Molecular mechanisms underlying the pro-inflammatory synergistic effect of tumor necrosis factor α and interferon γ in human microvascular endothelium

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European Journal of Cell Biology 88 (2009) 731–742 www.elsevier.de/ejcb

Molecular mechanisms underlying the pro-inflammatory synergistic effect of tumor necrosis factor a and interferon c in human microvascular endothelium Adriana Lombardia, Giulia Cantinia, Tommaso Melloa, Michela Francalancia, Stefania Gelminia, Lorenzo Cosmib, Veronica Santarlascib, Selene Degl’Innocentia, Paola Luciania, Cristiana Deleddaa, Francesco Annunziatob, Gianni Fortia, Andrea Gallia, Mario Serioa, Michaela Luconia, a

Department of Clinical Physiopathology, DENOthe Center of Excellence for Research, Transfer and High Education, University of Florence, Viale Pieraccini 6, I-50139 Florence, Italy b Department of Internal Medicine, DENOthe Center of Excellence for Research, Transfer and High Education, University of Florence, I-50139 Florence, Italy Received 5 June 2009; received in revised form 14 July 2009; accepted 14 July 2009

Abstract Tumor necrosis factor a (TNFa) and interferon g (IFNg) are among the most potent cytokines involved in orchestrating the inflammation response. The molecular mechanisms implicated in the synergism between cytokines are still poorly characterized. We demonstrate that both cytokines dose-dependently stimulate IFNg-inducibleprotein-of-10-kDa (IP-10) secretion in human microvascular endothelial cells (HMEC-1), showing a potent synergism which is not restricted to IP-10, but is also evident for monokine-induced-by-IFNg (MIG) and IL-6 secretion. Immunofluorescence analysis reveals that TNFa and IFNg converge on a rapid phosphorylation of ERK, which however results in a different subcellular compartmentalization of the activated enzyme in response to the two cytokines. Differences in the subcellular recruitment of ERK in response to IFNg and TNFa are responsible for generating different ERK downstream signaling, which can thus synergize on the secretion of IP-10 as well as of other cytokines/chemokines. The importance of ERK activation in mediating the synergism of the two cytokines is further confirmed by the inhibitory effect of the anti-diabetic drug rosiglitazone and ERK blockers on IP-10, MIG and IL-6 secretion. A further mechanism of synergism involving the reciprocal upregulation of TNFa-RII and of IFNg-R, in response to IFNg and TNFa, respectively, was revealed by flow cytometry and quantitative real time RT-PCR analysis. r 2009 Elsevier GmbH. All rights reserved. Keywords: ERK; Endothelium; Cytokines; Th1-response; Synergism

Introduction Corresponding author. Tel.: +39 055 4271369; fax: +39 055 4271371. E-mail address: [email protected]fi.it (M. Luconi).

0171-9335/$ - see front matter r 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2009.07.004

The strict crosstalk which occurs during inflammation between tissue-resident cells and the infiltrating immune

