IL-12 Regulates an Endothelial Cell-Lymphocyte Network: Effect on Metalloproteinase-9 Production

Stefania Mitola, Marina Strasly, Mauro Prato, Paolo Ghia and Federico Bussolino
J Immunol October 1, 2003, 171 (7) 3725-3733; DOI: https://doi.org/10.4049/jimmunol.171.7.3725

Abstract

IL-12 is key cytokine in innate immunity and participates in tumor rejection by stimulating an IFN-γ-mediated response characterized by CD8+ mediated-cytotoxicity, inhibition of angiogenesis, and vascular injury. We previously demonstrated that activated lymphocytes stimulated with IL-12 induced an angiostatic program in cocultured vascular endothelial cells. In this study, we have extended this observation showing that a reciprocal modulation of cellular responses occurs. Actually, the presence of endothelial cells enhanced the inhibitory effect of IL-12 on metalloproteinase-9 expression in activated PBMC as well as their ability to transmigrate across an extracellular matrix. IL-12 triggered intracellular signaling, as indicated by STAT-1 activation, appeared to mainly operative in activated CD4 + cells challenged with IL-12, but it was also initiated in CD8+ lymphocytes in the presence of endothelial cells. On the other hand, stimulated PBMC reduced the expression and the activity of metalloproteinase-9, up-regulated that of tissue inhibitor metalloproteinase-1, and stimulated the STAT-1 pathway in cocultured endothelial cells. We used neutralizing Abs to show that the IFN-inducible protein 10 (CXCL10) and monokine-induced by IFN-γ (CXCL9) chemokines produced by both PBMC and endothelial cells are pivotal in inducing these effects. Altogether these results suggest the existence of an IL-12-regulated circuit between endothelium and lymphocytes resulting in a shift of proteolytic homeostasis at site of tissue injury.

Digestion and remodeling of the extracellular matrix (ECM)3 are significant events in physiologic and pathologic settings, including cell motility and differentiation, organogenesis, angiogenesis, tumor progression, and tissue injury in inflammatory diseases.

During angiogenesis and vasculogenesis, endothelial cells (ECs) and their precursors secrete matrix metalloproteinases (MMPs), a family of enzymes characterized by their ability to remodel matrix proteins and their Zn2+ dependence. These enzymes allow ECs to modify the features of ECM, which becomes permissive to their migration, proliferation, and differentiation in new capillaries (1). Besides these effects, studies using MMP-deficient mice have revealed that MMPs may regulate angiogenesis by releasing matrix-sequestrated angiogenic growth factors (2, 3, 4).

The breakdown of connective tissue barriers mediated by MMPs actions is similar in physiologic and pathologic conditions but in the latter is highly deregulated. Transcription regulation of MMP genes is mediated by AP-1 regulatory elements in their proximal promoter regions (5, 6). In general, MMP genes are not constitutively expressed in vivo and their basal transcription is low in cell cultures (7). These genes can be induced by growth factors, cytokines, and cell-cell or cell-matrix interactions in several cell types, including mononuclear cells and ECs (8, 9, 10, 11, 12, 13, 14, 15, 16). The proteolytic activity of MMPs is repressed by nonspecific protease inhibitors, such as α1-antiprotease and α2-macroglobulin, and by the specific tissue inhibitors of the metalloproteinases (TIMPs) that form noncovalent stoichiometric complexes with the active zinc binding site (15, 17, 18). TIMP-2 is generally constitutively expressed, while TIMP-1 is produced when cells are challenged with growth factors, hormones, and cytokines (15, 17, 18).

IL-12 is an immunostimulatory cytokine made of p35 and p40 subunits. It is secreted by macrophages and APCs and is involved in the early stages of immune response. IL-12 stimulates secretion of several cytokines, in particular IFN-γ, by both T and NK cells (19, 20). IL-12 has been recently demonstrated to be a component of the complex signal network between leukocytes and neoplastic cells. Its systemic or local administration in tumor-bearing mice results in up-regulation of VCAM-1 on the EC surface, recruits leukocytes to the tumor site, alters tumor capillaries activated by polymorphonuclear cells, and leads to ischemic-hemorrhagic necrosis of the tumor (21, 22, 23, 24, 25, 26). Furthermore, an early effect of IL-12 on tumor behavior is inhibition of angiogenesis, resulting in ischemic necrosis (22, 24, 27, 28, 29, 30, 31). These effects are primarily dependent on the release of TNF-α and IFN-γ, which modulates the induction of downstream chemokines IFN-γ-inducible protein 10 (CXCL10) and monokine induced by IFN-γ (CXCL9) (26, 32, 33, 34). We recently demonstrated that coculture of human ECs in the presence of IL-12-stimulated lymphocytes resulted in the inhibition of in vitro angiogenesis and of induction of MMP9 in ECs. These results suggest that MMP9 could be a molecular target of IL-12-dependent cross-talk between immune and vascular cells (35). Along this line of evidences, experimental models showed that systemic administration of IL-12 reduces MMP9 expression in tumor tissues and that MMP inhibitors increased the therapeutic efficacy of IL-12 (36).

In this study, we investigated the effects of IL-12 on MMP9 activity in human lymphocytes and in ECs alone or cocultured in a Transwell system to avoid cellular contact. Our results demonstrate that IL-12 triggers both directly and indirectly the productions of CXCL9 and CXCL10 by PBMC and ECs. By suppressing MMP9 activity in both cell types, these chemokines induce an unbalance of the protease-antiprotease homeostasis that contributes in regulating PBMC transmigration across basement membrane.

