By
From the * Centenary Institute of Cancer Medicine and Cell Biology, Sydney, New South Wales 2050, Australia; and Department of Medicine, University of Sydney, Sydney, New South Wales 2006, Australia
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Abstract |
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Lymphotoxin (LT) is widely regarded as a proinflammatory cytokine with activities equivalent
to tumor necrosis factor (TNF). The contribution of LT to experimental autoimmune encephalomyelitis (EAE) was examined using TNF/LT/
mice, TNF
/
mice, and a new LT
/
line described here. All mice were generated directly in the C57BL/6 strain and used for the
preparation of radiation bone marrow chimeras to reconstitute peripheral lymphoid organs and restore immunocompetence. This approach overcame the problems related to the lack of
lymph nodes that results from LT
gene targeting. We show here that when LT is absent but
TNF is present, EAE progresses normally. In contrast, when TNF is absent but LT is present,
EAE is delayed in onset and inflammatory leukocytes fail to move normally into the central
nervous system parenchyma, even at the peak of disease. In the absence of both cytokines, the
clinical and histological picture is identical to that seen when TNF alone is deficient, including
demyelination. Furthermore, the therapeutic inhibition of TNF and LT
with soluble TNF
receptor in unmanipulated wild-type or TNF
/
mice exactly reproduces these outcomes. We
conclude from these studies that TNF and LT are functionally distinct cytokines in vivo, and
despite sharing common receptors, show no redundancy of function nor mutual compensation.
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Introduction |
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Lymphotoxin (LT)1, like TNF, is considered to be a proinflammatory, cytotoxic cytokine (1) and critical mediator of lymphocyte-dependent autoimmune pathologies such as multiple sclerosis (MS) and the MS animal model, experimental autoimmune encephalomyelitis (EAE; for review see reference 2). Evidence for a key role of LT in MS and EAE comes from four types of experiments. First, LT is identified within the lesions of MS (3, 4) and EAE (5). Second, the encephalitogenicity of T cells is associated with their ability to synthesize LT (6), although this association is not absolute (9). Third, LT is toxic to oligodendrocytes in culture (10). Fourth, LT and TNF blockade prevents or ameliorates disease (11).
LT in its secreted form (LT3) is thought to contribute
to pathologies of this kind by its ability to bind to TNFR1
and TNFR2 (1, 14), resulting in the promotion of inflammation by the upregulation of endothelial adhesion molecules (15) and the delivery of cytotoxic signals to target
cells (16). Furthermore, LT
in association with the related
cell surface molecule LT
(LT
1
2; references 17, 18),
binds the LT
receptor (19) in a system found to be essential for normal peripheral lymphoid development (20, 21),
and is capable of delivering a cytotoxic signal to target cells
(22).
Until recently, the means to test the relative contributions of LT and TNF to inflammatory processes have not
been available. TNF (23), TNF/LT (24), LT
, and LT
(25) gene-targeted mice have now been used to address this
question. In the latter two studies using the EAE disease
model, opposite conclusions have been drawn about the
role of LT. In the first of these (24), it was argued that neither LT nor TNF play any role in EAE, and in the second
(25), that secreted LT
3 is critical in the pathogenesis of this
disease. The direct use of LT
or LT
gene-deleted mice for studies of immune pathology, however, is compromised by the immune deficiencies that follow, for example,
the absence of peripheral lymph nodes in these mice (20,
26). The fact that control animals in these two studies had
normal immune systems necessarily implies that any differences observed in experimental outcomes between wild-type (WT) and LT-negative mice cannot be attributed to
the activities of the cytokine alone. The difficulty in interpretation of these experiments is compounded by the fact
that the TNF, LT
, and LT
genes are located within the
MHC (27). The backcrossing of 129 strain gene-deleted
mice onto EAE-susceptible strains, such as SJL or C57BL/6,
would create partial chromosome 17 congenics that differ
from WT controls in this fundamentally important disease susceptibility locus (28). For these reasons, we regard the question of the relative contribution of LT to EAE pathogenesis to be unresolved.
