Integrin 2Β X Α a Novel Osteopontin Receptor, the Osteopontin

Osteopontin Enhances Phagocytosis through a Novel Osteopontin Receptor, the αXβ2 Integrin

This information is current as Lotte Schack, Romualdas Stapulionis, Brian Christensen, of September 26, 2021. Emil Kofod-Olsen, Uffe B. Skov Sørensen, Thomas Vorup-Jensen, Esben S. Sørensen and Per Höllsberg J Immunol 2009; 182:6943-6950; ; doi: 10.4049/jimmunol.0900065

http://www.jimmunol.org/content/182/11/6943 Downloaded from

References This article cites 47 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/182/11/6943.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Osteopontin Enhances Phagocytosis through a Novel ␣ ␤ 1 Osteopontin Receptor, the X 2 Integrin

Lotte Schack,*†‡ Romualdas Stapulionis,*‡§ Brian Christensen,† Emil Kofod-Olsen,* Uffe B. Skov Sørensen,* Thomas Vorup-Jensen,*‡ Esben S. Sørensen,†‡ and Per Ho¨llsberg2*

Osteopontin (OPN) is a cytokine with multiple functions, including immune defense mechanisms against invading microorganisms. OPN-deficient mice are impaired in clearing intracellular pathogens, suggesting an important role of OPN during phagocytosis, but it remains to be defined how OPN may enhance this innate immune process. Here, we demonstrate that OPN binds to monocytes, but not resting T cells, NK cells, or B cells, and mediates chemoattraction of IL-1-activated human monocytes. Moreover, OPN binds in a specific manner to all known serotypes of the two bacterial species Streptococcus agalactiae and ␣ ␤ Staphylococcus aureus and opsonizes these bacteria for phagocytosis. We identify the integrin X 2 (CD11c/CD18), which is highly expressed on the cell surface of monocytes, as a novel OPN receptor. To eliminate the contribution from other molecular inter- Downloaded from ␣ ␤ actions between the bacteria and the phagocyte, we show that OPN-coated synthetic beads are phagocytosed in an X 2 integrin- dependent manner. The ligand recognition does not involve the RGD motif previously reported to support binding of OPN to ␣ ␤ integrins. Taken together, these data identify the X 2 integrin as a novel OPN receptor that is required for OPN-mediated phagocytosis, thereby elucidating an important mechanism of an innate immune function of OPN. The Journal of Immunology, 2009, 182: 6943–6950. http://www.jimmunol.org/ steopontin (OPN)3 is a multifunctional protein that is OPN is highly conserved among species in its primary structure synthesized by a variety of nonimmune and immune and distribution of posttranslational modifications (11–13), sug- O cells and is involved in interactions with cells medi- gesting an essential role of the protein throughout evolution. Thus, ating signaling, migration, and attachment (1). Within the im- an important function of OPN may be within the innate and most mune system, OPN is a cytokine secreted by activated T cells, conserved part of the immune system. Indeed, OPN favors the NK cells, dendritic cells, and macrophages (2–4). Despite its recruitment of innate cells by inducing migration of dendritic cells, ubiquitous presence, OPN-deficient mice develop normally and neutrophils, and macrophages (14–16). The attraction of macro- display no apparent functional deficits (5). However, when ho- phages to the site of infection can be inhibited by administration of by guest on September 26, 2021 meostasis is challenged, as seen during infections, OPN has neutralizing Abs to OPN (16–19). Likewise, silenced OPN ex- nonredundant functions in these mice revealed by their im- pression in macrophages results in impaired migration (20). While paired clearing of intracellular infections (6, 7). This is likely to OPN exerts important functional effects on macrophages, the im- be caused by a defective Th1-mediated response, since human pact on monocytes is less clear. OPN is chemotactic for monocytes herpes virus type 1- or Listeria monocytogenes-infected cells in nephritic and arthritic rodents, whereas similar findings are not from OPN-deficient mice have reduced secretion of IL-12 and seen in normal rodents (21–23). Additionally, a study with human ␥ IFN- , but an increased IL-10 production (8). Moreover, de- monocytes suggested that OPN is not a chemokine for monocyte velopment of sarcoid-type granulomas is virtually absent in recruitment (24). These apparently conflicting data could be rec- OPN-deficient mice (8), which is in agreement with the high onciled if OPN-induced chemotaxis is dependent on the activation level of OPN expression in patients with sarcoidosis and tuber- state of the monocytes. culosis (9, 10). Chemoattractance may not be the most important influence of OPN on macrophages, since OPN-deficient mice have impaired debridement in wounded areas even under conditions with normal *Department of Medical Microbiology and Immunology and †Department of Molec- ular Biology, ‡Interdisciplinary Nanoscience Center (iNANO), §Institute for Storage macrophage influx (25). This would be consistent with an impaired Ring Facilities, Aarhus University, Aarhus, Denmark phagocytosis, which is supported by the observation that OPN- Received for publication January 28, 2009. Accepted for publication March 31, 2009. coated beads enhance phagocytosis in a murine macrophage cell The costs of publication of this article were defrayed in part by the payment of page line (26). However, it is not known whether OPN is able to opso- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. nize bacteria. A putative receptor responsible for OPN-mediated phagocytosis 1 This work was supported by grants from the Danish Dairy Research Foundation, Arla Foods, the Milk Protein Research Consortium, the LEO Pharma Foundation, the has not been defined. OPN interacts with cells of the immune Carlsberg Foundation, the Novo Nordisk Foundation, and the Faculty of Health Sci- system in a complex manner through integrin and nonintegrin re- ences, Aarhus University. ceptors. An RGD sequence within OPN is responsible for binding 2 Address correspondence and reprint requests to Dr. Per Ho¨llsberg. Department of ␣ ␤ ␣ ␤ ␣ ␤ ␣ ␤ ␣ ␤ ␣ ␤ Medical Microbiology and Immunology, The Bartholin Building, Wilhelm Meyers to the integrins V 1, 5 1, 8 1, V 3, V 5, and V 6 (27–30), Alle´ 4, Aarhus University, DK-8000 Aarhus C, Denmark. E-mail address: ph@ whereas an N-terminal fragment resulting from thrombin cleavage microbiology.au.dk ␣ ␤ ␣ ␤ ␣ ␤ exposes the motif SVVYGLR, which binds 4 1, 9 1, and 4 7 3 Abbreviations used in this paper: OPN, osteopontin; MFI, mean fluorescence in- (31–33). Apart from integrin binding, OPN also interacts with tensity; MNC, mononuclear cell; OPN*, Alexa Fluor 488-conjugated OPN. CD44 and CD147 via RGD-independent motifs (34–36). Al- Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 though some of the previously reported receptors are likely to be www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900065 ␣ ␤ 6944 OPN ENHANCES PHAGOCYTOSIS VIA THE X 2 INTEGRIN

