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Prepublished online as a Blood First Edition Paper on April 17, 2002; DOI 10.1182/blood-2001-12-0229.
IMMUNOBIOLOGY
From the Departments of Microbiology-Immunology and
Dermatology and the Interdepartmental Immunobiology Center,
Northwestern University Medical School, Chicago, IL; and the Department
of Veterinary Anatomy and Public Health, College of Veterinary
Medicine, Texas A&M University, College Station.
Microglial cells and astrocytes are capable of processing and
presenting antigens for efficient activation of T cells. However, the
antigen-presenting function and role of cerebrovascular endothelial cells (CVEs) in central nervous system inflammatory responses remain
controversial. We compared the expression of necessary accessory
molecules and the functional antigen-presenting capacity of cloned
SJL/J CVEs and primary astrocytes in response to the pro-inflammatory
cytokines interferon- The brain has been considered an immune-privileged
site in the presence of an intact blood-brain barrier (BBB). The BBB
consists of tight junctions between cerebrovascular endothelial cells
(CVEs), and it serves as a physical barrier limiting T-cell and
antibody passage into the central nervous system (CNS).1
Few unactivated T cells are capable of extravasating through the BBB,
regardless of their antigen specificity, but low numbers of T cells can
be found in the CNS of healthy humans and rats, so there appears to be
a role for T-cell surveillance of the CNS in the absence of
inflammation. Under these conditions, the low expression of major
histocompatibility complex (MHC) class II and T-cell
costimulation/adhesion molecules on CNS-resident cells renders the CNS
an unsuitable site for T-cell priming. However, during an inflammatory
immune response, the BBB is disrupted and all T cells and mononuclear cells have the ability to traffic into the brain and spinal cord and to
recognize target antigens within the CNS.2-4 Under these conditions, potential antigen-presenting cells (APCs) residing in the
CNS are likely to play a major role in T-cell responses and may
contribute to the propagation or regulation of inflammatory immune
responses in the CNS, as they do in multiple sclerosis (MS).
The BBB is damaged by demyelinating diseases such as MS and its animal
model, experimental autoimmune encephalomyelitis (EAE). MS and EAE are
characterized by CNS infiltration of myelin protein-specific Th1 cells,
pro-inflammatory cytokine production of interferon- Astrocytes, the major glial cell type of the CNS, are known to play a
major role in maintaining the BBB.18,19 They provide nutritive functions and encapsulate inflammatory lesions, and they are
likely to influence immune responses in the CNS. Our laboratory has
reported that astrocytes exposed to the pro-inflammatory cytokine,
IFN- Little is known, however, concerning the role that CVEs may play in CNS
immune responses. It has been demonstrated that IFN- Mice
Media
Cerebrovascular endothelial cell isolation The procedure for isolating and deriving clonal populations of CVEs has been published elsewhere.27 Briefly, brains were removed from 2- to 4-week-old SJL/J mice, and the meninges and macroscopic pial vessels were removed by dissection. Brains were transferred to a second Petri dish, and the tissue was minced. Fragments were washed and resuspended in phosphate-buffered saline with 0.1% bovine serum albumin. The suspension was placed in a Wheaton homogenizer with 2 to 4 strokes of the B pestle to ensure the proper degree of homogenization to free the microvessels. The homogenate was passed by gravity through a succession of Nitex filters (Tetko, Switzerland) with 149-µm, 74-µm, and 20-µm pores. The filters were washed, and the 74-µm and 20-µm filters were placed in 50-mL conical tubes containing a 0.1% solution of collagenase-dispase (Boehringer, Indianapolis, IN) at 37°C and were digested through manual shaking for 3 hours. Then the filters were discarded. Digested material was centrifuged at 15g for 5 minutes, and the pellets were resuspended in 40 mL complete growth medium containing 20 µg/mL endothelial cell growth supplement (Collaborative Research, Bedford, MA), 50 µg/mL heparin (Elkins-Sinn, Cherry Hill, NJ), and 10% fetal bovine serum (FBS). Microvessel suspension was plated (1 mL suspension per 5 cm2) on plastic-ware coated with collagen