CD154/CD40-Mediated Expression of CD154 in Endothelial Cells: Consequences for Endothelial Cell–Monocyte Interaction
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《动脉硬化血栓血管生物学》
From the Department of Cardiovascular Physiology, University of Goettingen, Germany.
Correspondence Dr Markus Hecker, Department of Cardiovascular Physiology, University of Goettingen, Humboldtallee 23, D-37073 Goettingen, Germany. E-mail hecker@veg-physiol.med.uni-goettingen.de
ABSTRACT
Objective— CD40 ligand (CD154) expressed on activated T helper cells is a key costimulatory molecule for antigen-presenting cells expressing the corresponding receptor CD40. Moreover, CD40 stimulation in nonimmune cells, such as endothelial cells, may play an important role in atherogenesis. One gene product that is induced in endothelial cells on exposure to CD154 is CD154 itself.
Methods and Results— In human primary cultured endothelial cells, constitutive CD154 expression was virtually absent and insensitive to proinflammatory cytokines such as tumor necrosis factor and/or interferon-. However, CD154 expression was markedly induced, both on the mRNA and protein level, after CD40 stimulation. Moreover, CD40-positive human monocytes (THP-1 cell line) transmigrating through CD154-expressing endothelial cells responded with an increased expression of interleukin-1? (IL-1?) mRNA, indicative of their activation. This increase in IL-1? expression was confirmed on the protein level and could be abrogated by prior treatment of the endothelial cells with a neutralizing anti-CD154 antibody.
Conclusions— By way of CD154-induced CD154 expression, human endothelial cells thus seem capable of influencing the progression of proinflammatory reactions, including atherogenesis through activation of extravasating monocytes.
Key Words: CD154 expression ? atherosclerosis ? endothelial cells ? diapedesis ? monocyte activation
Introduction
Signaling between T helper cells and antigen-presenting cells (APC) through costimulatory molecules is crucial for the correct propagation of the immune response.1 Among these, CD40 and its ligand CD154 (CD40L or gp39) are required for T cell costimulation and, as a consequence, for T cell maturation and activation of APC effector function.2 CD154 is expressed on activated T cells but not on resting T cells, whereas its receptor CD40 is found on APC, including dendritic cells, monocytes/macrophages, and B lymphocytes, but also on nonimmune cells such as endothelial cells.3
Consequently, exaggerated CD40 signaling has been associated with the pathogenesis of various chronic inflammatory and autoimmune diseases such as asthma, rheumatoid arthritis or psoriasis, but also atherosclerosis (see review by Sch?nbeck and Libby4). Results of in vitro studies interrupting CD40 signaling in atheroma-associated endothelial cells, smooth muscle cells, and monocytes/macrophages suggest a central role for CD40/CD154 receptor-ligand interaction in atherogenesis, including early lesion formation, lesion progression, and plaque rupture.5 Ligation of CD40 on human endothelial cells, for example, results in a prominent stimulation of both chemokine6 and adhesion molecule expression.7–9
The molecular details of leukocyte transmigration through the endothelial barrier during an inflammatory response are not well understood. In the course of extravasation, the monocytes undergo further differentiation to become multifunctional tissue macrophages (see review by Muller and Randolph10). Cellular surface proteins such as adhesion molecules and chemokine receptors presented on endothelial cells and leukocytes, respectively, create the necessary surface topography for leukocyte migration.11 Here we have investigated the hypothesis that CD154-induced CD154 expression on endothelial cells enables these cells to costimulate CD40-expressing monocytes prior to extravasation. This investigation was prompted by preliminary findings of a CD154-induced CD154 mRNA expression in human cultured endothelial cells.
Methods
Cell Culture
Human umbilical vein endothelial cells (HUVEC) were isolated from freshly collected umbilical cords and cultured to a density of 26,000 cells per cm2 growth area (90% to 95% confluence) as described.12 The primary culture of HUVEC was essentially free of contaminating CD45+ (<0.1%) or CD14+ (<1.0%) leukocytes, as judged by fluorescence-activated cell sorter (FACS) analysis.12 The human premonocytic cell line (THP-1) (American Type Culture Collection, Rockville, MD) and the mouse myeloma cell line P3xTB.A7 (stably transfected with human CD154) or the corresponding control cells (P3x63Ag8.653) were cultured in RPMI 1640 medium (Invitrogen, Karlsruhe, Germany) containing 10% FBS and antibiotics as described.12 For coincubation experiments (1 to 24 hours), the CD154+ myeloma cells or the corresponding control cells were added to the cultured HUVEC at 1.5x106 cells per well in 6-well multi-well plates (growth area 9.6 cm2) for polymerase chain reaction (PCR)-analysis, or 4x106 cells per 60 mm diameter Petri dish (growth area 21.3 cm2) for Western blot and FACS analysis, respectively. Incubations were terminated by removing the supernatant, and thoroughly washing the HUVEC monolayer with medium M199 to remove the myeloma cells. In some experiments, CD154+ myeloma cells were preincubated for 90 minutes with a CD154-neutralizing antibody (TRAP-1, 40 μg/mL,6 BD Biosciences, Heidelberg, Germany) to block CD154-CD40 interaction. All reagents and media for cell culture were routinely tested for contamination with bacterial lipopolysaccharides (LPS) by using the colorimetric Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD) according to the manufacturer’s instructions. The endotoxin content of all reagents and media was lower than the detectable level by this method (<0.1 EU/mL).
Endothelial Cell–Leukocyte Interaction
Cell-cell interaction was monitored with a SPOT RT color CCD camera (Diagnostic Instruments, Burroughs St. Sterling Heights, MI) and a parallel plate flow chamber (Warner Instruments, Hamden, CT) that was placed on the stage of an Axiovert S100 TV microscope (Zeiss, Goettingen, Germany) as described.13
Reverse Transcription-PCR (RT-PCR) Analysis and Real-Time PCR
Total RNA isolation, reverse transcription, and polymerase chain reaction for vascular cell adhesion molecule-1 (VCAM-1), interleukin-12 p40 (IL-12p40), CD154, and 60S ribosomal protein L32 (RPL32) cDNA was performed essentially as described.13 Amplification of RPL32 served as an internal standard (house-keeping gene). For detection of human interleukin-1? (IL-1?) and interleukin-23 p19 (IL23p19) cDNA, the following primers with the respective GenBank accession No. and position of the PCR product in the coding sequence and predicted size were used for amplification: IL-1? (M54933, position 226 to 555, 329 bp fragment), 5'-GGCATCCAGCTACGAATCTCCG-3' (sense) and 5'-CACTTGTTGCTCCATATCCTGTCCC-3' (antisense); IL-23p19 (NM_016584, position 299 to 828, 529 bp fragment), 5'-TGGAGTGCACATCCACTAGT-3' (sense) and 5'-TAACCTGTTGGCTCACAGGT-3' (antisense).
