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T Cell Depletion on Cytokine Gene Expression in Experimental Allergic Encephalomyelitis1
Department of Pathology, Albert Einstein College of Medicine, Bronx, NY 10461
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T
cells using the mAb GL3 immediately before disease onset, or during the
chronic phase, significantly ameliorated clinical severity. We now
report on the effect of T cell depletion on expression of five
cytokine genes, IL-1, IL-6, TNF, lymphotoxin, and IFN- in spinal
cords of mice during the pre-onset, onset, height, and recovery phases
of EAE, and on expression of type II nitric oxide synthase. In control
animals, the mRNAs for IL-1 and IL-6 rose dramatically at disease onset
and peaked before disease height, whereas the mRNAs for TNF,
lymphotoxin, and IFN- rose more slowly and peaked with peak of
disease. In GL3-treated animals, a dramatic reduction in all five
cytokines was noted at disease onset, but only IFN- remained
significantly reduced at a time point equivalent to height of disease
in control animals. ELISA data confirmed the reduced levels of IL-1 and
IL-6 at disease onset in GL3-treated animals, and pathologic analysis
demonstrated a marked reduction in meningeal infiltrates at the same
time point. Studies of type II NOS also demonstrated a significant
reduction in both mRNA and protein expression at the height of disease
in GL3-treated animals. These results suggest that T cells
contribute to the pathogenesis of EAE by regulating the influx of
inflammatory cells into the spinal cord and by augmenting the
proinflammatory cytokine profile of the inflammatory infiltrates. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several studies now support the conclusion that T cells expressing the
TCR are also present in this inflammatory infiltrate, as well as
in multiple sclerosis lesions (2, 3, 4, 5, 6, 7). Although the exact function of
these cells remains unknown, studies in EAE have shown that their
numbers fluctuate in association with disease activity and that they
are principally localized to the edge of lesioned areas of the CNS,
particularly at sites in which altered expression of heat shock
proteins has been documented (3). T cells have been shown to
display in vitro cytotoxicity toward oligodendrocytes, and may thus
contribute to demyelinating activity within the lesion (8), and may
also be a source of Th1-type cytokines that could contribute to the
induction and/or maintenance of the proinflammatory activity of cells
within the lesion (9). Recently, three studies have attempted to define
a role for T cells in EAE either by depleting T cells
using Abs raised against the TCR, or by immunizing with a
peptide specific for V6, a V region gene that has been shown to
predominate in the CNS of animals with EAE. Although the results of
these studies gave conflicting results, since disease amelioration was
noted in two (10, 11), and disease exacerbation in the other (12), they
collectively support the conclusion that T cells contribute to
the inflammatory process in EAE.
In our own studies, we showed that in a chronic-relapsing mouse model
of EAE, induced by the passive transfer of myelin basic protein
(MBP)-activated lymph node cells, treatment with the mAb GL3 (10),
which transiently depletes T cells from the circulation,
significantly ameliorated disease expression when given either
immediately before the acute clinical episode, or during the more
chronic stages of the disease. FACS analysis showed that treatment with
this Ab resulted in a significant depletion of T cells both in
the spleen and in the CNS of affected animals, persisting for
approximately 15 days. In an attempt to define more precisely the
mechanism by which T cells contribute to the encephalitogenic
process during the acute phase of the disease, we have now studied
cytokine gene expression at varying stages of the disease in
GL3-treated and control animals using multiprobe ribonuclease
protection assays (RPA). The results obtained have been further
verified for some cytokines by studying protein levels in the spinal
cord, and by assessment of the activation of type II nitric oxide
synthase (iNOS), an enzyme that would be expected to be regulated by
proinflammatory cytokines in the lesion. The results support the
conclusion that T cells participate in the encephalitogenic
process by regulating the influx of inflammatory cells into the spinal
cord and by augmenting the proinflammatory cytokine profile of the
inflammatory infiltrates in the CNS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to eight-week-old female SJL/J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Animals were housed and maintained in a federally approved animal facility, and all protocols were approved by Institutional Animal Care and Use Committee of Albert Einstein College of Medicine (Bronx, NY).
