Critical points of tumor necrosis factor action in central nervous system autoimmune inflammation defined by gene targeting.

Abstract

Tumor necrosis factor (TNF)-dependent sites of action in the generation of autoimmune inflammation have been defined by targeted disruption of TNF in the C57BL/6 mouse strain. C57BL/6 mice are susceptible to an inflammatory, demyelinating form of experimental autoimmune encephalomyelitis (EAE) induced by the 35-55 peptide of myelin oligodendrocyte glycoprotein. Direct targeting of a strain in which EAE was inducible was necessary, as the location of the TNF gene renders segregation of the mutated allele from the original major histocompatibility complex by backcrossing virtually impossible. In this way a single gene effect was studied. We show here that TNF is obligatory for normal initiation of the neurological deficit, as demonstrated by a significant (6 d) delay in disease in its absence relative to wild-type (WT) mice. During this delay, comparable numbers of leukocytes were isolated from the perfused central nervous system (CNS) of WT and TNF-/- mice. However, in the TNF-/- mice, immunohistological analysis of CNS tissue indicated that leukocytes failed to form the typical mature perivascular cuffs observed in WT mice at this same time point. Severe EAE, including paralysis and widespread CNS perivascular inflammation, eventually developed without TNF. TNF-/- and WT mice recovered from the acute illness at the same time, such that the overall disease course in TNF-/- mice was only 60% of the course in control mice. Primary demyelination occurred in both WT and TNF-/- mice, although it was of variable magnitude. These results are consistent with the TNF dependence of processes controlling initial leukocyte movement within the CNS. Nevertheless, potent alternative mechanisms exist to mediate all other phases of EAE.

Full text

Page 1

1585

J. Exp. Med.
Volume 186, Number 9, November 3, 1997 1585–1590
http://www.jem.org





The R ockefeller University Press • 0022-1007/97/11/1585/06 $2.00

Brief Definitive Report

Critical Points of Tumor Necrosis Factor Action in Central
Nervous System Autoimmune Inflammation Defined by
Gene Targeting
By Heinrich Körner,
*
D. Sean R iminton,
Frances A. Lemckert,
*
John D. Pollard, and Jonathon D. Sedgwick




*

Deborah H. Strickland,

*
*





‡



From the
Sydney, New South Wales , 2050 Australia; and the
University of Sydney, Sydney, New South Wales , Australia

*

Centenary Institute of Cancer Medicine and Cell Biology, Ro yal Prince Alfred Hospital,
‡
Departm ent of Medicine (Neurology),



Summary
Tumor necrosis factor (TNF)–dependent sites of action in the generation of autoimmune in-
flammation have been defined by targeted disruption of TNF in the C57BL/6 mouse strain.
C57BL/6 mice are susceptible to an inflammatory, demyelinating form of experimental au-
toimmune encephalomyelitis (EAE) induced by the 35–55 peptide of myelin oligodendrocyte
glycoprotein. Direct targeting of a strain in which EAE was inducible was necessary, as the lo-
cation of the TNF gene renders segregation of the mutated allele from the original major histo-
compatibility complex by backcrossing virtually impossible. In this way a single gene effect was
studied. We show here that TNF is obligatory for normal initiation of the neurological deficit,
as demonstrated by a significant (6 d) delay in disease in its absence relative to wild-type (WT)
mice. During this delay, comparable numbers of leukocytes were isolated from the perfused
central nervous system (CNS) of WT and TNF
munohistological analysis of CNS tissue indicated that leukocytes failed to form the typical ma-
ture perivascular cuffs observed in WT mice at this same time point. Severe EAE, including pa-
ralysis and widespread CNS perivascular inflammation, eventually developed without TNF.
TNF
and WT mice recovered from the acute illness at the same time, such that the overall
disease course in TNF
mice was only 60% of the course in control mice. Primary demyeli-
nation occurred in both WT and TNF
mice, although it was of variable magnitude. These
results are consistent with the TNF dependence of processes controlling initial leukocyte
movement within the CNS. Nevertheless, potent alternative mechanisms exist to mediate all
other phases of EAE.


?

/

?

mice. However, in the TNF

?

/

?

mice, im-

?

/

?


?

/

?


?

/

?