In addition, real-time PCR was performed in a LightCycler (Roche Diagnostics) using the QuantiTect SYBR Green real-time RT-PCR kit (Qiagen) according to the manufacturer’s instructions. Primer sequences used for PCR amplification were as follows: CD154 (GenBank accession No. X67878, position 488 to 620, 132 bp fragment, 53°C annealing) 5'-TGAGCAACAACTTGGTAACCCTGG-3 (sense) and 5'-CTGGCTATAAATGGAGCTTGACTCG-3' (antisense); GAPDH (GenBank accession No. BC020308, position 563 to 700, 137 bp fragment, 60°C annealing) 5'-GACCACAGTCCATGCCATCACTGC-3' (sense) and 5'-ATGACCTTGCCCACAGCCTTGG-3' (antisense). Fluorescence was monitored (excitation at 470 nm and emission at 530 nm) at the end of the annealing phase (the LightCycler F1 channel). Recombinant standards were synthesized in vitro from the cloned PCR fragments in plasmid DNA (TOPO TA Cloning kit, Invitrogen). The identity of the amplification products for CD154, GAPDH, IL-1?, and IL-23p19 was verified by direct sequencing with an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Weiterstadt, Germany).
Western Blot Analysis
To detect CD154 expression, HUVEC were grown in 100-mm diameter Petri dishes (growth area 59.0 cm2). They were homogenized at 4°C by approximately 10 strokes through a cell cracker (EMBL) to achieve >95% cell disintegration. The resulting homogenate was centrifuged at 1,000g for 10 minutes to remove nuclei and unbroken cells, followed by preparation of a subcellular fraction enriched in membrane proteins (microsomes).14 Protein extracts (10 μg protein per lane) were separated by denaturing polyacrylamid gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) according to standard protocols.15 Transferred proteins were probed by a polyclonal rabbit anti-human CD154 antibody (1:500 dilution, Santa Cruz Biotechnology, Heidelberg, Germany) and, for normalization, a monoclonal anti-?-actin antibody (1:3000 dilution, Sigma-Aldrich) followed by densitometry.
Flow Cytometry
Flow cytometry of endothelial cells with a mouse anti-human CD154 monoclonal primary antibody (DakoCytomation) and a FITC-labeled polyclonal goat anti-mouse secondary antibody (BD Biosciences, Heidelberg, Germany) was performed as described.15
Endothelial cells were identified by positive staining for platelet endothelial cell adhesion molecule-1 (PECAM-1) using a fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD31 monoclonal antibody (BD Biosciences). Flow cytometry was performed with a Coulter Epics XL flow cytometer (Beckman Coulter, Krefeld, Germany). At least 10 000 cells were counted in each sample.
Transmigration Assay
For transmigration studies, cell culture inserts containing PET membranes (BD Falcon, Heidelberg, Germany) with a pore size of 8.0 μm, a density of 1x105 pores/cm2, and a growth area of 0.9 cm2 were fitted into 12-well multi-well plates (Techno Plastic Products, Trasadingen, Switzerland). Endothelial cells were allowed to grow on the PET membranes until reaching confluence as confirmed by the typical cobblestone morphology that could be visualized by light microscopy due to sufficient transparency of the PET membranes. Subsequently, CD154+ myeloma cells or control cells (0.5x106 cells per insert) were coincubated with the cultured HUVEC overnight (12 hours). Incubations were terminated by removing the supernatant and thoroughly washing the HUVEC monolayer with medium M199 to remove the myeloma cells.
To examine the presence of a humoral stimulatory factor for IL-1? expression, THP-1 cells (104-105 cells resolved in 100 μL medium) were exposed to 500 μL conditioned medium of the CD154-stimulated endothelial cells for 12 hours. For migration studies, 2x106/mL THP-1 cells were presented to the endothelial side of the membrane in the presence of 100 ng/mL recombinant monocyte chemoattractant protein-1 (MCP-1) (R&D Systems, Wiesbaden, Germany),16 added to the lower (ie, subendothelial) compartment, and allowed to migrate for 12 hours. In some experiments, CD154-expressing HUVEC were preincubated for 90 minutes with the CD154-neutralizing antibody (40 μg/mL) before addition of the THP-1 monocytes.
The THP-1 cells that had migrated through the HUVEC monolayer were quantified by cell counting, followed by RNA isolation and RT-PCR analysis or IL-1? immunoassay. Because the integrity of the endothelial monolayer is essential for this assay, endothelial cells were stained with toluidine blue after termination of the experiments and evaluated microscopically. Viability of THP-1 cells was assayed by using trypan blue exclusion, demonstrating no significant difference between THP-1 cells before and after transmigration.
IL-1? Immunoassay
Transmigrated THP-1 cells and cells exposed to the endothelial cell conditioned medium were quantified by protein determination after cell lysis for 15 minutes at 4°C in a homogenization buffer, consisting of 20 mmol/L Tris·HCl, 135 mmol/L sodium chloride, 1% NP-40, 10% glycerol, complete Mini protease inhibitor cocktail tablet according to the manufacturer’s instructions (Roche Diagnostics, Mannheim Germany), pH 8.0, and subsequent centrifugation for 80 minutes at 13 000 rpm and 4°C. For quantification of THP-1 cell-associated IL-1? protein, the human IL-1? QuantiGlo ELISA kit (R&D Systems, Wiesbaden, Germany) was used with 100 μL of the cell lysate each according the manufacturer’s instructions. The measured IL-1? concentrations (pg/mL) were normalized on the basis of the cell count of transmigrated THP-1 cells.
