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Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College of Science, Technology & Medicine, London, United Kingdom
Correspondence: Dr. Claudia Monaco, Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College of Science, Technology and Medicine, 1 Aspenlea Road, London W6 8LH, UK. E-mail: c.monaco{at}ic.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, interleukin (IL)-6, and IL-2, prior to fixation and coculture with human umbilical vein EC. Fixed, activated (anti-CD3- or cytokine-stimulated), but not unstimulated T cells, induced release of monocyte chemotactic protein-1, IL-8, and IL-6 by EC in a contact-dependent manner. Moreover, expression of tissue-factor antigen and activity was also significantly increased. Addition of anti-CD40 ligand antibody abolished T cell-induced activation of EC. Our data suggest that contact-mediated activation of EC by T cells, involving ligand:counter ligand interactions such as CD40:CD40 ligand, may represent a novel pathogenic mechanism of progression in inflammatory diseases such as atherosclerosis or rheumatoid arthritis.
Key Words: lymphocytes • endothelial cells • cell-cell interactions • CD40L
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-), have been studied extensively in the context of endothelial activation. However, more recently, the role of direct cell-cell contact-mediated interactions has also been proposed as an important mechanism for cellular activation. As the site for leukocyte adhesion and extravasation, endothelial cells (EC) are ideally positioned for responding to contact-mediated signaling by, for example, adherent lymphocytes. In addition, soluble mediators are likely to be removed rapidly by the circulating blood away from the endothelial lining, suggesting that cell-cell contact-dependent activation of EC might be an alternative and potentially important mechanism of perpetuating inflammation in vivo.
T lymphocytes have been shown to potently up-regulate a range of proinflammatory responses through contact-dependent signaling pathways. For example, fixed T lymphocytes, activated with phorbol 12-myristate 13-acetate (PMA) or phytohemagglutinin (PHA), were shown to increase expression of matrix metalloproteinases (MMPs) and tissue factor by monocytes [2
, 3
]. Monocytic cells were also found to be activated by stimulated HUT-78 T cells to produce TNF- [4
]. In terms of vascular endothelium, T lymphocytes stimulated with a combination of PHA and PMA were shown to up-regulate human brain microvascular EC expression of intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and E-selectin, as well as release of interleukin (IL)-6 and IL-8 [5
]. Peripheral blood lymphocytes have also been shown to increase production of prostaglandin I2 (PGI2) by EC [6
]. Similarly, T cells, resting or activated using PMA and ionomycin, induced expression of the adhesion molecules E-selectin and VCAM-1 on EC through a contact-dependent but CD40 ligand (CD40L)-independent mechanism [7
]. These observations suggest that T cell-mediated contact-dependent signaling may be an important mechanism for the induction of cellular activation.
In the present study, we have used fixed T lymphocytes, which do not release cytokines, to study cell-cell contact in vitro. Because stimulated but not resting T lymphocytes are more likely to cross the endothelial barrier at inflammatory sites spontaneously, T cells were activated through an antigen-dependent pathway with anti-CD3 monoclonal antibody (mAb) and in an antigen-independent manner using a combination of cytokines. These methods have been described previously in studies from our laboratory [8 , 9 ] and are more likely to reflect the in vivo milieu, avoiding the use of nonphysiological stimuli such as PMA or PHA. We investigated whether activated T cells could modulate EC responses in a cell-cell contact-dependent manner. In particular, we assessed the production of the proinflammatory cytokine IL-6, an inducer of acute phase protein synthesis, and of the chemoattractant cytokines IL-8 and monocyte chemotactic protein (MCP)-1. Moreover, we measured the expression of tissue factor, which is induced transiently by endothelial cells in response to various signals, and is a key initiator of coagulation, and hence clinical complications in vivo [10 , 11 ].