type IV (Sigma) at 1 mg/mL and human serum fibronectin (Boehringer) at 10 µg/mL and was incubated at 37°C with 5% CO2. Daily microscopic observations were performed, and the culture medium was changed every 2 to 3 days.Selective mobilization of CVE was accomplished through the use of porcine pancreatin (Sigma) during early passages. Most, if not all, muscle cells and many of the astrocytes and pericytes remained attached to the coated plates. Cells were cloned by limiting dilution onto coated 16-well glass chamber slides (Costar, Cambridge, MA). After 2 weeks of culture, wells with sufficient growth were passaged into 6-well plates (Costar) and then into 25 cm2 flasks (Costar). Three different fluorescent staining techniques were used to ensure that an endothelial cell phenotype was maintained. Cells were marked with antibodies to factor VIII-related antigen (abV ImmuneResponse, Derry, NH), antibodies to angiotensin-converting enzyme (gift of Dr R. Auerbach, University of Wisconsin), and acetylated low-density lipoprotein (Biomedical Technologies, Stoughton, MA). Cloned cells remained diploid and retained their differentiation markers for at least 20 passages. All experiments were performed with cells before passage 20. Astroglial cell isolation Tissue culture flasks were coated from 3 hours to overnight with 10 µg/mL poly-D-lysine (Sigma) and were rinsed with balanced salt solution supplemented with 3% fetal bovine serum (BSS-3%) before the addition of isolated cells. Brains were removed from 1- to 3-day-old neonatal mice, hindbrains were dissected away, and meninges were removed. Left and right hemispheres were transferred to a nylon mesh bag and gently dissociated. Cells in suspension were passed through #60 and #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 poly-D-lysine-coated tissue culture flasks and were incubated at 37°C, 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 hour at 100 rpm and for 24 hours at 300 rpm. Astroglial cells that adhered to the flask were trypsin treated and replated to ensure lack of microglial cell contamination.Purity of naive and IFN- Cell surface staining and analysis CVEs and astrocytes were incubated in media alone, mouse recombinant IFN- (rIFN-) (100 U/mL; R&D Systems, Minneapolis,
MN), mouse rTNF- (500 U/mL; R&D Systems), or both cytokines at
37°C, 7.5% CO2 for 48 hours. Cells were washed in the
flasks with cold PBS and then gently removed using cell scrapers. Cells
were washed and resuspended in 2.4 G2 supernatant and were incubated
for 30 minutes at 4°C with anti I-As-biotin (MKS4),
anti-ICAM-1-phycoerythrin (PE) (3E2) and anti-VCAM-1-biotin (429) (BD PharMingen, San Diego, CA), anti-B7-1-PE (16-10A1), anti-B7-2-PE (GL1), anti-CD40-PE (HM40-3), anti-CD95 (clone Jo2), anti-CD95L (clone Kay 10), or the appropriate isotype-matched controls.
Cells were washed in isotonic-buffered saline, and biotinylated antibodies were detected by subsequent staining with streptavidin-PE (BD PharMingen) for 20 minutes in the dark at 4°C. Cells were then
washed and resuspended in 200 µL isotonic-buffered saline. Data
collection and analysis were performed on a Becton Dickinson FACSCalibur fluorescence-activated cell sorter using CellQuest software. Nonspecific background staining was determined by incubating samples with isotype-matched control monoclonal antibodies. Mean fluorescence shifts were determined by subtracting the mean
fluorescence intensity of isotype controls from the mean fluorescence
intensity of the specific stain.
RT-PCR analyses CVEs and astrocytes were incubated in media alone, rIFN-,
rTNF-, or both cytokines for 48 hours. Cells were scraped from the
flasks and washed with PBS. Total RNA was isolated from the extract
using an RNeasy Total RNA Kit (Qiagen, Germany). 1-2 µg of total RNA
was used to make total cDNA using a Clontech cDNA kit (Clontech
Laboratories, Palo Alto, CA) per reaction and PCR was performed using
approximately 1/5 of total cDNA and Taq DNA polymerase (Qiagen GmbH,
Hilden, Germany). Primers for CIITA (sense, 5'-CAA GTC CCT GAA GGA TGT
GGA-3'; anti-sense, 5'-ACG TCC ATC ACC CGG AGG GAC-3') and actin
(sense, 5'-GTG GGC CGC TCT AGG CAC CAA; anti-sense, 5'-CTC TTT GAT GTC
ACG CAC GAT TTC) were synthesized at Northwestern University's
Biotechnology Center. Polymerase chain reaction (PCR) products were
visualized by ethidium bromide agarose (2%) gel electrophoresis.