Data Analysis
Unless indicated otherwise, results are expressed as mean±SEM of n observations. One-way analysis of variance followed by a Dunnett multiple comparisons test was used to determine differences between the means and the corresponding control value with P<0.05 considered statistically significant.
Results
CD154-Induced VCAM-1 Expression in Human Endothelial Cells and Effect on Endothelial Cell–Leukocyte Interaction
As previously reported,12 coincubation of the primary cultured HUVEC, which constitutively expresses CD40, with the CD154+ mouse myeloma cells, resulted in an increased expression of the adhesion molecules E-selectin, intercellular adhesion molecule-1 (ICAM-1), and VCAM-1 (not shown). Consequently, adhesion of the human monocytic cell line THP-1 to the CD154-stimulated HUVEC was significantly enhanced as compared with endothelial cells which were coincubated with CD154- myeloma cells (Figure 1A).
Figure 1. A, CD40–CD154 interaction leads to an increased endothelial cell-leukocyte interaction. Statistical summary demonstrating an increased THP-1 cell adhesion to the cultured HUVEC after a 12 hour coincubation with CD154+ myeloma cells, but not with CD154- control cells (n=5; *P<0.05 versus control, P<0.05 versus CD154+). B, mRNA expression of CD154, CD40, and VCAM-1 in primary cultured HUVEC (passage No. 0), and in later passages of these cells (passage Nos. 1 and 7). Constitutive CD154 mRNA expression seemed to increase in passaged cells while basal expression of CD40 and VCAM-1 appeared to decrease (exemplary RT-PCR analysis).
CD154-Induced CD154 Expression in Human Endothelial Cells
Primary cultured HUVEC showed a background expression of CD154 mRNA that was visualized by RT-PCR analysis with only a high number of cycles (>39 cycles). Interestingly, in passaged cells this constitutive CD154 mRNA expression seemed to rise, while basal expression of CD40 and VCAM-1, for example, appeared to fall (Figure 1B). CD154 expression was not inducible by calcium ionophore (1 μmol/L ionomycin) alone or in combination with phorbol 12-myristate 13-acetate (0.1 and 1 μmol/L; not shown, n=3). Neither IFN (1000 U/mL) nor TNF (100 U/mL), IL-1? (60 U/mL), or LPS (1 μg/mL) alone or in combination stimulated CD154 expression in the primary cultured HUVEC (n=3 to 6, not shown).
However, exposure of these cells to the CD154+ myeloma cells markedly enhanced CD154 expression both on the mRNA (Figure 2A) and protein level (Figure 2B and 2C). In addition to FACS analysis, CD154-induced CD154 protein expression was further verified by Western blot analysis (Figure 2E). Endothelial cell CD154 mRNA expression was inducible, albeit to a lesser degree (about 2% to 20% as compared with the effect of the CD154+ myeloma cells), by a stimulatory anti-CD40 antibody (5 μg/mL, Santa Cruz Biotechnology) or a recombinant CD154 fusion protein (3 μg/mL, Immunex, Seattle, WA; Figure 3A). Moreover, CD154-induced CD154 mRNA expression was strongly attenuated by coincubation with a neutralizing anti-CD154 antibody (Figure 3B) in a concentration-dependent manner (Figure 3B inset), whereas an unrelated control immunoglobulin had no such effect (not shown), and seemed to be sensitive to cotreatment with IFN- (Figure 3B). According to Western blot analysis after 24-hour stimulation, CD154-induced CD154 protein expression was inhibited by up to 70% through addition of the neutralizing anti-CD154 antibody, too (Figure 2C), while cotreatment with IFN did not affect CD154 protein abundance (not shown).
Figure 2. CD154-induced CD154 expression in human endothelial cells. Time-dependent effect of coincubation with CD154+ myeloma cells on A, CD154 mRNA expression in the cultured HUVEC, as judged by real-time PCR analysis (calculated copies per 5 ng RNA, n=4); and on B, C, cell surface expression of CD154 protein in HUVEC as judged by flow cytometry. C, Representative histograms depict isotype control (solid line), basal (dashed line), and stimulated endothelial CD154 expression after incubation with CD154+ cells for 16 hours (solid line, area under the curve highlighted in gray). B, Statistical summary of 7 experiments with different batches of cells with CD154 protein expressed as % of CD154 positive cells counted. D, Statistical summary (n=4) of the induction of endothelial cell CD154 protein expression by coincubation with CD154+ myeloma cells for 24 hours, and inhibition by addition of a neutralizing anti-CD154 antibody (CD154 mAb, 40 μg/mL). The inset shows an exemplary Western blot analysis. Protein extracts of CD154+ myeloma cells served as a positive control giving an immunoreactive band with a molecular mass of 32 kDa.
Figure 3. A, Induction of endothelial cell CD154 mRNA expression by an activating anti-CD40 antibody (anti-CD40 Ab, 5 μg/mL) and a recombinant CD154 fusion protein (CD154 fsp, 3 μg/mL) in comparison with the stimulatory effect of the CD154+ myeloma cells (CD154+ cells) after a 12-hour incubation period (n=4). The inset shows a representative RT-PCR analysis. B, Inhibition of CD154-induced CD154 mRNA expression in HUVEC by addition of a neutralizing anti-CD154 antibody (CD154 mAb, 40 μg/mL) or IFN- (1000 U/mL). Statistical summary (n=5) of the percentage of CD154-induced CD154 mRNA expression over 12 hours. The inset shows an exemplary RT-PCR analysis, demonstrating the concentration-dependent effect of the anti-CD154 antibody (10 to 40 μg/mL) on CD154 mRNA expression.
Transmigation Studies
To assess the functional relevance of CD154-stimulated CD154 gene expression, we employed a transmigration assay, monitoring THP-1 cell diapedesis through a monolayer of cultured HUVEC. The THP-1 cells constitutively express CD40 (compare
References 13 and15) but not CD154 (not shown). Therefore, a CD40/CD154-mediated costimulation between the two cell types seemed possible. Indeed, interaction of the monocytes with CD154-expressing endothelial cells resulted in a significant increase in IL-1? expression in the THP-1 cells, both on the mRNA and protein level, thus indicative of their activation (Figures 4A and 5A). Prior neutralization of endothelial cell CD154 completely abrogated this increase in IL-1? expression (Figures 4A and 5 A).