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system cells is essential for orchestrating the development and resolution of the inflammatory process. TNFa and IFNg are pleiotropic cytokines exerting a number of biological effects and involved in maintaining and potentiating this crosstalk during the endothelial response to inflammation (Marx et al., 2000). IFNg, which is a critical mediator of Th1 immunity, has been described as a potent activator of the endothelium through the induction of adhesion molecule expression (Lombardi et al., 2008) and cytokine/chemokine secretion (Sana et al., 2005). In particular, an upregulation of chemokines such as IFNg-inducible-protein-of-10-kDa (IP-10), monokine-induced-by-IFNg (MIG), IFNg-inducible-T-cell-a-chemoattractant (I-TAC), fractalkine and the CC-chemokine-monocyte-chemoattractantprotein-1 (MCP-1) has been described at the inflammation site (Marx et al., 2000; Lombardi et al., 2008; Mach et al., 1999; Rotondi et al., 2007). These chemokines appear as being deeply involved in the regulation of inflammation and vascular remodeling (Weber, 2008). In many biological systems, a synergism between IFNg and TNFa has been described as controlling the expression of a variety of cytokines and cell surface molecules (Piali et al., 1998; Cassatella et al., 1997; Ohmori et al., 1997; Ruggiero et al., 1986; Sanceau et al., 1992). An extensive microarray analysis has recently compared the clusters of cytokine, signaling and adhesion molecule genes modulated by IFNg and TNFa in endothelium, evidencing activation of different downstream pathways (Sana et al., 2005). Most of the intracellular signaling pathways elicited by many cytokines involve a rapid phosphorylation of cytoplasmic transcription factors (TFs), resulting in their recruitment to the nucleus where they interact with specific responsive elements in the promoter regions of target genes (Kishimoto et al., 1994). IFNg triggers phosphorylation of Stat-1, which translocates to the nucleus and binds to specific DNA sequence motifs, such as the gamma interferon-activated site (GAS) present in IFNginducible genes. On the other hand, the NF-kB-binding sequence motif has been shown to be a cis-acting regulatory element mediating TNFa-induced transcriptional activation of several genes encoding cytokines and surface molecules. In general, TNFa signaling triggers a rapid phosphorylation and degradation of the regulatory factor IKB, resulting in the consequent release of active NF-kB, which migrates into the nucleus and interacts with its binding sequence. It has been suggested that interaction between activated Stat-1 and NF-kB is the main mechanism underlying the synergism between IFNg and TNFa. However, a novel intracellular signaling pathway, involving the activated extracellular signal-regulated kinase (ERK) cascade, has been recently demonstrated as playing an essential role in mediating IFNg and TNFa effects in endothelial cells (Lombardi et al., 2008). In particular, a rapid

phosphorylation of ERK1/2 induced by both IFNg and TNFa underlies the cytokine-stimulated secretion of IP-10 in microvascular endothelial cells. This effect is counteracted through blocking ERK activation by rosiglitazone (RGZ) (Lombardi et al., 2008), an anti-diabetic drug acting as a ligand of the peroxisome-proliferator-activated receptor (PPARg) (Giannini et al., 2004). Thus, the present study was undertaken to investigate the effect of IFNg and TNFa and their synergism on the secretion of a panel of different chemo/cytokines, and to elucidate the molecular mechanisms underlying this synergism in an in vitro model of human microvascular endothelial cells (HMEC-1).

Materials and methods Cell culture Human microvascular endothelial cells (HMEC-1, Ades et al., 1992) were obtained from the Center for Disease Control and Prevention (Atlanta, USA). HMEC-1 were cultured under conditions described elsewhere (Lombardi et al., 2008). Starved cells were incubated with different stimuli as indicated in the figures. Rosiglitazone (Alexis Biochemicals; 10 mmol/l) was added simultaneously with TNFa and IFNg; 25 mmol/l U0126 and 30 mmol/l PD98059 (Calbiochem) were added 30 min before the two cytokines. IFNg concentration is given in U/ml throughout the manuscript (1 U/ml corresponds to 0.1 ng/ml).

ELISA Cells grown in 96-well plates were stimulated for 24 h and the conditioned media were analyzed with ELISA kits for IP-10, IL-6, IL-8 and MIG detection (R&D Systems). Each point was performed at least in quadruplicates in at least 3 different experiments. The assay sensitivity is 1.67 pg/ml, o0.70 pg/ml, 3.5 pg/ml and 3.84 pg/ml, for IP-10, IL-6, IL-8 and MIG, respectively. Each value was normalized to total protein as evaluated using Bradford Reagent (Sigma-Aldrich).

Quantitative real-time RT-PCR RNA isolation and quantitative real time RT-PCR were performed as detailed elsewhere (Lombardi et al., 2008) using specific primers/probes (Taqman Gene Expression Assay on Demand; Applied Biosystems). The amount of target normalized to GAPDH and relative to a calibrator was obtained by 2-DDCt calculation.