We have disrupted the TNF and LT genes directly in
C57BL/6 mice (29), a strain that is highly susceptible to
EAE induced by immunization with the 35-55 peptide of
myelin oligodendrocyte glycoprotein (MOG). This avoids
the problem of genetic heterogeneity introduced by backcrossing. In the first series of experiments using TNF
/
mice (23), direct immunization was possible because peripheral immunity in these mice was essentially intact. The
clinical course and pathological changes of EAE in TNF
/
mice were remarkable, revealing key activities for this cytokine in the initiation of inflammatory lesions and the
control of leukocyte movement within the central nervous
system (CNS).
To extend these experiments to the role of LT in EAE,
the problem of immunocompetence had to be accounted
for. Unlike TNF, LT is predominantly, if not exclusively, a
product of leukocytes (14) and particularly, Th1 T cells
(30). It is LT produced from these hemopoietically derived
sources that is thought to be crucial in CNS autoimmune
inflammatory lesions (6, 8). Bone marrow cells derived
from LT/
mice have the capacity to repopulate lymph
nodes in irradiated recipient animals (31). Therefore, chimeras may be generated that are LT deficient, but with a
reconstituted functional immune system.
In a second series of experiments reported here, bone
marrow cells from a newly generated LT/
C57BL/6
strain (see Results), in combination with previously described TNF
/
and TNF/LT
/
C57BL/6 mice (29),
were used to reconstitute peripheral lymphoid structures in
lethally irradiated recipient animals. This enabled the application of a standard EAE induction protocol to all mice and
the study of the activities of LT both in the presence and absence of TNF. Furthermore, inhibition of TNF and LT
3
by a soluble TNFR-human IgG fusion protein (TNFR-IgG)
in WT and TNF
/
mice represents the first direct comparison of the effects of gene targeting and therapeutic inhibition in a single disease model. Using these experimental
systems, we reveal the lack of any unique contribution by
LT to clinical manifestations of disease, CNS inflammation,
or demyelination. In addition, we demonstrate that the
profound role of TNF in the control of normal inflammatory cell movement within the CNS (23) is not shared by LT.
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Materials and Methods |
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Animals.
C57BL/6.TNFGeneration of C57BL/6 Strain LT/
Mice.
|
Southern Blot, Nested Set PCR, and Reverse Transcriptase PCR.
Genomic DNA was purified from ES cells according to standard procedures (38) or from white blood cells using a genomic DNA kit (Promega Corp., Madison, WI). To detect homologous recombination, nested set PCR was performed with an outer primer pair (5' sense: CTA GGA CAG GGT TCT CAA CCT TCC T; 3' antisense: CCA GTC CCT TCC CGC TTC AGT GAC AAC GTC; 20 cycles) followed by another round of amplification with an inner primer pair (5' sense: CAG TTC CTC ATG TTG TGG TGA CCC; 3' antisense: CCG ACT GCA TCT GCG TGT TCG A; 35 cycles). The annealing temperature was 60°C for both primer pairs. The Southern blot strategy used the introduction of an additional PstI site into the genome within the neomycin resistance cassette (29). Successfully targeted alleles showed a fragment length of 6.4 kb (WT 9.4 kb; Fig. 1 B). For analysis of cytokine mRNA transcripts, total RNA was extracted (39) from PMA-stimulated splenocytes (5 ng/ml for 12 h) or whole unperfused brain and spinal cord. oligo(dT) 12-18 (Boehringer Mannheim GmbH, Mannheim, Germany) primed cDNA was synthesized and analyzed by reverse transcriptase PCR (RT-PCR) using primers and methods as previously described (29). The PCR products were size fractionated by electrophoresis on 1.2% agarose gel containing ethidium bromide.Induction of EAE.
EAE was induced actively by subcutaneous tail base injection of 50 µg of MOG peptide (35-MEVGWYRSPFSRVVHLYRNGK-55) in CFA containing 1 mg of heat inactivated Mycobacterium tuberculosis (H37RA; DIFCO Labs., Detroit, MI). 200 ng of pertussis toxin (LIST Biological Labs., Inc., Campbell, CA) was injected intravenously on days 0 and 2. This disease induction protocol was optimized in WT C57BL/6 mice by pertussis toxin and MOG peptide dose-ranging experiments. Animals were observed daily and neurological deficits were quantified on an arbitrary clinical scale: 1+, flaccid tail; 2+, hind limb weakness or abnormal gait; 3+, severe hind limb weakness with loss of ability to right from supine; 4+, hind quarter paralysis; 5+, forelimb weakness or moribund; 6+, death. Supplementary food and water were provided on the cage floor for disabled animals.Generation of Radiation Bone Marrow Chimeras.