S
see reference 1). Experimental autoimmune encephalomy-
elitis (EAE), a central nervous system (CNS) autoimmune
inflammatory disease, is particularly well studied in this
context. EAE follows the recognition of myelin antigen in
the CNS by specific autoreactive TH1 CD4
This recognition leads to T cell and macrophage infiltration
of the CNS; cytokine secretion, including TNF, lympho-
toxin (LT)-
?
, and IFN-
?
(4); loss of blood brain barrier in-
tegrity; and, in some cases, antigen-specific tissue damage in
the form of demyelination (2).
Inhibitors of TNF consistently prevent or attenuate the
clinical course of EAE (5–8). The mechanism of inhibition
remains undefined, as TNF has the potential to contribute

tudies in vivo point to the importance of TNF in the
pathogenesis of autoimmune inflammation (for review

?

T cells (2, 3).




to CNS injury at many levels, including via effects on cell
adhesion (3), by macrophage activation (9), and by direct
cytolysis of oligodendrocytes, the myelinating cell of the
CNS (10). The complex interactions between TNF, its ho-
mologue LT-
?
, and their receptors (11), have prevented
the precise definition of the critical points of action for
TNF in any stage of CNS autoimmune inflammation, or
indeed in any inflammatory process.
Mice lacking TNF and/or LT-
this problem. In a recent report, 129 strain mice lacking
both TNF and LT-
?
, and interbred with C57BL/6-strain
mice or crossed to the EAE-susceptible SJL mouse strain
(12), were shown to be susceptible to a CNS inflammatory
disease after immunization with a number of different mye-
lin antigens. Some autoantigen combinations induced a
rapidly lethal atypical form of disease. The authors con-
cluded that neither TNF nor LT-
induction. Some properties of the mice used in Frei et al.



?

can now be applied to



?

was required for EAE

H. Körner, D.S. R iminton, and D.H. Strickland contributed equally to
this study.

Page 2

1586

Experimental Autoimmune Encephalomyelitis in TNF Gene Targeted Mice

(12) study render an interpretation of the experimental
outcome difficult. First, these mice lack all lymph nodes as
a result of the absence of LT-
?
gene loci reside within the MHC, a region strongly linked
to autoimmune disease susceptibility (14) and comprising
not only genes encoding for MHC class I and II and the
TNF and LT molecules, but also complement components
and molecules involved in antigen processing. Thus, at-
tempts to backcross the TNF/LT mutations onto another
strain will create individually variable congenic segments,
the majority of which will maintain the MHC locus profile
linked to the mutated allele. For this reason, the use of a di-
rectly targeted, disease-susceptible mouse strain represents a
major theoretical advantage.
To explore in detail the role of TNF in autoimmune in-
flammation in the CNS, TNF gene–deleted C57BL/6
mice normally susceptible to EAE induced by the 35–55
peptide of myelin oligodendrocyte glycoprotein (MOG)
were generated (15). Analysis of these mice points to a par-
ticular dependence on TNF for the early inflammatory
phase of EAE, and specifically to the processes involved in
the formation of perivascular cuffs within the CNS. The
existence of potent alternative pathways of CNS inflamma-
tion and demyelination is demonstrated.


(13). Second, the TNF/LT

Materials and Methods
Ge ne ratio n o f C57BL/6-strain TNF
use of the BL/6 III C57BL/6 embryonic stem cells, and genera-
tion and characterization of C57BL/6-strain TNF
been previously described (15). TNF
mice were maintained as a homozygous colony in the Centenary
Institute animal facility (Sydney, Australia). Control wild-type
(WT) C57BL/6J-strain mice were bred in-house or obtained
from CULAS Ltd. (Sydney, Australia).
Induction of EAE.EAE was actively induced in adult (8–12-
wk-old) TNF
and WT C57BL/6 mice by subcutaneous tail-
base injection of 50
?
g MOG peptide (35-MEVGWYR SPFSR –
VVHLYR NGK-55; reference 16) in CFA containing 1 mg of
heat-inactivated H37R A
Mycobacterium tuberculosis
ratories, Inc., Detroit, MI). 200 ng of pertussis toxin (LIST Biol.
Labs., Inc., Campbell, CA), was injected intravenously on days 0
and 2. Neurological deficits were quantified according to an arbi-
trary clinical scale: 0, normal; 1, flaccid tail; 2, hind limb weakness
or abnormal gait; 3, severe hind limb weakness, with loss of abil-
ity to right from supine; 4, hind quarter paralysis; 5, forelimb
weakness, moribund; 6, death. All animal procedures were ap-
proved by the Animal Care and Ethics Committee of the Univer-
sity of Sydney (Sydney, Australia).
Asse ssm e nt o f T Ce ll Ac tivatio n and Antibo dy Pro duc tio n.
WT and TNF
mice were immunized (day 0) with MOG
peptide/CFA as above, but without the pertussis toxin injection.
For assessment of T cell proliferation and IFN-
draining lymph node cells were harvested on days 9 and 5, re-
spectively, after MOG peptide/CFA. Proliferation was assessed
using 2
?
10 viable cells/well in the presence of MOG peptide
(10
?
g/ml final concentration) or control antigens. T cell prolif-
erative responses were quantified at 96 h after a 14-h pulse with
[
H]thymidine. Supernatants for IFN-
erated by culture of cells at 0.5–1


?

