By
From the Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, Connecticut 06520-8034
The lymphotoxin (LT)/tumor necrosis factor (TNF) family has been implicated in the neurologic inflammatory diseases multiple sclerosis (MS) and experimental allergic encephalomyelitis
(EAE). To determine the role of individual family members in EAE, C57BL/6 mice, LT--deficient (LT-
/
mice), or LT-
-deficient (LT-
/
mice), and their wild-type (WT) littermates were immunized with rat myelin oligodendrocyte glycoprotein (MOG) peptide 35-55. C57BL/6 and WT mice developed chronic, sustained paralytic disease with average maximum
clinical scores of 3.5 and disease indices (a measure of day of onset and sustained disease scores)
ranging from 367 to 663 with central nervous system (CNS) inflammation and demyelination.
LT-
/
mice were primed so that their splenic lymphocytes proliferated in response to MOG
35-55 and the mice produced anti-MOG antibody. However, LT-
/
mice were quite resistant to EAE with low average clinical scores (<1), an average disease index of 61, and the negligible CNS inflammation and demyelination. WT T cells transferred EAE to LT-
/
recipients. LT-
/
mice were susceptible to EAE, though less than WT, with an average maximum
clinical score of 1.9 and disease index of 312. These data implicate T cell production of LT-
in MOG EAE and support a major role for LT-
3, a minor role for the LT-
/
complex, and by inference, no role for TNF-
.
Several studies have implicated members of the lymphotoxin(LT)1/TNF family in multiple sclerosis (MS) and
its animal model, experimental allergic encephalomyelitis
(EAE). LT- Recently, mice selectively deficient in LT- Because we wished to study EAE in LT-deficient mice,
an immunization regimen that would induce EAE in mice
of the H-2b haplotype was necessary. We did not believe it
was practical to use the more commonly studied methods
of MBP or PLP immunization of H-2s or H-2u mice because the knockout mice were developed from 129 (H-2b)
embryonic stem cells, and it was necessary to retain chromosome 17 because the LT/TNF genes map to the MHC.
Thus, if the mice were crossed to SJL, WT littermates
would of necessity differ at the H-2 locus. Recently, a
method of inducing EAE in C57BL/6 (H-2b) mice has
been described (16, 17). C57BL/6 mice injected with rat
myelin oligodendrocyte glycoprotein (MOG) peptide 35- 55 in CFA and pertussis toxin (Pt) and boosted with peptide in CFA develop a chronic sustained paralysis. These
authors reported that the disease could be transferred to naive recipients with MOG peptide-specific T cell lines.
MOG peptide immunization also induces EAE in marmosets and rats (18, 19). However, passive transfer of EAE in
these models is effected by a combination of specific T cells
and antibody, suggesting that the neurologic lesions are initiated by T cells and sustained by antibody. None of the
previous studies with MOG in mice, rats, or marmosets
have addressed the role of cytokines in this disease, with
the exception of a report by Willenborg et al. (20) in IFN- Mice.
C57BL/6 (H-2b) mice were obtained from the Jackson
Laboratory (Bar Harbor, ME) or from a breeding colony maintained at Yale University School of Medicine (New Haven, CT).
A breeding pair of LT- MOG Peptide.
MOG peptide 35-55 of rat origin was synthesized by the W.M. Keck Biotechnology Resource Center at Yale
University. The sequence was MEVGWYRSPFSRVVHLYRNGK (16, 17). In most experiments, material had been subjected
to reverse phase (C18) column HPLC and a TFA/acetonitrile gradient.
Active Induction of EAE with MOG Peptide.
Mice of 8-10 wk
of age were injected with 300 µg MOG peptide in CFA (Difco,
Detroit, MI) with 500 µg of Mycobacterium tuberculosis. The 0.2-ml emulsion was injected subcutaneously in one flank. Mice received 500 ng Pt (List Biological, Campbell, CA) in 200 µl PBS
in the tail vein immediately after the first immunization and 48 h
later and were boosted with peptide with CFA subcutaneously in
the other flank 1 wk later. The mice were observed daily for clinical signs and scored on a scale of 0-5 with gradations of 0.5 for
intermediate scores: 0, no clinical signs; 1, loss of tail tone; 2, wobbly gait; 3, hind limb paralysis; 4, hind and fore limb paralysis; 5, death. The average day of disease onset was calculated by
averaging the first day of clinical signs for each mouse in the
group. The average maximum disease score was similarly calculated by averaging the highest individual score for each mouse.
