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-Activated SJL/J Astrocytes1
*
Department of Microbiology-Immunology and the Interdepartmental Immunobiology Center, Northwestern University Medical School, Chicago, IL 60611; and the
Division of Immunobiology, Alexion Pharmaceuticals, Inc., New Haven, CT 06511
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-treated astrocytes from
EAE-susceptible SJL/J mice to process and present myelin epitopes.
IFN- activation up-regulated ICAM-1, VCAM-1, MHC class II, invariant
chain, H2-M, CD40, and B7-1 as determined by FACS and/or RT-PCR
analyses. B7-2 expression was only marginally enhanced on SJL/J
astrocytes. Consistent with the expression of these accessory
molecules, IFN--treated SJL/J astrocytes induced the B7-1-dependent
activation of Th1 lines and lymph node T cells specific for the
immunodominant encephalitogenic proteolipid protein (PLP) epitope
(PLP139–151) as assessed by proliferation and
activation for the adoptive transfer of EAE. Interestingly,
IFN--activated astrocytes efficiently processed and presented
PLP139–151, but not the subdominant
PLP178–191, PLP56–70, or
PLP104–117 epitopes, from intact PLP and a recombinant
variant fusion protein of PLP (MP4). The data are consistent with the
hypothesis that astrocytes in the proinflammatory CNS environment have
the capability of activating CNS-infiltrating encephalitogenic T cells
specific for immunodominant epitopes on various myelin proteins that
may be involved in either the initial or the relapsing stages of EAE. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The extensive study of the autoantigens involved in this disease in the SJL/J mouse has led to the identification of proteolipid protein (PLP) as a major encephalitogen in the disease pathogenesis (10, 11). Specifically, it has been shown that the dominant encephalitogenic epitope within PLP lies between amino acid residues 139 and 151 (PLP139–151) (12, 13). Thus, R-EAE can be reliably induced in SJL/J mice either by the active immunization of mice with PLP139–151 in CFA or by the adoptive transfer of in vitro-activated, PLP139–151-specific T cells. Additionally, it has been shown that residues 178 to 191 (PLP178–191), 56 to 70 (PLP56–70), and 104 to 117 (PLP104–117) are all encephalitogenic determinants of PLP in the SJL/J mouse, albeit with varying degrees of severity (13). Specifically, our laboratory has shown that PLP178–191 is the major epitope to which autoimmune T cell responses spread during the course of disease via the phenomenon of "epitope spreading" (14, 15, 16, 17).
While significant attention has been devoted to elucidating the immunopathology of R-EAE and the potential regimens that can be utilized to prevent and/or treat ongoing disease (10, 15, 18, 19, 20, 21), little information exists as to the roles that might be played in disease pathogenesis by the nonprofessional APCs of the CNS. Several reports have demonstrated that microglia are competent to process and present autoantigens in vitro (22, 23, 24). In addition, microglia are thought to initiate the effector limb of EAE (25, 26) and express MHC class II in normal white matter and in MS lesions (27). On the other hand, significant controversy exists as to the ability of neural crest-derived astrocytes to process and present protein Ags, let alone participate in the autoimmune pathogenesis (28, 29, 30, 31). While it is known that astrocytes play a major role in maintaining the blood-brain barrier, provide potential nutritive functions, and seal off damaged areas in the CNS (32, 33), the ability of astrocytes to process and present Ag, particularly self Ags, is still in dispute (28).
Histopathologic examination of the demyelinating plaques of R-EAE shows
areas of inflammatory monocytic infiltration that are surrounded by
astroglial cells (34). In light of their proximity to the infiltrating
autoimmune T cells, it is a continuing mystery as to the role
astrocytes may play in the disease initiation and/or progression. We
have recently demonstrated that subsequent to IFN- activation,
astrocytes isolated from neonatal BALB/c mice up-regulate B7-2
costimulatory molecules and process and present native OVA to naive
transgenic T cells, as well as to Th1 clones and hybridomas (30). In
this study, we report that astrocytes from EAE-susceptible SJL/J
neonates, activated with IFN-, up-regulate all the necessary
accessory molecules to initiate and/or perpetuate a T cell response. In
addition, we show that SJL/J astrocytes process and present the
immunodominant 139–151 epitope from either MP4, a fusion protein
containing a recombinant variant of PLP, or whole PLP. In addition,
IFN--treated astrocytes were able to process MP4 and activate
PLP139–151-specific T cells for the adoptive transfer of
R-EAE.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pregnant (15–17 days) SJL mice and 5- to 6-wk-old female mice were purchased from Harlan Labs, Bethesda, MD. Mice were housed in the Northwestern University animal facility. One- to three-day-old neonates were used for the isolation of astroglial cells.