important for OPN binding to cells of the immune system, other Cells integrin receptors abundantly expressed on, for example, mono- PBMC (mononuclear cells, MNCs) were prepared from buffy coat by Fi- cytes might also play a role. These include the complement recep- coll-Paque Plus (GE HealthCare) gradient centrifugation. To isolate mono- ␣ ␤ ␣ ␤ tors M 2 (CD11b/CD18) and X 2 (CD11c/CD18) integrins, cyte-enriched cells for migration assay and flow cytometry studies, MNCs ϩ which are known to enhance phagocytosis (reviewed in Refs. 37, were depleted for CD2 cells by incubation with 50 ␮l of CD2 panT beads ␣ ␤ (Dynal Biotech) per 1 ϫ 107 MNCs for 30 min at 4°C with head-over-end 38). Whereas M 2 integrin receptors bind to unfolded, positively ␣ ␤ rotation. The remaining cells, which were almost completely monocytes as charged residues (39), the X 2 integrin receptor binds to unfolded judged by microscopic examination, were resuspended in AIM-V media proteins that carry a high negative charge (40). As OPN is highly (Invitrogen) to a final concentration of 1 ϫ 106 cells/ml. Isolation of lym- phosphorylated and has a high content of acidic residues and a phocyte-enriched cells was performed by a similar procedure using CD14 flexible structure (1, 41), it is possible that the protein could be a beads for negative selection (Dynal Biotech). For cell adhesion studies, ␣ ␤ ␤ monocytes were isolated from MNCs using the Dynal Biotech monocyte- ligand for the X 2 integrin. However, it is unknown whether 2 negative isolation kit and stored at Ϫ135°C in culture medium (RPMI 1640 integrins are important for OPN-mediated functions. with 2 mM glutamine, 10 U/ml penicillin, 10 ␮g/ml streptomycin, and 10 Here, we demonstrate that OPN binds in a specific manner to mM HEPES (pH 7.4) supplemented with 15% (v/v) FBS and 10% (v/v) bacteria and opsonizes them for phagocytosis. Moreover, we iden- DMSO (Sigma-Aldrich). In general, cell viability of the monocytes was ␣ ␤ Ͼ90% after thawing. tify the X 2 integrin as a novel OPN receptor and show that this ␣ ␤ ␣ ␤ K562 cells and the M 2 integrin and X 2 integrin stably transfected receptor is required for OPN-mediated phagocytosis. lines of K562 (43, 44) were cultured in RPMI 1640 medium with 2 mM glutamine, 10 U/ml penicillin, 10 ␮g/ml streptomycin, and 10% FBS. The ␣ ␤ ␮ Materials and Methods M 2 integrin-transfected cells were grown in the presence of 4 g/ml ␣ ␤ Purification and thrombin cleavage of bovine OPN puromycin (Sigma-Aldrich), whereas X 2 integrin-transfected cells were Downloaded from grown in the presence of 200 ␮g/ml hygromycin (Invitrogen). Native OPN was purified from bovine milk and prepared as described (42). U937 cells were cultured in suspension in RPMI 1640 media supple- For generation of N- and C-terminal fragments, OPN was digested with mented with extra glutamine (0.146 g/L), 10 mM HEPES, streptomycin thrombin (GE Healthcare; 0.01 U/␮g OPN) in 0.1 M ammonium bicar- (0.2 g/L), penicillin (0.2 IE/L), and 10% heat-inactivated FBS. bonate at 37°C for 1 h. The resulting fragments were separated on a Su- perdex Peptide HR 10/30 column (GE Healthcare) equilibrated with 0.1 M Flow cytometric assay of OPN binding ammonium bicarbonate. This resulted in two peaks containing the N-ter- ϩ ϩ

