WO1995028962A1 - Inhibition of myelin destruction by interfering with the interaction between mac-2 and myelin - Google Patents

Abstract

A method for inhibiting myelin destruction including the steps of identifying the type of myelin destruction and determining if MAC-2 expression has been induced on the cells involved. Compounds are then administered to act at the MAC-2 site thereby inhibiting interactions between MAC-2 and myelin.

Description

INHIBITION OF MYELIN DESTRUCTION BY INTERFERING WITH THE INTERACTION BETWEEN MAC-2 AND MYELIN

TECHNICAL FIELD

The present invention relates to a novel method of treating demyelination by directly interfering with myelin destruction.

BACKGROUND OF THE INVENTION

Demyelination, i.e. destruction of myelin and thereby loss of function, can occur through genetic and non-genetic mechanisms (for a review see Morris, 1992) . In humans, the leu odystrophies are a major source of inherited demyelination in the central nervous system (CNS) . Inherited forms of chronic demyelination in the peripheral nervous system (PNS) are seen in humans in Charcot-Marie-Tooth Disease, Dejerine-Sottas Disease and Refsum/s Disease.

Non-genetic forms of demyelination, loss of the myelin sheath, are associated with multiple causes and diverse etiologies such as infectious agents, nutritional deficiencies, environmental (toxic) factors, metabolic disturbances and trauma. Examples of demyelinating diseases in the CNS are multiple sclerosis, progressive multifocal leukoencephalopathy (slow-virus disease) , and central pontine myelinolysis (toxic metabolic disorder) . In the PNS there are three degenerative processes: allerian, axonal and segmental demyelination. The peripheral neuropathies are an example of demyelination in the PNS. These neuropathies can be either an acute demyelinating neuropathy or an axonopathy which includes a demyelinating component. Acute demyelinating neuropathies include Landry-Guillain-Barre syndrome and the neuropathies associated with infectious mononucleosis, hepatitis, diphtheria, and certain toxic agents.²⁶ Further, there are other peripheral neuropathies with demyelination components including those associated with alcoholism, diabetes, AIDS, leprosy, zoster, and palsies. allerian degeneration follows peripheral transection of the axon. Distal to the transection there is degeneration of both the axon and its myelin sheath accompanied by Schwann cell proliferation. In the PNS, regeneration can occur by the outgrowth of multiple sprouts from the distal ends of the surviving segments of the axons. If there is no obstruction to regrowth, the axon grows back along the same pathway with the proliferated Schwann cells providing a myelin sheath. If, as is frequently the case in Wallerian degeneration secondary to a traumatic injury, hematoma or fibrous scar tissue prevents the regenerating sprouts from proper growth and the regenerating axons form a tangled, often painful mass of intertwined nerve fibers call an amputation, or traumatic neuroma. In segmental demyelination, after an episode of demyelination, re yelination can be accomplished by the remaining Schwann cells. Repeated episodes of demyelination and remyelination can occur and generally concentric arrangements of alternating Schwann cell processes and collagen (hypertrophic neuropathies) are formed. The prevention of demyelination sequelae in the above listed demyelinating-associated syndromes and diseases would be useful. For example, in syndromes with chronic demyelination the formation of hypertrophic neuropathies could be reduced. Certainly, the prevention of demyelination in multiple sclerosis would be beneficial. In demyelinating syndromes that are caused by infectious agents, toxic substances, nutritional deficiencies or metabolic disturbances, the prevention or reduction of demyelination during treatment of the underlying disease would be beneficial to the patient.

During demyelination, phagocytosis occurs as part of the process. The macrophages which are known to mediate the phagocytosis process express MAC-1 (the C3b compliment receptor) , MAC-2 (a galactose specific lectin) , the Fc receptor and the F4/80 (a marker of mature macrophages) . MAC-1, MAC-2 and the Fc receptor are of particular interest since each can be instrumental in phagocytosis.²⁸

SUMMARY OF THE INVENTION AND ADVANTAGES

According to the present invention, a method is provided for inhibiting myelin destruction including the steps of identifying the type of myelin destruction and determining if MAC-2 expression has been induced in the cells involved. Compounds are then administered to act at the MAC-2 receptor thereby interfering with the interaction between MAC-2 and myelin.

BRIEF DESCRIPTION OF THE DRAWINGS

Other advantages of the present invention will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein:

FIGURE 1 is an electron micrograph of a seven day, in vivo degenerating C57/BL/6NHSD mouse nerve cross section, Schwann cells (S) are present within their basal lamina sheath (small arrow heads) , their ribosome rich cytoplasm contains phagocytized myelin (large arrow heads) and lipid droplets, a macrophage (m) with its characteristic filopodia is present within a Schwann cells basal lamina sheath, its dark cytoplasm contains phagocytized myelin and lipid droplets (scale - 2 μm) ; FIGURE 2 A-D are electron micrographs of cross sections through seven day C57/BL/6NHSD mice frozen nerve segments, macrophages that are present either inside (A & B) or outside (C & D) Schwann cells'^" basal lamina sheaths contain phagocytized myelin and lipid droplets, macrophages adhere to the internal surface of basal laminae through their filopodia (A & B) (Scale - 2 μm) ;

FIGURE 3 A-J are photomicrographs showing the immunocytochemical detection of MAC-2 by mAb M3/38 (A, B, C, D & E) and the F4/80 antigen (F, G, H, I & J) in intact (A & F) , neuroma (B & G) , in vivo degenerating (C & H) , frozen (D & I) and in vitro degenerating (E & J) C57/BL/6NHSD mice nerves, immunoreactivity to MAC-2 is not detected in intact peripheral nerves, it is abundant in seven day neuroma (B) , in vivo degenerating (C) , frozen (D) , and in vitro degenerating (E) nerves, immunoreactivity to the F4/80 antigen, a marker specific of macrophages, is scarce in intact (F) and in seven days in vitro degenerating (J) nerves, and is abundant in seven days neuroma (G) , in vivo degenerating (H) and frozen (I) nerves, the distribution of immunoreactivity to MAC-2 and the F4/80 antigen was similar all along in vivo degenerating nerves that were sampled from the lesion site and up to 10 mm distal to it (Scale - 400 μm) ; FIGURE 4 A-C are photomicrographs showing the immunocytochemical detection of MAC-2 by mAb M3/38 (A & C) and the F4/80 antigen (B) in 2.5 days in vivo degenerating C57/BL/6NHSD mice nerves, the stain for MAC-2 is densely and widely distributed, it assumes tube (A) and ring (C) like structures in longitudinal and cross sections, respectively, the stain for the F4/80 molecule is not as dense as the stain for MAC-2 (Scale - 400 μm) ;

FIGURE 5 is an immunoblot of the detection of MAC-2 by mAb M3/38 in extracts of intact (A) , and seven day, neuroma (B) , in vivo degenerating (C) , frozen (D) , and in vitro degenerating (E) C57/BL/6NHSD mice nerves, MAC-2 (arrow) and its lower molecular weight degradation products are detected in all extracts but that of intact nerve;