Isolation of Thy1.2+ T cells Spleens were harvested from syngeneic SJL/J or allogeneic A/J mice and were mashed through 100 mesh screens into single-cell suspensions. Red blood cells were lysed with 2 mL/spleen Tris NH4Cl, and remaining live cells were counted. Cells were labeled with 10 µL anti-CD90.2 conjugated to microbeads per 107 total cells for 15 minutes on ice. After thorough washing, labeled cells were collected using the AutoMACS (Miltenyi Biotec, Auburn, CA) possel program.Mixed lymphocyte cultures Astrocytes were removed from tissue culture flasks using trypsin, washed, resuspended in DMEM-F12 complete medium, and cultured in poly-D-lysine-treated 96-well flat-bottom tissue culture plates (Becton Dickinson Labware) (2 × 104/well) in the presence or absence of rIFN- (100 U/mL), TNF- (500 U/mL), or both
and were incubated for 48 hours before the assay. CVEs were cultured in
T75 flasks in the presence or absence of rIFN- (100 U/mL), TNF-
(500 U/mL), or both for 48 hours before the assay. CVEs were
trypsinized, washed, and plated (5 × 104/well) in
96-well flat-bottom tissue culture plates (Corning). Immediately
preceding the addition of T cells and antigen, the CVEs or astrocytes
were irradiated with 30 Gy, then gently, but extensively,
washed with BSS-3% FCS to remove residual cytokine. Cells
(4 × 105 CD90.2+) from A/J spleens were
added to the cultures. Irradiated (30 Gy) SJL/J splenocytes
(4 × 105/well) were used as a control allogeneic APC
population in separate wells. Proliferation assays were carried out in
DMEM-10% FCS supplemented with 40 µM indomethacin (Sigma) and 2 mM
aminoguanidine (Sigma). Culture wells were pulsed with 0.037 MBq/well 3[H]-TdR (ICN Radiochemicals, Irvine, CA) for
the final 24 hours of the 72-hour incubation period.
3[H]-TdR uptake was detected using a top-count microplate
scintillation counter (Packard Instruments, Meriden, CT), and results
are expressed as the mean of triplicate cultures ± SEM.
Long-term T-cell lines Long-term PLP139-151 Th1 lines and Th2 lines were established from the lymph nodes of SJL/J mice, primed 10 days prior with 100 µg peptide in complete Freund adjuvant supplemented with 200 µg Mycobacterium tuberculosis H37Ra. Every 3 to 4 weeks, live T cells were isolated on Ficoll-Histopaque (Amersham Pharmacia Biotech AB, Uppsala, Sweden) by centrifugation at 1200 rpm at room temperature for 15 minutes and were propagated by in vitro stimulation of 1 × 106 T cells with 5 × 106 irradiated syngeneic splenic APCs and 25 µM appropriate peptide for 96 hours. Th2 lines were skewed by repeated stimulations in the presence of 10 µg/mL anti IL-12 (R&D), 10 µg/mL anti IFN- (R&D), and 0.1 ng/mL
recombinant IL-4 (R&D). All stimulation assays were performed in
DMEM-10:DMEM (Sigma) supplemented with 10% FBS (Sigma),
2 × 10 3 M L-glutamine (GIBCO BRL, Grand Island, NY),
100 U/mL penicillin (GIBCO BRL), 100 µg/mL streptomycin (GIBCO BRL),
5 × 105 M 2-mercaptoethanol, and 0.1 mM nonessential
amino acids. After stimulation, T cells were rested in DMEM-10
supplemented with 2 U/mL recombinant IL-2 (Boehringer Mannheim
Biochemicals, Indianapolis, IN). All antigen presentation assays using
T-cell lines were conducted 14 to 30 days after stimulation.