Bone marrow cells, harvested from the long bones of matched donor mice by flushing with cold PBS, were gently deaggregated through 70 µm nylon cell strainers (Becton Dickinson, Franklin Lakes, NJ), washed, and counted. Recipient animals were preconditioned with 5.5 Gy gamma radiation on day 2, and again on day 0. Bone marrow cells (2 × 107 cells/recipient) were injected intravenously on day 0. Cage water was supplemented with trimethoprim (50 µg/ml)-sulphamethoxazole (0.25 mg/ml) between weeks 2 and 3 after transplantation. As a means of tracking engraftment, reciprocal transplantations between TNFAntibodies.
The origins of mAbs specific for mouse CD4, Mac-1 (CD11b), vascular cell adhesion molecule 1 (VCAM-1), CD45, TCR-MOG-specific IgG Determination.
Relative serum IgG anti-MOG 35-55 responses were quantified by ELISA as described (23).Flow Cytometric Analysis.
Flow cytometric analysis of bone marrow engraftment in chimeras was performed on peripheral blood leukocytes after red blood cell lysis. Cells were incubated with biotinylated anti-Ly5.1 mAbs and one of the following directly PE-conjugated mAbs: anti-B220 (B cells), anti-TCR-Immunohistology and Neuropathology.
Dissected specimens of nonperfused whole brain were embedded in OCT compound (Tissue Tek, Miles, Inc., Elkhart, IN), frozen in liquid nitrogen vapor, and stored atTreatment of Mice with TNFR-IgG.
The activities of LT ![]() |
Results |
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The injection of
one targeted C57BL/6 ES cell clone into blastocysts of the
BALB/c strain resulted in the birth of chimeric mice, all of
which transmitted through the germ line after mating with
C57BL/6 mice. Founder heterozygous mice were chosen,
interbred, and screened by Southern blot. LT/
mice
were born in the expected Mendelian ratio of 1:2:1 and
were sustained as a homozygous line on the C57BL/6
background (Fig. 1 B). The successful disruption of the
gene encoding LT
was confirmed by RT-PCR. LT
-specific mRNA was not detectable in PMA-activated splenocytes (Fig. 1 C), whereas expression of TNF mRNA was
readily detected. By contrast, LT
transcripts were detected in stimulated cells from TNF
/
mice, but neither
TNF nor LT
transcripts from TNF/LT
/
mice (Fig. 1
C). LT
/
mice bred well and were grossly normal.
Closer inspection of the lymphoid organs showed the expected absence of peripheral lymph nodes and Peyer's
patches, a disturbed splenic architecture, and peripheral
blood lymphocytosis (20, 21, data not shown). LT
/
mice lack both secreted LT
3 and membrane LT
1
2 forms
of LT because functional surface expression of LT
depends on the presence of LT
(17).
Subcutaneous immunization of WT
C57BL/6 mice with 50 µg of MOG 35-55 in CFA, and
pertussis toxin intravenously on days 0 and 2, resulted in a
highly reproducible severe acute meningoencephalomyelitis, with clinical signs of rapidly progressing, ascending
symmetrical motor deficits, appearing on or around day 10. Clinical disease was present for ~15 d and spontaneously
remitted (Fig. 2) to leave a mild, chronic, nonrelapsing deficit (approximately disease score 1). Disease in TNF/
mice (Fig. 2) was delayed in onset by ~6 d. Once established, however, clinical deficits in these mice progressed in
a parallel course to an equivalent mean peak severity, but
were of a reduced overall duration when compared with
WT, as we have previously described (23).
|
C57BL/6 TNF/LT/
mice, which lack peripheral
lymph nodes and exhibit a range of immune deficiencies
(29, 45), were entirely resistant to clinical disease induction
by our standard immunization protocol (Fig. 2). Similarly,
immunization of RAG 1
/
mice, which lack mature lymphocytes, also resulted in no clinical syndrome (Fig. 2 and
Table 1, A-C). Thus, disease resistance in TNF/LT
/
mice could indicate either a critical role for LT, or the consequences of a broadly deficient immune system as exemplified by the RAG-1
/
mice. Determination of the role
of LT in EAE required the reconstitution of immunocompetent peripheral lymphoid organs by other means.