Materials and Methods

Cells

Human ECs from umbilical cord veins, prepared and characterized as previously described (37), were grown in M199 medium (BioWhittaker, Walkersville, MD) supplemented with 20% FCS (Life Technologies, Paisley, Scotland), EC growth factor (100 μg/ml; Sigma-Aldrich, St. Louis, MO), and porcine heparin (100 μg/ml; Sigma-Aldrich). They were used at second passage and grown on plastic surfaces coated with porcine gelatin (Sigma-Aldrich) or as otherwise specified.

Human PBMC were isolated from buffy coats of healthy blood donors through the courtesy of “Centro Trasfusionale AVIS” (Torino, Italy). Blood was washed twice with cold PBS at 400 × g to remove plasma and platelets and then centrifuged on Histopaque 1077 (Sigma-Aldrich) at 600 × g for 30 min at room temperature. Cells were collected at the interface, washed twice with PBS, and resuspended in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% FCS. Cells (1.5 × 106/ml) were activated for 24 h with Con A (1.5 μg/ml; Sigma-Aldrich) in the absence (activated PBMC) or in the presence of 10 ng/ml human IL-12 (R&D Systems, Wiesbaden-Nordenstadt, Germany) (stimulated PBMC).

In selected experiments, PBMC were depleted of adherent cells by two 1-h rounds of adherence to plastic on tissue culture dishes at 37°C. To purify CD4 + and CD8+ cells, adherent cell-depleted PBMC were then incubated overnight at 4°C on a disk rotator with a combination of Abs against lineage-specific markers (HLA class II, CD19, CD14, CD56, CD11b, glycophorin, and CD4 or CD8; BD Biosciences, Mountain View, CA). Subsequently, cells were washed and counted and incubated magnetic beads were coated with anti-mouse IgG mAbs (Dynal, Oslo, Norway; final dilution, 40 beads/cell). After incubation, CD4+ or CD8+ T cells were separated by negative immunomagnetic selection. The enrichment in CD4+ or CD8+ T cells was evaluated by FACS analysis performed with a FACScan flow cytometer (FACSCalibur; BD Biosciences). The purity of the cellular subset was >95%.

Cell treatment

Coculture experiments were performed in six-well plate Transwell systems (0.4 μm, Falcon; BD Labware, Plymouth, U.K.) with EC (6 × 104) plated at the bottom of the wells. Human PBMC or purified CD4+ or CD8+ cells (1.5–3 × 106/ml) were seeded onto the inserts. RPMI 1640 medium with 10% FCS was used. In selected experiments, cocultures were performed in the presence of rabbit serum against CXCL9 (1/100 final dilution), against CXCL10 (1/200 final dilution; both kindly provided by Dr. J. Farber, National Institutes of Health, Bethesda, MD), a mAb anti-IFN-γ (B27 clone 10 μg/ml; BD PharMingen, San Diego, CA), or rabbit serum (1/50 final dilution; Sigma-Aldrich) or a mouse IgG1 as control Ab (10 μg/ml; BD PharMingen). After 48 h, ECs or PBMC were washed twice with RPMI 1640 and further processed. Alternatively PBMC or CD4+ and CD8+ T cells were activated by Con A and stimulated with IL-12 as described above and then stimulated for 24 h with recombinant protein CXCL9 (10 ng/ml; PeproTech, Rocky Hill, NJ), CXCL10 (10 ng/ml; PeproTech), and IFN-γ (10 ng/ml; R&D Systems). After washes, cells were submitted to zymography or motility assays.

After coculture, the morphologic aspects of ECs were recorded by an inverted photomicroscope (model DM IRB HC; Leica Microsystems, Bensheim, Germany).

Western blot analysis

Cell were washed and lysed at 4°C in 25 mM HEPES buffer (pH 7.6; 150 mM NaCl, 1% Triton X-100, and protease and phosphatase inhibitors: pepstatin, 50 ng/ml; leupeptin, 50 ng/ml; aprotinin, 10 μg/ml; and sodium orthovanadate, 1 mM). Proteins (15 μg) were separated by SDS-PAGE and transferred to Immobilon-P sheets (Millipore, Bedford, MA) and probed with Ab against MMP9 (1/50 final dilution; Santa Cruz Biotechnology, Santa Cruz, CA) and TIMP-1 (1/50 final dilution; Santa Cruz Biotechnology). Immunoblot analysis performed with mAb anti-VE-cadherin (1/ 500 final dilution; Chemicon International, Temecula, CA), Ab anti-CD-31 (1/200 final dilution; Santa Cruz Biotechnology), Ab anti-bovine serum albumin (1/1000 final dilution; US Biological, Swampscott, MA), or anti-actin (1/250 final dilution; Sigma-Aldrich) was used as loading control when specified. To evaluate STAT-1 activation, PBMC, CD4+, or CD8+ T cells were stimulated by Con A and IL-12 as previously detailed and then maintained in RPMI 1640 with 10% FCS or cocultured with ECs. Lysed proteins (30 μg) from different cells were immunoblotted with a mAb anti-STAT-1 or anti-tyrosine phosphorylated STAT-1 (1/1000 final dilution; Santa Cruz Biotechnology). The ECL (PerkinElmer/Cetus, Norwalk, CT) was used for detection.

