1 Department of Immunology, The Forsyth Institute, 140 Fenway, Boston, MA 02115, USA
2 Mitsubishi-Tokyo Pharmaceuticals, 1000 Kamoshida-cho, Aoba-ku, Yokohama 227, Japan
3 Department of Periodontology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku,Tokyo 113, Japan
Correspondence to: M. A. Taubman
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Abstract |
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(ECC/B7-1) were developed to elucidate the influence of B7 co-stimulation by EC. ECC/B7-1 induced proliferation of Th1 clone cells, whereas ECC did not induce proliferation in co-culture of Th1 clone cells and EC stimulated with IFN- and antigen. In the transmigration assay, ECC/B7-1 did not induce transmigration anergy of Th1 clones or Th1 lines unless anti-rat B7-1 blocking mAb was added. Therefore, in rats, the T cell anergy induced during transmigration across a layer of EC seemed to be due to antigen presentation in the absence of B7 on the EC. We introduce the concept of transmigration anergy in this manuscript. Thus, EC can play a critical immune regulatory role in the context of antigen presentation by MHC class II to transmigrating T cells.
Keywords: anergy, B7, MHC class II, T cell migration, vascular endothelium
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Introduction |
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MHC class II molecules can be induced on EC by cytokines such as IFN- (1,3). MHC class II expression has also been found on EC in cutaneous inflammatory skin disease (4), gingival tissue of periodontal disease (5), synovial membrane of rheumatoid arthritis (6), atherosclerotic plaques (7) and mucosa of ulcerative colitis (8). However, the functional significance of MHC class II expression on the EC at the inflammation site has not been elucidated.
IFN- stimulation of EC not only induces MHC class II, but also enhances Th1-type selective transmigration across the EC layer (9). This enhancement of Th1-type selective migration is responsible for diapedesis induced by interaction between preferentially expressed CC chemokine receptor (CCR)-5 on Th1-type cells (10) and RANTES (regulated on activation normal T cell expressed and secreted) produced by EC (9). Since adherent T cells begin diapedesis very shortly (~ 6 min) after EC are stimulated with IFN-
in vitro (9), it would be interesting to determine if MHC class II signaling by EC can affect the transmigrating T cells in such a short period.
Human T cells can proliferate in response to autoantigen presentation by human umbilical cord vein EC (HUVEC) (11) or to allogeneic MHC molecules of HUVEC (12,13), hence MHC class II expression by EC can transduce TCR signaling. Interestingly, proliferation of human T cells by EC could be initiated by accessory signaling from CD2, but not by co-stimulatory signaling from CD28 (11,14), because human EC express lymphocyte function-associated antigen (LFA)-3 which interacts with CD2, but do not express B7 which is necessary for interaction with CD28 (15). Complete T cell activation requires two signals: one from the TCR and the other from a co-stimulatory molecule(s) (16). TCR occupancy in the absence of co-stimulatory signal B7/CD28 that renders T cells into a long-term non-responsive state is termed `anergy' (17,18). However, the influence of antigen-specific stimulation of T cells by EC in the absence of B7 is still unclear, especially for T cells transmigrating across the EC. To gain insight into the potential pathophysiological role of MHC class II expressed by EC in inflammation, we examined the T cell response in the course of transmigration across antigen-presenting EC to determine if such transmigration could affect T cell responsiveness to subsequent antigen presentation by professional antigen-presenting cells (APC). We found that Th1 lymphocytes that transmigrated across antigen-presenting EC were rendered anergic (transmigration assay), indicating that EC can regulate antigen responsiveness of T lymphocytes that migrate into tissue.
Methods
Antigens and reagents
Actinobacillus actinomycetemcomitans ATCC43718 (strain Y4), which has been implicated as a periodontal disease pathogen (19,20), was formalin-fixed and served as a T cell antigen (21). Rat recombinant IFN- was purchased from Life Technologies (Gaithersburg, MD). Rat recombinant IL-2 was obtained from Serotec (Oxford, UK).