|
|
Chimeras were generated using a split-dose irradiation protocol
and large bone marrow inoculum to ensure complete conversion of the recipient hemopoietic compartment to donor type. Two types of radiation bone marrow chimeras
were generated. The first involved the transfer of WT or
LT/
bone marrow to RAG-1
/
mice (WT
RAG,
LT
RAG). This chimeric system tested whether the absence of both forms of LT, but the presence of TNF, affected susceptibility to EAE. The absence of lymphocytes
in RAG-1
/
mice guaranteed that any T or B cells in
these chimeras were LT negative. These mice were used
for EAE studies 8 wk after bone marrow reconstitution. A
further chimera, generated by the transfer of TNF
/
bone
marrow to RAG-1
/
recipients (TNF
RAG), was used
as a control for the CNS cytokine mRNA expression analysis (see below).
The second series of chimeras involved the transfer of
TNF/LT/
bone marrow (completely deficient in both
TNF and LT) to TNF
/
recipient mice (TNF/LT
TNF)
or appropriate controls (WT
WT, TNF
TNF). This arrangement tested how the absence of both TNF and LT affected susceptibility to EAE and, in particular, if LT was responsible for the EAE observed in TNF
/
mice (23). Each
type of radiation bone marrow chimera was examined for
the presence of lymphoid tissue after experimental use. As expected, lymph nodes were repopulated in all mice (not shown). The efficacy of the irradiation and reconstitution protocol
for engraftment was examined in parallel using a CD45 congenic marker (Ly5.1.WT
TNF, and TNF
Ly5.1.WT)
and serial flow cytometric analyses of peripheral blood leukocytes (Fig. 3 A). At the time of immunization, in excess
of 95% of circulating
/
-TCR+, B220+, and CD11b+
(Mac-1) cells were of donor type. Importantly, engraftment
proceeded irrespective of the TNF status of the donor or
recipient (Fig. 3 A).
Antibody is important in the development of demyelination (46). To determine whether antibody responses in chimeric mice had been normalized, serum samples were collected from WT WT, TNF
TNF, and TNF/LT
TNF
mice at day 23 after immunization with MOG 35-55/
CFA/pertussis toxin and were assayed by ELISA. Peptide-specific IgG responses were observed in all mice (Fig. 3 B)
with responses in TNF/LT-deficient mice at least as great
as in WT mice.
WT RAG and
LT
RAG mice were immunized according to the standard protocol. Disease onset, rate of progression, and peak
severity were equivalent in both groups (Fig. 3 C and Table 1) indicating no unique role for LT in the clinical
neurological deficit. Disease course is illustrated up to day
22 only, due to the ethical requirement to kill severely affected animals. The day 15 onset of disease in both groups
(rather than day 10 in unmanipulated animals, see Fig. 2) is
probably due to the use of these mice only 8 wk after transplantation when T cell reconstitution to normal levels may not have occurred (see Fig. 3 legend). Irrespective of the
time of use, all lymphocytes in these chimeras were of donor type.
To explore
the possibility that the activity of LT in EAE was obscured
by the compensatory influence of normal TNF expression in Fig. 3 C, the role of LT was examined in a TNF-deficient system (TNF/LT TNF). The clinical course of disease in WT
WT and TNF
TNF mice followed that
predicted by previous experiments (Fig. 3 D and Table 1
E), reproducing the delay in disease onset seen in Fig. 2 and
validating this experimental approach. Remarkably, TNF/
LT
TNF mice followed a course of disease that was indistinguishable from TNF
TNF mice, with no augmentation of the delay in onset, normal slope of the disease progression curve, and normal peak severity of disease (Fig. 3
D). Of those mice allowed to continue on to recovery, a
normal resolution of clinical deficits occurred (data not
shown). This result excluded the possibility that an important activity of LT in the course of EAE in the LT
RAG
experiment (Fig. 3 C) was obscured by the compensations
of TNF.
The use of the Ly5 markers established the
validity of this approach for effective reconstitution of the
hemopoietic compartment in recipients. The most rigorous
determination of cytokine expression within the CNS itself
is RT-PCR analysis, and this was applied to mRNA extracted from inflamed CNS tissue (Fig. 4). LT message was abundantly expressed in the CNS of all EAE-affected
animals with the exception of the TNF/LT
TNF and
LT
RAG chimeras, consistent with the hemopoietic origin of this cytokine. Likewise, LT
expression was seen in
the unperfused normal WT CNS (Fig. 4), but not in perfused WT tissue (data not shown). TNF mRNA expression
was clearly evident in the CNS of all experimental groups,
with the exception of the TNF
TNF and TNF/LT
TNF
chimeras (data not shown). As expected, abundant expression of LT
was found in the TNF
RAG chimera (Fig.