minal part of OPN (residues 1–147), termed OPN , and different C- Purified MNCs were depleted for either CD14 or CD2 cells. For each http://www.jimmunol.org/ 1–147 ␮ terminal fragments. The C-terminal fragments were further separated by condition, 200,000 cells were incubated with 35 g/ml OPN* in binding buffer (10 mM HEPES-KOH (pH 7.4), 150 mM NaCl, 5 mM KCl, 1 mM reverse-phase HPLC on a Vydac C18 column (Separations Group). Sepa- ration was conducted in 0.1% trifluoroacetic acid and fragments were MgCl2, 1.8 mM CaCl2,5mMD-glucose, 1 mM MnCl2) in the absence or presence of 100-fold excess of unlabelled OPN, BSA, or casein. For ex- eluted with a linear gradient of 75% 2-propanol in 0.1% trifluoroacetic ϩ acid. Hereby, a pure C-terminal fragment (residues 148–204), termed amination of OPN binding to lymphocytes, CD14 -depleted cells were further stained with either PE-conjugated anti-CD2, anti-CD56, or anti- OPN148–204, was obtained. The endotoxin content in the purified OPN was ϩ measured using the Limulus amebocyte lysate assay (Associates of Cape CD19 Abs (Dako). For examination of OPN binding to monocytes, CD2 - Cod). The endotoxin content was measured to be 0.0005 EU/␮g OPN. depleted cells were further stained with anti-CD14-ECD (Coulter/Immu- notech). After the end of incubation, the cells were washed three times in Preparation of bacterial cells binding buffer and OPN* binding was examined by flow cytometry and

expressed as MFI. A total of 25,000 events were collected from each by guest on September 26, 2021 The following bacterial strains were used in the assays: Streptococcus aga- sample. lactiae serotypes Ia, Ib, II–VII (strain nos. O90, SS618, 18RS21, 3782/67, 3139, 1169, 10214, and 7271) and a noncapsulated strain (O90R) (Statens Migration assay Serum Institut, Copenhagen, Denmark); and Staphylococcus aureus serotypes 1–13 (strain numbers T1–T13) and a noncapsulated variant (Wood) Migration assays were performed in 24-well plates using inserts with poly- (National Institutes of Health, Bethesda, MD). The strains were grown in ethylene terephthalate membranes of 8-␮m pore size (BD Biosciences) coated with fibronectin (2.5 ␮g/ml). AIM-V medium (800 ␮l) (Invitrogen) Todd-Hewitt broth medium (Oxoid) overnight in 5% CO2 at 37°C. Fol- lowing incubation, formaldehyde (Sigma-Aldrich) was added to the broth containing either 1 mg/ml BSA (Sigma-Aldrich), 10% normal human se- cultures to a final concentration of 1% (w/v) and the cultures were kept at rum (NHS) (v/v), or OPN at a concentration of 0.1–10 ␮g/ml was added to room temperature until the next day. This treatment stabilizes the cells but the lower well. Highly purified monocytes (2 ϫ 105) were added to the does not alter the polysaccharide Ags. Bacterial cells were harvested from upper side of the inserts in 200 ␮l of AIM-V medium and incubated at the formalin-stabilized cultures by centrifugation (1500 ϫ g, 15 min), and 37°C overnight. For some conditions, 10 ng/ml IL-1 (Hoffmann-LaRoche) residual reactive aldehyde groups were blocked by suspending the precip- was added to the media for activation of the cells. At the end of the incu- itates in 100 mM ethanolamine (pH 9.0) for 1 h and subsequently washed bation, the inserts were washed and bound cells were fixed in 3.7% glu- taraldehyde and stained with 0.1% crystal violet for 15 min. Nonmigrating three times in 20 mM Tris-HCl, 140 mM NaCl, 1.5 mM NaN3 (pH 7.4) (TBS) and stored at 4°C until use. The concentrations of bacteria were cells on the upper surface of the inserts were removed with a cotton swab estimated by measuring OD at 600 nm, and they were further adjusted by and the numbers of migrating cells in the lower surfaces of the inserts were flow cytometry by measuring the number of events of the bacterial determined by microscopically examination at ϫ20 magnification, and six suspension. randomly chosen areas were counted per condition. The measurements were performed in duplicates. OPN binding to bacteria Cell adhesion assay Native bovine OPN was labeled (OPN*) using an Alexa Fluor 488 mi- croscale protein labeling kit (Molecular Probes/Invitrogen). Stabilized bac- Cellular adhesion was measured essentially as described (40, 45). Costar terial cells (2.3 ϫ 108) of each S. aureus or S. agalactiae strain were V-well microtiter plates (Corning) were coated with either full-length ␮ ␮ incubated with 6 g of labeled OPN in a total volume of 300 l of TBS OPN, OPN1–147,orOPN148–204 for 20 min at 37°C in 20 mM Tris-HCl (pH containing 30 mM CaCl2 and 0.05% (v/v) Tween 20 (TBS/Ca/Tween) for 9.4), 150 mM NaCl. Then, the plate was blocked in PBS containing 0.05% 1 h at room temperature with end-over-end rotation followed by incubation (v/v) Tween 20 for 20 min at 37°C. Purified monocytes were fluorescently for1hat37°C. Competitive inhibition experiments were performed with labeled by incubation with 2Ј,7Ј-bis-(2-carboxyethyl)-5(6)-carboxyfluores- 100-fold molar excess of unlabelled OPN, BSA (Sigma-Aldrich), or human cein acetoxymethyl ester (BCECF-AM; Sigma-Aldrich) for 15 min at milk casein (Sigma-Aldrich). As an additional negative control, bacterial 37°C. Cells were washed and resuspended in adhesion buffer (10 mM

