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The Journal of Immunology, 1999, 163: 3636-3641.
Copyright © 1999 by The American Association of Immunologists

MHC Class II Engagement in Brain Endothelial Cells Induces Protein Kinase A-Dependent IL-6 Secretion and Phosphorylation of cAMP Response Element-Binding Protein1

Sandrine Etienne2,*, Sandrine Bourdoulous*, A. Donny Strosberg* and Pierre-Olivier Couraud*,{dagger}

* Laboratoire d’Immuno-Pharmacologie Moléculaire, Institut Cochin de Génétique Moléculaire, Centre National de la Recherche Scientifique, Unité Propre de Recherche 0415, Université Paris VII, Paris, France; and {dagger} Neurotech SA, Evry, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Activated endothelial cells can directly participate in immune responses by interacting with immunocompetent cells via class II MHC proteins. We show here that, after induction of MHC class II molecule expression by IFN-{gamma}, rat brain endothelial cells responded to MHC class II ligands, anti-MHC class II Abs, or superantigens by expression of IL-6 transcript and IL-6 secretion. This response was not affected by protein kinase C depletion but was mimicked by the cAMP-elevating agent forskolin and completely blocked by H89, an inhibitor of cAMP-dependent protein kinase (PKA). Involvement of a cAMP/PKA signaling pathway in response to MHC class II ligands was further demonstrated by measure of a dose-dependent increase in cAMP level and phosphorylation of the transcription factor cAMP response element-binding protein (CREB). Our results indicate that MHC class II engagement in brain endothelial cells is directly coupled to IL-6 production via a cAMP/PKA-dependent intracellular pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Antigen-specific activation of CD4+ T lymphocytes is mediated by interaction between the TCR and MHC class II-peptide Ag complex. Although constitutive expression of MHC class II genes is restricted to "professional" APCs like B cells and monocytes, expression can also be induced, in inflammatory situations, in a variety of MHC class II-negative cells, such as smooth muscle cells, fibroblasts, keratinocytes, and endothelial cells (1, 2). Accordingly, MHC class II expression has been reported in brain microvessel endothelial cells during inflammatory diseases of the CNS (3), like multiple sclerosis and its animal model experimental allergic encephalomyelitis (3, 4) or, in vitro, after IFN-{gamma} treatment (5). Induced expression of MHC class II molecules on these cells allows them to contribute to the recruitment and activation of cytotoxic CD4+ T lymphocytes in the brain (6).

The capacity of MHC class II molecules to transduce signals was initially suggested by studies demonstrating that anti-MHC class II mAbs can induce cellular response in human hemopoietic cells. Alternatively, TCR activation can proceed after MHC class II engagement by microbial superantigens, which bring TCR and MHC class II in close proximity by binding to amino acid residues outside of the conventional Ag-binding groove, on both Vß and MHC class II molecules (7). Indeed, binding of microbial superantigens, such as staphylococcal enterotoxins (SE)3 A, B, or E or toxic shock syndrome toxin 1 (TSST-1), to MHC class II molecules was initially demonstrated by their ability to stimulate many T cells, in both mice and humans, in the presence of MHC class II-positive cells (for review, see 7). Although binding of SE to MHC class II molecules appears to be much less restricted than to Vß, important differences in their binding affinity and specificity to various isotypes and allotypes have been reported in mice and humans (8, 9).

In response to MHC class II ligands, downstream signaling occurs through MHC class II molecules, resulting in cytokine secretion and/or gene transcription. Such events have been reported in B lymphocytes (10, 11) or in cells induced to transiently express MHC class II molecules, such as synoviocytes and keratinocytes (12, 13). The signaling pathways involved depend on cell type or activation level. Surprisingly, little is known about endothelial cell response to MHC class II engagement.