Immunization and induction of EAE
Mice were immunized with MBP (Sigma, St. Louis, MO), as described previously (10). Draining axial, brachial, and inguinal lymph nodes were removed after 10 days, and a single cell suspension was prepared in RPMI 1640 medium containing 10% FCS, 100 µg/ml penicillin/streptomycin, 2 mM glutamine, 5 x 10-5 M 2-ME, 1 mM sodium pyruvate, 0.1 M HEPES, and nonessential amino acids (Sigma). Cells were plated at a density of 4 x 106 cells/ml in 24-well plates and activated in vitro with 50 µg/ml MBP at 37°C in 8% CO2 for 4 days. For adoptive transfer, 5 x 107 cells in 0.1 to 0.2 ml were injected into naive syngeneic recipients via the lateral tail vein. Clinical expression of disease was graded on a clinical index (CI) scale of 0 to 5, as follows: grade 1, limp tail; grade 2, hind limb weakness; grade 3, plegia of both hind limbs; 4, plegia of three or four limbs; grade 5, moribund. Recipient mice first showed signs of EAE 6 to 8 days posttransfer. Animals in recovery were defined as a clear clinical improvement of at least one grade persisting for 24 to 48 h following a paralytic incident. Animals were studied from 0 to 20 days posttransfer.
In vivo depletion of T cells
Culture supernatants of the hamster IgG mAb against pan
TCR- (GL3) (gift of Dr. Lefrancois, University of Connecticut,
Farmington, CT) were collected, and the IgG was affinity purified using
an Affigel protein A MAPS II Kit (Bio-Rad, Hercules, CA). Mice were
injected i.p. on 2 consecutive days with 1 mg of purified GL3 Ab (13)
given in two equal doses twice daily. Control group mice were injected
with an equivalent dose and amount of normal hamster IgG (NHIgG;
Accurate Chemicals, Westbury, NY).
Neuropathology
For pathologic analysis of the tissue, mice were anesthetized by ether inhalation and perfused through the left ventricle with 20 ml cold PBS. The spinal cord was removed, and sections of the lumbar cord were immersion fixed in Trump’s fixative (4% paraformaldehyde, 1% glutaraldehyde, in 0.1 M phosphate buffer, pH 7.4), or snap frozen in optimal cooling compound. For light microscopy, the tissue fixed in Trump’s solution was dehydrated through a graded series of ethanol, cleared in propylene oxide, and embedded in Epon 812 (Electron Microscopy Sciences, Fort Washington, PA). One-micrometer epoxy sections were placed on glass slides and stained with 1% toluidine blue.
Quantitation of cells in the CNS by FACS analysis
Sensitized mice (n = 4 per group) that had been treated with NHIgG or GL3 as above were perfused through the ascending aorta with 30 ml ice-cold PBS on day 6 or day 7 posttransfer of MBP-reactive T cells. The spinal cord was dissected free from the vertebral column and dissociated by passing through a stainless steel mesh grid. Leukocytes were isolated by Percoll density-gradient centrifugation, and single cell suspensions were stained for identification of the total leukocyte population using Abs to CD45 and CD3 (PharMingen, San Diego, CA), as described previously (10). FACS analysis was performed using a Becton Dickinson FACScan (Becton Dickinson, Mountain View, CA). A total cell count was obtained following gating of the appropriately labeled cell populations.
Immunohistochemistry
For immunohistochemistry, 10-µm sections were prepared from the snap-frozen tissue. Sections were air dried for 30 min and fixed in ice-cold acetone for 10 min at -20°C. Slides were rinsed in PBS, followed by incubation in 0.3% H2O2 in methanol for 15 min. After washing with PBS three times, the sections were blocked with 10% normal goat serum for 1 h. For staining of IL-1, slides were then incubated with an Ab to IL-1ß (Genzyme, Cambridge, MA) overnight at 4°C, washed three times in PBS, and incubated with biotinylated secondary Ab to hamster IgG (PharMingen) for 2 h at room temperature. Control tissues were incubated with hamster NHIgG and processed as above. Slides were washed three times in PBS and incubated with an avidin-biotin horseradish peroxidase complex (Vector Laboratories, Burlingame, CA) diluted 1/100, mixed, and incubated for 1 h at room temperature. Diaminobenzidine tetrahydrochloride (3,3') (Vector Laboratories) was used for chromogenic detection of peroxidase staining. After the final wash with PBS, sections were dehydrated and mounted in Permount. For analysis of type II NOS, slides were blocked as above and incubated with a polyclonal Ab to type II NOS (Transduction Laboratories, Lexington, KT) diluted 1/200 overnight at +4°C. They were then washed three times with PBS and incubated with a Cy3-coupled anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) for 2 h at room temperature in the dark. Control slides were incubated with PBS. Slides were then washed, mounted in aqua-mount (Lerner Laboratories, Pittsburgh, PA), and examined by fluorescence microscopy.