/

?

Mic e .

Construct design,

?

/

?

mice have

?

/

?

specific pathogen free


?

/

?



(DIFCO Labo-

Groups of

?

/

?


?

production,



5




3


?

determinations were gen-
10 /ml with MOG peptide

?


7

(20
sandwich ELISA. For examination of humoral responses, groups
of MOG peptide/CFA–challenged mice were secondarily immu-
nized with MOG/IFA on day 9 and were bled for serum collec-
tion on day 14. MOG peptide–specific IgG responses were evalu-
ated by ELISA using plates coated with avidin and biotinylated
peptide and by detection of antibody with alkaline-phosphatase–
conjugated sheep anti–mouse IgG (Sigma Chemical Co., St.
Louis, MO).
Flow Cytom etry.Flow cytometric analysis of CNS-associated
leukocytes was performed on collagenase-digested tissue after he-
parin–saline perfusion of animals. Isolation and purification steps
were adapted from a previously described method (17). Antibod-
ies used in flow cytometry were FITC-conjugated hamster anti–
murine
?
/
?
-TCR (H57-597), PE-conjugated rat anti-CD45
(30F11.1; PharMingen, San Diego, CA) and relevant isotype
control reagents (to set compensation levels and analysis gates).
Flow cytometry was performed on a FACStar
analysis software (Becton Dickinson, San Jose, CA).
Im m uno histo lo gy and Ne uro patho lo gy.
ous regions of the CNS of nonperfused mice were embedded in
Tissue Tek OCT compound (Sakura Finetek, Torrance, CA) and
6-
?
m serial cryostat sections were prepared. Sections were
stained via the immunoperoxidase technique using HR P-conju-
gated rabbit anti–rat Ig (DAKO, Carpenteria, CA), and counter-
stained with hematoxylin. R at mAb reactive with mouse cell–
surface antigens were: GK1.5 (CD4); M1.70 (CD11b/Mac-1);
YBM142.2.2 (CD45, supplied by Dr. S. Cobbold, Oxford Uni-
versity, Oxford, UK); 429 (vascular cellular adhesion molecule
[VCAM]–1; PharMingen); and isotype control reagents R -35-95
(rat IgG2a, PharMingen) and YKIX 16.13 (rat IgG2b, supplied by
Dr. S. Cobbold). Neuropathological assessment for demyelina-
tion was performed on CNS tissue obtained from animals per-
fused with warm PBS, followed by 4% paraformaldehyde and
2.5% glutaraldehyde in PBS. Tissue was post-fixed in Dalton’s
chrome osmium solution and dehydrated in graded concentra-
tions of ethanol and acetone. Transverse sections of spinal cord
(0.25
?
m) were stained with toluidine blue. All sections were ex-
amined and photographed using standard bright-field optics.

?

g/ml), or no antigen, for 60 h. IFN-

?

was quantitated by






PlusTM

using CellQuest

Tissue specimens from vari-





Results
TNF
m e diate d and Hum oral Im m une Re sponse s.
cell-mediated and humoral immune responses is considered
important for the full manifestation of EAE, particularly in
relation to demyelination, in which a clear role for anti-
body to myelin components, including MOG (18), has
been demonstrated. To determine whether mice lacking
TNF were deficient in their immune responsiveness to
MOG peptide, mice were immunized and analyzed for
MOG peptide–specific T cell proliferation, IFN-
tion, and IgG production, as detailed in Materials and
Methods. No significant differences in any of these param-
eters were observed between TNF
not shown).
Onset of EAE in TNF C57BL/6 Mice Is Substantially
De laye d.
Upon challenge with the encephalitogenic MOG
35–55 peptide, WT mice exhibited signs of clinical disease
from day 10 (Fig. 1
A ), manifest as symmetrical ascending
motor deficits. Disease severity then increased rapidly to


?

/

?

Mice Exhibit Norm al MOG Peptide–specific Cell-

A combination of

?

produc-

?

/

?

and WT mice (data


?

/

?