Antigens
PLP56–70 (DYEYLINVIHAFQYV), PLP104–117 (KTTICGKGLSATVT), and PLP178–191 (NTWTTCQSIAFPSK) were synthesized on an Applied Biosystems 432A automated peptide synthesizer. PLP139–151 (HSLGKWLGHPDKF) was synthesized by the Peptide Facility at University of North Carolina, Chapel Hill, NC. All peptides were analyzed for correct m.w. by laser desorption mass spectrometric analyses. MP4 (a recombinant fusion protein comprising the 21.5-kDa isoform of human myelin basic protein and a recombinant variant of human PLP was produced and purified as previously described (35, 36). Intact bovine PLP was prepared from chloroform-methanol (2:1) extracts of bovine white matter as previously described (37). Chloroform-methanol-soluble PLP was converted to the water-soluble form by gradual replacement of the organic solvent with water, according to the method of Sherman and Folch-Pi (38).
Media
T cell lines were maintained in DMEM (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (Sigma), 2 x 10-3 M L-glutamine (Life Technologies, Gaithersburg, MD), 100 U/ml penicillin (Life Technologies), 100 µg/ml streptomycin (Life Technologies), 5 x 10-5 M 2-ME, 0.1 mM nonessential amino acids (Sigma), 1 mM sodium pyruvate (Life Technologies), MEM essential vitamins (Life Technologies), 0.1 mM asparagine (Life Technologies), 0.1 mg/ml folic acid (Life Technologies), 0.8% T STIM (Collaborative Biomedical Research, Bedford, MA), and recombinant IL-2 (Boehringer Mannheim, Mannheim, Germany). T cell hybridomas were propagated and proliferation assays were performed in DMEM supplemented with 7% FCS, 2 x 10-3 M L-glutamine, 0.1 mM nonessential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin, and 5 x 10-5 M 2-ME (DMEM-7% FCS). Astroglial cultures were maintained in DMEM/Ham’s F-12 (1:1) (Sigma) supplemented with 10% FCS and adjusted to a final concentration of 6 g/L glucose, 2.4 g/L NaHCO3, 0.37 g/L L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (DMEM-F12). Media and fetal bovine serum were free of endotoxin contamination.
Astroglial cell isolation
Tissue culture flasks were coated from 3 h to overnight with 10 µg/ml polyD-lysine (Sigma) and rinsed with balanced salt solution supplemented with BSS-3% FCS before addition of isolated cells. The brains were removed from 1- to 3-day-old neonatal mice, the hind brains were dissected away and the meninges were removed. The left and right hemispheres were transferred to a nylon mesh bag and gently dissociated. Cells in suspension were passed through No. 60 and No. 100 stainless steel screens (Sigma) to remove large pieces of debris and tissue. Cells were pelleted, resuspended in DMEM-F12 complete medium, and seeded in the polyD-lysine-coated tissue culture flasks and incubated at 37°C and 7.5% CO2. Fresh medium was added every 3 to 4 days. After 12 to 14 days, microglia and oligodendrocytes were removed from the astroglial bed layer by shaking the flasks on an orbital shaker for 1 h at 100 rpm and 24 h at 300 rpm. Astroglial cells remaining adhered to the flask were treated with trypsin and replated.
The purity of normal and IFN--treated astrocytes was determined by
intracellular staining with Abs to glial fibrillary acidic protein
(GFAP). Cells were adhered overnight to LabTek chambered glass slides
(Nunc, Glostrup, Denmark) at 2 x 105 cells/chamber.
Cells were fixed for 10 min at room temperature with 10% neutral
buffered formalin (Sigma), rinsed with PBS, and permeabilized with
PBS-0.2% fish skin gelatin (FSG, Sigma)-0.5% saponin for 20 min.
Cells were rinsed with PBS, and nonspecific binding was blocked with
PBS-0.2% FSG for 30 min and then incubated with primary anti-GFAP
(1:200, Dako, Carpinteria, CA) for 1 h at 37°C. Cells were
rinsed with PBS-0.2% FSG and incubated with FITC-conjugated goat
anti-rabbit Ig (1:200, Calbiochem, La Jolla, CA) for 1 h at
37°C. Cells were washed with PBS-FSG, covered with glass coverslips,
and examined under a fluorescent microscope. Astrocyte cultures were
>98% GFAP+, and showed no staining with I-As
indicating no contamination with microglia cells.
Cell surface staining
Astroglial cells were incubated in the presence or absence of
mouse rIFN- (100 U/ml) at 37°C and 7.5% CO2 for
the indicated amount of time. Cells were washed with cold PBS, and the
cells were gently removed with cell scrapers. Cells were washed then
incubated for 30 min at 4°C with anti I-As-biotin
(PharMingen, San Diego, CA), anti-ICAM-1-phycoerythrin (PE)
(PharMingen), anti-VCAM-1-PE (PharMingen), anti-B7-1-biotin,
anti-B7-2-biotin, anti-CD40-biotin, or the appropriate
isotype-matched controls. Biotinylated Abs were detected by subsequent
staining with avidin-PE (Vector Laboratories, Burlingame, CA). Cells
were analyzed using a Becton Dickinson FACScan FACS.