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Epifluorescence and confocal immunofluorescence microscopy Cells grown up to 80% confluence on glass coverslips were differently treated, fixed, permeabilized and subjected to immunofluorescence analysis with anti-NF-kB (Santa Cruz Biotechnology), anti-phosphoStat1 and anti-phosphoERK1/2 (Cell Signaling Technology) antibodies. Cell images were acquired with a Leica DM4000 epifluorescence microscope or with a Leica TCS-SP2 AOBS laser scanning confocal microscope (Leica Microsystems GmbH). Z-stack images of the whole thickness of the cells were acquired sampling every 122 nm. Z-axis projections were obtained by summing every plane in the data set with Image J software, while 3D volume section views were realized with Volocity x 64 software (Perkin Elmer). Negative controls (omitting the primary antibodies; data not shown) were performed.

Flow cytometric analysis Flow cytometry was performed as detailed elsewhere (Cosmi et al., 2006). Briefly, following stimulation, 106 cells/sample were analyzed using anti-IFNg-R and anti-TNFa-RII antibodies on a BDLSRII cytofluorometer, using the Diva software (BD Biosciences). The area of positivity was determined using an isotype-matched control mAb. Ten thousand events for each sample were acquired. In some experiments amplifying flow cytometry technique (FASER) was applied, as previously described (Romagnani et al., 2005).

In silico analysis of the human TNFa- and IFNc-receptor gene promoters The gene sequences were localized using the database NCBI Map-viewer (http://www.ncbi.nlm.nih.gov/mapview/maps.cgi). A 5-kb region upstream from the TNFa-RII and the IFNg-R open reading frames was analyzed using the promoter identification programs promoter predictions (http://www.fruitfly.org/seq_tools/ promoter) and TSSG (http://softberry.com/berry.phtml? topic=tssg&group=programs&subgroup=promoter), and by the CpG-finder software (http://www.softberry. com/berry.phtml?topic=cpgfinder&group=programs &subgroup=promoter) (Luciani et al., 2008). The TFSEARCH (http://www.cbrc.jp/research/db/ TFSEARCH.html) and Alibaba 2.1 (http://www.generegulation.com/pub/programs/alibaba2/index.html) software were used to identify transcription factor-binding sites (Kast et al., 2003).

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Statistical analysis Statistical analysis was performed using the SPSS 12.0 software package (SPSS for Windows 12.0; SPSS Inc.). The Kolmogorov–Smirnov test was used to verify normal distribution of the data. One-way analysis of variance (ANOVA) followed by the Dunnett’s post hoc test was applied for multiple comparisons, whereas Student’s t-test was used for comparisons of two classes of data. A P value o0.05 was considered significant.

Results TNFa and IFNc synergistically stimulate IP-10 secretion in HMEC-1 Treatment of HMEC-1 for 24 h with increasing concentrations of IFNg (Fig. 1A) or TNFa (Fig. 1B) resulted in a significant stimulation of IP-10 secretion at all doses used compared to the undetectable levels observed in untreated control cells. The effect of TNFa was more pronounced compared to the corresponding equimolar doses of IFNg. Interestingly, the receptor system seemed to be saturable for the TNFa response only (Fig. 1B), whereas the IP-10 response curve for IFNg (Fig. 1A) did not reach a plateau at any dose. The simultaneous addition of a fixed dose of TNFa (1 ng/ml) to increasing doses of IFNg (1 to 5000 U/ml, Fig. 1C) as well as of a fixed dose of IFNg (100 U/ml) to increasing doses of TNFa (0.1 to 100 ng/ml, Fig. 1D) resulted in a tremendously increased secretion of IP-10 compared to the levels obtained with singly used cytokines, suggesting a strong synergistic effect on IP-10 secretion. Combination of 1 ng/ml TNFa and 100 U/ml IFNg (hatched bars) yielded the maximum increase of IP-10 secretion versus the respective cytokine singly used (a 60- and 700-fold increase versus TNFa and IFNg alone, respectively). The synergism occurring between TNFa and IFNg was not confined to IP-10 regulation but was also involved in the upregulation of other chemokines/ cytokines characterizing the inflammatory response. Fig. 2 shows the levels of IL-6 (A), MIG (B) and IL-8 (C) secretion in response to TNFa and IFNg alone or together. Of note, IFNg exerted an inhibitory effect on IL-8 secretion both in basal conditions and in combination with TNFa+IFNg (Fig. 2C). Different scalar combinations of TNFa+IFNg exponentially stimulated IP-10 production at both protein (Fig. 2D) and mRNA level (Fig. 2E) as well as the expression of MIG (Fig. 2F), fractalkine (Fig. 2G) and IL-6 (Fig. 2H). Interestingly, the scalar combination of the two cytokines induced an exponential increase also in IL-8 gene expression (Fig. 2I), suggesting that the stimulation