Page 3

1587

Körner et al.Brief Definitive R eport

reach a peak at day 20, followed by gradual recovery over
the next 20 d to a relatively mild deficit that persisted for
the life of the mouse (data not shown). Although TNF
mice did develop EAE after MOG peptide challenge, the
onset of clinical disease was substantially and reproducibly
delayed (Fig. 1
A). The rate of progression once disease be-
came established and the eventual peak severity of disease
in TNF
mice were comparable to WT mice. Both WT
and TNF
mice recovered simultaneously despite the
initial delay in disease onset in mice lacking TNF. Thus,
the overall disease course was reduced to 60% of the course
in control mice in the absence of TNF (Fig. 1
mice, mice lacking TNF maintained a mild level of disabil-
ity for an extended time after resolution of the initial peak
clinical deficit.
The clear conclusion that can be drawn from these re-
sults is that TNF is required for the normal initiation of the
neurological deficit in EAE, but is not a necessary factor for
disease progression or for recovery from the acute clinical
illness.
Leukocytes Accum ulate in the CNS in the Absence of TNF
b ut Fo rm atio n o f Pe rivasc ular Cuffs is De laye d.
peared to be an essential participant in the early events lead-
ing to the development of EAE, a series of experiments was
performed to establish how the absence of TNF affected
the CNS inflammatory process, using the time point at which
the discrepancy between the clinical scores of TNF
WT mice was maximal (Fig. 1
bar
). Immunohistological analysis of mice at this time re-
vealed small but frequently detectable accumulations of
leukocytes (CD45
) in TNF
els
), but a marked reduction in discrete perivascular cuffs of
leukocytes relative to WT. On the other hand, no detect-
able accumulations of leukocytes (CD45
the CNS of normal nonimmunized mice (data not shown).
Clear evidence for the existence of immunological activa-
tion within the CNS was revealed by staining for the adhe-

?

/

?


?

/

?


?

/

?

B

). Like WT

Since TNF ap-

?

/

?

and

A

, days 13–15,

horizontal


?


?

/

?

mice (Fig. 2

A

,

upper pan-


?

) were found in

sion molecule VCAM-1 (Fig. 2
substantially upregulated on vascular endothelium through-
out the CNS of TNF
mice.
When this observation was extended to an investigation
of the immune response throughout the entire CNS, the ki-
netics of total inflammatory cell accumulation in the CNS
were found to be remarkably similar in WT and TNF
mice (Fig. 2, B and C ). Cells isolated from PBS-perfused
CNS tissue of WT and TNF
intervals after immunization (Fig. 2
flow cytometry using criteria previously developed (17).
Flow-cytometric analysis at day 15 (Fig. 2
presence of equivalent numbers of
1 , CD45
?
/
?
TCR ), non–T inflammatory cells (popula-
tion
2, CD45
?
/
?
TCR ), the majority of which were
macrophages (data not shown), and microglia (population
CD45low?/?TCR?). R elative to other populations isolated
(T cells, and microglia), there were more inflammatory
macrophages in the CNS of WT than TNF?/? mice (Fig. 2
C, population 2). A small number of T cells and macro-
phages were isolated from the CNS of nonimmunized WT
mice (Fig. 2 C, upper panel), as expected, although resident
microglial cells (population 3, CD45low?/?TCR?) were
readily detectable (17).
These studies support the view that MOG peptide–reac-
tive T cells are generated normally in TNF?/? mice, mi-
grating to and distributing throughout the CNS vasculature
and leading to endothelial activation, as evidenced by
VCAM-1 upregulation—all outcomes that are not depen-
dent on TNF. However, in the absence of TNF, formation
of discrete mature perivascular cuffs of inflammatory cells is
significantly retarded.
Peak Disease Inflam m ation and Dem yelination in WT and
TNF?/? Mic e . At the peak of the disease there was extensive
inflammation in the CNS of both WT and TNF?/? mice
(Fig. 3 A), characterized by perivascular and submeningeal
infiltrates of CD45? cells and microglial activation (Fig. 3
A, arrows). Serial section staining revealed a predominance
of macrophages and CD4? T cells (data not shown). A
general feature of the immunopathology in TNF?/? mice
was a more limited expansion of cells from the perivascular
cuff into the parenchyma. Primary demyelination, involv-
ing loss of myelin from otherwise viable axons, is a hall-
mark of the human disease multiple sclerosis, for which
EAE serves as an experimental model (2). Primary demyeli-
nation was a relatively late event, detected in WT and
TNF?/? mice from ?30 d after MOG peptide/CFA im-
munization and most clearly apparent after the bulk of in-
flammatory cells had dissipated (Fig. 3 B, day 35). Naked
axons of otherwise normal appearance were seen, consis-
tent with the specificity of the immune insult (Fig. 3 B, ar-
rows). A degree of variability in magnitude of demyelina-
tion was observed in TNF?/? mice with from one of five
mice examined exhibiting few if any demyelinated axons
(data not shown), to the one mouse illustrated here (Fig. 3
B, day 40) with a level of demyelination indistinguishable
from WT mice. A more extensive comparison of WT and
TNF?/? mice is currently underway to determine whether

A

,

lower panels

), which was

?