Antigen presentation assays Astrocytes were removed from tissue culture flasks using trypsin, washed, resuspended in DMEM-F12 complete medium, and cultured in poly-D-lysine-treated 96-well flat bottom tissue culture plates (Becton Dickinson Labware, Bedford, MA) (1 × 104/well) in the presence or absence of rIFN- (100 U/mL), TNF- (500 U/mL),
or both and were incubated for 48 hours before the assay. CVEs were
cultured in T75 flasks in the presence or absence of rIFN- (100 U/mL), TNF- (500 U/mL), or both for 48 hours before the assay. CVEs
were trypsinized, washed, and plated (1 × 104/well) in
96-well flat-bottom tissue culture plates (Corning, NY). Immediately
preceding the addition of T cells and antigen, the CVEs or astrocytes
were irradiated with 30 Gy, then gently, but extensively, washed with
BSS-3% to remove residual cytokine. Myelin-specific T cells
(5 × 104) or CD90.2+ T cells
(4 × 105) were added to the 96-well plates in the
presence of peptide or staphylococcal enterotoxin B (SEB). Irradiated
(30 Gy) syngeneic splenocytes (4 × 105/well) were used
as a control APC population in separate wells. Proliferation assays
were carried out in DMEM-10 supplemented with 40 µM indomethacin
(Sigma) and 2 mM aminoguanidine (Sigma). Culture wells were pulsed with
0.037 MBq/well 3[H]-TdR (ICN Radiochemicals, Irvine, CA)
for the final 24 hours of the 72-hour incubation period.
3[H]-TdR uptake was detected using a top-count microplate
scintillation counter (Packard Instruments, Meriden, CT), and results
are expressed as the mean of triplicate cultures ± SEM.
T-cell hybridoma stimulation PLP139-151-specific T-cell hybridomas were generated. Briefly, lymph node T cells from previously primed mice were restimulated in vitro with PLP139-151 and were fused with BW5147 fusion partners using polyethylene glycol (Sigma). Successful fusions were selected in DMEM-10 containing HAT (Sigma). Long-term T-cell hybridomas were cultured in DMEM-10, and T-cell receptor engagement was measured through IL-2 production as determined by enzyme-linked immunosorbent assay (ELISA; Endogen, Cambridge, MA). CVEs were cultured in T75 flasks in the presence or absence of rIFN- (100 U/mL), TNF- (500 U/mL),
or both for 48 hours before assay. CVEs were trypsinized, washed and
plated (1 × 104/well) in 96-well flat-bottom tissue
culture plates (Corning). Immediately preceding the addition of T-cell
hybridomas and antigen, the CVEs were gently, but extensively, washed
with BSS-3% FCS to remove residual cytokine. PLP139-151-specific T
cell hybridomas (5 × 104) were added to the 96-well
plates in the presence or absence of peptide. SJL/J splenocytes
(4 × 105/well) were used as a control APC population in
separate wells. Hybridoma assays were carried out in DMEM-10. After 24 hours, culture supernatants were collected and IL-2 levels were
determined using an ELISA Minikit (Endogen). Data are represented as
average absorbance of triplicate cultures at 490 nm ± SD.
Coculture analysis CVE clones were allowed to grow to confluence in a 48-well tissue culture plate or chamber slide. T cells (2.5 × 105) ± peptide were added to the monolayers overnight. Pictures were taken using a Nikon N90 AF 35-mm camera and were scanned into Photoshop using Polaroid Insight. For fluorescence analysis of live versus dead cells, chamber slides were washed in PBS, stained with propidium iodide (Molecular Probes, Eugene, OR), 1:2000 in PBS, washed in PBS again, stained with DAPI (Sigma), 1:25 000 in PBS, washed in PBS again, dried, and cover-slipped using Vectashield mounting medium (Vector, Burlingame, CA). Slides were examined, and images were acquired by epifluorescence using the SPOT RT camera (Diagnostic Instruments, Sterling Heights, MI) and Metamorph Imaging Software (Universal Imaging, Downingtown, PA).
TNF- in vitro.21 However, the
inflammatory environment of the CNS during immune-mediated
demyelinating diseases and infection is characterized by the expression
of multiple cytokines, including the major pro-inflammatory cytokines,
IFN- and TNF-.35 TNF- has been suggested to play
a necessary role in the development of EAE.36-38 Thus, we
first sought to determine whether MHC class II expression on BBB cells
was differentially affected by incubation with IFN- or TNF- alone
or with both cytokines in combination.