The disease index was calculated by adding all the daily average
disease scores, dividing by the average day of disease onset, and
multiplying by 100. The day on which the index was calculated is
indicated in individual experiments. For those animals which
showed no clinical signs, the onset is arbitrarily calculated as one
day after the experiment was terminated.
Histology.
Mice were perfused with 50 ml of cold PBS and
with 40 ml of cold Z-fix (formaldehyde, ionized zinc, buffer).
The brain and spinal cords were removed. Paraffin sections were
prepared and stained with hematoxylin and eosin or Luxol fast
blue counterstained with nuclear fast red by the Yale Pathology
Services. In other experiments, tissue was embedded in epon,
sectioned at 2 microns, and stained with toluidine blue.
ELISA.
Serum samples were prepared from peripheral blood
obtained by retroorbital or cardiac puncture immediately before
perfusion at days 6 and 30 after the first immunization. ELISA
was performed using purified rat MOG 35-55 peptide. The peptide was diluted to 10 µg/ml in carbonate buffer, pH 9.3, and
coated onto 96-well microtiter plates (Dynex Technologies,
Chantilly, VA). After incubation at 37°C overnight, the plates
were washed with TBS, pH 7.5, containing 0.1% Tween 20 and
blocked with 5% BSA in TBS (TBS-BSA) for 2 h at 37°C. Twofold serial dilutions of serum were made in TBS-BSA and applied
to preblocked wells for 2 h at 37°C. Appropriately diluted alkaline-phosphatase-conjugated goat anti-mouse IgG (H + L) antibody (Pierce, Rockford, IL) was added to the plates and incubated for 1 h at 37°C. The plates were washed and incubated with p-nitrophenyl phosphate substrate for 30 min before optical density was measured at 405 nm. The end-point titer was defined as the dilution at which the OD was 0.05 above background.
MOG-specific T Cell Lines.
Two mice were immunized with
purified MOG peptide (150 µg) in CFA with Mycobacterium tuberculosis subcutaneously in the tail. After 9 d, the lymph nodes were
removed and cultured in the presence of 40 µg/ml MOG peptide. T cell lines were derived and maintained as previously described (4) by feeding MOG peptide (40 µg/ml) with irradiated
spleen cells in RPMI media (GIBCO BRL, Gaithersburg, MD)
supplemented with 1 mM L-glutamine, penicillin-streptomycin, 10% FCS, fungizone, and 2-mercaptoethanol every 14 d. Cells
were fed IL-2 every 7 d. They were incubated in a humidified
37°C, 10% CO2 incubator and routinely tested for antigen specific proliferation.
Proliferative Response of T Cells to MOG Antigen.
The assays
were set up in triplicate in 96-well flat-bottomed plates in 250 µl/well with 5 × 104 T cells, 1.5 × 105 irradiated spleen cells,
and 30 µg of MOG peptide in RPMI medium supplemented as
above. [3H]thymidine, 1 µCi/well, specific activity 5 Ci/mmol
(Amersham, Arlington Heights, IL) per well, was added 24 h after
culture initiation. Plates were harvested at 48 h using a PHD harvester (Cambridge Technology, Cambridge, MA) and counted
by scintillation counter.
Passive Transfer of EAE.
WT MOG-specific T cell lines and
spleen cells from MOG immunized mice were used in passive
transfer experiments. Cells were injected intraperitoneally into
recipient mice which were irradiated (550 rads) or not and treated
or not with Pt (500 µg) as indicated in individual experiments.
For passive transfer of MOG T cell lines, cells were stimulated
with IL-2, irradiated spleen cells, and MOG peptide 4 d before
injection, and were fed IL-2 1 d before the transfer. The number
of cells injected varied from 2.5-3.8 × 107. For passive transfer of
MOG spleen cells, donor mice were immunized with 300 µg of
MOG peptide in CFA in both flanks. 14 d later, the spleens were
removed and the cells isolated and placed in 20-ml tissue culture
dishes at 5 × 106 cells/ml with 20 µg/ml of MOG peptide antigen in RPMI. 4 d later, the cells were washed with HBSS,
counted, and 6 × 107 cells were injected into recipient mice.