In the present study, we used the nontransformed immortalized rat brain microvessel endothelial RBE4 cells, which have been extensively characterized by us and others and maintained in culture the phenotype of the blood-brain barrier endothelium (14, 15, 16, 17, 18, 19). We report that in RBE4 cells, MHC class II engagement by mAb or microbial superantigens induces an increase in cAMP level that leads to transcriptional regulation and secretion of IL-6. This is the first demonstration that MHC class II molecules ectopically expressed on brain endothelial cells can transduce intracellular signals leading to cytokine production and suggest that their engagement with TCR directly contributes to gene regulation in endothelial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Recombinant rat IFN-{gamma} was purchased from Life Technologies (Eragny, France). Human IL-1ß and TNF-{alpha} and mouse IL-6 were from Genzyme (Cambridge, MA). Escherichia coli LPS, the calcium ionophore A23187, PMA, and forskolin were from Sigma (St. Louis, MO). The bacterial superantigens SEA, SEB, SEE, and TSST-1 were from Toxin Technologies (Sarasota, FL). The mAbs IgG1 anti-rat MHC class II RT1-B (OX6) and anti-ICAM-1 1A29 were obtained from Serotec (Wiesbaden, Germany), the polyclonal Abs directed against CREB and Ser133-phosphorylated CREB were from Euromedex (Souffelweyersheim, France). The highly selective cAMP-dependent protein kinase (PKA) inhibitor H89 was purchased from Calbiochem (San Diego, CA).

Cell culture and treatment

RBE4 cells were grown onto type I collagen-coated dishes in {alpha}-medium/Ham’s F-10 (1:1; Life Technologies Gibco-BRL, Eragny, France), supplemented with 10% FBS and 1 ng/ml basic fibroblast growth factor (Boehringer Mannheim, Mannheim, Germany), and used between the 30th and 60th passage. RBE4 cells were seeded at 104 cells/cm2 in 6-well dishes and incubated for 3 days in a 37°C, 5% CO2 humidified incubator, until confluent. Cytokine or LPS treatments were performed in fresh medium for 24 h; cell supernatants were then collected and processed for IL-6 bioassay as described below. Alternatively, cells were pretreated for 48 h in presence of 100 U/ml IFN-{gamma} to induce the expression of MHC class II molecules, before incubation in presence of OX6 Ab or bacterial superantigens followed by IL-6 bioassay, PCR analysis, or cAMP accumulation assay.

Flow cytometry analysis

Subconfluent RBE4 cells were incubated in the presence of increasing concentrations of recombinant rat IFN-{gamma}, for 48 h in fresh complete medium. Single-cell suspensions were prepared by short trypsin-EDTA treatment. The cells were incubated in 100 µl PBS containing 3% FBS and 0.1% sodium azide, at a final density of 2 x 105 cells/ml, in the presence or absence (negative control) of OX6 Ab for 30 min on ice. Afterward, cells were washed in the same buffer and resuspended in 100 µl in the presence of FITC-labeled goat anti-mouse Igs for another 30 min on ice. After three washes, the cells were fixed in a final volume of 500 µl 1% paraformaldehyde, and 5000 cells were analyzed on an Epics-Elite flow cytometer (Coulter Electronics, Hialeah, FL). Fluorescence intensity is expressed in a logarithmic scale.

IL-6 bioassay

IL-6-dependent 7TD1 cells (20) were routinely grown, in DMEM (1 g/l glucose) supplemented with 10% FBS, 2 mM glutamine, 0.1 mM hypoxanthine, 16 µM thymidine, nonessential amino acids, 2-ME, and 200 U/ml IL-6. For IL-6 bioassay, 7TD1 cells were resuspended in the same (IL-6-free) medium at 2 x 104 cells/ml, and 100 µl of this cell suspension were added to each well of a 96-well microtiter plate, together with 100 µl serial dilutions of test samples. Dilutions of IL-6 (2–200 U/ml) were used as standard. Plates were incubated for 72 h in a 37°C, 5% CO2 humidified incubator. After centrifugation, the cells were washed twice with 200 µl PBS, and 60 µl of a solution containing 3.75 mM p-nitrophenyl N-acetyl-ß-D-glucosaminide from Sigma, 50 mM sodium citrate (pH 5), and 0.25% Triton X-100 were added to each well. The reaction was stopped after 6–8 h at 37°C by addition of 90 µl of glycine buffer (100 mM, pH 10.4). Optical densities were measured at 405 nm in a Titertek Multiskan spectrophotometer. Results were expressed in picograms IL-6/106 cells. Preliminary experiments indicated that 7TD1 proliferation was not affected by any of the bioactive agents (cytokines, LPS, A23187, MHC class II ligands) used for stimulation of RBE4 cells.