Preparation of total RNA
Following administration of a lethal dose of sodium pentobarbital, mice were perfused through the ascending aorta with 20 ml ice-cold PBS. The spinal cord was then dissected free from the spinal column, and total RNA was extracted using TRIREAGENT (Molecular Research Center, Cincinnati, OH).
Ribonuclease protection assay
RPA using either the ML-11 multiprobe template set (kindly
provided by Dr. Monte Hobbs) (14) or the mCK-1 template set
(PharMingen) was performed essentially as described (14). Briefly,
[
-32P]UTP-labeled antisense RNA transcripts for
TNF-, lymphotoxin (LT), IL-1 and ß, IL-6, IFN-, and L-32
(large ribosomal subunit protein 32) were generated using the template
sets and T7 RNA polymerase. Ten to twenty micrograms of total RNA from
each sample were allowed to hybridize to the labeled probe for 20
h at 45°C. ssRNA was digested with an RNase A/T1 mixture (Ambion,
Austin, TX), and the hybrids were analyzed on denaturing
urea/polyacrylamide gels. Protected fragments were visualized by
autoradiography and quantified using Image Quant (Molecular Dynamics,
Sunnyvale, CA). The ML-11 probe set was used to measure levels of IL-1,
IL-6, TNF-, and LT, and the mCK1 for IFN-. For each sample, a
ratio of the intensity of the cytokine band was obtained using the band
for ml32.
Competitive RT-PCR/MIMIC PCR
mRNA for iNOS was quantified by competitive RT-PCR or MIMIC
PCR, according to the manufacturer’s instructions (Clontech
Laboratories, Palo Alto, CA). Briefly, total RNA from spleen and spinal
cord was isolated as above, and 5 µg of RNA was reverse transcribed
using Ready-to-go you-prime first-strand beads (Pharmacia Biotech,
Piscataway, NJ). A series of 10-fold dilutions of known iNOS MIMIC
cDNA, a commercial DNA that is amplified with similar efficiency as
target cDNA for iNOS by the specific primers, but produces a product of
smaller size, was added to a constant amount (0.75 µg) of target cDNA
containing a final concentration of 2 U/50 µl Amplitaq DNA polymerase
(Perkin-Elmer/Cetus, Norwalk, CT), 0.4 µM 5' primer, 0.4 µM 3'
primer (Clontech Laboratories), 0.2 mM of each dNTP, 1.5 mM magnesium
chloride, and 10 mM PCR buffer (Perkin-Elmer/Cetus). PCR amplifications
were performed for 35 cycles, each cycle consisting of 94°C for
45 s, 65°C for 45 s, and 72°C for 2 min, followed by a
10-min extension at 72°C. PCR products were separated by
electrophoresis on a 1.6% agarose gel and visualized by UV light after
staining with ethidium bromide. A fine-tuned competitive PCR was
performed using twofold serial dilutions after determination of the
10-fold dilution in which the PCR MIMIC and target cDNA template gave
bands of equal intensity. PCR amplifications were done as before,
except that one of the dNTP was [-32P]dCTP. The PCR
products were then resolved on a 1.6% ethidium bromide agarose gel,
and the bands corresponding to the iNOS target and MIMIC were excised
from the agarose gel and the amount of radioactivity was determined by
scintillation counting. The log of the ratio of the radioactivity of
the target and MIMIC DNA was plotted against the log of the MIMIC
molecules added to the PCR. Results were derived from a linear
regression of the data. Statistical analysis was performed using
Student’s t test.