Our data indicate that activated T cells can stimulate EC expression of cytokines, chemokines, and tissue factor in a contact-dependent manner. Antibody studies revealed that CD40:CD40L interactions were involved in the induction of EC responses by T cells activated in an antigen-dependent and -independent manner. This suggests that such intercellular interactions may play a major role in the pathogenesis of inflammatory diseases such as atherosclerosis, where leukocyte infiltration and thrombosis are major contributors to disease progression and complications.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(Prof. W. Stec, Center of Molecular and Macromolecular Studies, Lodz, Poland), IL-6 (Dr. P. Ramage, Sandoz, Switzerland), and IL-2 (Dr. U. Gubler, Hoffman-La Roche, Nutley, NJ). Cell-culture medium was RPMI 1640 containing 100 U/ml penicillin and 100 µg/ml streptomycin (BioWhittaker, UK). Hybridoma secreting the anti-CD3 mouse mAb OKT3 was obtained from American Type Culture Collection (Manassas, VA). All reagents and medium were shown to contain <0.1 U/ml lipopolysaccharide (LPS), measured using the Limulus amoebocyte lysate assay (BioWhittaker, UK). The following fluorochrome-conjugated reagents were used for flow cytometry: anti-CD3, anti-CD4, anti-CD8, anti-CD19, anti-CD14, anti-CD25, anti-CD69, anti-CD154 (anti-CD40L; Becton Dickinson, Cowley, UK), anti-CD11a, anti-CD11b, and anti-CD18 (ImmunoKontact, Switzerland). Isotype controls were fluorescein isothiocyanate (FITC)-mouse immunoglobulin G (IgG)1 (Becton Dickinson) and FITC-mouse IgG2a (Sigma Chemical Co., Dorset, UK). Propidium iodide (PI) was purchased from Sigma Chemical Co. Anti-CD154 blocking antibody was purchased from Alexis Corporation (Switzerland).
T cell isolation and activation protocol
Human peripheral blood T lymphocytes were isolated from single-donor plateletphoresis residues purchased from the North London Blood Transfusion Service (Colindale, UK). Mononuclear cells were isolated by Ficoll/Hypaque centrifugation (specific density, 1.077 g/ml; Nycomed, Pharma A.S., Oslo, Norway) prior to separation in a Beckman JE6 elutriator. Elutriation was performed in culture medium with 1% fetal calf serum (FCS). Lymphocyte purity was assessed by flow cytometry. T lymphocyte fractions typically contained >80% CD3-expressing cells, >3% CD19-expressing cells, and <2% CD14-expressing cells.
Elutriation-enriched T lymphocytes were resuspended at 106 cells/ml in culture medium, plus 10% heat-inactivated AB+ human serum (BioWhittaker). T cells were cultured for 48 h with immobilized anti-CD3 mAb OKT3 that had been coated previously onto culture plates at a concentration of 10 µg/ml for 24 h, together with IL-2 (25 ng/ml), as described previously [12
]. Alternatively, T cells were cultured in the presence of a combination of 25 ng/ml TNF-, 100 ng/ml IL-6, and 25 ng/ml IL-2 for 8 days. All of these are saturating concentrations [8
, 13
]. In all instances, control T lymphocytes were cultured in the absence of any stimulus. Initial experiments were performed using T cells that had been stimulated with OKT3 mAb and IL-2 for 24 or 48 h. However, because no marked differences were observed between the EC-activating capacity of these two sets of cells, T cells were stimulated for 48 h in later experiments. Similarly, in cytokine-activated T cell/EC experiments, the greatest EC-activating capacity was observed with T cells stimulated with cytokines for 8 days (unpublished results), as was observed previously for monocyte/T cell studies [9
]. As a consequence, T cells stimulated with cytokines for 8 days were used in further experiments. Following stimulation, T cells were harvested and washed three times in medium before fixation for 1 min in phosphate-buffered saline containing 0.05% glutaraldehyde. Following an additional three washes, fixed cells were resuspended and stored for up to 3 days at 4°C until use. This method of T cell fixation was shown to inhibit T cell proliferation and interferon-
(IFN-) production in response to anti-CD3 stimulation and also prevents the risk of a mixed lymphocyte response of T cells to cells from different donors (unpublished results).