RT-PCR analyses
Astroglial cells were incubated in the presence or absence of
rIFN- (100 U/ml) for 6 to 24 h. Cells were scraped from the
flasks, washed with PBS, and solubilized with 1 ml of 4.0 M guanidium
thiocyanate. Chromosomal DNA was sheared using a 1-ml syringe equipped
with a 23-gauge needle. Total RNA was isolated from the extract using
an RNeasy Total RNA Kit (Qiagen, Germany). One microgram of total RNA
was typically used per RT reaction. Oligo(dT)12–18
primers were used for cDNA synthesis using a 1st Strand Synthesis Kit
(Clontech Laboratories, Palo Alto, CA). PCR was performed using primers
for class II trans-activator (CIITA) (sense, 5'-CAA GTC CCT
GAA GGA TGT GGA-3'; anti-sense, 5'-ACG TCC ATC ACC CGG AGG GAC-3'),
H-2M
(sense, 5'-GTG CTC GAA GCA TCT ACA CC;
anti-sense, 5'-GAC ACT GTG TGC TCT AAC TG-3'), B7-1 (sense, 5'-TGC
TGT CTG TCA TTG CTG GGA AAC T-3'; anti-sense, 5'-CCC AGG TGA AGT
CCT CTG ACA CGT G-3'), B7-2 (sense, 5'-GGG GGA TCC ATG GGC TTG GCA ATC
CTT AT-3'; anti-sense, 5'-TCG GGT GAC CTT GCT TAG ACG TGC AGG-3')
and ß-actin (sense, 5'-GTG GGC CGC TCT AGG CAC CAA-3';
anti-sense, 5'-CTC TTT GAT GTC ACG CAC GAT TTC-3'). Primers for
B7-1, B7-2, and ß-actin were synthesized at the Northwestern
University Biotechnology Center, Chicago, IL. Primers for the CIITA and
H-2Ma were synthesized by Life Technologies Custom Primers,
Gaithersburg, MD. Matched actin controls were run for all samples, and
PCR products were visualized by ethidium bromide-agarose (2%) gel
electrophoresis.
Western blot analysis of invariant chain
Astroglial cells (4 x 106) were incubated
in the presence or absence of rIFN- (100 U/ml) for 24 h. Cells
were scraped from the flasks, washed with PBS, and solubilized with 1%
Nonidet P-40 supplemented with the following protease inhibitors: PMSF;
N-tosyl-L-phenylalanine chloromethyl ketone
(both from Boehringer Mannheim); and leupeptin (Sigma). Lysates were
centrifuged for 20 min at 14,000 rpm in a refrigerated Eppendorf
centrifuge, and supernatants were collected, leaving behind the nuclear
pellet. Proteins were analyzed by 12% SDS-PAGE and were transferred
from SDS-PAGE gels onto nitrocellulose membranes at 70 V for 5 h
in Towbin transfer buffer. Membranes were blocked for 1 h at room
temperature in 5% dry milk and rinsed with Tris-buffered saline
containing 0.1% Tween-20 (TBST). Invariant chain (Ii) molecules were
detected by an overnight incubation of the membrane in a 1:20 dilution
of the anti-Ii mAb In-1 (the kind gift of Dr. Andrea Sant,
University of Chicago, Chicago, IL) in 1.5% dry milk in distilled
water. Blots were then extensively washed with TBST and incubated with
a 1:5000 dilution of horseradish peroxidase-conjugated goat
anti-rat (KPL Laboratories, Gaithersburg, MD) in distilled water
containing 2.5% dry milk for 1 h. Membranes were then washed
extensively and visualized by enhanced chemiluminescence (Amersham
ECL).
Long term Th1 T cell lines
Long term PLP56–70, PLP104–117, PLP139–151, PLP178–191 Th1, and PLP104–117 Th2 lines were established from the lymph nodes of SJL/J mice primed 10 days earlier with 100 µg of the respective peptide emulsified in IFA supplemented with 200 µg of Mycobacterium tuberculosis H37Ra. Lines were propagated by repeated in vitro stimulation of 1 x 106 T cells with 4 x 106 irradiated syngeneic splenic APCs and 25 µM concentrations of the respective peptide. T cells were restimulated every 3 to 4 wk with fresh spleen cells and Ag.