RT-PCR analysis

Cell monolayers were lysed in guanidinium isothiocyanate, and total RNA was isolated by phenol-chloroform extraction and precipitation with isopropanol as previously described (21). RNA (3–5 µg) was reverse transcribed using an oligo(dT) primer and Moloney murine leukemia virus reverse transcriptase, according to the manufacturer’s instructions (Life Technologies Gibco-BRL, Eragny, France). The cDNAs were subjected to 30 cycles of amplification (92°C, 1 min; 45°C, 1.5 min; and 72°C, 1.5 min) using 2.5 U Thermus aquaticus polymerase (Perkin-Elmer Cetus, Norwalk, CT) in 50 µl of a buffer consisting of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 100 µg/ml gelatin, 250 µM concentrations of each deoxynucleotide triphosphate, and 250 nM concentrations of each primer. For the rat IL-6 cDNA amplification, a sense primer (5'-CAAGAGACTTCCAGCCAGTTGC-3') corresponding to nucleotides 81–102 and an antisense primer (5'-TTGCCGAGTAGACCTCATAGTGACC-3') corresponding to nucleotides 694–670 were purchased from Clontech (Palo Alto, CA). As an internal standard of reverse transcriptase efficiency, a set of primers specific for the human G3PDH were purchased from Clontech. All pairs of primers hybridize to different exons, allowing detection of contaminating genomic DNA in the cDNA preparations.

Amplification products were electrophoresed through a 2% agarose gel and blotted onto nylon membrane. The membrane was prehybridized for 24 h at 42°C in 4x SSC, 5x Denhardt’s solution, 40% deionized formamide, 50 mM sodium phosphate (pH 6.8), and 100 µg/ml heat-denatured salmon sperm DNA. Hybridization was performed in the same solution in the presence of 32P-labeled murine IL-6 cDNA probe, a 1.1-kb EcoRI restriction fragment of pHP1B5, kindly provided by Dr J. Van Snick (Ludwig Institute for Cancer Research, Brussels, Belgium). Final washes were at 42°C in 1x SSC, 0.1% SDS.

cAMP accumulation assay

RBE4 cells were grown until they reached confluence in 6-well dishes and pretreated for 48 h in the presence of 100 U/ml IFN-{gamma}. Cells were lysed in cold ethanol for 10 min. Amounts of cAMP were quantified with an Amersham (Les Ulis, France) cAMP ELISA determination kit.

Immunoblot analysis

After treatment, cells (106) were washed in ice-cold PBS and then immediately lyzed in 80 µl SDS-sample buffer. Cellular extracts were then loaded on polyacrylamide gel and submitted to electrophoresis, as previously described (19). For serial incubation of membranes, bound Abs were stripped out by incubation for 10 min in 0.1 M glycine, pH 2.5, and the membranes were reprobed with different Abs as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Surface expression of MHC class II molecules

To identify the conditions for MHC class II expression on RBE4 cell surface flow cytometry analysis was assessed with the use of OX6 mAb. As shown in Fig. 1Go, RBE4 cells did not constitutively express MHC class II molecules. During inflammatory diseases of the CNS, IFN-{gamma} concentration largely increases until reaching >1000 U/ml in cerebrospinal fluid (22, 23). Therefore we determined MHC class II expression in IFN-{gamma}-treated RBE4 cells. MHC class II expression was detected on virtually all RBE4 cells after 48 h treatment with 50 U/ml IFN-{gamma} (Fig. 1GoA). Higher concentrations of IFN-{gamma} further increased MHC class II surface expression (Fig. 1GoB).



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  		FIGURE 1. Flow cytometry analysis of MHC class II surface expression on RBE4 cells. Cells were either not treated or treated (bold line) for 48 h with 50 U/ml IFN-{gamma} (A) or indicated concentrations of IFN-{gamma} (B) and stained by anti-MHC class II mAb OX6 and FITC-labeled goat anti-mouse Igs. Results are expressed in fluorescence intensities (mean ± SEM) from four independent determinations.