T cell clones and lines
CD4+ T cell clones were developed from the cervical lymph nodes of Rowett rats injected with A. actinomycetemcomitans, as previously described (21). An A. actinomycetemcomitans outer membrane protein (Omp) 29 kDa-specific T cell clone [Th1-type: G23 (22)] was used in this study. The Th1-type clone G26, which responded to an unknown antigen of A. actinomycetemcomitans and did not respond to Omp29, was also employed. T clones were maintained by weekly stimulation with irradiated (3300 rad) syngeneic rat spleen APC and formalin-fixed whole A. actinomycetemcomitans antigen. The A. actinomycetemcomitans Omp29-specific Th1-type lines were developed from primary culture of whole A. actinomycetemcomitans immunized (s.c.) Rowett rat lymph node cells. The Omp29-specific lines were primed in vitro in the presence of irradiated spleen APC and Omp29 antigen with recombinant mouse IL-12 (2 ng/ml; gift from Genetic Institute, Cambridge, MA). Tetanus toxoid (TT)-specific T cell lines were developed by immunization with TT (Wyeth, Marietta, PA) (50 µg/time) first in complete Freund's adjuvant (s.c.), then in incomplete Freund's adjuvant (s.c.) and finally in saline (i.v.) at intervals of 2 weeks respectively. The T cells were isolated from local lymph nodes, and re-stimulated in culture with irradiated spleen APC and TT (10 µg/ml).
EC culture
The culture and characteristics of rat EC lines and clones were previously described (9,22,23). Briefly, EC were maintained in RPMI 1640 (Life Technologies) complete medium containing 10% FBS (Sigma, St Louis, MO), 10 mM HEPES (Sigma), 2 mM L-glutamine (Life Technologies), 100 U/ml of penicillin and 100 µg/ml of streptomycin (Life Technologies), 5x105M 2-mercaptoethanol (Sigma) and 1 mM sodium pyruvate (Sigma), supplemented with 2.5% rat brain conditioned medium. EC were removed with 0.02% EDTA/PBS as they were passaged. Four to six passages of freshly isolated rat aorta EC were used for experiments as primary cultured EC.
B7-1-transfected EC clone (MAT-171)
Rat B7-1 cDNA provided by Dr Turka (24) was subcloned into the pcDNA3/CMV expression vector (Invitrogen, Portland, OR). MAT-1 B7 negative ECC cells (6x105 cells) were transfected with 2.5 µg pcDNA3/CMV/B7-1 plasmid and 15 µl Lipofectamine (Life Technologies) in 1 ml OPTI-MEM I Reduced-Serum Medium (Life Technologies) for 12 h. Transfected cells were selected in RPMI 1640 complete medium supplemented with 2.5% rat brain conditioned medium with 250 µg/ml G418 (Life Technologies). After G418 drug selection, an EC culture which expressed B7-1 on its surface was designated as MAT-171.
Cellular ELISA
A direct ELISA assay procedure, as modified from the method of Piela et al. (25), was utilized to detect the expression of surface markers on the EC. Confluent EC in 96-well plates were stimulated with IFN- for various time periods and fixed with 2% formalin/saline for 10 min. After extensive washing with PBS, EC were reacted individually with several mouse mAb (10 µg/ml in PBS with 2% rat serum) to rat surface markers, including MHC class II (OX6; Serotec), intercellular adhesion (molecule (ICAM)-1; (1A29; Serotec), vascular cell adhesion [molecule (VCAM)-1 [5F10; gift of Dr R. Lobb (26)], and B7-1 and B7-2 [CD80; 3H5 and CD86; 24F; gift from Dr Okumura, Juntendo University, Tokyo, Japan (27)]. Biotin-conjugated anti-mouse IgG (Boehringer Mannheim, Indianapolis, IN) was applied and followed by horseradish peroxidase (HRP)-conjugated avidin (Boehringer Mannheim). Colorimetric reactions were developed with o-phenylenediamine (OPD) (Sigma) in the presence of 0.02% H2O2. The reactions were stopped with 2 N H2SO4 and were measured at 490 nm.
Flow cytometry
Subconfluent monolayers of MAT-1 and MAT-171 were cultured for 3 days with or without IFN- (1000 U/ml). Cells were harvested after EDTA treatment, and resuspended at 15x105 cells/ml in PBS with 1% rat serum and 0.01% sodium azide. The resulting suspension was incubated with anti-MHC class II (OX6) mAb, anti-CD80 (B7-1) mAb or anti-CD86 (B7-2) mAb followed by FITC-labeled rat F(ab')2 anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA). Th1 clone G23 was stimulated with irradiated splenic APC and antigen for 3 days. T clone cells were isolated by Isolymph (Gallard-Schlesinger, Carle Place, NY) gradient centrifugation and were stained with mAb to CD28 (JJ319) (PharMingen, San Diego, CA), CD80, CD86,
ß TCR (R73; gift of Dr T. Hünig, University of Würzburg, Germany) or with isotype-matched control mAb PA20 (IgG1) or PF18 (IgG2a) (28). FITC-labeled rat F(ab')2 anti-mouse IgG was employed to verify specific binding of each mAb.
