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The Journal of Immunology, 2006, 176: 3098-3107.
Copyright © 2006 by The American Association of Immunologists

Vascular Endothelial Growth Factor-Induced Signaling Pathways in Endothelial Cells That Mediate Overexpression of the Chemokine IFN-{gamma}-Inducible Protein of 10 kDa In Vitro and In Vivo1

Gwénola Boulday, Zdenka Haskova, Marlies E. J. Reinders, Soumitro Pal2 and David M. Briscoe2,3

Division of Nephrology, Department of Medicine, and Transplantation Research Center, Children’s Hospital Boston and Brigham and Woman’s Hospital, and Department of Pediatrics, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Vascular endothelial growth factor (VEGF), an angiogenesis factor, has recently been found to have potent proinflammatory properties in vivo. However, the mechanism by which it mediates inflammation is poorly understood. In this study, we have evaluated the function of VEGF on the induced expression and function of the T cell chemoattractant chemokine IFN-{gamma}-inducible protein of 10 kDa (IP-10). In vitro, we find that VEGF augments the effect of IFN-{gamma} on the induction of IP-10 mRNA and protein expression in endothelial cells. Moreover, we show that VEGF and IFN-{gamma} regulate the activation of the IP-10 promoter, and that the kinases PI3K, phosphoinositide-dependent kinase 1, and Akt act as intermediary signaling molecules for cytokine-inducible IP-10 transcriptional activation in endothelial cells. To examine whether VEGF is functional for IP-10 expression in vivo, Chinese hamster ovary cells that were designed to secrete VEGF were injected s.c. into the skin of nude mice and were found to mediate a time-dependent increase in IP-10 mRNA. This response was reduced in animals treated systemically with the PI3K inhibitor wortmannin. When the Chinese hamster ovary cells expressing VEGF plasmid were injected s.c. into C57BL/6 wild-type or CXCR3–/– mice, they elicited an inflammatory reaction in wild-type but not in CXCR3–/– mice. Collectively, these findings indicate that VEGF-induced augmentation of IP-10 expression is a major mechanism underlying its proinflammatory function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The importance of vascular endothelial growth factor (VEGF)4 is well established in developmental, physiological, and pathological vasculogenesis and angiogenesis (1, 2, 3, 4, 5, 6). VEGF-induced angiogenesis occurs experimentally in the absence of inflammation, but its expression is frequently associated with inflammatory conditions (6, 7, 8, 9). Moreover, the in vivo expression of VEGF has been reported to be functional for the development of some inflammatory diseases including arthritis (10), atherosclerosis (11), and allograft rejection (12, 13, 14, 15, 16). However, the intracellular signals and mechanisms by which VEGF regulates inflammation are poorly understood.

Activated microvascular endothelial cells (EC) promote the local trafficking of leukocytes into sites of inflammation (17, 18, 19, 20). This process is mediated by a series of adhesion events that involve receptor-counterreceptor interactions among multiple adhesion molecules and chemokine receptors/ligands expressed by both leukocytes and EC (21, 22, 23, 24, 25). VEGF-VEGF receptor interactions have been found to result in the induced EC expression of E-selectin, ICAM-1, and VCAM-1 that mediate leukocyte-EC adhesion in vitro and in vivo (26, 27, 28). Also, stimulation of EC in vitro with VEGF results in the expression of the chemokines MCP-1 and IL-8 (16, 29, 30). Because the expression of both adhesion molecules and chemokines by EC has been demonstrated to promote leukocyte recruitment (19, 20, 24, 25), this function likely provides VEGF with significant proinflammatory properties. In addition because the infiltration of tissues with T cells and monocytes is known to mediate VEGF expression (9, 31), it is possible that these proinflammatory effects of VEGF promote positive feedback loops that amplify cell-mediated immune inflammatory reactions.

IFN-{gamma}-inducible protein of 10 kDa (IP-10, also called CXCL10) is a major T cell chemoattractant chemokine (32). It was originally cloned from an IFN-{gamma}-treated monocyte cell library and was identified as a member of the CXC chemokine family expressed and secreted by monocytes, fibroblasts, T cells, and EC after IFN-{gamma} stimulation (23, 32, 33). Its expression has been reported to be associated with T cell infiltrates at sites of acute and chronic inflammation (34, 35, 36, 37, 38), and it has been demonstrated that the local expression of IP-10 in allografts is of critical importance for the development of acute rejection in vivo (39). The IP-10 receptor CXCR3 is expressed predominantly on activated T cells, although it is also known to be expressed on other cell types (23, 40, 41). CXCR3-deficient recipients of cardiac allografts are protected from acute rejection and have prolonged graft survival, suggesting that local IP-10 on EC within an allograft interacts with CXCR3 on infiltrating leukocytes to mediate trafficking and acute rejection (39, 42). However, little is known of the regulation of IP-10 in EC, and no study has evaluated the mechanisms or functional interrelationship between VEGF and IP-10 expression in vitro or in vivo.