FIGURE 6 A-F are photomicrographs showing the immunocytochemical detection of MAC-2 by mAb M3/38 in non-neuronal cells that migrated out from in vitro degenerating C57/BL/6NHSD mice nerves during seven days of explantation, the micrographs display two fields, (A, B & C) and (D, E & F) , (A & D) combined immunofluorescence and phase, (B & E) phase, and (C & F) immunofluorescence light microscopy, in the first field, (A, B & C) , macrophages are the only cells stained by mAb M3/38 (A & C) , they contain granules (B) and vacuoles (C) in their cytoplasm, none of the many fibroblasts that appear in the background are stained by mAb M3/38, in the second field (D, E & F) many Schwann cells, fibroblasts and one macrophage are present, Schwann cells (S) appear in phase as dark, bipolar, spindle shaped and cytoplasm poor cells (E) , most of them are stained by mAb M3/38 (D & F) , in this field the single macrophage (m) , and one out of the many fibroblasts are also stained by the mAb (Scale - 400 μm) ;

FIGURE 7 A-D are photomicrographs showing Schwann cells co-expressing the S-100 molecule and MAC-2, cells were dissociated from C57/BL/6NHSD mice nerves and double stained immunohistochemically by antiserum raised against the S-100 molecule (A & C) and mAb M3/38 (B & D) (Scale - 400 μm) ; FIGURE 8 is an immunoblot showing that MAC-2 is present in isolated macrophages and Schwann cells but not fibroblasts that were obtain from five days in vivo degenerating nerves, immunoblot detection of MAC-2 by mAb M3/38 in extracts of single cell cultures of macrophages (M) , fibroblasts (F) and Schwann cells (S) dissociated from C57/BL/6NHSD mice nerves;

FIGURE 9 is a photomicrograph showing macrophages

(A) and Schwann cells (B) that reside in in vivo degenerating C57/BL/6NHSD mice nerves express MAC-2 on their surface, the cells were allowed to migrate out of five days in vivo degenerating nerve segments, and then stained with mAb M3/38 in the absence of Triton x-100 (Scale - 400 μm) ; FIGURE 10 A-D are photomicrographs showing cross sections through C57/BL/6NHSD mice nerves: intact (A) , and in vitro degenerating nerves that were explanted into culture for six days in the presence of 25 mM of mannose (B) , galactose (C) , and lactose (D) , intact myelin figures assume the classical doughnut shape morphology in intact nerve (A) , there is a marked reduction in the density of intact myelin in the various nerve explants, but more so in the mannose

(B) than in the galactose (C) and lactose (D) treated ones (Scale - 900 μm) ;

FIGURE 11 is a graph showing that galactose and lactose significantly and specifically inhibit myelin phagocytosis and consequent destruction in cultures of intact C57/BL/6NHSD mice nerve explants, myelin destruction was studied by morpho etry to determine the density (in arbitrary units) of intact unphagocytized residual myelin, intact nerves were explanted into culture for six days, and incubated in either medium (Med) , or medium supplemented by 25 mM of D-mannose (Man) , D-galactose (Gal) , or lactose (Lac) , for comparison, density of intact myelin in intact nerves (Int) and nerves undergoing in vivo Wallerian degeneration (Deg) for six days are also shown, values which are smaller than those found in intact nerves express extent of myelin destruction by phagocytosis, bars represent the average values of residual (intact, unphagocytized) myelin, error bars one standard error of the mean, n = number of nerve cross sections examined, each representing a separate nerve taken from different mice, each one of the same 12 mice contributed to the Med, Man, and Gal experiments, and seven of these to the Lac experiments, levels of significance of differences were calculated by the Mann-Whitney U test: p <0.02 for Gal over Med, p <0.002 for Gal over Man, p <0.002 for Lac over Med and Man, and p <0.02 for Lac over Gal;

FIGURE 12 A-C are photomicrographs showing cross sections through C57/BL/6-WLD/0LA/NHSD mice peripheral nerves after six days of in vivo (A & B) and in vitro (C) degeneration, the nerve segment located distal to the site of nerve transection was divided into two domains: (A) the distal domain (over 5 mm from the site of transection) that displays a morphology which is indistinguishable from normal, and (B) the injury region (less than 3 mm from the site of transection) where myelin destruction occurred, (C) intact nerves that were explanted into culture and allowed to degenerate in vitro exhibit low frequency of myelin destruction (Scale - 725 μm) ; FIGURE 13 A-C are graphs that show that myelin destruction occurs in nerves that express MAC-2, the extent of myelin phagocytosis and MAC-2 expression were studied in peripheral nerves of mice undergoing rapid (N) and slow (W) Wallerian degeneration in vivo , (A) myelin destruction was studied by morphometry to determine the density (in arbitrary units) of intact unphagocytized residual myelin, tissues were examined six days after surgery, intact nerves (Int) , nerve explants undergoing in vitro degeneration (Expl) , and nerve segments located farther distal than 5 mm from sites of nerve transection and thus allowed to undergo in vivo Wallerian degeneration (Deg) , values which are smaller than those found in intact nerves express extent of myelin destruction by phagocytosis, bars represent average values, error bars 1 SEM, n = number of sections examined each taken from a different animal, (B) similar tissues were assayed for MAC-2 content by ELISA, levels of MAC-2 are expressed in optical density units, all samples were tested simultaneously and thus subject to quantitative comparison, bars represent average values, error bars 1 SEM, n = number of independent experiments, (C) higher levels of MAC-2 expression are correlated with a larger extent of myelin destruction, values of residual myelin were taken from (B) and plotted against values of MAC-2 taken from (A) ;

FIGURE 14 A-J are photomicrographs showing the immunocytochemical detection of MAC-2 by mAb M3/38 (A, B, C, D & E) and the F4/80 antigen (F, G, H, I & J) in C57/BL/6-WLD/OLA/NHSD mice peripheral nerves, intact nerves (A & F) , in in vivo degenerating nerve segments located at the injury region (<3mm from the site of transection) (B & G) , in vivo degenerating nerve distal segments located beyond the injury region (>5mm from the site of transection) (C & H) , in frozen nerves (D & I) and in in vitro degenerating nerves

(E & J) , immunoreactivity to MAC-2 is not detected in intact peripheral nerves (A) , it is abundant six days after surgery at the injury region (B) , in frozen nerves (D) , and in in vitro degenerating (E) nerves, there is no MAC-2 immunoreactivity in vivo at distal regions (C) , immunoreactivity to the F4/80 antigen, a marker specific of macrophages, is scarce in intact nerves (F) , in in vivo degenerating distal nerve segments six days after nerve transection (H) , and in six days in vitro degenerating (J) nerves, the F4/80 antigen is abundant six days after surgery at the injury region of in vivo degenerating nerves (G) and in frozen nerves (I) (Scale - 400 μm) ; and

FIGURE 15 A-B are photomicrographs of Schwann cells (A) and macrophages (B) dissociated from C57/BL/6/WLD/0LA/NHSD mice peripheral nerves, in phase light microscopy the morphology of cells originating from C57/BL/6/WLD/OLA/NHSD mice and C57/BL/6NHSD mice is indistinguishable, (A) cultured Schwann display bipolar spindle shape morphology and express MAC-2 as revealed by immunocytochemistry, (B) macrophages express the F4/80 antigen (Scale - 400 μm) .

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention provides a method for inhibiting myelin destruction, i.e. demyelination, the loss of the myelin sheath in both the PNS and CNS.

The demyelination can occur due to any mechanism that involves the binding to the myelin through the MAC-2 receptor. Underlying courses of demyelination such as multiple sclerosis, peripheral neuropathies and

Wallerian degeneration are all susceptible to treatment by the present invention.

The steps include first identifying the type of myelin destruction and if MAC-2 expression has been induced on the cells involved in the demyelination.