The protocol of active immunization, virtually identical to that of Mendel and
co-workers (16, 17) which induces a chronic EAE in
C57BL/6 (H-2b) mice was used. C57BL/6 and LT- Table 1.
Spleen Cells of LT (also known as TNF-
) is a member of the
immediate MHC-linked TNF family, which consists of
TNF-
, LT-
, and LT-
. LT can be secreted as an LT-
3
homotrimer, which can bind to either the p55 TNFRI or
p75 TNFRII, where it emulates many of the activities of
TNF-
. It is also present as a cell surface complex in association with a type II transmembrane protein, LT-
. The
most common form, LT-
1
2, binds to the LT-
R, and
the less common form, LT-
2
1, can bind to p55 TNFRI
(1). Studies that implicate members of the family in the
pathogenesis of MS and EAE include the presence of LT-
and TNF-
in MS plaques (2) and the correlation of T cell
clones' encephalitogenicity with their production of LT-
and/or TNF-
(3). Even more compelling evidence for
the role of members of the family in EAE includes the ability of antibodies that neutralize LT-
and TNF-
to inhibit passive transfer of both acute (4, 5) and relapsing remitting disease (6). On the other hand, other studies do not
support the conclusion that TNF-
is pathogenic in EAE.
One study suggests that administration of a rabbit anti-TNF antibody at the time of immunization of mice with
myelin basic protein (MBP) does not affect the development of EAE (7) and another indicates that administration
of TNF-
, through a vaccinia virus vector delivery system,
inhibits the development of EAE (8). Furthermore, a study
published while this manuscript was being prepared indicated that mice deficient in both LT-
and TNF-
and
backcrossed to SJL mice (though retaining the H-2b knockout
chromosome) developed clinical signs of EAE after immunization with MBP or mouse spinal cord homogenate (9).
In view of the conflicting data, and the fact that none of
the previous studies clearly distinguished between LT and
TNF-
, nor did any address the role of secreted LT-
3 as
opposed to the membrane associated LT-
/
heterotrimer,
we systematically addressed the role of the individual members of the LT family (and by inference TNF-
) in EAE in
C57BL/6 H-2b mice.
or LT-
have been produced (10). LT-
-deficient mice (LT-
/
mice) have profound defects in lymphoid organ development (10, 11). They are missing all LNs and Peyer's
patches (PP), exhibit marked splenic disorganization, and
lack germinal centers (13). Despite these anatomic defects,
humoral immune responses can be elicited. The mice produce normal levels of anti-nitrophenyl antibody when challenged with a high dose of nitrophenyl-ovalbumin and their
immunoglobulin genes undergo somatic hypermutation resulting in antibody affinity maturation (14). LT-
/
mice
produce normal or enhanced levels of IgM to SRBC immunization, although IgG levels are reduced compared to
wild-type (WT) littermates (12). Delayed type hypersensitivity to ovalbumin is markedly impaired although LT-
/
splenic T cells proliferate to that antigen (Bergman, C.M.,
and N.H. Ruddle, manuscript in preparation; 15). LT-
/
mice exhibit some, but not all, of the defects of LT-
/
mice (12). They lack PP and exhibit splenic disorganization with an absence of germinal centers and antibody responses
essentially comparable to those of LT-
/
mice. LT-
/
mice differ from LT-
/
mice in that, although most peripheral LNs are absent, the mesenteric and cervical LNs
are present and some members of the lumbar group are
variably apparent (12).
R
/
mice. However, in that study, WT mice were not
susceptible to MOG-induced EAE (although the IFN-
R
/
mice were) probably because human rather than rat
MOG peptide was used. In our study, the rat MOG peptide immunization regimen was precisely followed to induce EAE in C57BL/6 and WT controls as we evaluated
mice that were selectively deficient in LT-
or LT-
to
identify the role of those individual cytokines, and by inference TNF-
, in EAE.
/
mice were originally obtained from
Dr. David Chaplin (Washington University, St. Louis, MO) and
have been previously described (10). They have been maintained
as a breeding colony under specific pathogen-free conditions at
Yale University for ~2 yr. LT-
/
mice were developed in collaboration with Drs. Richard Flavell (Yale University School of
Medicine) and Jeffrey Browning (Biogen, Cambridge, MA) as
previously described (12) and were also maintained under specific
pathogen-free conditions at Yale University School of Medicine.
