Perforin Lysis of Oligodendrocytes in vitro, Relevance to Multiple Sclerosis

1. Journal of Neuroscience Research 64:380 –391 (2001)
Structural Dynamics of Oligodendrocyte
Lysis by Perforin in Culture: Relevance to
Multiple Sclerosis
Rana Zeine,1* Wendy Cammer,2,3 Elisa Barbarese,4 Chau-Ching Liu,5 and
Cedric S. Raine1–3
1
Department of Pathology (Neuropathology), Albert Einstein College of Medicine, Bronx, New York
2
Department of Neurology, Albert Einstein College of Medicine, Bronx, New York
3
Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York
4
Departments of Neuroscience and Neurology, University of Connecticut Health Center, Farmington,
Connecticut
5
Division of Rheumatology, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
The mechanism by which oligodendrocytes are de- Multiple Sclerosis (MS) is considered to be an auto-
pleted from active lesions in multiple sclerosis (MS) is immune demyelinating disease of the central nervous sys-
not clear but many reports implicate a cytolytic pro- tem (CNS) in which oligodendrocytes and myelin be-
cess. The most applied animal model for MS, chronic come exposed to components of the immune system,
relapsing experimental autoimmune encephalomyelitis including infiltrating T-cells, killer cells and soluble cyto-
(EAE), has been established in inbred strains of mice, lytic factors (Brosnan and Raine, 1996; Pouly and Antel,
especially SJL and PL. Studies on oligodendrocytes 1999), and oligodendrocytes are selectively depleted from
from these strains in vitro have been hampered to date lesions by as-yet-unrecognized mechanisms (Raine 1997a;
by an inability to grow these cells from mouse CNS Lucchinetti et al., 1999). Numerous causes of injury to
tissue. We report here a successful method to culture oligodendrocytes have been explored and proposed as
SJL mouse oligodendrocytes and have analyzed lysis possible mechanisms underlying the damage to myelin in
of these cells in vitro mediated by the pore-forming the MS lesion. Oligodendrocytes synthesize and maintain
protein, perforin, a candidate effector molecule in in- CNS myelin and, depending on the nature of the insult,
flammatory demyelination. Cultures were exposed to are known to be susceptible to death via both apoptosis
murine perforin, 36 –72 hemolytic U, for up to 2.5 hr and necrosis, (D’Souza et al., 1996; Ludwin, 1997; Merrill
and examined using the oligodendrocyte phenotypic and Scolding, 1999). Their documented susceptibility to
markers O4, galactocerebroside and myelin basic pro- injury by components of the immune system renders them
tein (MBP), in addition to a membrane dye (DiI) and a vulnerable to damage in the vicinity of immune responses.
marker of necrosis, propidium iodide, (PI). Cultures In active MS, some features of the lesion are sugges-
were imaged chronologically by phase contrast, immu- tive of cytolytic damage (Prineas and McDonald, 1997;
nofluorescence, digital, light and electron microscopy. Raine, 1997b), specifically oligodendrocyte swelling, cy-
Findings showed that the majority of oligodendrocytes toplasmic vacuolation and myelin vesiculation (Suzuki et
were killed within 60 –90 min via pore expansion and al., 1969; Rodriguez and Scheithauer, 1994; Genain et al.,
ultimately, membrane disruption. The structural fea- 1999; Raine et al., 1999). Although initial studies on
tures of the cellular damage comprised swelling of the demyelination invoked serum factors (Raine, 1984; Ruijs
cell body, fenestration and fragmentation of mem- et al., 1990), subsequent studies emphasized damage to
branes and processes, cytoplasmic vacuolation and myelin and oligodendrocytes induced by lymphocytes and
breakdown of the nuclear envelope. Astrocytes in the their soluble products (Selmaj and Raine, 1988; Ruijs et
same system were relatively resistant to cell lysis. The
above patterns of oligodendrocyte damage in SJL oli-
Contract grant sponsor: National Multiple Sclerosis Society; Contract grant
godendrocytes were reminiscent of patterns in the MS numbers: NMSS RG 1001-J-10, NMSS RG 2971, and NMSS RG 2843;
lesion, leaving us to conclude that perforin may play an Contract grant sponsor: American Heart Association; Contract grant spon-
important role in the human disease. J. Neurosci. Res. sor: HHS; Contract grant numbers: NS 08952, NS 11920, and NS 19943.