As shown in Figure 1A, neither astrocytes
nor CVEs constitutively expressed MHC class II, but both cell types
up-regulated MHC class II when incubated with IFN-
Differential expression of MHC class II on APCs is regulated by the
CIITA molecule.13,14,39 To better understand the mechanism underlying the differential effects of TNF- Limited expression of costimulatory/adhesion molecules by SJL/J CVEs compared to SJL/J astrocytes So-called professional APCs express MHC class II and numerous costimulatory and adhesion molecules. One factor that might influence the ability of SJL/J CVEs to serve as APCs is their ability to express costimulatory or adhesion molecules that are critical for T-cell activation. Therefore, we examined constitutive and cytokine-induced expression of various costimulatory molecules on SJL/J CVEs compared to that of SJL/J astrocytes using flow cytometry. B7-1 and B7-2 molecules expressed on APCs interact with CD28 expressed on T cells to induce critical second signals necessary for antigen-specific T-cell activation.8 In SJL/J astrocytes, for example, blocking either B7-1 or B7-2 can diminish the magnitude of T-cell proliferation in vitro, though B7-1 is functionally dominant.21 Previous experiments examining the ability of endothelial cells to express B7 costimulatory molecules have yielded disparate results.43-45 We found that B7-1 was constitutively expressed at a low level on the surfaces of the cloned CVEs, and this level was unaffected by incubation with TNF- (Figure 2). However,
B7-1 expression was significantly increased upon stimulation with
IFN-, and the level was further enhanced by incubation with IFN-
and TNF- in combination. B7-2 was not constitutively expressed, nor
was it detected by flow cytometric analysis on SJL/J CVEs stimulated
with IFN- or TNF-. Similar results were observed on determination
of B7-1 and B7-2 mRNA levels measured by semiquantitative PCR (data not
shown). The B7-1-dominant expression pattern on SJL CVEs was similar
to what we observed on examination of SJL/J astrocytes before and after
activation with Th1 cytokines (Figure 2). Interestingly, CD40, another
important costimulatory molecule,46 was neither
constitutively nor inducibly expressed on CVEs or astrocytes by the
various cytokines. VCAM-1 and ICAM-1 have also been shown to decrease
the activation threshold of T cells.47-49 VCAM-1 was
expressed constitutively on the SJL CVE clones and was further
up-regulated by the combined effects of IFN- and TNF-, whereas
ICAM-1 was not expressed on CVEs under any cytokine conditions tested.
In contrast, VCAM-1 and ICAM-1 were both constitutively expressed on SJL/J astrocytes, and the expression of each adhesion molecule was significantly up-regulated with Th1 cytokine activation. Therefore, SJL/J CVEs inducibly express B7-1, which normally can act
alone as a potent costimulatory molecule for the induction of Th1
proliferation,50 but they do not express the
full-repertoire of costimulatory/adhesion molecules normally seen in
professional APCs. In contrast, astrocytes express a more complete
repertoire with constitutive B7-1, VCAM-1, and ICAM-1 expression and
inducible expression of B7-2.
SJL astrocytes, but not CVEs, elicit MHC class II-restricted T-cell responses I-As has been detected on IFN--stimulated CVEs by
flow cytometry and RT-PCR (Figure 1 and data not shown). To measure the functional expression of I-As on the surfaces of SJL/J
astrocytes and CVEs, mixed lymphocyte cultures were set up using these
CNS-resident APC populations as stimulators and T cells from A/J
(H-2k) mice as responders. Using this assay,
functional I-As expression on the surfaces of different
stimulator cell populations can be detected as a function of allogeneic
T-cell proliferation. Astrocytes and CVEs were pretreated with media
alone or with media containing IFN-, TNF-, or both cytokines. As
seen in Figure 3A, bulk SJL splenocytes
elicited a vigorous alloresponse, indicating that the A/J T cells
recognized the presence of allogeneic I-As-bearing cells.
Untreated astrocytes did not elicit a significant alloresponse,
suggesting that these cells expressed an insufficient level of
I-As. All cytokine-pretreated groups of astrocytes elicited
significant allogeneic T-cell responses, confirming the presence of MHC
class II on their surfaces (Figure 3A). Interestingly, the T-cell
proliferative responses correlated closely with the levels of
I-As detected by flow cytometry (Figures 1A, 3A). In
contrast, none of the CVE stimulators elicited significant allogeneic
T-cell responses (Figure 3A), suggesting that CVEs are not competent APCs or stimulators, despite their surface expression of MHC class II.