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Fig. 1. IP-10 secretion is induced by IFNg and TNFa and potentiated by a synergism between the two cytokines. Increasing concentrations of IFNg (A) or TNFa (B) dose-dependently induced IP-10 secretion versus almost undetectable levels in control cells (0). Data represent mean7S.E.M. of 3 separate experiments, **Po0.005 and ***Po0.001 versus respective Ctrl (0). (C) A fixed dose of TNFa (1 ng/ml) enhanced IP-10 secretion induced by IFNg versus 0 U/ml IFNg (*Po0.05, **Po0.005, ***Po0.001) or versus the corresponding doses of IFNg alone (11Po0.001). (D) A fixed dose of IFNg (100 U/ml) enhanced IP-10 secretion induced by TNFa versus 0 ng/ml TNFa (*Po0.05, **Po0.005, ***Po0.001) or versus the corresponding doses of TNFa alone (B, 1Po0.005 and 11Po0.001). Hatched bars indicate the combined dose of TNFa (1 ng/ml) and IFNg (100 U/ml) used in the rest of the study. Data represent mean7S.E.M. of 3 experiments.

exerted by TNFa was more pronounced than inhibition by IFNg.

TNFa and IFNc stimulate different intracellular signaling pathways To investigate the molecular mechanism underlying the synergism occurring between TNFa and IFNg on cytokine secretion in HMEC-1, we first evaluated the two main signaling pathways known to mediate TNFa and IFNg effects, namely the NF-kB and Stat-1 pathways. A rapid stimulation (30 min) with TNFa and IFNg resulted in nuclear translocation of NF-kB (Fig. 3A) and pStat-1 (Fig. 3B), respectively. In addition to these classic signaling pathways, a rapid phosphorylation/activation of ERK1/2 by TNFa and IFNg was demonstrated to partially mediate IP-10 upregulation, which was reduced not only by a well known inhibitor of ERK signaling, U0126, but also by the anti-diabetic drug RGZ (Lombardi et al., 2008). Table 1 shows that U0126 and RGZ significantly affected both TNFa- and IFNg-stimulated secretion of IL-6, IL-8 and MIG, in particular when the two cytokines were combined.

In order to understand how phosphorylation of the same kinase could be involved in TNFa and IFNg synergism, we have now investigated the intracellular localization of activated ERKs following a 15-min stimulation of HMEC-1 with TNFa and IFNg alone and combined (Fig. 4). In fact, since no synergism at ERK phosphorylation level had been evident following TNFa and IFNg stimulation (Lombardi et al., 2008), we investigated ERK localization and we found a different subcellular compartmentalization of activated enzyme in response to the two cytokines singly used (Fig. 4). IFNg predominantly stimulated a nuclear shift of phosphorylated ERKs (Fig. 4A), whereas a diffuse cellular distribution of phosphorylated enzyme was evident in response to TNFa (Fig. 4B): the combination of the two stimuli resulted in both nuclear and extra nuclear immunostaining (Fig. 4C). RGZ significantly inhibited ERK phosphorylation and compartmentalization (Fig. 4D–F), although to a lesser extent than the two ERK inhibitors, PD98059 (Fig. 4G) and U0126 (Fig. 4H). A more detailed confocal microscopy investigation, also using 3D inspection of the cell volume (Fig. 5C, D), localized pERK diffusely inside the cell (cytosol, plasma membrane and nucleus) following TNFa stimulation (Fig. 5A, C), whereas IFNg induced a predominant