64:380 –391, 2001. © 2001 Wiley-Liss, Inc. *Correspondence to: Dr. Rana Zeine, Department of Pathology (Neuro-
pathology), Albert Einstein College of Medicine, 1300 Morris Park Ave-
Key words: demyelination; cytotoxicity; autoimmunity; nue, F-140, Bronx, NY 10461. E-mail: harkzen@aol.com
oligodendrocyte; perforin; experimental autoimmune en- Received 13 November 2000; Revised 8 January 2001; Accepted 9 January
cephalomyelitis; multiple sclerosis 2001
© 2001 Wiley-Liss, Inc.

2. Oligodendrocyte Lysis by Perforin 381
al., 1990; Selmaj et al., 1991a; Freedman et al., 1991; Antel toplasmic damage that are reminiscent of the oligodendro-
et al., 1998; Dal Canto et al., 2000). Several lines of cyte pathology encountered in MS lesions.
evidence support the hypothesis that oligodendrocyte pa-
thology and depletion from the CNS in MS may be METHODS AND MATERIALS
related to perforin-mediated lysis. This is particularly a Animals
propos to lysis of human oligodendrocytes by human Lactating female SJL mice with newborn pups were ob-
T-cells that has been shown to occur in vitro via a pre- tained from either Jackson Labs (Bar Harbor, ME) or Charles
dominantly perforin-based cytotoxicity mechanism (Zeine River (Wilmington, MA), and housed in an AAALAC-
et al., 1998). accredited facility at the Albert Einstein College of Medicine.
Perforin has been demonstrated in vivo in inflam-
matory lesions of a variety of autoimmune diseases (Liu et Oligodendrocyte Cultures
al., 1996; Kappeler and Mueller, 2000). In the CNS, Oligodendrocyte-enriched cultures were prepared as de-
perforin expression has been detected in astrocytes in scribed previously (Barbarese, 1991). Primary CNS cultures
normal brain tissue, and in astrocytes and infiltrating lym- were obtained by dissociation from the cerebral hemispheres of
phocytes within demyelinative lesions of MS and its 1–3-day-old SJL mouse pups and plated onto poly-L-lysine-
model, experimental autoimmune encephalomyelitis coated tissue culture dishes (Nunc, Fisher Scientific, Pittsburgh,
(EAE) (Held et al., 1993; Gasque et al., 1998; Zeine et al., PA), for 12 days in Dulbecco’s modified Eagle’s medium
1999). Furthermore, perforin mRNA has been detected at (DMEM) (Gibco BRL, Rockville, MD), supplemented with
elevated levels in the CSF of patients with MS (Matusevi- 10% newborn bovine serum (Gibco BRL). Enriched cultures of
cius et al., 1998; Kivisakk et al., 1999). This potent mem- oligodendrocytes were obtained by mild trypsinization of the
branolytic molecule, perforin, is a 70 kD pore-forming mixed cultures followed by depletion of adherent cells though
protein (PFP) that is stored within cytolytic granules of differential adhesion to plastic substrata. Oligodendrocyte-
cytotoxic T-cells and natural killer (NK) cells (Liu et al., enriched populations were seeded onto 24-well plates at 1 105
1995a). Perforin-mediated cytotoxicity is a calcium- cells per well and grown on either Thermanox plastic coverslips
dependent mechanism as calcium ions are required both (Nunc) or glass D coverslips (Fisher), precoated with poly-L-
for effective granule exocytosis and for the appropriate ornithine (Sigma, St. Louis, MO). Alternatively, Lab-Tek glass
perforin conformation. Mechanistically, perforin mono- chamber slides (Nunc) were used. The feeding medium
mers released by killer cells bind to phosphorylcholine (DMEM-N1-B104) was composed of a freshly prepared mix-
lipid moieties and insert into target cell membranes where ture of DMEM/F12 (Gibco BRL), with pyruvic acid, HEPES,
they polymerize and assemble to form homopolymeric albumin and the following N1 supplements: apo-transferrin
transmembrane pore structures (Tschopp et al., 1989; (0.05 mg/ml), sodium selenite (30 nM) and biotin (10 ng/ml).