Human umbilical vein endothelial cell (HUVEC) isolation and coculture protocol
EC were isolated from human umbilical veins by digestion for 10 min at 37°C with 0.25 mg/ml collagenase (from Clostridium histolyticum; Boehringer Mannheim GmbH, Mannheim, Germany) and cultured in RPMI 1640 containing 10% FCS, 10% newborn calf serum, heparin (90 µg/ml), and EC growth supplement (20 µg/ml). For the experiments, cells before fourth passage were used.
HUVEC were cultured at 100% confluence in 200 mm2 wells in the presence of fixed T cells at varying T cell/EC ratios, ranging from 3:1 to 10:1 T cells/EC for between 6 and 48 h as indicated. EC are not major histocompatibility complex-restricted and would be expected to respond equally well to a contact-mediated signal from autologous and nonautologous T cells. In some experiments, semipermeable (0.2 µm) anopore-membrane inserts (Nunc Life Technologies, Paisley, UK) were fitted into the culture wells to separate HUVEC (lower chamber) physically from T cells (upper chamber). In addition, in some experiments, anti-CD154 (anti-CD40L) antibody or murine anti-human IgG1 isotype control was added to the fixed T cells at a concentration of 10 µg/ml for 30 min at 4°C, prior to addition of T cells to HUVEC at a ratio of 10:1. Efficient fixation of T lymphocytes was tested by incubating an aliquot of T cells in the absence of EC and by assaying the amount of TNF- and IL-6 released by the cells under these conditions. No release of TNF- and IL-6 by fixed, unstimulated or stimulated T cells was detected in our studies (not shown). TNF- (10 ng/ml) was included in all experiments as a positive control.
Measurement of tissue factor antigen
After coincubation of T cells and HUVEC for between 6 and 48 h, supernatants were removed, and HUVEC were lysed using 1% Triton X-100 in Tris-buffered saline, pH 8.5, at 4°C for 1 h. The cell lysates were stored at -80°C before use. Before using the cell lysates in the assay, it was necessary to determine their protein concentration to ensure that equivalent amounts of protein were present in each well. The total protein content of the cell lysates was determined using a bicinchoninic acid protein assay (PerBio Science UK Ltd., Cheshire, UK). To measure tissue factor, a commercially available enzyme-linked immunosorbent assay (ELISA) kit was used (IMUBIND tissue factor ELISA kit, American Diagnostica, Greenwich, CT). The limit of the assay was >50 pg/ml. All values are expressed as ng tissue factor/106 HUVEC.
Measurement of tissue factor activity
To determine tissue factor activity, a commercially available assay was used (ACTICHROME tissue factor activity assay, American Diagnostica), which measures the activity of tissue factor toward peptidyl substrates. Cells were lysed by repeated cycles of freezing and thawing. Samples were then mixed with sample buffer and human factor VII and incubated at 37°C, allowing formation of the tissue factor/factor VII complex. This complex becomes activated allosterically, and its activity is measured as cleavage of a specific chromogenic substrate (Spectrozyme® factor VIIa). The absorbance of the paranitroaniline chromophore was read at 405 nm and compared with values obtained from a standard curve generated using known amounts of lipidated tissue factor standard. The sensitivity of this method was 0.015 nM lipidated tissue factor.
Measurement of cytokines
For measurement of IL-6, IL-8, and MCP-1 release, T cells and HUVEC were coincubated for between 24 and 48 h. Subsequently, HUVEC supernatants were harvested and stored prior to assay at -20°C.
Cytokines were measured by sandwich ELISA with matched antibody pairs as described previously [14 ]. Reagents for IL-8 and MCP-1 ELISAs were purchased from Becton Dickinson and R&D Systems (Abingdon, UK), respectively. Reagents for IL-6 ELISA were a generous gift from Dr. F. Di Padova, Novartis (Bern, Switzerland). The minimal sensitivity of the ELISA was <13 pg/ml IL-8 and IL-6 and <7 pg/ml MCP-1.
Statistical analysis
Statistical analyses were performed using the GraphPad Prism software package (GraphPad Software, San Diego, CA). To compare antigen expression on T cells exposed to different conditions, Student’s t-test was used. Two-way analysis of variance (ANOVA) was used to compare the effects of different methods of T-cell activation (antigen-dependent vs. antigen-independent) at ratios of between 3:1 and 10:1 T cells:HUVEC.