Compounds are then administered that act at the MAC-2 receptor thereby interfering with the interaction between MAC-2 and myelin.

Once a patient is diagnosed as having a particular disease in which myelin dysfunction and, more particularly demyelination, is a component, the type of myelin destruction that is occurring is evaluated if not already known. Evaluation occurs through a combination of electron microscopy, immunocytochemical, immunoblot and ELISA techniques as described hereinbelow. Briefly, tissue samples from patients with the particular disease are removed and the presence of MAC-2 receptors on the Schwann cells, oligodendroglia, macrophages and microglia is determined utilizing an immunohistological morphometric analysis. Similarly, the tissue biopsy is evaluated using both immunoblot and ELISA assays for the increased presence of MAC-2 receptors indicative of a demyelinating syndrome that includes the binding of phagocytic cells to the myelin sheath through the MAC-2 receptor.

Once it is known that a particular disease or syndrome with associated demyelination involves binding of phagocytic cells to the myelin through MAC- 2, this interaction can be interrupted with administration of the appropriate, compounds. Lactose, galactose and cerebrosides and other agents that act at the MAC-2 receptor to interrupt binding of the receptor to myelin will be administered. In one embodiment, antibodies, either polyclonal or monoclonal, raised against the MAC-2 receptor will be used.

Antibodies raised against the MAC-2 receptor are available commercially. Alternatively, the antibodies can be raised against isolated human MAC-2 receptors, portions thereof, or a synthetic peptide based on the sequence of MAC-2 may be used as the immunogen. Such proteins or peptides can be used to produce antibodies by standard antibody production technology well known to those skilled in the art. -ll-

For producing polyclonal antibodies, a host, such as a rabbit or goat, is immunized with the protein or peptide, generally with an adjuvant and, if necessary, coupled to a carrier; antibodies to the protein are collected from the sera. Monoclonal antibodies can also be produced by standard techniques well known to those skilled in the art. Basically, the technique involves hyperimmunization of an appropriate donor with the protein or peptide fragment, generally a mouse, and isolation of splenic antibody producing cells. These cells are fused to a cell having immortality, such as a myeloma cell preferably from a human, to provide a fused cell hybrid which has immortality and secretes the required antibody. The cells are then cultured, in bulk, and the monoclonal antibodies harvested from the culture media for use.

The demyelinating interrupting compounds such as lactose, galactose and cerebrosides, anti-MAC-2 and other compounds which bind to MAC-2 or interrupt binding at MAC-2 are administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, and other factors known to medical practitioners. The "effective amount" for purposes herein is thus determined by such considerations as are known in the art. In the method of the present invention, the demyelinating interrupting compounds can be administered in various ways. It should be noted that they can be administered as the compound or as pharmaceutically acceptable salt and can be administered alone or in combination with pharmaceutically acceptable carriers. The compounds can be administered orally or parenterally, including intravenous, intraperitoneally, intranasal, and subcutaneous administration. Implants of the compounds are also useful. The patient being treated is a warm-blooded animal and, in particular, mammals including man. When administering the demyelinating interrupting compounds parenterally, the pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions. The carrier can be a solvent or dispersing medium containing, for example, water, ethanol, polyol (such as glycerol, propylene glycol, liquid polyethylene glycol, and the like) , suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Nonaqueous vehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and esters, such as isopropyl myristate, may also be used as solvent systems for compound compositions. Additionally, various additives which enhance the stability, sterility, and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. In many cases, it will be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the compounds. Sterile injectable solutions can be prepared by incorporating the compounds utilized in practicing the present invention in the required amount of the appropriate solvent with various of the other ingredients, as desired.

A pharmacological formulation of the demyelinating interrupting compounds can be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicle, adjuvants, additives, and diluents; or the compounds utilized in the present invention can be administered parenterally to the patient in the form of slow-release subcutaneous implants or targeted delivery systems such as polymer matrices, liposomes, and microspheres. An implant suitable for use in the present invention can take the form of a pellet which slowly dissolves after being implanted or a biocompatible delivery module well known to those skilled in the art. Such well known dosage forms and modules are designed such that the active ingredients are slowly released over a period of several days to several weeks. Examples of well-known implants and modules useful in the present invention include: U.S. Patent No. 4,487,603, which discloses an implantable micro- infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are well known to those skilled in the art.

A pharmacological formulation of the demyelinating interrupting compounds utilized in the present invention can be administered orally to the patient. Conventional methods such as administering the compounds in tablets, suspensions, solutions, emulsions, capsules, powders, syrups and the like are usable. Known techniques which deliver the compounds of the present invention orally or intravenously and retain the biological activity are preferred.

For delivery within the CNS, pharmacological formulations that cross the blood-brain barrier can be administered.⁵ Such formulations can take advantage of methods now available to produce chimeric peptides in which the present invention is coupled to a brain transport vector allowing transportation across the barrier.^30,31'³²

In one embodiment, the demyelinating interrupting compounds that act at MAC-2 can be administered initially by intravenous injection to bring blood levels of the compounds to a suitable level. The patient's levels are then maintained by an oral dosage form, although other forms of administration, dependent upon the patient's condition and as indicated above, can be used. The quantity of demyelinating interrupting compounds to be administered will vary for the patient being treated and will vary from about 100 ng/kg of body weight to 100 mg/kg of body weight per day and preferably will be from 10 μg/kg to 10 mg/kg per day. EXPERIMENTAL STUDIES Applicants hypothesized that the MAC-2 receptor that is on macrophages is also induced on Schwann cells or oligodendroglia during at least some types of demyelination and that these cells participate in phagocytizing myelin along with cells from the macrophage lineage. Applicants based this hypothesis, in part, on lectinophagocytosis. Lectinophagocytosis is a mechanism of phagocytosis dependent on the ability of a phagocyte to identify its specific target by molecular fitting between a cell surface associated carbohydrate binding protein (the lectin) and a complementary sugar on the target.²⁷ In this context, MAC-2 is a galactose specific lectin which is associated with macrophage and Schwann cell surfaces, and myelin, the phagocytized target, is rich in galactolipids such as cerebroside and sulfatide.²⁸ The prior art did not disclose whether MAC-2 is involved in lectin mediated phagocytosis. Applicants hypothesized that this could be the case, and, therefore, tested for the possibility that MAC-2 mediates phagocytosis of myelin.

Peripheral nerve injury was used to test the hypothesis. Peripheral nerve injury is followed by Wallerian degeneration of the nerve segment distal to the lesion site and by axonal regeneration at the neuroma nerve segment just proximal to it.⁷ Molecular and cellular events that take place during the course of Wallerian degeneration turn the degenerating segment into an environment which supports the regeneration of peripheral and central adult neurons.^3,11 Among the identified molecular changes are increases in the productions of Interleukin-1 activity,³⁸ nerve growth factor,¹³'³⁶ and apolipoproteins.¹'¹⁶'³⁷ Cellular events that characterize Wallerian degeneration are the breakdown of axons, disintegration of myelin sheaths, proliferation of Schwann cells, and the recruitment of macrophages from the circulation. There is a general agreement that macrophages play a major role in the phagocytosis and metabolism of myelin. There is, however, a long standing debate whether Schwann cells play a similar role. Recent studies reflect this controversy. On the one hand, no phagocytosis of myelin by Schwann cells was detected in mouse and cat peripheral nerves undergoing axonal degeneration in either diffusion chambers⁴ or tissue culture.¹⁰ On the other hand, phagocytosis of myelin by rat Schwann cells was reported during the course of in vivo, Wallerian degeneration,⁴² and by neonate Schwann cells in culture.⁶

With this model system, applicants determined that the failure to degenerate in vivo in mice that genetically display a slow progression of Wallerian degeneration is associated with deficient MAC-2 production. Further, degeneration that occurs in vitro is associated with MAC-2 production and there is a strong correlation between levels of MAC-2 and the extent of myelin destruction by phagocytosis.