LT-
/
and LT-
/
mice were screened by their respective
PCR assays as previously described (12). The colonies of LT-
/
and LT-
/
mice are of mixed 129 × C57BL/6 origin and
were studied in the course of backcrossing to C57BL/6. Each
colony was at least at the level of the fourth backcross at the time
of these experiments.
LT-/
Mice Are Primed to MOG.
/
mice were bled at various times after immunization and
their sera tested for total IgG by ELISA. There were no detectable titers of IgG against MOG 35-55 6 d after immunization. 30 d after immunization, WT mice (n = 9) had a
mean titer of 1420 (200-3,300) and LT-
/
mice (n = 7)
had a mean titer of 380 (130-1,600). Nonimmunized mice
(n = 5), mice immunized with adjuvant alone (n = 5), and mice immunized with other antigens (n = 5) did not show
any antibody response to MOG 35-55. Spleen cells of
MOG immunized LT-
/
mice proliferated in vitro in
response to MOG peptide (Table 1). These results, in
agreement with previous studies, indicate that, despite the
absence of LNs, LT-
/
mice can be sensitized to exogenous antigen.
/
Mice Proliferate in Response
to MOG
Source of spleen cells*
No antigen
MOG peptide
SI
cpm
C57BL/6
74.5
616.4
8.2
LT
/
74
374.0
5
*
Mice were immunized with 300 µg MOG 35-55 in CFA and 14 d
later cells cultured in the presence or absence of 30 µg MOG 35-55. [3H]Thymidine incorporation was evaluated after 48 h in culture.
We next tested whether MOG-immunized
LT-/
mice could develop EAE. C57BL/6 and LT-
+/+
littermates exhibited clinical signs by ~12 d after the initial immunization. The clinical manifestations were similar to
those previously reported (16, 17) with a chronic and severe paralysis (Fig. 1) and weight loss. In some cases, an altered gait was apparent a day or two before the tail was affected. However, in general the tail eventually became
flaccid. Most mice progressed to hindlimb paralysis, and
several progressed to hind- and forelimb paralysis or death.
The clinical signs continued for the duration of the observation period and beyond (for >60 d). A similar clinical course was seen in 129 mice (data not shown). In contrast,
LT-
/
mice were markedly resistant to active induction
of EAE (Fig. 2; Table 2). In two separate experiments involving a total of nine mice, only one animal attained a
score >0.5, and this was much later (at day 23) than the
usual peak scores in the other two groups (Table 2). The
mean maximum clinical score was significantly less than
any of the control groups, including the LT-
+/
mice.
Interestingly, LT-
+/
mice, despite their full complement
of lymphoid organs, exhibited a sensitivity intermediate between LT-
+/+ and LT-
/
mice, suggesting a dose related effect of LT.
|
The brain and cervical, thoracic and lumbar spinal cord of individual C57BL/6,
LT-+/+, LT-
+/
, and LT-
/
mice were evaluated histologically at various times after MOG immunization. All positive control mice exhibited extensive mononuclear infiltration in all areas examined, including the choroid plexus and
all levels of the spinal cord. The mononuclear infiltrate, consisting of T cells, B cells, and macrophages (Suen, W.E.,
C.M. Bergman, and N.H. Ruddle, manuscript in preparation) was diffuse and not restricted to cuffing around the
vessels but extended into the parenchyma, predominantly
in the white but also in the gray matter. Demyelination was
apparent (Fig. 3). In contrast, there was very little inflammation in any sections obtained from LT-
/
mice. The
minimal inflammation was limited almost exclusively to the
meninges. The most extensive inflammation detected is
shown in Fig. 3 C. There was no evidence of demyelination. Thus, the minimal clinical signs of LT-
/
mice
were mirrored in the very minor histological involvement.