Because we have been unable to detect functional MHC class II
expression on the surfaces of CVEs as determined by allogeneic T-cell
proliferation (Figure 3A), the relative abilities of astrocytes and
CVEs to present the superantigen SEB to stimulate syngeneic T cells was
tested. Superantigen presentation was chosen because SEB binds to MHC
class II outside the peptide-binding groove and also
to V CVEs do not activate myelin epitope-specific Th1 and Th2 lines To better understand the role CVEs may play during an immune response within the CNS, we investigated whether these cells were capable of presenting antigen for the activation of Th1 cells involved in CNS demyelination, as we have previously reported for astrocytes.21 Purified cultures of CVE clones were incubated with media alone or media containing IFN-, TNF-, or
both cytokines for 48 hours. The cells were then cultured with
PLP139-151, the immunodominant encephalitogenic epitope on proteolipid
protein in SJL/J mice, and a peptide-specific Th1 line. SJL/J CVEs
could not stimulate significant proliferation of the
PLP139-151-specific T-cell line, even after pre-incubation with
IFN- alone, a condition that leads to significant up-regulation of
MHC class II (Figures 1, 4A). Albeit low,
it is notable that CVEs pretreated with IFN- did elicit Th1
proliferation to a greater degree than the other 3 treatment conditions
(SI of 2.5 compared with SIs of 0.8, 0.6; 1.0 µM) (Figure 4A). To
verify this lack of responsiveness to CVE antigen presentation, the
experiment was repeated numerous times. Varying culture conditions,
including the concentration of peptide (1 µM to 100 µM) and numbers
of CVE clones in culture (1 × 103/well to
1 × 105/well), yielded consistently negative results.
CVEs failed to activate Th1 lines specific for either PLP178-191 or
PLP56-70 (data not shown). Additionally, supernatants were collected
from all the cultures to measure cytokine production by ELISA, but none
of the CVE + Th1 cultures produced cytokines (data not shown). Thus, despite the expression of significant levels of MHC class II on
the surfaces of CVEs, these cells were unable to serve as efficient
APCs to activate Th1 lines to proliferate or produce cytokines.
Fabry et al29 reported the activation of Th2 but not of Th1 cells as a consequence of antigen presentation by primary BALB/c brain microvessel endothelial cells. Based on that report, the relative abilities of cytokine-stimulated SJL/J CVE clones to activate PLP139-151-specific Th2 cells were tested (Figure 4B). Similar to the Th1 lines, CVE clones were not capable of activating Th2 cells to proliferate (Figure 4B) or produce IL-5 and IL-10 (data not shown). In contrast to the earlier report, we find that CVEs are not capable of serving as APCs for the activation of myelin-specific Th1 or Th2 cells. CVE clones are not capable of activating T-cell hybridomas to secrete IL-2 One possible explanation for the failure of CVEs to activate Th1 and Th2 cells is the low level of costimulatory molecule expression on CVEs compared to that on astrocytes and professional APCs (Figure 2). Therefore, we asked whether CVEs could activate costimulation-independent PLP139-151-specific T-cell hybridomas. Following treatment with media alone or with IFN-, TNF-, or both
cytokines, CVE clones were unable to activate T-cell hybridomas to
produce IL-2 (Figure 4C), but irradiated bulk splenocytes efficiently activated these hybridomas to produce IL-2 in an antigen-dependent manner (Figure 4C). Again, these experiments led to the same
conclusion: despite the expression of MHC class II, CVE clones are
poor APCs.
T cells damage CVE and astrocyte monolayers CVEs do not appear to serve as competent APCs in vitro despite their expression of surface molecules necessary for antigen presentation. Although CVEs may not activate T cells in the CNS, they are involved in T cell entry into the CNS. Previous reports have indicated that encephalitogenic and nonencephalitogenic CD4+ T cells can cause damage to Lewis rat CVE monolayers,51 suggesting that EAE induction may involve T-cell-mediated damage to CNS vasculature.To determine whether SJL/J CVEs are susceptible to
T-cell-mediated damage, CVE monolayers were cultured with
PLP139-151-specific T cells and were examined 18 hours later. Figure
5A displays healthy, confluent CVEs that
were cultured alone. After coculture with T cells overnight, there was
clear evidence that the CVE monolayer had been damaged, as indicated by
fewer, more constricted cell processes (Figure 5B). These data are
representative of 5 experiments with a variety of myelin
peptide-specific T-cell lines cultured in the presence and absence of
antigen. Consequently, rested T cells used in antigen presentation
assays (Figure 4) were capable of damaging CVEs, which may, in part,
explain why CVEs cannot activate T cells as measured by proliferation.