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Fig. 2. TNFa and IFNg synergism is not restricted to IP-10 expression and secretion. Following a 24-h treatment of HMEC-1 with the indicated stimuli (1 ng/ml TNFa and 100 U/ml IFNg), IL-6 (A), MIG (B) and IL-8 (C) protein levels were measured. Results represent mean7S.E.M. from n ¼ 12 experiments. **Po0.01 and 1Po0.001 versus Ctrl, yPo0.001 versus TNFa or IFNg. Following 24 h stimulation with increasing scalar combinations of TNFa+ IFNg, IP-10 secretion (D) as well as IP-10 (E), MIG (F), fractalkine (G), IL-6 (H) and IL-8 (I) mRNA expression were evaluated by ELISA and TaqMan analyses, respectively. Results represent cytokine/chemokine secretion mean7S.E.M. (n ¼ 3, 1Po0.001 versus unstimulated Ctrl), or the mean7S.E.M. of mRNA fold increase versus expression in unstimulated control cells (n ¼ 3, *Po0.05; **Po0.01;11Po0.001 versus unstimulated Ctrl taken as 1).

recruitment of (Fig. 5B, D).

activated

ERKs

to

the

nuclei

IFNc and TNFa reciprocally upregulate TNFa-RII and IFNc-R In addition to the above described mechanisms, we then investigated whether the potentiated activity of the two cytokines added together could be partly due to a reciprocal upregulation of their receptors, since a positive loop between TNFa and IFNg on the expression of the receptor for the other cytokine has already

been described (Ruggiero et al., 1986; Krakauer and Oppenheim, 1993). Fig. 6 shows that IFNg induced a slight increase in TNFa-RII surface expression, as evaluated by flow cytometry (Fig. 6A), and in its encoding mRNA, as evaluated by quantitative real time RT-PCR (Fig. 6B). An increase in the expression of IFNg-R, at both protein (Fig. 6C) and mRNA (Fig. 6D) level, was detected following TNFa stimulation, although the increase in the protein was higher than the one in the encoding mRNA. The effect was specific since no alteration in the TNFa-RI following TNFa and IFNg addition was observed (not shown). Simultaneous addition of the two cytokines had no synergistic effect

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Fig. 3. TNFa and IFNg stimulate the nuclear shift of NF-kB and pStat-1, respectively. Treatment of HMEC-1 with 1 ng/ml TNFa (A) or 100 U/ml IFNg (B) for 30 min resulted in a marked nuclear localization of the p65 subunit of NF-kB (A) and pStat-1 (B), respectively, compared to untreated Ctrl samples (C, D) as evaluated by immunofluorescence microscopy. The image is representative of 3 similar experiments.

Table 1. Inhibition of ERK1/2 activation by U0126 and RGZ significantly reduces TNFa and IFNg stimulation of different cytokines. RGZ

U0126

TNFa

IFNg

TNFa+IFNg

TNFa

IFNg

TNFa+IFNg

IL-6

13.676.7# n¼8

30.2710.4* n¼8

13.3711.7* n ¼ 10

45.375.7** n¼8

53.9714.1* n¼8

59.673.1# n ¼ 12

MIG

100.070.0 n¼6

53.8716.0* n¼6

36.577.5# n ¼ 10

100.070.0 n¼6

100.070.0** n¼6

90.673.4# n ¼ 10

IL-8

23.0718.3y n ¼ 12

-

39.279.8# n ¼ 12

61.574.1** n ¼ 10

-

54.376.8# n ¼ 10

The mean percentage7S.E.M. of inhibition of cytokine secretion following 24 h stimulation with TNFa (1 ng/ml), IFNg (100 U/ml), TNFa+IFNg, RGZ (10 mmol/l) and U0126 (25 mmol/l) is shown for the indicated number of experiments. Statistical analysis versus respective controls without inhibitors: *Po0.05; **Po0.01; yPo0.005; #Po0.001. (-) Inhibition could not be evaluated since IL-8 secretion is not stimulated by IFNg alone.

on either receptor. Cytofluorometric histograms show surface expression of both TNFa-RII (Fig. 6E–H) and IFNg-R (Fig. 6I–L) in basal conditions and in response to TNFa and IFNg alone or combined. No effect of RGZ or U0126 was evident on either receptor (not shown). The apparently low percentages of cells expressing the receptors in basal conditions confirm previous data obtained in HUVEC (Mackay et al., 1993) and are

due to the non-amplifying conventional FACScan technique used. When amplifying FACScan technique (FASER) was applied to better estimate basal expression of the cytokine receptors, the modulation following cytokine stimulation could not be detected, as almost the entire cell population appeared positive for the receptors already in basal conditions (data not shown).