Based on this hypothesis, applicants examined whether or not galactose and lactose would inhibit myelin phagocytosis and thereby inhibit demyelination. Applicants reasoned that inhibition is to be expected based on the competition between the free sugars (galactose and lactose) and the myelin galactolipids on binding to MAC-2. Furthermore, it was predicted based on the hypothesis that lactose could be a more potent inhibitor than galactose since the prior art showed its higher binding affinity to MAC-2.¹² The following experimental studies were undertaken. Phagocytosis by Schwann cells and macrophages was examined electron microscopically in four different preparations of injured mouse peripheral nerves. First, cultured explants of intact nerves: In culture in vitro axonal degeneration takes place in the presence of the "intact nerve non-neuronal cell population" (i.e., Schwann, fibroblasts, and endothelial cells)^40,44 from which blood born macrophages are excluded. Second nerve segments undergoing degeneration in vivo , containing the entire array of non-neuronal cells characteristic of Wallerian degeneration: the "intact nerve non- neuronal cell population" joined by macrophages which are recruited from the circulation.⁴ Third, nerve segments that were first frozen and then returned in situ : in frozen nerves, the "intact nerve non- neuronal cell population" that had been killed by freezing is replaced by blood born macrophages.¹⁵ Fourth, neuroma nerve segments: the site where axonal regeneration commences.⁷

Two strains were used to test applicants' hypothesis: (1) the C57/BL/6NHSD normal mouse (N mice) that displays the normal rapid progression of Wallerian degeneration; and (2) the C57BL/6-WLD/OLA/NHSD mutant mouse (W mice; formerly known as C57BL/01a) that displays a very slow progression of Wallerian degeneration.²¹'³³ Using

N mice, it was observed electron microscopically, that Schwann cells phagocytized myelin, thus displaying a phenotype characteristic of macrophages. To test the possibility that additional macrophage phenotypes are expressed by Schwann cells, the expression of molecules that inflammatory and mature murine macrophages characteristically display were studied by immunocytochemistry and immunoblot analysis: MAC-1, MAC-2, the Fc receptor, and the F4/80 antigen.^25,29,4S MAC-1 and the Fc receptor take part in immune related, opsonin dependent phagocytosis in which complement and antigens act as opsonins. The galactose specific lectin MAC-2 applicants hypothesize can be instrumental in the non-immune form, opsonin independent phagocytosis: lectinophagocytosis (lectin mediated phagocytosis) . Cell surface and cytoplasmic MAC-2 was detected in Schwann cells and macrophages. Further, it was observed that galactose and lactose specifically inhibited myelin phagocytosis, indicating that MAC-2 was involved in lectinophagocytosis, confirming applicants' hypothesis that demyelination can be inhibited by interfering with the MAC-2 receptor. Nerve injury thus induces Schwann cells to express MAC-2 and is followed by the recruitment of macrophages that also express MAC-2. In turn, MAC-2 enables Schwann cells and macrophages to exhibit lectinophagocytosis of myelin. By interrupting the interaction of MAC-2 and myelin, demyelination can be interrupted.

The role of MAC-2 in mediating myelin phagocytosis was tested in W mice that display a slow progression of Wallerian degeneration in vivo as a further test of the hypothesis. Retarded Wallerian degeneration has been attributed to the slow pace recruitment of macrophages distal to the lesion site.^21,33 The failure to recruit macrophages provides only a partial explanation for the lack of myelin destruction if it is accepted that Schwann cells and macrophages exercise MAC-2 mediated myelin phagocytosis. Thus, an additional requirement should be fulfilled: W mice Schwann cells should fail to express MAC-2 in vivo after nerve transection, which indeed was the case. Some extent of myelin destruction occurred during in vitro degeneration of W mice nerve explants. As predicted, this event was associated with MAC-2 expression. Further, there was a positive correlation between levels of MAC-2 expression and extent of myelin destruction by phagocytosis over a wide range of values obtained from N and W mice.

The above discussion provides a factual basis for the inhibition of myelin destruction. The methods used with and the utility of the present invention can be shown by the following examples.

METHODS Animals and surgical procedures. Two strains of mice, 2 to 4 months old, were used. First, the C57/BL/6NHSD normal strain (Harlan Sprague Dawley Inc. , USA) that displays a rapid progression of Wallerian degeneration. Second, the C57/BL/6- WLD/OLA/NHSD mutant strain (Harlan, Olac, England) that displays a slow progression of Wallerian degeneration.²¹ This strain was formally referred to as C57/BL/01a by the supplier who has now assigned the new name to it. The two strains are referred to herein as N and W, respectively. All surgical procedures were performed under anesthesia. The sciatic nerve was transected after leaving the pelvis. Frozen nerve segments were obtained by subjecting 1 cm nerve segments to 3 cycles of freeze/thaw in distilled water. Frozen nerves were returned in situ into the donor animals. Nerve explants were obtained by placing 5 mm nerve segments in Dulbecco's modified Eagle medium (DMEM) , supplemented by 10% FCS (Beit-Hae ek, Israel) . Cultures were kept in a humidified incubator, saturated by 5% C0₂ at 37°C. Cell dissociation and culture. The method described by Scarpini et al.³⁹ was used with some modifications. Briefly, nerves were cut into small pieces and incubated for 18 to 20 hours in DMEM, 1.2 units/ml Dispase, 0.05% Collagenase, and 0.1%

Hyaluronidase (Sigma) . To obtain cultures containing mixed cell populations, the washed cell suspension was plated on poly-L-lysine or laminin (Sigma) coated plastic culture dishes (Nunc) . To obtain Schwann cell cultures, cells were plated on plastic dishes for 1 day. Occasionally this procedure was repeated twice. Then, the non-adherent cells, primarily Schwann, were plated on poly-L-lysine or laminin (Sigma) coated dishes. In instances where fibroblasts were still present, the cells were exposed for 3 days to 10^"5 M cytosine arabinoside (Sigma) .

Immunocytochemistry. Peripheral nerves were sectioned (8μm) in a freezing microtome. Sections were blocked in 20% normal rabbit serum (NRS) for 20 min, exposed for 60 min to either one of the rat anti-mouse monoclonal antibodies (mAb) raised against MAC-1, MAC-2, F4/80 antigen and the Fc receptor (diluted 1:5 in 10% NRS/2.5% BSA) , washed in PBS for 30 min, fixed in 1% neutral formalin in PBS for 5 min, washed in PBS for 10 min, incubated for 60 min in FITC conjugated rabbit anti-rat IgG (diluted 1:500 in 10% NRS/2.5% BSA), and finally washed in PBS for 30 min. The entire staining procedure was carried out at room temperature.

Cells were washed in serum free DMEM, blocked in 20% NRS in DMEM for 15 min, exposed for 40 min to either one of the mAb raised against MAC-1, MAC-2, F4/80 antigen, and Fc receptor (diluted 1:5 in 10% NRS in DMEM), washed in Ca-Mg PBS, fixed in PLP²⁰ for 15 min, washed in PBS for 20 min, incubated in FITC conjugated rabbit anti-rat IgG for 30 min, and finally washed in PBS for 30 min. Procedures up to fixation were carried out at 37°C, and thereafter at room temperature.

