antigen, passive transfer of antibodies against the antigen, or transfer of antibodies from patients with the disease to naive animals should induce disease. Finally, reduction of serum antibody levels, by plasma exchange or by immunosuppression, generally leads to clinical improvement in patients, whereas an increase in antibody levels induces a return of clinical symptoms. Autoantibodies may also play a role in MS. When antibodies to myelin oligodendrocyte glycoprotein (MOG) are injected after the induction of EAE, the severity of the disease is dramatically increased and large demyelinating lesions develop (Genain et al., 1995; Linington et al., 1992; Schluesener et al., 1987). In addition, antibodies to MOG have been detected in association with disintegrating myelin in both human MS patients and in a marmoset model of EAE (Genain et al., 1999). Likewise, EAE-like Type II MS lesions, responsible for 30% to 50% of lesions in patients, are characterized by the deposition of immunoglobulin and complement (Lucchinetti et al., 1998, 2000). Finally, we have shown that plasma exchange is effective in approximately 40% of cases with fulminant MS exacerbations, suggesting the presence of pathogenic autoantibodies in these patients (Weinshenker et al., 1999).

In a novel application of many of the same criteria used to define pathogenic antibodies, we have tried to define autoantibodies that promote tissue repair. A direct demonstration that autoreactive antibodies can enhance endogenous myelin repair came from our studies using Theiler's virus to induce chronic demyelinating disease in mice (Rodriguez et al., 1987a). As described previously, intra-cerebral infection of susceptible mice with Theiler's virus results in a disease course characterized by acute encephalitis that is resolved in 14 to 21 days, followed by chronic viral persistence in spinal cord white matter. Persistent Theiler's virus infection eventually leads to chronic demyelination and progressive loss of motor function, a clinical pattern similar to that observed for progressive MS in humans. Myelin pathology in Theiler's virus infected mice is immune-mediated, with chronically infected animals demonstrating a wide range of disease phenotypes depending on their specific genetic background. In the SJL strain, demyelination is evident within 30 days after infection and by 1 to 3 months the animals develop neurological deficits including spasticity and gait abnormalities, weakness of the lower extremities, and bladder incontinence. Paralysis eventually occurs by 6 to 9 months after infection. Spontaneous remyelination is common in many mouse strains, but is limited in the SJL strain; often less than 10% of the total demyelinated lesion area is repaired. The low background of spontaneous repair in these animals makes them an excellent model for the study of strategies to promote endogenous remyelination (Rodriguez et al., 1987b).

Our initial observation of a beneficial humoral immune response occurred when significant remyelination was demonstrated in SJL mice that were chronically infected with Theiler's virus and immunized with spinal cord homogenate (SCH) prepared in incomplete Freund's adjuvant. Histological examination of spinal cord lesions from immunized animals revealed substantial remyelination compared to control animals. Passive transfer of antiserum (Rodriguez et al., 1987a) or purified immunoglobulin (Rodriguez and Lennon, 1990) from uninfected animals that had been immunized with SCH also enhanced remyelination in infected SJL mice, directly demonstrating for the first time a beneficial role of the humoral immune response in promoting myelin repair. This enhancement of remyelination was associated with proliferation or preservation of mature oligodendrocytes (Ludwin and Bakker, 1988; Prayoonwiwat and Rodriguez,

To further explore the nature of this beneficial immune response, hybridomas were generated from SJL mice after SCH immunization, in an attempt to identify monoclonal antibodies that promote remyelination. Two mouse monoclonal antibodies that enhance remyelination were identified and designated SCH94.03 and SCH79.08 (Miller et al.,

1994) (Fig. 17). Both of these monoclonal antibodies are polyreactive IgMs that bind to antigens on the surface of oligodendrocytes, suggesting that the activity of these monoclonal antibodies may involve direct stimulation of the myelin-producing cells (Asakura et al., 1996). Four additional oligodendrocyte-specific mouse IgMs (O4, O1, A2B5, and HNK1) were also shown to promote CNS remyelination (Asakura et al., 1998), suggesting that this phenomenon was a general principle rather than an isolated immunological aberration.

Figure 17 Antibody-mediated remyelination. (A) Normal myelin in an uninfected SJL/J mouse. (B) Severe demyelination is observed in a chronically infected SJL/J mouse. (C) Five weeks after a single treatment with the mouse IgM 94.03 there is significant remyelination within demyelinated lesions of a chronically infected SJL/J mouse. (D) Likewise, 5 weeks after a single treatment with the human antibody rHIgM22 there is a dramatic increase in remyelinated axons within demyelinated lesions. Scale bar in (D) is 50 |m and refers to all panels.