In this study, we have evaluated a mechanism by which VEGF synergizes with IFN-{gamma} for the overexpression of IP-10 in EC. We show that VEGF and IFN-{gamma} regulate IP-10 expression by transcriptional mechanisms and show that the kinases PI3K, phosphoinositide-dependent kinase (PDK)1, and Akt are critical for VEGF-inducible transcription of IP-10 in vitro. Moreover, we find that VEGF mediates the expression of IP-10 in vivo and that local VEGF-inducible IP-10 is functional to mediate CXCR3-dependent trafficking of leukocytes into skin. Together, these observations for the first time identify a functional interrelationship between VEGF and IP-10 of pathophysiological significance in several inflammatory disease processes.


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

Human VEGF165 and IFN-{gamma} were purchased from R&D Systems. Mouse IFN-{gamma} was purchased from PeproTech, wortmannin and PD 98059 were purchased from Calbiochem, and rapamycin was gifted to the laboratory from Wyeth-Ayerst Research (Princeton, NJ). Abs used for flow cytometry were a PE-conjugated mouse anti-human IP-10 Ab (mAb) and a PE-conjugated mouse IgG2a isotype Ab (both purchased from BD Pharmingen). The [{alpha}-32P]UTP was purchased from PerkinElmer.

Cell culture

Human EC were isolated from umbilical cords and cultured as described (43). The cells were cultured in M199 (Cambrex), supplemented with 20% decomplemented FBS, 1% EC growth factor, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin were subcultured and used at passages 3 and 4. Before all studies, cultured EC were starved for 5 h in medium supplemented with 5% FBS M199 medium, and all experiments were performed in this medium. Chinese hamster ovary (CHO) cells stably transfected with a mouse VEGF-expressing plasmid (CHO-VEGF) (as described in Refs.16 and 44) were cultured in MEM (Sigma-Aldrich) containing 100 U/ml penicillin, 100 µg/ml streptomycin, 10% tetracycline-free FBS, 4 mM glutamine, 100 µg/ml G418, and 100 µg/ml hygromycin B. Control transfected CHO cells were cultured in the same medium without hygromycin B.

RNase protection assay (RPA)

RNA was extracted using the TRIzol isolation system (Invitrogen Life Technologies). Equal amounts of mRNA (from 3 to 8 µg) were analyzed by RPA using the mouse or human CK-5 cytokine RiboQuant multiprobe template set (BD Pharmingen) according to the manufacturer’s instructions. Chemokine transcripts were analyzed by autoradiography and were quantified by densitometry (Alpha Innotech) and were normalized to the density of the housekeeping gene L32.

Real-time PCR

Quantitative real-time PCR was performed using the 7300 real-time PCR System and the Assays-on-Demand Gene Expression product (TaqMan, Mammalian Gene Collection probes) according to the manufacturer’s instructions (Applied Biosystems). Primers for human IP-10, Mig (monokine induced by IFN-{gamma}), I-TAC (IFN-inducible T cell {alpha} chemoattractant), GAPDH, and mouse IFN-{gamma} and GAPDH were also obtained from Applied Biosystems.

Plasmids

The IP-10 promoter-luciferase construct (45) containing the 5'-flanking region of the human IP-10 gene from –875 to +97 (relative to the transcription start site) was obtained as a generous gift from R. M. Ransohoff (Cleveland Clinic, Cleveland, OH). The Myc-PDK1, Myc-PDK1.K/N, and GST-{Delta}p85 were gifts from A. Toker (Beth Israel Deaconess Medical Center, Boston, MA) (46). Wild-type Akt (myr-Akt) cloned into pcDNA3 (47) was obtained from D. Mukhopadhyay (Mayo Clinic, Rochester, MN). The dominant-negative (DN) mutant Akt (T308A, S473A, Adeno-dnAkt) (48) was obtained from K. Walsh (Boston University, Boston, MA).

Transfection

EC were cultured at 2.5 x 105 cells/well in 6-well cell culture plates and were transfected using the Effectene transfection reagent (Qiagen), according to the manufacturer’s instructions. Luciferase activity was measured using a standard assay kit (Promega) and was read using a MicroBeta Trilux luminometer (Wallac PerkinElmer). Also, as a positive control for transfection efficiency, in occasional cultures, we used the beta-galactosidase gene under control of CMV immediate-early promoter, and we assessed beta-galactosidase activity as described (49).