T cell proliferation
Astroglial cells were removed from tissue culture flasks using
trypsin, washed, resuspended in DMEM-F12 complete medium, and titrated
in polyD-lysine-treated 96-well flat-bottom tissue
culture plates (Costar) in the presence or absence of rIFN- (100
U/ml) and incubated for 48 h before assay. Immediately preceding
the addition of T cells or hybridomas and Ag, the plates were
irradiated with 3000 rads and then gently, but extensively, washed with
BSS-3% FCS to remove residual rIFN-. PLP peptide specific cell
lines were added to the 96-well plates in the presence of PLP peptides,
intact PLP, or MP4. Irradiated (3000 rads) syngeneic splenocytes
(4 x 105/well) were used as a control APC population
in separate wells. Culture wells were pulsed with [3H]TdR
(1 µCi/well, ICN Radiochemicals, Irvine, CA) for the final 24 h
of a 72 h incubation period. Radioactivity was detected using a
Packard Topcount microplate scintillation counter (Packard Instruments,
Meriden, CA). With T cell hybridomas, assay plates were frozen after a
24 h incubation and IL-2 activity in the supernatants was assayed
using the IL-2 sensitive CTLL-2 cell line. Briefly, 1 x
104 CTLL-2 cells were added in 100 µl of DMEM-7%FCS to
100 µl of the culture supernatants from the proliferation assay and
incubated at 37°C, 7.5% CO2 for a total of 48 h.
Cultures were pulsed for the final 24 h with [3H]TdR
and radioactivity was detected as described above.
Adoptive transfer of R-EAE
Astrocytes (2.5 x 106) were plated onto
T-75 tissue culture flasks (coated overnight with
polyD-lysine) in the presence or absence of rIFN-
(100U/ml) for 48 h. The astrocytic cultures were irradiated with
3000 rads and washed with BSS-3%FCS to remove residual rIFN-.
PLP139–151-specific T cells (35 x 106),
isolated from the draining lymph nodes of SJL/J mice primed 10 days
before with PLP139–151, were added to each of the flasks
in a total of 12 ml of DMEM-7%FCS in the presence of either 50 µM
PLP139–151 peptide or 25 µg/ml MP4. Following a 4-day
incubation at 37°C and 7.5% CO2, T cells were harvested
and washed twice, and 5 x 106 blasts were injected
i.p. into naive SJL/J mice. As a control, 35 x 106
PLP139–151 T cells were activated with 18 x
107 irradiated syngeneic spleen cells (3000 rads) in the
presence of 50 µM PLP139–151 peptide, and 5 x
106 blast cells from these cultures were also transferred
into naive SJL/J recipients. Recipient mice were observed for clinical
signs of disease.
Clinical evaluation
Mice were observed daily for clinical signs of disease for 
30
to 35 days posttransfer and, thereafter, every 1 to 7 days for the
duration of the experiment. Mice were scored according to their
clinical severity as follows: grade 0, no abnormality; grade 1, limp
tail; grade 2, limp tail and hind limb weakness (waddling gait); grade
3, partial hind limb paralysis; grade 4, complete hind limb paralysis;
and grade 5, moribund. The scores of the asymptomatic mice (score
= 0) were included in the calculation of the daily mean clinical score
for each group.
Statistical analyses
Comparison of the percentage of animals showing clinical disease
were analyzed by 
2 and comparisons of T cell
proliferative responses were analyzed by Student’s t test.
p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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treatment. To study the effects of a
proinflammatory environment on the cell surface expression of MHC class
II and the expression of its chaperone molecules, Ii and H2-M, we
cultured astrocytes isolated from the brains of SJL/J neonates in the
absence or presence of 100 U/ml rIFN- for varying periods of time.
Following the incubation, cells were washed and stained with a
monoclonal anti-I-As Ab. As can be seen in Figure 1
A, which is representative of
three separate experiments, there is a slight constitutive expression
of I-As on the astrocytes, which may be due to partial
activation of the cells by adherence to plastic. However, incubation of
the astrocytes for increasing periods in rIFN- results in a
significant increase in MHC class II expression. This is initially
visualized by the appearance of a small I-As bright
population followed by a general shift in the class II brightness of
entire population. Maximal expression of class II is reached between 24
and 48 h and persists up to 72 h postinduction.
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on the expression of Ii in astrocytes. Figure 1
B
indicates that in the absence of IFN-, there is little or no
detectable Ii in the SJL/J astrocytes. However, following treatment
with 100 U/ml IFN- for 24 h, there is a dramatic increase in
the amount of detectable p31 and p41 isoforms of Ii. Treatment with
a combination of 100 U/ml IFN- and 500 U/ml TNF- resulted in even
higher levels of Ii expression. Additionally, while there does not
appear to be any alteration in the ratio of the p31-p41 isoforms with
IFN- treatment, it appears that there is more of the p41 isoform
than the 1:9 ratio of p41-p31 isoforms reported for traditional APCs
(41, 42). Regardless, rIFN- treatment increases the expression of Ii
and MHC class II, suggesting that they may be competent for MHC class
II peptide loading.
We next examined the effects of IFN- on the expression H2-M. This
class II homologue is important in catalyzing the removal of the Ii
CLIP peptide from the MHC class II Ag-binding groove or serves as a
unique molecular chaperone for MHC class II, thereby facilitating the
loading of foreign antigenic peptides (43, 44). IFN- treatment of
SJL/J astrocytes resulted in an increase in the message levels for the
gene for the -chain of H2-M, Ma. Ma message
first appeared at 6 h IFN- treatment and peaked at 24 h
IFN- treatment (Fig. 1C).