/

?


?

/

?


?

/

?

mice were quantified at
B) and phenotyped by

C

) revealed the
T cells (population

?

/

?


hi





?


hi





?

3,
Figure 1.
(A) Mean clinical EAE scores (? SEM) of WT mice (?, n ? 6) and
TNF?/? mice (?, n ? 8) after immunization with MOG/CFA. The
horizontal bar at days 13–15 indicates a time point of closer examination,
referred to in Fig. 2 and in the text. (B) As a measure of the relative sus-
ceptibility of WT and TNF?/? mice to neurological deficits induced by
immunization with MOG, areas under the curves in A were determined.
R esults are representative of four separate time course studies.
Natural history of EAE in WT and TNF?/? C57BL/6 mice.

Page 4

1588
Experimental Autoimmune Encephalomyelitis in TNF Gene Targeted Mice
the levels of demyelination in mice lacking TNF are re-
duced overall. Nevertheless, TNF is not an obligatory me-
diator in the demyelinating process.
Discussion
From this study of the course of EAE, clear conclusions
can be derived regarding the critical roles for TNF. Of the
Figure 2.
WT and TNF?/? mice. (A) Leukocyte inflammation (CD45?) and
VCAM-1 expression at day 15 (Fig. 1 A, horizontal bar). Tissue sections
were derived from brain stem and cerebellum and are representative of
tissues throughout the CNS of several WT and TNF ?/? mice at this time
point. (Inset) VCAM-1 expression of unimmunized C57BL/6J-strain CNS.
Bar ? 60 ?m. (B) Total cell recoveries from the perfused CNS of normal
and immunized mice over the course of disease. R eplicates concentrated
on the early disease phase when TNF?/? mice were not showing signs of
clinical disease. For simplicity, individual mice from days 13–15 are all
shown at the day 15 time point. Each data point represents a single
mouse. ?, WT unimmunized. ?, WT MOG immunized. ?, TNF?/?
MOG immunized. (C) T cell accumulation in the CNS of normal and
MOG-immunized WT and TNF?/? mice at day 15. Pooled cells ob-
tained from two mice in each case were stained and analyzed by flow cy-
tometry. Percentage of total cells in each preparation as defined by popu-
lations 1, 2, and 3 (see text) are shown in the inset. CD45? TCR? cells
are undefined but would represent nonhematopoietically derived cells in-
cluding neurons, endothelial cells, astrocytes, and oligodendrocytes.
Characterization of the early CNS inflammatory infiltrate in
three stages of the inflammatory process: initiation, tissue
injury, and recovery, TNF appears to play a unique role
only in the first. Unexpectedly, the altered characteristics of
the inflammatory disease process in TNF?/? mice are con-
sistent with the inefficient movement of cells within the
CNS, while normal upregulation of VCAM-1 and the
identification of recruited cells at the time of disease delay
suggest there are no major deficiencies in vascular adhesion
in the absence of TNF. While the precise location of leu-
kocytes within the CNS of TNF?/? mice is unknown dur-
ing the delayed preclinical phase of EAE (Fig. 2 A, upper
right panel), the data nevertheless support the view that ex-
travasation of leukocytes, localization to the perivascular
space throughout the CNS, and antigen recognition by
MOG-reactive T cells at that site (19, 20) have occurred
normally. This conclusion is based on the observation of
VCAM-1 upregulation as well as accumulation of leuko-
cytes other than T cells in TNF?/? mice during the pre-
clinical phase (Fig. 2, A and C), events almost certainly ne-
cessitating CNS antigen recognition by infiltrating T cells.
A variety of TNF-dependent processes may underlie this
unusual phenotype. However, a likely explanation is that
the delayed movement of leukocytes within the tissue to
form perivascular cuffs reflects a general inability of leuko-
cytes to move correctly in the absence of TNF-inducible
chemotactic factors, notably chemokines (21). Consistent
with this concept is the defect of cell movement manifest
in the altered microarchitectural T and B cell arrangements
in lymphoid tissues of TNF?/? mice (15, 22). The poten-
tial role of secondary mediators in this process is high-
lighted by a recent description of mice in which deletion of
the gene encoding a putative chemokine receptor, blr1, re-
sulted in splenic B cell architecture not unlike that seen in
TNF?/? mice (23).
An important outcome of these studies is the demonstra-
tion of a potent, but TNF-independent, mechanism pro-
ducing tissue injury in EAE. The results of studies of col-
lagen arthritis in mice administered TNFR -IgG fusion
protein or lacking TNFR -1 (24) are strikingly similar to
those reported here in TNF?/? mice with EAE. In particu-
lar, in TNFR 1-deficient mice, arthritis was of a reduced
overall severity, but, once established in an individual joint,
progressed in a manner similar to WT mice. Therefore, the
processes that lead to tissue damage in EAE and collagen
arthritis, once it is initiated, and the eventual peak severity
of the diseases, are not due to the actions of TNF alone.
Soluble LT-? homotrimer, a predominantly T cell cy-
tokine with some functional similarities to TNF and bind-
ing the same receptors as TNF (11), remains a possible me-
diator of demyelination as well as of the acute EAE phase.
A full analysis of the role of LT-? and -? in EAE in gene-
targeted C57BL/6 mice prepared in parallel to the TNF?/?
mice is ongoing. These studies are cumbersome, requiring
the use of irradiation bone marrow chimeras (13) to gener-
ate mice which carry lymphoid tissues but are deficient in
LT-?. Lymph nodes were lacking in TNF/LT-? double-
deficient mice shown recently to be susceptible to autoim-
mune CNS inflammation (12). Likely alterations to normal

