Th1 transmigration anergy: a new concept of endothelial cell–T cell regulatory interaction

Toshihisa Kawai1, Makoto Seki2, Hisashi Watanabe3, Jean W. Eastcott1, Daniel J. Smith1 and Martin A. Taubman1

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


    Abstract
 Top
 Abstract
 Introduction
 Discussion
 References
 
Stimulation of endothelial cells (EC) with IFN-{gamma} generates selective enhancement of Th1 cell transmigration and induction of MHC class II expression on EC. In the present study, we tested whether antigen presentation by EC could influence transmigrating T cells in an in vitro system. Bacterial antigen presentation by EC from primary culture and after cloning induced antigen-specific anergy of transmigrating Th1 clone cells in a MHC class II-dependent manner as characterized by non-responsiveness to subsequent antigen presentation and inability to produce IL-2. This T cell transmigration anergy induced by EC was abrogated by anti-rat CD28 mAb, suggesting that lack of B7 co-stimulatory signals by EC might be related to the induction of anergy. While MHC class II expression on primary and cloned EC was observed after IFN-{gamma} stimulation, these cells never expressed B7. B7-1 gene-transfected endothelial clone cells

(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-{gamma} 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


    Introduction
 Top
 Abstract
 Introduction
 Discussion
 References
 
Endothelial cell (EC) enrollment in the immune system has been recently elucidated (1). Vascular endothelium is not only the duct which transports lymphocytes in the blood stream, but also these are elements which can regulate lymphocyte traffic to particular effector sites (2).

MHC class II molecules can be induced on EC by cytokines such as IFN-{gamma} (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-{gamma} 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-{gamma} 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-{gamma} 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), 5x10–5M 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-{gamma} 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-{gamma} (1000 U/ml). Cells were harvested after EDTA treatment, and resuspended at 1–5x105 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, {alpha}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-{gamma} 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-4–Ig 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-{gamma}. 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-{gamma}. 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-{gamma}-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. 1AGo), Omp29-specific Th1 (Fig. 1BGo) line and a TT-specific T cell line (Fig. 1CGo) 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-4–Ig, 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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Fig. 1. Influence of B7 on T cell proliferative response to professional APC or EC, transmigration capacity across antigen-presenting EC and induction of antigen-specific non-responsiveness (transmigration anergy) after transmigration across antigen-presenting EC. B7- and MHC-dependent T cell proliferation by professional APC. All T cells (2x104 cells/well) were cultured with irradiated spleen APC and antigen (formalin-killed A. actinomycetemcomitans, in A and B; TT, in C) for 3 days with anti-CD80 mAb (xCD80), anti-CD86 mAb, anti-MHC class II mAb, control mAb, CTLA-4–Ig or control fusion protein L6 (10 µg/ml) and [3H]thymidine (0.5 µCi/well) incorporation during the last 16 h was measured (A, G23; B, Omp29 specific-Th1 line; C, TT-specific T line). EC can induce T cell proliferation in the presence of anti-CD28 stimulatory mAb (D, G23; E, Omp29-specific Th1 line; F, TT-specific T line). T cells (2x104 cells/well) were cultured with MMC-treated EC which was pre-stimulated with IFN-{gamma} in the presence or absence of antigen. T cells were cultured with anti-CD28 stimulatory mAb, control mAb, CTLA-4–Ig or control fusion protein L6 (10 µg/ml), for 4 days and [3H]thymidine (0.5 µCi/well) incorporation during the last 16 h was measured. Anti-CD28 mAb increased T cell proliferation by EC pre-stimulated with IFN-{gamma} and antigen, but not by IFN-{gamma} alone (not shown). *Significantly different from EC stimulated with antigen and IFN-{gamma} by Student's t-test (P < 0.05). CD28 stimulation enhanced T cell transmigration across an EC layer. The confluent EC layer on a PET filter was pre-stimulated with IFN-{gamma} and/or antigen. T cells (5x105 cells/filter) were overlaid with anti-CD28 stimulatory mAb (10 µg/ml) for 3 h (G, G23; H, Omp29-specific Th1 line; I, TT-specific T line). The number of transmigrated T cells was counted. *Significantly different from EC medium control by Student's t-test (P < 0.01). **Significantly different from EC stimulated with IFN-{gamma} alone by Student's t-test (P < 0.05). Induction of antigen non-responsiveness of T cells by transmigration across an antigen and IFN-{gamma}-stimulated EC layer. The transmigrated T cells shown above (G–I) were subsequently stimulated (5x103 cells/well) with spleen APC with or without antigen (J, G23; K, Omp29-specific Th1 line; L, TT-specific T line). Proliferation of T cells was detected by the [3H]thymidine incorporation assay. *Significantly different from EC stimulated with IFN-{gamma} alone by Student's t-test (P < 0.01). All results are expressed as mean ± SD of triplicate wells.