Figure 4. Interaction of THP-1 monocytes with CD154-expressing endothelial cells results in a significant increase in IL-1? (A) and IL-23p19 (B) mRNA expression in the THP-1 monocytes after diapedesis. Transmigration assays and the IL-1? immunoassay were performed as described in Methods. In brief, cultured HUVEC were coincubated overnight with CD154+ myeloma cells for CD40 stimulation or CD154- control cells. Incubations were terminated by removing the supernatant and thoroughly washing the HUVEC monolayer with medium to remove the myeloma cells. Subsequently, THP-1 cells were presented to the endothelial cells with 100 ng/mL recombinant MCP-1 in the subendothelial compartment and allowed to migrate for 12 hours. For CD154 neutralization, CD154-expressing HUVEC were preincubated for 90 minutes with the CD154-neutralizing antibody (CD154 mAb, 40 μg/mL) before addition of the THP-1 monocytes (n=4 each; *P<0.05 and P<0.05 versus CD154+ cell stimulation).
Figure 5. Only direct cell–cell contact between CD154-expressing endothelial cells and THP-1 monocytes (A) and not the release of a humoral factor (B) results in a significant increase in IL-1? protein expression in the THP-1 cells. Transmigration assays and CD154 neutralization were performed as described in the legend to Figure 4 (3 different batches of cells with n=2 to 4 each; *P<0.05 versus control and control cells, P<0.05 versus CD145 mAb).
To examine the possible presence of a humoral stimulatory factor for IL-1? expression, THP-1 cells were exposed to the conditioned medium of CD154-stimulated endothelial cells for 12 hours. As shown in Figure 5B, this incubation had as little effect on IL-1? synthesis by the THP-1 cells as the physical interaction of the monocytes with CD154-expressing endothelial cells, in which the ligand had been neutralized through prior treatment with the anti-CD154 antibody.
Moreover, CD40/CD154-dependent costimulation also resulted in the induction of IL-23p19 expression in the THP-1 cells. Again, this effect was blocked by prior treatment of the cultured HUVEC with the neutralizing anti-CD154 antibody (Figure 4B).
Discussion
The working hypothesis underlying the present study was that during an inflammatory response, CD40-CD154 mediated T helper cell–endothelial cell interaction results not only in an increased recruitment of circulating monocytes, but also an activation of these cells on their crossing of the endothelial cell barrier through CD154-mediated costimulation.
As shown, ligation of endothelial cell CD40 triggered a marked increase in expression of the adhesion molecules E-selectin, ICAM-1, and VCAM-17–9,12 as well as that of the chemokines IL-8 and MCP-1.6,12 Whereas MCP-1 is presumably the most potent chemoattractant for circulating monocytes, VCAM-1 represents the primary integrin for these cells on the endothelial cell surface to which they bind through VLA-4.17 It did not come as a surprise, therefore, that monocyte adhesion to the cultured endothelial cells was markedly elevated after CD40 ligation.
However, to costimulate and hence activate the adherent monocytes, which in their capacity as APC constitutively express CD40, the endothelial cells, in addition, must express CD154 in a biologically active form on their surface. The present findings demonstrate that this is indeed the case, provided that the endothelial cells are appropriately stimulated. In contrast to quiescent T helper cells, which express CD154 in response to calcium ionophore and/or a phorbol ester stimulation,18 these stimuli had just as little effect on endothelial cell CD154 expression as various proinflammatory cytokines such as IFN and/or TNF. Only when exposed to CD154, either in a cell membrane–bound form or as a fusion protein, was significant induction of CD154 expression detected on both the mRNA and protein level, suggesting that prior interaction with activated T helper cells is mandatory for endothelial cell CD154 expression to occur.
In this context, it should be pointed out that primary cultured endothelial cells revealed virtually no constitutive expression of CD154, while this increased over subsequent passages. Similar findings have been described for PECAM-1 and endothelial nitric oxide synthase expression in rat cultured endothelial cells due to activation or silencing of nuclear transcription factors during this process.19 Moreover, in the presence of IFN-, which typically originates from activated T helper cells, CD154-induced endothelial cell CD154 mRNA expression was attenuated. The observed lack of effect of IFN- on CD154 protein expression might be explained by increased protein stability. Thus, in T helper cells, continuous stimulation maintains CD154 ligand expression for up to 48 hours.20 The extent of reciprocal T helper cell stimulation by endothelial cell–derived IL-1212 may therefore direct the course of the subsequent inflammatory response; that is, restricting it to Th1 cell–endothelial cell interaction or facilitating monocyte recruitment and activation in the vessel wall.
Does endothelial cell-monocyte costimulation really proceed through the CD154/CD40 ligand/receptor dyad? To address this question, a transmigration assay was established revealing that only monocytes that physically interact with CD154-expressing endothelial cells respond with a significant increase in the synthesis of IL-1?, which is considered to be a reliable marker of monocyte activation.21 In addition, the transmigrated cells also exhibited an increased expression of IL-23 p19 mRNA. Like IL-12, IL-23 is an integral part of the reciprocal Th1 cell costimulatory function of APC, preferentially targeting memory CD4+ T cells; whereas, IL-12 acts on naive CD4+ T cells.22 Exposing the monocytes to the conditioned medium of CD154-stimulated endothelial cells (indicative of the presence of a humoral stimulatory factor), on the other hand, did not affect IL-1? synthesis.
Collectively, these data demonstrate that by way of CD154-induced CD154 expression, human endothelial cells not only actively recruit circulating monocytes but also activate them, possibly through a dual lock-key principle (eg, endothelial VCAM-1 interaction with monocyte VLA-4 plus endothelial CD154 interaction with monocyte CD40) during their transmigration into the subendothelial space. This may have a significant impact on the severity and possibly on the chronicity of the ensuing inflammatory response, not only in atherosclerosis but also in other forms of chronic inflammatory or autoimmune diseases. Proofing this assumption would render CD154-induced CD154 expression in the vascular endothelium an interesting therapeutic target.
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 402/C9).