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Fig. 4. ERK phosphorylation/activation is stimulated by TNFa and IFNg (alone or combined) and can be inhibited by ERK blockers. Confocal immunofluorescence analysis of cells treated for 15 min with 1 ng/ml TNFa, 100 U/ml IFNg, 10 mmol/l RGZ, 30 mmol/l PD98059 and 25 mmol/l U0126 revealed a marked localization of activated ERK1/2 in the nuclei, following IFNg treatment (A), whereas a more diffuse localization was present following TNFa (B). Both nuclear and diffuse localization of phospho-ERK was induced by TNFa+ IFNg (C). Both RGZ (D-F) and ERK blockers (PD98059, G, and U0126, H) prevented the cytokine stimulation of ERK activation. Untreated control cells (Ctrl) are shown in (I). Bar: 25 mm.

Finally, we investigated whether TNFa-RII and IFNg-R gene promoters possessed putative NF-kB-binding and and GAS-like elements which might confer gene responsiveness to TNFa and IFNg through NF-kB and Stat-1. Table 2 lists two NF-kB-responsive elements in the IFNg-R gene promoter and a GAS-like element in the TNFa-RII promoter.

Discussion Synergism between IFNg and TNFa has been described in many biological systems: in particular, the concomitant presence at the inflammation site of both cytokines is essential for orchestrating cell response. Indeed, not only the infiltrating cells but also

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Fig. 5. Differential compartmentalization of activated ERKs following TNFa and IFNg stimulation. Z-axis projection and 3D volume section views obtained by confocal immunofluorescence analysis of cells treated for 15 min with 1 ng/ml TNFa (A, C) or with 100 U/ml IFNg (B, D) revealed that the distribution of activated ERK was predominantly diffuse following TNFa treatment (A: Z-axis projection, C: 3D volume cross section view; inset: XY-plane view), while IFNg stimulated a defined nuclear localization of activated ERK (B: Z-axis projection, D: 3D volume cross section view; inset: XY-plane view).

the tissue-resident cells as well as the vascular endothelial and muscle cells do respond locally to TNFa and IFNg (Lombardi et al., 2008; Appel et al., 2005). Here, we demonstrate for the first time that not only IFNg can dose-dependently stimulate a Th1-recruiting response in endothelial cells, but also increasing doses of TNFa induce a more marked IP-10 secretion, differently from what has previously been found in HUVEC, where the two cytokines could stimulate IP-10 secretion only if combined and not if singly used (Piali et al., 1998). These findings indicate that even a low local TNFa release could initiate the endothelial mediated Th1 inflammatory response, which is then amplified by the specific cytokine secretion of the recruited Th1 lymphocytes. Interestingly, the IFNg response does not reach a plateau at any dose used, suggesting that IP-10 secretion is a non saturable response. Moreover, the cooperative effect of the two cytokines on IP-10 secretion is higher when TNFa is added to IFNg (a 700-fold increase versus IFNg alone compared to a 60-fold increase versus TNFa alone), although, again, the response is not saturable. We also demonstrate that TNFa upregulates IFNg-R expression, thus enabling cells to respond to even low levels of IFNg. This synergism may be of extreme importance especially in the very first phases of the inflammatory process, when TNFa is the locally produced driving cytokine. In this situation, TNFa may sensitize the endothelium to the still low levels of IFNg produced by the very few infiltrating lymphocytes present.