 
Induction of IL-6 secretion by MHC class II ligands

On the basis of these data, RBE4 cells were pretreated for 48 h with IFN-{gamma} (100 U/ml), before incubation for 24 h in the presence of OX6 mAb. Treatment with OX6 mAb (10 µg/ml) induced IL-6 secretion that could be detected as early as 4 h after stimulation. The maximum level of IL-6 secretion induced by OX6 mAb treatment for 24 h was higher than that those obtained in response to IL-1ß, TNF-{alpha}, IFN-{gamma}, or LPS (Table IGo) and was not further increased by OX6 cross-linking with a second Abs (data not shown).


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  		Table I. IL-6 induction in RBE4 cells

 
SEs have been well characterized as human, murine, and rat MHC class II ligands, and their potential ability to induce IL-6 secretion by RBE4 cells was investigated. Treatment with 10 µg/ml SEA induced IL-6 secretion by IFN-{gamma}-activated RBE4 cells at a slightly higher level than treatment with OX6 mAb (Fig. 2GoA). SEE and SEB also induced IL-6 secretion, although to a lower extent, whereas TSST-1 had no effect. As expected, no significant secretion was detected when RBE4 cells were not pretreated with IFN-{gamma} (<10 pg IL-6/106 cells), and thus did not express MHC class II molecules (Fig. 2GoA). No secretion of IL-6 was detected when IFN-{gamma}-pretreated cells were left untreated during the 24-h incubation time of the assay (Fig. 2GoA, NT) or were treated with an Ab directed toward an unrelated adhesion molecule, ICAM-1 (not shown). As reported in Fig. 2GoB, these effects were dose dependent, between 0.5 and 10 µg/ml of MHC class II ligands. IL-6 secretion was detectable 4 h after MHC class II engagement and increased until 24 h of incubation (Fig. 2GoC).



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  		FIGURE 2. IL-6 secretion by IFN-{gamma}-activated RBE4 cells in response to MHC class II engagement. A, RBE4 cells, either nonpretreated (-) or pretreated (+) by IFN-{gamma} (100 U/ml), were stimulated by the MHC class II ligand (10 µg/ml) OX6 mAb or the superantigens SEA, SEB, SEE, or TSST-1 for 24 h or left untreated (NT). B, Dose-dependent stimulation of IL-6 secretion by OX6, SEA, SEB, and SEE. C, IFN-{gamma}-activated-RBE4 cells were stimulated by mAb OX6 (10 µg/ml) ({square}) or by LPS (10 µg/ml) +A23187 (5 µM) ({blacklozenge}) for the indicated periods of time. IL-6 in cell supernatants was quantitated by bioassay. Results are the means ± SEM of four to six independent determinations.

 
Taken together, these results indicate that engagement of MCH class II molecules on brain endothelial cells can induce IL-6 secretion.

Induction of IL-6 mRNA by MHC class II ligands

To assess whether regulation of IL-6 secretion occurs at the transcriptional level, RT-PCR analysis was performed on total RNA samples from unstimulated, OX6- stimulated, or SEA-stimulated RBE4 cells at different time points. As shown in Fig. 3Go, a cDNA fragment of the expected size (614 bp) and hybridizing with an IL-6 specific probe was amplified as soon as 2 h after treatment with either OX6 or SEA and was still detected at 24 h. G3PDH amplification was used as an internal standard. These data establish that the induction of IL-6 production by MHC class II ligands in RBE4 cells is correlated with the elevation of IL-6 mRNA level in RBE4 cells.



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  		FIGURE 3. RT-PCR analysis of IL-6 mRNA, followed by hybridization with IL-6-specific probe, in RBE4 cells treated by mAb OX6 or SEA (10 µg/ml) for the indicated times. Reverse transcriptase efficiency was assessed by G3PDH amplification of the same samples and UV staining of amplified fragments. Results are representative of four independent experiments.