Antigen presentation to T cell clones by EC
The T cells were stimulated with splenic APC and formalin-fixed A. actinomycetemcomitans or TT for 3 days in advance. EC (primary culture, clone MAT-1 or clone MAT-171) in 96-well plates were stimulated with IFN- and formalin-fixed A. actinomycetemcomitans or TT for 3 days. T cells (2x104 cells/well) were applied to the EC treated with mitomycin C (MMC; 25 µg/ml for 30 min; Sigma) on day 0 and cultured for 3 or 4 days. T cell proliferation was assayed by [3H]thymidine (0.5 µCi/well) incorporation during the last 16 h of total culture. In some experiments, mAb (10 µg/ml) to CD2 (OX34; PharMingen), MHC class II, ICAM-1, VCAM-1, B7-1, B7-2, control mAb PA20, CTLA-4Ig fusion protein or control fusion protein L6 (both fusion proteins were a gift from Dr P. S. Linsley, Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA) were selectively added to the culture on day 0.
T lymphocyte transmigration across an EC layer
The T cell transendothelial migration assay has been previously reported (23). EC (5x104/insert) were applied onto 0.2% gelatin-coated Falcon cell culture insert polyethylene terephthalate (PET) filter, 3 µm pore size, 24-well format (Becton Dickinson, Franklin Lakes, NJ). After 1 or 2 days of culture, confluent EC on the PET filter membrane were stimulated with rat recombinant IFN-. After washing the EC on the PET filter membrane, T cell clones (5x105 cells/filter) were overlaid with or without mAb (10 µg/ml) to MHC class II, B7-1, B7-2 or control mAb and incubated for 3 h at 37°C in a 5% CO2 atmosphere. Lymphocytes transmigrating to the bottom of the individual wells of a 24-well plate were harvested and the number of cells was counted with the aid of a hemocytometer.
Antigen responsiveness of T cells after transmigration across an EC layer
Transmigrated T cells were cultured (5x103 cells/well) with irradiated splenic APC with or without antigen and/or recombinant rat IL-2. The other group of transmigrated T cells was maintained in the medium alone for 4 days and re-stimulated with antigen and irradiated splenic APC. [3H]Thymidine (0.5 µCi/well) was applied for the last 16 h of a total of 3 days culture and the radioactivity was detected by scintillation spectrometry (LS-100C) (Beckman, Irvine, CA).
Detection of IL-2
The CTLL-2 bioassay (29) is specific for rat IL-2 and does not react to rat recombinant IL-4 or IFN-. The supernatant of T cells, which were cultured for 24 h, was harvested and stored for the IL-2 bioassay. CTLL-2 cells (2x104 cells/well) were cultured with the supernatant of T cells or serial dilution of control recombinant rat IL-2. After 24 h culture of CTLL-2 cells, each well was incubated with 0.5 µCi/well of [3H]thymidine and the radioactivity was detected by scintillation spectrometry. The IL-2 concentration in the samples was titered and compared to the control recombinant rat IL-2.
Electron microscopic analysis
The analytical methods have been previously described (9). Briefly, the G23 clone cells were incubated with no stimulation or IFN--stimulated EC clone (ECC) on the PET filter for 8 min. The samples on the filter were fixed in 2.5% glutaraldehyde/1% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.4), followed by 1% osmium tetroxide in 0.1 M sodium cacodylate buffer (pH 7.4) and 1% uranyl acetate in 0.1 M maleate buffer (pH 5.2). After dehydration, the membranes were immersed in 100% propylene. Subsequently, the samples were embedded in Spurr's resin (Electron Microscopy Science, Fort Washington, PA). Finally, thin sections were stained with 3% uranyl acetate in 50% methanol for 10 min and Reynolds lead citrate for 30 s. Electron micrographs were obtained with a Jeol 100 CX TEM operated at an accelerating voltage of 80 kV.