Flow cytometry

Cell suspensions of EC were permeabilized using the Cytofix/Cytoperm kit (BD Pharmingen), and the cells were stained using a PE-conjugated Ab using standard techniques. Negative control cells were stained with an isotype control Ab. Stained cells were subsequently analyzed using a FACSCalibur cell sorter (BD Biosciences).

ELISA

EC were cultured in 96-well cell culture plates and untreated or treated with cytokines. Secreted IP-10 protein was quantified in culture supernatants using the Quantikine human IP-10 ELISA kit (R&D Systems), according to the manufacturer’s instructions.

In vivo model for the assessment of VEGF-induced IP-10 expression

CHO-VEGF cells (1 x 105 cells in 10 µl of 0.9% NaCl solution) were injected into the ears of nude mice (NCRNU-M; Taconic Farms) as previously described (16). Control mice received identical injections of mock-transfected CHO cells that do not express VEGF. Groups of mice received injections s.c. with either CHO-VEGF cells alone or CHO-VEGF cells with IFN-{gamma} (1000 U). Mice were untreated or were treated with i.p. injections of wortmannin (Calbiochem) at a dose of 1 mg/kg on days –1, 0, and 1 followed by 0.5 mg/kg on days 2–4. At the indicated times, the skin was photographed (to assess visible VEGF-induced angiogenesis) and was harvested and processed for RPA or for real-time PCR. As a control for this experiment we confirmed that wortmannin had no effect on the secretion of VEGF by the CHO-VEGF cells.

CHO-VEGF cells were also injected intradermally into C57BL/6 mice (Taconic Farms) or CXCR3 knockout mice (CXCR3–/–, BL/6; gifted to the laboratory by Dr. C. Gerard, Children’s Hospital Boston, Boston, MA) as described earlier. Skin samples were harvested 1, 2, and 3 days following administration of the CHO-VEGF cells, and were fixed in Formalin, paraffin embedded, and processed by H&E staining.

Statistics

Data were analyzed by the Student’s t test. Values for p < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Effect of VEGF on the induced expression of IP-10 in human EC

We previously reported that VEGF induces the mRNA expression of MCP-1 and IL-8, and that it is most notable to augment the effect of IFN-{gamma} on IP-10 mRNA expression in human EC (16). In this study, we first determined whether VEGF also augments the expression of Mig and I-TAC, which are the other IFN-{gamma}-inducible and CXCR3 binding chemokines. EC were untreated or treated with VEGF, IFN-{gamma}, or VEGF in combination with IFN-{gamma}, and the expression of IP-10, Mig, and I-TAC were evaluated by real-time PCR. Although we found that IFN-{gamma} induced the expression of all three chemokines, coadministration of VEGF only enhanced IFN-{gamma}-inducible expression of IP-10 (Fig. 1A). By intracellular FACS and ELISA we also determined that VEGF enhances IFN-{gamma}-inducible expression of IP-10 at the protein level (Fig. 1, B and C).


Figure 1
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  		FIGURE 1. The effect of VEGF on IFN-{gamma}-mediated induction of IP-10 in EC. Confluent cultures of human EC were treated with VEGF (20 ng/ml) or IFN-{gamma} (1000 U/ml), alone or in combination. A, RNA was extracted from EC after 1 h, and real-time PCR was performed for the assessment of IP-10, Mig, and I-TAC. The expression of each chemokine was calculated relative to the quantity of the housekeeping gene GAPDH obtained in each respective sample. Results are expressed as fold induction for each chemokine as compared with untreated cells. B, FACS analysis of intracellular IP-10 expression following 6 h of treatment. IP-10 staining (solid line histogram) is compared with that of an isotype-matched control Ab (dotted line histogram); the difference ({Delta}) in mean fluorescence was calculated as the difference in staining for IP-10 compared with the control Ab. C, IP-10 secretion measured by ELISA in culture supernatant of EC (diluted 1/20) incubated for 24 h with VEGF or IFN-{gamma} alone and in combination is illustrated. A and C, Represents the mean of two independent experiments performed in duplicate wells. B, A representative experiment of three with similar results.