cells incubated without OPN were included for flow cytometric adjust- HEPES-KOH (pH 7.4), 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM ϫ 6 ments. After incubation, the bacterial cells were washed three times and CaCl2,5mMD-glucose) to 0.5 10 cells/ml. When indicated, the cells ␮ resuspended in TBS/Ca/Tween. The samples were examined with a Cy- were preincubated for 30 min at 37°C with either 1 mM MnCl2 or 5 g/ml tomics FC500 flow cytometer (Beckman Coulter) and a total of 25,000 KIM127 mAb (46). The KIM127 mAb was produced from a hybridoma events were collected from each sample. The data were analyzed using cell line (catalog no. CRL-2838, American Type Culture Collection/LCG FlowJo software (Tree Star) and are expressed as mean fluorescence in- Promochem) and purified on a protein G-Sepharose column. For blocking ␣ ␮ tensity (MFI). of X integrin function, cells were incubated in the presence of the 5 g/ml The Journal of Immunology 6945

3.9 mAb (clone 3.9; Biodesign International) or an IgG1 isotype control mAb (Dako) and the KIM127 Ab. Subsequently, cells (100 ␮l) were added to each well and incubated for 20 min at 37°C, followed by centrifugation for 5 min, and the ﬂuorescent signal in the bottom of the well was measured (Victor 3TM; Wallac Oy). The process was repeated with increasing centrifugation levels until all cells were pelleted, which occurred above 40 ϫ g. The fraction (F) of adhering cells was estimated below 40 ϫ g by comparing the signal from the ligand-coated wells with the signal from the detergent-blocked uncoated wells according to the formula: Cell adhesion ϭ ϫ (%) 100% (Funcoated – Fcoated)/Funcoated. Adhesion studies with the K562 cell line were performed as described

above with 1 mM MnCl2 preincubation. Cellular adhesion was measured in triplicate wells and expressed as mean values Ϯ SEM.

Labeling of bacteria with CypHer5E mono-N-hydroxysuccinimide ester S. aureus serotype T5 was labeled with CypHer5E (GE Healthcare) essentially as described (47). Brieﬂy, the bacteria were washed in 0.1 M sodium carbonate (pH 9.0) and resuspended to a ﬁnal concentration of ϳ1 ϫ 109/ml by measuring the absorbance at 600 nm. CypHer5E mono-N-hydroxysuccinimide ester was dissolved in 0.1 M sodium carbonate (pH 9.0)

to a concentration of 100 mg/ml. The labeling reaction was initiated by FIGURE 1. Monocytes, but not lymphocytes, bind OPN. Binding of Downloaded from adding 0.12 mg of CypHer5E mono-N-hydroxysuccinimide ester per mil- Alexa Fluor 488-conjugated OPN (OPN*) to lymphocytes and monocytes liliter of bacteria suspension and the mixture was incubated by head-over- was examined by flow cytometry. CD14- or CD2-depleted mononuclear end rotation for2hatroom temperature. After the incubation the bacteria cells were incubated with OPN* (35 ␮g/ml) and (A) lymphocyte subsets were washed and stored at 4°C until use. were defined by binding of PE-conjugated Abs against CD2 (T cells), CD56 (NK cells), and CD19 (B cells), and (B) the monocyte subset was Protein coating of fluorescent latex beads defined by binding of PE-conjugated Abs to CD14. C, The binding of