Results
B7 co-stimulation dependent T clone and lines
An A. actinomycetemcomitans Omp29-specific Th1 clone (G23) (Fig. 1A), Omp29-specific Th1 (Fig. 1B
) line and a TT-specific T cell line (Fig. 1C
) were tested for stimulation with antigen and APC (irradiated spleen). The proliferation of all T clone cells and lines was significantly enhanced by antigen and APC stimulation as compared to APC alone. The enhanced proliferation was abrogated by anti-CD80 (B7-1), anti-CD86 (B7-2), anti-MHC class II mAb (blocking antibodies) and by the fusion protein CTLA-4Ig, but not by control mAb or control fusion protein L6. These results demonstrated that antigen-specific proliferation of all T clone cells and T lines was dependent on MHC class II and B7 co-stimulation.
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Enhanced T cell trans-EC migration by CD28 signaling
Previously we have reported that IFN- stimulation of EC can up-regulate Th1-specific transmigration due to the interaction between CCR5 on Th1 cells and RANTES produced by the EC (9). RANTES functions on T cell transmigration very rapidly by inducing diapedesis through the phosphotidylinositol 3-kinase-associated pathway. Since CD28 stimulation also induces phosphotidylinositol 3-kinase activation (30), we tested whether CD28 stimulation of the transmigrating T cells could affect their ability to transmigrate (Fig. 1
: G, Omp29-specific T clone; H, Omp29-specific Th1 line; I, TT-specific T cell line). Stimulation of EC with IFN-
in the presence or absence of antigen up-regulated T cell transmigration (Fig. 1G and H
), which was inhibited by anti-RANTES (data not shown) (9). There was no significant difference between the presence and the absence of antigen in the number of transmigrated T cells (Fig. 1GI
). However, the number of T cells transmigrated across EC stimulated with IFN-
and antigen was further enhanced by the presence of anti-CD28 mAb (Fig. 1GI
).
Induction of antigen non-responsiveness of T cells (anergy) by transmigration across the antigen- and IFN--stimulated EC layer
We addressed the effect of antigen presentation by EC to the transmigrating T cells. T cells that had undergone transendothelial migration were subsequently stimulated with splenic APC and antigen (Fig. 1: J, Omp29-specific T clone; K, Omp29-specific Th1 line; L, TT-specific T cell line). The proliferative response of T cells that transmigrated across IFN-
- and antigen-stimulated EC was significantly reduced as compared to the IFN-
-alone-stimulated EC. The reduced proliferative response was abrogated by the presence of anti-CD28 mAb, suggesting that absence of B7 co-stimulatory signal seemed to be responsible for the reduction of T cell proliferation (anergy).
Role of transmigration for the induction of T cell nonresponsiveness
It has been reported that activated T cells also express B7-1 and B7-2 co-stimulatory molecules, and that B7-expressing T cellT cell interaction can exert a T cell stimulatory effect (31,32). T cells used in this study were tested for expression of ßTCR, CD28, B7-1 and B7-2. Flow cytometry analyses of G23 (Fig. 2
) indicated expression of
ßTCR, CD28, B7-1 and B7-2. Omp29-specific T line cells and TT-specific T line cells expressed B7-1 and B7-2, but naive T cells from lymph nodes did not (data not shown).
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Expression of MHC class II, but not B7 molecules, on EC
The kinetics of expression of MHC class II, ICAM-1, VCAM-1 and B7-1 on both primary EC culture and cloned EC (MAT-1) after IFN- stimulation were studied by cellular ELISA (Fig. 4A
, MAT-1, primary culture EC; similar to MAT-1, not shown). MAT-1 did not express MHC class II in the absence of stimulation (0 h). However, MHC class II expression on MAT-1 was induced after 24 h stimulation with IFN-
. ICAM-1 was constitutively expressed on MAT-1 and was enhanced earlier (3 h stimulation with IFN-
) than MHC class II expression. VCAM-1 expression was also induced by IFN-
, although expression was relatively low compared to MHC class II and ICAM-1. Importantly, primary cultures of EC and the EC clone MAT-1 showed the same expression patterns of all molecules described above. The B7-1 transfectant MAT-171 also maintained the same characteristics as the parent ECC MAT-1, except for the constitutive expression of B7-1 (Fig. 4A
). Expression of B7-2 was not observed on either cell type after stimulation with IFN-
for as long as 96 h (data not shown).