Gel zymography

MMPs activity was measured by zymography assay on EC and PBMC proteins or on cell supernatants according to Hyuga et al. (38). In selected experiments, ECs were washed (2 min in ice bath) with a high salt buffer (500 mM NaCl and 10 mM Na2HPO4, pH 11.0) to remove the noncovalently bound molecules. Briefly ECs and PBMC were lysed in 50 mM Tris, 300 mM NaCl, and 1% Triton X-100 (pH 7.5) or cell supernatants were collected and centrifuged at 500 × g for 15 min at 4°C. Ten micrograms of proteins were separated by 10% SDS-PAGE impregnated with gelatin (1 mg/ml; Sigma-Aldrich) in nonreducing conditions. Gels were washed twice for 20 min with 50 mM Tris and 2.5% Triton X-100 (pH 7.5), and incubated overnight at 37°C in 40 mM Tris, 200 mM NaCl, 10 mM CaCl2, and 0.02% NaN3 (pH 7.5) with or without 5 mM EDTA. Clear bands were visualized on the blue background after staining with 0.25% Coomassie blue R250 and destained with 50% methanol and 10% acetic acid. Densitometric analysis of the bands was performed with Phoretix 1D standard software (Nonlinear USA, Durham, NC).

Motility assays

After IL-12 activation and ECs cocultured as detailed above, 5 × 105 PBMC suspended in 50 μl of RPMI 1640 containing 2% FCS were put in the upper chamber of a Transwell apparatus (12-μm diameter) coated with Matrigel (50 μl; BD Biosciences) to study the ability to transmigrate across the ECM (chemoinvasion assay). After a 16-h incubation at 37°C, migrated cells were stained with trypan blue and counted in a Burker chamber. Alternatively, PBMC chemotaxis was assayed in a Boyden chamber as previously described (39). In both assays, RPMI 1640 containing 10% FCS was used in the lower chamber as chemotactic stimulus.

Northern blot analysis and RT-PCR

Total cellular RNA was isolated by RNAzol (Tel-Test, Friendswood, TX) according to manufacturer’s instruction. Equal amounts of total RNA (15 μg/lane) were electrophoresed in 1% agarose gel containing 6.3% formaldehyde in 3-morpholino-propanesulfonic acid buffer (Sigma-Aldrich) and blotted onto a Nylon Duralon-UV membrane (Sigma-Aldrich). Hybridization was conducted overnight at 42°C with an [α-32P]dCTP-labeled (3000 Ci/mmol; Amersham Biotech Pharmacia, Milan, Italy) human MMP9 probe (a 1.3-kb EcoR1-PstI fragment of MMP9) and GAPDH probe (a 1.2-kb PstI-PstI fragment). For RT-PCR, 10 μg of total RNA was reverse transcribed using an RNase H-RT (Promega, Madison, WI) according to the manufacturer’s protocol. Resultant cDNA was amplified using specific primers for CXCL9, CXCL10, and IFN-γ (40) by a semiquantitative PCR. Amplifications were performed in a thermocycler (PerkinElmer, Boston, MA) by using 1.25 U of Taq polymerase (Roche Applied Science, Monza, Italy) after heating at 94°C for 3 min, followed by 35 amplification cycles: 45 s at 94°C, 45 s at primer annealing temperature (57°C for IFN-γ and CXCL10, 61°C for CXCL9), and 60 s at 72°C.

Results

IL-12 modulates MMP9 during the interaction between ECs and PBMC

To study a possible modulation of MMP activity during the interaction between ECs and PBMC, we analyzed MMP9 released by ECs cocultured in the presence of PBMC, Con A-activated PBMC (activated PBMC) or activated PBMC challenged with IL-12 (stimulated PBMC).

MMP9 activity was readily detectable in the supernatants obtained from PBMC alone and further increased in activated PBMC (Fig. 1⇓A, lanes 1 and 2). The addition of IL-12 to activated PBMC abolished the Con A effect (Fig. 1⇓A, lane 3).

FIGURE 1.
FIGURE 1.

MMP9 and TIMP-1 activity and expression are regulated by IL-12 in a coculture system with ECs and PBMC. ECs were cultured alone or with PBMC, activated or stimulated PBMC, for 48 h. At the end of incubation, proteins in the supernatants (10 μg of proteins; A and B) or from EC lysates (15 μg of proteins; C) were separated by SDS-PAGE (10%) and submitted to zymography or immunoblot analysis. A, Gelatin zymography of supernatants obtained from either PBMC alone (lanes 1–3), ECs alone (lanes 4–6), or in coculture (lanes 7–9). The band of 97 kDa corresponds to pro-MMP9. The right panel shows densitometric analysis of zymography (mean ± SD of five independent experiments). Data were analyzed by one-way ANOVA (p < 0.001) and Student-Newman-Keuls test. ∗, p < 0.05 vs stimulated PBMC cocultured with ECs; §, p < 0.05 vs PBMC alone or cocultured with ECs. To simplify the photograph, statistical analysis taking on data of ECs alone is not indicated. B, Immunoblot of MMP9 in the supernatant of ECs alone (lane 1) or cocultured (lanes 2–4) with PBMC (top panel). The bottom panel shows the immunoblot of bovine albumin as loading control. The right panel shows densitometric analysis of MMP9 (mean ± SD of three independent experiments). Data were analyzed by one-way ANOVA (p < 0.0001) and Student-Newman-Keuls test. ∗, p < 0.05 vs ECs cocultured with stimulated PBMC. C, Immunoblot of MMP9 (top panel), TIMP-1 (middle panel), and VE-cadherin (bottom panel) in cell lysates of ECs alone (lane 1) or cocultured (lanes 2–4) with PBMC. The right panel shows densitometric analysis of MMP9 (□) and TIMP-1 (▪) (mean ± SD of three independent experiments). MMP9 and TIMP-1 data were analyzed by one-way ANOVA (MMP9, p < 0.0005; TIMP-1, p < 0.0001) and Student-Newman-Keuls test. ∗, p < 0.05 vs ECs cocultured with stimulated PBMC; §, p < 0.05 vs ECs alone.