Figure 17 Antibody-mediated remyelination. (A) Normal myelin in an uninfected SJL/J mouse. (B) Severe demyelination is observed in a chronically infected SJL/J mouse. (C) Five weeks after a single treatment with the mouse IgM 94.03 there is significant remyelination within demyelinated lesions of a chronically infected SJL/J mouse. (D) Likewise, 5 weeks after a single treatment with the human antibody rHIgM22 there is a dramatic increase in remyelinated axons within demyelinated lesions. Scale bar in (D) is 50 |m and refers to all panels.

Using oligodendrocyte binding as a screening assay, we identified candidate human monoclonal antibodies that might also promote remyelination (Warrington et al., 2000). Human monoclonal antibodies were isolated from patients with monoclonal gammopathy, a relatively common condition characterized by high concentrations of monoclonal serum antibody. A total of 52 serum-derived human monoclonal IgMs (sHIgMs) were screened, and six were found to bind to the surface of morphologically mature rat oligoden-drocytes in culture. In contrast, none of the 50 serum-derived human monoclonal IgGs (sHIgG) bound to oligodendrocytes. The six oligodendrocyte-binding sHIgMs were tested in vivo, and two, designated sHIgM22 and sHIgM46, were found to promote substantial remyelination (Warrington et al., 2000) (Fig. 17). More recently, we have engineered a recombinant form of sHIgM22, designated rHIgM22, that exhibits the same pattern of oligodendrocyte binding (Fig. 18) and induces the same level of remyelination as sHIgM22 (Mitsunaga et al., 2002) (Fig. 17). Generation of this recombinant monoclonal human IgM marked an important step forward in the production of a potential therapeutic agent aimed at ameliorating demyeli-nation in patients with MS, as this antibody is available in essentially unlimited quantities and will not induce cross-species reactivity if administered to humans.

As outlined previously, there are several properties used to define autoantibodies as pathogenic agents. Similar criteria can be applied to autoreactive antibodies involved in tissue repair. For example, recognition of appropriate tissues or molecules is an important defining characteristic. All of the antibodies that promote remyelination bind to antigens on the surface of oligodendrocytes, suggesting that these antibodies function through direct stimulation of the myelin-producing cells. It is important to note that these antibodies are polyreactive, recognizing a variety of chemical haptens and proteins as measured by enzyme-linked immunosorbent assay, and also robustly recognizing intracellular proteins in permeablized cells. Despite this antigenic promiscuity, however, remyelination-promoting antibodies bind only to a limited number of antigens on the surface of live oligo-dendrocytes. The surface antigens bound by several of these monoclonal antibodies have been characterized and are generally lipid or carbohydrate in nature (Asakura et al., 1998; Sommer and Schachner, 1981).

Remyelination-promoting antibodies appear to be naturally occurring autoantibodies. Antibodies of this type are present in the serum of normal individuals and are often polyreactive IgMs capable of binding to a variety of structurally unrelated, self and non-self antigens (Lacroix-Desmazes et al., 1998). Such antibodies may represent a primordial form of the immune system that evolved to perform largely physiological functions rather than classical immune functions (Bouvet and Dighiero, 1998; Stewart, 1992). One of these nonimmunological functions may be to promote tissue repair, serving effectively as trophic factors for specific cell types (Bieber et al., 2001).

The concept of an endogenous antibody-mediated repair system is consistent with our initial observation of enhanced remyelination after SCH or myelin antigen immunization (Rodriguez et al., 1987a). Immunization may mimic immune system exposure to CNS antigens that occurs after injury, resulting in an increased titer of anti-CNS antibodies. Human IgMs from patients with macroglobulinemia were found to bind at high frequency to myelin antigens, suggesting that anti-CNS antibodies are common in the serum of individuals with no history of neurological damage (Warrington et al., 2000). Therefore, increasing the serum concentration of specific anti-CNS monoclonal antibodies may be a novel therapeutic treatment for human neurological injury and disease.