Ojcius et al., 1991a). These pores perturb membrane Insulin (0.016 mg/ml), hydrocortisone (3.6 ng/ml), T3
permeability and result in target cell death by osmotic lysis, (15 nM), -ME and gentamicin (0.5%) were also added, and the
a mechanism most closely related to necrosis. final mixture was supplemented with B104 neuroblastoma-
conditioned medium in an 87:13 proportion. Chemicals and
Among the mouse models of demyelination that
hormones were obtained from Sigma, and B104 was supplied by
most closely mimic the human disease, those most applied
Dr. Barbarese. The final composition of these cultures was ca.
to MS have been established in inbred mouse strains (SJL, 80% oligodendrocytes, 15% astrocytes and 5% microglia plus
PL), strains that are susceptible to remitting-relapsing precursor cells.
forms of EAE (Brown et al., 1982; Cross et al., 1987). The
susceptibility of oligodendrocytes from mouse strains to Perforin-Mediated Lysis
perforin-mediated lysis has never been examined because Adherent oligodendrocyte-enriched cultures were incu-
mouse oligodendrocytes have been difficult to culture. In bated at 37°C with a preparation of the pore forming protein
the early 1990s, the susceptibility of rat oligodendrocytes (PFP) perforin, provided by Dr. C.-C. Liu, in the range of
to perforin-mediated injury was described (Scolding et al., 36 –72 hemolytic units (HU) in the presence of 2 mM Ca2 and
1990), and more recently, using a chromium-51 release in a final volume of 400 l serum-free medium. Control cul-
cytotoxicity assay (Malipiero et al., 1997). To explore tures received either Ca2 Cl2 alone or PFP alone. Cell lysis was
perforin-mediated CNS injury in a mouse model, we have evaluated over a period of 2.5 hr by phase contrast microscopy,
developed a method to culture oligodendrocytes from SJL indirect immuno-fluorescence, LM and EM, or time-lapse dig-
mice and have documented for the first time the selective ital imaging.
effects of perforin on these cells using chronologic digital
techniques. For this, we have examined the kinetics of Immunocytochemistry
perforin-induced osmotic lysis of oligodendrocytes and At 30 min intervals after exposure to perforin, propidium
have employed immunofluorescence, digital imaging and iodide (PI) (50 g/ml), an indicator of membrane disruption,
light and electron microscopy (LM and EM), to define the was added to duplicate cultures for 3 min and the cells washed
structural features of this process. Our analysis has revealed in PBS (Sigma) and fixed in 4% paraformaldehyde at 4°C
that SJL mouse oligodendrocytes in vitro display several overnight. Fixed cells were then rinsed in PBS and incubated
perforin-induced structural patterns of membrane and cy- with the following primary antibodies for 1 hr at room temper-

3. 382 Zeine et al.
ature: mouse monoclonal anti-galactocerebroside-GalC anti- glutaraldehyde in Millonig’s buffer, pH 7.4. The cultures were
body (IgG3; Roche/Boehringer-Mannheim, Indianapolis, IN) then post-fixed for 45 min in 1% OsO4 in PBS, pH 7.4,
at 1:10 dilution; mouse monoclonal anti-O4 antibody (IgM; dehydrated though a graded series of ethyl alcohol, washed in a
Roche/Boehringer-Mannheim) at 1:5 dilution; mouse mono- 1:1 mixture of absolute alcohol and Epon 812, and embedded in
clonal anti-myelin basic protein-MBP antibody (IgG1; Roche/ Epon 812. After polymerization, the flat sheets of epoxy were
Boehringer-Mannheim) at 1:100 dilution. After gentle rinsing in removed from the plastic coverslips. For sectioning perpendic-
PBS, coverslips were treated with the appropriate secondary ular to the plane of growth, the blocks were re-embedded
antibodies for 1 hr at room temperature at 1:100 dilution: end-on in capsules of fresh epoxy (Norton et al., 1983). One
FITC-conjugated goat anti-mouse IgG (Rockland, Gilberts- micron sections stained with toluidine blue were examined by
ville, PA), and FITC-conjugated goat anti-mouse IgM light microscopy and for electron microscopy, thin sections
(Rockland). The coverslips were washed and mounted on slides were double-stained with uranium and lead salts, carbon-coated
using 50% glycerol containing the anti-fading agent, n-propyl and scanned in an Hitachi H 600.
gallate (Sigma). The cells were viewed using a Nikon Optiphot
and an Olympus IX 70 inverted microscope, both equipped for RESULTS
epifluorescence with filters for rhodamine and FITC. SJL Mouse Oligodendrocytes in Culture
DiI Membrane Staining and Imaging Oligodendrocytes were successfully isolated from
Living cells were labeled with a fluorescent lipidophilic primary CNS cultures from newborn SJL mice and con-
membrane tracer, the long-chain dialkylcarbocyanine, DiI (Mo- sisted of bipolar, tripolar and multipolar populations.
lecular Probes, Eugene, OR). Oligodendrocyte-enriched cul- These cells differentiated and expressed the oligodendro-
tures were incubated with 10 M DiI for 2 hr at 37°C. The cyte markers, GalC (Fig. 1A), O4 (Fig. 1J) and MBP (Fig.