 
To evaluate whether the effect of VEGF on IP-10 expression involves transcriptional mechanisms, we transfected EC with a full-length IP-10 promoter-luciferase construct and cultured the cells in the absence or presence of VEGF or IFN-{gamma}, alone or in combination (Fig. 2A). We found that treatment of EC with VEGF alone had a weak but consistent effect to augment IP-10 promoter activation as compared with the untreated controls (p < 0.01 vs untreated cells). Treatment of EC with IFN-{gamma} alone also induced IP-10 promoter activation (as expected from Refs.32 and 50), and treatment with both VEGF and IFN-{gamma} in combination induced IP-10 promoter activation to a greater extent than that observed following treatment with each agent alone (Fig. 2A). These findings are consistent with a major but selective effect of VEGF on the expression of chemokines in EC and indicate that the combined effect of VEGF and IFN-{gamma} on IP-10 overexpression involves transcriptional mechanisms.


Figure 2
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  		FIGURE 2. The PI3K pathway mediates IP-10 transcriptional activation in vitro. A, Confluent cultures of EC were transfected with an IP-10 promoter-luciferase construct (1 µg) and were treated with VEGF (20 ng/ml), IFN-{gamma} (1000 U/ml), or a combination of the two for 5 h. B, Wortmannin (30 nM), a pharmacological inhibitor of PI3K, was added to cell cultures ({blacksquare}). Cells were also treated with cytokines alone ({square}). C, EC were cotransfected with the IP-10 promoter-luciferase construct (1 µg) and a PI3K DN mutant construct (20 ng), or an empty vector as a control, before treatment with VEGF and/or IFN-{gamma}. A–C, The fold change in IP-10 promoter-luciferase activity was calculated as the fold increase or decrease in treated cells compared with untreated cells. D, EC were transfected with a PI3K DN mutant construct (1 µg), or an empty vector as a control, before treatment with VEGF and/or IFN-{gamma} for 6 h. Cells were permeabilized and stained for intracellular IP-10 (solid line histogram) or an isotype control Ab (dotted line histogram) by FACS. The difference ({Delta}) in mean fluorescence was calculated as the difference in staining for IP-10 compared with the control Ab. The mean results (±1 SE) of five (A) and three (B) independent experiments are shown. C and D, Representative experiment of three with similar results. *, p < 0.01 and **, p < 0.005 vs nontreated cells.

 
Role of the PI3K signaling pathway in IP-10 transcription

VEGF postreceptor events result in the activation of several signaling pathways, including the MAPK and the PI3K pathways that elicit several pluripotent responses following VEGF treatment of EC (6, 51). However, it is not known whether these pathways are functional for IP-10 transcriptional activation. We next evaluated the effect of VEGF and IFN-{gamma} on IP-10 promoter activation in EC pretreated with pharmacological inhibitors of each of these kinases. Consistently, we failed to find any effect of the MAPK inhibitor PD 98059 on VEGF- and IFN-{gamma}-induced IP-10 overexpression (data not shown). However, we found that the PI3K inhibitor wortmannin markedly inhibited IP-10 promoter activation in response to VEGF or IFN-{gamma} alone or in combination (Fig. 2B). To further define the involvement of the PI3K pathway in IP-10 promoter activation, we cotransfected EC with the full-length IP-10 promoter-luciferase construct and a PI3K DN mutant construct. Cotransfection with the IP-10 promoter-luciferase construct and an empty expression vector served as a control. We found that the PI3K DN mutant significantly inhibited the ability of VEGF and IFN-{gamma}, either alone or in combination, to increase IP-10 transcriptional activity (Fig. 2C). In addition, the expression of IP-10 protein, induced by VEGF and IFN-{gamma} as assessed by intracellular FACS and ELISA (data not shown), was significantly inhibited in EC transfected with the PI3K DN construct (Fig. 2D, right panels), compared with empty vector-transfected EC (Fig. 2D, left panels). Thus, VEGF-induced and PI3K-mediated signals are critical for maximal IP-10 expression in human EC.

Role of PDK1- and Akt-induced signals for IP-10 overexpression in EC

PDK1 and Akt are functional downstream signaling molecules in the PI3K pathway (46, 52). To examine whether these kinases are functional for inducible IP-10 expression, EC were cotransfected with the IP-10 promoter-luciferase construct and cDNA plasmids encoding the active form of either PDK1 or Akt. We found that PDK1 or Akt induced IP-10 transcriptional activation in a dose-dependent manner (Fig. 3, A and C). Moreover, IP-10 promoter activity in response to VEGF and IFN-{gamma} was significantly reduced in EC cotransfected with the IP-10 promoter-luciferase construct and a DN mutant of each kinase (Fig. 3, B and D).


