Cutting Edge: IL-1β Mediates the Proangiogenic Activity of Osteopontin-Activated Human Monocytes

Reagents

Recombinant human OPN, recombinant fibroblast growth factor-2 (FGF-2), and mAb to IL-1β, IL-8, and TNF-α were obtained from R&D Systems. Recombinant human IL-1β was purchased from Endogen. The serum-free medium HYQCCM1 and FBS were obtained from HyClone. Mouse OPN and its RGD deletion mutant (ΔRGD-OPN) were expressed in Escherichia coli as GST fusion proteins, as described previously (11). Endotoxin levels were always <0.06 EU/ml (6 pg/ml), as determined by the Limulus amebocyte lysate method (Cambrex). LPS was purchased from Euroclone.

Monocyte preparation and culture

Human monocytes were obtained from buffy coats of healthy blood donors (through the courtesy of the Blood Center, Siena Medical Center) by Ficoll (Lympholyte-H; Cederlane Laboratories) and Percoll (Amersham Biosciences) gradients, as described previously (11). Where indicated, cells were incubated for 4–24 h with different concentrations of OPN in HYQCCM1 and 1% FBS. Then, conditioned medium (CM) was collected and evaluated either for cytokine content or for its angiogenic activity in the gelatin sponge/chick embryo chorioallantoic membrane (CAM) assay. In parallel, monocytes were lysed, and total RNA was extracted for quantitative RT-PCR (qRT-PCR) analysis.

Measurement of cytokines by ELISA

IL-1β, TNF-α, IL-6, IL-8, and IL-10 concentrations were assessed on monocytes supernatants by ELISA using high-performance ELISA reagents (Euroclone). None of the assays showed cross-reactivity with other cytokines. The minimum detectable doses were for IL-1β, TNF-α, IL-6, and IL-10 < 5 pg/ml and for IL-8 < 25 pg/ml.

RT-PCR and qRT-PCR

IL-1RI mRNA expression in control unstimulated CAM was analyzed by RT-PCR. Total RNA was extracted from CAM fragments using the TRIzol Reagent (Invitrogen Life Technologies). RNA samples (2 μg) were retrotranscribed with Ready-To-Go You-Prime First Strand Beads (Amersham Biosciences). PCR were performed on cDNA samples using the forward primer, 5′-TGCCATCTTGATCCTCAATG-3′, and the reverse primer, 5′-TGGAAGCAAGCCATACACAC-3′ (chicken IL-1RI: GenBank accession no. NM_205485). Amplified products were subjected to electrophoresis on agarose gel and visualized by ethidium bromide staining.

IL-1β mRNA expression in OPN-treated monocytes was determined by qRT-PCR using a MJ MiniOpticon Cycler (Bio-Rad). Total RNA was isolated using RNAwiz (Ambion). First-strand cDNA synthesis was performed using a iScript cDNA Synthesis kit (Bio-Rad). qRT-PCR was performed using iTaq SYBR Green Supermix with ROX (Bio-Rad) and the following primers: IL-1β forward, 5′-TGATGGCTTATTACAGTGGCAATG-3′, and IL-1β reverse, 5′-GTAGTGGTGGTGGGAGATTCG-3′; and β-actin forward, 5′-CGCCGCCAGCTCACCATG-3′, and β-actin reverse, 5′-CACGATGGAGGGGAAGACGG-3′ (GenBank accession no. for IL-1β, NM000576, and for β-actin, NM001101). Data were quantitatively analyzed on an MJ OpticonMonitor detection system (Bio-Rad). All values were expressed as fold increase relative to the expression of β-actin.

Immunohistochemistry and Western blot analysis

IL-1RI immunodetection was performed on deparaffinized 5-μm CAM sections (15), using a goat polyclonal anti-mouse IL-1RI Ab (R&D Systems) and a preimmune goat serum as negative control.

For Western blot analysis, monocytes were treated with 100 nM OPN, and at different time points, cells were properly lysed. Aliquots (40 μg) of the extracted material were analyzed by Western blotting using rabbit polyclonal anti-phospho-p38 and anti-pan-p38 Abs (Cell Signaling Technology).