We are indebted to Kathrin Schreiber, Henrike Struwe, and Martina Ohme for expert technical assistance, and to Dr Robert Krzesz for providing the IL-23 p19 primer.
References
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Correspondence Dr Markus Hecker, Department of Cardiovascular Physiology, University of Goettingen, Humboldtallee 23, D-37073 Goettingen, Germany. E-mail hecker@veg-physiol.med.uni-goettingen.de
ABSTRACT
Objective— CD40 ligand (CD154) expressed on activated T helper cells is a key costimulatory molecule for antigen-presenting cells expressing the corresponding receptor CD40. Moreover, CD40 stimulation in nonimmune cells, such as endothelial cells, may play an important role in atherogenesis. One gene product that is induced in endothelial cells on exposure to CD154 is CD154 itself.
Methods and Results— In human primary cultured endothelial cells, constitutive CD154 expression was virtually absent and insensitive to proinflammatory cytokines such as tumor necrosis factor and/or interferon-. However, CD154 expression was markedly induced, both on the mRNA and protein level, after CD40 stimulation. Moreover, CD40-positive human monocytes (THP-1 cell line) transmigrating through CD154-expressing endothelial cells responded with an increased expression of interleukin-1? (IL-1?) mRNA, indicative of their activation. This increase in IL-1? expression was confirmed on the protein level and could be abrogated by prior treatment of the endothelial cells with a neutralizing anti-CD154 antibody.
Conclusions— By way of CD154-induced CD154 expression, human endothelial cells thus seem capable of influencing the progression of proinflammatory reactions, including atherogenesis through activation of extravasating monocytes.
Key Words: CD154 expression ? atherosclerosis ? endothelial cells ? diapedesis ? monocyte activation
Introduction
Signaling between T helper cells and antigen-presenting cells (APC) through costimulatory molecules is crucial for the correct propagation of the immune response.1 Among these, CD40 and its ligand CD154 (CD40L or gp39) are required for T cell costimulation and, as a consequence, for T cell maturation and activation of APC effector function.2 CD154 is expressed on activated T cells but not on resting T cells, whereas its receptor CD40 is found on APC, including dendritic cells, monocytes/macrophages, and B lymphocytes, but also on nonimmune cells such as endothelial cells.3
Consequently, exaggerated CD40 signaling has been associated with the pathogenesis of various chronic inflammatory and autoimmune diseases such as asthma, rheumatoid arthritis or psoriasis, but also atherosclerosis (see review by Sch?nbeck and Libby4). Results of in vitro studies interrupting CD40 signaling in atheroma-associated endothelial cells, smooth muscle cells, and monocytes/macrophages suggest a central role for CD40/CD154 receptor-ligand interaction in atherogenesis, including early lesion formation, lesion progression, and plaque rupture.5 Ligation of CD40 on human endothelial cells, for example, results in a prominent stimulation of both chemokine6 and adhesion molecule expression.7–9
The molecular details of leukocyte transmigration through the endothelial barrier during an inflammatory response are not well understood. In the course of extravasation, the monocytes undergo further differentiation to become multifunctional tissue macrophages (see review by Muller and Randolph10). Cellular surface proteins such as adhesion molecules and chemokine receptors presented on endothelial cells and leukocytes, respectively, create the necessary surface topography for leukocyte migration.11 Here we have investigated the hypothesis that CD154-induced CD154 expression on endothelial cells enables these cells to costimulate CD40-expressing monocytes prior to extravasation. This investigation was prompted by preliminary findings of a CD154-induced CD154 mRNA expression in human cultured endothelial cells.
Methods
Cell Culture
Human umbilical vein endothelial cells (HUVEC) were isolated from freshly collected umbilical cords and cultured to a density of 26,000 cells per cm2 growth area (90% to 95% confluence) as described.12 The primary culture of HUVEC was essentially free of contaminating CD45+ (<0.1%) or CD14+ (<1.0%) leukocytes, as judged by fluorescence-activated cell sorter (FACS) analysis.12 The human premonocytic cell line (THP-1) (American Type Culture Collection, Rockville, MD) and the mouse myeloma cell line P3xTB.A7 (stably transfected with human CD154) or the corresponding control cells (P3x63Ag8.653) were cultured in RPMI 1640 medium (Invitrogen, Karlsruhe, Germany) containing 10% FBS and antibiotics as described.12 For coincubation experiments (1 to 24 hours), the CD154+ myeloma cells or the corresponding control cells were added to the cultured HUVEC at 1.5x106 cells per well in 6-well multi-well plates (growth area 9.6 cm2) for polymerase chain reaction (PCR)-analysis, or 4x106 cells per 60 mm diameter Petri dish (growth area 21.3 cm2) for Western blot and FACS analysis, respectively. Incubations were terminated by removing the supernatant, and thoroughly washing the HUVEC monolayer with medium M199 to remove the myeloma cells. In some experiments, CD154+ myeloma cells were preincubated for 90 minutes with a CD154-neutralizing antibody (TRAP-1, 40 μg/mL,6 BD Biosciences, Heidelberg, Germany) to block CD154-CD40 interaction. All reagents and media for cell culture were routinely tested for contamination with bacterial lipopolysaccharides (LPS) by using the colorimetric Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD) according to the manufacturer’s instructions. The endotoxin content of all reagents and media was lower than the detectable level by this method (<0.1 EU/mL).
Endothelial Cell–Leukocyte Interaction
Cell-cell interaction was monitored with a SPOT RT color CCD camera (Diagnostic Instruments, Burroughs St. Sterling Heights, MI) and a parallel plate flow chamber (Warner Instruments, Hamden, CT) that was placed on the stage of an Axiovert S100 TV microscope (Zeiss, Goettingen, Germany) as described.13
Reverse Transcription-PCR (RT-PCR) Analysis and Real-Time PCR
Total RNA isolation, reverse transcription, and polymerase chain reaction for vascular cell adhesion molecule-1 (VCAM-1), interleukin-12 p40 (IL-12p40), CD154, and 60S ribosomal protein L32 (RPL32) cDNA was performed essentially as described.13 Amplification of RPL32 served as an internal standard (house-keeping gene). For detection of human interleukin-1? (IL-1?) and interleukin-23 p19 (IL23p19) cDNA, the following primers with the respective GenBank accession No. and position of the PCR product in the coding sequence and predicted size were used for amplification: IL-1? (M54933, position 226 to 555, 329 bp fragment), 5'-GGCATCCAGCTACGAATCTCCG-3' (sense) and 5'-CACTTGTTGCTCCATATCCTGTCCC-3' (antisense); IL-23p19 (NM_016584, position 299 to 828, 529 bp fragment), 5'-TGGAGTGCACATCCACTAGT-3' (sense) and 5'-TAACCTGTTGGCTCACAGGT-3' (antisense).