 
Antigen-specific T cell proliferation induced by primary cultured EC in the presence of stimulatory anti-CD28 mAb
The ability of primary cultures of EC to present antigen to T cells was tested (Fig. 1Go: D, Omp29-specific T clone; E, Omp29-specific Th1 line; F, TT-specific T cell line). Significant enhancement of proliferation was observed by T cells co-cultured with IFN-{gamma}- and antigen-stimulated EC in the presence of anti-CD28 mAb. The fusion protein CTLA-4–Ig did not affect the T cell proliferative response to IFN-{gamma}- and antigen-stimulated EC, nor did other mAb anti-CD2, anti-CD80 (B7-1), anti-CD86 (B7-2), anti-ICAM-1 or anti-VCAM-1 (not shown). EC stimulated with IFN-{gamma} alone or antigen alone did not induce T cell proliferation even in the presence of anti-CD28 mAb (not shown).

Enhanced T cell trans-EC migration by CD28 signaling
Previously we have reported that IFN-{gamma} 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. 1Go: G, Omp29-specific T clone; H, Omp29-specific Th1 line; I, TT-specific T cell line). Stimulation of EC with IFN-{gamma} in the presence or absence of antigen up-regulated T cell transmigration (Fig. 1G and HGo), 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. 1G–IGo). However, the number of T cells transmigrated across EC stimulated with IFN-{gamma} and antigen was further enhanced by the presence of anti-CD28 mAb (Fig. 1G–IGo).

Induction of antigen non-responsiveness of T cells (anergy) by transmigration across the antigen- and IFN-{gamma}-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. 1Go: 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-{gamma}- and antigen-stimulated EC was significantly reduced as compared to the IFN-{gamma}-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 cell–T cell interaction can exert a T cell stimulatory effect (31,32). T cells used in this study were tested for expression of {alpha}ßTCR, CD28, B7-1 and B7-2. Flow cytometry analyses of G23 (Fig. 2Go) indicated expression of {alpha}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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Fig. 2. Flow cytometry analyses of the surface phenotype of the Th1 clone G23 after 3 days stimulation with spleen APC and antigen. Single-cell suspensions of T clone cells isolated from APC were stained with control mAb (open histogram) or mAb (shaded histogram) anti-rat {alpha}ßTCR (A), CD28 (B), CD80 (C; B7-1) and CD86 (D; B7-2). The data scales are logarithmic. One representative experiment of three is shown.

 
Interaction between T cells and EC
Electron microscopic analyses were carried out to view the physical interaction between the T cell and the EC (Fig. 3A and BGo). T cells (G23) were incubated for 8 min on the EC layer stimulated with IFN-{gamma} (Fig. 3BGo) or in medium alone (Fig. 3AGo). T cells did not demonstrate diapedesis in the medium control (Fig. 3AGo). However, it appeared that T cells which adhered to the EC had simultaneously contacted bystander T cells (Fig. 3AGo; also seen with IFN-{gamma}, but not shown), suggesting that adherent T cells on EC can receive two different stimuli from either EC or from other T cells (Fig. 3AGo). At 8 min incubation T cells began to transmigrate across EC layers pre-stimulated with IFN-{gamma} (Fig. 3BGo). When T cells are transmigrating across EC layers, the T cells seemed to contact only the EC. Therefore, transmigrating T cells may receive more stimuli from EC and may be less influenced by other T cells than the adherent T cells on the EC.