As expected (41, 42, 43), MMP9 activity in the supernatant of ECs alone was negligible and was not modified by the addition of IL-12 alone or in combination with Con A (Fig. 1⇑A, lanes 4–6). Interestingly, MMP9 was detected in the supernatants of ECs cocultured with PBMC and its level increased with activated PBMC but was dramatically fallen with stimulated PBMC (Fig. 1⇑A, lanes 7–9). Similar results were obtained by evaluating the protein level of MMP9 in the cell supernatants (Fig. 1⇑B). PBMC depleted of adherent cells showed a similar behavior in our experimental system (data not shown).

To define the specific contribution of ECs and PBMC in the secretion of MMP9 in the coculture system, we studied the enzyme expression in lysates of both populations alone or after coculture in the different conditions. The level of MMP9 in ECs increased after 48 h of coculture with activated PBMC, but not with stimulated PBMC (Fig. 1⇑C, lanes 3 and 4), paralleling data previously obtained in the analysis of gelatinolytic activity (35). The amount of MMP9 detected in ECs was not modified by washing the cell surface with a high salt buffer to remove noncovalently bound proteinases (44) (data not shown). This excludes that MMP9 found in ECs was secreted by PBMC and subsequently adsorbed on the cell surface. Interestingly, the presence of stimulated PBMC induced in ECs the expression of TIMP-1, the natural inhibitor of MMP9 catalytic activity (17) (Fig. 1⇑C), suggesting that IL-12 affects the proteolytic-antiproteolytic balance.

The same approach conducted on PBMC lysates shows that in stimulated PBMC a marked decrease of MMP9 activity (Fig. 2⇓, A and B) and transcript expression occurred (Fig. 2⇓C). ECs reduced MMP9 activity in PBMC and activated PBMC, but greatly augmented the observed effect of IL-12 (Fig. 2⇓, A–C). This suggests that MMP9 in PBMC may be regulated by both IL-12 and ECs. In contrast, MMP2 activity detected by the zymography assay was not modified by the different treatments (data not shown), suggesting a rather specific regulation on MMP9.

FIGURE 2.
FIGURE 2.

EC coculture modulates IL-12 activity on PBMC: effects on MMP9 and chemoinvasion. PBMC, activated or stimulated PBMC, were cultured alone or with ECs for 48 h. At the end of the incubation, PBMC were lysed or used in a chemoinvasion assay. A, Gelatin zymography of cell lysates (10 μg of proteins) was performed as detailed in Materials and Methods and the densitometric analysis is shown in B (mean ± SD of five experiments; data were analyzed by one-way ANOVA (p < 0.0001) and Student-Newman-Keuls test. ∗, p < 0.05 vs stimulated PBMC cocultured with ECs; §, p < 0.05 vs stimulated PBMC). C, Northern blot analysis of MMP9 and GAPDH expression in different PBMC populations. Photographs are representative of three individual experiments. D, Chemoinvasion assay of PBMC (5 × 105 cells) was performed in a Transwell system with Matrigel-coated membrane. Cells were allowed to migrate for 16 h at 37°C (mean ± SD of five experiments; data were analyzed by one-way ANOVA (p < 0.0001) and Student-Newman-Keuls test. ∗, p < 0.05 vs stimulated PBMC cocultured with ECs; §, p < 0.05 vs stimulated PBMC).

ECs affect the chemoinvasive, but not chemotactic properties of PBMC

We previously reported that the abrogation of MMP9 production in ECs induced by stimulated PBMC participates in the indirect antiangiogenic activity of IL-12 in tumors (35). The production of MMPs by lymphocytes is likely to be an important factor in facilitating trafficking of lymphocytes through the endothelial barrier and their recruitment at the site of tissue injury, including neoplastic transformation (45). To study the biologic relevance of MMP9 regulation in PBMC (Fig. 2⇑), we evaluated their motility in a chemoinvasion assay, where cells have to degrade ECM before migrating across the filter. This assay summarizes two independent functions of circulating cells, namely, the secretion of matrix metalloproteinases to make selective clips in ECM and the movement along a gradient of a chemotactic agent (46). PBMC showed a high capacity to migrate through ECM that was reduced after Con A activation (p < 0.05). PBMC stimulation by IL-12 did not significantly modify the effect of Con A. Interestingly, the coculture of PBMC or activated PBMC with ECs already reduced their motility to levels comparable to the ones observed in stimulated PBMC. A further strong reduction could be selectively observed in stimulated PBMC when cocultured in the presence of ECs (p < 0.002; Fig. 2⇑D). On the contrary, the chemotaxis of PBMC induced by FCS was not significantly modified by Con A and IL-12 treatment. The coculture of differently treated PBMC with ECs slightly increased their motility (Fig. 3⇓).