DiI-containing medium was then removed and the cells washed 1G). They extended multiple processes and elaborated flat
before exposure to perforin. Control cells and cells exposed to membranes. These membrane sheets expanded with time
perforin were viewed under an Olympus IX 70 microscope. and covered larger areas, remaining intact and exhibiting
The temperature of the specimen was maintained at 37°C an homogeneous consistency and a well-demarcated edge
within an environmental chamber equipped with air and stage (Figs. 1A,J and 2A). DiI revealed cytoplasmic ribs within
heaters (Olympus, Melville, NY). Images were collected at 0.5 the flat membrane veils (Fig. 2A). O4 and GalC gave
and 1 min intervals with a Censys-cooled CCD camera (Pho- intense membrane staining (Fig. 1A,J), whereas MBP
tometrics, Tucson, AZ), and the light was shuttered (Ludl, staining was more cytoplasmic, denser in perinuclear re-
Hawthorne, NY) between exposures. Software control was gions and diffuse over the membrane sheets (Fig. 1G).
provided by I.P. Lab Spectrum (Scanalytics, Fairfax, VA), and
deconvolution was performed at 80% to 90% haze reduction Perforin-Mediated Injury of SJL Mouse
with PowerHazebuster (Vaytek, Fairfield, IA) on a Macintosh Oligodendrocytes
G3. Oligodendrocyte-enriched cultures were exposed to
36, 48, 60 and 72 HU of perforin in the presence of 2 mM
TUNEL Method Ca2 . Light and phase contrast microscopy showed swell-
In some experiments, cell death by apoptosis was assessed ing of the cells within 15 min and rearrangement of the
using the In Situ Cell Detection Kit with Fluorescein, (Enzo/ cytoplasm within 30 min of exposure. Examination of
Boehringer-Mannheim, Indianapolis, IN), according to the membrane integrity in living cells stained with DiI re-
manufacturer’s instructions. vealed the formation and expansion of distinct membrane
holes, probably the result of coalescence of multiple sub-
Dead Cell Ratio microscopic (15–20 nm) pores (Liu et al., 1995a), between
The ratio of PI positive cells to total cells was calculated by 30 and 90 min after exposure to perforin (Fig. 2B,C). By
counting approximately 300 cells in each of seven randomly- indirect immunofluorescence, the nuclei of dying and
selected low power fields. Cells were exposed to 72 HU of dead cells stained bright red with PI after exposure to PFP
perforin for 1.5 hr and then stained with PI and counted unfixed for 0.5 hr (Fig. 1D,K), 1 hr (Fig. 1H), and 2.5 hr (Fig.
at 10 magnification. 1F,I,L). The pattern of distribution of O4 and GalC re-
vealed distortion of the cellular architecture suggestive of
Membrane Hole Expansion (Fenestration) swelling of the cell body and fragmentation of membranes
The size of membrane holes (fenestrae) was measured at and processes seen by 30 min (Fig. 1C,K) Complete lysis
the LM level by manually tracing with I.P. Lab Spectrum most frequently involved reduction of the cytoplasm to
(Scanalytics), the areas of 16 individual fenestrae at each of 30 fragmented processes and droplets (Fig. 2D). At 2.5 hr,
time points. The rate of hole expansion was determined by some cells could be found that had not proceeded though
plotting the average areas at each time point against time. completion of osmotic lysis, but had arrested at a stage that
exhibited marked swelling with abundant membrane holes
Light and Electron Microscopy (Figs. 1E,L; 2C). These structural features of perforin-
At 30 min intervals after exposure to perforin, monolayers induced oligodendrocyte damage were also clearly dem-
of oligodendrocyte- enriched cells were fixed for 30 min di- onstrated in experiments where cultures were fixed and
rectly on Thermanox coverslips by immersion in cold 2.5% stained with toluidine blue (Fig. 3). As compared to con-

4. Oligodendrocyte Lysis by Perforin 383
Fig. 1. Indirect immunofluorescence of oligodendrocytes in culture, godendrocytes that are negative for PI. H: One hour PFP exposure. A
before and after exposure to PFP. A: Control culture. Two GalC- group of MBP-positive , PI-positive oligodendrocytes can be seen.
