Osteopontin-Activated Human Monocytes Proangiogenic Activity

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

This information is current as Antonella Naldini, Daria Leali, Annalisa Pucci, Emilia of September 28, 2021. Morena, Fabio Carraro, Beatrice Nico, Domenico Ribatti and Marco Presta J Immunol 2006; 177:4267-4270; ; doi: 10.4049/jimmunol.177.7.4267

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JOURNAL OF IMMUNOLOGY CUTTING EDGE

Cutting Edge: IL-1␤ Mediates the Proangiogenic Activity of Osteopontin-Activated Human Monocytes1 Antonella Naldini,2* Daria Leali,† Annalisa Pucci,* Emilia Morena,* Fabio Carraro,* Beatrice Nico,‡ Domenico Ribatti,‡ and Marco Presta†

Inflammation plays an important role in the onset of an- matrix component and as a soluble molecule implicated in in- giogenesis. In the present study, we show that osteopontin flammation, cell-mediated immunity, tissue remodeling, and (OPN), a proinflammatory mediator involved in tissue tumor metastases (8–10). OPN acts as a proinflammatory cy- repair, induces IL-1␤ up-regulation in human mono- tokine that plays important roles in monocyte/macrophage cytes. This was accompanied by the enhanced production functions (11–13). Experiments performed on OPN-null mice of TNF-␣, IL-8, and IL-6, a decreased release of IL-10, implicate OPN in Th1 cell-mediated immunity during infec- Downloaded from and increased p38 phosphorylation. The supernatants of tion, autoimmune demyelinating disease, rheumatoid arthritis, wound healing, and bone resorption (14). OPN-treated monocytes were highly angiogenic when de- In keeping with the tight cross-talk among angiogenic livered on the chick embryo chorioallantoic membrane. growth factors and cytokines, we have shown that OPN up- The angiogenic response was completely abrogated by a regulation in endothelial cells may represent a mechanism of neutralizing anti-IL-1 Ab, thus indicating that this cytokine amplification of growth factor-induced neovascularization http://www.jimmunol.org/ represents the major proangiogenic factor expressed by OPN- (11). The experimental evidence suggests that OPN may affect activated monocytes. Accordingly, rIL-1␤ mimicked the angiogenesis indirectly via mononuclear phagocyte recruitment proangiogenic activity of OPN-treated monocyte superna- and up-regulation of the expression of monocyte-derived tants, and IL-1R (type I) was found to be expressed in the proangiogenic cytokine(s). chorioallantoic membrane. In conclusion, OPN-activated In the present study, we demonstrate that OPN activates a monocytes may contribute to the onset of angiogenesis novel monocyte-mediated mechanism of neovascularization through a mechanism mediated by IL-1␤. The Journal of triggered by IL-1␤. Immunology, 2006, 177: 4267–4270. by guest on September 28, 2021 Materials and Methods he causal relationship between inflammation and an- Reagents giogenesis is now widely accepted (1); however, many Recombinant human OPN, recombinant fibroblast growth factor-2 (FGF-2), ␤ ␣ of the molecular and cellular mechanisms mediating and mAb to IL-1 , IL-8, and TNF- were obtained from R&D Systems. Re- T combinant human IL-1␤ was purchased from Endogen. The serum-free me- this relationship remain unresolved. The term angiogenic dium HYQCCM1 and FBS were obtained from HyClone. Mouse OPN and its switch describes the expression of specific genes that alter the RGD deletion mutant (⌬RGD-OPN) were expressed in Escherichia coli as GST balance between pro- and antiangiogenic molecules that partic- fusion proteins, as described previously (11). Endotoxin levels were always Ͻ0.06 EU/ml (6 pg/ml), as determined by the Limulus amebocyte lysate ipate in blood vessel formation (2). Monocytes/macrophages method (Cambrex). LPS was purchased from Euroclone. produce direct and indirect inducers of angiogenesis, as well as angiogenic inhibitors (3, 4). Also, recent observations have Monocyte preparation and culture shown that IL-1␤ may act as a potent proangiogenic cytokine Human monocytes were obtained from buffy coats of healthy blood donors (5) and that administration of IL-1 receptor antagonist inhibits (through the courtesy of the Blood Center, Siena Medical Center) by Ficoll (Lympholyte-H; Cederlane Laboratories) and Percoll (Amersham Biosciences) tumor growth and neovascularization (6). gradients, as described previously (11). Where indicated, cells were incubated Osteopontin (OPN)3, also known as early T lymphocyte-ac- for 4–24 h with different concentrations of OPN in HYQCCM1 and 1% FBS. tivating gene-1 (7), is a phosphorylated acidic RGD-containing Then, conditioned medium (CM) was collected and evaluated either for cytokine content or for its angiogenic activity in the gelatin sponge/chick embryo glycoprotein that binds certain CD44 variants and integrin re- chorioallantoic membrane (CAM) assay. In parallel, monocytes were lysed, and ceptors (8). OPN exists both as an immobilized extracellular total RNA was extracted for quantitative RT-PCR (qRT-PCR) analysis.