Because all of the remyelination-promoting monoclonal antibodies that we have identified so far bind to oligoden-drocytes or myelin, it seems reasonable to suggest a direct effect on the recognized cells. Other laboratories have demonstrated that oligodendrocyte-specific antibodies can induce biochemical and morphological changes in glial cells. For example, Dyer and colleagues showed that antibodies directed against oligodendrocyte surface epitopes, including antibodies to galactocerebroside, sulfatide, and myelin/oligodendrocyte-specific protein, can induce changes in the organization of oligodendrocyte plasma membrane and can alter cytoskeletal structure (Dyer and Benjamins, 1988; 1989; Dyer and Matthieu, 1994). These changes in cellular structure were preceded by antibody-induced calcium influx (Dyer, 1993; Dyer and Benjamins, 1990; 1991), suggesting that the gating of calcium may play an important role in the regulation of oligodendrocyte structure and function, and may play a role in antibody-induced remyelination. In support of this hypothesis, we recently reported similar changes in intracellular calcium concentration in oligodendrocytes after treatment with remyelination-promoting monoclonal antibodies (Howe et al., 2004; Paz Soldan et al., 2003). We assessed the direct effect of mouse and human monoclonal IgMs on CNS glial cells by using ratiometric fluorescent monitoring of intracellular calcium concentration ([Ca2+];). A change in [Ca2+] was used as a marker for physiological activation. We found that remyeli-nation-promoting antibodies stimulated a transient elevation of [Ca2+]i in both astrocytes and oligodendrocytes. Of importance, all remyelination-promoting antibodies tested evoked Ca2+ transients in mixed glial cultures; isotype- and species-matched control antibodies did not (Paz Soldan et al., 2003) (Fig. 19).

We observed calcium responses of two types: a rapid transient calcium increase that occurred immediately upon exposure to antibody, and a slower transient calcium increase that was slightly delayed after antibody exposure. Morphological examination of the responding cells revealed

Figure 18 rHIgM22 stains white matter and human oligodendrocytes. (A) Phase contrast image of a slice of mouse cerebellum. The phase dark material is white matter. (B) rHIgM22 specifically stains the white matter tracts of the same cerebellar section shown in (A). (C) Higher magnification phase contrast view of a white matter tract in the cerebellum. (D) rHIgM22 staining in the same section shown in (C). Note the finely stained myelinated axons. (E) Phase contrast image of human glial cells, with a mature, highly branched oligodendrocyte present in the center of the field. (F) rHIgM22 only labels the oligodendrocyte. Note the extensive labeling of the elaborate myelin branches.

Figure 18 rHIgM22 stains white matter and human oligodendrocytes. (A) Phase contrast image of a slice of mouse cerebellum. The phase dark material is white matter. (B) rHIgM22 specifically stains the white matter tracts of the same cerebellar section shown in (A). (C) Higher magnification phase contrast view of a white matter tract in the cerebellum. (D) rHIgM22 staining in the same section shown in (C). Note the finely stained myelinated axons. (E) Phase contrast image of human glial cells, with a mature, highly branched oligodendrocyte present in the center of the field. (F) rHIgM22 only labels the oligodendrocyte. Note the extensive labeling of the elaborate myelin branches.

that the immediate type response occurred in astrocytes, of differentiation in oligodendrocytes that showed the whereas the delayed type response occurred in oligodendro- delayed calcium response, and found that approximately cytes (Paz Soldan et al., 2003). We also investigated the state 60% of delayed response cells showed surface immunoreac-

Time (seconds)

Time (seconds)

Figure 19 sHIgM22 induces transient calcium influx in mixed glial cells in culture as detected by ratiometric imaging using Fura-2. (A) Immediately after treatment with sHIgM22, there is a brief influx of calcium in astrocytes, followed by a slower but transient influx of calcium in oligodendrocytes. Ionophore (Bromo-A23187) induces maximal calcium influx in all cells. (B) Fluorescent image of two cells (8, 9) fluxing calcium in response to sHIgM22 treatment. It is important to note that after treatment with antibody, calcium levels return to baseline, suggesting that the calcium influx is not toxic but is rather an element in a specific signal transduction cascade elicited by the antibody.

Figure 19 sHIgM22 induces transient calcium influx in mixed glial cells in culture as detected by ratiometric imaging using Fura-2. (A) Immediately after treatment with sHIgM22, there is a brief influx of calcium in astrocytes, followed by a slower but transient influx of calcium in oligodendrocytes. Ionophore (Bromo-A23187) induces maximal calcium influx in all cells. (B) Fluorescent image of two cells (8, 9) fluxing calcium in response to sHIgM22 treatment. It is important to note that after treatment with antibody, calcium levels return to baseline, suggesting that the calcium influx is not toxic but is rather an element in a specific signal transduction cascade elicited by the antibody.

tivity to A2B5, a marker for preoligodendrocytes. The remaining 40% of cells exhibiting the delayed type response showed surface immunoreactivity to O4, an antisulfatide antibody that serves as a marker for more mature oligoden-drocytes (Paz Soldan et al., 2003).