positive oligodendrocytes are shown. B: Same field as (A), stained with I: PFP exposure (2.5 hr). A group of MBP-positive/PI-positive oligo-
PI. Note lack of PI staining in control oligodendrocytes shown in (A). dendrocytes. J: Control culture. Superimposition of O4 on a phase
C: Thirty-minute PFP exposure. Four oligodendrocytes stain positively contrast image reveals a normal oligodendrocyte with a flattened mem-
for GalC. D: Same field as (C) showing PI positivity of the four brane. K: Thirty minute PFP exposure. The pattern of O4 distribution
oligodendrocytes. E: PFP exposure (2.5 hr). A group of GalC-positive suggests membrane fragmentation. The nucleus is slightly PI positive.
oligodendrocytes is shown. F: Same field as (E) showing PI positivity L: PFP exposure (2.5 hr). O4 staining reveals multiple membrane holes
and distorted nuclei of the oligodendrocytes. G: Control culture. and fragmentation. The nucleus is intensely positive for PI. Magnifi-
Superimposition of MBP and PI staining showing MBP-positive oli- cation 450 in A–I; 700 in J–L.

5. 384 Zeine et al.
Fig. 2. Living oligodendrocytes stained with DiI. A: Control oligodendrocyte. Note the well-
demarcated edge and homogeneous nature of the membranous veil of this oligodendrocyte.
B: Forty-five minute exposure to PFP. By DiI staining, distinct membrane holes are visible. C: Same
cell, 90 min exposure to PFP. The number and size of the holes have increased considerably.
D: End-Stage lysis. A collection of cytoplasmic fragments is arranged around a distorted nucleus.
Magnification 800.
trols (Fig. 3A–C), cells exposed to PFP for 60 min man- areas of 16 individual fenestrae and the rate of expansion
ifested swelling, cytoplasmic disintegration and vacuola- was determined from the slope of the plot of average areas
tion (Fig. 3D–F). The nuclei became asymmetrically against time (data not shown). For this particular cell,
distorted but did not exhibit signs of apoptosis (Figs. 1F,L, fenestrae expanded at a rate of 55 m2/min over a period
3D–F). TUNEL staining revealed no increase in TUNEL of 5 min after which most ceased to change. This pattern
positive cells over control cultures (data not shown). of cellular arrest was not uncommon and probably repre-
sented death with incomplete lysis. Some oligodendro-
Rate of Membrane Fenestration cytes that were fixed as late as 2.5 hr after exposure to
With DiI staining, distinct holes (fenestrae) became perforin exhibited a pattern of O4 distribution suggestive
visible by EM in the membranes of oligodendrocytes after of numerous expanded fenestrae delineating vacuoles
exposure to perforin for 30 min. The progression of throughout the damaged cell (Fig. 1L).
fenestration was followed in one cell by time-lapse imag-
ing starting at 45 min and ending at 90 min (Figs. 2B–C). Pattern of End-Stage Lysis
Images were collected at 30 sec intervals. Hole size was A more severe pattern of destruction was evident by
measured by tracing with I.P. Lab Spectrum software, the LM in oligodendrocytes that proceeded rapidly to com-

6. Oligodendrocyte Lysis by Perforin 385
Fig. 3. Light microscopy of 1 micron toluidine blue-stained epoxy swollen nucleus surrounded by cytoplasmic remnants (arrow) lies ad-
sections. A: Control culture. A flattened oligodendrocyte attached to jacent to a cell with vacuolated cytoplasm. E: Sixty minutes PFP. Two
the substrate (arrow), lies next to an astrocyte. B: Control culture. sick, vacuolated oligodendrocytes lie beneath a layer of astrocytes.
Three oligodendrocytes (arrows) lie on layer of astrocytic processes. F: Sixty minutes PFP. A fragmented culture shows a layer of degen-
C: Control culture. A large flattened, healthy astrocyte with a single erating oligodendrocytes overlying a layer of minimally affected astro-
nucleus is shown. D: Sixty minutes PFP. An oligodendrocyte with a cytes. Magnification 750 in B; 1,000 in A,C–F.
plete lysis. Examples of such cells showed complete loss of forin for 1.5 hr and then stained with PI. For each field, PI
all membrane veils and were captured by both immuno- images were superimposed on corresponding phase con-
fluorescence and DiI staining. Some were found displaying trast micrograph and the cells counted unfixed. In cultures
PI positive nuclei associated with few GalC positive rem- exposed to perforin, the ratio of PI-positive cells ranged
nants of cytoplasmic fragments and processes (data not between 42 and 100% (mean 77%, SD 24), whereas the
shown), whereas others presented as arrays of cytoplasmic range in control cultures remained between 9 and 15%
fragments around an asymmetrically-enlarged and dis- (mean 11% , SD 2).