4), but also abundant TNF message (data not shown), consistent with TNF production by nonhemopoietic, radiation-resistant CNS structures, such as microglia and astrocytes (47). Similarly, a trace amount of LT
message was
sometimes observed in the CNS from TNF/LT
TNF
and LT
RAG chimeras at the peak of disease (Fig. 4),
most likely due to low level LT
production by activated
glia (47). It is improbable that LT
expression of this nature is significant within the terms of these experiments, an
assertion supported by further studies using TNFR-IgG
(see below).
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The influence of individual cytokines on
the number of inflammatory cells recruited to the entire
CNS was examined by flow cytometric analysis of leukocytes extracted from the whole perfused CNS of chimeras
(Fig. 5, A and B). The magnitude of inflammatory cell recruitment was comparable in WT WT, TNF
TNF, and
TNF/LT
TNF chimeras (Fig. 5 A). Immunophenotyping was then performed to evaluate the possibility that
TNF or LT influenced the relative proportions of individual inflammatory cell subsets. Fig. 5 B shows that, in the
absence of TNF, there was a relative increase in the proportion of
/
-TCR+ cells as a percentage of total CD45+
cells, comprising populations of monocyte-macrophages, T
cells, and microglia (42). Most importantly, differences
were not observed between the TNF
TNF and TNF/
LT
TNF chimeras (Fig. 5 B, middle and right) indicating
no additional alteration of the infiltrate imposed by the absence of LT.
|
To examine the influence of TNF and LT on the formation of
CNS inflammatory lesions, immunohistochemical studies
using CD45 staining of cells within CNS tissue were performed (Fig. 5, C-F). In all specimens, large numbers of
CD45+ cells could be identified at the peak of disease. Lesions within the CNS of WT WT chimeras exhibited the
normal appearance of cells moving freely from the vessel
lumen into the perivascular space, and beyond into the
CNS parenchyma (Fig. 5 C). In contrast, the perivascular
cuffs of TNF
TNF and TNF/LT
TNF chimeras (Fig. 5, D and E) showed a striking perivascular congestion of
cells. Significantly, the diffuse appearance of lesions within
the CNS of LT
RAG chimeras (Fig. 5 F) clearly indicated that this role is not shared by LT. Moreover, despite
abundant LT expression in the TNF
TNF mice (Fig. 4),
normal cell movement within the CNS parenchyma failed
to occur (Fig. 5 D) in the absence of TNF. No alteration in
the magnitude or distribution of cells expressing MHC class
II, Mac-1 (CD11b), CD4, iNOS, or vascular cell adhesion molecule 1 could be observed in the absence of LT (data
not shown). The normal expression of iNOS in the TNF/
LT
TNF brain indicated that the activation of this critical
macrophage effector was not dependent on the activities of
either TNF or LT (not shown).
A neuropathological study of fixed tissues from TNF/
LT TNF CNS at day 40 demonstrated clear primary demyelination, despite the lack of both TNF and LT (Fig. 5
G). Lipid-filled glitter cells (phagocytic macrophages), which
are pathognomonic of inflammatory demyelinating disease,
were also present.
Collectively, these findings confirm normal histopathological features of EAE, despite the lack of LT. The abnormal features described above are explained by the absence of TNF alone.
TNFR-IgG Alters the Course of EAE in WT, but Not TNFThe effect of TNF and LT3 blockade on
EAE susceptibility in otherwise unmanipulated WT mice
was examined. Very high doses of TNFR-IgG administered immediately before the onset of clinical neurological
deficits resulted in a delay in the onset of disease when
compared with animals treated with PBS (Fig. 6 A and Table 1), in a manner that was analogous to findings in the
TNF
/
animals (Fig. 2). Similarly, the rate of disease progression and the eventual peak severity of disease was not
affected by treatment, even when treatment was continued
through the emergence of clinical signs. This effect depended exclusively on the TNFR component of the fusion
protein (Fig. 6 B).
|
As an alternative means of studying the activities of LT3
in a TNF-deficient system, LT
3 was blocked in TNF
/
mice by administration of TNFR-IgG (Fig. 6 C and Table
1). Disease onset and disease severity in both human IgG
control- and TNFR-IgG-treated mice was identical, and
although some divergence of the disease curves was observed, there was no indication that LT
3 inhibition influenced its course. Again, most mice were killed at the peak of disease to conform with ethical requirements. Mice from
both groups that were followed through to disease resolution progressed similarly.