Page 5

1589
Körner et al.Brief Definitive R eport
immunological regulation, and the absence of a switched
humoral response in these mice (25) have the potential to
significantly influence the disease outcome after immuniza-
tion. As the experiments here have shown, TNF appears to
play a critical role in the early inflammatory process in
EAE. This same TNF dependency was not revealed in
mice lacking both TNF and LT-? (12). Thus, it is difficult
to say with certainty at this stage that LT-? plays no role in
the EAE disease process in immunologically intact mice.
The requirement for TNF in the normal initiation of au-
toimmune inflammation, as demonstrated in this study,
may help to explain the therapeutic effectiveness of TNF
blocking agents when administered before rather than after
disease onset in several disease models (7, 24, 26, 27). Con-
versely, there is evidence from other models (notably in the
rat) that TNF may act as a downstream effector of tissue in-
jury (8, 28, 29), while in this study, once the disease was
established, it progressed normally without TNF. R econ-
ciling these apparent discrepancies must await a more de-
tailed understanding of the role of TNF in cell movement
within tissues, but also an appreciation of the role that TNF
may play in induction of alternative effector pathways.
Figure 3.
mation and demyelination. (A)
Leukocyte infiltration and forma-
tion of perivascular cuffs. Sections
were derived from spinal cord of
animals harvested at day 19 (WT)
and day 23 (TNF?/?) and stained
for CD45. CD45? filamentous
processes within the CNS paren-
chyma (arrows) indicate activated
microglia. Comparable infiltra-
tion was found at all levels of the
spinal cord, brain stem, and cere-
bellum. Bar ? 60 ?m. (B) Pri-
mary demyelination in WT and
TNF?/? mice. Sections were de-
rived from spinal cord of mice
harvested at day 35 (WT) and
day 40 (TNF?/?). Tissues from
WT and TNF?/? mice show a
region of comparable perivenous
(v) demyelination and gliosis be-
neath the meningeal surface (m ).
Arrows indicate naked (demyeli-
nated) axons. In these mice, a
similar histological picture was
obtained at all levels of the spinal
cord. Bar ? 12 ?m.
Peak disease inflam-
These studies were supported by grants from the National Health and Medical R esearch Council
(NHMR C) and the National Multiple Sclerosis Society of Australia. D.H. Strickland is supported by an
Elizabeth Albiez Fellowship from the National Multiple Sclerosis Society of Australia, D.S. R iminton by an
NHMR C postgraduate Scholarship, and J.D. Sedgwick by a Wellcome Trust Senior R esearch Fellowship in
Australia (1992–1996) and an NHMR C Fellowship.
Thanks are extended to Professor Antony Basten for critical analysis of the manuscript, to Dr. Philip
Hodgkin for providing reagents for IFN-? analysis, Mr. Jim Bonner for technical assistance, and Ms. Karen
Knight and Mr. James Crozer for outstanding Animal Facility organization and animal husbandry.
Address correspondence to Dr. Jonathon D. Sedgwick, Centenary Institute of Cancer Medicine and Cell Bi-
ology, Bldg 93, R oyal Prince Alfred Hospital, Missenden R d., Camperdown, Sydney NSW 2050, Australia.
Phone: 61-2-9565-6116; FAX : 61-2-9565-6103; E-mail: j.sedgwick@centenary.usyd.edu.au
Received for publication 17 June 1997 and in revised form 18 August 1997.
References
1. Körner, H., and J.D. Sedgwick. 1996. Tumour necrosis fac-
tor and lymphotoxin: molecular aspects and role in tissue-
specific autoimmunity. Im m unol. Cell Biol. 74:465–472.
2. R aine, C.S. 1984. Biology of disease. Analysis of autoim-
mune demyelination: its impact upon multiple sclerosis. Lab.
Invest. 50:608–635.
3. Baron, J.L., J.A. Madri, N.H. R uddle, G. Hashim, and C.A.
Janeway, Jr. 1993. Surface expression of ?4 integrin by CD4