*Unit of Neuroimmunophysiology, Department of Physiology, University of Siena, Siena, G. Berlucchi, Istituto Superiore Sanita`(Progetto Oncotecnologico), and Associazione Italy; †Unit of General Pathology and Immunology, Department of Biomedical Sciences Italiana per la Ricerca sul Cancro (Italy) (to M.P.). and Biotechnology, University of Brescia, Brescia, Italy; and ‡Department of Human 2 Address correspondence and reprint requests to Dr. Antonella Naldini, Unit of Neu- Anatomy and Histology, University of Bari, Bari, Italy roimmunophysiology, Department of Physiology, University of Siena, Via Aldo Received for publication April 6, 2006. Accepted for publication July 26, 2006. Moro, 53100 Siena, Italy. E-mail address: [email protected] The costs of publication of this article were defrayed in part by the payment of page charges. 3 Abbreviations used in this paper: OPN, osteopontin; FGF-2, ﬁbroblast growth fac- This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. tor-2; CM, conditioned medium; CAM, chorioallantoic membrane; qRT-PCR, quan- Section 1734 solely to indicate this fact. titative RT-PCR; RGD, Arg-Gly-Asp. 1 This work was supported by grants from MIUR (Cofin 2004) and Fondazione MPS (to A.N.) and from MIUR (Centro di Eccellenza IDET, FIRB 2001, Cofin 2004), Fondazione