torted nucleus (Fig. 2D). This type of cellular fragmenta-
tion most probably represented end-stage osmotic lysis. Ultrastructural Features of Perforin-Induced
Damage
Kinetics of Perforin-Mediated Lysis By EM, the cytoplasm of normal oligodendrocytes in
Experiments were repeated using either 36, 48, 60 or vitro was more electron-dense than that of astrocytes,
72 HU of perforin. In all cases, the kinetics of perforin- possessed short segments of rough ER and contained
induced injury proceeded systematically though cell swell- microtubules but no intermediate filaments (Fig. 4A,B).
ing (15–30 min), membrane fenestration and expansion After perforin-induced injury, these cells became edema-
(30 –90 min), membrane fragmentation (30 –90 min), and tous and displayed extensive cytoplasmic vacuolation of
end-stage lysis (90 –150 min). As mentioned above, het- the cell body and processes within 30 min of exposure. On
erogeneity occurred in the extent of lysis achieved by occasion, oligodendrocytes featured an extremely large
individual cells under the same culture conditions, with some single cytoplasmic vacuole, which most probably evolved
cells becoming arrested at a stage before end-stage lysis. In from the confluence of several vacuoles during the process
most cells, however, death was complete within 3 hr. of osmotic lysis (Fig. 5A). Fragmentation of unit mem-
branes and the presence of myelin-like formations were
Ratio of Cell Killing by Perforin noted at the perimeter of such vacuoles (Fig. 5B). Another
The ratio of PI-positive cells was calculated by change common to the cytoplasm of affected oligoden-
counting by LM approximately 300 cells in each of seven drocytes was evident at the 0.5 hr time-point, when
randomly-selected low power fields. For this, cultures formation of villus-like profiles, rich in microtubules, was
consisting of a mixture of early differentiating oligoden- encountered (Fig. 4C). This appearance probably corre-
drocytes and astrocytes, were exposed to 72 HU of per- sponded to images seen by LM (Fig. 3D).

7. 386 Zeine et al.
Fig. 4. EM of control cells and early perforin-induced cytoplasmic 30,000. C: Thirty minutes PFP. The cytoplasm of this oligodendro-
injury. A: Control culture. A normal oligodendrocyte lies to the left of cyte protrudes as villus-like processes into the extracellular space. Note
an astrocyte. The cytoplasm of the oligodendrocyte is distinctly denser. the numerous microtubules with the cell body and the villus-like
Magnification 12,000. B: Detail of the oligodendrocyte cytoplasm in profiles (arrows). Magnification 75,000.
(A). Note the microtubules in cross section (arrows). Magnification

8. Oligodendrocyte Lysis by Perforin 387
Fig. 5. Ultrastructural patterns of membrane damage in oligodendro- 30,000. C: Sixty minutes PFP. End-stage lysis is seen in an oligo-
cytes exposed to PFP. A: Thirty minutes PFP. This oligodendrocyte dendrocyte that has lost its cell membrane, cytoplasmic integrity and
has developed a large vacuole around the perimeter of which myelin- intact nuclear membrane. Magnification 14,000. D: Detail of (C) to
like figures are located. Magnification 12,000. B: Detail from A to show beginning disruption of the nuclear membrane. Magnification
show the lamellar patterns of the myelin droplets. Magnification 30,000.

9. 388 Zeine et al.
Fig. 6. Sixty minutes PFP. Two
interdigitating, apparently healthy
astrocytes display an abundance of
intermediate filaments and ro-
settes of ribosomes. The nucleus
of the cell on the right is normal
and a hemidesmosome (typical of
subpial astrocytes) is indicated (ar-
row). Magnification 19,000.