To double stain cells for MAC-2 and S-100, cell cultures were washed in serum free medium, fixed for

15 min in PLP, washed in PBS for 20 min, incubated for 10 min in 0.05% Triton in PBS, washed in PBS, blocked in 20% NGS/5% BSA for 15 min, incubated for 1 hour at 37°C in polyclonal rabbit anti-bovine antibodies raised against S-100 (diluted 1:500-1000 in 10% NGS/2.5% BSA in PBS), washed in PBS, incubated in

Rhodamine conjugated goat anti-rabbit IgG for 30 min (diluted 1:500 in 10% NGS/2.5% BSA in PBS), and washed in PBS. Cells were then exposed to anti-MAC-2 for 30 min (dilution of 1:200 in 10% NRS/2.5% BSA in PBS), washed in PBS, incubated in FITC conjugated rabbit anti-rat IgG (diluted 1:1000 in 10% NRS/2.5% BSA in PBS) , and finally washed in PBS.

The mAbs raised against MAC-1, F4/80 antigen and Fc receptor were obtained from Serotec (USA) , the hybrido a cell line M3/38 producing anti-MAC-2 from the American Type Culture Collection, FITC conjugated rabbit anti-rat IgG, Rhodamine conjugated goat anti- rabbit IgG, and S-100 from Bio-Makor (Israel) .

Immunoblot analysis. Tissues were extracted in PBS (100 μl/mg wet weight) , containing 1% Triton

X-100, and the protease inhibitors PMSF (100 μg/ml) and Aprotinin (1 μg/ml) . Of each tissue, 6 μg protein were separated by 12.5% SDS PAGE, and transferred electrophoretically to Immobilon PVDF transfer membrane (Millipore, USA) , in Towbin, 20% methanol,

2.5% isopropanol for 1 hour at 100 volts using a Mini Trans Blot Cell (BioRad, USA) . Nonspecific binding was blocked by incubation with 2% gelatin in Tris buffered saline (TBS) for 2 hour at 37°C, followed by 2 washes with TBS containing 0.1% Tween 20 (TBST) . Membranes were incubated overnight at 4°C with supernates of hybridoma cells producing the rat anti-mouse MAC-2 (mAb M3/38) , and then washed 4 times with TBST. Alkaline phosphatase conjugated anti-rat IgG and NBT/BCIP substrate were used to detect the primary antibody (Promega, USA) . Determining levels of MAC-2 in tissues by ELISA.

Nerves were homogenized for 2 min in 50 mM sodium carbonate buffer pH 10.0 in a volume of 0.3 ml/nerve, and further incubated for 1 hour at 37°C. 0.1 ml/nerve of 0.5M sodium carbonate pH 9.6 was added to samples that were then vortexed and centrifuged for 10 min at 15,000 g. Protein contents of supernates were determined (Bio-Rad protein assay reagent) , protein concentration adjusted to 12.5 μg/ml with sodium carbonate buffer pH 9.6, and 50 μL of serial dilutions were used to coat 96 well plates (Maxisorb Immuno Plates 96F Nunc) over night at 4°C. Wells were washed x2 with Tris buffered saline (TBS) , blocked for 2 hours at 37°C with 2% gelatin (Sigma) in TBS, washed x2 with 0.1% Tween 20 in TBS (TBST), incubated over night at 4°C with mAb M3/38 (diluted 1:50 in 1% BSA in PBS) , washed x2 with TBST, incubated for 1 hour in a 1:5000 dilution (in 1% BSA in 50mM Tris, lOOmM NaCl, pH 8.0) of alkaline phosphatase conjugated goat anti- rat IgG (Jackson) , washed x4 in PBS, incubated for as long as required at room temperature with substrate solution (1 mg/ml p-nitrophenyl phosphate sodium in 10% diethanolamine, pH 9.8; Sigma). The reaction product was read in a Dynatec Elisa reader at 405 n wave length.

Electron microscopy. Tissues were fixed for 2 hours in Karnovsky, 1 hour in 2.5% glutaraldehyde, 1 hour in 2% Osmium, dehydrated, and embedded in Araldite. Thin sections were examined in the electron microscope.

Morphometric analysis of unphagocytized residual myelin. Nerve cross sections were obtained from intact, in vivo degenerating, in vitro degenerating and frozen nerves. The sections from in vivo degenerating nerves were taken 5 mm distal to sites of nerve transection. One micron thick sections were photographed and printed at a total magnification of 700. A matrix composed of 450 cross points (9 vertical lines spaced 2 cm apart, over 50 horizontal lines spaced 0.5 cm apart) was used.

The number of unphagocytized myelin profiles that overlapped matrix cross points were counted, thus giving a measure for the density of unphagocytized, residual myelin. Unphagocytized, residual myelin profiles were characterized as round shaped myelin profiles enclosing a translucent space. In nerve explants, some of these profiles were not associated with Schwann cells and thus regarded as rejected myelin. Frequently, these figures were collapsed and distorted from a perfect doughnut shape. Fragments of compact and dense myelin were considered phagocytized and/or processed. These criteria are based on applicants' electron microscopy studies as detailed herein below. The validity of this methodology is evident from the reproducibility of the results and the differences between intact and in vivo degenerating N mice nerves. The person performing the counting was unaware of the source tissue.

EXAMPLES

Myelin phagocytosis in injured N mice peripheral nerves. Intact and injured nerves were examined electron microscopically. In intact nerves, Schwann cells outnumbered all other non-neuronal cell types. Fibroblasts were associated, primarily, with the epineural sheath at the perimeter of nerve trucks. Macrophages were rarely present. In contrast, macrophages were frequently observed 5 to 7 days after nerve section in nerve segments undergoing Wallerian degeneration (Fig. 1) . They were identified as macrophages by their dark cytoplasm which was relatively poor in free ribosomes and abundant in rough endoplasmic reticulum, irregular nuclear surfaces, and characteristic filopodia (see also Fig. 2; reviewed in Papadimitriou and Ashman.²⁹ Macrophages were present either inside or outside Schwann cells' basement membranes. Macrophages contained fragmented myelin and lipid droplets, indicating their involvement in the phagocytosis and metabolism of myelin. Schwann cells were distinguished from macrophages by their translucent cytoplasm which was rich in free ribosomes and poor in rough endoplasmic reticulum, smooth nuclear surface, and their presence inside basement membranes (Figs. 1) . Myelin fragments and lipid droplets were frequently detected within their cytoplasm. Thus

Schwann cells also phagocytized and processed myelin.

Intact nerves were explanted into culture and allowed to degenerate in vitro for 5 to 7 days. Axons disintegrated, and Schwann cells became separated from their myelin. Schwann cells and rejected myelin were contained within basement membranes. Macrophages were rarely present, and fibroblasts were associated with the epineural sheath at the perimeter of nerve trunks. As in in vivo degenerating nerves, myelin fragments and lipid droplets were detected in the cytoplasm of

Schwann cells, further indicating their involvement in myelin phagocytosis.

Nerve segments that have been first freeze- thawed, to kill non-neuronal cells, became populated by macrophages 5 to 7 days thereafter (Fig. 2) .