LT-
Several protocols of passive
transfer were used to test whether LT-/
mice, although
resistant to active induction of EAE, could act as recipients
of passive transfer of EAE by cells from WT donors. Several experiments were performed using either T cell lines, lymph node cells, or spleen cells from C57BL/6 mice immunized with MOG 35-55 in CFA. Recipients were irradiated or not and/or injected or not with Pt. In five separate experiments (three of which are shown in Table 3),
C57BL/6 mice showed rather mild clinical signs after
transfer of as many as 4 × 107 T cells. It is likely that a
combination of MOG-specific T cells and anti-MOG antibody will result in more dramatic clinical signs. Nevertheless, in three out of three experiments, LT-
/
mice were
susceptible to passive transfer with WT MOG T cells, even
more so than the control recipients. The enhanced susceptibility was manifested as a slightly higher maximum clinical score, and especially as a much earlier day of onset. The
enhanced susceptibility of LT-
/
mice to transfer could
be due to the fact that their TNF receptors are not occupied by endogenous LT and hence would be more readily triggered.
|
To determine
whether the effects of LT in EAE are mediated by the soluble
LT-3 form or as a membrane-associated LT-
/
complex, we tested whether LT-
/
mice are susceptible to
EAE. These mice were also of interest because they lack PP
and most LNs (although, in contrast to LT-
/
mice,
they still have mesenteric and cervical LNs). LT-
/
mice
exhibit the splenic disorganization and minimal defects in
immunoglobulin switching that are seen in LT-
/
mice.
LT-
/
mice were susceptible to EAE in that they
showed clinical signs and histologic manifestations, although they were somewhat less susceptible than WT mice
(Table 4). These data indicate that peripheral LNs, PP, and
an organized spleen are not required for EAE. The data also
suggest that the activities of LT in EAE are mediated predominantly as soluble LT-
3, rather than as an LT-
/
membrane-associated complex.
|
In this study, we evaluated mice selectively deficient in
LT- or LT-
in order to identify the role of those individual cytokines, and by inference, TNF-
, in MOG-induced
EAE. C57BL/6 and WT littermates of LT-deficient mice
were susceptible to EAE with a chronic, sustained paralysis
and CNS inflammation and demyelination. Despite evidence that LT-
/
mice were primed to MOG in that
they produced antipeptide antibody and their spleen cells
proliferated in vitro in response to challenge with MOG
35-55, LT-
/
mice were quite resistant to EAE with regard to clinical signs and histologic manifestations. LT-
/
mice were susceptible to passive transfer of EAE with WT
MOG T cells. LT-
/
were susceptible to both the clinical signs and CNS pathology, but less so than WT mice.
These data suggest that the reason LT-
/
mice do not
develop EAE is because they lack T cell production of LT
which is crucial for pathogenesis. They implicate the secreted form of LT-
3 more strongly than the LT-
/
complex and, by inference, suggest a very little compensatory role for TNF-
in this inflammatory demyelinating
disease. The data suggest that the inability of LT-
/
mice
to develop EAE is largely because of the fact that their antigen-specific T cells do not produce LT, rather than because of the anatomic problems of their lack of LN and PP and
their splenic disorganization. LT-
+/
mice were less susceptible than LT-
+/+ mice to the clinical signs of EAE
despite the fact that they have normal lymphoid organs. An
anatomic problem is also less likely since LT-
/
mice
were susceptible to EAE even though they have many of
the same lymphoid organ anomalies as LT-
/
mice, that
is absence of PP, disorganized spleen, and the absence of
most LNs.
We are currently investigating the role of nonlymphoid
cells and antibody in MOG EAE. It is possible that LT production, both by infiltrating T cells and by cells in the CNS
such as astrocytes, is necessary for full manifestation of the
disease. The data presented here, which demonstrate that
LT-/
mice make low, but detectable amounts of anti-MOG peptide antibody that do not differ significantly from
that made by controls, are consistent with the interpretation that antibody alone is not sufficient for manifestations
of this disease, although it may be necessary. This concept
is in agreement with the conclusions reached in studies of
MOG-induced EAE in rats and marmosets (19, 21) and
suggests that LT production by antigen-specific T cells initiates the lesion that is sustained by antibody.
There are several possible avenues through which LT
may exert its effects in EAE. The LT- secreted by T cells
may contribute to the initial entrance of inflammatory cells
into the CNS. We have recently shown that murine LT induces the expression of E-selectin on murine endothelial
cells in vitro (Schwartz, J., C.M. Bergman, K. Russell, J. Bender, and N.H. Ruddle, manuscript in preparation). LT-
could also contribute to recruitment in EAE. It induces expression of vascular cellular adhesion molecule (VCAM)
and intracellular adhesion molecule (ICAM) in vitro.