Further characterization showed that the immediate astrocyte and delayed oligodendrocyte responses use distinct Ca2+ entry pathways. We found that the immediate Ca2+ response occurred in either the presence or absence of extracellular calcium, but that thapsigargin-induced depletion of endoplasmic reticulum calcium stores abrogated this response. In contrast, the delayed type response was pre vented by removal and chelation of extracellular Ca2+ (Paz Soldan et al., 2003). Moreover, although the number of cells exhibiting an immediate response to treatment with the O4 monoclonal IgM was not affected by blocking several classes of plasma membrane Ca2+ channels, pharmacologically blocking the activity of phospholipase C (PLC) significantly decreased the percentage of astrocytes that exhibited elevated intracellular Ca2+. In contrast, the delayed Ca2+ response in oligodendrocytes was not dependent on intracel-lular stores, but was sensitive to pharmacological inhibition of Ca2+ flux through a-amino-3-hydroxy-5-methyl-4-isoxa-zolepropionic acid (AMPA)-sensitive glutamate channels (Paz Soldan et al., 2003). These data support the conclusion that the delayed response in oligodendrocytes is dependent on Ca2+ entry through AMPA receptor Ca2+ channels, whereas the immediate response in astrocytes is dependent on Ca2+ release from endoplasmic reticulum stores, likely through PLC-mediated generation of IP3 and gating of IP3-sensitive calcium channels.

We also determined that the antigen-binding domain of remyelination-promoting antibodies is required for Ca2+ signaling in glial cells. As described previously, Theiler's virus-infected mice treated with mouse monoclonal IgM antibody 94.03 exhibit enhanced remyelination. In contrast, mice treated with species- and isotype-matched control monoclonal antibody CH12 exhibit fourfold to sixfold less myelin repair, similar to saline treatment; 94.03 and CH12 have greater than 99% amino acid identity, differing only in the third complementarity determining region (CDR3) by five amino acids (Asakura et al., 1998). The CDR3 is critical in the formation of the antigen-binding domain, and the difference in remyelination potential engendered by the sequence difference between 94.03 and CH12 suggests that antigen specificity, rather than some other component of antibody structure, is required for remyelination. Finally, mixed primary glial cultures exposed to SCH 94.03 exhibited a transient rise in [Ca2+]i, while similar cultures exposed to CH12 showed no calcium changes. Therefore, remyelination-pro-moting antibody-induced Ca2+ signaling in glial cells depends on antigen specificity defined by the CDR3 region of the antibody.

Of primary importance, antibody-mediated Ca2+ signaling in vitro correlated statistically with remyelination promotion in vivo. To establish a relationship between the ability of an antibody to elicit a Ca2+ response and its ability to promote remyelination, we studied several remyelination-promoting and control antibodies. Mouse remyelination-promoting antibodies SCH 94.03, SCH 79.08, and O4 all mediated Ca2+ responses in astrocytes and oligodendrocytes. The control mouse antibody CH12, which did not promote remyelination, also did not evoke Ca2+ responses. Likewise, human remyelination-promoting antibodies sHIgM22, sHIgM46, and CB2BG8 all mediated Ca2+ responses in astrocytes and oligodendrocytes, whereas the control human

antibodies sHIgM12, sHIgM14, sHIgM47, and AKJR4, which did not promote remyelination, did not evoke Ca2+ responses (Paz Soldan et al., 2003). Thus, there appears to be a high degree of correlation between the ability of monoclonal IgM antibodies to promote remyelination and the stimulation of calcium influx, suggesting a potential signaling connection between these two phenomena.

Further evidence in support of a causal link between calcium signaling and remyelination is provided by experiments showing that rHIgM22 elicits a transient elevation in intracellular calcium in CG4 cells, a model of premyelinat-ing oligodendrocytes (Howe et al., 2004). As with primary oligodendrocytes, we found that this calcium signal was dependent on the influx of extracellular calcium through AMPA receptor calcium channels. More importantly, we showed that rHIgM22 was able to rescue CG4 cells from death induced by either hydrogen peroxide or TNFa, and that this rescue was dependent on calcium flux through CNQX-sensitive calcium channels (Howe et al., 2004). Biochemically, rHIgM22 appears to block cell death by preventing c-jun N-terminal kinase (JNK) signaling and cas-pase-3 activation. In support of this model for rHIgM22 survival signaling, we also found that antibody treatment of SJL mice chronically infected with Theiler's virus led to a significant decrease in caspase family gene expression and caspase-3 activity within demyelinated lesions of the cervical spinal cord (Howe et al., 2004). As rHIgM22 induced substantial remyelination in these animals, we concluded that rHIgM22-induced calcium signaling blocked caspase-3 activation in premyelinating oligodendrocytes and thereby promoted their survival and consequent differentiation into myelin-producing oligodendrocytes (Howe et al., 2004).