Page 6

1590
Experimental Autoimmune Encephalomyelitis in TNF Gene Targeted Mice
T cells is required for their entry into brain parenchyma. J.
Exp. Med. 177:57–68.
4. Olsson, T. 1995. Critical influences of the cytokine orches-
tration on the outcome of myelin antigen–specific T-cell au-
toimmunity in experimental autoimmune encephalomyelitis
and multiple sclerosis. Im m unol. Rev. 144:245–268.
5. R uddle, N.H., C.M. Bergman, K.M. McGrath, E.G., Lin-
genheld, M.L. Grunnet, S.J. Padula, and R .B. Clark. 1990.
An antibody to lymphotoxin and tumor necrosis factor pre-
vents transfer of experimental allergic encephalomyelitis. J.
Exp. Med. 172:1193–1200.
6. Selmaj, K., C.S. R aine, and A.H. Cross. 1991. Anti–tumor
necrosis factor therapy abrogates autoimmune demyelination.
Ann. Neurol. 30:694–700.
7. Baker, D., D. Butler, B.J. Scallon, J.K. O’Neill, J.L. Turk,
and M. Feldmann. 1994. Control of established experimental
allergic encephalomyelitis by inhibition of tumor necrosis
factor (TNF) activity within the central nervous system using
monoclonal antibodies and TNF receptor-immunoglobulin
fusion proteins. Eur. J. Im m unol. 24:2040–2048.
8. Körner, H., A.L. Goodsall, F.A. Lemckert, B.J. Scallon, J. Ghra-
yeb, A.L. Ford, and J.D. Sedgwick. 1995. Unimpaired au-
toreactive T-cell traffic within the central nervous system
during tumor necrosis factor receptor–mediated inhibition of
experimental autoimmune encephalomyelitis. Pro c . Natl. Ac ad.
Sci. USA. 92:11066–11070.
9. Philip, R ., and L.B. Epstein. 1986. Tumour necrosis factor as
immunomodulator and mediator of monocyte cytotoxicity
induced by itself, ?-interferon and interleukin-1. Nature
(Lond.). 323:86–89.
10. Selmaj, K.W., and C.S. R aine. 1988. Tumor necrosis factor
mediates myelin and oligodendrocyte damage in vitro. Ann.
Neurol. 23:339–346.
11. Bazzoni, F., and B. Beutler. 1996. The tumor necrosis factor
ligand and receptor families. N. Engl. J. Med. 334:1717–1725.
12. Frei, K., H.P. Eugster, M. Bopst, C.S. Constantinescu, E.
Lavi, and A. Fontana. 1997. Tumor necrosis factor ? and
lymphotoxin ? are not required for induction of acute exper-
imental autoimmune encephalomyelitis. J. Exp. Med. 185:
2177–2182.
13. De Togni, P., J. Goellner, N.H. R uddle, P.R . Streeter, A.
Fick, S. Mariathasan, S.C. Smith, R . Carlson, L.P. Shornick,
J. Strauss-Schoenberger et al. 1994. Abnormal development
of peripheral lymphoid organs in mice deficient in lympho-
toxin. Science (Wash. DC). 264:703–707.
14. Theofilopoulos, A.N. 1995. The basis of autoimmunity: part
II. Genetic predisposition. Im m unol. Today. 16:150–159.
15. Körner, H., M. Cook, D.S. R iminton, F.A. Lemckert, R .M.
Hoek, B. Ledermann, F. Köntgen, B. Fazekas de St. Groth,
and J.D. Sedgwick. 1997. Distinct roles for lymphotoxin-?
and tumor necrosis factor in organogenesis and spatial organi-
sation of lymphoid tissue. Eur. J. Im m unol. In press.
16. Gardinier, M.V., P. Amiguet, C. Linington, and J.M. Mat-
thieu. 1992. Myelin/oligodendrocyte glycoprotein is a unique
member of the immunoglobulin superfamily. J. Neurosci. Res.
33:177–187.
17. Ford, A.L., A.L. Goodsall, W.F. Hickey, and J.D. Sedgwick.
1995. Normal adult ramified microglia separated from other