VCAM expression correlates with clinical signs in EAE and
its upregulation is inhibited by treatment with anti-LT/
TNF antibody (22) and the interaction between VLA4 on
antigen-specific T cells and VCAM on endothelial cells is
crucial for EAE (23). LTs may also contribute to the pathogenesis of EAE through an effect on oligodendrocytes,
since it has been shown that LT-
is even more effective
than TNF-
in inducing apoptosis of this myelin-producing cell (24). LTs may contribute to the determinant
spreading that is apparent in some forms of EAE. That this
could also very well be an aspect of the mouse MOG-induced EAE is supported by the chronic manifestation of
clinical signs in this model. LT-
and the LT-
/
complex
induce lymphoid organs in embryogenesis, and an LT-
transgene induced inflammation represents a phenomenon
we have termed lymphoid neogenesis (25). It is possible
that the role of LT in EAE includes this organizational aspect which may contribute to perpetuation through the
presentation of new antigens.
The studies presented here should be considered in light
of the different but overlapping roles of the members of the
LT/TNF family with regard to inflammation and lymphoid organ development. LT- appears to be primarily
concerned with lymphoid organ development as an LT-
/
complex. Its role in inflammation is less crucial. It does
not induce expression of VCAM or ICAM (26) and plays a
minor role in EAE (as shown in this paper). LT-
appears
to be crucial in lymphoid organ development both as an LT-
3
homotrimer and an LT-
/
heterotrimer LT-
is also a
major participant in inflammation, which it induces in transgenic mice (27). It is crucial for delayed type hypersensitivity
(Bergman, C.M., and N.H. Ruddle, manuscript in preparation) and for EAE, as demonstrated here. TNF-
plays
almost no role in lymphoid organ development in that
TNF-
/
mice have normal LNs and PP, though they
exhibit some disorganization of B cell areas in the spleen.
TNF-
has generally been thought of as a major participant
in the inflammatory process (28) and it induces expression
of ICAM and VCAM in vitro. Thus, we were surprised that it
did not compensate for the absence of LT-
in LT-
/
mice and we eagerly await the results of MOG immunization of TNF-
/
mice. It appears from the data presented
here that its role may have been misinterpreted in experiments in which it appeared to be essential for EAE.
While this manuscript was being prepared for publication, a communication was published that indicated that
TNF- and LT-
were not required for EAE induced by
proteolipid protein or mouse spinal cord homogenate (9).
Mice in that study had a simultaneous deletion of the tnfa
and lta genes and the intervening sequences and had been
backcrossed to SJL. These animals developed clinical signs
of EAE after immunization with antigen. It is difficult to
reconcile those observations with the ones reported here,
but there are many differences in the systems. They differ
with regard to antigen, the H-2 haplotype of the controls,
background genes, and the fact that this study analyzed
mice defective in single genes, whereas Frei et al. (9) involved a large chromosomal deletion. An additional possibility is that LT and TNF-
play different roles in EAE,
i.e., LT-
and TNF-
both induce inflammation, but, at
high concentrations, TNF-
downmodulates the activities of LT-
. Perhaps in the model of Frei et al. (9) other cytokines compensate for LT-
in the absence of TNF-
.
The data presented here and elsewhere indicate that the
members of the LT/TNF family, through their various
physical presentations as soluble or membrane forms and by
means of their multiple receptors, can each contribute to
many different aspects of inflammation and development.
They occasionally compensate for each other, and in that
way exhibit some redundancy, and in some transgenic systems LT- and TNF-
appear to have similar activities
(29). Nevertheless, in some instances, it is clear that these
cytokines have unique functions that cannot be compensated for by other members of the family. Such appears to
be the case described here in which LT-
is crucial for
MOG-induced EAE.
Address correspondence to Dr. Nancy H. Ruddle, Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT 06520-8034. Phone: 203-785-2915; FAX: 203-785-6130; E-mail: nancy.ruddle{at}yale.edu
Received for publication 22 July 1997.
1 Abbreviations used in this paper: CNS, central nervous system; EAE, experimental allergic encephalomyelitis; ICAM, intracellular adhesion molecule; LT, lymphotoxin; MBP, myelin basic protein; MOG, myelin oligodendrocyte glycoprotein; PP, Peyer's patches; Pt, pertussis toxin; VCAM, vascular cell adhesion molecule; WT, wild-type.This work was supported by grant RG 2394 from the National Multiple Sclerosis Society. P. Hjelmström was supported by a fellowship from the Swedish Foundation for International Cooperation in Research and Higher Education.