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Induction of T cell proliferation by B7-1-transfected ECC but not by B7 ECC
MAT-1 stimulated with IFN- alone or with IFN-
in the presence of A. actinomycetemcomitans did not activate the Th1 clone G23 (Fig. 5A
) as primary cultured EC shown in Fig. 1
(D) did not. However, the B7-1 transfectant MAT-171 was able to activate G23, which could only be inhibited by mAb anti-B7-1 (CD80) and anti-MHC class II (Fig. 5A
). Blocking of the adhesion molecules ICAM-1 or VCAM-1 did not inhibit G23 proliferation, which was induced by antigen presentation of MAT-171 (not shown). Thus, proliferation of T cells induced by MAT-171 seemed to be dependent on B7.
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The absence of B7 on ECC is responsible for transmigration anergy on Th1-type cells
To determine whether transmigration anergy of T cells could be related to the absence of B7-1 or B7-2 co-stimulation, the Th1-type clone (G23) was subjected to transmigration across MAT-171, which expresses B7-1 molecules as compared with the B7 negative MAT-1. Transmigrated G23 cells were re-stimulated with APC and antigen (A. actinomycetemcomitans). Proliferation and IL-2 production by transmigrated G23 to subsequent APC and A. actinomycetemcomitans antigen stimulation was monitored (Fig. 6). G23 transmigrated across IFN-
- and antigen-stimulated MAT-1 did not respond to subsequent APC and antigen stimulation, and also did not produce IL-2 (Fig. 6A
). The induction of anergy was inhibited by anti-MHC class II mAb, but not by any control antibodies, suggesting that the single signal of MHC class II on MAT-1 to Th1 cells was responsible for the induction of anergy. After maintenance of transmigrated T cells in medium alone for 2 days, the viability of T cells transmigrating across IFN-
- and antigen-stimulated EC or EC stimulated with IFN-
-alone showed no differences (82 ± 5 versus 74 ± 7% respectively; mean ± SD), but still showed differences in proliferative response to APC and antigen stimulation (3092 ± 568 or 696 ± 228 c.p.m. respectively; mean ± SD). Interestingly, G23 cells that transmigrated across IFN-
- and antigen-stimulated MAT-171, responded to subsequent antigen presentation by APC and produced IL-2 (Fig. 6B
). When anti-B7-1 mAb was added at the time of G23 transmigration across MAT-171, G23 was rendered anergic. IL-2 production by G23 after re-stimulation with APC and A. actinomycetemcomitans, was always correlated with proliferation.
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Discussion |
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In this manuscript we introduce a novel regulatory consequence of T cellEC interaction which can result in anergic T cell entry into inflamed tissue. In humans, CD45RO+/CD29+ (memory/effector) T cells traffic into inflammatory tissue (33) or exhibit preferential transmigration across EC layers in vitro (34) when compared to CD45RA+ (naive) T cells. Naive T cells preferentially migrate into peripheral lymph nodes. We have demonstrated that the presence of antigen in gingival tissue can attract and retain rat antigen-specific T clone cells in gingival tissues (22). Importantly, the Th1 clone (G23) and Omp29-specific Th1 line cells that we used in this study were memory-type T cells (CD44high/CD45RC/CD62L). The Th1 clone G23 has shown dramatic transendothelial migration compared to naive T cells (9), and has migrated into lipopolysaccharide- and antigen-challenged gingival tissue (22). These cells also caused inflammatory bone resorption dependent on local B7 co-stimulation by macrophages (35). Thus, we believe our transmigration model reflects memory/effector lymphocyte migration into peripheral inflammatory lesions, but not necessarily lymphocyte traffic to lymphoid organs.
With respect to memory T cells such as in vitro cultured T clone cells, TCR single occupancy in the absence of B7/CD28 signals renders the T cells anergic (16,36,37) and CD28 signaling prevents the induction of anergy (18). By contrast, TCR single occupancy without B7 co-stimulatory molecules induces Fas-dependent apoptosis in naive T cells (38). However, exposing naive T cells to joint ligation of both TCR and CD28 in vitro causes exponential growth of the cells for several weeks, even though the cells express high levels of Fas and Fas ligand (FasL) (39). Therefore, although EC express FasL constitutively (40), mature T cells (experienced antigen presentation by professional APC) seem to be independent from the FasL influence. Actually, transmigration events caused the memory T cells used in this study to become anergic, but not apoptotic, because transmigrated T cells exhibited no difference in their viability irrespective of antigen presentation by EC and only showed differences in antigen responsiveness.