In addition, real-time PCR was performed in a LightCycler (Roche Diagnostics) using the QuantiTect SYBR Green real-time RT-PCR kit (Qiagen) according to the manufacturer’s instructions. Primer sequences used for PCR amplification were as follows: CD154 (GenBank accession No. X67878, position 488 to 620, 132 bp fragment, 53°C annealing) 5'-TGAGCAACAACTTGGTAACCCTGG-3 (sense) and 5'-CTGGCTATAAATGGAGCTTGACTCG-3' (antisense); GAPDH (GenBank accession No. BC020308, position 563 to 700, 137 bp fragment, 60°C annealing) 5'-GACCACAGTCCATGCCATCACTGC-3' (sense) and 5'-ATGACCTTGCCCACAGCCTTGG-3' (antisense). Fluorescence was monitored (excitation at 470 nm and emission at 530 nm) at the end of the annealing phase (the LightCycler F1 channel). Recombinant standards were synthesized in vitro from the cloned PCR fragments in plasmid DNA (TOPO TA Cloning kit, Invitrogen). The identity of the amplification products for CD154, GAPDH, IL-1?, and IL-23p19 was verified by direct sequencing with an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Weiterstadt, Germany).
Western Blot Analysis
To detect CD154 expression, HUVEC were grown in 100-mm diameter Petri dishes (growth area 59.0 cm2). They were homogenized at 4°C by approximately 10 strokes through a cell cracker (EMBL) to achieve >95% cell disintegration. The resulting homogenate was centrifuged at 1,000g for 10 minutes to remove nuclei and unbroken cells, followed by preparation of a subcellular fraction enriched in membrane proteins (microsomes).14 Protein extracts (10 μg protein per lane) were separated by denaturing polyacrylamid gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) according to standard protocols.15 Transferred proteins were probed by a polyclonal rabbit anti-human CD154 antibody (1:500 dilution, Santa Cruz Biotechnology, Heidelberg, Germany) and, for normalization, a monoclonal anti-?-actin antibody (1:3000 dilution, Sigma-Aldrich) followed by densitometry.
Flow Cytometry
Flow cytometry of endothelial cells with a mouse anti-human CD154 monoclonal primary antibody (DakoCytomation) and a FITC-labeled polyclonal goat anti-mouse secondary antibody (BD Biosciences, Heidelberg, Germany) was performed as described.15
Endothelial cells were identified by positive staining for platelet endothelial cell adhesion molecule-1 (PECAM-1) using a fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD31 monoclonal antibody (BD Biosciences). Flow cytometry was performed with a Coulter Epics XL flow cytometer (Beckman Coulter, Krefeld, Germany). At least 10 000 cells were counted in each sample.
Transmigration Assay
For transmigration studies, cell culture inserts containing PET membranes (BD Falcon, Heidelberg, Germany) with a pore size of 8.0 μm, a density of 1x105 pores/cm2, and a growth area of 0.9 cm2 were fitted into 12-well multi-well plates (Techno Plastic Products, Trasadingen, Switzerland). Endothelial cells were allowed to grow on the PET membranes until reaching confluence as confirmed by the typical cobblestone morphology that could be visualized by light microscopy due to sufficient transparency of the PET membranes. Subsequently, CD154+ myeloma cells or control cells (0.5x106 cells per insert) were coincubated with the cultured HUVEC overnight (12 hours). Incubations were terminated by removing the supernatant and thoroughly washing the HUVEC monolayer with medium M199 to remove the myeloma cells.
To examine the presence of a humoral stimulatory factor for IL-1? expression, THP-1 cells (104-105 cells resolved in 100 μL medium) were exposed to 500 μL conditioned medium of the CD154-stimulated endothelial cells for 12 hours. For migration studies, 2x106/mL THP-1 cells were presented to the endothelial side of the membrane in the presence of 100 ng/mL recombinant monocyte chemoattractant protein-1 (MCP-1) (R&D Systems, Wiesbaden, Germany),16 added to the lower (ie, subendothelial) compartment, and allowed to migrate for 12 hours. In some experiments, CD154-expressing HUVEC were preincubated for 90 minutes with the CD154-neutralizing antibody (40 μg/mL) before addition of the THP-1 monocytes.
The THP-1 cells that had migrated through the HUVEC monolayer were quantified by cell counting, followed by RNA isolation and RT-PCR analysis or IL-1? immunoassay. Because the integrity of the endothelial monolayer is essential for this assay, endothelial cells were stained with toluidine blue after termination of the experiments and evaluated microscopically. Viability of THP-1 cells was assayed by using trypan blue exclusion, demonstrating no significant difference between THP-1 cells before and after transmigration.
IL-1? Immunoassay
Transmigrated THP-1 cells and cells exposed to the endothelial cell conditioned medium were quantified by protein determination after cell lysis for 15 minutes at 4°C in a homogenization buffer, consisting of 20 mmol/L Tris·HCl, 135 mmol/L sodium chloride, 1% NP-40, 10% glycerol, complete Mini protease inhibitor cocktail tablet according to the manufacturer’s instructions (Roche Diagnostics, Mannheim Germany), pH 8.0, and subsequent centrifugation for 80 minutes at 13 000 rpm and 4°C. For quantification of THP-1 cell-associated IL-1? protein, the human IL-1? QuantiGlo ELISA kit (R&D Systems, Wiesbaden, Germany) was used with 100 μL of the cell lysate each according the manufacturer’s instructions. The measured IL-1? concentrations (pg/mL) were normalized on the basis of the cell count of transmigrated THP-1 cells.