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Fig. 3. Role of transmigration in the induction of anergy. Homotypic T cell–T cell interaction occurs more readily on adherent T cells than transmigrating T cells. Electron microscopic analyses were carried out on the T cells (G23) after incubation for 8 min incubated on non-stimulated (A) or IFN-{gamma}-stimulated EC layers (B). The transmigration assay system is shown (C). The T cells (G23) were added into the upper compartment of the filter insert with or without CTLA-4–Ig (10 µg/ml) or anti-MHC class II (10 µg/ml). After 3 h incubation, the T cells adherent on the EC layer (D) or the transmigrated T cells (E) were harvested and subjected to subsequent APC antigen presentation and the antigen-specific proliferation assay. The adherent T cells (D) and transmigrated T cells (E) (5x103 cells/well) were cultured with spleen APC and antigen in the presence or absence of recombinant IL-2 and/or anti-MHC class II mAb (xMHC class II) for 4 days. T cell proliferation was enumerated by [3H]thymidine incorporation (mean c.p.m. ± SD of triplicate wells). *Significantly different from EC stimulated with IFN-{gamma} alone by Student's t-test (P < 0.001). **Significantly different from T cell proliferation stimulated with APC + antigen + IL-2 by Student's t-test (P < 0.01).

 
Transmigration is required for the induction of anergy in vitro
We hypothesized that T cells adherent on EC may not become anergic because these T cells can receive both MHC class II and B7 signals from EC and also from other T cells. Transmigration may result in the induction of anergy due to the single signal from MHC class II by EC layers. In order to investigate this hypothesis, T cells adherent on EC or T cells that had transmigrated across EC were tested for antigen-specific proliferative response (Fig. 3Go: D, non-transmigrated; E, transmigrated T cells). Three hours after G23 T clone cells were incubated in the transmigration system shown in Fig. 3Go(C), adherent T cells which remained on the EC and the transmigrated T cells were separately recovered (Fig. 3Go: D, adherent T cells; Fig. 3EGo, transmigrated T cells). It is noteworthy that recovered adherent T cells showed transmigration across EC when tested in other fresh EC layers. The adherent T cells recovered from IFN-{gamma}-alone-stimulated EC or IFN-{gamma}- and antigen-stimulated EC responded to the APC plus antigen compared to the APC alone (Fig. 3DGo). This antigen-specific T cell proliferation was significantly reduced when CTLA-4–Ig was added to the upper compartment of the transmigration system with T cells (Fig. 3DGo). The T cells that transmigrated across IFN-{gamma}- and antigen-stimulated EC showed significantly diminished antigen-specific proliferation as compared to EC stimulated with IFN-{gamma} alone (Fig. 3EGo). This diminished antigen-specific proliferation was abrogated by exogenous IL-2. The proliferation regained in the presence of IL-2 was also antigen specific, because anti-MHC class II mAb significantly inhibited the T cell proliferation regained in the presence of IL-2 (Fig. 3EGo). T cell responsiveness was not affected by anti-MHC class II mAb at T cell adhesion or T cell transmigration across IFN-{gamma}- and antigen-stimulated EC (Fig. 3D and EGo). These lines of evidence suggested that transmigration was required for the induction of anergy in the in vitro T cell–EC interaction. Also, cognate co-stimulation by B7 expressed on activated T cells can inhibit the induction of transmigration anergy.

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-{gamma} stimulation were studied by cellular ELISA (Fig. 4AGo, 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-{gamma}. ICAM-1 was constitutively expressed on MAT-1 and was enhanced earlier (3 h stimulation with IFN-{gamma}) than MHC class II expression. VCAM-1 expression was also induced by IFN-{gamma}, 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. 4AGo). Expression of B7-2 was not observed on either cell type after stimulation with IFN-{gamma} for as long as 96 h (data not shown).



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Fig. 4. B7 and other surface molecule expression on ECC after stimulation with IFN-{gamma}. Kinetic analyses of surface markers on MAT-1 and MAT-171, the B7-1 gene transfectant of MAT-1, (A) were detected by cellular ELISA. Confluent ECC in 96-well plates were stimulated with recombinant IFN-{gamma} for the time periods indicated. Then, ECC were fixed with formalin and reacted with the mAb anti-rat MHC class II (xMHC class II), B7-1, ICAM-1 or VCAM-1. The y-axis shows the optical density at 490 nm when OPD was developed to the HRP-conjugated anti-mouse IgG. Results are expressed as the mean optical density of triplicate wells (±SD). (B) Flow cytometry analysis of MHC class II, B7-1 and B7-2 on ECC MAT-1 and MAT-171. MAT-1, unstimulated or stimulated with IFN-{gamma} for 3 days or unstimulated MAT-171 were isolated from culture by treatment with 0.02 % EDTA/PBS. Single-cell suspensions were stained with control mAb (open histograms) or mAb to MHC class II, B7-1 or B7-2 (shaded histograms) followed by FITC-conjugated rat anti-mouse IgG. The data scales are logarithmic.

