| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Is Critical to the Control of Murine Autoimmune Encephalomyelitis and Regulates Both in the Periphery and in the Target Tissue: A Possible Role for Nitric Oxide1
*
Neurosciences Research Unit, Canberra Hospital, and
University of Sydney Canberra Clinical School, Canberra, Australian Capital Territory, Australia; and
John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory, Australia
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
have normally been considered cytotoxic and
proinflammatory molecules, respectively, in the setting of the central
nervous system inflammatory disease autoimmune encephalomyelitis (EAE).
Using mice lacking the ligand binding chain of the IFN- receptor
(IFNR-/-), we have previously shown that IFN- is
not essential for myelin oligodendrocyte glycoprotein peptide
(MOG35–55) induced EAE expression but is in fact essential
for its down-regulation. Here we examined the downstream molecular and
cellular mechanism(s) of IFN- regulation and demonstrate that
neither IL-4 nor IL-10 appear to play a role in down-regulation nor do
various lymphoid cell populations. Cells of the macrophage lineage are
key to down-regulation as evidenced by the fact that peritoneal exudate
cells from IFNR+/+ mice inhibit Ag-driven proliferation
of IFNR-/- lymphocytes, whereas
IFNR-/- peritoneal exudate cells do not. High levels
of reactive nitrogen intermediates are detected in the former cultures
but not the latter, and the inhibition of proliferation is reversible
with an inhibitor of inducible NO synthase, indicating a key role for
NO in down-regulation. Studies with bone marrow chimeras indicate that
down-regulation occurs not only systemically but also within the target
tissue. These data suggest that IFN- down-regulates EAE by inducing
inducible NO synthase and subsequently NO production, both by
macrophages in the periphery and, by inference, microglia and
astrocytes in the target tissue. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
With the exception of a study in immunocompromised animals
(3), EAE has been described as being a Th1 T cell-mediated
disease (4, 5, 6), and therefore a pivotal role for the Th1
proinflammatory cytokine IFN- in EAE pathology has been suggested.
Numerous effects of IFN- in promoting inflammation have been
described, such as: macrophage activation; up-regulation of both class
I and class II MHC molecules necessary for (self) Ag presentation;
induction of adhesion molecules such as ICAM-1 and VCAM, perhaps
promoting homing of inflammatory cells; and induction of expression of
receptors for other cytokines, in particular TNF receptors (for a
review, see Ref. 7). Therefore, the implication has been
that IFN- is an essential contributor to EAE pathology.
We have previously shown, however, that mice lacking the ligand binding
chain of the IFN- receptor (IFNR-/-) and
therefore unable to respond to IFN-, develop severe and usually
fatal EAE when immunized with human myelin oligodendrocyte glycoprotein
peptide 35–55 (MOG35–55), whereas control mice
expressing the gene are resistant to disease induction, indicating that
IFN- is not essential for disease induction (8).
Furthermore, passive transfer of disease with
MOG35–55-specific lymphoid cells from
IFNR-/- mice produces, in knockout
(IFNR-/-) mice, severe EAE from which the
recipients fail to recover. The same cells produce equally severe
disease in IFNR+/+ control mice, but
importantly all the recipients recover fully. These results provide
definitive evidence that IFN- is not essential for the generation or
function of anti-MOG35–55 effector cells,
but that it does play an obligatory role in down-regulating the
disease.
We have suggested that, at least in the current model, this down
regulation must act indirectly through a secondary mediator.
IFNR-/- effector cells produce extremely
high levels of IFN- (8) but cannot respond to it
because of the lack of the receptor. When these cells are transferred
into wild-type (WT) recipients the recipient cells can and do respond
to IFN- with the production of some mediator(s) which ultimately
feeds back and down-regulates the effector cells.
In this paper we examine possible mechanisms by which IFN-
down-regulates disease. We provide evidence that neither IL-4 nor IL-10
appear to be involved nor are CD8+ T cells. In
fact, lymphoid cells do not appear to play a role as evidenced by the
fact that transfer of large numbers of IFNR+/+
spleen cells into IFNR-/- animals fails to
confer on the IFNR-/- mice the ability to
recover from passively induced disease. In vitro studies of inhibition
of Ag-specific proliferation of IFNR-/- lymphoid cells
show that while lymphoid cells from WT mice do not inhibit
proliferation, peritoneal exudate cells (PEC) readily do so in a
dose-dependent manner and high levels of reactive nitrogen
intermediates (RNI) can be detected in such cultures. Inhibition of
proliferation is reversible with an inhibitor of inducible NO synthase
(iNOS), indicating a key down-regulatory role for NO. Furthermore, the
use of chimeric mice indicates that down-regulation occurs not only
systemically but also at the level of the target tissue, the
CNS.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
129/Sv, H2b mice of either sex, homozygous
for disrupted gene (IFNR-/-) and for null
mutations (IFNR+/+), were obtained from Dr.
Michel Aguet (University of Zurich, Zurich, Switzerland). Disruption of
the IFNR gene was verified using PCR and primers as described
(9). Mice were maintained in pathogen-free conditions and
used between the ages of 8 and 14 wk. All animal experimentation was
approved by the Animal Ethics Committee of the Australian National
University.
The peptide corresponding to amino acids 35–55 of human MOG (Met-Glu-Val-Gly-Trp-Tyr-Arg-Pro-Pro-Phe-Ser-Arg-Val-Val-His-Leu-Tyr-Arg-Asn-Gly-Lys) was synthesized by standard Fmoc chemistry, and the purity was determined by reverse-phase HPLC (Biomolecular Resource Facility, John Curtin School of Medical Research, Australian National University). MOG35–55 (1.67 mg/ml in saline) was emulsified in an equal volume of CFA containing 0.5 mg/ml Mycobacterium butyricum and 4 mg/ml Mycobacterium tuberculosis (H37Ra). Each mouse received 120 µl emulsion distributed in footpads of both hind feet and in the nape of the neck. The total dose of MOG35–55 was 100 µg/mouse. Immediately before and 2 days after injection of emulsion the mice received an i.v. injection of 300 ng pertussis toxin (List Biological Laboratories, Campbell, CA) in 250 µl PBS.