Occasionally, fibroblasts and polymorphonuclear cells were also detected. The vast majority of macrophages were loaded with myelin fragments and lipid droplets. Some macrophages were present inside, and others outside Schwann cells' basement membranes.

Interestingly, macrophages attached to basement membranes by filopodia at the internal but not external surfaces of basal lamina. Many profiles of vacant, collapsed basement membranes were observed. Neuroma nerve segments contained regenerating axons, growth cones, Schwann cells, macrophages and fibroblasts. Interestingly, yet unexplained, close appositions were observed between axonal growth cones and macrophages. Fibroblasts, identified by their translucent, rough endoplasmic reticulum rich cytoplasm, appeared in large numbers at tips of neuromata. Some fibroblasts had myelin fragments within their cytoplasm, suggesting their possible involvement in phagocytic activity at this site.

MAC-1_/ MAC-2, the F4/80 antigen and Fc receptor in normal and injured N mice peripheral nerves. The molecules MAC-1, MAC-2, the F4/80 antigen and the Fc receptor are characteristically expressed by inflammatory and mature murine macrophages.²,²⁵,²⁹,⁴⁵ Their expression was studied in intact and lesioned nerves by immunofluorescence light microscopy. The

F4/80 antigen is considered to be unique to all mature monocytes/macrophages and, hence, can be used as a reliable marker for the presence or absence of macrophages. In agreement with applicants' electron microscopical observations, macrophages (F4/80 positive cells) are scarce in intact and in in vitro degenerating nerves, but are numerous in neuroma, in vivo degenerating and frozen nerves (Fig. 5 F-I) . Immunoreactivity to MAC-1, MAC-2 and the Fc receptor was rarely detected in intact nerves. In contrast, immunoreactivity was detected in 5 to 7 days in vivo degenerating, neuroma, and frozen nerve segments (Fig. 3 A through D for MAC-2) . Since macrophages can express the three molecules, the findings correlate well with the electron microscopical and immunocytochemical (F4/80) observations of the rare occurrence of macrophages in intact nerves, and their presence in in vivo degenerating, frozen, and neuroma nerve segments. Surprisingly, a substantial increase in MAC-2 immunoreactivity was detected at 5 to 7 days in in vitro degenerating nerve explants where macrophages are few (Fig. 3 E & J) . The same nerve explants that displayed increased MAC-2 expression, did not display similar increases in immunoreactivity to either the F4/80 antigen, MAC-1 or the Fc receptor: there was either no change, or very little increase in immunoreactivity to these molecules.

Several conclusions can be drawn from these observations. Non-neuronal cells that reside in intact normal peripheral nerves, Schwann cells included, do not express MAC-2. Macrophages that are scarce in in vitro degenerating nerves cannot account for the wide distribution of MAC-2 presentation in this tissue. By comparison to the electron microscopical observations, the wide distribution of MAC-2 in in vitro degenerating nerves correlates with the anatomical distribution of Schwann cells and no other cell type. The same is the case in 2 to 3 days in vivo degenerating nerves (Fig. 4) . During this period of time, macrophage recruitment has just begun, not yet reaching their highest density as in later days. Immunoreactivity to MAC-2, however, is densely and widely distributed in ring-like shapes, correlating with the anatomical distribution of

Schwann cells, strongly suggesting that they express MAC-2 during in vivo degeneration as they do during in vitro degeneration.

It was further verified that the lesion induced increase in MAC-2 expression by immunoblot analysis

(Fig. 5) . The M3/38 mAb identified a major 31/32 kDa protein band and some lower molecular weight protein bands. The 31/32 kDa protein is MAC-2, and the lower molecular weight proteins are its degradation products.¹⁴ MAC-2 was barely detected in extracts of intact nerves. It was clearly detected in substantial, though varying, amounts in neuroma, in vivo degenerating, frozen, and in vitro degenerating explants.

MAC-2 expression by isolated and identified N mice macrophages and Schwann cells. To determine which cell types express MAC-2 following nerve injury, the presence of the molecule in cells that migrated out of, or were dissociated from injured peripheral nerves was examined. Three cell types dominated cultures of cells that originated from in vivo degenerating nerves: macrophages, Schwann cells and fibroblasts. When in vitro degenerating nerves were the source tissue, macrophages constituted only a very small proportion of the cell population. Cells were examined after immunocytochemical staining by phase and fluorescence light microscopy (Fig. 6) . The three cell types differed from each other by their distinct morphology and pattern of staining. Macrophages appeared as cytoplasm rich cells, containing numerous vacuoles and granules. Soon after plating, they displayed a round shape. With time they assumed diverse shapes, extending large diameter processes and/or very fine filopodia. These cells were stained by the mAb F4/80 antigen, as expected (not shown for N mice macrophages, but see Fig. 15B for W mice macrophages) . Schwann cells appeared as spindle shaped, bipolar/tripolar, cytoplasm poor cells. On laminin coated dishes, Schwann cells extended particularly very long and thin processes. In many, vacuoles were present at their center portion where cytoplasm was relatively more abundant. These cells were further identified as Schwann cells immunohistochemically by antisera raised against the S-100 molecule (Fig. 7) , which in the peripheral nerves is specific to Schwann cells.¹⁸'²³'⁴¹ The possibility that these cells are macrophages of some peculiar morphology was further ruled out by the fact that they did not stain for the F4/80 antigen. Fibroblasts appeared as flat, cytoplasm rich cells. Their nuclei were very distinct, and often, two nucleoli were associated with each nucleus.

To visualize both surface associated and cytoplasmic MAC-2, cells were stained immunohistochemically by mAb M3/38 in the presence of Triton X-100. Macrophages, Schwann cells and fibroblasts differed in their frequency and intensity of staining. Differences could best be studied when all three cell types were present in the same culture, and thus stained simultaneously. Fig. 6 displays cells that migrated out of nerve explants that were left in culture for one week. All macrophages stained moderately to most intense by the mAb. The majority of Schwann cells were also stained by the mAb. Some displayed intense and others moderate staining. In contrast, the vast majority of fibroblasts did not stain by the mAb. Of those stained, most displayed weak staining intensity. A similar pattern of mAb staining was obtained in cells originating from nerve segments that underwent Wallerian degeneration in vivo . The immunocytochemical detection of MAC-2 in Schwann cells and macrophages in cell cultures was further confirmed by immunoblot analysis of extracts of single cell type cultures (Fig. 8) .

Using Triton X-100, the mAb stain appeared throughout the stained cells (Figs. 6 & 7) . In contrast, when Triton X-100 was omitted and only cell surface associated MAC-2 was visualized, the staining pattern of macrophages and Schwann cells was not uniform but rather patchy (Fig. 9) . The extent of surface membrane associated with the stain differed depending on the procedure used to obtain cells. A large proportion of surface membrane was stained when cells were allowed to migrate out of in vivo or in vitro degenerating nerve segments. Minute portions of membrane were stained following enzymatic dissociation. The extent of cell surface staining was also extensive in the tissue itself. This was evident from staining of mechanically teased fibers from 8 day nerve explants.