ELISA
The ELISAs for IL-1ß and IL-6 were performed using kits purchased from R&D Systems (Minneapolis, MN). Affinity-purified polyclonal Abs for IL-1ß or IL-6 were precoated onto microtiter plates. Animals were perfused with ice-cold PBS, as above, and the spinal cord and spleen were homogenized with 1 ml PBS containing 0.1 M PMSF. Duplicate 50 µl of standards, controls, and spinal cord or spleen homogenates were added to the wells and incubated at room temperature for 2 h. After washing, enzyme-linked polyclonal Abs specific for mouse IL-1ß, or IL-6 were added and incubated for 2 h at room temperature. The plates were then washed and reacted with substrate solution for 30 min, stop solution was added, and the plates were read at an OD of 450 nm. The sample values were then calculated from the standard curve.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cell-depleted mice
To determine the effect of T cell depletion on cytokine
gene expression in the CNS of animals with EAE, mice were sensitized by
the passive transfer of MBP-reactive lymph node cells and treated with
the GL3 Ab, or with NHIgG as a control, on days 4 and 5 posttransfer.
At varying times in the disease process, animals were perfused with
PBS, the spinal cords were removed, total RNA was extracted, and
cytokine gene expression was assessed using the multiprobe template RPA
system. Since preliminary data indicated that changes occurring at the
early stages of the disease process were critical to this analysis, we
took tissue immediately before (day 6) and at the time of (day 7)
disease onset, as well as at the height (days 10/11) and during the
recovery phase (day 20) of the disease. A total of four animals from
two separate experiments was analyzed for each time point. The mean CI
for the NHIgG animals taken on day 6 was 0, day 7 was 1.3 ± 0.6,
days 10/11 was 2.8 ± 0.8, and day 20 was 1.3 ± 0.7. For the
animals treated with GL3, the mean CI for day 6 was 0, day 7 was 0,
days 10/11 was 0.75 ± 0.6, and day 20 was 0.8 ± 0.2. Using
the ML-11 RPA multiprobe template set (14), we were able to distinguish
signals for IL-1, IL-6, TNF-, and LT. The results are shown in
Figure 1
and are expressed as a ratio of
cytokine mRNA to mL-32. In the NHIgG animals, levels of mRNA for IL-1
rose abruptly at the time of disease onset, then fell gradually over
the course of the disease. Treatment with GL3 dramatically reduced the
levels of IL-1 mRNA at the pre-onset and onset phases, but had
essentially no effect on levels detected at the height and recovery
phases. A similar pattern of cytokine mRNA expression was observed for
IL-6 in the NHIgG-treated animals, with peak levels noted at the time
of disease onset. Treatment with GL3 also dramatically changed the IL-6
mRNA pattern of expression, with low levels noted at the expected time
of disease onset, but with levels that were actually higher than the
controls during the recovery phase. In contrast to the data for IL-1
and IL-6, cytokine mRNA levels for TNF- and LT peaked at the height
of disease. Treatment with GL3 led to significantly lower levels of
mRNA for these cytokines at disease onset, but at later stages of the
disease no differences were noted between treated and control animals.
The levels of mRNA for IFN- in these same samples were determined
using a separate RPA kit (Fig. 1E). The results show
that, like TNF and LT, IFN- levels peaked with the height of
disease. However, in contrast to the results obtained with TNF- and
LT, treatment with GL3 led to reduced expression of mRNA for IFN- at
all stages of the disease process.
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T cell-depleted
mice
To determine whether the changes noted in the mRNA levels for
IL-1 and IL-6 in the T cell-depleted animals were reflected in
the amounts of cytokine present in the CNS, we used IL-1ß- and
IL-6-specific ELISAs to assess protein levels present in the spinal
cord on day 6 and day 7 postsensitization. The results are shown in
Table I. On day 6, only low levels of
IL-1 and IL-6 were detected in spinal cord homogenates from both the
NHIgG- and GL3-treated animals. However, on day 7, coincident with
disease onset, there was a dramatic increase in the levels of both of
these cytokines in the NHIgG animals that was not detected in the
GL3-treated animals. No differences were detected in IL-1 or IL-6
levels in the spleen cell samples harvested from the same animals (data
not shown). These data indicate, therefore, that the results obtained
with the RPA were reflected in the total levels of protein present in
the CNS, and indicate that depletion of T cells led to
significantly reduced levels of the proinflammatory cytokines IL-1 and
IL-6 at the time of disease onset.