Data Analysis
Unless indicated otherwise, results are expressed as mean±SEM of n observations. One-way analysis of variance followed by a Dunnett multiple comparisons test was used to determine differences between the means and the corresponding control value with P<0.05 considered statistically significant.
Results
CD154-Induced VCAM-1 Expression in Human Endothelial Cells and Effect on Endothelial Cell–Leukocyte Interaction
As previously reported,12 coincubation of the primary cultured HUVEC, which constitutively expresses CD40, with the CD154+ mouse myeloma cells, resulted in an increased expression of the adhesion molecules E-selectin, intercellular adhesion molecule-1 (ICAM-1), and VCAM-1 (not shown). Consequently, adhesion of the human monocytic cell line THP-1 to the CD154-stimulated HUVEC was significantly enhanced as compared with endothelial cells which were coincubated with CD154- myeloma cells (Figure 1A).
Figure 1. A, CD40–CD154 interaction leads to an increased endothelial cell-leukocyte interaction. Statistical summary demonstrating an increased THP-1 cell adhesion to the cultured HUVEC after a 12 hour coincubation with CD154+ myeloma cells, but not with CD154- control cells (n=5; *P<0.05 versus control, P<0.05 versus CD154+). B, mRNA expression of CD154, CD40, and VCAM-1 in primary cultured HUVEC (passage No. 0), and in later passages of these cells (passage Nos. 1 and 7). Constitutive CD154 mRNA expression seemed to increase in passaged cells while basal expression of CD40 and VCAM-1 appeared to decrease (exemplary RT-PCR analysis).
CD154-Induced CD154 Expression in Human Endothelial Cells
Primary cultured HUVEC showed a background expression of CD154 mRNA that was visualized by RT-PCR analysis with only a high number of cycles (>39 cycles). Interestingly, in passaged cells this constitutive CD154 mRNA expression seemed to rise, while basal expression of CD40 and VCAM-1, for example, appeared to fall (Figure 1B). CD154 expression was not inducible by calcium ionophore (1 μmol/L ionomycin) alone or in combination with phorbol 12-myristate 13-acetate (0.1 and 1 μmol/L; not shown, n=3). Neither IFN (1000 U/mL) nor TNF (100 U/mL), IL-1? (60 U/mL), or LPS (1 μg/mL) alone or in combination stimulated CD154 expression in the primary cultured HUVEC (n=3 to 6, not shown).
However, exposure of these cells to the CD154+ myeloma cells markedly enhanced CD154 expression both on the mRNA (Figure 2A) and protein level (Figure 2B and 2C). In addition to FACS analysis, CD154-induced CD154 protein expression was further verified by Western blot analysis (Figure 2E). Endothelial cell CD154 mRNA expression was inducible, albeit to a lesser degree (about 2% to 20% as compared with the effect of the CD154+ myeloma cells), by a stimulatory anti-CD40 antibody (5 μg/mL, Santa Cruz Biotechnology) or a recombinant CD154 fusion protein (3 μg/mL, Immunex, Seattle, WA; Figure 3A). Moreover, CD154-induced CD154 mRNA expression was strongly attenuated by coincubation with a neutralizing anti-CD154 antibody (Figure 3B) in a concentration-dependent manner (Figure 3B inset), whereas an unrelated control immunoglobulin had no such effect (not shown), and seemed to be sensitive to cotreatment with IFN- (Figure 3B). According to Western blot analysis after 24-hour stimulation, CD154-induced CD154 protein expression was inhibited by up to 70% through addition of the neutralizing anti-CD154 antibody, too (Figure 2C), while cotreatment with IFN did not affect CD154 protein abundance (not shown).
Figure 2. CD154-induced CD154 expression in human endothelial cells. Time-dependent effect of coincubation with CD154+ myeloma cells on A, CD154 mRNA expression in the cultured HUVEC, as judged by real-time PCR analysis (calculated copies per 5 ng RNA, n=4); and on B, C, cell surface expression of CD154 protein in HUVEC as judged by flow cytometry. C, Representative histograms depict isotype control (solid line), basal (dashed line), and stimulated endothelial CD154 expression after incubation with CD154+ cells for 16 hours (solid line, area under the curve highlighted in gray). B, Statistical summary of 7 experiments with different batches of cells with CD154 protein expressed as % of CD154 positive cells counted. D, Statistical summary (n=4) of the induction of endothelial cell CD154 protein expression by coincubation with CD154+ myeloma cells for 24 hours, and inhibition by addition of a neutralizing anti-CD154 antibody (CD154 mAb, 40 μg/mL). The inset shows an exemplary Western blot analysis. Protein extracts of CD154+ myeloma cells served as a positive control giving an immunoreactive band with a molecular mass of 32 kDa.
Figure 3. A, Induction of endothelial cell CD154 mRNA expression by an activating anti-CD40 antibody (anti-CD40 Ab, 5 μg/mL) and a recombinant CD154 fusion protein (CD154 fsp, 3 μg/mL) in comparison with the stimulatory effect of the CD154+ myeloma cells (CD154+ cells) after a 12-hour incubation period (n=4). The inset shows a representative RT-PCR analysis. B, Inhibition of CD154-induced CD154 mRNA expression in HUVEC by addition of a neutralizing anti-CD154 antibody (CD154 mAb, 40 μg/mL) or IFN- (1000 U/mL). Statistical summary (n=5) of the percentage of CD154-induced CD154 mRNA expression over 12 hours. The inset shows an exemplary RT-PCR analysis, demonstrating the concentration-dependent effect of the anti-CD154 antibody (10 to 40 μg/mL) on CD154 mRNA expression.
Transmigation Studies
To assess the functional relevance of CD154-stimulated CD154 gene expression, we employed a transmigration assay, monitoring THP-1 cell diapedesis through a monolayer of cultured HUVEC. The THP-1 cells constitutively express CD40 (compare
References 13 and15) but not CD154 (not shown). Therefore, a CD40/CD154-mediated costimulation between the two cell types seemed possible. Indeed, interaction of the monocytes with CD154-expressing endothelial cells resulted in a significant increase in IL-1? expression in the THP-1 cells, both on the mRNA and protein level, thus indicative of their activation (Figures 4A and 5A). Prior neutralization of endothelial cell CD154 completely abrogated this increase in IL-1? expression (Figures 4A and 5 A).