Scoring of clinical disease
Disease severity was scored on a scale of 0 (asymptomatic) to 5 (moribund). Where there were no detectable signs of EAE, the score was designated 0; slight weakness of the tail, 1; definite tail and partial hind limb paralysis, 2; tail paralysis and moderate hind limb paralysis, 3; complete paralysis of the tail and hind limbs often associated with incontinence, 3.5; paralysis of tail and hind limbs with moderate forelimb weakness, 4; and total paralysis of hind and forelimbs, 5. The mean maximal clinical score and mean day of onset was calculated for each group of animals.
Abs and treatment schedules
The anti-murine IL-4 was the rat IgG1 mAb 11B11
(10) and the anti-IL-10 (2A5) was also an IgG1 mAb and
was a gift of Dr. K. Moore (DNAX, Palo Alto, CA). Control Ab was an
IgG1 anti-ß-galactosidase, a gift from Dr. John Abrams (DNAX).
Anti-IFN- was R46A2 from the American Type Culture Collection (ATCC,
Manassas, VA). All Abs were given on days -1, 0, +1, 2, 4, 6, 8, 10,
and 12 in relation to passive transfer of cells on day 0. Depletion of
CD8+ cells was done using an anti-CD8 Ab
(ATCC TIB-210) kindly donated by Dr. Guna Karupiah (John Curtin School
Medical Research, Canberra, Australia). Mice were given 1 mg of Ab,
i.p., at 3-day intervals for a total of four doses. Treatment was begun
on day 6 following passive transfer. Spleen and lymph nodes assayed 2
days after the last treatment had 
2% CD8+
cells as assessed by FACS analysis.
Passively transferred EAE
IFNR-/- mice were immunized with
MOG35–55 as above. Fourteen days later they were
killed, and spleen cell suspensions were prepared and cultured in RPMI
1640 + 10% FCS with 10 µg/ml MOG35–55 for 4
days at 37°C. Cultures were harvested, cells washed three times with
HBSS and transferred at 5–8 x 107 cells
i.v./recipient.
Cell culture and proliferation assay
Spleens were removed from mice 15 days after sensitization, and proliferation assays were performed in 96-well microtiter plates. Cells, 1 x 106/ml (200 µl/well), were cultured with RPMI 1640 + 10% FCS, 5 x 10-5 M 2-ME, penicillin, streptomycin, and neomycin (PSN), and 10 µg/ml MOG35–55. [3H]Thymidine was added at 72 h and cultures harvested 18–20 h later. When inhibiting cells were added to the cultures, the lymphoid cells were at a concentration of 2 x 106 and 100 µl was added/well. Inhibitors were then added in 100 µl.
Peritoneal exudates were induced by injecting 0.5 ml 3% thioglycollate broth i.p. 3 days before harvest. At harvest, 8 ml cold HBSS was injected i.p. and then removed; cells were washed three times, counted, and added to lymphoid cultures in the numbers shown in the Tables. When PEC were added 48 h after initiation of cultures, lymphoid cells were washed extensively (three times) before addition of the PEC. N-methyl-L-arginine acetate (L-NMA) was prepared essentially according to the method of Patthy et al. (11) and added to cultures at a final concentration of 50 µM.
Measurement of RNI
The concentration of nitrate plus nitrite ions were used as an indirect measure of the amount of NO being produced. Measurement was performed using a microplate assay essentially according to the method of Rockett et al. (12). Briefly, nitrite was measured by addition of 100 µl of Griess reagent to 30 µl of test sample. Protein was removed by addition of 100 µl of trichloroacetic acid followed by centrifugation, and the OD of the sample read at 540 nm with a reference at 650 nm using a microplate reader (Molecular Devices,, Menlo Park, CA). For nitrate measurements, the nitrate was first converted to nitrite by incubation with nitrate reductase and NADPH (Boehringer Mannheim, Mannheim, Germany) for 20 min. The results were quantified by reading against appropriate nitrite and nitrate standard curves.
Generation of radiation bone marrow chimeras
Bone marrow cells were harvested from the long bones of either
IFNR+/+ or IFNR-/-
mice by flushing with cold RPMI. Cells were deaggregated by gently
cycling through a 26-guage needle and then through a 400 mesh stainless
steel strainer. Cells were washed three times, and nucleated cells were
counted and transferred, i.v., at 7 x
106/recipient. Recipients had been prepared by
two doses of whole body irradiation with 5.5 Gy/dose from a Cobalt 60
source, given 48 h apart. Bone marrow cells were transferred
within 5 h of the last dose of irradiation. Mice were maintained
on antibiotics via the drinking water for the first 4 wk posttransfer.
At 8 wk posttransfer, all mice were tail bled and the extent of
engraftment was determined. Initially we attempted to define the
genotype of peripheral leukocytes using Abs to the IFN- receptor and
flow cytometric analysis. Interesting, using two different Abs (rabbit
polyclonal IgG (Santa Cruz Biotechnology, Santa Cruz, CA) and hamster
monoclonal IgG (Genzyme Diagnostics, Cambridge, MA)), we were unable to
detect more that 25% positive cells in the circulation of
IFNR+/+ control mice even if the cells had
been previously activated with mitogen in vitro. This was unexpected in
light of reports stating that all cells express the IFN- receptor
(13). We did not feel that 25% was an adequate window
with which to examine for chimerism, so we therefore chose to do
cellular PCR on peripheral blood of each of the chimeras and for
comparison three animals each of the control WT or KO mice.