FIGURE 3.
FIGURE 3.

Effect of ECs on PBMC chemotaxis. PBMC, activated or stimulated PBMC, were cultured alone or with ECs for 48 h. After washes, chemotaxis of PBMC (5 × 105 cells) was evaluated in a Boyen chamber by putting 10% FCS in the bottom chamber as chemotactic stimulus. Cells were allowed to migrate for 1 h at 37°C. Mean ± SD of five experiments; data were analyzed by one-way ANOVA (p < 0.0005) and Student-Newman-Keuls test. ∗, p < 0.05 vs the same corresponding PBMC population alone.

CXCL9 and CXCL10 alter MMP9 activity in ECs and chemoinvasion of PBMC

The indirect biologic effects of IL-12 on ECs are mainly exerted by the production of IFN-γ, which in turn stimulates that of CXCL10 and CXCL9 (23, 29, 32, 33, 34, 47, 48, 49, 50, 51). In our system, stimulated PBMC either alone or cocultured with ECs produced IFN-γ transcript as evaluated by RT-PCR. IFN-γ mRNA was not found in ECs differently treated. Stimulated PBMC show also transcript for CXCL9 and CXCL10 without being affected by the presence of ECs. ECs also expressed both chemokines when cocultured with stimulated PBMC (Fig. 4⇓) and no modification occurred in all of the other culture conditions.

FIGURE 4.
FIGURE 4.

RT-PCR analysis of IFN-γ, CXCL9, and CXCL10 RNA expression in ECs and PBMC. RNA isolated from ECs and the differently treated PBMC maintained alone or in coculture was processed as detailed in Materials and Methods. The expected size of the mRNA-derived PCR products for IFN-γ, CXCL9, and CXCL10 is 121, 123 and 108 bp, respectively. The small vertical bars at the bottom of the figure mark the lane of electrophoresis gel. These panels are representative of three individual experiments.

To evaluate the effect of CXCL9 and CXCL10 in the coculture system, we added to the cultures specific neutralizing Abs. MMP9 activity in ECs increased when cocultured with activated PBMC and was down-modulated by stimulated PBMC (Fig. 5⇓, A and B, and Ref. 35). The addition of anti-CXCL9 and anti-CXCL10 Abs completely abrogated the effect of stimulated PBMC, suggesting that these chemokines are the final activating molecules of ECs (Fig. 5⇓, A and B).

FIGURE 5.
FIGURE 5.

Anti-CXCL9 and anti-CXCL10 Abs abrogate IL-12 activities on EC-PBMC coculture: effect on MMP9 production in ECs and chemoinvasion ability of PBMC. Anti-CXCL9 and anti-CXCL10 Abs or nonimmune Ig were added to the coculture of ECs with stimulated PBMC. After 48 h (A), the activity of MMP9 in ECs was assayed by gel zymography (10 μg of proteins) and quantified by densitometric analysis (mean ± SD of four experiments; data were analyzed by one-way ANOVA (p < 0.0001) and Student-Newman-Keuls test. ∗, p < 0.05 vs stimulated PBMC). B, PBMC chemoinvasion was evaluated in a Transwell system with Matrigel-coated membrane (mean ± SD of four experiments; data were analyzed by one-way ANOVA (p < 0.0001) and Student-Newman-Keuls test. ∗, p < 0.05 vs stimulated PBMC).

Interestingly, only the concomitant addition of both Abs completely restored the ability of cocultured stimulated PBMC to cross the ECM, meaning the single addition of Ab anti-CXCL9 or anti-CXCL10 was ineffective (Fig. 5⇑C). Similar results have also been obtained with PBMC depleted of adherent cells (data not shown).

In an attempt to reproduce the coculture condition, recombinant CXCL9 and/or CXCL10 and/or IFN-γ were added to stimulated PBMC. Each chemokine used alone did not alter the MMP9 activity in activated (data not shown) or stimulated PBMC, while IFN-γ was inhibitory (Fig. 6⇓, A and B). In stimulated PBMC, the combined action of CXCL9 and CXCL10 resulted in a similar reduction of MMP9 activity. The addition of IFN-γ resulted in a stronger effect, in particular when associated with CXCL10.

FIGURE 6.
FIGURE 6.

CXCL9 and CXCL10 mimic the presence of EC and modulate MMP9 activity in PBMC and their chemoinvasion ability. Activated or stimulated PBMC were treated for 24 h with the indicated molecules, washed, and then processed for zymography (A and B) and chemoinvasion (C) (mean ± SD of four experiments; data were analyzed by one-way ANOVA (p < 0.0001) and Student-Newman-Keuls test. ∗, p < 0.05 vs stimulated-PBMC] as detailed in the legend to Fig. 2⇑. B, The densitometric analysis of zymography is shown (mean ± SD of four experiments; data were analyzed by one-way ANOVA (p < 0.0001) and Student-Newman-Keuls test. ∗, p < 0.05 vs stimulated PBMC; §, p < 0.05 vs stimulated PBMC in the presence of CXCL10 and IFN-γ).