 
Expression of MHC class II, B7-1 and B7-2 on ECC was further examined by flow cytometry analysis (Fig. 4BGo). MHC class II was only expressed on MAT-1 after stimulation with IFN-{gamma}, whereas B7-1 and B7-2 were not expressed irrespective of IFN-{gamma} stimulation. However, B7-1, but not B7-2, expression was observed on MAT-171 in the absence of IFN-{gamma} stimulation.

Induction of T cell proliferation by B7-1-transfected ECC but not by B7 ECC
MAT-1 stimulated with IFN-{gamma} alone or with IFN-{gamma} in the presence of A. actinomycetemcomitans did not activate the Th1 clone G23 (Fig. 5AGo) as primary cultured EC shown in Fig. 1Go(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. 5AGo). 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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Fig. 5. Antigen presentation by the B7-1-transfected EC to T cells in the co-culture system and influence on the T cell transmigration by B7-1+ EC. Confluent MAT-1 or MAT-171 in a 96-well plate were stimulated with IFN-{gamma} in the presence or absence of antigen for 3 days. After 3 days pre-stimulation with spleen APC and antigen, G23 T clone cells were isolated and applied (2x104 cells/well) onto MMC-treated MAT-1 or MAT-171. T clone cells were cultured for 3 days in the presence of several mAb (10 µg/ml) indicated on the ordinate and proliferation was determined by [3H]thymidine incorporation assay (A). The results are expressed as the mean c.p.m. of triplicate wells (±SD). One representative experiment of three is shown in each panel. EC antigen presentation does not affect the level of T cell transmigration in the absence of B7 but enhances transmigration in the presence of B7 (B). Confluent MAT-1 on the PET filter were stimulated with IFN-{gamma} in the presence or absence of antigen for 3 days. After 3 days stimulation with spleen APC and formalin-fixed A. actinomycetemcomitans, G23 clone cells (5x105 cells/filter) were applied onto MAT-1 on the filter and the number of transmigrated T clone cells in a well were counted after 3 h incubation. Results are expressed as the mean number of transmigrated cells of triplicate wells (±SD). *Significantly different from the non-stimulated EC (medium alone) by Student's t-test (P < 0.05). **Significantly different from all other IFN-{gamma}-stimulated EC (*) by Student's t-test (P < 0.01).

 
Antigen presentation by EC does not affect the level of transmigration of T cells in the absence of B7 signal but enhances transmigration in the presence of B7 co-stimulation
The efficiency of transendothelial migration of rat Th1 clone cells (G23) was examined (Fig. 5BGo). Enhancement of transmigration of G23 was observed after stimulation of ECC with IFN-{gamma} irrespective of the presence of antigen as with the primary culture of EC shown in Fig. 1Go(G). However, only MAT-171 (not MAT-1) presentation of antigen enhanced transmigration (Fig. 5BGo). This enhanced transmigration could be inhibited with CTLA-4–Ig or antibody to MHC class II.

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. 6Go). G23 transmigrated across IFN-{gamma}- and antigen-stimulated MAT-1 did not respond to subsequent APC and antigen stimulation, and also did not produce IL-2 (Fig. 6AGo). 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-{gamma}- and antigen-stimulated EC or EC stimulated with IFN-{gamma}-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-{gamma}- and antigen-stimulated MAT-171, responded to subsequent antigen presentation by APC and produced IL-2 (Fig. 6BGo). 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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Fig. 6. Transmigration anergy is due to the absence of the co-stimulatory molecule B7. Th1-type clone G23 was subjected to transmigration across either MAT-1 (A) or MAT-171; B7-1 transfectant (B) stimulated with IFN-{gamma} in the presence of antigen. Blocking mAb (10 µg/ml) to MHC class II (xMHC class II), B7-1 or B7-2 or control mAb was applied to the upper compartment of the transmigration assay system at the time of G23 transmigration across ECC. After transmigration, G23 was re-stimulated with APC alone or APC and antigen. Proliferation and IL-2 production were monitored. Results are expressed as the mean c.p.m. (proliferation) or U/ml (IL-2) of triplicate wells (±SD).