Carboxylate-modified fluorescent latex beads (Sigma-Aldrich) were coated OPN* to monocytes was examined in the presence of 100-fold excess of http://www.jimmunol.org/ with OPN, BSA, or IgG1 essentially as described by Pedraza et al. (26). unlabelled OPN or BSA. The data are representative of three experiments. Briefly, 25 ␮l (1.4 ϫ 108) of beads was transferred to silicone-coated Eppendorf tubes and washed in an appropriate buffer. The buffer compo- sition varied depending on the protein used to coat the beads. Acetate ϫ buffer (25 mM (pH 4.5)) was used for OPN, phosphate buffer (25 mM (pH laser on an upright Leica TCS SL confocal microscope with a 40 oil- 7.2)) was used for BSA, and borate buffer (25 mM (pH 8.5)) was used for immersion objective. IgG1. The buffers were chosen to keep the ionic strength as low as possible and to be close to the isoelectric point of the protein. The proteins were Statistical analysis diluted to a final concentration of 1 mg/ml and incubated with the beads Statistical analyses of data were performed by Student’s t test. A significant overnight by head-over-end rotation at room temperature (in 1 ml of total difference was defined as p Ͻ 0.05. by guest on September 26, 2021 buffer). The following day the beads were washed twice with the appropriate buffer and used immediately in the phagocytosis assay. Results Phagocytosis assay Monocytes, but not lymphocytes, bind OPN U937 cells (2 ϫ 105) were added to a 24-well plate in a total volume of 400 It is well known that OPN is secreted from several types of im- ␮l of RPMI 1640 medium and stimulated with 25 ng/ml PMA (Sigma- mune cells. To demonstrate a direct interaction between OPN and Aldrich) for 48 h. After the stimulation the cells were washed in serum-free immune cells, we examined the ability of fluorochrome-labeled medium. For phagocytosis of latex beads, the protein-coated fluorescent OPN (OPN*) to bind freshly isolated lymphocytes and monocytes latex beads (30 beads/cell) were dissolved in 300 ␮l of prewarmed serum- free RPMI 1640 medium. For the bacterial phagocytosis assay, the purified from human blood. Mononuclear cells were incubated CypHer5E-labeled bacteria (50 bacteria/cell) coated with protein were dis- with OPN* and the potential binding of cellular subsets was de- solved in 300 ␮l of prewarmed serum-free RPMI 1640 medium modified termined by flow cytometry. Virtually no binding of OPN* was to contain 30 mM calcium. When indicated, the cells were preincubated for observed to resting T cells (CD2ϩ), NK cells (CD56ϩ), or B cells 10 min with 10 ␮g/ml of an azide-free CD11c-blocking Ab (clone CBR- ϩ p150/4G1, MCA2786Z; AbD Serotec) or an isotype control (MCA929XZ; (CD19 ) (Fig. 1A). However, we cannot exclude that low-affinity AbD Serotec) before addition of the beads. Then, the beads/bacteria were binding of OPN to lymphocytes might occur. In contrast, OPN* added to the cells and the plate was centrifuged for 10 min at 250 ϫ g to bound to monocytes (CD14ϩ) (Fig. 1B) in a dose- and Mn2ϩ- pellet the beads/bacteria before a 30-min incubation at 37°C. At the end of dependent manner (data not shown). The OPN* binding to mono- ␮ the incubation 300 l of ice-cold medium was added to the cells to stop the cytes was not caused by pinocytosis of OPN*, as the binding was reaction. The cells were detached from the well by gentle pipetting and transferred to a flow tube for examination by flow cytometry. A total of significantly inhibited by excess OPN, but not by BSA (Fig. 1C)or 20,000 events were collected from each sample. casein (data not shown).