We also tested the ability of CXCL9 and CXCL10 to reduce PBMC chemoinvasion. As shown in Fig. 6⇑C, both chemokines reduced the chemoinvasion capacity of both activated and stimulated PBMC, thus mimicking the presence of ECs.

IL-12 response is mostly mediated by CD4+ T cells

We were interested in identifying which subcellular population of PBMC was the major target of IL-12, allowing the above described effects orchestrated on PBMC themselves and ECs. We took advantage by the unambiguous observation that ECs totally change their shape during cell-mediated hypersensitivity reaction or when stimulated by IFN-γ (52, 53). Preliminary experiments demonstrated that PBMC depleted of mononuclear-adhering cells maintained the same features described above (data not shown). Therefore, we cocultured purified peripheral CD4+ and CD8+ cells, both known to be responsive to IL-12 (19, 20), with ECs. Usually, cultured ECs showed a polygonal morphology, with a cobblestone-like pattern (Fig. 7⇓A); the presence of stimulated PBMC or stimulated CD4+ cells transformed ECs in elongated cells similar to fibroblasts, with a reduced planar surface (Fig. 7⇓, B and D). PBMC alone (data not shown), activated PBMC, activated CD4+, and activated and stimulated CD8+ lymphocytes were unable to alter the cobblestone-like morphology of ECs (Fig. 7⇓, A, C, E, and F). These observations indicate that activated CD4+ cells are the major targets of IL-12 in our coculture system.

FIGURE 7.
FIGURE 7.

Morphology of ECs cocultured with PBMC, purified CD4+, or purified CD8+ cells. ECs were cocultured for 48 h in the presence of activated (A) and stimulated PBMC (B), activated (C) and stimulated CD4+ cells (D), and activated (E) and stimulated CD8+ cell (F). Final magnification, ×100. Photographs are representative of four different experiments.

STAT-1 expression and phosphorylation is modulated by IL-12 in cooperating EC and lymphocyte populations

To further demonstrate that CD4+ cells were the main cell population stimulated by IL-12 in our coculture system, we studied the expression and the activation of STAT-1, which is a downstream molecule of the IL-12R in lymphocytes (19, 20, 54).

We first evaluated the STAT-1 expression in total PBMC, purified CD4+, and purified CD8+ cells treated with or without Con A and IL-12 as detailed above. Cells were lysed and proteins were separated by SDS-PAGE. Western blot analysis showed an increase in STAT-1 expression in stimulated PBMC and stimulated CD4+ cells (Fig. 8⇓A). The same samples were probed with anti-phosphorylated tyrosine STAT-1, showing that the expressed protein was also heavily phosphorylated in stimulated PBMC and CD4+ cells (Fig. 8⇓B). The same analysis was conducted on PBMC cocultured with ECs and showed that the presence of ECs did not modify the pattern of STAT-1 in total PBMC under the different culture conditions (Fig. 8⇓D). In clear contrast, the presence of ECs increased STAT-1 expression in activated CD4+ cells and induced it in both activated and stimulated CD8+ cells (Fig. 8⇓D). In addition, the presence of ECs increased the phosphorylation rate of STAT-1 in both CD4+ and CD8+ lymphocytes (Fig. 8⇓E).

FIGURE 8.
FIGURE 8.

Activation of STAT-1 in activated and stimulated PBMC, CD4+, and CD8+ cells. Cells were maintained in RPMI 1640 medium with 10% FCS (A–C) or cocultured with ECs (D–F). Cells were lysed, 20 μg of proteins was separated by SDS-PAGE (7%), and Western blot analysis was performed with mAb anti-STAT-1 (A and D) or antiphosphotyrosine STAT-1 (B and E). As loading control, the same amount of protein was separated and immunoblotted with Ab anti-actin (C and F). Photographs are representative of three different experiments.

Then we analyzed the level of STAT-1 protein in ECs. We observed only a small increment in STAT-1 expression in ECs cocultured with PBMC or activated PBMC. A similar STAT-1 modulation was also observed in ECs cocultured with purified CD4+ or purified CD8+ cells. In contrast, stimulated PBMC, CD4+, or CD8+ induced a definitely higher expression (Fig. 9⇓A) and phosphorylation of STAT-1 protein in cocultured ECs (Fig. 9⇓B). The presence of a neutralizing Ab anti-IFN-γ abolished the up-regulation of the STAT-1 level in ECs cocultured with stimulated PBMC, suggesting that this cytokine produced by PBMC is the final molecule that activates STAT-1 in ECs (Fig. 9⇓D).

FIGURE 9.
FIGURE 9.

Activation of STAT-1 in ECs cocultured with PBMC, purified CD4+, or purified CD8+ cells. Twenty micrograms of proteins of lysed ECs alone (lane 1) or 48-h cocultured with PBMC (lanes 2–4), CD4+ (lanes 5–7), or CD8+ cells (lanes 8–10) differently treated were separated by SDS-PAGE (7%), and Western blot analysis was performed with mAb anti-STAT-1 (A and D), antiphosphotyrosine STAT-1 (B), or anti-CD-31 (C and E). D and E, ECs were cultured alone (lanes 1 and 5) or with PBMC differently treated (lanes 2–4 and 6–8) in the presence of neutralizing Ab anti-human IFN-γ (lanes 5–8) or irrelevant IgG (lanes 1–4). Photographs are representative of three different experiments.