Three drops of peripheral blood were obtained from the tails of mice
and DNA extracted following standard techniques. Using primers
described previously (9), DNA was amplified by 25 cycles
of PCR under conditions that result in a single amplification product
of the correct size: 1 min denaturation at 94°C, 1 min annealing at
54°C, and 2 min extension at 72°C. Amplified DNA was analyzed by
gel electrophoresis. Under these conditions 9 of 12 WT recipients of
IFNR-/- bone marrow appeared to have only
IFNR-/- peripheral cells (in three the
sample was apparently inadequate), and similarly 6 of 12 of the
IFNR-/- recipients of WT bone marrow showed
only the 200-bp product representing the IFNR gene, indicating
certainly a great majority of IFNR+/+
peripheral cells (Fig. 1
). These animals
were used in subsequent experiments. The chimeras and seven control
IFNR+/+ and IFNR-/-
mice received 5 x 107 Ag activated lymphoid
cells from MOG35–55-immunized
IFNR-/- donor mice and were observed for
disease for 45 days.
|
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
IFNR+/+ mice receiving
MOG35–55-specific effector cells from
IFNR-/- mice develop severe disease from
which they recover. To determine whether IL-4 or IL-10 play a role in
this recovery process we treated the IFNR+/+
recipients with neutralizing Ab to either of the cytokines beginning
the day before cell transfer and observed the mice for the possible
loss of the ability to recover. Table I
shows that inhibiting IL-4 or IL-10 had no effect on the ability of
IFNR+/+ animals to recover. Another group of
recipient mice were treated with anti-IFN- Abs, and these
animals failed to begin to recover until the Ab treatment was
discontinued at day 8. The mice then recovered by day 18 (data not
shown). This again indicates the essential nature of IFN- in the
recovery process.
|
To examine the function of CD8+ cells in
recovery, IFNR+/+ mice were depleted of
CD8+ cells according to the treatment described
in Materials and Methods. This treatment of mice with EAE
resulted in <2% CD8+ cells in spleen and lymph
nodes at day 17 post-cell transfer. The data in Table II, representing one of two experiments,
demonstrate that such depletion of CD8+ cells did
not alter the ability of IFNR+/+ mice to
recover from disease.
|
R-/- recipients
In an attempt to identify a cell type responsible for contributing
to the recovery process we transferred into
IFNR-/- mice one spleen equivalent of
lymphoid cells (1 x 108) from naive
IFNR+/+ animals 24 h before the induction
of EAE passively with IFNR-/- effector
cells. These animals developed severe EAE as did
IFNR-/- animals receiving no cells or cells
from naive IFNR-/- mice (to control for
possible inhibition of disease due to crowding); none of the recipients
recovered from disease by day 14 and some had in fact died
indicating the absence or insufficient numbers of a cell population in
the IFNR+/+ spleen lymphoid population capable
of orchestrating recovery (data not shown).
In vitro Ag-specific proliferation of
IFNR-/- lymphoid cells and its inhibition by
IFNR+/+ cells
No inhibition with IFNR+/+ lymphoid
cells. Splenic lymphoid cells from
MOG35–55-immunized
IFNR-/- mice proliferate readily in vitro
when stimulated with specific Ag. Using this in vitro assay system we
attempted to define a cell population in WT animals able to inhibit
this proliferation. Neither spleen nor lymph node lymphocytes from
IFNR+/+ mice, when mixed at various
concentrations with IFNR-/- spleen cells,
inhibited Ag-specific proliferation of the latter (data not shown).
Inhibition with IFNR+/+PEC. Naive mice were given i.p. injections of thioglycollate and
PEC harvested 3 days later. These PEC were added at various
concentrations to IFNR-/- spleen cells from
MOG35–55 immunized mice;
MOG35–55 was also added and proliferation
determined 4 days later (Table III). At
high concentrations (8 x 104), PEC from
both IFNR+/+ and
IFNR-/- mice gave inhibition of
proliferation, indicating perhaps a nonspecific crowding effect. At
lower concentrations however (4 x 104 and
2 x 104) IFNR+/+
PEC gave 100% and 75% inhibition, respectively. The same number of
PEC from naive IFNR-/- mice had no effect.
Similar results were also obtained when the addition of
IFNR+/+ PEC to cultures of
IFNR-/- spleen cells plus
MOG35–55 was delayed until 48 h. after the
initiation of such cultures (Table IV).
|
|
R+/+ PEC
IFNR-/- spleen cells from immunized
mice were cultured with IFNR+/+ or
IFNR-/- PEC in the presence or absence of
specific Ag (MOG35–55) for 3 days at which time
supernatants were harvested and assayed for nitrate and nitrite.
Proliferation of spleen cells was also assessed. As shown in Fig. 2A only those cultures
containing Ag-stimulated spleen cells plus
IFNR+/+ PEC produced high levels of RNI.