Another important clue as to the mechanism of remyeli-nation-promoting antibody action was provided by experiments showing that the ability of rHIgM22 to induce calcium influx and rescue CG4 cells from death was dependent on the integrity of lipid rafts in the plasma membrane of these cells (Howe et al., 2004). Treatment with P-methylcyclodextrin, a cholesterol-chelating compound that effectively destroys lipid raft structure, prevented rHIgM22-induced survival signaling, suggesting that perhaps redistribution or aggregation of lipid raft domains is necessary for antibody-mediated signal transduction (Howe et al., 2004) (Fig. 20). This hypothesis is further supported by evidence that monovalent forms of sHIgM22 are unable to elicit remyelination or calcium signaling (Ciric et al., 2003), suggesting that multivalency, and therefore oligomerization of cognate antigen, is necessary for the function of remyelination-promoting antibodies. In fact, on the basis of the evidence at hand regarding antibody-induced signaling, we hypothesize that remyelination-pro-moting antibodies function as soluble initiators of the type of clustering and consequent signaling described for integrins and proteoglycans involved in cell-cell adhesion and contact-mediated signaling (Fig. 21).

Integrins and cell surface proteoglycans are associated with plasma membrane lipid raft microdomains and with the oligomeric scaffolding protein caveolin (Baron et al., 2003). Therefore, lipid rafts, enriched in a number of receptor and nonreceptor kinases, serve as a locus of integrin- and pro-teoglycan-related signaling. As a result of clustering induced by binding to extracellular matrix elements, integrin-associ-ated structural and signaling molecules are also clustered within lipid raft domains, and the resulting increase in density and proximity of these molecules results in the initiation of several important signals (Harder et al., 1998; Harder and Simons, 1997). The reorganization of actin filaments into stress fibers occurs at the focal point of this clustering activity, and results in a positive feedback loop that encourages further integrin and proteoglycan clustering and signaling. These focal adhesions are highly enriched in signaling molecules such as FAK (focal adhesion kinase) and members of the Src-family of nonreceptor tyrosine kinase (Hanks et al., 2003). FAK is recruited to integrin clusters either through direct interaction with the integrin P subunit or via interaction with reorganized actin fibers. Activated FAK in turn activates phosphoinositide-3 kinase (PI3-K), Src, and elements of the mitogen-activated protein kinase (MAPK) cascades (Bernfield et al., 1999; Bokoch, 2003; Juliano, 2002). Likewise, lipid raft clustering leads to signaling through the myristoylated and palmitoylated Src-family kinases Src, Fyn, Yes, and Lck (Hoessli et al., 2000). These nonreceptor tyrosine kinases are activated by clustering, and they initiate signaling cascades that result in activation of the MAPK Erk1/2. Erk1/2 signaling results in phosphorylation of elements of the ternary complex factor, and thus promotes transcription of the immediate early gene c-fos (Sofroniew et al., 2001). In parallel, lipid raft clustering of integrins and pro-teoglycans engages the MAPK JNK, leading to phosphory-lation of the transcription factor c-jun, and association of c-jun with c-fos to form the AP-1 transcription factor complex. This complex is a critical regulator of genes involved in cell proliferation (Lin, 2003). Likewise, clustering-induced activation of FAK and PI3-K leads to signaling through Akt, a protein kinase that promotes survival by phosphorylating and inactivating the proapoptotic proteins Bad and caspase-9 (Sofroniew et al., 2001). Finally, lipid raft clustering of integrins in response to extracellular matrix binding results in the activation of the Rho family of small guanine nucleotide-binding proteins. These molecules include Rac, Cdc42, and RhoA, which are involved in induction of filopodia and formation of focal adhesions (Kaibuchi et al., 1999).

An additional signaling mechanism invoked by clustering of integrins within lipid rafts involves the concomitant aggregation of growth factor receptors and ion channels. Aggregation of growth factor receptors results in partial activation via proximity-dependent transphosphorylation (Ferguson, 2003). This partial activation brings the growth

A

B

-, v-'" !

>

/ / " _

- ;

\

{ t ' < ' ■ "V

C

D

\

>

\

E

*

* *

! " 'A

S

A

st

i t ■

r*

rHIgM22 p-MCD rHIgM22

How To Bolster Your Immune System

How To Bolster Your Immune System

All Natural Immune Boosters Proven To Fight Infection, Disease And More. Discover A Natural, Safe Effective Way To Boost Your Immune System Using Ingredients From Your Kitchen Cupboard. The only common sense, no holds barred guide to hit the market today no gimmicks, no pills, just old fashioned common sense remedies to cure colds, influenza, viral infections and more.

Get My Free Audio Book


Post a comment