Oligodendrocytes examined after exposure to per- of perforin exposure, cell membranes were lost and nu-
forin for 1 hr exhibited complete loss of the cell membrane clear envelopes began to degenerate (Fig. 5C). This is the
with only remnants of cytoplasm remaining around the first report demonstrating that SJL mouse oligodendro-
nucleus (Fig. 5C). In addition, evidence of degeneration of cytes are susceptible to perforin-induced osmotic lysis, a
the nuclear envelope was noted (Fig. 5D). Similar pathol- highly conserved, classical cell-death mechanism (Ojcius
ogy, albeit delayed and to a lesser degree, was seen in et al., 1991a; Liu et al., 1995b) and this occurred at a dose
astrocytes in the same culture system but on most occa- level similar to that which kills other eukaryotic cells.
sions, astrocytes were relatively resistant to damage and Interestingly, oligodendrocyte changes observed
maintained a normal ultrastructure well into the experi- here in vitro held much in common with appearances
mental period (Fig. 6). described in MS lesions, ultrastructural studies of which
DISCUSSION have consistently documented that oligodendrocyte pa-
thology exhibits features suggestive of immune osmotic
The mechanisms underlying oligodendrocyte deple- lysis or necrosis (Prineas, 1985; Prineas and McDonald,
tion from the MS plaque remain to be clarified although
cytolytic mechanisms have repeatedly been implicated 1997). In contrast, evidence for oligodendrocyte apoptosis
(Raine, 1997b). One candidate molecule in this process is in MS and EAE has been lacking or, at most, rare
the pore-forming membranolytic molecule, perforin, and (D’Souza et al., 1996; Bonetti et al., 1997; Bonetti and
in this report, we have studied the effects of perforin on Raine, 1997; Waldner et al., 1997). Perforin is a potent
oligodendrocytes cultured from a strain of mouse (SJL) mediator of osmotic lysis and perforin-mediated cytotox-
most frequently applied to studies on MS and a strain from icity is induced by killer T-cells (Lowin et al., 1994; Kagi
which oligodendrocytes have been difficult to grow in the et al., 1994). The presence of killer cells and perforin in
past. Our results have shown that during normal develop- demyelinative lesions of EAE and MS is well-known
ment in culture, oligodendrocytes extended processes and (Selmaj et al., 1991b; Held et al., 1993; Olive, 1996; Rajan
elaborated flattened myelin-like membranes, as described et al., 1996; Gasque et al., 1998; Zeine et al., 1999), and
previously for the murine system (Barry et al., 1996). After a dual role has been proposed for these cell types whereby
exposure to perforin, the integrity of the membrane veils injury to oligodendrocytes and neurons is detrimental
was compromised as microscopic fenestrae appeared and whereas injury to immune cells is protective to the CNS
expanded rapidly, transforming the flat membrane sheets (Battistini et al., 1995; Malipiero et al., 1997; Zhang et al.,
into a delicate lacework (Fig. 2B,C). The cell bodies 1997; Matsumoto et al., 1998; Murray et al., 1998; Zeine
swelled and the cytoplasm became vacuolated and dis- et al., 1999). Most likely, the balance between the effector
rupted into fragments rich in microtubules. Within 60 min and immunoregulatory arms of the immune response de-

10. Oligodendrocyte Lysis by Perforin 389
termines the magnitude of CNS damage inflicted by the Kinase inhibitors block the mobilization and orientation of
perforin-based cytotoxicity pathway. In a study on MOG- perforin-containing granules toward the contact zone
induced EAE in perforin knockout mice, oligodendrocyte (Wei et al., 1998); and calreticulin blocks perforin lytic
pathology was not discussed. activity by stabilizing target cell membranes (Fraser et al.,
Ultrastructurally, injured oligodendrocytes in MS le- 2000). Thus, the development of strategies to block per-
sions appear swollen, with vacuolated cytoplasm and wid- forin activity in vivo holds promise for rescuing oligoden-
ened, degenerating or ruptured membranes (Field and drocyte targets during acute episodes of inflammatory de-
Raine, 1964; Perier and Gregoire, 1965; Prineas, 1985;
` ` myelination.
Rodriguez et al., 1993; Rodriguez and Scheithauer, 1994; The observation that astrocytes were more resistant
Raine, 1997b; Raine et al., 1999). Previous studies have than oligodendrocytes to perforin was not surprising con-
shown that myelin in acute lesions is transformed into sidering their general resilience to disease. We and others
vesicular networks (Kirk, 1979; Lee et al., 1990; Rodri- have shown the expression of perforin within the cyto-
guez and Scheithauer, 1994; Genain et al., 1999; Raine et plasm of astrocytes in vivo and in culture (Gasque et al.,
al., 1999). In this study, some images of oligodendrocytes 1998; Zeine et al., 1999). Astrocytes are the only cells
and their membranes undergoing lysis by perforin-induced known to harbor free cytosolic perforin, and they must
membrane fragmentation were reminiscent of changes therefore have some protective mechanism to guard their
described in acute MS lesions (viz. Suzuki et al., 1969; Fig. own membranes against perforin lysis. Furthermore, as-
11, loc.cit.; Rodriguez and Scheithauer, 1994; Fig. 4A, trocytes may be a source of perforin in the CNS and may
loc.cit.; Raine, 1997b; Fig. 5, loc. cit.; Prineas and Mc- play an effector or regulatory role during inflammation.