Finally, we examined the mRNA levels for CIITA. This protein is
involved in the activation of both class II and Ii transcription. As
seen in Figure 1C and in support of the data presented
above, there is an increase in the message levels of CIITA, which peaks
at 6 h of rIFN- incubation. These levels appear to decline by
24 h. Thus, the increase in I-As and Ii expression
correlates with an earlier increase in the levels of the MHC CIITA
mRNA. Thus, a proinflammatory environment as simulated by rIFN-
results in the up-regulation of all the components necessary for the
loading of antigenic peptide onto MHC class II.
CNS astrocytes up-regulate expression of costimulatory molecules
following IFN- treatment. To study the effects of a proinflammatory
environment on the cell surface expression of costimulatory molecules
necessary for T cell activation, neonatal SJL/J astrocytes were
cultured in the absence or presence of 100 U/ml rIFN- for varying
periods of times. The cells were then washed and stained with Abs
against B7-1, B7-2, and CD40. As can be seen in Figure 2, which is representative of three
separate experiments, there is constitutive expression of B7-1 on a
subpopulation of CNS astrocytes, which increases steadily with
increasing incubation periods in rIFN- (Fig. 2A).
Optimum up-regulation of cell surface expression of B7-1 occurs at
48 h of rIFN- treatment. As seen in Figure 2B, B7-2
constitutive cell surface expression is undetectable in untreated
astrocytes and increases only slightly during the incubation periods
examined. This is in contrast with our previous studies in BALB/c
astrocytes, which show little constitutive or induced expression of
B7-1 but an increase in B7-2 expression with increasing exposure to
IFN- (30). CD40 expression is not apparent in untreated astrocytes,
but with increasing incubation periods in rIFN-, there is a dramatic
enhancement of CD40 expression that is first visible at 6 h
IFN- treatment peaking at 24 h and then rapidly declining (Fig. 2C). This is interesting in light of reports that
CD40 ligation may play a role in the events that regulate expression of
B7-1 and B7-2 in APCs and may also participate directly in the
activation of distinct subsets of T cells (45, 46, 47). Finally, ICAM-1 and
VCAM-1 show large increases in cell surface expression, reaching an
optimum at 24–48 h of IFN- treatment (data not shown). Thus, it
appears that in the presence of a proinflammatory cytokine like
IFN-, astrocytes up-regulate expression of the requisite
costimulatory molecules necessary for activation of naive and memory T
cells.
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treatment
To confirm the effects of IFN- on the cell surface expression
of B7-1 and B7-2 in SJL/J astrocytes, we examined the steady-state mRNA
levels of B7-1 and B7-2 following a 6- or 24-h incubation with IFN-.
As can be seen in Figure 3, there is
detectable message for B7-1 in the absence of rIFN- treatment, and
incubation in rIFN- results in an increase in the mRNA levels for
B7-1 which is visible by 6 h of incubation and maximal by 24
h (Fig. 3A). B7-2 mRNA levels are also visible at
6 h and peak by 24 h (Fig. 3B) but never
reach those of B7-1 consistent with the FACS data showing higher
constitutive and induced levels of cell surface expression of B7-1 than
those of B7-2. Thus, there is correlation in mRNA levels of B7-1 and
B7-2 with the cell surface expression levels of the proteins. Taken
together with the data for MHC class II expression, SJL/J astrocytes
appear to properly up-regulate/express all of the necessary molecules
required for the processing and presentation of Ag for the initiation
and maintenance of T cell activation.
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To functionally test the T cell activating capacity of SJL/J
astrocytes, we examined the ability of astrocytes treated for 24 or
48 h with 100 U/ml rIFN- to process and present
encephalitogenic PLP epitopes from purified PLP and from the
recombinant fusion protein, MP4, to a panel of PLP epitope-specific T
cell lines and hybridomas. MP4 has been demonstrated to be
encephalitogenic in SJL/J mice (36). Representative results compiled
from six experiments using separate astrocyte isolations are shown in
Figure 4. As can be seen,
IFN--activated astrocytes processed intact MP4 and whole PLP for
activation of a T cell line specific for the dominant encephalitogenic
PLP139–151 determinant. However, astrocytes failed to
present either intact MP4 or PLP to several different T cell lines
specific for the subdominant encephalitogenic determinants of PLP,
residues 56 to 70, 104 to 117, and 178 to 191. In all instances,
presentation of the autologous peptide was normal. While this
observation may not be surprising for the cryptic 104–117 epitope, it
was somewhat unexpected for the subdominant 178–191 and 56–70
epitopes. Naive SJL/J splenocytes show a pattern similar to those of
the IFN--treated astrocytes wherein PLP139–151 is
processed most effectively, although small responses are observed in T
cells specific for the subdominant PLP56–70 and
PLP178–191 epitopes. In contrast, LPS-activated
splenocytes efficiently process the dominant and both the subdominant
PLP epitopes. Since the 178–191 epitope has been implicated in the
phenomenon of epitope spreading in R-EAE induced by priming with
PLP139–151 (14), it would appear that astrocytes, at least
under the proinflammatory conditions that we have examined, may not be
capable of efficiently processing subdominant epitopes from intact PLP
but still may be able to process proteolyzed myelin fragments available
in the CNS inflammatory milieu.