Cultures without Ag or with IFNR-/- PEC with
or without Ag showed only background levels of RNI. There was also an
inverse relationship between increased RNI production and lymphoid cell
proliferation (Fig. 2B).
|
Cultures of spleen cells from
MOG35–55-immunized
IFNR-/- mice were set up as before with
specific Ag in the presence of various numbers of PEC from either
IFNR+/+ or IFNR-/-
mice. L-NMA was also added to some of the cultures with
IFNR+/+ PEC. As seen in Table V IFNR+/+ PEC
totally inhibited proliferation of the spleen cells whereas equal
numbers of PEC from IFNR-/- mice had no
inhibitory effect. L-NMA restored the proliferative
response to between 25 and 100% depending on the number of
IFNR+/+ PEC that had been added to the
cultures. The inhibition seen when IFNR+/+ PEC
are added 48 h after initiation of culture was also shown to be
reversible by the addition of L-NMA when added at the same
time as the PEC (data not shown).
|
R+/+ and
IFNR-/- mice behave differently than either the
IFNR+/+ or IFNR-/- mice with respect to
recovery from disease
Having shown that IFNR+/+ mice
have cells, probably of the macrophage lineage, that are capable of
shutting off IFNR-/- lymphoid cell
proliferation, we addressed the question as to where in the
IFNR+/+ mice the down-regulation of disease
was occurring. In other words, was it in the peripheral lymphoid
organs or in the target tissue or in both? To examine this we
generated chimeric mice as described in Materials and
Methods. Mice thus constructed have
IFNR+/+ (WT) cells in the periphery (i.e., the
ability to respond to IFN-) and IFNR-/-
(KO cells) in the CNS (the inability to respond to IFN-), or KO
cells in the periphery and WT cells in the CNS. EAE effector cells were
generated in IFNR-/- mice as before, and
5 x 107 cells were transferred to four
groups of mice: IFNR+/+,
IFNR-/-, and the two types of chimeras. Fig. 3 shows individual clinical scores for
animals in the four groups. As seen previously,
IFNR+/+ WT recipients developed severe EAE
from which they recovered, whereas IFNR-/-
recipients developed severe disease and died. Interesting, both types
of chimeras developed severe disease from which they neither died nor
recovered. Instead, they all developed a chronic course of disease with
some animals showing modest recovery with subsequent relapse.
|
R+/+) group. Sera were pooled (to obtain
an adequate volume for assay) in each group, and RNI determinations
were performed. The samples from the intact
IFNR+/+ mice and the
IFNR-/- mice with
IFNR+/+ reconstituted periphery had 7.2 and
8.7 µM RNI, respectively. There were no detectable RNI in the sample
from the IFNR+/+ mice with an
IFNR-/- periphery. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
receptor
develop severe EAE and usually die following immunization with human
MOG35–55 peptide, whereas their WT counterparts
possessing the receptor are resistant to EAE induction. This
demonstrates that IFN- is not essential as a proinflammatory
molecule in EAE and suggests that it may in fact protect against
disease. Evidence for the essential nature of IFN- in
down-regulation of disease is shown by the fact that
IFNR-/- mice die following severe disease
induced by passive transfer of effector cells from
MOG35–55 immunized
IFNR-/- donor mice whereas
IFNR+/+ mice (which differ only in their
ability to respond to IFN-) develop equally severe passive disease
but recover fully. We have suggested that recovery occurs as a result
of cells in the IFNR+/+ mice responding to
IFN- being produced by the IFNR-/-
effector cells and producing a factor(s) which feeds back to the
effector cell and shuts them off (8).
In the current investigation we have examined a number of possible
cellular and molecular mechanisms that might contribute to the IFN-
down-regulation of disease. The Th1 and Th2 CD4+
T cell populations cross-regulate one another because their respective
cytokines act antagonistically (14), and therefore IL-4
and IL-10 both have been implicated in down-regulation of EAE
(15, 16, 17). We treated IFNR+/+ mice
with Abs to either IL-4 or IL-10 to determine whether this would alter
their ability to recover from passively induced EAE. Neither treatment
altered the course of disease in these animals, indicating that
neither molecule appears to be the feedback effector shutting off
disease. That treatment with an anti-cytokine Ab in such a model can
alter the course of disease is seen by the fact that treating the
IFNR+/+ mice with anti-IFN- Ab resulted
in augmented disease until the Ab was discontinued. It could of course
be argued that one or both of these cytokines play a role in
down-regulation and do so at the level of the target tissue and that
the anti-cytokine Abs in these experiments did not cross the
blood-brain barrier. However, this seems unlikely in light of the
results obtained from the chimeric mice suggesting that down-regulation
occurs at the level of both the target tissue and the periphery. If the
cytokines in question were playing a role, then Ab treatment should
have at the very least caused a more chronic disease in the
IFNR+/+ mice by its ability to block cytokine
function in the periphery.
From a cellular perspective we examined the role of
CD8+ cells in down-regulation of disease and
found that depleting IFNR+/+ mice of
CD8+ cells did not impair their ability to
recover from passively induced disease suggesting a negligible role for
this cell type in regulation. We could not in fact demonstrate any
involvement of lymphoid cells in recovery in this model. Transferring
one spleen equivalent of lymphoid cells from
IFNR+/+ mice into
IFNR-/- failed to alter the rapidly lethal
outcome of passively induced disease in these animals.
In vitro studies, based on the observation that lymphoid cells from
MOG35–55 immunized
IFNR-/- mice proliferate extensively when
stimulated with specific Ag, suggested both a cellular and a molecular
mechanism for down-regulation of EAE by IFN-. PEC harvested from
thioglycollate stimulated IFNR+/+ mice and
added to IFNR-/- spleen cultures at the time
of addition of Ag inhibited proliferation in a dose-dependent manner.
Equal numbers of PEC from IFNR-/- mice had
no such effect, indicating that inhibition was not merely a matter of
crowding. Similar inhibition was observed when PEC were added 48 h
after the initiation of culture, demonstrating that ongoing
proliferation as well as initiation of proliferation is sensitive to
down-regulation.