Donald, 1997; Fig. 13.35, loc. cit.; Raine et al., 1999; Figs. Thus, we have shown that oligodendrocytes cultured
6 – 8, loc. cit.). from the CNS of SJL mice are selectively vulnerable to
The search for molecules that readily lyse oligoden- cytolytic damage by perforin, and that early-stage cell lysis
drocytes has been difficult. Perforin-based cytotoxicity is involves the formation of fenestrae in the cell membranes,
most akin to complement toxicity. Although the C9 com- probably as a result of the coalescence of numerous sub-
ponent of complement is deposited in MS and EAE le- microscopic perforin-induced pores and the influx of wa-
sions, some studies have shown that human oligodendro- ter into the cell. Subsequently, cells either disintegrated
cytes are not sensitive to complement damage in culture into cytoplasmic droplets, developed large cytoplasmic
(Zajicek et al., 1995). Combining antibodies with com- vacuoles, formed disrupted membranous networks or be-
plement results in a reduction in the survival of oligoden- came totally devoid of cell membrane - all these changes
drocytes in culture via a toxicity mechanism that can be primary to nuclear disintegration. As stated above, many of
inhibited by the addition of normal immunoglobulins these morphologic patterns have been described in MS
(Scolding et al., 1989; Scolding et al., 1990; Stangel et al., and EAE previously and are in support of oligodendrocyte
2000). It is most unlikely, however, that immunoglobulin damage in MS being effected by a non-apoptotic process.
would be protective in the case of perforin-mediated lysis. In ongoing studies on tissue from EAE and MS (Zeine et
We therefore reaffirm a unique role for perforin in irre- al., in preparation), we have documented the upregulation
versible oligodendrocyte injury relevant to the pathogen- of perforin by western blotting during acute exacerbations
esis of MS and extend previous findings by providing and downregulation during remission. Perforin was
present within infiltrating cytotoxic cells and astrocytes by
detailed timed images from oligodendrocytes undergoing immunohistochemistry, and the extent of remyelination
osmotic lysis. correlated with the absence of perforin. Thus, in the series
With regard to previous works on perforin-mediated of molecular events leading to the formation of the MS
lysis, our results are in agreement with the findings of lesion, it seems that a role for perforin may very well be
Jones et al. (1991) showing that “not all PI-positive cells found at the acute end of the spectrum.
proceeded to lysis.” Thus, the possibility of sub-lethal
attack by perforin on individual cells remains open and ACKNOWLEDGMENTS
raises interest in the mechanism of resistance to perforin We are indebted to Michael Cammer for expert
( Jiang et al., 1990; Ojcius et al., 1990, 1991b). Of ther- technical assistance with digital imaging at the Analytical
apeutic relevance would be the conferring of resistance to Imaging Facility of the Albert Einstein College of Medi-
selected target cells, e.g., oligodendrocytes, in EAE and cine; to Dr. Hin Hark Gan of New York University and
MS. Such a prospect awaits the isolation of ‘L-protectin,’ Howard Hughes Medical Institute for discussion; to Dr.
a membrane-bound glycosylated molecule that interferes Jorge Larocca of the Albert Einstein College of Medicine,
with perforin function in resistant target cell membranes Department of Neurology, for advice; and to Patricia
(Liu et al., 1995b). Currently, some experience has been Cobban-Bond for administrative assistance. This research
gained by blocking perforin-mediated lysis via four classes was supported in part by grants from the National Multiple
of chemical reagents in vitro. Calcium chelators inhibit Sclerosis Society of the USA (NMSS RG 1001-J-10
exocytosis and perforin binding to membranes (Liu et al., [CSR], NMSS RG 2971 [WC] and NMSS RG 2843
1995b); H -ATPase inhibitors raise the pH within cyto- [EB]), the American Heart Association (CCL), and by
toxic granules leading to accelerated degradation of per- HHS Grants NS 08952 (CSR), NS 11920 (CSR), and NS
forin (Kataoka et al., 1996; Zeine et al., 1998); MAP- 19943 (EB).

11. 390 Zeine et al.
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