A number of studies have discussed antigen presentation to T cells by EC in vitro in co-culture systems (11,12,41). Most studies in humans demonstrated that antigen presentation by EC resulted in T cell proliferation dependent on CD2, but not CD28 (11,14). However, rat microvascular EC induced a non-responsive state of a syngeneic encephalitogenic T cell line (42) similar to that shown in the present study. There may be a species difference in CD2 ligand between human and rats, because the most potent ligand of CD2 in humans is CD58, whereas in rats it is CD48 (43). Although both molecules are structurally quite similar, they are different at least in affinity for CD2 (44). However, this still falls under the category of speculation and requires further investigation. It is noteworthy that T cell proliferation induced by MAT-171 was partially reduced by anti-CD2 mAb in the present experiment (not shown).
Although transmigration across antigen-presenting EC could induce T cell anergy, we could not induce anergy of adherent T cells on EC that had not transmigrated (Fig. 3). This seemed to be related to expression of both B7 and CD28 by activated T cells (Fig. 2
). Interestingly, Denton, et al. (45) demonstrated that EC can up-regulate CD86 expression of alloreactive CD4+ T cells associated with signaling by MHC class II and LFA3. Also, B7 expressed on T cells can function as co-stimulatory in TT interaction (31,32,45). These lines of evidence supported our finding that adherent T cells on EC are incapable of becoming anergic because of the B7 signaling of bystander T cells combined with TCR single signaling by MHC class II on EC. If TT interaction plays a key role in antigen presentation by EC to T cells, the number of T cells added into the co-culture with EC seems to be an important factor. As in most experiments in which nearly confluent T cells (35x105/well of 96-well plate) are used to test T cell response to antigen-presenting EC (41,45), it is noteworthy that the same number of T cells (5x105 cells/filter 9 mm diameter = diameter of 96-well plate) were used in this study.
RANTES produced by EC seems to be responsible for enhancement of Th1 specific transmigration across IFN--stimulated EC (9), because RANTES receptor CCR5 is exclusively expressed on Th1-type cells (10). In the present study, IFN-
stimulation of EC also enhanced T cell transmigration because of the RANTES produced by EC. However, bacterial stimulation of EC in the presence of IFN-
did not alter the transmigration of T cells unless stimulatory anti-CD28 mAb was added. Thus, TCR single signaling by EC seemed not to affect the transmigration capacity of T cells, and both signals from MHC class II and B7-1 on MAT-171 appeared to enhance T cell transmigration (Fig. 5B
).
The biofunctional role of MHC class II expression on EC in the inflammatory lesion is unknown. We first proposed that T cell transmigration anergy would be a protective mechanism to eliminate autoreactive T cells in the inflammation (23). The autoreactive T cells, which may not be deleted by thymus selection, would hypothetically traffic to inflamed tissue and could cause tissue destruction. Th1-type cells seem to play a key role in the induction of autoimmune disease. The activation of Th1-type cells is correlated with the induction of autoimmune disease and tolerance is often associated with a block in the development of self-antigen-specific Th1-type cells (46). Antigen-specific peripheral tolerance is associated with blockade of expansion of Th1-type cells, but not of Th2-type cells (47,48). Thus, MHC class II expression on the EC in inflammatory lesions would be a mechanism to generate peripheral T cell tolerance, limiting proliferation of autoreactive Th1-type cells.
EC involvement in the immune system has been recently recognized (1). The vascular endothelium is not only the duct that transports lymphocytes in the blood stream, but also these are the elements which regulate lymphocyte traffic to particular effector sites. In this study, we illustrated a further major immune regulatory function of EC; induction of anergy on transmigrating Th1 cells by antigen presentation in the absence of co-stimulatory signal.
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Acknowledgments |
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Abbreviations |
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APC antigen-presenting cells |
CCR CC chemokine receptor |
EC endothelial cells |
ECC endothelial clone cells |
FasL Fas ligand |
HRP horseradish peroxidase |
HUVEC human umbilical cord vein EC |
ICAM intercellular adhesion molecule |
LFA lymphocyte function-associated antigen |
MAT-1 B7 negative ECC |
MAT-171 B7-1 transfected ECC |
MMC mitomycin C |
Omp outer membrane protein |
OPD o-phenylenediamine |
PET polyethylene terephthalate |
RANTES regulated on activation normal T cell expressed and secreted |
TT tetanus toxoid |
VCAM vascular cell adhesion molecule |
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Notes |
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Received 3 December 1999, accepted 28 February 2000.
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References |
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