Measurement of RNI in these mixed cultures revealed high levels in
those containing PEC from IFNR+/+ mice plus
MOG35–55. In the absence of specific Ag, no
significant levels of RNI were produced nor were RNI detected when PEC
from IFNR-/- mice were added to spleen
cells, with or without Ag. Finally, the inhibition of proliferation,
both early and late, was totally reversible by the addition of
L-NMA, an inhibitor of NO production via inhibition of
iNOS. Taken together, these data provide strong support for a central
role for IFN--driven NO production by macrophages in down-regulating
effector cell proliferation leading to recovery from disease.
NO mediates a great variety of biological functions. For example, it regulates vascular tone (18), causes platelet activation (19), acts as a neurotransmitter of nonadrenergic, noncholinergic innervation (20), is tumoricidal and microbicidal (21, 22), and plays the role of trans-synaptic retrograde messenger in the brain, thus participating in synaptic plasticity (23, 24). It has also been implicated in a number of immunopathologies including EAE and MS (25, 26, 27). In fact, until rather recently the majority of reports concerning NO and autoimmunity have focused on NO as a contributor to the pathogenesis of disease mainly due to the observations that autoimmune diseases correlate with an overproduction of NO and that often administration of NO inhibitors prevented development of disease, e.g., glomerulonephritis (28), arthritis (29, 30), diabetes (31), and of course EAE (27, 32, 33, 34). Contrary evidence is now appearing, however, that despite its demonstrated role in certain tissue pathologies, NO plays a role in down-regulation of adaptive immune responses (35, 36, 37, 38). For example, we have shown that in vivo inhibition of iNOS not only exacerbates EAE in the Lewis rat but also converts the relatively resistant PVG rat to high susceptibility (38). Susceptibility in the PVG rats correlated with the reduction of serum NO levels following immunization from 3-fold greater than the Lewis rats to levels similar to or below those found in the Lewis. Furthermore, inhibition of iNOS in vivo led to a 3-fold increase in Ag-specific proliferation by lymphocytes from immunized PVG rats. These data indicate that NO might regulate the development of autoimmune reactivity by limiting proliferation of autoreactive lymphocytes.
Stimuli that induce NO production via iNOS include cytokines and
microbial products (22), and the cells that respond to
these stimuli are widely distributed and include peripheral macrophages
as well as resident CNS cells such as microglia and astrocytes
(39). In the context of the present work, the cytokine
IFN- is of central importance, and this cytokine has been shown to
be the major inducer of iNOS (40). IFN- induces iNOS in
peripheral macrophages as well as cells in the CNS. With respect to CNS
cells, IFN- is sufficient to induce NO release from microglial cells
but astrocytes are provoked into NO production only by a combination of
IFN- and TNF-
(39).
We have suggested previously (8) that recovery of
IFNR+/+ mice from passively induced
MOG35–55 EAE is driven by IFN- and that this
happens as a result of some cell type(s) in the
IFNR+/+ mice responding to the high levels of
IFN- being produced by the transferred
IFNR-/- effector cells with the production
of a factor(s) that then feeds back and silences the effector cells. We
now suggest that the factor responsible for down-regulation of disease
is NO, and that the cells producing NO under the upstream influence of
IFN- are most likely macrophages in the periphery and, based on the
experiments reported here on chimeric mice, the resident glial cells in
the target tissue. This latter suggestion derives from the observation
that the chimeras with WT (IFNR+/+) reactivity
to IFN- expressed only in the CNS did not succumb to the lethal
effects of disease as did the intact IFNR-/-
mice, indicating a very definite and significant contribution to
immunoregulation by the target tissue itself. A role for NO, at least
in the periphery, is supported not only by the in vitro experiments
showing NO production by PEC from IFNR+/+ mice
but also from the in vivo observation in the chimera experiment that
sera from mice with IFNR+/+ periphery, either
intact WT or chimera had significant levels of RNI, whereas mice with
IFNR-/- periphery did not.
It is interesting to note that both types of chimeras developed a
moderately severe chronic disease from which none had recovered at the
termination (for ethical reasons) of the experiment 45 days after
initiation. Chronicity could be explained on the basis that effector
cells in an IFNR+/+ periphery are being
down-regulated and/or eliminated by NO production in the periphery, but
the effector cells that have entered the
IFNR-/- CNS, where the regulating signal
cannot be made, continue to orchestrate disease. Conversely, effector
cells in an IFNR-/- periphery are
continually proliferating, recirculating, and entering the
IFNR+/+ CNS where they sustain pathology but
are also continually eliminated because of the ability of
IFNR+/+ cells to respond to IFN- with NO
production. With respect to the cell type making NO, in this latter
case we would suggest that because the effectors are making large
quantities of both IFN- and TNF (8) that both
astrocytes and microglia are making NO (39). This is
currently under investigation.
There are a number of possible mechanisms by which NO down-regulates the immune response. Certainly, as demonstrated here and elsewhere (41), NO inhibits lymphocyte proliferation, and the suggestion has been made that this is by preventing the activation of Janus kinases (42), molecules critical in cytokine receptor signal transduction (43). More recent studies have described a NO-induced bias of cytokine gene expression in which NO preferentially down-regulates Th1-type cytokines specifically while up-regulating Th2-associated molecules resulting in a Th2 bias (44). Such bias could shut off the Th1 pathology of EAE. A similar regulatory effect might be exerted by NO at the level of Ag presentation, as it has been shown that NO induces transcription of the IL-12 p40 gene in macrophages but not the p35 gene. Because the p40 homodimer acts as an antagonist for IL-12 (45), this might lead to less Th1 reactivity in the presence of NO.