Figure 3
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  		FIGURE 3. PDK1 and Akt mediate IP-10 transcriptional activation in vitro. Confluent cultures of EC were cotransfected with an IP-10 promoter-luciferase construct (1 µg) and increasing concentrations of wild-type PDK1 (A) or wild-type Akt (C and E) as described in Materials and Methods. EC were also cotransfected with the IP-10 promoter-luciferase construct and DN constructs of either PDK1 (100 ng) (B) or Akt (1 ng) (D). B and D, The transfected cells were treated with VEGF (20 ng/ml) and IFN-{gamma} (1000 U/ml) for 5 h. E, Transfected cells were treated with rapamycin (1–100 ng/ml). Control cells for each experiment were cotransfected with the IP-10 promoter-luciferase construct and the respective empty vector such that the total DNA content in all cell groups was identical. F, EC were transfected with the IP-10 promoter-luciferase construct and were treated with VEGF and IFN-{gamma} in combination (B and D), in the absence or presence of 24-h treatment with rapamycin. Fold increase in IP-10 luciferase activity was calculated as the fold change in luciferase activity in treated cells compared with untreated cells. Data shown represent the mean of three independent experiments. *, p < 0.05 vs empty vector-transfected cells; **, p < 0.05 vs empty vector-transfected cells after treatment.

 
The mammalian target of rapamycin (mTOR) kinase is reported to be a major (and perhaps critical) Akt-associated effector molecule that functions in the control of translational initiation and in the proliferation of several cell types (53, 54, 55, 56). mTOR kinases can regulate the transcription of genes, for instance, via the activation of HIF-1 and STAT-3 (56, 57, 58). To assess the effect of mTOR in Akt-inducible expression of IP-10, EC were cotransfected with the IP-10 promoter-luciferase construct and either a cDNA plasmid encoding the active form of Akt or an empty vector as a control. Transfected cells were subsequently cultured in the absence or presence of rapamycin (Fig. 3E). Although IP-10 transcription was induced by Akt, this Akt-induced response was not significantly inhibited by rapamycin. In addition, EC were transfected with the IP-10 promoter-luciferase construct and were cultured with VEGF and IFN-{gamma} in the absence or presence of increasing concentrations of rapamycin. Again, inhibition of mTOR with rapamycin had no significant effect on IP-10 promoter activation (Fig. 3F). Altogether, these results indicate that PDK1 and Akt signals are functional for VEGF-induced IP-10 expression in EC and that the mTOR pathway is not significantly associated with this response.

Effect of VEGF on IP-10 expression in vivo

The in vitro studies we have described indicate that although VEGF alone is sufficient to induce some degree of IP-10 transcriptional activation, maximal expression of IP-10 requires additional signals such as those resulting from activation by IFN-{gamma}. To evaluate whether IP-10 is regulated by VEGF in vivo, we developed a model in which CHO cells, designed to secrete VEGF (CHO-VEGF cells), were injected s.c. into nude mice (16). This model allows for the analysis of the direct effects of VEGF in vivo in the absence of a T cell-mediated immune response. Following injection, the skin was harvested as a time course, and we evaluated the expression of IP-10 and MCP-1 by RPA. In addition, the effect of VEGF or VEGF in combination with IFN-{gamma} on angiogenesis was assessed by direct visualization (Fig. 4A). Both IP-10 and MCP-1 were induced in expression as early as 8 h following injection of CHO-VEGF cells, and expression increased and persisted through day 4 (Figs. 4B and 5A). In contrast, injection of the control CHO cells resulted in an early weak induction of IP-10 (at 8 h) that returned to basal levels after 24 h (Fig. 4B).


Figure 4
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  		FIGURE 4. The effect of VEGF on the induced expression of IP-10 in vivo. CHO cells (1 x 105 cells) stably transfected with a VEGF-expressing plasmid (CHO-VEGF cells) or mock-transfected cells (CHO cells) were injected s.c. into the skin of nude mice, with or without IFN-{gamma} (1000 U). A, Photograph of the neovascularization reaction 4 days following injection of each cell type into the ears of mice (magnification, x20). B, Representative RPA is shown for the expression of IP-10 as a time course in skin samples following injection of either CHO-VEGF or CHO cells. C, The fold change in IFN-{gamma} expression is depicted following injection of CHO or CHO-VEGF cells, as evaluated by real-time PCR. IFN-{gamma} expression was calculated relative to the quantity of GAPDH obtained in each respective sample. D, Expression of IP-10 by RPA in skin following the s.c. injection of CHO-VEGF cells alone or in combination with IFN-{gamma} (1000 U). B and D, The graphs (top panels) represent the densitometric quantification of IP-10 mRNA expression compared with the housekeeping gene L32 as shown in the blot (bottom panels). A, B, and D, Data are representative of five independent experiments with identical results. C, Data represent the average expression in two skin samples from each group of a total of four skins at each time point.