All data presented are from a representative experiment, and the total number of experiments performed is indicated. Each experiment was analyzed separately for levels of statistical significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, IL-2, and IL-6 (unpublished results). More than 90% of cells expressed CD11a, and more than 65% of cells expressed CD18 in all T cell subsets. There were no significant differences in expression of CD154, CD54, CD11a, CD11b, CD11c, and CD18 between TTCR and TCK.
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Fixed T cells, activated through the T cell receptor (TCR)/CD3 complex or in an antigen-independent manner, stimulate endothelial cells to induce production of proinflammatory cytokines and chemokines
When cultured in the absence of T cells, unstimulated HUVEC ("EC only") released relatively low levels of MCP-1 (Fig. 2a
and b
), IL-6 (Fig. 3a
and b
), and IL-8 (Fig. 4a
and b
). There was no detectable release of MCP-1, IL-6, or IL-8 by fixed T cells TNS, TTCR, or TCK ("T cells only"; Fig. 2a
and 2b
; Fig. 3a and 3b
; Fig. 4a
and 4b
). The addition of fixed T cells (cultured in the absence of any stimulus for 48 h for comparison with TTCR or for 8 days for comparison with TCK; "TNS") failed to up-regulate production of any of these cytokines by HUVEC (Fig. 2a
and 2b
; Fig. 3a
and 3b
; Fig. 4a
and 4b
).
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T cells incubated for 8 days in the presence of a combination of cytokines (IL-2, IL-6, and TNF-; TCK) and fixed to preclude any effects of soluble factors also induced significant production of MCP-1, IL-8, and IL-6. Addition of fixed TCK cells to HUVEC at a ratio of 10:1 for 48 h induced production of 5.7 ± 0.7 ng/ml IL-6 (Fig. 3b)
in one experiment. Production of MCP-1 (Fig. 2b)
and IL-8 (Fig. 4b)
was up-regulated in a comparable manner. Similar induction of MCP-1, IL-6, and IL-8 release by TCK cells was observed in a total of six out of six experiments. Release of MCP-1 (Fig. 2b)
, IL-6 (Fig. 3b)
, and IL-8 (Fig. 4b) increased progressively as a function of an increasing TCK cell:EC ratio. Although in most experiments, 48 h coculture was used, we observed that even after 24 h coculture, there was significant induction of cytokine and chemokine release by TCK cells (Figs. 2c
3c
and 4c)
.
Differential patterns of cytokine and chemokine production induced by T cells activated in an antigen-dependent versus antigen- independent pathway
Although stimulated T cells, activated through the TCR receptor (TTCR) or using cytokines (TCK), are able to induce endothelial production of chemokines and cytokines, the relative amounts of different cytokines were dependent on the method of T cell stimulation. For example, at a ratio of 10:1, TTCR cells induced relatively greater amounts of MCP-1 (59.1±8.0 ng/ml) than did TCK cells (24.8±6.6 ng/ml; Fig. 2
). The difference between the amounts of MCP-1 release induced by TCK cells and TTCR cells was statistically significant (TCK vs. TTCR; P<0.05 by ANOVA in the experiment illustrated).
In contrast, production of IL-6 was up-regulated to a greater extent in the presence of TCK cells (Fig. 3 ; TCK vs. TTCR; P<0.001 by ANOVA in the experiment illustrated). The amounts of IL-8 induced by TCK and TTCR cells were approximately equal (Fig. 4 ; TCK vs. TTCR; no significant difference by ANOVA in the experiment illustrated).
These differences in the relative amounts of cytokine and chemokine production in response to stimulation with TTCR and TCK were reproduced in a total of three experiments.
Stimulation of chemokine and cytokine release from HUVEC induced by activated T cells is dependent on cell-cell contact and on CD40L
To confirm that T cell induction of EC activation was a result of a contact-mediated signal, T cell/EC cultures were set up in the presence and absence of a porous membrane insert to separate the two populations physically. A typical experiment is illustrated in Figure 5
, in which fixed T cells, TTCR (Fig. 5a)
or TCK (Fig. 5b)
at a ratio of 5:1 T cells/EC, induced endothelial production of IL-8, IL-6, and MCP-1 directly. However, such up-regulation of cytokine secretion was abrogated when the two cell populations were separated.