 
Role of protein kinase C (PKC) and PKA in MHC class II-induced IL-6 secretion

Previous studies addressing the molecular basis of MHC class II signaling in B cells have pointed out the role of PKC and intracellular cAMP (24, 25, 26).

In RBE4 cells, the PKC activator PMA (160 nM) appeared as a weak inducer of IL-6 secretion (data not shown). Furthermore, cell pretreatment for 16 h with 160 nM PMA, a condition known to deplete PKC in various cell type (27) including RBE4 cells (data not shown), failed to affect the capacity of OX6 mAb to induce IL-6 secretion. These observations strongly suggest that the OX6-associated signaling pathway leading to IL-6 secretion is PKC independent.

A potential role of PKA was investigated by comparing RBE4 cell responses to MHC class II ligands and to forskolin, a direct activator of adenylyl cyclase and PKA pathway. As shown in Fig. 4GoA, forskolin induced IL-6 secretion in a dose-dependent manner. In addition, the highly selective PKA inhibitor H89 (28) completely blocked not only the forskolin-induced IL-6 secretion but also IL-6 secretion induced by MHC class II ligands, OX6 or SEA (Fig. 4GoB). At the concentrations used (30–60 µM), H89 did not affect the viability of RBE4 or 7TD1 cell lines and did not induce per se any IL-6 secretion by RBE4 cells (Fig. 4GoB).



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  		FIGURE 4. Role of PKA in OX6-induced IL-6 secretion by IFN-{gamma}-activated RBE4 cells. A, Cells were stimulated by increasing concentrations of forskolin. IL-6 was quantitated in cell supernatants by bioassay after 24 h of stimulation. Results are the means ± SEM of four independent determinations. B, Effect of PKA inhibitor on SEA-induced IL-6 secretion. RBE4 cells were treated by H89 at the indicated concentrations and stimulated by forskolin (FK; 10-7 M), OX6 (5 µg/ml) or SEA (5 µg/ml). IL-6 was quantitated in cell supernatants by bioassay after 24 h of stimulation. Results are the means ± SEM of three independent experiments.

 
These results indicate that PKA activation is sufficient to induce IL-6 secretion and is necessary for MHC class II-induced IL-6 secretion by RBE4 cells.

To further demonstrate the induction of the PKA pathway by MHC class II ligands, cAMP level quantification was performed in RBE4 cells. OX6 as well as SEA induced cAMP accumulation in a dose-dependent manner (Fig. 5Go): cAMP level was maximum after treatment with 10 µg/ml OX6 mAb or SEA. Interestingly, a similar level was measured in response to 10-7 M forskolin, all three effectors also inducing, at those concentrations, similar levels of secreted IL-6 (Fig. 5Go). No cAMP accumulation was detected when IFN-{gamma} pretreatment was omitted or when cells were treated with irrelevant isotype-matched Abs (not shown).



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  		FIGURE 5. cAMP accumulation in RBE4 cells. Cells were treated with increasing concentrations of OX6 ({square}), SEA (left (•)) or forskolin (FK) (right ({blacksquare})). After 10 min of incubation, cAMP accumulation assay was performed as described in Materials and Methods. Results are means ± SEM of two independent experiments.

 
These results demonstrate that MHC class II engagement induces cAMP accumulation in RBE4 cells and strongly suggest that this is the major pathway responsible for MHC class II- induced IL-6 secretion.

MHC class II stimulation induces phosphorylation of the cAMP response element-binding protein (CREB)