 
The expression of endogenous IFN-{gamma} (as evaluated by real-time PCR) was not increased at 8 h following injection of either the control CHO cells or the CHO-VEGF cells, but was transiently increased at later times (day 2) only following injection of the CHO-VEGF cells (Fig. 4C). This result suggests that the early expression of IP-10 following injection of both cell types was not mediated by endogenous IFN-{gamma}. Moreover, we found that the coadministration of IFN-{gamma} together with the CHO-VEGF cells resulted in an enhancement of IP-10 expression (Fig. 4D), suggesting that the level of IFN-{gamma} produced by the cells alone may not be of major physiological significance. Interestingly, by direct visualization, the coadministration of IFN-{gamma} with the CHO-VEGF cells led to a reduction in the angiogenesis response, compared with that seen with the CHO-VEGF cells alone (Fig. 4A). Thus, VEGF-inducible IP-10 may have angiostatic effects and negatively regulate VEGF-induced angiogenesis. Collectively, these findings indicate that VEGF alone is sufficient to induce IP-10 in vivo, and that the presence of both VEGF and IFN-{gamma} together at sites of cell-mediated immunity is likely a major stimulus for IP-10 expression.

Involvement of the PI3K signaling pathway for IP-10 overexpression in vivo

To next establish whether the PI3K signaling pathway mediates IP-10 expression in vivo, we treated nude mice with wortmannin, before and following s.c. injection of the CHO-VEGF cells (as previously described). Wortmannin, a well-established pharmacological PI3K inhibitor, was administered by daily i.p. injection commencing on the day before the injection of the CHO-VEGF cells. Although IP-10 mRNA was significantly induced in untreated animals, these same cells failed to induce detectable levels of IP-10 in mice treated with wortmannin (Fig. 5A). We also observed that the administration of wortmannin inhibited the ability of VEGF in combination with IFN-{gamma} to induce IP-10 expression (Fig. 5B). However, VEGF-inducible expression of MCP-1 was not significantly reduced in animals treated with wortmannin (Fig. 5A). Together, these observations define a critical role for the PI3K pathway in the local inducible expression of IP-10 in vivo.


Figure 5
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  		FIGURE 5. Systemic treatment with wortmannin inhibits local VEGF and IFN-{gamma}-mediated induction of IP-10 in vivo. CHO-VEGF cells (1 x 105 cells) were injected s.c. into the skin of nude mice either alone (A) or in combination with 1000 U of IFN-{gamma} (B). The mice were either untreated or were treated with wortmannin daily (1 mg/kg on days –1, 0, and 1 and 0.5 mg/kg on days 2–4) by i.p. injection. The skin was harvested as a time course, and RNA was extracted and evaluated by RPA for the inducible expression of a panel of chemokines. A, Representative RPA showing the expression of IP-10 and MCP-1 as a time course in skin samples following injection of CHO-VEGF cells in untreated animals and in animals treated with wortmannin. B, The effect of wortmannin on the expression of IP-10 2 days following the injection of CHO-VEGF cells in combination with IFN-{gamma}. The graphs (top panels) represent the densitometric quantification of IP-10 mRNA expression compared with the housekeeping gene L32 as shown in the blot (bottom panels).

 
Function of VEGF-inducible expression of IP-10 in leukocyte recruitment in vivo

We next wished to examine whether VEGF-inducible expression of IP-10 is functional for leukocyte recruitment and inflammation in vivo. CHO-VEGF cells were injected s.c. either into C57/BL6 wild-type mice or into mice deficient in the IP-10 receptor CXCR3 (CXCR3–/– mice). We found that the CHO-VEGF cells mediated an inflammatory response in wild-type mice (Fig. 6, A and B) that peaked between 48 (data not shown) and 72 h following injection. By contrast, injection of CHO-VEGF cells into CXCR3–/– mice failed to elicit a similar inflammatory reaction (Fig. 6, C and D). However, the CHO-VEGF cells mediated similar levels of IP-10 mRNA expression in skin harvested from both wild-type and CXCR3–/– animals (Fig. 6E). This finding clearly demonstrates that VEGF-inducible expression of IP-10 is functional for leukocyte recruitment and inflammation in vivo.