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, decreasing at 24 h and undetectable at 48 h (Fig. 6c)
. In contrast, addition of fixed TTCR or TCK cells to HUVEC at a ratio of 10:1 resulted in a slower induction of tissue factor, which peaked 24 h after addition of T cells and was decreased by 48 h (Fig. 6c)
.
The induction of tissue factor antigen by fixed, activated T cells was paralleled by induction of tissue factor activity (Fig. 6c
, inset). Addition of T cells stimulated using anti-CD3 antibody (TTCR) or using cytokines (TCK) for 24 h induced comparable tissue factor activity on HUVEC. Fixed, unstimulated T cells (TNS) failed to induce tissue factor activity. The levels of tissue factor activity induced by TTCR and TCK coculture were similar to TNF--induced tissue factor activity at the same 24-h time point (Fig. 6c
, inset).
As was the case for up-regulation of cytokine and chemokine release, induction of tissue factor required cell-cell contact (Fig. 6d) and was dependent on CD40-CD154 interactions (Tables 2 and 3) .
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and to the imbalance of proinflammatory cytokines over anti-inflammatory cytokines [8
, 9
]. Similarly, in the context of atherosclerotic disease, acknowledged recently as an inflammatory disease, activated T cells (expressing HLA-DR, VLA-1, CD25, and CD40L) accumulate early in atheroma formation and persist at any stage of disease [16
, 17
]. Thus, T cell EC interactions could contribute to the development of the atherosclerotic plaque [18
]. Our study describes the ability of stimulated but not resting T lymphocytes to modulate EC activation by direct cell-cell contact. Fixed T cells, activated through the CD3/TCR pathway (TTCR) or through an antigen-independent pathway using cytokines (TCK), induced substantial release from EC of MCP-1, IL-8 and IL-6, and expression of tissue factor. Activation of EC was dependent on cell-cell contact. Because stimulation of T cells by cytokines or through the TCR is likely to occur in pathological situations in vivo, such T cell contact-mediated activation of endothelium could contribute to perpetuation of the inflammatory response.
Cell-cell contact (cognate) interactions are emerging as being important in the up-regulation of a range of responses, including T cell-induced up-regulation of adhesion-molecule expression, as well as production of cytokines and PGI2 by EC [5 6 7 ]. However, in these previous studies, T lymphocytes were activated with nonphysiological stimuli such as PHA or a combination of PMA and ionomycin. In contrast, in our study T cells were activated through the TCR using anti-CD3 antibody as an antigen surrogate or with cytokines. Such methods of T cell activation are based on a previously established model of T cell monocyte interactions [8 , 9 ] and are more likely to mimic the physiological milieu.
Possible candidate ligand:counter ligand interactions involved in T cell contact-mediated EC activation include membrane-bound TNF- [5
] or other members of this family, such as CD40L or OX-40. In our study, we have examined the role CD40:CD40L interactions in TTCR- and TCK-induced EC activation. Expression on endothelial cells of CD40 and its up-regulation by cytokines has been shown in a number of studies [19
, 20
]. Anti-CD40L antibody treatment significantly reduced evolution of established atherosclerotic lesions in mice, suggesting that the CD40:CD40L pathway is important in atherosclerosis and its complications [21
]. In a series of in vitro studies, engagement of CD40 up-regulated expression of adhesion molecules and tissue factor, as well as release of chemokines [17
, 19
, 20
, 22
, 23
]. In addition, ligation of CD40 on EC has been shown to increase expression of MMPs (MMP-1, -2, -3, and -9) and vascular endothelial growth factor [24
, 25
]. More interestingly with respect to our own study, platelets have been found to activate EC in a CD40:CD40L-dependent manner to secrete chemokines and to express adhesion molecules and tissue factor [26
, 27
]. In contrast, PHA/ionomycin-activated T cells up-regulated expression of E-selectin and VCAM-1 on EC through a CD40L-independent mechanism [7
]. In our study, anti-CD40L antibody attenuated EC responses induced by fixed T cells, irrespective of whether T cells were activated through an antigen-dependent or -independent manner. This observation underlines the importance of the CD40:CD40L pathway in T cell contact-mediated EC activation. In particular, we believe that this is the first study to demonstrate that T cells activated in a manner likely to reflect the in vivo milieu (e.g., through CD3 or using cytokines present in inflammatory lesions) induce contact-dependent EC responses through endothelial CD40. However, the involvement of other ligand pairs, such as TNF- and its receptors, cannot be excluded at this stage.