Inhibition of myelin phagocytosis by galactose and lactose during in vitro degeneration of N mice nerve explants. Lectinophagocytosis is a mechanism of phagocytosis dependent on the ability of a phagocyte to identify its specific target by molecular fitting between a cell surface associated carbohydrate binding protein (the lectin) and a complementary sugar on the target.²⁷ In this context, MAC-2 is a galactose specific lectin which is associated with macrophage and Schwann cell surfaces, and myelin, the phagocytized target, is rich in galactolipids such as cerebroside and sulfatide.²⁸ The prior art did not disclose whether MAC-2 is involved in lectin mediated phagocytosis. Applicants hypothesized that this could be the case, and, therefore, tested for the possibility that MAC-2 mediates phagocytosis of myelin. Based on this hypothesis, applicants examined whether or not galactose and lactose will inhibit myelin phagocytosis. Inhibition could be expected based on the competition between the free sugars (galactose and lactose) and the myelin galactolipids on binding to MAC-2. Furthermore, it could be expected that lactose will be a more potent inhibitor than galactose based on its higher binding affinity to MAC-2.¹²

Intact nerves from individual mice were distributed between regular medium (DMEM + 10% FCS) , and medium to which 25 mM of either mannose, galactose, or lactose were added. Nerves were kept in culture for 6 days, and then analyzed by morphometry to assess the density of unphagocytized, residual myelin. Unphagocytized, residual myelin was defined morphologically as round shaped myelin figures enclosing translucent spaces (Fig. 10) . The validity of the morphometric methodology used is evident from the values obtained for intact (374) and in vivo degenerating (13) nerves. The average value of residual myelin in nerve explants incubated in medium (144) did not differ significantly from that in nerve explants exposed to 25mM mannose (130) . Levels of residual myelin in explants exposed to 25mM galactose (211) or lactose (289) were significantly higher than those in explants bathed in regular medium or medium supplemented by mannose (Fig. 11) . Galactose and lactose, the competing sugars, but not mannose, the non- competing sugar, increased residual myelin. Furthermore, lactose was significantly more effective than galactose in this respect, as predicted from the hypothesis. Therefore, inhibition of phagocytosis by galactose and lactose is specific and not due to some unspecific effect of sugars.

Myelin phagocytosis in injured W mice peripheral nerves. W mice display a slow progression of Wallerian degeneration.²¹ At post injury time intervals that N mice already exhibit profound destruction of axons and phagocytosis of myelin, W mice display normal morphology. Applicants have repeated those observations and extended them.

Sciatic nerves of W mice were examined by light and electron microscopy 5 to 7 days after injury (Fig. 12) . Nerve segments situated distal to sites of transection were divided into two domains. From the site of transection to about 3 mm, and the more distally located portion up to additional 10 mm. The first domain will be referred to as the injury/lesion site/region. In W mice, the two domains differed in their morphological appearance. Break down of axons, recruitment of macrophages, and phagocytosis of myelin were detected at the lesion site (Fig. 12B) . The more distal domain appeared normal (Fig. 12A) ; axons and myelin were intact, and macrophages were as scarce as in intact nerves. In contrast to the findings during in vivo degeneration, some break down of axons and myelin phagocytosis were detected throughout W mice nerve explants that degenerated in vitro for 5 to 7 days (Fig. 12C) .

Applicants further examined W mice nerves that were damaged by cycles of freeze and thaw to kill all non- neuronal cells present (e.g. Schwann) . The frozen nerves were placed back into the donor animals and examined 5 to 7 days later. Macrophages invaded the frozen nerves and phagocytized myelin.

The extent of myelin destruction was assessed by estimating by morphometry the density of unphagocytized residual myelin in intact nerves, in 6 days in vitro degenerating nerves, and in 6 days in vivo degenerating nerves (> 5mm distal to injury sites) . N and W mice were examined (Fig. 13A) . In N mice, myelin destruction was partial during in vitro degeneration (about 64%) and almost complete during in vivo Wallerian degeneration (>95%) In W mice, myelin destruction that occurred during in vitro degeneration was less pronounced than in N mice (about 23%) . In marked contrast to N mice, there was no reduction in the density of unphagocytized myelin during the first 6 days of in vivo degeneration of W mice nerves at regions located farther distal than 5 mm from sites of transection.

The expression of MAC-1, MAC-2, the Fc receptor and the F4/80 antigen in injured W mice peripheral nerves. The slow progression of Wallerian degeneration in W mice has been attributed to the slow pace recruitment of macrophages distal to the lesion site. The failure to recruit macrophages provides only a partial explanation for the lack of axonal breakdown and myelin phagocytosis. This conclusion is based on the observation that some degree of phagocytosis occurred in vitro but not in vivo although the same arrays of non-neuronal cells were involved in the two instances. Applicants have shown that MAC-2 is involved in the mediation of myelin phagocytosis by Schwann cells and macrophages (see above) . Two questions thus arise. First, does the failure to show any degree of myelin destruction in vivo result from a deficient production of MAC-2 in vivol Second, is the in vitro destruction of myelin associated with MAC-2 production? Applicants studied, therefore, in W mice, as in N mice (see above) , the expression of the molecules MAC-1, MAC-2, the Fc receptor, and the F4/80 antigen using immunohistochemistry (see Fig. 14 for MAC-2 and the F4/80 antigen) . Very little and often no immunoreactivity to the four molecules was observed in intact nerves. In vivo degenerating nerves were examined at three locations along their length: at the site of injury and 5 and 10 mm distal to it. Increased immunoreactivity to the four molecules was detected at the site of injury but neither at 5 nor 10 mm distal to it. This finding was in marked contrast to findings in N nerves where increased immunoreactivity to the four molecules was detected at the three locations as of the third day after nerve transection. MAC-1, MAC-2, the Fc receptor and the F4/80 antigen were expressed throughout frozen

W nerves. W nerves that were explanted into culture and allowed to undergo in vitro degeneration for 5 to 7 days expressed higher than normal levels of MAC-2, but normal levels of the F4/80 antigen, the Fc receptor and MAC-1. Thus in W mice, macrophages

(the F4/80 positive cells) were recruited in vivo to the site of nerve injury and into frozen nerves (Fig. 14 G & I) . Macrophages were largely excluded in vitro from nerve explants and in vivo from intact nerves and injured nerve segments situated from 3 mm and further distal to injury sites (Fig. 14F, H & J) . The distribution of MAC-2 bearing cells paralleled that of macrophages except for in the in vitro degenerating nerve explants where the expression of MAC-2 increased although macrophages remained scarce (Fig. 14 E & J) . Thus MAC-2 was present where myelin destruction occurred and was absent where myelin destruction did not take place.

In W, as in N, mice nerve explants the increase in the expression of MAC-2 was not accompanied by similar increases in the expression of the F4/80 antigen, the Fc receptor and the MAC-1. This suggests that a cell type other than macrophages displayed MAC-2. Applicants demonstrated herein that N mice Schwann cells express MAC-2 after nerve injury. To determine whether W mice Schwann cells do the same, cell cultures were stained immunohistochemically for MAC-2 either in the presence of Triton X-100 to visualize cytoplasmic and cell surface MAC-2, or in the absence of Triton X-100 to visualize only cell surface MAC-2. Only a proportion of W mice Schwann cells stained for MAC-2 (Fig. 15A) . In the presence of Triton X-100, the entire cell stained. In the absence of Triton

X-100, the stain was very faint and patchy. Thus, the Schwann cells from W mice displayed MAC-2 primarily in their cytoplasms and relatively very little on their surface. The few macrophages that were present (identified as F4/80 positive cells; Fig. 15B) stained well for MAC-2 in the presence as in the absence of Triton X-100. Fibroblasts stained neither for F4/80 nor for MAC-2.