Figure 6
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  		FIGURE 6. Function of VEGF-induced IP-10 for CXCR3-dependent trafficking of leukocytes. Representative H&E staining of skins harvested from normal C57BL/6 wild-type mice (A and B) or CXCR3 knockout (CXCR3–/–) mice (C and D) 72 h following the intradermal injection of CHO-VEGF cells (as described in Fig. 4). The identical pattern of inflammation was found in skins harvested from four animals in each group. E, IP-10 expression in skins harvested from C57BL/6 and CXCR3–/– mice 1–3 days following injection of the CHO-VEGF cells, as assessed by RPA. Housekeeping genes L32 and GAPDH serve as internal controls. Data are representative of two animals for each time point. Magnification to x100 (A and C) and x400 (B and D).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we define signals in EC mediating the synergism between VEGF and IFN-{gamma} for the overexpression of the potent T cell chemoattractant chemokine IP-10 in vitro. We also show that VEGF induces the expression of IP-10 in vivo and mediates CXCR3-dependent trafficking of leukocytes into skin. We suggest that these findings are of pathophysiological significance in cell-mediated immunity and in inflammatory diseases in which VEGF and IP-10 are known to be expressed in abundance.

A relatively underappreciated aspect of VEGF function is its role as a potent proinflammatory cytokine. It has been shown to promote T cell and monocyte recruitment into sites of inflammation, and inhibition of VEGF function with Abs has been shown to limit leukocyte trafficking in several disease models (6, 8, 10, 11, 12, 13, 16). VEGF is a direct monocyte chemoattractant (59) and it induces the expression of adhesion molecules and chemokines in EC (16, 26). Moreover, VEGF is expressed by EC at early times in an immune response, perhaps as a result of early T cell or platelet-EC interactions (8, 9), or it may be delivered into the local inflammatory site by infiltrating VEGF-expressing mononuclear cells (16). Thus, the abundant expression of VEGF within tissues in association with cell-mediated immune reactions likely provides positive feedback loops to amplify leukocyte trafficking and the inflammatory reaction.

The chemokines IP-10 and MCP-1 are expressed in some diseases in association with VEGF (15, 25, 37). VEGF has been found to induce the expression of MCP-1 via direct interactions with EC, and our findings in this study suggest that it may also play a major role to augment the expression of IP-10 in conditions in which T cells (and IFN-{gamma}) are characteristic (23, 36, 37, 39). We also find that VEGF is selective to augment IFN-{gamma}-mediated expression of IP-10, as it fails to alter the expression of the other CXCR3 ligands Mig and I-TAC. Because VEGF and IP-10 alone can mediate inflammation, the interrelationship between these proinflammatory mediators is likely of importance for the development of immune inflammation. Consistent with this possibility, we find that the s.c. injection of CHO cells expressing VEGF into skin results in the induced expression of IP-10 and mediates a CXCR3-dependent inflammatory reaction. In animals deficient in CXCR3, the trafficking of leukocytes into the local VEGF-injected site is more limited than that observed in wild-type animals. These observations are suggestive that the coexpression of VEGF and IP-10 in vivo is mechanistically interrelated, and that together these mediators have a major effect to augment leukocyte trafficking.

Another possibility is that the coexpression and interrelationship between VEGF and IP-10 has biological effects to regulate immune-mediated angiogenesis. Angiogenesis is a well-established component of cell-mediated immunity and in its own right has the ability to mediate inflammation (60). Angiogenic vessels are "sticky," express adhesion molecules and chemokines, and provide entry points for the trafficking of leukocytes into tissues (28, 61). Recently, it was reported that the IP-10 receptor CXCR3 is expressed by microvascular EC, and in general it is reported that IP-10 inhibits EC proliferation (40, 62, 63). Consistent with this observation, we find that the coadministration of IFN-{gamma} with VEGF (to augment IP-10 expression) has an inhibitory effect on VEGF-induced angiogenesis. The IP-10 receptor CXCR3 exists in two distinct isoforms, CXCR3A and CXCR3B, which have distinct functions (41). The selective binding of IP-10 to the CXCR3B isoform mediates angiostasis, whereas it is reported that the binding of IP-10 to the CXCR3A isoform promotes an angiogenesis response (41). Thus, the relative expression of CXCR3B vs CXCR3A on EC could dictate the angiostatic/angiogenesis potential of IP-10. Because CXCR3B expression increases during cell cycle (40, 62, 63), VEGF-inducible proliferation of EC may be associated with a relative increase in its expression. Moreover, the local expression of VEGF at sites of inflammation (or following injection in our studies) may result in both VEGF-induced proliferation of EC and, perhaps, the induction of CXCR3B. In this scenario, VEGF-induced IP-10 will facilitate a regulatory angiostasis mechanism, and one could anticipate that VEGF-induced angiogenesis might be augmented in CXCR3-deficient mice. However, in circumstances in which CXCR3A is expressed on EC, VEGF-inducible IP-10 could provide a positive feedback loop for VEGF-induced angiogenesis (41). Thus, the relative expression of CXCR3 isoforms on EC may explain why VEGF, a most potent angiogenesis factor, is expressed in some acute inflammatory conditions in the absence of angiogenesis (16), but in other conditions (4, 10) is associated with active angiogenesis.