Measurement of cytokines by ELISA IL-1␤, TNF-␣, IL-6, IL-8, and IL-10 concentrations were assessed on monocytes supernatants by ELISA using high-performance ELISA reagents (Euro- clone). None of the assays showed cross-reactivity with other cytokines. The minimum detectable doses were for IL-1␤, TNF-␣, IL-6, and IL-10 Ͻ 5 pg/ml and for IL-8 Ͻ 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 Re- agent (Invitrogen Life Technologies). RNA samples (2 ␮g) were retrotranscribed with Ready-To-Go You-Prime First Strand Beads (Amersham Biosciences). PCR were performed on cDNA samples using the forward primer, 5Ј-TGCCATCTTGATCCTCAATG-3Ј, and the reverse primer, 5Ј- TGGAAGCAAGCCATACACAC-3Ј (chicken IL-1RI: GenBank accession no. NM_205485). Amplified products were subjected to electrophoresis on agarose gel and visualized by ethidium bromide staining. IL-1␤ 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-1␤ Downloaded from forward, 5Ј-TGATGGCTTATTACAGTGGCAATG-3Ј, and IL-1␤ reverse, 5Ј-GTAGTGGTGGTGGGAGATTCG-3Ј; and ␤-actin forward, 5Ј-CGC FIGURE 1. OPN induces a proinﬂammatory response in human mono- CGCCAGCTCACCATG-3Ј, and ␤-actin reverse, 5Ј-CACGATGGAGGG cytes. A, OPN induces p38-MAPK activation in human monocytes. Human GAAGACGG-3Ј (GenBank accession no. for IL-1␤, NM000576, and for monocytes were treated with 100 nM OPN at different time points, and ␤ -actin, NM001101). Data were quantitatively analyzed on an MJ Opticon- cellular extracts were probed with anti-phospho-p38 Ab and an anti-pan- Monitor detection system (Bio-Rad). All values were expressed as fold increase ␤ p38 Ab, as a loading control. B–F, OPN modulates cytokine production in relative to the expression of -actin. http://www.jimmunol.org/ human monocytes. Human monocytes were cultured at 106 cells/ml in the Immunohistochemistry and Western blot analysis presence of 100 nM OPN. After 18–24 h (4–6 h for IL-8) of culture, cell-free supernatants were obtained, and IL-1␤, TNF-␣, IL-8, and IL-6 ␮ IL-1RI immunodetection was performed on deparaffinized 5- m CAM sec- concentration was determined by ELISA. IL-10 determination was as- tions (15), using a goat polyclonal anti-mouse IL-1RI Ab (R&D Systems) and sessed from monocytes cultured in the presence of 100 nM OPN and LPS a preimmune goat serum as negative control. (0.1 ␮g/ml). The levels of IL-10 in the absence of LPS were undetectable For Western blot analysis, monocytes were treated with 100 nM OPN, and Ͻ ء Ϯ ϭ at different time points, cells were properly lysed. Aliquots (40 ␮g) of the ex- (data not shown). Data are expressed as mean SEM (n 3–6); , p tracted material were analyzed by Western blotting using rabbit polyclonal anti- 0.05 by Student’s t test. phospho-p38 and anti-pan-p38 Abs (Cell Signaling Technology). CAM assay by guest on September 28, 2021 diators TNF-␣, IL-8, and IL-6 (Fig. 1, B–E). Also, OPN inhib- The CAM of fertilized White Leghorn chicken eggs were added, on day 8, with its the release of the anti-inflammatory/antiangiogenic cytokine 1-mm3 sterilized gelatin sponges (Gelfoam Upjohn), as described previously (16). The sponges were loaded with 3 ␮l of PBS as negative control; 3 ␮lofPBS IL-10 when cells were activated with LPS (Fig. 1F), whereas it with 500 ng of FGF-2; 3 ␮l of CM from human monocytes pretreated with 100 has no effect on IL-10 production in resting monocytes (data nM OPN and added or not with a saturating dose (400 ng) of neutralizing not shown). ␤ ␣ ␮ ␤ anti-IL-1 , anti-IL-8, or anti-TNF- mAb; and 3 l of PBS with 5 ng of IL-1 . The capacity of OPN to up-regulate IL-1␤ in human mono- CAM were photographed in ovo with a stereomicroscope equipped with a cam- era and image analyzer system (Olympus Italia). At day 12, the angiogenic re- cytes was further investigated. Fig. 2 shows that the effect of sponse was evaluated as the number of vessels converging toward the sponge. OPN on IL-1␤ protein production is dose dependent with an ED equal to ϳ50 ng/ml (Fig. 2A) and paralleled by an in- Endothelial cell migration assay 50 crease in steady-state levels of IL-1␤ mRNA, as assessed by The CM from control and OPN-treated monocytes were diluted (1/3 v/v) in qRT-PCR (Fig. 2B). The effect was statistically significant HYQCCM1 medium plus 1.0% FCS and placed in the lower compartment of a chemotaxis microchamber (Neuroprobe). Murine aortic endothelial cells 16–18 h after stimulation, but it was already detectable at 4–6 (50,000 cells/well) were added in the upper compartment. After4hat37°C, h (data not shown). IL-1␤ was similarly induced by the rGST- cells migrated through the gelatin-coated polycarbonate filter (8-␮m pore size; OPN fusion protein and the GST-⌬RGD-OPN mutant de- Neuroprobe) were stained with Diff-Quik (Baxter) and counted in triplicate leted for the integrin-binding RGD sequence (11), whereas (five fields per well). control GST was inactive (Fig. 2, C and D), indicating that in- Statistical analysis tegrin engagement is not responsible for IL-1␤ up-regulation Statistical significance between the experimental groups was determined using triggered by OPN in human monocytes. unpaired Student’s t test or one-way ANOVA with Dunnett’s post hoc test Collectively, these results show that OPN induces the release where appropriate. of IL-1␤ and other proinflammatory/proangiogenic cytokines, such as TNF-␣, IL-8, and IL-6, and inhibits the production of Results antiinflammatory/antiangiogenic IL-10 in human monocytes. OPN triggers proinflammatory cytokine production in human monocytes ␤ Human rOPN causes the rapid phosphorylation of p38 stress OPN-activated monocytes induces angiogenesis via IL-1 production kinase when administered to freshly isolated monocytes from The above observations are in agreement with the hypothesis healthy blood donors (Fig. 1A). Activation of p38 is commonly that OPN may cause a switch of the angiogenic balance in hu- associated with a proinflammatory response (17). Accordingly, man monocytes that favors the neovascularization process. Ac- OPN causes a significant increase in the levels of secreted IL-1␤ cordingly, when tested in a Boyden chamber assay, the CM protein, as well as of the proinflammatory/proangiogenic me- from OPN-treated monocytes exerted a chemotactic response The Journal of Immunology 4269

FIGURE 2. OPN enhances IL-␤ production in human monocytes. Downloaded from Monocytes were cultured with either different doses of OPN or 100 nM GST, GST-OPN, or GST-⌬RGD-OPN for 18–24 h. IL-1␤ present in cell- free supernatants was determined by ELISA (A and C). Evaluation of IL-1␤ mRNA expression in OPN-treated monocytes was determined by qRT-PCR (B and D). All values were expressed as fold increase relative to ,ء ;(the expression of ␤-actin. Data are expressed as mean Ϯ SEM (n ϭ 3 p Ͻ 0.05 by one-way ANOVA with Dunnett’s post hoc test. http://www.jimmunol.org/