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T cell-depleted mice
It is now well recognized that the commencement of clinical
expression of EAE in susceptible animals is marked by an abrupt onset
of inflammation in the CNS, particularly localized to the lumbar spinal
cord. In our previous studies, we showed that T cell depletion
led to a significant reduction in the extent of inflammation in the CNS
detectable at the height of the acute clinical episode (10) but, in
those experiments, we did not study the early phases of the disease
process. Therefore, we repeated the T cell depletion experiments
and took tissue for pathology from the day 6 and day 7 time points. The
results are shown in Figure 2. They
indicated that in the animals treated with NHIgG on day 6, immediately
before disease onset, a low level of inflammation could be observed in
the lumbar spinal cord sections that was restricted to the meninges
(Fig. 2A). On day 7, the extent of this inflammation
had increased considerably, although the bulk of the inflammatory
infiltrate was still restricted to the meningeal vessels (Fig. 2B). In contrast, in animals treated with GL3, only
small foci of inflammation could be detected in the meninges on day 6
(Fig. 2C) that was not measurably increased by day 7
(Fig. 2D). To provide a more quantitative analysis of
these events, an additional set of animals was sensitized and treated
as before. On days 6 (n = 4 per group) and 7
(n = 4 per group), the spinal cord was removed
after transcardial perfusion with PBS, and a total leukocyte count was
performed using FACS analysis. In animals treated with NHIgG, the total
number of leukocytes isolated from the spinal cord on day 6 was
6,308 ± 1,959, and on day 7 was 622,990 ± 8,290. In animals
treated with GL3, the total number of leukocytes isolated on day 6 was
2,426 ± 762, and on day 7 was 9,476 ± 3,027. Thus, T
cell depletion led to a striking decrease in the extent of infiltration
during the early phases of the inflammatory episode.
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T cell depletion on type II NOS expression
The most persistent effects of T cell depletion on
cytokine gene expression in EAE were noted for IFN- in which reduced
levels of mRNA for this cytokine were detected at each of the time
points tested. IFN- is thought to contribute to the pathology of EAE
by activating macrophages to produce a range of toxic factors involved
in the disruption of the blood-brain barrier, and the loss of myelin
from the axon. One of the factors that has been strongly implicated in
this process is the activation of type II NOS, which, once activated,
leads to the prolonged release of large amounts of nitric oxide (15).
In EAE, both activated macrophages and reactive astrocytes have been
implicated as the major sources of type II NOS (16). To study the
effects of T cell depletion on type II NOS expression in EAE, we
treated animals as before with NHIgG and GL3 and, at the height of the
acute clinical episode, harvested spleens and spinal cords and
performed a semiquantitative analysis for type II NOS using a MIMIC
PCR-based assay (see Materials and Methods). No PCR
products for type II NOS were detected in any of the spleen samples.
However, in spinal cord tissues, a strong signal for type II NOS was
obtained. As shown in Figure 4, all of
the animals (n = 5) treated with NHIgG had
detectable product for type II NOS with a mean level of 1.3 ± 0.7
attomoles/spinal cord, ranging from 0.94 (CI +0.5) to 2.5 (CI +3.5) in
individual animals, consistent with the data obtained by Cross et al.
(17). In contrast, in the animals treated with GL3, the mean level of
activity was 0.36 ± 0.5 attomoles activity
(n = 5), ranging from 0 (CI +0.5) to 1.25 (CI
+1), indicating a significant (p < 0.02)
reduction in the levels of type II NOS activity associated with the
depletion of T cells.
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A, animals treated
with NHIgG showed prominent staining for type II NOS associated with
inflammatory foci and glia that was considerably reduced in the animals
treated with GL3 (Fig. 5B). Thus, both mRNA and
protein for type II NOS were found to be reduced in the animals
depleted of T cells. These data would be consistent with the low
levels of IFN- detected in these animals, since this cytokine has
been strongly implicated in synergizing with other cytokines in the
production of high levels of type II NOS.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cell
depletion on cytokine gene expression in the spinal cords of mice in
which an acute attack of EAE was induced by the passive transfer of
MBP-reactive T cells. Two different patterns of response were observed.
For IL-1, IL-6, TNF, and LT, T cell depletion markedly reduced
the levels of mRNA for these cytokines at disease onset but, at a time
point that corresponded to the height of disease in control animals, no
differences were detected in the mRNA levels between depleted and
control animals. In contrast, mRNA levels for IFN- were reduced in
T cell-depleted animals at all stages of the disease process.