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Discussion |
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It is widely accepted that the secreted (LT3) or cell
membrane (LT
1
2) forms of LT exhibit TNF-like properties, particularly since LT
3 can bind to the TNFRs (14).
A feature common to gene-targeted mice lacking TNF
(29, 48), LT
(20), or LT
(26), or of mice in which the
action of these cytokines is blocked during gestation (49), is
that all exhibit changes in lymphoid structures. Nevertheless, the magnitude and precise nature of anatomical
changes in TNF
/
versus LT
/
or LT
/
mice are
largely nonoverlapping, indicating distinct in vivo functions for these molecules. The results of this study, in
which the roles of LT and TNF in autoimmune inflammation were compared, are entirely consistent with the view
that TNF and LT are functionally distinct entities in vivo.
In these studies, the clinical and pathological course of
EAE was influenced in a striking way by the absence of
TNF (Fig. 2 and Table 1), an outcome that appears to be
explained by the abnormal movement of inflammatory cells
within the target tissue early in the clinical course (23).
This phenotype is not due to the failure of inflammatory
cell recruitment per se, because normal numbers of CD45+
cells can be extracted from the perfused whole CNS of
mice lacking TNF at any given time point after immunization (Fig. 5 A; reference 23). TNF-dependent cell adhesion
events are not, therefore, a satisfactory or complete explanation. The importance of TNF in the control of cell
movement within the tissue is supported by the observation that even when disease is established in TNF-deficient mice, leukocytes fail to move freely from a perivascular location into the CNS parenchyma (compare Fig. 5 C
[WT WT] with Fig. 5 D [TNF
TNF]). This consolidates the impression that the normal formation of the inflammatory lesion depends upon an activity of TNF on the
abluminal side of the CNS vascular endothelium as suggested previously (23). Possible mechanisms to explain this effect include a role for TNF in the generation of chemoattractant gradients (50), in the digestion of the extracellular
matrix (51, 52), or in the final effector phase activation of
the recruited inflammatory cells. The relative reduction in
the proportion of non-T inflammatory cells in mice lacking
TNF (Fig. 5 B) or in rats treated with TNFR-IgG (12)
may also indicate that the T cell is less dependent on TNF
for normal tissue infiltration. Irrespective of these considerations, primary demyelination, which is the characteristic
end point of the pathological process and which depends
upon many of the coordinated processes of acquired immunity including antibody production, proceeds without
inhibition in the absence of TNF (Fig. 5 G; reference 23).
Clearly, LT could not compensate for the abnormal
course of disease in TNF
/
mice (Fig. 2), although it was
possible that the emergence of disease, when it occurred,
was indeed a LT-dependent process. To pursue this further, we adopted a combination of approaches, the results
of which are summarized in Table 1. The use of direct immunization in LT-deficient mice was precluded by the failure of these mice to respond to a standard disease induction
protocol (Fig. 2 and Table 1), an explanation being the
absence of peripheral lymphoid organs. Thus, we sought to
examine the role of LT in this model by the use of gene-targeted radiation bone marrow chimeric mice.
The first set of experiments (LT RAG; Fig. 3 C and
Table 1) demonstrated that LT has no unique role in the
clinical manifestations of disease. It was possible, however,
that TNF expression compensated for the absence of LT in
this experiment. It would follow, therefore, that in a TNF-deficient mouse, the role of LT might become apparent, although such an activity would then be regarded as fully
redundant to TNF. Thus, a second set of studies was performed using TNF/LT
TNF chimeras (Fig. 3 D and Table 1). These experiments showed that the absence of LT
had no additional influence on the clinical course of EAE
in TNF-deficient mice. The concept that TNF and LT
have redundant functions in autoimmunity therefore fails
to be supported by these observations, which have been
made despite the knowledge that both cytokines are abundantly expressed in the inflammatory lesion (Fig. 4 and
text) and share common receptor binding (14). Although
LT
1
2-LT
R interactions do seem to elicit a cytotoxic
signal (22), it seems likely that the importance of the LT
3-
TNFR interaction in cytotoxicity in the mouse has been
overestimated (43). The use of LT
/
mice in this study,
which lack both the secreted and membrane forms of LT, indicates that neither plays an indispensable role in the processes leading to clinical manifestations of EAE.