Intercellular signaling, such as that demonstrated in the present study between activated T cells and EC, might play a key role in disease situations, such as development of the atherosclerotic plaque. MCP-1, IL-8, and IL-6 are all present in abundance in atherosclerotic lesions [28
, 29
], and our studies suggest that their induction may, at least in part, be a result of T cell contact-mediated signaling to EC. This mechanism may also play a role, via IL-6, in the induction of a systemic low-grade inflammatory response, which is a feature of atherosclerotic disease and is able to predict future cardiovascular events, including nonfatal myocardial infarction, stroke, and the progression of peripheral arterial disease [30
, 31
]. Such contact-dependent induction of IL-6 production may also contribute to the overt up-regulation of IL-6 and C-reactive protein in patients with unstable angina [32
]. Direct cell-cell contact might act in addition or synergistically with soluble factors such as TNF- and IFN-, which are produced by stimulated T lymphocytes [15
].
In addition to inflammation and leukocyte infiltration, expression of tissue factor is found in human atherosclerotic plaques [33 ] and more abundantly in unstable plaques [34 ]. Exposure of tissue factor to circulating blood, as a result of plaque rupture or erosion and/or endothelial activation, is thought to be a major contributor to thrombus formation leading to acute coronary syndromes [35 ] by initiating the extrinsic and intrinsic coagulation pathways [36 ]. The induction of EC activation by T cells may thus play a significant role in promoting the procoagulant state of the endothelium and promote atherosclerotic plaque rupture. Furthermore, increased expression of tissue factor has been shown recently in an animal model of arthritis [37 ], suggesting that T cell-mediated induction of endothelial tissue factor could promote the formation of fibrin, which is observed in the joints of individuals with rheumatoid arthritis.
An additional, interesting observation in our study was the finding that different pathways of T cell activation result in differential responses of EC. In our system, T cells activated through the CD3/TCR pathway preferentially induced production of MCP-1. Antigen-specific T lymphocytes are likely to be present in the human atherosclerotic plaque [38
], and over-expression of MCP-1 induced by TTCR/EC contact interaction might thus preferentially regulate the influx of monocytes [28
]. However, like other cell types, lymphocytes also respond to cytokines. It has been shown that the cocktail of cytokines (IL-6, TNF-, and IL-2) used in our system, could activate T cells in the apparent absence of antigenic stimulation to proliferate and stimulate B cells and to induce monocyte secretion of proinflammatory cytokines [8
, 9
, 13
]. Furthermore, T cells activated by cytokines have been demonstrated to have a role in rheumatoid arthritis, where such cells may drive the excess production of TNF- [8
, 9
]. In atherosclerotic disease, conditions for generating TCK (namely, production of TNF- [39
], IL-6 [29
], and IL-2 [40
]) are likely to be present. In our system, TCK preferentially induced IL-6 production, which is the main inducer of the acute phase response. It is interesting that release of IL-8 and expression of tissue factor were up-regulated to approximately the same levels by both T-cell subsets.
In conclusion, our results demonstrate that stimulated T lymphocytes activate EC in a contact-dependent manner, inducing the production of proinflammatory cytokines and chemokines and expression of tissue factor. We propose that such direct cell-cell contact between stimulated T lymphocytes and endothelium represents a novel pathogenic mechanism in inflammatory diseases such as atherosclerosis and rheumatoid arthritis.
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ACKNOWLEDGEMENTS |
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Received May 26, 2001; revised November 24, 2001; accepted November 24, 2001.
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