The differences noted in the levels of IL-1 and IL-6 at disease onset
were confirmed by ELISA, and the reduction in the levels of mRNA for
IFN- was supported by reduced expression of type II NOS, an enzyme
for which IFN- functions as a major inducing cytokine. The fact that
each of these proinflammatory cytokines is up-regulated in EAE tissues
has been amply documented in various different models (18, 19, 20, 21, 22, 23, 24, 25). The
results presented in this work are substantially in agreement with
these other studies, but also suggest that the timing and sequence of
release of these cytokines may be as important as their relative
levels, since T cell-depleted mice had clearly elevated levels
of IL-1, IL-6, TNF, and LT at a time when they expressed little to no
clinical evidence of disease (mean CI 0.75 ± 0.6), although
pathologic evidence of inflammation could be readily detected
(10).
The results for IL-1, IL-6, TNF, and LT suggest a regulatory role for
T cells at disease onset, a concept that was supported by
reduced levels of inflammation at this time point in the cord meninges
in depleted animals. Several studies have demonstrated that the onset
of EAE is accompanied by an abrupt onset of inflammation in the CNS,
and clinical disease has been shown to correlate with the extent of
this inflammatory infiltrate (26, 27). A critical role for TNF in the
initiation of the CNS inflammatory process has been shown using
anti-TNF Abs as well as soluble forms of the TNF receptor (28, 29, 30).
Similarly, a role for IL-1 in the initiation of inflammation is
supported by the exacerbating effect of administration of IL-1, as well
as by the ameliorating effect of the IL-1R antagonist (31, 32), in EAE.
Recently, several studies have documented a role for T cells in
the regulation of inflammation at various tissue sites. Thus, it has
been shown that T cells control inflammatory reactivity and
prevent excessive liver damage in mice infected with Listeria
monocytogenes by regulating the influx of neutrophils into the
liver (33). T cells have also been shown to be required for the
accumulation of eosinophils in the pleural cavity of mice following
challenge with LPS, an effect that was postulated to occur via
amplification of the role of macrophages in this response (34). In mice
with acquired immunity to Mycobacterium tuberculosis,
T cells have also been shown to regulate cellular traffic into the
pleural cavity, promoting the influx of lymphocytes and monocytes
and inhibiting the trafficking of neutrophils, possibly through
the secretion of specific chemokines (35).
The role of T cells as a source of chemokines has not been
documented extensively, but intraepithelial T cells can be
induced to express MIP-1, MIP-1ß, RANTES, and lymphotactin, but
not monocyte-chemotactic protein-1, with lymphotactin being the most
abundantly produced (36). In these cells, the expression of RANTES
remained elevated following activation in contrast to the kinetics of
RANTES expression in ß T cells, suggesting that T cells may
be a potent source of this chemokine during chronic inflammatory
episodes (36). In early Listeria infection, T cells
have been found to be a major source of a monocyte-chemotactic factor,
a cytokine/chemokine known to be crucial for protection against
Listeria infection (37). Chemokines are expressed abundantly
in EAE lesions (38), and ongoing studies of chemokine expression in our
T cell-depleted mice have shown a dramatic reduction in the
levels of monocyte-chemotactic protein-1, MIP-1, MIP-1ß, and
RANTES in the spinal cord, particularly in the early phases of the
disease process (Rajan et al., manuscript in preparation). These data
would be consistent with an important role for T cells in
orchestrating the influx of inflammatory cells into the CNS lesions
during the early stages of lesion formation.
The results for IFN- suggest that T cells themselves are
either a major source of this cytokine or regulate its expression in
other cells in inflamed tissues. Both T cells and NK cells are known to
be major sources of IFN- in vivo, and our results would be
consistent with the observations of others that T cells share
with NK cells a propensity toward high IFN- release (39, 40, 41, 42, 43). The
role of IFN- in EAE, however, has been shown to be complex with both
pro- and antiinflammatory effects noted (44, 45, 46). Although the reasons
for these discrepancies remain unclear, they most likely reflect the
fact that IFN- plays a key regulatory role in both the inductive and
protective phases of the disease process.
A major effect of IFN- is thought to be its role in activation of
cells of the monocyte/macrophage series. As a measure of this in our
experiments, we chose to investigate the expression of type II NOS, an
enzyme that is induced in many cell types in response to combinations
of cytokines, but for which IFN- has been recognized as the major
regulatory cytokine. The fact that T cell-depleted mice showed
lower levels of type II NOS would be consistent with observations in
experimental mucosal candidiasis, in which T cells have been
shown to enhance macrophage nitric oxide production in vitro via
IFN- secretion, as well as in vivo, in which it was shown that
depletion of T cells abrogated NOS expression in affected
mucosal sites (47). However, again like IFN-, a role for this enzyme
in EAE remains controversial, with both exacerbating and beneficial
effects of inhibitors of NOS having been documented extensively
(reviewed in 1 .