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. The data follow the trend observed for
activation of conventional T cell lines. Only a hybridoma specific for
PLP139–151 responded to both autologous peptide and intact
MP4 or PLP. In contrast, PLP178–191-specific hybridomas
failed to respond to intact MP4 or PLP presented by IFN--activated
astrocytes but responded well to autologous peptide. This phenomenon
does not appear to be dependent on the dose of intact Ag since when
equimolar concentrations of intact PLP and autologous peptide were
used, similar results were obtained (data not shown). These data and
those shown in Figure 4 may therefore reflect a deficiency in the
astrocytes to effectively process MP4 and PLP into the encephalitogenic
56–70, 104–117, and 178–191 determinants. Thus, epitope spreading
may be due to the presentation of subdominant epitopes on other
CNS (e.g., microglia or more likely, infiltrating macrophages) or
peripheral APCs. It is also possible that the local CNS
microenvironment that may result in astrocyte processing and
presentation of the "spread" 178–191 determinant is not mimicked
by the in vitro proinflammatory conditions examined in this study.
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We have previously demonstrated that activation of naive
OVA-specific TCR-transgenic T cells and Th1 lines by BALB/c astrocytes
is predominantly B7-2 dependent. In light of the current data
suggesting that SJL/J astrocytes can process and present Ag to T cell
lines, we examined whether the observed differences in B7-1 and B7-2
expression in the SJL/J astrocytes, as compared with BALB/c astrocytes,
would translate into a different costimulation dependence. In Figure 6, we assessed the ability of
anti-B7-1 and anti-B7-2 mAbs to inhibit SJL/J astrocyte-induced
T cell activation. As can be seen, anti-B7-1 almost completely
inhibited proliferative responses of the PLP139–151,
PLP104–117, and PLP178–191 specific Th1 lines
and partially inhibited the response of the
PLP104–117-specific Th2 line. Anti-B7-2 was generally less
effective than anti-B7-1 for inhibiting the Th1 responses and the
combination of anti-B7-1 and anti-B7-2 completely eliminated
responses of the Th1 lines. Activation of the PLP104–117
Th2 cell line was only partially inhibited by anti-B7-1 and/or
anti-B7-2 reflecting the reduced costimulatory dependency of this
Th2 line. Thus, these results suggest that B7-1 is the dominant
functional costimulatory molecule for SJL/J astrocyte-induced
activation of Th1 lines. This is consistent with the predominant cell
surface expression of B7-1 on SJL/J astrocytes activated for 48 h
with rIFN-.
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-activated SJL/J astrocytes can activate
PLP139–151-specific T cells for adoptive transfer of
R-EAE
Since SJL/J astrocytes can process PLP and MP4 leading to
activation of a PLP139–151-specific Th1 line, we
tested whether IFN--treated SJL/J astrocytes had the capacity to
process and present MP4 in vitro to activate
PLP139–151-primed T cells for the adoptive transfer of
R-EAE. As can be seen in Figure 7,
IFN--treated astrocytes pulsed with 25 µg/ml MP4 were able to
activate PLP139–151-primed LN T cells to efficiently
transfer EAE to 100% of naive recipients. MP4-activated T cells were
somewhat more efficient for disease induction than T cells activated
with IFN--activated astrocytes or naive splenic APCs pulsed with the
autologous peptide. The lack of disease relapses during the observation
period in recipients of T cells activated with
PLP139–151-pulsed, IFN--treated astrocytes likely
reflects the protracted day of onset and reduced severity of acute
disease. On the other hand, astrocytes not treated with IFN- were
incapable of activating the PLP139–151-primed T cells for
adoptive transfer. Therefore, it appears that under the proper
inflammatory conditions, SJL/J astrocytes may participate in the
disease process.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We recently demonstrated that BALB/c astrocytes activated for 24 to
48 h with IFN-, up-regulated cell surface expression of
I-Ad (30). Following IFN- treatment, SJL/J astrocytes
also significantly up-regulated surface expression of I-As
(Fig. 1A). As expected, untreated astrocytes
expressed only trace amounts of MHC class II. The minimal constitutive
level of I-As which we observed may be due to interaction
of the astrocytes with the plastic culture wells because we noted that
their adherence was enhanced after incubation with IFN- for extended
culture periods. Maximal up-regulation of MHC class II occurred 48 h
following addition of IFN-, a time course similar to that for
expression of I-Ad on BALB/c astrocytes.