CAM assay

The CAM of fertilized White Leghorn chicken eggs were added, on day 8, with 1-mm³ sterilized gelatin sponges (Gelfoam Upjohn), as described previously (16). The sponges were loaded with 3 μl of PBS as negative control; 3 μl of PBS with 500 ng of FGF-2; 3 μl of CM from human monocytes pretreated with 100 nM OPN and added or not with a saturating dose (400 ng) of neutralizing anti-IL-1β, anti-IL-8, or anti-TNF-α mAb; and 3 μl of PBS with 5 ng of IL-1β. CAM were photographed in ovo with a stereomicroscope equipped with a camera and image analyzer system (Olympus Italia). At day 12, the angiogenic response was evaluated as the number of vessels converging toward the sponge.

Endothelial cell migration assay

The CM from control and OPN-treated monocytes were diluted (1/3 v/v) in HYQCCM1 medium plus 1.0% FCS and placed in the lower compartment of a chemotaxis microchamber (Neuroprobe). Murine aortic endothelial cells (50,000 cells/well) were added in the upper compartment. After 4 h at 37°C, cells migrated through the gelatin-coated polycarbonate filter (8-μm pore size; Neuroprobe) were stained with Diff-Quik (Baxter) and counted in triplicate (five fields per well).

Statistical analysis

Statistical significance between the experimental groups was determined using unpaired Student’s t test or one-way ANOVA with Dunnett’s post hoc test where appropriate.

OPN triggers proinflammatory cytokine production in human monocytes

Human rOPN causes the rapid phosphorylation of p38 stress kinase when administered to freshly isolated monocytes from healthy blood donors (Fig. 1⇓A). Activation of p38 is commonly associated with a proinflammatory response (17). Accordingly, OPN causes a significant increase in the levels of secreted IL-1β protein, as well as of the proinflammatory/proangiogenic mediators TNF-α, IL-8, and IL-6 (Fig. 1⇓, B–E). Also, OPN inhibits the release of the anti-inflammatory/antiangiogenic cytokine IL-10 when cells were activated with LPS (Fig. 1⇓F), whereas it has no effect on IL-10 production in resting monocytes (data not shown).

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FIGURE 1.

OPN induces a proinflammatory response in human monocytes. A, OPN induces p38-MAPK activation in human monocytes. Human monocytes were treated with 100 nM OPN at different time points, and cellular extracts were probed with anti-phospho-p38 Ab and an anti-pan-p38 Ab, as a loading control. B–F, OPN modulates cytokine production in human monocytes. Human monocytes were cultured at 10⁶ cells/ml in the presence of 100 nM OPN. After 18–24 h (4–6 h for IL-8) of culture, cell-free supernatants were obtained, and IL-1β, TNF-α, IL-8, and IL-6 concentration was determined by ELISA. IL-10 determination was assessed from monocytes cultured in the presence of 100 nM OPN and LPS (0.1 μg/ml). The levels of IL-10 in the absence of LPS were undetectable (data not shown). Data are expressed as mean ± SEM (n = 3–6); *, p < 0.05 by Student’s t test.

The capacity of OPN to up-regulate IL-1β in human monocytes was further investigated. Fig. 2⇓ shows that the effect of OPN on IL-1β protein production is dose dependent with an ED₅₀ equal to ∼50 ng/ml (Fig. 2⇓A) and paralleled by an increase in steady-state levels of IL-1β mRNA, as assessed by qRT-PCR (Fig. 2⇓B). The effect was statistically significant 16–18 h after stimulation, but it was already detectable at 4–6 h (data not shown). IL-1β was similarly induced by the rGST-OPN fusion protein and the GST-ΔRGD-OPN mutant deleted for the integrin-binding RGD sequence (11), whereas control GST was inactive (Fig. 2⇓, C and D), indicating that integrin engagement is not responsible for IL-1β up-regulation triggered by OPN in human monocytes.

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FIGURE 2.

OPN enhances IL-β production in human monocytes. Monocytes were cultured with either different doses of OPN or 100 nM GST, GST-OPN, or GST-ΔRGD-OPN for 18–24 h. IL-1β present in cell-free supernatants was determined by ELISA (A and C). Evaluation of IL-1β mRNA expression in OPN-treated monocytes was determined by qRT-PCR (B and D). All values were expressed as fold increase relative to the expression of β-actin. Data are expressed as mean ± SEM (n = 3); *, p < 0.05 by one-way ANOVA with Dunnett’s post hoc test.

Collectively, these results show that OPN induces the release of IL-1β and other proinflammatory/proangiogenic cytokines, such as TNF-α, IL-8, and IL-6, and inhibits the production of antiinflammatory/antiangiogenic IL-10 in human monocytes.