That T cells play an important role in immunity toward bacterial
and viral infections has been documented extensively, but their
potential role in autoimmune conditions has not been so widely studied.
However, there is a growing body of evidence supporting a role for
T cells as immunoregulatory cells. T cells have been
shown to be involved in the control of ß T cell activation (48),
and to both propagate and regulate murine lupus in the
MRL/Mp+/+ strain (49). In addition, T cells have
been shown to be critical mediators of tolerance induction in a number
of different model systems, including those induced by either
aerosolized or ingested soluble Ags and involving humoral and
cell-mediated immune responses (41, 42, 50, 51). Although the
mechanisms involved in these complex regulatory pathways have not yet
been defined, they have been shown to be mediated by remarkably low
numbers of T cells, ranging from 
2 x 105
cells/animal in the studies of McMenamin et al. (41), to 2 x
103 cells/animal in the studies of Szczepanik et al. (50),
which would be within the range of T cells found in the spinal
cord of animals with EAE.
The results presented in this work could be interpreted in one of two
ways: either T cells promote the inflammatory process providing
additional cytokines that facilitate the activation and migration of
myelin-reactive T cells across the blood-brain barrier, or T
cells function to down-regulate a regulatory cell that blocks the
activation of these autoimmune cells. Studies in TCR-transgenic mice
specific for MBP have clearly shown that only a few mice spontaneously
express disease, unless they are challenged with Ag, pertussis toxin,
agents present in a nonsterile environment (52), or are crossed with
Rag-/- animals (53). A critical step in this
process appears to be transfer into the CNS, which is known to require
cell activation and expression of certain sets of adhesion molecules
(27). We propose, therefore, that T cells facilitate the
activation and transfer of myelin-reactive T cells into the CNS by
providing appropriate proinflammatory cytokines and chemokines. As
noted above, these effects may not require large numbers of cells and
suggest that interpretations made from the relative numbers of
different cell populations may not necessarily reflect the role of
these cells in the disease process (10). We further propose that when
autoantigen-reactive T cell lines and/or clones are driven in vitro by
repeated stimulation with Ag and cytokines, they become sufficiently
activated that they function independently of factors supplied by
T cells, and under these conditions are able to transfer disease
in the absence of T cells, as has been shown in the TCR-
knockout mouse (54). An alternative possibility is that T cells
function to regulate a regulatory cell, but the results obtained in the
TCR- knockout mouse would tend to argue against this.
In conclusion, the results of this study suggest that T cells
participate in the development of EAE by functioning to regulate
leukocyte transfer across the blood-brain barrier and acting as a
source of proinflammatory cytokines. As such, these data form part of a
growing awareness of the role of T cells in regulating a number
of inflammatory events. However, in many of these conditions, the
effect of T cell depletion has led to conflicting results, with
amelioration or exacerbation of disease noted in often closely related
animal model systems. Although some of these differences may be
attributable to differences in the various strategies used, the role of
T cells in these conditions remains confusing. As such, these
data are reminiscent of the plethora of effects attributable to
CD4+ ß TCR+ T cells before the
establishment of the Th1 vs Th2 paradigm (55). Whether or not T
cells can be subdivided into different functional subsets remains to be
determined, but it is clear that the regulatory pathways involved in
T cell activation differ from that found in ß T cells.
However, given the growing number of Ag-specific immune responses that
can now be attributed to T cells, it is to be expected that
progress in our understanding of the biology of this subset of T cells
will proceed more rapidly.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Alice J. Rajan, Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461.
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; CI, clinical index; CNS, central nervous system; iNOS, inducible nitric oxide synthase; LT, lymphotoxin; MBP, myelin basic protein; MIP, macrophage-inflammatory protein; NHIgG, normal hamster immunoglobulin G; NOS, nitric oxide synthase; RANTES, regulated upon activation, normal T cell expressed and secreted; RPA, ribonuclease protection assay; RT-PCR, reverse-transcriptase polymerase chain reaction.
Received for publication September 30, 1997. Accepted for publication February 18, 1998.
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