Confocal microscopy OPN induces migration of IL-1-activated human monocytes Phagocytosis assay for confocal microscopy examination was performed as Our observation that OPN interacted directly with monocytes sug- decribed above with a few modifications. The U937 cells were transferred gests that OPN may have an influence on monocyte function. Al- to a 24-well plate containing poly-L-lysine-coated slides and stimulated though OPN affects migration of macrophages, its function on with PMA (25 ng/ml). At the end of the assay the cells were fixed in 4% formalin/PBS (pH 7.5), washed in PBS, and subsequently incubated with monocyte migration is less explored. To address whether OPN has the DNA dye TO-PRO-3 iodide 642/661 (1/1000) (Molecular Probes) to the ability to induce human monocyte migration, we used a trans- visualize the nucleus during microscopy. When indicated, internalization well chamber assay with fibronectin-coated membranes containing of the beads was visualized by staining of acidic organelles using Lyso- pores of 8 ␮m. During 16 h of incubation, OPN induced a minor Tracker Green (Molecular Probes) at a concentration of 50 nM. This procedure was performed in the absence of formalin fixation. The imaging was but significant transmigration of monocytes when compared with performed using the 488-nm line of the multiline argon laser, the 543-nm BSA ( p ϭ 0.005). Nevertheless, this migration was significantly line of the green helium-neon laser, and the 633-nm line of the helium-neon less than that seen by addition of NHS (Fig. 2A). As an important ␣ ␤ 6946 OPN ENHANCES PHAGOCYTOSIS VIA THE X 2 INTEGRIN

FIGURE 2. OPN induces migration of IL-1-activated human monocytes. Human monocytes were analyzed for migration through a ﬁbronectin-coated membrane in a transwell chamber. Cells were added to the upper side of the insert and proteins, as indicated, in the lower chamber. Cells were allowed to migrate for 16 h toward a gradient within the membrane pores. A, Migration of IL-1␤-stimulated or unstimulated monocytes in response to 10 ␮g/ml

OPN, 1 mg/ml BSA, or 10% NHS is shown. The number of migrating cells was determined microscopically by counting six random fields at ϫ20 Downloaded from magnification. Results are expressed as relative migration in percentage of monocytes migrating to NHS in the absence of IL-1␤. Mean values and error bars are representative of four independent experiments. B, Photographs at ϫ40 magnification of migrating human monocytes are shown. Cells were activated by IL-1␤ unless otherwise indicated. Thin arrows indicate migrating monocytes; thick arrows indicate membrane pores. Photographs are representative of four experiments. http://www.jimmunol.org/ part of the inflammatory response to infections, monocytes secrete 100-fold excess of unlabelled OPN, BSA, or casein as competitive IL-1. To mimic the in vivo activation during infections, we stim- inhibitors (Fig. 3, B–D). Unlabeled OPN, but not BSA or casein, ulated monocytes with 10 ng/ml IL-1␤. The OPN-induced migra- was able to competitively displace the binding of OPN* to the tion was increased considerably upon activation with IL-1␤ when bacteria (Fig. 3D), indicating that OPN binds specifically to the compared with nonactivated cells ( p ϽϽ 0.001). In contrast, IL-1␤ bacteria in a Ca2ϩ-dependent interaction. activation did not further enhance migration induced by NHS or ␣ ␤ BSA (Fig. 2A). Similar data were obtained when monocytes were The X 2 integrin is a receptor for OPN incubated with IL-1␣ (data not shown). At OPN concentrations as The binding of OPN to monocytes (Fig. 1C) was dependent on low as 0.1 ␮g/ml, OPN induced migration of IL-1␤-activated Mn2ϩ, indicating that integrin activation was involved. Monocytes by guest on September 26, 2021 ␤ ␣ ␤ monocytes (Fig. 2B). The migration of IL-1 -activated monocytes express high levels of the X 2 integrin receptor, which is able to to 1 ␮g/ml OPN equaled 56% of the chemotactic effect obtained bind acidic, unfolded proteins (40). Since OPN is an acidic and ␣ ␤ with 1 ng/ml monocyte chemotactic and activating factor (MCAF/ intrinsically unfolded protein, we hypothesized that the X 2 in- MCP-1) (data not shown). tegrin could potentially be a novel receptor for OPN. To examine this, purified monocytes were examined for their ability to bind to OPN binds to capsulated and noncapsulated bacteria an OPN-coated surface in the presence or absence of integrin Since OPN is capable of forming a gradient that chemotactically activation. attracts monocytes, we speculated whether OPN could target in- In the absence of integrin activation, no monocyte binding to vading pathogens by binding directly to them. To examine whether OPN was observed. However, upon activation with 1 mM Mn2ϩ, OPN binds to bacteria, S. aureus (strain Wood) was incubated with which is known to activate all integrins for ligand binding, mono- OPN* and binding was measured by flow cytometry. This dem- cyte binding to the OPN-coated surface was induced (Fig. 4A). To ␣ ␤ onstrated that OPN* bound to S. aureus in a dose-dependent man- demonstrate that the X 2 integrin receptor can mediate binding to ner (Fig. 3A). Concentrations of OPN Ͼ10 ␮g/ml (0.3 ␮M) did not OPN, the monocytes were preincubated with the KIM127 mAb, ␤ enhance binding further (data not shown). which stabilizes all integrins of the 2 family in an open/active We then speculated whether an innate immune function of OPN conformation. In this case, 40% of the cells were able to adhere to targeting invading pathogens may require the binding of OPN to the OPN-coated surface (Fig. 4A). In contrast to the control IgG1 common structures on bacterial surfaces. To test this hypothesis, Ab, KIM127-induced monocyte adhesion to the OPN-coated sur- we investigated the ability of OPN to bind to all known serotypes face was completely inhibited when the monocytes were preincu- ␣ of the two bacterial species S. aureus and S. agalactiae. Polysac- bated with a blocking Ab (mAb 3.9) against the X chain of the ␣ ␤ ␣ ␤ charide capsules did not prevent OPN* binding, as S. aureus se- X 2 integrin receptor. This indicates that the X 2 integrin is a rotypes 1–13 (strain numbers T1–T13) all bound OPN* (data not receptor for OPN. ␣ ␤ shown). Similar findings were made for the different strains of To ascertain that the X 2 integrin is a major player in binding ␣ ␤ ␣ ␤ group B streptococci. The serotypes Ia, Ib, II-VII, and a noncap- to OPN, we compared the binding of X 2 or M 2 integrin-trans- sulated strain O90R all bound OPN (data for serotypes O90R, Ia, fected or untransfected K562 cells to OPN-coated surfaces. Only ␣ ␤ II, IV, and V are shown in Fig. 3B). The binding was found to be the X 2 integrin-expressing cells bound the OPN-coated surface calcium dependent, as binding assays performed in a calcium-free (Fig. 4B), and this binding was dependent on divalent cations (Fig. buffer (Fig. 3C) or a buffer containing EDTA (data not shown) 4, C and D). significantly reduced OPN* binding to bacteria. The primary structure of bovine OPN contains an RGD motif To address whether the binding of OPN* to bacteria was spe- at residues 135–137 that supports binding of several integrins ␤ ␤ ␣ ␤ cific, the binding assays were also performed in the presence of (e.g., 1 and 3 integrins). To examine the location of X 2 The Journal of Immunology 6947 Downloaded from http://www.jimmunol.org/