FIGURE 3. OPN-treated monocytes induce endothelial cell chemotaxis and angiogenesis in the CAM through IL-1␤. A, Murine aortic endothelial for murine aortic endothelial cells significantly higher than that cells were assessed for their capacity to migrate in response to CM from triggered by the CM of control monocytes (Fig. 3). To assess control and OPN-treated monocytes in a Boyden chamber assay. After 4 h, this hypothesis in vivo, the CM from OPN-treated monocytes cells that migrated through the filter were counted in triplicate (five fields per well) at ϫ250 magnification. Control values obtained in fresh medium Significantly ,ء .was delivered onto the CAM at day 8 of development. At day (52 Ϯ 8 cells/field) were subtracted from all the data 12, implants were surrounded by numerous allantoic neovessels different from control CM, p Ͻ 0.01 by Student’s t test. B, Gelatin sponges by guest on September 28, 2021 developing radially toward the implant in a “spoked-wheel” were adsorbed with: vehicle (PBS); rFGF-2; CM from OPN-treated mono- pattern (Fig. 3B). The response was similar to that elicited by cytes in the absence (CM) or in the presence of neutralizing anti-IL-1␤ the delivery of human rFGF-2, here used as a positive control (CM ϩ anti-IL-1␤), anti-IL-8 (CM ϩ anti-IL-8), or anti-TNF-␣ (CM ϩ (16) (mean number of vessels equal to 35 Ϯ 5 and 32 Ϯ 5 for anti-TNF-␣) mAb; and rIL-1␤. Sponges were then implanted onto the CM and FGF-2, respectively). It must be pointed out that no CAM. At day 12, blood vessels entering the sponges were counted. Data Significantly different from CM, p Ͻ ,ء .are the mean Ϯ SD of 20 embryos significant angiogenic response was observed when fresh me- 0.05 by Student’s t test. C–E, Macroscopic images of CAM treated with dium containing 100 nM OPN, the same dose used to stimu- CM from OPN-treated monocytes in the absence (C) or in the presence (D) late human monocytes, was applied directly onto the CAM of a neutralizing anti-IL-1␤ mAb. Note the significantly reduced angio- (data not shown). Indeed, this treatment results in the delivery genic response in D, similar to that observed in the CAM treated with PBS of 18 ng of OPN per embryo, a dose five times lower than the (E). F and G, IL-1RI expression in the CAM. RT-PCR was performed using specific chicken IL-1RI primers on retrotranscribed CAM mRNA minimal angiogenic dose of the cytokine (11). Thus, these ob- ϩ Ϫ servations confirm the hypothesis that OPN treatment triggers ( RT). Nonretrotranscribed CAM mRNA ( RT) was used as control (F). Paraffin-embedded CAM sections were nuclear counterstained and deco- a proangiogenic phenotype in human monocytes. rated with affinity-purified goat polyclonal anti-IL-1RI Ab on day 12. IL- To assess the contribution of IL-1␤ to this response, CM 1RI immunoreactivity was evident in the endothelium lining the blood from OPN-treated monocytes was preincubated with a saturat- vessels (G). Original magnification, ϫ400. No specific signal was observed ing dose of neutralizing anti-IL-1␤ mAb before being delivered in CAM in which primary Ab was replaced by preimmune rabbit serum onto the CAM. As shown in Fig. 3, the Ab completely abolished (data not shown). the neovascular response triggered by the CM, the mean number of blood vessel converging toward the implant being similar (10 Ϯ 3) to that observed in control CAM treated with PBS- 1RI protein was localized on chick embryo endothelium, as loaded implants (7 Ϯ 3). In contrast, neutralizing anti-IL-8 or shown by immunostaining of CAM sections (Fig. 3). anti-TNF-␣ Abs did not affect the neovascular response induced by the CM. Discussion In keeping with these observations, recombinant human In the present article, we demonstrate that OPN induces a IL-1␤ (5 ng per embryo) caused a potent angiogenic response in proangiogenic switch in human monocytes characterized by IL- the CAM, similar to that induced by the CM from OPN- 1␤,TNF-␣, IL-8, and IL-6 up-regulation and by IL-10 down- treated monocytes (Fig. 3). IL-1␤ exerts its biological effects by modulation. This extends previous observations about the ca- interacting with cognate IL-1R (18). Accordingly, IL-1RI pacity of OPN to induce proangiogenic cytokines and mRNA was detectable in CAM extracts by RT-PCR, and IL- chemokines (13). Accordingly, the CM from OPN-treated 4270 CUTTING EDGE: IL-1␤ AND OPN INTERACTION IN ANGIOGENESIS monocytes is endowed with a potent angiogenic activity that is 2. 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