OPN-activated monocytes induces angiogenesis via IL-1β production

The above observations are in agreement with the hypothesis that OPN may cause a switch of the angiogenic balance in human monocytes that favors the neovascularization process. Accordingly, when tested in a Boyden chamber assay, the CM from OPN-treated monocytes exerted a chemotactic response for murine aortic endothelial cells significantly higher than that triggered by the CM of control monocytes (Fig. 3⇓). To assess this hypothesis in vivo, the CM from OPN-treated monocytes was delivered onto the CAM at day 8 of development. At day 12, implants were surrounded by numerous allantoic neovessels developing radially toward the implant in a “spoked-wheel” pattern (Fig. 3⇓B). The response was similar to that elicited by the delivery of human rFGF-2, here used as a positive control (16) (mean number of vessels equal to 35 ± 5 and 32 ± 5 for CM and FGF-2, respectively). It must be pointed out that no significant angiogenic response was observed when fresh medium containing 100 nM OPN, the same dose used to stimulate human monocytes, was applied directly onto the CAM (data not shown). Indeed, this treatment results in the delivery of 18 ng of OPN per embryo, a dose five times lower than the minimal angiogenic dose of the cytokine (11). Thus, these observations confirm the hypothesis that OPN treatment triggers a proangiogenic phenotype in human monocytes.

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FIGURE 3.

OPN-treated monocytes induce endothelial cell chemotaxis and angiogenesis in the CAM through IL-1β. A, Murine aortic endothelial cells were assessed for their capacity to migrate in response to CM from control and OPN-treated monocytes in a Boyden chamber assay. After 4 h, cells that migrated through the filter were counted in triplicate (five fields per well) at ×250 magnification. Control values obtained in fresh medium (52 ± 8 cells/field) were subtracted from all the data. *, Significantly different from control CM, p < 0.01 by Student’s t test. B, Gelatin sponges were adsorbed with: vehicle (PBS); rFGF-2; CM from OPN-treated monocytes in the absence (CM) or in the presence of neutralizing anti-IL-1β (CM + anti-IL-1β), anti-IL-8 (CM + anti-IL-8), or anti-TNF-α (CM + anti-TNF-α) mAb; and rIL-1β. Sponges were then implanted onto the CAM. At day 12, blood vessels entering the sponges were counted. Data are the mean ± SD of 20 embryos. *, Significantly different from CM, p < 0.05 by Student’s t test. C–E, Macroscopic images of CAM treated with CM from OPN-treated monocytes in the absence (C) or in the presence (D) of a neutralizing anti-IL-1β mAb. Note the significantly reduced angiogenic response in D, similar to that observed in the CAM treated with PBS (E). F and G, IL-1RI expression in the CAM. RT-PCR was performed using specific chicken IL-1RI primers on retrotranscribed CAM mRNA (+RT). Nonretrotranscribed CAM mRNA (−RT) was used as control (F). Paraffin-embedded CAM sections were nuclear counterstained and decorated with affinity-purified goat polyclonal anti-IL-1RI Ab on day 12. IL-1RI immunoreactivity was evident in the endothelium lining the blood vessels (G). Original magnification, ×400. No specific signal was observed in CAM in which primary Ab was replaced by preimmune rabbit serum (data not shown).

To assess the contribution of IL-1β to this response, CM from OPN-treated monocytes was preincubated with a saturating dose of neutralizing anti-IL-1β mAb before being delivered onto the CAM. As shown in Fig. 3⇑, the Ab completely abolished the neovascular response triggered by the CM, the mean number of blood vessel converging toward the implant being similar (10 ± 3) to that observed in control CAM treated with PBS-loaded implants (7 ± 3). In contrast, neutralizing anti-IL-8 or anti-TNF-α Abs did not affect the neovascular response induced by the CM.

In keeping with these observations, recombinant human IL-1β (5 ng per embryo) caused a potent angiogenic response in the CAM, similar to that induced by the CM from OPN-treated monocytes (Fig. 3⇑). IL-1β exerts its biological effects by interacting with cognate IL-1R (18). Accordingly, IL-1RI mRNA was detectable in CAM extracts by RT-PCR, and IL-1RI protein was localized on chick embryo endothelium, as shown by immunostaining of CAM sections (Fig. 3⇑).