Because the IL-6 promoter is under the control of a cAMP response element (29), we intended to assess the role of the transcription factor CREB in the response to MHC class II engagement. The ability of CREB to activate transcription in response to cAMP is regulated by its phosphorylation at residue Ser133 (30). Western blot analysis of RBE4 cell extracts from untreated or OX6 (or SEA)-treated cells was thus performed using Abs that specifically recognize the Ser133-phosphorylated form of CREB. CREB was found to be phosphorylated within minutes of MHC class II stimulation by either OX6 or SEA (Fig. 6GoA, upper panels). Phosphorylation reached a maximum between 5 and 10 min after binding and then gradually decreased over the next 20 min. The same blot was subjected to Western blot analysis by using anti-CREB Abs that do not discriminate between phosphorylated and unphosphorylated forms (Fig. 6GoA, lower panels), indicating that similar CREB quantity was loaded in all lanes. In contrast, when cells were treated with an irrelevant isotype-matched Ab, no change in CREB phosphorylation was observed (not shown). OX6 cross-linking did not induce significantly stronger CREB phosphorylation than treatment with OX6 alone (data not shown). Pretreatment of the cells with the specific PKA inhibitor H89 prevented the phosphorylation of CREB by MHC class II engagement, as well as by forskolin (Fig. 6GoB, upper panels), illustrating the major role of PKA in MHC class II-induced CREB phosphorylation. In the same conditions, increase in cAMP level was still observed, confirming the viability of the cells and their ability to respond normally to stimulation (not shown).



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  		FIGURE 6. Induction of CREB phosphorylation by MHC class II ligands. A, Cells were stimulated with OX6 (5 µg/ml) or SEA (5 µg/ml) for the indicated periods of time. B, Cells were stimulated with OX6 (5 µg/ml), SEA (5 µg/ml), or forskolin (FK; 10-8 M) or left untreated (NT) for 10 min; when indicated, cells were pretreated with H89 (30 µM) for 1 h. After lysis, cell extracts were analyzed by SDS-PAGE and submitted to immunoblot analysis with anti-Ser133-phosphorylated CREB (anti-P-CREB, upper panels). After stripping, immunoblotting with Abs that recognize CREB regardless of its phosphorylation status (anti-CREB) was performed on the same membrane (anti-CREB, lower panels). Results are representative of three independent experiments.

 
These results indicate that the PKA-dependent IL-6 secretion observed in response to MHC-class II engagement is associated with CREB phosphorylation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In our study, we demonstrate that after MHC class II induction by IFN-{gamma}, MHC class II ligands induce IL-6 secretion by brain endothelial RBE4 cells. Moreover, we show that this response occurs via a cAMP/PKA-dependent pathway and is associated with phosphorylation of the transcription factor CREB.

These data indicate differences between biological activity of the various superantigens tested, which might be directly related to variations in fine specificity of binding. Our results suggest that cross-linking of MHC class II molecules is not required for induction of MHC class II-coupled signaling events and that ligand binding to only selected epitopes of MHC class II molecules can induce IL-6 secretion by brain endothelial cells. Differences in the affinity of enterotoxins for MHC class II may also explain the higher response observed with SEA (31). Our results indicate that MHC class II engagement in IFN-{gamma}-pretreated rat brain microvessel endothelial cells can also induce cytokine secretion, as previously reported in hemopoietic cells. Together with the previous observation of IL-6 and IL-8 production by human rheumatoid synoviocytes in response to SEA (12), this study demonstrates that MHC class II molecules ectopically expressed in nonhemopoietic cells can trigger cytokine secretion.

Two distinct signaling pathways have been shown to be activated by MHC class II engagement in professional APCs: increase in cAMP accumulation together with activation of PKC in resting B cells (25) and protein tyrosine kinase activation followed by phosphoinositide hydrolysis and calcium mobilization in monocytes and in IL-4 (or CD40 ligand)-primed B cells (32, 33). Our results indicate that IL-6 secretion by RBE4 cells, induced by MHC class II ligands, is PKC independent. In addition, investigating a putative role of protein tyrosine kinases in MHC class II signaling in these cells, we failed to detect any change in the tyrosine phosphorylation pattern of cellular proteins, or in the activity of the cytosolic protein tyrosine kinases src, lyn, or fgr, at any time after MHC class II binding (data not shown). In contrast, we show here that forskolin, a cAMP-elevating agent, can induce IL-6 production in RBE4 cells. Moreover, our observation that the highly selective PKA inhibitor H89 completely abrogates the SEA- or OX6-induced IL-6 secretion strongly suggests that activation of PKA is necessary to the induction of IL-6 production by MHC class II ligands. In agreement with this observation, we show that MHC class II engagement by OX-6 or SEA induces an increase in intracellular cAMP level. Taken together, these results demonstrate that cAMP/PKA signaling in RBE4 cells is the major pathway involved in MHC class II-induced IL-6 secretion.