Using phase-contrast microscopy, we observed morphologic changes
associated with apoptosis in CVEs. To determine whether CVEs actually
undergo apoptosis as a result of T-cell interactions, live versus dead
cells were examined. After culturing CVEs in the presence or absence of
T cells overnight, the monolayers were washed and stained with DAPI and
propidium iodide (PI) to analyze total versus dead cells, respectively.
When CVEs were cultured alone, no PI-positive cells were detected,
indicating that all the cells in the culture were alive (Figure 5D).
Surprisingly, no PI-positive cells were found in CVE monolayers
following overnight coculture with T cells (Figure 5E). As a
positive control, CVEs treated with Triton-X stained positive for
PI (Figure 5F). In summary, T cells induce morphologic changes in CVE
monolayers without causing apoptosis, as indicated by the lack of PI
staining.
CNS-resident cells may play an important role in the inflammation
seen in autoimmune demyelinating diseases such as MS and EAE, during
either disease initiation or disease progression. Microglial cells and
astrocytes can activate T cells following exposure to pro-inflammatory
cytokines, such as IFN- Our first goal was to better understand the regulation of MHC class II
on the CVE surface. Interestingly, the ability of CVEs to up-regulate
MHC class II on the cell surface in response to IFN- To serve as efficient APCs to CD4+ T cells, CVEs must
be able to up-regulate MHC class II and costimulatory molecules. Using flow cytometry, we compared surface adhesion and costimulatory molecule
expression on cytokine-stimulated CVEs and astrocytes. We found that
CVEs constitutively expressed low levels of B7-1 and VCAM-1 on their
surfaces. Following IFN- Various groups have shown that endothelial cells treated with IFN- Previous reports have indicated that encephalitogenic and nonencephalitogenic CD4+ T cells can cause damage to Lewis rat CVE monolayers,51 suggesting that EAE induction may involve T-cell-mediated damage to CNS vasculature. On close analysis, CVEs did not look particularly healthy following in vitro culture with T cells (Figure 5B). Further analysis revealed that CVEs undergo morphologic changes after coculture with myelin peptide-specific T cells. Myelin peptide-specific T cells do not appear to induce apoptosis of CVEs, but the CVE monolayer is significantly changed by T-cell interactions. CVEs express high levels of Fas (CD95), but blockade of Fas-FasL interactions by inclusion of rFas-Fc in T-cell-CVE cultures did not inhibit the development of T-cell-induced changes in CVE monolayer architecture (data not shown). Experiments are under way to determine the mechanism(s) underlying T-cell-induced damage to the CVE monolayers. It is possible that CD4+ T cells activated in vivo by neuroantigen-CFA may interact with and damage endothelial cells of the BBB to allow inflammatory cells to nonspecifically enter the CNS. Others have demonstrated that CD4+ T cells can have cytotoxic function.66-68 In particular, Sedjwick et al68 demonstrated that encephalitogenic and nonencephalitogenic CD4+ T-cell lines cause antigen-specific damage to Lewis rat brain microvascular endothelial cells. Furthermore, they found that rats developed hemorrhage in the spinal cord and brain following the transfer of MBP-activated splenocytes. These data suggest that CNS endothelial cells are primary targets of activated T cells. In conclusion, the observations outlined in this paper have led us to conclude that CVEs are not inducible APCs. Instead, we speculate that T-cell-CVE interactions damage the BBB, facilitating T-cell entry into the CNS during neuro-inflammation.
We thank S. Mark Tompkins, PhD, for his discussions and critical review of this manuscript.
Submitted December 13, 2001; accepted January 2, 2002.
Prepublished online as Blood First Edition Paper, April 17, 2002; DOI 10.1182/blood-2001-12-0229.
Supported in part by United States Public Health Service National Institutes of Health grants NS-26543 and NS-30871. K.B.G. was funded by NIH Training Grant IT32AB0759301. A.M.G. was supported by NIH Training Grant AI-07476.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Stephen D. Miller, Department of Microbiology-Immunology, Northwestern University Medical School, 303 East Chicago Ave, Chicago, IL 60611; e-mail: s-d-miller{at}northwestern.edu.
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© 2002 by The American Society of Hematology.
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Top
Abstract
Introduction
mean fluorescence intensity of the isotype control) for each marker on
cells following cytokine pretreatment.
plays a role in down-regulating this protein at the promoter
level
implications for their role in neurologic disease.
Neuroscience.
1993;54:15-36.