The ability of therapeutic inhibition of TNF and LT to
modulate, and in some cases ablate, the clinical manifestations of EAE is well described (11) and has represented
the most persuasive source of experimental evidence for a
key role for these molecules in the pathogenesis of CNS
autoimmune inflammation. In the Lewis rat, inhibition of
TNF and LT3 resulted in abrogation of disease, but did
not impede T lymphocyte accumulation within the CNS
(12, 53). The results obtained after the administration of
TNFR-IgG to WT C57BL/6 mice (Fig. 6 A and Table 1
F) precisely reproduced the clinical outcome observed in
TNF
/
mice (Fig. 2 and Fig. 3 D). Differences in the extent of disease inhibition by TNFR-IgG between other
models and MOG 35-55-induced EAE in C57BL/6 mice
may be due to the severity of the clinical disease and particularly the development of substantial primary demyelination in the MOG model. These findings reinforce the need for a reevaluation of the influences of TNF inhibition in
other autoimmune disease models, particularly with respect
to the nature of the inflammatory process.
In summary, once factors such as immune competence
and genetic heterogeneity are accounted for, EAE in the
absence of LT progresses in a manner that is indistinguishable from controls, as measured by the clinical manifestations of disease, the generation of inflammatory lesions, and
specific target damage in the form of demyelination. On
the other hand, the lack of TNF alone results in profound
changes in the movement of leukocytes within the CNS
(Fig. 5) and LT appears unable to compensate for its absence (Fig. 4 and Fig. 5 D). These findings do not support
the concept that LT is an independent mediator of acute
autoimmune inflammatory processes. Therefore, the purpose of abundant LT expression within these lesions is unexplained. The capacity of transgenic overexpression of
LT to generate organized ectopic lymphoid structures
within tissues (54), raises the possibility that LT establishes
the cellular architecture of chronic inflammatory lesions.
Further studies investigating the role of LT in the late
phases of autoimmune inflammation are warranted.
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Footnotes |
---|
Address correspondence to Jonathon D. Sedgwick, Centenary Institute of Cancer Medicine and Cell Biology, Bldg. 93, Royal Prince Alfred Hospital, Missenden Rd., Camperdown, Sydney, NSW 2050, Australia. Phone: 61-2-9565-6116; Fax: 61-2-9565-6103; E-mail: j.sedgwick{at}centenary.usyd.edu.au
Received for publication 22 December 1997 and in revised form 18 February 1998.
Ms. Lisa Galli's role in the generation of the LTThese studies were supported by the National Health and Medical Research Council (NHMRC) and the National Multiple Sclerosis Society of Australia. D.S. Riminton is supported by a NHMRC postgraduate scholarship, D.H. Strickland by an Elizabeth Albiez fellowship from the National Multiple Sclerosis Society of Australia, and J.D. Sedgwick by a Wellcome Trust Senior Research fellowship in Australia (1992-1996) and an NHMRC fellowship.
Abbreviations used in this paper
CNS, central nervous system;
EAE, experimental autoimmune encephalomyelitis;
ES, embryonic stem;
iNOS, inducible nitric oxide synthase;
LT, lymphotoxin;
MOG, myelin oligodendrocyte glycoprotein;
MS, multiple sclerosis;
RAG, recombinase activation
gene;
RT-PCR, reverse transcriptase PCR;
TNFR-IgG, TNFR-human
IgG fusion protein;
WT, wild type. Radiation bone marrow chimeras:
WT WT, WT bone marrow transplanted into WT recipients ;
WT
RAG, WT bone marrow transplanted into RAG-1
/
recipients;
LT
RAG, LT
/
bone marrow transplanted into RAG-1
/
recipients;
TNF
RAG, TNF
/
bone marrow transplanted into RAG-1
/
recipients;
TNF
TNF, TNF
/
bone marrow transplanted into TNF
/
recipients;
TNF/LT
TNF, TNF/LT
/
bone marrow transplanted
into TNF
/
recipients.
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References |
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