The synergism occurring between TNFa and IFNg is not confined to the stimulatory effect on IP-10 and fractalkine secretion, as already described (Lombardi et al., 2008), but it is also involved in the upregulation of other chemokines/cytokines characterizing the inflammatory response. In particular, we demonstrate a potentiated effect of TNFa and IFNg on IL-6 and MIG secretion. This stimulatory effect on Th1 chemokines (IP-10, MIG, fractalkine) and IL-6 seems to be specific, as demonstrated by its dose dependence in response to scalar combinations of increasing doses of TNFa and IFNg. IP-10 and MIG secretion in response to TNFa and IFNg combined stimulation of endothelial cells is not only important for lymphocyte recruitment but also for lymphocyte adhesion to the endothelium itself (Piali et al., 1998). As we have already demonstrated in our previous paper (Lombardi et al., 2008), combined doses of TNFa and IFNg also resulted in a synergism in the endothelial expression of adhesion molecules, such as ICAM-1 and VCAM-1, the upregulation of which functionally correlates with leukocyte adhesion and migration through the activated endothelium. The cooperative IFNg/TNFa effect may act at different signaling levels. Here, we confirm that IFNg and TNFa independently activate distinct transcription pathways involving Stat-1 and NF-kB, respectively. Activation of these separate signaling pathways finally results in regulation of different clusters of cytokine and adhesion molecule genes containing IFNg- or TNFaresponsive elements (Sana et al., 2005). These pathways

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Fig. 6. TNFa and IFNg reciprocally upregulate their receptors. Protein and gene expression of TNFa-RII and IFNg-R were evaluated by flow cytometry (A and C, n ¼ 7) and by quantitative real time RT-PCR (B and D, n ¼ 4) in HMEC-1 treated for 24 h with 1 ng/ml TNFa and 100 U/ml IFNg, alone or combined. All data are expressed as mean fold increase over untreated Ctrl 7S.E.M. *Po0.05, **Po0.01 versus respective controls taken as 1. A marked stimulation of IFNg-R was evident following TNFa administration (C, D). Solid histograms represent the staining obtained with anti-TNFa-RII (E-H) or anti-IFNg-R (I-L) antibodies while dashed histograms represent the staining obtained with the appropriate isotype-matched control monoclonal antibodies. Cell counts and fluorescence intensity are indicated in ordinate and abscissa. Cell treatments and the percentage of positive cells are indicated in each panel. One representative experiment is depicted.

Table 2. Putative responsive element sequences and distance from translation start site as identified by IFNg and semiempirical analysis. Promoter

Stat-1

NF-kB

TNFa-RII IFNg-R

TTCCCCTAA (-355) -

TTTTCCCT (-1019) TATTCCCCA (-3031)

(-) not evaluated.

do not seem to be so independent any more, since a cooperative interaction between NF-kB and Stat-1 has been described on promoters of genes possessing responsive elements for both factors (Marx et al., 2000; Ohmori et al., 1997; Sanceau et al., 1995). A number of these responsive genes are themselves encoding for TFs, required for the amplification of the

transcription response. Among them, the interferon regulatory factor-1 (IRF-1) is transcriptionally regulated by both GAS and NF-kB sites (Sims et al., 1993; Harada et al., 1994) and is in turn responsible for the activation of a large number of IFNg- and TNFaresponsive genes (Sanceau et al., 1995; Chang et al., 1992; Boehm et al., 1997; Hobart et al., 1997). Among the responsive genes, MIG and IP-10 (Marx et al., 2000; Ohmori et al., 1997) as well as IL-8 (Mori et al., 1999; Gharavi et al., 2007) have been demonstrated to share a similar organization of the promoter, containing NFkB-binding and GAS sequences together with the directrepeat IFNg-stimulated responsive elements (ISREs), which are targeted by members of the IFNg regulatory factor (IRF) family other than Stats, such as IRF-1 (Levy, 1998). Besides the activation of NF-kB and Stat-1 signaling, our results confirm that TNFa and IFNg converge on a