Expression of adhesion molecules has been found to be down-regulated by NO and hence lymphocyte migration into the CNS might be impaired and contribute to down-regulation of the disease process. Finally, NO can, depending on concentration, be an inducer or suppressor of apoptosis and/or necrosis in a number of different systems (46). Several studies have indicated that elimination of inflammatory T cells and macrophages from the CNS during EAE might be the result of apoptosis (47, 48, 49). There is also recent evidence that Th1 cells are more prone to apoptosis than are Th2 cells (50), which would again be beneficial in regulating EAE, which is essentially a Th1-mediated disease.
In the present system it is likely that NO acts at any number of these levels to down-regulate CNS inflammation. The observation that, at least in vitro, rapidly proliferating cells can be shut off when NO-producing PEC are added at the height of proliferation suggests an important role for cell death—either apoptosis or necrosis—in regulation. Because regulation is occurring at both the systemic level and at the level of the target tissue, it will be of interest to determine whether cell death of effector cells is occurring by the same mechanism in both places.
We have demonstrated that the normally considered proinflammatory
cytokine IFN- is, in the context of MOG-induced EAE, essential in
down-regulation of the disease and that reactivity to IFN- in the
target tissue itself, i.e., the CNS, as well as the periphery plays a
critical role in this down-regulation. The beneficial role of IFN-
in the CNS is in contrast to recent work described by Horwitz et al.
(51). These investigators expressed IFN- in the CNS of
mice under the myelin basic protein (MBP) promoter such that the
cytokine was produced by oligodendrocytes. They found demyelination in
a proportion of their mice that was both age and gender related. The
effect of any cytokine is undoubtedly dose, time and site dependent and
these parameters in the work of Horwitz et al. are vastly different
from what would be found for IFN- produced during an inflammation of
the CNS where the cytokine is produced mainly by T cells under control
of the TCR, i.e., upon Ag recognition. The prolonged over-expression of
IFN- in the CNS with subsequent production of demyelination does not
preclude the possibility of IFN- in more physiological circumstances
(an inflammatory episode) contributing to down regulation of
inflammation.
With respect to mechanism of action of IFN-, we present in vitro
data which show clearly that reactivity to IFN- results in the
production of large amounts of RNI by peripheral mononuclear cells with
subsequent inhibition of effector cell proliferation. We suggest that
this IFN--driven NO production in the periphery, and by inference in
the CNS as well, is responsible for limiting disease. In light of the
robust nature of the results presented here, we suggest that perhaps
the data on the therapeutic use of IFN- in the clinical setting of
the human CNS inflammatory disease MS should be re-examined with an eye
to the possible retrial of IFN- or perhaps another inducer of the
downstream molecule NO, in MS.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. David O. Willenborg, Neurosciences Research Unit, Canberra Hospital, P.O. Box 11, Woden, ACT 2606, Australia. E-mail address:
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; iNOS, inducible NO synthase; L-NMA, N-methyl-L-arginine acetate; RNI, reactive nitrogen intermediates; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; IFNR+/+, IFN- receptor wild type; WT, wild type; IFNR-/-, IFN- receptor knockout; KO, knockout; PEC, peritoneal exudate cells.
Received for publication May 25, 1999. Accepted for publication September 1, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
4 integrin by CD4 T cells is required for their entry into brain parenchyma. J. Exp. Med. 177:57. plays a critical down-regulatory role in the induction and effector phase of myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis. J. Immunol. 157:3223.[Abstract]
receptor. Science 259:1742. and its receptor. Annu. Rev. Immunol. 11:571.[Medline]
and nitric oxide may exacerbate CNS inflammation in experimental autoimmune encephalomyelitis. J. Neuroimmunol. 89:122.[Medline]
. Nat. Med. 3:1037.[Medline]
This article has been cited by other articles:
|
|
 |
|
  A. J. Canonigo-Balancio, C. Fos, T. Prod'homme, S. Becart, and A. Altman SLAT/Def6 Plays a Critical Role in the Development of Th17 Cell-Mediated Experimental Autoimmune Encephalomyelitis J. Immunol., December 1, 2009; 183(11): 7259 - 7267. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Annunziato and S. Romagnani Do studies in humans better depict Th17 cells? Blood, September 10, 2009; 114(11): 2213 - 2219. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Fujita, T. Otsuka, M. Mizuno, C. Tomi, T. Yamamura, and S. Miyake Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 Modulates Experimental Autoimmune Encephalomyelitis via an iNKT Cell-Dependent Mechanism Am. J. Pathol., September 1, 2009; 175(3): 1116 - 1123. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Lees, Y. Iwakura, and J. H. Russell Host T Cells Are the Main Producers of IL-17 within the Central Nervous System during Initiation of Experimental Autoimmune Encephalomyelitis Induced by Adoptive Transfer of Th1 Cell Lines J. Immunol., June 15, 2008; 180(12): 8066 - 8072. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Mangalam, D. Luckey, E. Basal, M. Behrens, M. Rodriguez, and C. David HLA-DQ6 (DQB1*0601)-Restricted T Cells Protect against Experimental Autoimmune Encephalomyelitis in HLA-DR3.DQ6 Double-Transgenic Mice by Generating Anti-Inflammatory IFN-{gamma} J. Immunol., June 1, 2008; 180(11): 7747 - 7756. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Tian, J. Lappalainen, M. Autero, S. Hanninen, H. Rauvala, and C. G. Gahmberg Shedded neuronal ICAM-5 suppresses T-cell activation Blood, April 1, 2008; 111(7): 3615 - 3625. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Balabanov, K. Strand, R. Goswami, E. McMahon, W. Begolka, S. D. Miller, and B. Popko Interferon-{gamma}-Oligodendrocyte Interactions in the Regulation of Experimental Autoimmune Encephalomyelitis J. Neurosci., February 21, 2007; 27(8): 2013 - 2024. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Minguela, S. Pastor, W. Mi, J. A. Richardson, and E. S. Ward Feedback Regulation of Murine Autoimmunity via Dominant Anti-Inflammatory Effects of Interferon {gamma} J. Immunol., January 1, 2007; 178(1): 134 - 144. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Mana, D. Linares, S. Fordham, M. Staykova, and D. Willenborg Deleterious Role of IFN{gamma} in a Toxic Model of Central Nervous System Demyelination Am. J. Pathol., May 1, 2006; 168(5): 1464 - 1473. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Liu, D. Huang, M. Matsui, T. T. He, T. Hu, J. DeMartino, B. Lu, C. Gerard, and R. M. Ransohoff Severe Disease, Unaltered Leukocyte Migration, and Reduced IFN-{gamma} Production in CXCR3-/- Mice with Experimental Autoimmune Encephalomyelitis J. Immunol., April 1, 2006; 176(7): 4399 - 4409. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Dawes, S. Petrova, Z. Liu, D. Wraith, P. C. L. Beverley, and E. Z. Tchilian Combinations of CD45 Isoforms Are Crucial for Immune Function and Disease J. Immunol., March 15, 2006; 176(6): 3417 - 3425. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Chen and C. F. Brosnan Exacerbation of Experimental Autoimmune Encephalomyelitis in P2X7R-/- Mice: Evidence for Loss of Apoptotic Activity in Lymphocytes. J. Immunol., March 1, 2006; 176(5): 3115 - 3126. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Galazka, M. Stasiolek, A. Walczak, A. Jurewicz, A. Zylicz, C. F. Brosnan, C. S. Raine, and K. W. Selmaj Brain-Derived Heat Shock Protein 70-Peptide Complexes Induce NK Cell-Dependent Tolerance to Experimental Autoimmune Encephalomyelitis J. Immunol., February 1, 2006; 176(3): 1588 - 1599. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Sercan, G. J. Hammerling, B. Arnold, and T. Schuler Cutting Edge: Innate Immune Cells Contribute to the IFN-{gamma}-Dependent Regulation of Antigen-Specific CD8+ T Cell Homeostasis J. Immunol., January 15, 2006; 176(2): 735 - 739. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-L. Hsieh Effects of Ultrasound and Diclofenac Phonophoresis on Inflammatory Pain Relief: Suppression of Inducible Nitric Oxide Synthase in Arthritic Rats Physical Therapy, January 1, 2006; 86(1): 39 - 49. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. P. Zehntner, C. Brickman, L. Bourbonniere, L. Remington, M. Caruso, and T. Owens Neutrophils That Infiltrate the Central Nervous System Regulate T Cell Responses J. Immunol., April 15, 2005; 174(8): 5124 - 5131. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Mekala, R. S. Alli, and T. L. Geiger IL-10-Dependent Suppression of Experimental Allergic Encephalomyelitis by Th2-Differentiated, Anti-TCR Redirected T Lymphocytes J. Immunol., March 15, 2005; 174(6): 3789 - 3797. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. K. Wensky, G. C. Furtado, M. C. Garibaldi Marcondes, S. Chen, D. Manfra, S. A. Lira, D. Zagzag, and J. J. Lafaille IFN-{gamma} Determines Distinct Clinical Outcomes in Autoimmune Encephalomyelitis J. Immunol., February 1, 2005; 174(3): 1416 - 1423. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Bettelli and V. K. Kuchroo IL-12- and IL-23-induced T helper cell subsets: birds of the same feather flock together J. Exp. Med., January 18, 2005; 201(2): 169 - 171. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Staykova, J. T. Paridaen, W. B. Cowden, and D. O. Willenborg Nitric Oxide Contributes to Resistance of the Brown Norway Rat to Experimental Autoimmune Encephalomyelitis Am. J. Pathol., January 1, 2005; 166(1): 147 - 157. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Abromson-Leeman, R. Bronson, Y. Luo, M. Berman, R. Leeman, J. Leeman, and M. Dorf T-Cell Properties Determine Disease Site, Clinical Presentation, and Cellular Pathology of Experimental Autoimmune Encephalomyelitis Am. J. Pathol., November 1, 2004; 165(5): 1519 - 1533. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. D. Ponomarev, M. Novikova, M. Yassai, M. Szczepanik, J. Gorski, and B. N. Dittel {gamma}{delta} T Cell Regulation of IFN-{gamma} Production by Central Nervous System-Infiltrating Encephalitogenic T Cells: Correlation with Recovery from Experimental Autoimmune Encephalomyelitis J. Immunol., August 1, 2004; 173(3): 1587 - 1595. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Bettelli, B. Sullivan, S. J. Szabo, R. A. Sobel, L. H. Glimcher, and V. K. Kuchroo Loss of T-bet, But Not STAT1, Prevents the Development of Experimental Autoimmune Encephalomyelitis J. Exp. Med., July 6, 2004; 200(1): 79 - 87. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B.-G. Xiao, X.-C. Wu, J.-S. Yang, L.-Y. Xu, X. Liu, Y.-M. Huang, B. Bjelke, and H. Link Therapeutic potential of IFN-{gamma}-modified dendritic cells in acute and chronic experimental allergic encephalomyelitis Int. Immunol., January 1, 2004; 16(1): 13 - 22. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Gran, G.-X. Zhang, S. Yu, J. Li, X.-H. Chen, E. S. Ventura, M. Kamoun, and A. Rostami IL-12p35-Deficient Mice Are Susceptible to Experimental Autoimmune Encephalomyelitis: Evidence for Redundancy in the IL-12 System in the Induction of Central Nervous System Autoimmune Demyelination J. Immunol., December 15, 2002; 169(12): 7104 - 7110. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G-X Zhang, M Kishi, H Xu, and A Rostami Mature bone marrow-derived dendritic cells polarize Th2 response and suppress experimental autoimmune encephalomyelitis Multiple Sclerosis, December 1, 2002; 8(6): 463 - 468. [Abstract] [PDF]