To further compare between W and N mice, an ELISA was used to quantify levels of MAC-2 in W and N mice nerves (Fig. 13B) . Intact nerves, 6 days in vivo degenerating nerves (>5mm distal to injury sites) and 6 days in vitro degenerating nerve explants were examined. In intact nerves, MAC-2 was barely detected in the two strains of mice. W and N mice differed dramatically in their in vivo response to nerve transection, but much less so in their in vitro response. The relationship between the extent of myelin destruction and levels of MAC-2 over the entire range represented by N and W nerves (Fig. 13C) was also explored. It is evident that a strong positive correlation is displayed between the two: higher levels of MAC-2 are associated with an increasing extent of myelin destruction.

The above examples provide evidence that injury of normal N mice peripheral nerves induces Schwann cells to express the galactose specific lectin MAC-2 and to phagocytize myelin. This allows for treatment of demyelination by interrupting the interaction between MAC-2 and myelin in vivo . The MAC-2 receptor is a phenotype characteristic of blood born macrophages that reside in degenerating nerves.

Evidence showed a functional role for MAC-2 in lectin mediated myelin phagocytosis. These findings and conclusions are consistent with several situations. The intactness of myelin in normal nerves is associated with the lack of MAC-2 expression by Schwann cells and by the few macrophages present in the tissue. In contrast, during the normal course of Wallerian degeneration, Schwann cells and recruited macrophages express MAC-2 that enables the two cell types to exhibit lectinophagocytosis of myelin. Of particular interest is the understanding of the ability of Schwann cells to phagocytize a specific portion of their own membrane, the myelin. This becomes possible secondary to the appearance of MAC-2. In mutant W mice, the in vivo intactness of myelin in nerve segments situated distal to sites of nerve transection is associated with the failures to recruit macrophages and to induce MAC-2 production by Schwann cells. In both normal and mutant mice nerves, the in vitro destruction of myelin is correlated with the production of MAC-2 in the tissues.

Myelin phagocytosis by Schwann cells was clearly observed in the electron microscope in nerve segments undergoing in vivo and in vitro axonal degeneration. Based on the electron microscopical identification of, and distinction between, Schwann cells and macrophages, these findings confirm those documenting myelin phagocytosis by rat Schwann cells.⁶'⁴² In further agreement with these findings are two type of observations. First, in quantitative electron microscopical studies of intact peripheral nerves, there is no positive identification of cell as macrophages.⁴⁰'⁴³ The macrophage population in intact nerves must, therefore, be very small. Second, only a small proportion of tissue macrophages are capable of cell division, and then of one cycle only.²⁹'⁴⁵ Several lines of evidence indicate that nerve injury induces Schwann cells to express MAC-2 which normally they do not. Immunocytochemistry and immunoblot analysis of intact peripheral nerves resulted in very little, if any, MAC-2 immunoreactivity. Thus, Schwann cells in intact and normal peripheral nerves do not express MAC-2. During in vitro degeneration of nerve explants, MAC-2 bearing cells become numerous. This increase in MAC-2 presentation in nerve explants was not accompanied by similar, concomitant increases in MAC-1, Fc receptor and F4/80 antigen presentations. Thus, macrophages cannot account for the increase in MAC-2 expression in nerve explants. If macrophages were responsible for the increase in the display of MAC-2, one would expect substantial increases in the expression of the F4/80 antigen, if not all, as was the case in the macrophage rich tissues (the in vivo degenerating and frozen nerves) . The dense and wide anatomical distribution of ring/tube-like shaped structures revealed by the immunocytochemical stain for MAC-2 in in vitro and early in vivo degenerating nerves correlates well with the anatomical distribution of Schwann cells and no other cell type. Finally, MAC-2 was detected by immunocytochemistry and immunoblot analysis in cells isolated from in vivo and in vitro degenerating nerves that were identified as Schwann by their characteristic morphology, S-100 positive and F4/80 negative immunocytochemistry. The difference in the pattern of MAC-2 staining between cells that were either, or not, treated with Triton X-100 further indicates that MAC-2 is associated with the cytoplasm and the external surface of Schwann cells.

MAC-2 was first identified as a cell surface molecule expressed by inflammatory and mature murine macrophages¹⁴ and thereafter also by human macrophages.⁹ Some, but not all, macrophage activating factors up-regulate MAC-2 expression.¹⁴ Interestingly, an identical/homologous molecule is also produced by transformed, malignant cells: the fibroblast 3T3 cell line,¹⁷'²⁴ rat basophilic leukemia¹⁹ and metastasis associated malignancies.³⁴'³⁵ In view of these observations, the detection of MAC-2 in Schwann cells which, on one hand, differ in their lineage from macrophages and, on the other hand, are not malignant cells is unexpected.

MAC-2 has already been identified as a galactose specific lectin, a non-integrin laminin binding protein, an IgE binding molecule, and a component of heterogeneous nuclear binding protein.⁹'¹⁷'¹⁹'^24,46 Applicants have shown a novel role for MAC-2: a cell surface associated lectin which can mediate myelin phagocytosis through the non-immune, opsonin independent mechanism of phagocytosis, lectinophagocytosis.²⁷ In lectinophagocytosis, the ability of a phagocyte to identify its specific target is dependent on a molecular fitting between a cell surface associated carbohydrate binding protein (the lectin) and a complementary sugar on the target. In this context, MAC-2 is a galactose specific lectin which is associated with macrophage and Schwann cell surfaces and myelin, the phagocytized target, is rich in galactolipids.²⁸ Present, therefore, is the molecular fitting required by lectinophagocytosis. MAC-2 mediated myelin phagocytosis was observed using a methodology most commonly used to test for lectin mediated phagocytosis, competitive inhibition of phagocytosis by specific sugars.²⁷ Applicants' observations that galactose and lactose, but not mannose, inhibited myelin phagocytosis indicates, therefore, that MAC-2 can provide a molecular basis for myelin lectinophagocytosis by Schwann cells and that blocking the MAC-2 receptor will interrupt the pathological process of demyelination.

The observations made in W mice further support applicants' hypothesis that MAC-2 is mediating myelin phagocytosis. There was in W mice a mutual absence in vivo and mutual presence in vitro of MAC-2 expression and myelin phagocytosis. In addition, there was a quantitative correlation between levels of MAC-2 expression and extent of phagocytosis over a wide range of values obtained from N and W mice nerves. Increasing levels of MAC-2 production were accompanied by extended destruction of myelin by phagocytosis.

The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims the invention may be practiced otherwise than as specifically described.

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Claims

CLAIMSWhat is claimed is:

1. A method for inhibiting myelin destruction including the steps of identifying the type of demyelination associated with a patient's disease or syndrome, determining if MAC-2 expression has been induced as part of demyelination, administering compounds that act at the MAC-

2 receptor thereby interferring with binding between MAC-2 and myelin.

2. A method as set forth in claim 1 wherein the step of determining MAC-2 expression is for cells selected from the group consisting of Schwann cells, macrophages, oligodendroglia and microglia.

3. A method as set forth in claim 1 wherein the step of administering compounds that act at the MAC-2 receptor is for compounds selected from the group consisting of lactose, galactose and cerebrosides.

4. A method as set forth in claim 1 wherein the step of administering compounds that act at the MAC-2 receptor is for compounds selected from the group consisting of polyclonal or monoclonal antibodies directed against the MAC-2 receptor.

5. A method as set forth in claim 1 wherein the step of determining if MAC-2 expression has been induced as part of demyelination includes determining if MAC-2 expression has been induced on cells that phagocytize myelin.