To further examine the capacity of astrocytes to serve as potential
APCs, we investigated the effect of the IFN- treatment on the
expression of two additional molecules required for Ag processing and
presentation, Ii and H2-M. Ii is not expressed at detectable levels in
untreated astrocytes (Fig. 1B) as determined by
Western blotting. However, following a 24-h treatment with IFN-, Ii
expression was dramatically up-regulated. The ratio of the p31 to the
p41 isoforms of Ii was clearly lower than the expected 9:1 ratio
previously reported for B cells (41, 42). This is interesting in light
of speculation that the p41 isoform may play a role in differential Ag
processing within endocytic compartments (42, 51). We are currently
exploring the possibility that this difference might be related to the
heightened susceptibility of the SJL/J strain to R-EAE. Using RT-PCR,
we also determined that the mRNA levels of the MHC CIITA, the protein
involved in the transcriptional regulation of both MHC class II and Ii,
were significantly up-regulated and peaked within 6 h of IFN-
treatment (Fig. 1C). Thus, as would be expected,
CIITA mRNA levels peaked before the maximal expression of
I-As and Ii. Similarly, transcription of the -chain
(Ma) of the H-2 M molecule, which is involved
in the removal of the Ii CLIP peptide from the MHC class II binding
groove (43), was also up-regulated within 6 h and peaked at
24 h of IFN- treatment (Fig. 1C). Thus, it
appears that SJL/J astrocytes possess all the necessary molecules
required for efficient Ag processing and presentation following IFN-
treatment.
As efficient T cell activation requires two signals, TCR occupancy and
delivery of the appropriate costimulatory signals (52), we next
examined the constitutive and IFN--induced expression levels of
B7-1, B7-2, CD40, ICAM-1, and VCAM-1 on primary SJL/J astrocytes. B7-1
is constitutively expressed at low levels and significantly
up-regulated on IFN--treated SJL/J astrocytes (Fig. 2A), in contrast to our earlier finding that
IFN--treated BALB/c astrocytes up-regulated B7-2 but not B7-1 (30).
This is supported by RT-PCR data, which showed detectable constitutive
expression of B7-1 mRNA that was significantly up-regulated upon
IFN- treatment (Fig. 3A). Additionally, functional
activation of Th1 lines specific for several different PLP
encephalitogenic epitopes was dependent primarily on B7-1-CD28
interactions, although the combination of B7-1 and B7-2 Abs resulted in
maximal inhibition (Fig. 6). On the other hand, B7-2 was not
constitutively expressed on SJL/J astrocytes and only marginally
expressed following treatment with IFN- for 24 h (Figs. 2B and 3B). IFN- treatment of SJL/J
astrocytes was also shown to up-regulate expression of CD40 (Fig. 3C) compatible with the findings that CD40-CD40L
interactions are involved in inducing the expression of B7-1 and B7-2
(45, 46). Additionally, expression of ICAM-1 and VCAM-1, adherence
molecules also critical for T cell activation (53, 54), are also
increased following 24 h of IFN- treatment (data not shown).
Thus, SJL/J astrocytes in an inflammatory environment such as exists in
the CNS of mice with ongoing R-EAE can express the requisite molecules
necessary for costimulation of T cell activation.
It is tempting to speculate that the differential susceptibility to active EAE of SJL/J vs BALB/c mice may, in part, relate to the differential CNS expression of B7-1 and B7-2 costimulatory molecules. There are several salient observations that support this hypothesis. First, we have shown that B7-1 is preferentially up-regulated on F4/80+ microglia/macrophages and B cells in the CNS of SJL/J mice undergoing active R-EAE and that clinical relapses can be ameliorated by blockading B7-1 during the remission period after the acute phase of disease (55). Secondly, preliminary experiments with a genome scan of an F2 cross between EAE-susceptible SJL/J and EAE-resistant B10.S mice have revealed a linkage of susceptibility to B7-1/B7-2 on mouse chromosome 16.5
We have previously demonstrated that IFN--treated BALB/c astrocytes
had the capacity to process and present OVA to naive transgenic T cells
derived from the DO11.10 mouse which are specific for the
immunodominant OVA323–339 epitope and to memory Th1 lines
derived from these mice (30). Thus, we also wanted to investigate the
functional ability of SJL/J astrocytes to process and present various
encephalitogenic PLP epitopes. We used whole PLP purified from bovine
brain and MP4 as sources of Ag and a panel of T cell lines and
hybridomas specific for the four described SJL/J encephalitogenic
epitopes, PLP139–151, PLP56–70,
PLP104–117, and PLP178–191. Interestingly,
SJLJ astrocytes fed intact PLP or MP4 efficiently activated lines and
hybridomas specific for the immunodominant PLP139–151
epitope (Figs. 4 and 5). However, T cell lines specific for the less
immunodominant self encephalitogenic epitopes (PLP56–70,
PLP104–117, and PLP178–191) were not
activated by IFN--treated astrocytes fed intact PLP but were
activated by astrocytes pulsed with the relevant autologous peptide.