FIGURE 3. OPN binds to bacteria in a specific manner. The binding of Alexa Fluor 488-conjugated OPN (OPN*) to S. aureus or S. agalactiae was examined by flow cytometry. A, The binding of OPN* at concentrations of 0, 0.8, 3.3, and 10 ␮g/ml to S. aureus (strain Wood) is shown. The data are representative of four experiments. B, The binding of OPN* (10 ␮g/ml) to the capsular strains Ia, II, IV, and V and the noncapsulated strain O90R of by guest on September 26, 2021 S. agalactiae is presented. The binding was examined in the presence or absence of 100-fold excess of unlabelled OPN or BSA, as indicated. Bind- ing is presented as MFI. A representative of two experiments is shown. C, Divalent cation requirement for OPN* binding (10 ␮g/ml) to S. aureus was examined in the presence or absence of Ca2ϩ or 100-fold excess of unlabelled OPN, as indicated. Binding is presented as MFI. A representative of four experiments is shown. D, Binding of OPN* (10 ␮g/ml) to S. aureus FIGURE 4. The ␣ ␤ integrin is a receptor for OPN. Percentage cell was tested in TBS/Tween 20 with 30 mM CaCl . The binding was inhibited x 2 2 Materials and Methods A by the presence of 100-fold molar excess of unlabeled OPN, while the adhesion was calculated as described in . , Adhe- addition of BSA or casein in similar concentrations showed no inhibition. sion of human monocytes to OPN-coated surfaces was tested in the presence of integrin activation (Mn2ϩ), ␤ integrin activation (KIM127) with Binding is presented as MFI. A representative of four experiments is 2 and without ␣ blocking Ab (anti-␣ ), or isotype control IgG1. B, Cell shown. X X ␣ ␤ ␣ ␤ adhesion of K562 cells and K562 cells stably expressing X 2 or M 2 integrin to OPN-coated surfaces is shown. The binding was tested under integrin-binding epitopes in the OPN sequence, an N-terminal integrin-activating conditions in the presence of Mn2ϩ. C, The adhesion to fragment (OPN1–147, comprising residues 1–147) and a fragment surfaces coated with OPN is shown for K562 cells stably expressing the ␣ ␤ 2ϩ from the C-terminal part (OPN148–204, comprising residues 148– X 2 integrin in the presence (1 mM Mn ) or absence (5 mM EDTA) of ϩ 204) of OPN were generated. In the absence of Mn2ϩ, control and divalent cations ions. Data in the presence of Mn2 are identical to data in ␣ ␤ B but are shown for comparison. D, The adhesion to OPN or the OPN X 2 integrin-expressing K562 cells showed a similar degree of fragments OPN or OPN is shown for K562 cells or K562 cells binding to full-length OPN (10–15% binding) and OPN , 1–147 148–204 1–147 ␣ ␤ stably expressing the X 2 integrin. Cell adhesion was examined under whereas binding to OPN was negligible (Fig. 4D). In the ϩ ϩ ϩ ϩ 148–204 integrin-activating conditions in the presence of Mn2 (Mn2 /Ca2 /Mg2 ) 2ϩ ␣ ␤ presence of Mn , the X 2 integrin-expressing cells showed en- or in a buffer with divalent cations not supporting activation of the integrin hanced binding to full-length OPN and the derived fragments (Fig. receptors (Ca2ϩ/Mg2ϩ). Mean values and error bars are calculated from ␣ ␤ 4D) compared with the control cells. The binding of X 2 integrin- three independent experiments. ␣ ␤ expressing cells to OPN148–204 indicated that the binding of X 2 integrin to OPN was RGD independent. Increased binding could also be observed to the fragment containing RGD, indicating that ␣ ␤ ␣ ␤ OPN enhances phagocytosis through the X 2 integrin receptor the binding site for the X 2 integrin is not a specific sequence in ␣ ␤ ␣ ␤ the C-terminal fragment. This would be consistent with X 2 in- The X 2 integrin is known to be an enhancer of phagocytosis (37, ␣ ␤ tegrin binding to negatively charged residues, which are distrib- 38). Since OPN can bind to both the monocytic integrin X 2 as ␣ ␤ uted throughout the OPN sequence. Thus, our data clearly dem- well as to bacteria, we hypothesized that the X 2 integrin may be ␣ ␤ onstrate that OPN is a novel ligand for the X 2 integrin. a novel receptor for OPN-mediated phagocytosis. To examine this ␣ ␤ 6948 OPN ENHANCES PHAGOCYTOSIS VIA THE X 2 INTEGRIN