Discussion

In a previous work (35), we described an intercellular circuit between endothelium and stimulated PBMC resulting in reduction of proliferation and morphogenesis of ECs. In this study, we have extended these observations and demonstrate that this circuit has a bidirectional effect on both PBMC and ECs. In particular, we show in our experimental conditions that 1) the presence of ECs augments the inhibitory effect of IL-12 on MMP9 expression and activity in activated PBMC as well as their ability to migrate through an ECM. IL-12 and ECs seem to specifically inhibit the proteolytic component of the transmigration process across ECM, because they do not affect the ability of PBMC to migrate under a chemotactic stimulus. 2) The presence of stimulated PBMC reduces simultaneously the expression and activity of MMP9 in ECs while up-regulating TIMP-1 expression. 3) Neutralizing Abs anti-CXCL9 or -CXCL10 entirely abrogate the above effects on ECs and on PBMC. 4) The primary target of IL-12 appears to be CD4+ lymphocytes, even if in the presence of ECs CD8+ cells are also stimulated.

We used a coculture model where cells are separated by a semipermeable membrane to exclude that our observations are dependent on molecular interactions requiring PBMC-EC physical interaction (55, 56, 57), including the up-regulation of MMPs in ECs through a T cell contact-dependent activation of CD40 (58). However, in a physiopathologic setting we cannot exclude that our observation may be influenced by signals coming from cell-cell contact. The complex cross-talks between these two cell populations emerging from our experimental system may represent the biologic basis for the observed reduction of vascularization in IL-12-treated tumors. In murine models of invasive tumors treated with IL-12, there is a reduction of the tumor mass flanked by a huge amount of infiltrating leukocytes and inhibition of angiogenesis (21, 22, 23, 24, 26, 29, 48, 49, 59). A paradigmatic scenario occurring in IL-12-mediated tumor rejection involves the cytotoxicity of CD8+ cells, the Th1 differentiation of naive CD4+ cells that can subsequently provide help for tumor-specific priming of CTLs, and an antiangiogenic effect. It has been shown that the IL-12 effect is largely mediated in vivo by IFN-γ and its correlate molecules CXCL10 and CXCL9 (26, 32, 33, 34). Our experiments confirm the key role of the IFN-γ-CXCL9/CXCL10 axis in the effect of IL-12, but also add new details describing a reciprocal paracrine loop existing between ECs and stimulated PBMC that is regulated by CXCL9 and CXCL10 and does not require the presence of tumor cells as the source of these chemokines (24, 49, 60, 61). This is deduced by the transcription of CXCL9 and CXCL10 genes in both cocultured cell populations, by the mimicry activity of CXCL9 and CXCL10 in inhibiting MMP9 production and chemoinvasion in activated PBMC, and by the abrogation of biologic events observed in both costimulated cells by neutralizing Abs anti-CXCL9 and CXCL10. Although the inhibition of stimulated PBMC on ECs is reverted by a single Ab, the total effect on PBMC is obtained when the two Abs are used in combination. This discrepancy suggests a possible redundancy in the activity of the two chemokines on PBMC that share CXCR3 (62, 63). The restoring effect on ECs by either anti-CXCL9 or anti-CXCL10 may be explained by the recent evidence that ECs express two isoforms of CXCR3 having different affinities for both ligands and triggering independent biologic responses (64). The possibility that the two CXCR3 isoforms heterodimerize (65) or cooperate differently with a new identified CXCL10-specific receptor expressed on ECs (66) has to take into account and could contribute to explain the discrepant effect of anti-CXCL9 and anti-CXCL10 on PBMC and ECs.

In our experimental model, we show that IL-12 induces a modification of protease-antiprotease homeostasis. This is suggested by the evident reduction of MMP9 in both ECs and PBMC along with the concomitant induction of TIMP-1 expression in ECs. These results mirror an in vivo experimental model where that tumor-based IL-12 therapy causes inhibition of MMP9 expression and induction of TIMP-1 (36). They also explain the synergistic effect between IL-12 and the MMP inhibitor Batimastat in inducing tumor regression (67).

The observed reduction of MMP9 in PBMC and the reduced ability to migrate through the protein matrix seem to be contradictory to the final effect of IL-12 on tumors in vivo. Actually the treatment with IL-12 is characterized by induction of a robust innate immune response and infiltration of lymphomononuclear cells (21, 22, 23, 24, 26, 30, 48). We may speculate that circulating lymphomononuclear cells may be recruited into tumors by an IL-12-dependent mechanism, which in turn favors their location. Systemically administered IL-12 activates circulating leukocytes, which become responsive to chemokines (e.g.CCR5 ligands (68)), and up-regulates vascular adhesion molecules (24), thus favoring inflammatory infiltrate. There, leukocytes interact with the tumor microenvironment and that may further change their functions. For instance, tumor-specific activated mouse T cells greatly up-regulate IL-13 and IL-10 (69) that in turn may reduce T cell motility (70, 71, 72). Accordingly, our data may suggest that in the tumor microenvironment where, among other cells, ECs are present, CXCL10 and CXCL9 may down-modulate MMP9 in PBMC. Both CXCL9 and CXCL10 are also able to induce adhesion of activated T cells to immobilized matrix by up-regulating integrin (73), likely explaining the augmented presence of lymphomononuclear cells in the tumor. It is intriguing to speculate on a possible scenario characterized by a first step in which IL-12 favors PBMC migration into tumors. A second step takes into account the interaction of IL-12-stimulated PBMC with the tumor microenvironment (e.g., ECs), which results in blocking PBMC at the site of immune-mediated tumor rejection. A mirrored situation occurs during mobilization of bone marrow precursors into the bloodstream, in which MMP9 plays a pivotal role. It has been clearly shown that placental growth factor-mediated mobilization of leukocytes and stem cells is reduced in MMP9−/− mice (74). Similarly, Ab anti-MMP9 reduces CXCL8-induced mobilization of hemopoietic precursors from the bone marrow of rhesus monkeys (75).