Similar results were seen with multiple independently derived PLP
peptide-specific T cell lines and hybridomas specific for the less
dominant epitopes and upon the use of astrocytes activated with a
combination of TNF- and IFN-, which enhances MHC class II and Ii
expression above the levels seen for astrocytes stimulated with IFN-
alone (data not shown). In light of these results, it appears that
astrocytes, under the proinflammatory conditions that we have examined,
may not play a major role in the phenomenon of epitope spreading
wherein relapses in SJL/J mice with PLP139–151-induced
R-EAE are mediated primarily by T cells specific for the
PLP178–191 determinant (14). However, it is also possible
that in the local milieu of the CNS, additional cytokines may play a
role in activating astrocytes to more effectively process and present
the subdominant PLP epitopes (56, 57, 58, 59). Alternatively, in the
inflammatory environment within the CNS of mice with acute R-EAE,
extracellular proteases may cleave PLP into smaller fragments, which
may result in enhanced presentation of subdominant determinants by
astrocytes (60, 61, 62).
Significantly, IFN--treated SJL/J astrocytes pulsed with either
intact MP4 or PLP139–151 were also capable of activating
PLP139–151-specific T cells for the adoptive transfer of
R-EAE (Fig. 7), indicating that they can induce the up-regulation of
the appropriate integrins and cytokines necessary for CD4+
T cells to home to the CNS and initiate the demyelinating process. This
is consistent with an earlier report showing that rat astrocytes could
induce proliferation of encephalitogenic MBP-specific T cell lines
(63). Regardless of the role of astrocytes in initiating or maintaining
CNS presentation of myelin epitopes, our recent data indicate that
myelin peptides related to the epitope spreading process are generated
within the CNS of SJL/J mice undergoing immune-mediated demyelination.
CNS MHC class II+, F4/80+ microglia/macrophages
isolated from the spinal cords of mice with ongoing
PLP139–151-induced R-EAE or from mice with Theiler’s
virus-induced demyelinating disease both have the capacity to directly
activate T cell lines and hybridomas specific for both immunodominant
and subdominant epitopes on myelin and virus proteins in the absence of
addition of exogenous proteins/peptides to the in vitro cultures (Y.
Katz-Levy, L. J. Tan, and S.D. Miller, unpublished data).
Collectively, these and our previous results (30) clearly indicate that
astrocytes exposed to proinflammatory cytokines have the capacity to
up-regulate the necessary molecules, including MHC class II, Ii, and
H2-M and B7-1, enabling them to efficiently process and present self
Ags and activate CD4 T cells. Astrocytes clearly have the functional
capacity to process and present immunodominant antigenic determinants
of conventional foreign proteins (30, 64) and self myelin Ags (
Figs. 4–6) to both naive and memory T cells. Most significantly, T cells
specific for the immunodominant PLP139–151 epitope could
be activated in vitro by SJL/J astrocytes for the adoptive transfer
R-EAE. Astrocytes may thus play a role in the process of chronic
demyelination and epitope spreading by presenting dominant
encephalitogenic epitopes from endogenous myelin proteins to
encephalitogenic T cells. However, it is also possible that in response
to different cytokine stimuli in the CNS, or at the activation state of
the autoreactive T cells that astrocytes could also participate in
down-regulating disease (28, 50). The definitive proof of a pathologic
role of astrocytes in generating and presenting myelin epitopes will
have to await the conclusion of ongoing experiments examining the Ag
presenting capacity of astrocytes directly purified from the CNS of
adult mice with ongoing R-EAE.
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Footnotes |
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3 Address correspondence and reprint requests to Dr. Stephen D. Miller, Department of Microbiology-Immunology, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago, IL 60611. E-mail address:
4 Abbreviations used in this paper: R-EAE, relepsing experimental autoimmune encephalomyelitis; CNS, central nervous system; Ii, invariant chain; MS, multiple sclerosis; PLP, proteolipid protein; GFAP, glial fibrillary acidic protein; BSS, balanced salt solution; PE, phycoerythrin; FSG, fish skin gelatin; CIITA, class II trans-activator.
5 C. Teuscher and E. Blankenhorn. Submitted for publication.
Received for publication October 10, 1997. Accepted for publication January 5, 1998.
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activated primary murine astrocytes express B7 costimulatory molecules and prime naive antigen-specific T cells. J. Immunol. 158:614.[Abstract]
, interleukin-1ß, and interferon- in astrocytes. J. Neuroimmunol. 51:209.[Medline]
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cpm
(x10-3) ± SEM of triplicate wells. Stimulation indices
over background counts are indicated in parentheses. An experiment
representative of six separate astrocyte preparations is shown.
*Proliferative responses significantly above no Ag controls,
p < 0.05.
, IFN-
, IFN-
, 3
mice) and transfer of 5 x 106 blast cells activated
by irradiated syngeneic spleen cells (3000 rads) in the presence of 50
µM PLP139–151 peptide as a positive control (
, 4
mice). Recipient mice were observed for clinical signs of disease for
49 days. Disease incidence in each group is indicated in the
parentheses. These results are representative of two separate
experiments.