IL-6 expression is regulated via various signal transduction pathways because of the presence of several cis-regulatory elements in the IL-6 gene promoter (34), including a cAMP response element, which is bound by the transcription factor CREB. Multiple pathways have recently been shown to be able to regulate CREB phosphorylation and activity, including cAMP/PKA, calcium signaling, MAP kinases, Erk, and p38 (35, 36, 37). In the present study, specific inhibitors of pathways other than cAMP/PKA did not affect MHC class II-induced CREB phosphorylation. No activation of Erk in response to MHC class II ligands was detected in RBE4 cells (not shown). Moreover, CREB phosphorylation induced by MHC class II engagement was abolished by inhibition of PKA, strongly suggesting that PKA was entirely responsible for CREB phosphorylation. Once it is phosphorylated, CREB can contribute directly to the transcription of late response gene. In addition, CREB plays a key role in the activation of immediate early genes, such as c-fos, and may therefore be indirectly responsible for IL-6 transcription (38). In addition to CREB, transcription factors such as AP-1, NF-IL-6, and NF-{kappa}B may also transmit cAMP-mediated signals to the transcriptional machinery (37, 39, 40, 41). Thus, cAMP accumulation observed after MHC class II engagement could activate IL-6 transcription via multiple transcription factors.

To our knowledge, these results indicate, for the first time, that MHC class II molecules can induce intracellular signal transduction in endothelial cells and trigger transcriptional regulation of IL-6 secretion, whereas other inflammatory cytokines such as IL-1ß and TNF-{alpha} were not induced (data not shown). Moreover, they suggest that brain endothelium could release IL-6 in response to MHC class II engagement during lymphocyte adhesion and thus may participate to the enhanced IL-6 production observed in various inflammatory diseases of the CNS (42, 43). IL-6 expression in the brain plays an important role in the global regulation of neurons, astrocytes, and microglia (44, 45). Moreover, inflammatory cytokines like IL-6 affect brain endothelium permeability to macromolecules in vitro (46), and extensive breakdown of the blood-brain barrier was observed in transgenic mice expressing IL-6 in astrocytes (47). Using human brain endothelial cells in culture, Persidsky et al. (48) recently pointed out the role of these inflammatory cytokines in activation of cerebral endothelium and opening of intercellular junctions, leading to enhanced migration of HIV-infected monocytes. Induction of IL-6 secretion by brain endothelial cells in response to MHC class II engagement with T lymphocytes may thus participate to the local opening of the blood-brain barrier and enhancement lymphocyte infiltration during experimental allergic encephalomyelitis or other inflammatory diseases of the CNS (49).


   Acknowledgments
 
We thank Dr. J. Van Snick for his kind gift of 7TD1 cells and murine IL-6 cDNA; A. Moser, Dr. O. Durieu-Trautman, and N. Chaverot for their contribution to the initiation of this work; and Dr. S. Cazaubon for helpful comments on the manuscript.


   Footnotes
 
1 This work was supported by grants from the Centre National de la Recherche Scientifique, the Institut National de la Santé et de Recherche Médicale, the Association pour le Développement de la Recherche sur le Cancer, the Ligue Nationale Française contre le Cancer, the Université Paris VII, the Ministère de la Recherche et de l’Enseignement Supérieur. Back

2 Address correspondence and reprint requests to Dr. S. Etienne, Laboratoire d’Immuno-Pharmacologie Moléculaire, Centre National de la Recherche Scientifique, Unité Propre de Recherche 0415, Institut Cochin de Génétique Moléculaire, 22 rue Méchain, 75014 Paris, France. E-mail: Back

3 Abbreviations used in this paper: SE, staphylococcal enterotoxin; CREB, cAMP-response-element binding protein; PKA, cAMP-dependent kinase; PKC, protein kinase C; TSST-1, toxic shock syndrome toxin 1. Back

Received for publication February 8, 1999.
   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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