We thank Dr. Matthew Hanson and Irene Visintin for instruction and assistance in the perfusion experiments, Dr. Jeffrey Kocsis for assistance in epon embedding and Toluidine blue staining, and Dr. Kocsis and Dr. Jung Kim for assistance in histological evaluation.
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11. | Banks, T.A., B.T. Rouse, M.K. Kerley, P.J. Blair, V.L. Godfrey, N.A. Kuklin, D.M. Bouley, J. Thomas, S. Kanangat, and M.L. Mucenski. 1995. Lymphotoxin-alpha-deficient mice. Effects on secondary lymphoid organ development and humoral immune responsiveness. J. Immunol. 144: 1685-1693 . |
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14. |
Matsumoto, M.,
S.F. Lo,
C.J.L. Carruthers,
J. Min,
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G. Huang,
D.R. Plas,
S.M. Martin,
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M.N. Nahm, and
D.D. Chaplin.
1996.
Affinity maturation without
germinal centres in lymphotoxin-![]() |
15. | Sacca, R., S. Turley, L. Soong, I. Mellman, and N.H. Ruddle. 1997. Transgenic expression of lymphotoxin restores
lymph nodes to lymphotoxin ![]() |
16. | Kerlero de Rosbo, N., I. Mendel, and A. Ben-Nun. 1995. Chronic relapsing experimental autoimmune encephalomyelitis with a delayed onset and an atypical clinical course, induced in PL/J mice by myelin oligodendrocyte glycoprotein (MOG)-derived peptide: preliminary analysis of MOG T cell epitopes. Eur. J. Immunol. 25: 985-993 [Medline]. |
17. | Mendel, I., N. Kerlero de Rosbo, and A. Ben-Nun. 1995. A myelin oligodendrocyte glycoprotein peptide induces typical chronic experimental autoimmune encephalomyelitis in H-2b mice: fine specificity and T cell receptor Vb expression of encephalitogenic T cells. Eur. J Immunol. 25: 1951-1959 [Medline]. |
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19. | Genain, C.P., M.-H. Nguyen, N.L. Letvin, R. Pearl, R.L. Davis, M. Adelman, M. Lees, C. Linington, and S.L. Hauser. 1995. Antibody facilitation of muliple sclerosis-like lesions in a nonhuman primate. J. Clin. Invest. 96: 2966-2974 [Medline]. |
20. | Willenborg, D.O., S. Fordham, C.C.A. Bernard, W.B. Cowden,
and I.A. Ramshaw. 1996. IFN-![]() |
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22. | Barten, D.M., and N.H. Ruddle. 1994. Vascular cell adhesion molecule-1 modulation by TNF in experimental allergic encephalomyelitis. J. Neuroimmunol. 51: 123-133 [Medline]. |
23. |
Baron, J.L.,
J.A. Madri,
N.H. Ruddle,
G. Hashim, and
C.A. Janeway.
1993.
Surface expression of ![]() |
24. |
Selmaj, K.,
C.F. Raine,
M. Farooq,
W.T. Norton, and
C.F. Brosnan.
1991.
Cytokine cytotoxicity against oligodendrocytes: apoptosis induced by lymphotoxin.
J. Immunol.
147:
1522-1529
|
25. | Kratz, A., A. Campos-Neto, M.S. Hanson, and N.H. Ruddle. 1996. Chronic inflammation caused by lymphotoxin is lymphoid neogenesis. J. Exp. Med. 183: 1461-1472 [Abstract]. |
26. | Hochman, P.S., G.R. Majeau, F. Mackay, and J.L. Browning. 1996. Proinflammatory responses are efficiently induced by homotrimeric but not heterotrimeric lymphotoxin ligands. J. Inflamm. 46: 220-234 . |
27. |
Picarella, D.E.,
A. Kratz,
C.-B. Li,
N.H. Ruddle, and
R.A. Flavell.
1992.
Insulitis in transgenic mice expressing TNF-![]() |
28. | Paul, N.L., and N.H. Ruddle. 1988. Lymphotoxin. Annu. Rev. Immunol. 6: 407-438 [Medline]. |
29. |
Picarella, D.E.,
A. Kratz,
C.-B. Li,
N.H. Ruddle, and
R.A. Flavell.
1993.
Transgenic TNF-![]() ![]() ![]() |