|
|
|
|
|
|
K. Nicolson, S. Freland, C. Weir, B. Delahunt, R. A. Flavell, and B. T. Backstrom Induction of experimental autoimmune encephalomyelitis in the absence of c-Jun N-terminal kinase 2 Int. Immunol., August 1, 2002; 14(8): 849 - 856. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Marchetti, M. C. Morale, J. Brouwer, C. Tirolo, N. Testa, S. Caniglia, N. Barden, S. Amor, P. A. Smith, and C. D. Dijkstra Exposure to a Dysfunctional Glucocorticoid Receptor from Early Embryonic Life Programs the Resistance to Experimental Autoimmune Encephalomyelitis Via Nitric Oxide-Induced Immunosuppression J. Immunol., June 1, 2002; 168(11): 5848 - 5859. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-G. Yang, J. Qi, M.-G. Wang, and M. Sykes Donor-derived interferon gamma separates graft-versus-leukemia effects and graft-versus-host disease induced by donor CD8 T cells Blood, May 13, 2002; 99(11): 4207 - 4215. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G.-X. Zhang, H. Xu, M. Kishi, D. Calida, and A. Rostami The Role of IL-12 in the Induction of Intravenous Tolerance in Experimental Autoimmune Encephalomyelitis J. Immunol., March 1, 2002; 168(5): 2501 - 2507. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. A. Arnett, R. P. Hellendall, G. K. Matsushima, K. Suzuki, V. E. Laubach, P. Sherman, and J. P.-Y. Ting The Protective Role of Nitric Oxide in a Neurotoxicant- Induced Demyelinating Model J. Immunol., January 1, 2002; 168(1): 427 - 433. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. D. Brand, L. K. Myers, K. B. Whittington, K. A. Latham, J. M. Stuart, A. H. Kang, and E. F. Rosloniec Detection of Early Changes in Autoimmune T Cell Phenotype and Function Following Intravenous Administration of Type II Collagen in a TCR-Transgenic Model J. Immunol., January 1, 2002; 168(1): 490 - 498. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Liu, Y. Yu, M. Zhang, W. Wang, and X. Cao The Involvement of TNF-{{alpha}}-Related Apoptosis-Inducing Ligand in the Enhanced Cytotoxicity of IFN-{{beta}}-Stimulated Human Dendritic Cells to Tumor Cells J. Immunol., May 1, 2001; 166(9): 5407 - 5415. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L.-Y. Xu, J.-S. Yang, H. Link, and B.-G. Xiao SIN-1, a Nitric Oxide Donor, Ameliorates Experimental Allergic Encephalomyelitis in Lewis Rats in the Incipient Phase: The Importance of the Time Window J. Immunol., May 1, 2001; 166(9): 5810 - 5816. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. E. Juedes and N. H. Ruddle Resident and Infiltrating Central Nervous System APCs Regulate the Emergence and Resolution of Experimental Autoimmune Encephalomyelitis J. Immunol., April 15, 2001; 166(8): 5168 - 5175. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Grohmann, F. Fallarino, S. Silla, R. Bianchi, M. L. Belladonna, C. Vacca, A. Micheletti, M. C. Fioretti, and P. Puccetti CD40 Ligation Ablates the Tolerogenic Potential of Lymphoid Dendritic Cells J. Immunol., January 1, 2001; 166(1): 277 - 283. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Weishaupt, S. Jander, W. Bruck, T. Kuhlmann, M. Stienekemeier, T. Hartung, K. V. Toyka, G. Stoll, and R. Gold Molecular Mechanisms of High-Dose Antigen Therapy in Experimental Autoimmune Encephalomyelitis: Rapid Induction of Th1-Type Cytokines and Inducible Nitric Oxide Synthase J. Immunol., December 15, 2000; 165(12): 7157 - 7163. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. S. Cauley, E. E. Miller, M. Yen, and S. L. Swain Superantigen-Induced CD4 T Cell Tolerance Mediated by Myeloid Cells and IFN-{gamma} J. Immunol., December 1, 2000; 165(11): 6056 - 6066. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Matthys, K. Vermeire, H. Heremans, and A. Billiau The protective effect of IFN-{gamma} in experimental autoimmune diseases: a central role of mycobacterial adjuvant-induced myelopoiesis J. Leukoc. Biol., October 1, 2000; 68(4): 447 - 454. [Abstract] [Full Text]
|
|
|
|
|
|
U. Grohmann, R. Bianchi, M. L. Belladonna, S. Silla, F. Fallarino, M. C. Fioretti, and P. Puccetti IFN-{gamma} Inhibits Presentation of a Tumor/Self Peptide by CD8{alpha}- Dendritic Cells Via Potentiation of the CD8{alpha}+ Subset J. Immunol., August 1, 2000; 165(3): 1357 - 1363. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-Q. Chu, S. Wittmer, and D. K. Dalton Failure to Suppress the Expansion of the Activated Cd4 T Cell Population in Interferon {gamma}-Deficient Mice Leads to Exacerbation of Experimental Autoimmune Encephalomyelitis J. Exp. Med., July 3, 2000; 192(1): 123 - 128. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Vermeire, L. Thielemans, P. Matthys, and A. Billiau The effects of NO synthase inhibitors on murine collagen-induced arthritis do not support a role of NO in the protective effect of IFN-{gamma} J. Leukoc. Biol., July 1, 2000; 68(1): 119 - 124. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