central nervous system macrophages by flow cytometric sort-
ing. Phenotypic differences defined and direct ex vivo anti-
gen presentation to myelin basic protein-reactive CD4? T
cells compared. J. Im m unol. 154:4309–4321.
18. Linington, C., M. Bradl, H. Lassmann, C. Brunner, and K.
Vass. 1988. Augmentation of demyelination in rat acute aller-
gic encephalomyelitis by circulating mouse monoclonal anti-
bodies directed against a myelin/oligodendrocyte glycopro-
tein. Am . J. Pathol. 130:443–454.
19. Hickey, W.F., and H. Kimura. 1988. Perivascular microglial
cells of the CNS are bone marrow–derived and present anti-
gen in vivo. Science (Wash. DC). 239:290–292.
20. Ford, A.L., E. Foulcher, F.A. Lemckert, and J.D. Sedgwick.
1996. Microglia induce CD4 T lymphocyte final effector
function and death. J. Exp. Med. 184:1737–1746.
21. Butcher, E.C., and L.J. Picker. 1996. Lymphocyte homing
and homeostasis. Science (Wash. DC). 272:60–66.
22. Pasparakis, M., L. Alexopoulou, V. Episkopou, and G. Kollias.
1996. Immune and inflammatory responses in TNF-?-defi-
cient mice: a critical requirement for TNF? in the formation
of primary B cell follicles, follicular dendritic cell networks
and germinal centers, and in the maturation of the humoral
immune response. J. Exp. Med. 184:1397–1411.
23. Forster, R ., A.E. Mattis, E. Kremmer, E. Wolf, G. Brem, and
M. Lipp. 1996. A putative chemokine receptor, blr1, directs
B cell migration to defined lymphoid organs and specific ana-
tomic compartments of the spleen. Cell. 87:1037–1047.
24. Mori, L., S. Iselin, G. De Libero, and W. Lesslauer. 1996. At-
tenuation of collagen-induced arthritis in 55-kDa TNF re-
ceptor 1 (TNFR1)–IgG1-treated and TNFR1-deficient mice. J.
Im m unol. 157:3178–3182.
25. Eugster, H.-P., M. Muller, U. Karrer, B.D. Car, B. Schny-
der, V.M. Eng, G. Woerly, M. Le Hir, F. di Padova, M. Au-
get et al. 1996. Multiple immune abnormalities in tumor ne-
crosis factor and lymphotoxin ? double-deficient mice. Int.
Im m unol. 8:23–36.
26. Yang, X .D., R . Tisch, S.M. Singer, Z.A. Cao, R .S. Liblau,
R .D. Schreiber, and H.O. McDevitt. 1994. Effect of tumor
necrosis factor ? on insulin-dependent diabetes mellitus in
NOD mice. I. The early development of autoimmunity and
the diabetogenic process. J. Exp. Med. 180:995–1004.
27. Körner, H., F.A. Lemckert, S. Etteldorf, B.J. Scallon, and
J.D. Sedgwick. 1997. Tumor necrosis factor blockade in ac-
tively-induced experimental autoimmune encephalomyelitis
prevents clinical disease despite activated T-cell infiltration to
the central nervous system. Eur. J. Im m unol. 27:1973–1981.
28. Dick, A.D., P.G. McMenamin, H. Körner, B.J. Scallon, J.
Ghrayeb, J.V. Forrester, and J.D. Sedgwick. 1996. Inhibition
of tumor necrosis factor–activity minimizes target organ dam-
age in experimental autoimmune uveoretinitis despite quan-
titatively normal activated T cell traffic to the retina. Eur. J.
Im m unol. 26:1018–1025.
29. Klinkert, W.E.F., K. Kojima, W. Lesslauer, W. R inner, H.
Lassmann, and H. Wekerle. 1997. TNF-? receptor fusion
protein prevents experimental auto-immune encephalomy-
elitis and demyelination in Lewis rats: an overview. J. Neu-
roim m unol. 72:163–168.