Figure 4. Interaction of THP-1 monocytes with CD154-expressing endothelial cells results in a significant increase in IL-1? (A) and IL-23p19 (B) mRNA expression in the THP-1 monocytes after diapedesis. Transmigration assays and the IL-1? immunoassay were performed as described in Methods. In brief, cultured HUVEC were coincubated overnight with CD154+ myeloma cells for CD40 stimulation or CD154- control cells. Incubations were terminated by removing the supernatant and thoroughly washing the HUVEC monolayer with medium to remove the myeloma cells. Subsequently, THP-1 cells were presented to the endothelial cells with 100 ng/mL recombinant MCP-1 in the subendothelial compartment and allowed to migrate for 12 hours. For CD154 neutralization, CD154-expressing HUVEC were preincubated for 90 minutes with the CD154-neutralizing antibody (CD154 mAb, 40 μg/mL) before addition of the THP-1 monocytes (n=4 each; *P<0.05 and P<0.05 versus CD154+ cell stimulation).
Figure 5. Only direct cell–cell contact between CD154-expressing endothelial cells and THP-1 monocytes (A) and not the release of a humoral factor (B) results in a significant increase in IL-1? protein expression in the THP-1 cells. Transmigration assays and CD154 neutralization were performed as described in the legend to Figure 4 (3 different batches of cells with n=2 to 4 each; *P<0.05 versus control and control cells, P<0.05 versus CD145 mAb).
To examine the possible presence of a humoral stimulatory factor for IL-1? expression, THP-1 cells were exposed to the conditioned medium of CD154-stimulated endothelial cells for 12 hours. As shown in Figure 5B, this incubation had as little effect on IL-1? synthesis by the THP-1 cells as the physical interaction of the monocytes with CD154-expressing endothelial cells, in which the ligand had been neutralized through prior treatment with the anti-CD154 antibody.
Moreover, CD40/CD154-dependent costimulation also resulted in the induction of IL-23p19 expression in the THP-1 cells. Again, this effect was blocked by prior treatment of the cultured HUVEC with the neutralizing anti-CD154 antibody (Figure 4B).
Discussion
The working hypothesis underlying the present study was that during an inflammatory response, CD40-CD154 mediated T helper cell–endothelial cell interaction results not only in an increased recruitment of circulating monocytes, but also an activation of these cells on their crossing of the endothelial cell barrier through CD154-mediated costimulation.
As shown, ligation of endothelial cell CD40 triggered a marked increase in expression of the adhesion molecules E-selectin, ICAM-1, and VCAM-17–9,12 as well as that of the chemokines IL-8 and MCP-1.6,12 Whereas MCP-1 is presumably the most potent chemoattractant for circulating monocytes, VCAM-1 represents the primary integrin for these cells on the endothelial cell surface to which they bind through VLA-4.17 It did not come as a surprise, therefore, that monocyte adhesion to the cultured endothelial cells was markedly elevated after CD40 ligation.
However, to costimulate and hence activate the adherent monocytes, which in their capacity as APC constitutively express CD40, the endothelial cells, in addition, must express CD154 in a biologically active form on their surface. The present findings demonstrate that this is indeed the case, provided that the endothelial cells are appropriately stimulated. In contrast to quiescent T helper cells, which express CD154 in response to calcium ionophore and/or a phorbol ester stimulation,18 these stimuli had just as little effect on endothelial cell CD154 expression as various proinflammatory cytokines such as IFN and/or TNF. Only when exposed to CD154, either in a cell membrane–bound form or as a fusion protein, was significant induction of CD154 expression detected on both the mRNA and protein level, suggesting that prior interaction with activated T helper cells is mandatory for endothelial cell CD154 expression to occur.
In this context, it should be pointed out that primary cultured endothelial cells revealed virtually no constitutive expression of CD154, while this increased over subsequent passages. Similar findings have been described for PECAM-1 and endothelial nitric oxide synthase expression in rat cultured endothelial cells due to activation or silencing of nuclear transcription factors during this process.19 Moreover, in the presence of IFN-, which typically originates from activated T helper cells, CD154-induced endothelial cell CD154 mRNA expression was attenuated. The observed lack of effect of IFN- on CD154 protein expression might be explained by increased protein stability. Thus, in T helper cells, continuous stimulation maintains CD154 ligand expression for up to 48 hours.20 The extent of reciprocal T helper cell stimulation by endothelial cell–derived IL-1212 may therefore direct the course of the subsequent inflammatory response; that is, restricting it to Th1 cell–endothelial cell interaction or facilitating monocyte recruitment and activation in the vessel wall.
Does endothelial cell-monocyte costimulation really proceed through the CD154/CD40 ligand/receptor dyad? To address this question, a transmigration assay was established revealing that only monocytes that physically interact with CD154-expressing endothelial cells respond with a significant increase in the synthesis of IL-1?, which is considered to be a reliable marker of monocyte activation.21 In addition, the transmigrated cells also exhibited an increased expression of IL-23 p19 mRNA. Like IL-12, IL-23 is an integral part of the reciprocal Th1 cell costimulatory function of APC, preferentially targeting memory CD4+ T cells; whereas, IL-12 acts on naive CD4+ T cells.22 Exposing the monocytes to the conditioned medium of CD154-stimulated endothelial cells (indicative of the presence of a humoral stimulatory factor), on the other hand, did not affect IL-1? synthesis.
Collectively, these data demonstrate that by way of CD154-induced CD154 expression, human endothelial cells not only actively recruit circulating monocytes but also activate them, possibly through a dual lock-key principle (eg, endothelial VCAM-1 interaction with monocyte VLA-4 plus endothelial CD154 interaction with monocyte CD40) during their transmigration into the subendothelial space. This may have a significant impact on the severity and possibly on the chronicity of the ensuing inflammatory response, not only in atherosclerosis but also in other forms of chronic inflammatory or autoimmune diseases. Proofing this assumption would render CD154-induced CD154 expression in the vascular endothelium an interesting therapeutic target.
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 402/C9).
We are indebted to Kathrin Schreiber, Henrike Struwe, and Martina Ohme for expert technical assistance, and to Dr Robert Krzesz for providing the IL-23 p19 primer.
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