In in vivo models of IL-12-mediated tumor rejection, both CD4+ and CD8+ cells seem to be essential in the onset of the immune response. CD8+ cells are plentifully recruited in the tumors, but also the number of CD4+ lymphocytes are constantly increased after IL-12 treatment, as well as that of dendritic and NK cells (21, 24, 60, 76, 77). Elimination of CD4+ lymphocytes or NK or NKT cells has a contradictory effect on the therapeutic outcome of IL-12 depending on the model used (21, 24, 48, 49, 78, 79, 80, 81). The necessary role of CD8+ cells is largely recognized (24, 48, 49, 61, 82), but their function may be replaced by other cellular circuits (22). The evidence that IL-12 reduces angiogenesis in SCID mice, NK cell-deficient beige mice, and ν/ν mice indicates that this inhibitory effect is not necessarily mediated by a single immune cell population (27), as also supported by the observation that the maximal block of the EC cycle induced by IL-12-stimulated murine mononuclear cells is exerted when both CD4+ and CD8+ cells are present (35). Furthermore, many evidences indicate that tumor rejection and angiogenesis inhibition require cooperation between several immune cell populations, each necessary but not itself sufficient (21, 22, 24, 27, 35, 50). Therefore, it remains controversial which lymphocyte population is first involved in mediating the antitumor activities of IL-12. Because depletion of adherent cells from PBMC did not alter the observed inhibition of MMP9 activity, we looked at which lymphocyte population was the primary target of IL-12 in our coculture system. CD4+ and CD8+ cells were enriched from mononuclear cells by negative immunodepletion. This technique avoids cell activation, but does not permit a significant cell recovery. Because the change of shape is a morphologic sign of EC activation by cell-mediated immune response (52, 53), this approach has been used to determine the primary target of IL-12. The morphologic observation of ECs cocultured with stimulated cells indicates that only CD4+ lymphocytes induce change of their shape, implying that in vitro CD4+ cells could be the first to be activated by this cytokine.

To gain insight in these early circuits, we studied the expression and the activation of STAT-1, which is in an intracellular target of IL-12-induced biologic activities (19, 20) and negatively regulates MMP9 transcription (83). In our experimental conditions, we observe that STAT-1 is expressed and tyrosine phosphorylated in stimulated PBMC and CD4+ cells regardless of the presence of ECs but not in stimulated CD8+, corroborating the morphologic observations discussed above. The STAT-1 pathway is triggered in CD8+ cells when cocultured with ECs. These data are in agreement with those of Gollob et al. (54, 84, 85), which demonstrated that IL-12 induced the STAT-1 pathway in CD4+ cells and in CD18bright cells, a subset of CD8+ cells. It is intriguing to speculate that molecules produced by ECs may render all subsets of CD8 cells able to activate STAT-1-dependent machineries or expand the CD8+CD18bright population.

Mirroring the EC requirement for triggering STAT-1 in CD8+ cells, stimulated PBMC are necessary for the activation of the same transcriptional pathway in ECs, through a IFN-γ-dependent mechanism. The activation of the STAT-1 pathway in ECs has been demonstrated to be pivotal in rapid transcription of TAP1 and IFN regulatory factor 1 (86, 87). The coordinated expression of these molecules by stimulated PBMC may enhance the capacity of ECs to present foreign peptides bound to class I MHC molecules (88). By this way, IL-12 may link innate to immune-mediated host defense.

In conclusion, the study presented here brings new insights into the mechanisms of cooperation between ECs and lymphocytes, highlighting a cellular circuit regulated by the IL-12-IFN-γ-CXCL9/CXCL10 axis. Given the relevance of the collaboration between vascular and immune systems, this circuit may be operative not only in tumor regression but also in the pathogenesis of vascular diseases where chronic inflammatory injury occurs (56, 89).

Footnotes

  • ↵1 This study was supported by Associazione Italiana per la Ricerca sul Cancro, Istituto Superiore di Sanità (IV Programma Nazionale di Ricerca sull’AIDS-2001 and Progetto “Tumour therapy”), Compagnia di San Paolo, Ministero dell’ Università e della Ricerca (60%, Cofin 2002 and FIRB), and Centro Nazionale della Ricerca C.N.R. (Progetto Strategico Oncologia).

  • ↵2 Address correspondence and reprint requests to Dr. Federico Bussolino, Department of Genetics, University of Torino, IRCC, Sp. 142, Km 3.95, 10060 Candiolo (Torino), Italy. E-mail address: federico.bussolino{at}ircc.it

  • ↵3 Abbreviations used in this paper: ECM, extracellular matrix; EC, endothelial cell; MMP, metalloproteinase; TIMP, tissue inhibitors of the metalloproteinases.

  • Received March 31, 2003.
  • Accepted July 22, 2003.

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