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rapid activation of ERK. Since TNFa and IFNg both stimulate tyrosine phosphorylation of ERK1/2, we cannot expect any synergistic cooperation on this enzyme. However, ERK activation results in a different relocation of the enzyme, either mainly diffuse in the cell or recruited to the nucleus in response to TNFa or IFNg, respectively, as clearly shown by confocal immunofluorescence analysis. In general, upon activation, ERKs migrate to the nucleus where they phosphorylate specific nuclear TF, involved in proliferation and differentiation. However, activated ERKs are sometimes retained in the cytosol to phosphorylate kinases, phosphatases and cytoskeletal proteins or are recruited to the plasma membrane in multimeric complexes associated with activated membrane receptors (Ebisuya et al., 2005; Harding et al., 2005; Ramos, 2008). Over 100 putative substrates in different cell compartments have been described so far as being phosphorylated upon ERK1/2 activation (Yoon and Seger, 2006). Therefore, differences in the magnitude and subcellular compartmentalization of ERKs in response to IFNg and TNFa may be responsible for generating different ERK downstream signaling which can thus synergize on IP-10 and other cytokine/ chemokine secretion or result in distinct cellular outcomes. Furthermore, since a strict crosstalk has been described (Junttila et al., 2008) between the three main MAPK cascades, a potential role of the other two members of the MAPK family, JNK and p38, in contributing to IFNg and TNFa synergism may be also hypothesized. In particular, the ability of TNFa in stimulating p38 and JNK pathways has already been reported in HUVEC cells (Yoshizumi et al., 2003). Another mechanism supporting the TNFa and IFNg synergism may be the reciprocal upregulation of the expression (2- to 3-fold) of their receptors as described in several cell types (Ruggiero et al., 1986; Sanceau et al., 1992; Kast et al., 2003; Aggarwal et al., 1985; Crescioli et al., 2007, 2008). In HMEC-1, we clearly show that IFNg only slightly increases membrane expression of TNFa-RII, while TNFa upregulates up to five times IFNg-R. Since IFNg-R does not seem to be saturable, such an increase in the receptor expression, although modest, may be crucial for mediating the stronger effect observed when IFNg is combined with TNFa. These findings suggest that TNFa might make cells more capable of responding to IFNg stimulation. It is not clear whether the small increase in TNFa-RII observed in response to IFNg is relevant for amplifying the cell response to the two cytokines, although both TNFa-RI and -RII have been described as concurring to TNFa-induced endothelial cell activation (Slowik et al., 1993). In particular, TNFa-RII has been hypothesized to improve TNF binding to the TNFa-RI, rather than to initiate a direct intracellular signaling (Tartaglia et al., 1993). Finally, although the TNFa/IFNg effect on

cytokine/chemokine secretion is synergistic, the reciprocal stimulatory effect on the receptors is not, and once the receptor has been modulated by the single cytokine, there is no further upregulation. An in silico analysis of the promoter regions of the two cytokine receptor genes revealed putative NF-kB elements in the IFNg-R gene promoter and a GAS-like element in the TNFa-RII gene promoter. The identified sequences might directly interact with NF-kB and Stat1, suggesting that the two genes are potentially able to respond to the cytokine stimulus resulting in the upregulated expression of the receptor for the other cytokine. However, evaluation of the functional responsiveness to the two TF, is beyond the aim of our study and will need further investigations. In conclusion, our study elucidates the possible intracellular mechanisms underlying the synergistic effects exerted by TNFa and IFNg on secretion of different cyto/chemokines involved in mediating the endothelial response in a Th1 inflammatory process. In particular, we describe a novel mechanism by which the same event of a rapid ERK phosphorylation may result in the synergistic action of TNFa and IFNg by a different subcellular relocation of activated ERKs in response to the two cytokines. Moreover, a long-term mechanism of reciprocal upregulation of the expression of the two cytokine membrane receptors, in particular of IFNg-R, may contribute to the synergistic effects of TNFa and IFNg. In addition to the well known synergism of TFs downstream from cytokine signaling pathways, such newly discovered mechanisms may not be confined to the activated endothelium, but may also play a pivotal role in supporting the synergism involved in cytokine action in resident and immune cells during the inflammatory process, suggesting potential targets for the development of a new generation of antiinflammatory drugs.

Acknowledgements We thank the CDCP (Atlanta, Georgia, USA) for providing HMEC-1 cells. This study has been supported by the Tuscany Regional Study On Rosiglitazone (TRESOR) project.

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