Although the post-VEGF receptor-mediated signals resulting in the activation of EC are well characterized, the signaling mechanism for chemokine expression in EC is not very clear. VEGF binds three receptors expressed by EC, of which two are related tyrosine-kinase receptors (VEGF receptor 1 or Flt-1 and VEGF receptor 2 or KDR) and the third (called neuropilin) is a member of the semaphorin family (4, 64). Most of the effects of VEGF on EC activation are reported to be mediated by interactions with the KDR receptor that is well established to activate PI3K- and Akt-induced signals in EC (4, 27, 51, 65). The other VEGF receptors Flt-1 and neuropilin are also known to mediate activation signals and have been found to regulate KDR-inducible responses (4, 51). Thus, in future studies it will be important to determine the relative effect and function of each VEGF receptor for chemokine expression.

VEGF receptor-induced signals in EC result in a sequence of protein phosphorylations, beginning with autophosphorylation of both receptors and an increase in inositol 1,4,5 triphosphate, which results in the accumulation of intracellular Ca2+ concentration (6, 51). Subsequent steps in the signaling cascade are only partially understood but are known to involve tyrosine phosphorylation of phospholipase C{gamma} and the activation of protein kinase C (66). Activated protein kinase C is a strong inducer of NF-{kappa}B activation (67), which is known to mediate the transcription of several proinflammatory genes in EC (68, 69). As detailed in this report, the stimulation of EC with VEGF results in the activation of the PI3K-Akt pathway (6, 70, 71), and our studies detailed in this report indicate that PI3K and Akt signaling in EC may promote VEGF-induced inflammation. Targeting PI3K in vitro and in vivo with wortmannin inhibits VEGF-inducible (as well as VEGF plus IFN-{gamma}-inducible) expression of IP-10. This observation is important because the activation of the PI3K-Akt pathway is classically thought to promote antiapoptotic genes and proliferative responses in EC. Thus, a conclusion from these studies is that Akt signaling in EC is also of importance for EC-dependent mechanisms of proinflammation. Because pharmacologic inhibition of NF-{kappa}B inhibits the effect of Akt on chemokine expression (our unpublished observations), we suggest that activation of NF-{kappa}B is a common postreceptor mechanism for the activation of IP-10 in EC by VEGF.

In summary, our studies establish for the first time an inducible signaling pathway in EC mediating IP-10 expression, and we identify PI3K and Akt as a functional intermediary molecules for VEGF and/or IFN-{gamma}-induced responses in vitro and in vivo. These findings establish a mechanistic association among VEGF, a known angiogenesis factor, and IP-10, a known T cell chemoattractant chemokine. We suggest that these observations define a major proinflammatory mechanism by which VEGF is of importance in cell-mediated immunity.


   Acknowledgments
 
We thank Dr. Richard Ransohoff for the gift of the IP-10 promoter-luciferase construct, Dr. Alex Toker for the Myc-PDK1, Myc-PDK1.K/N, and GST-{Delta}p85 vectors, Dr. Debabrata Mukhopadhyay for the wild-type Akt (Mayo Clinic, Rochester, MN) and the CHO cells, and Dr. Kenneth Walsh for the DN mutant Akt. We also thank Christopher Geehan, Evelyn Flynn, and Kerrith Koss for expert technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants R01 HL 74436 (to D.M.B.) and K01 DK64182 (to S.P.). G.B. was supported by a fellowship grant from the Fondation pour la Recherche Médicale. Back

2 S.P. and D.M.B. are co-senior authors. Back

3 Address correspondence and reprint requests to Dr. David M. Briscoe, Division of Nephrology, Children’s Hospital Boston, 300 Longwood Avenue, Boston, MA 02115. E-mail address: david.briscoe{at}childrens.harvard.edu Back

4 Abbreviations used in this paper: VEGF, vascular endothelial growth factor; CHO, Chinese hamster ovary; EC, endothelial cell; PDK, phosphoinositide-dependent kinase; IP-10, IFN-{gamma}-inducible protein of 10 kDa; DN, dominant negative; RPA, RNase protection assay; mTOR, mammalian target of rapamycin. Back

Received for publication July 27, 2005. Accepted for publication December 15, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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J. D. Joo, M. Kim, V. D. D'Agati, and H. T. Lee
Ischemic Preconditioning Provides Both Acute and Delayed Protection against Renal Ischemia and Reperfusion Injury in Mice
J. Am. Soc. Nephrol., November 1, 2006; 17(11): 3115 - 3123.
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