 
Induction of anergy in T cell clones and lines after transmigration across IFN-{gamma}- and antigen-stimulated EC
To determine if T cell anergy could generally be induced at transendothelial migration, we examined other T cells (Fig. 7Go). All T clones (G23, Omp29-specific; G26, unknown antigen of A. actinomycetemcomitans) and line cells (Line #1, Omp29-specific T cell line (shown in Fig. 1Go); Line #2; Omp29-specific T cell line developed in animals different from Line #1] were rendered anergic when they transmigrated across IFN-{gamma}- and A. actinomycetemcomitans-stimulated MAT-1 (Fig. 7AGo). The anergic state of these T cells was abrogated by the addition of exogenous IL-2. Conversely, anergy was not induced in the cells, which transmigrated across IFN-{gamma}- and antigen-stimulated MAT-171 (Fig. 7BGo), because all T cells that transmigrated across MAT-171 responded with antigen-specific proliferation.



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Fig. 7. The anergic state of Th1-type cells induced after transmigration across antigen-presenting EC can be extended to include antigen-specific T cell clones and T cell lines, suggesting universality of the phenomenon. T cells were subjected to transmigration across either MAT-1 (A) or MAT-171 (B) which were stimulated with IFN-{gamma} in the presence of antigen for 3 days. Before the transmigration experiment, all T clone and line cells were pre-stimulated with APC and antigen for 3 days. Transmigrated T cells were re-stimulated (5x103 cells/well) for 4 days in the presence of irradiated spleen APC, with or without antigen and/or recombinant IL-2. Results of T cell proliferation are expressed as the mean c.p.m. of triplicate wells (±SD).

 

    Discussion
 Top
 Abstract
 Introduction
 Discussion
 References
 
To gain insight into the potential pathophysiological role of MHC class II expressed by EC in inflammation, we examined T cell transmigration across antigen-presenting EC for effects on the T cell responsiveness to succeeding antigen presentation by professional APC. Antigen presentation to T cells by primary cultured EC or cloned EC was observed in the presence of stimulatory anti-CD28 mAb. The number of T cells migrating across antigen-presenting EC was enhanced in the presence of anti-CD28 mAb suggesting that both TCR and CD28 signaling were required for enhancement of T cell transmigration. By contrast, the number of transmigrated T cells was not affected by antigen presentation by EC in the absence of anti-CD28 mAb. However, the T cells that transmigrated across antigen-presenting EC became non-responsive to subsequent antigen presentation by professional APC and could recover response with exogenous IL-2. This induction of the state of `anergy' seemed to be consistent with the lack of expression of B7 co-stimulatory molecules. Transmigration seemed to be necessary for the induction of anergy, otherwise cognate B7 co-stimulation by the bystander-activated T cells prohibited induction of anergy in the EC–T cell co-culture system. The significance of the deficiency of B7 co-stimulatory molecules on EC was confirmed by B7-1 gene-transfected EC. Anergy was not observed on T cells that transmigrated across antigenpresenting EC which express B7-1. However, transmigration anergy was regained in the presence of anti-B7-1 mAb. These results suggested that T cells that transmigrated across antigen-presenting EC were rendered anergic due to the absence of B7 co-stimulation, indicating that EC can regulate antigen responsiveness of T lymphocytes that migrate into tissue.

In this manuscript we introduce a novel regulatory consequence of T cell–EC 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. 3Go). This seemed to be related to expression of both B7 and CD28 by activated T cells (Fig. 2Go). 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 T–T 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 T–T 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 (3–5x105/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-{gamma}-stimulated EC (9), because RANTES receptor CCR5 is exclusively expressed on Th1-type cells (10). In the present study, IFN-{gamma} 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-{gamma} 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. 5BGo).

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.


    Acknowledgments
 
We thank Dr L. Turka for the rat B7-1 cDNA, Dr R. Lobb for mAb 5F10, Dr K. Okumura for mAb 3H5 and 24F, Dr P. Linsley for CTLA-4–Ig and L6, and Dr T. Hünig for the R73 hybridoma. We also thank Ms Jan Schafer for expert secretarial assistance. This work was supported by grant DE-03420 from the National Institute for Dental and Craniofacial Research.


    Abbreviations
 
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

    Notes
 
Transmitting editor: H. Bazin Back

Received 3 December 1999, accepted 28 February 2000.


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