FIGURE 5. OPN enhances phagocytosis of bacteria. S. aureus was labeled with the pH-sensitive dye CypHer5E, which is fluorescent only at low pH found in acidic compartments of the cell. U937 cells were stimulated with PMA for 48 h and examined for phagocytosis of (A) uncoated, (B) OPN-coated, or (C) BSA-coated CypHer5E-labeled S. aureus serotype T5 by flow cytometry. The bars indicate the percentage of cells with internalized bacteria. The MFI value of the total population is shown in the upper left corner. A representative of four experiments is shown. hypothesis, S. aureus was labeled with CypHer5E. This dye is pH cubated with U937 cells that were differentiated to adhere and ␣ sensitive and emits fluorescence only at low pH, as found in express high levels of X integrin by pretreatment with PMA for acidic compartments of the cell, such as phagosomes and lyso- 48 h (data not shown). When the latex beads were opsonized with somes. After labeling with CypHer5E, the bacteria were opso- OPN, a specific induction of phagocytosis was observed in 33.7% nized with OPN or BSA and added to PMA-activated U937 of the cells corresponding to a MFI value of the total population of

cells. Bacterial opsonization with OPN (S. aureus-OPN) en- 36.2. In comparison, after opsonization with IgG1, which is a Downloaded from hanced phagocytosis, as 78.8% (MFI of 12.0) of the cells emit- known inducer of phagocytosis, 29.0% (MFI of 23.7) of the cells ted fluorescence compared with S. aureus alone or opsonization had internalized beads, whereas only 8.4% (MFI of 5.3) and 10.9% with BSA (S. aureus-BSA) (67.4%, MFI of 11.0 and 68.7%, (MFI of 7.2) of the cells had internalized BSA-coated or uncoated MFI of 11.2, respectively) (Fig. 5). beads (Fig. 6, A–D). This strongly suggests that OPN is an en- ␣ To rule out a contribution of other proteins on the bacteria, we hancer of phagocytosis. We hypothesized that the X integrin re- wanted to confirm the phagocytosis using latex beads. OPN or ceptor could be responsible for the OPN induced phagocytosis, as http://www.jimmunol.org/ control proteins were coated onto fluorescent latex beads and in- this integrin is known to be involved in phagocytosis. As shown in

FIGURE 6. OPN enhances phagocytosis of ␣ latex beads via the X integrin receptor. The phagocytosis of protein-coated ﬂuorescent latex beads was examined by ﬂow cytometry and con-

focal microscopy. U937 cells were stimulated by guest on September 26, 2021 with PMA for 48 h before addition of the protein-coated latex beads. A–F, The bars indicate the percentage of cells with internalized beads measured by flow cytometry. The MFI value of the total population is shown in the upper left corner. U937 cells were exposed to (A) IgG1- coated beads as a positive control, (B) BSA- coated beads as a negative control, (C) uncoated beads, or (D) OPN-coated beads. In E and F, the cells were preincubated with (E) a blocking Ab ␣ against the X integrin subunit or (F) an isotype control before addition of OPN-coated beads. Confocal microscopy was used to visualize association of OPN-coated beads with (G) untreated cells or (H) cells preincubated with an ␣ anti- X Ab or (I) isotype control. The nucleus was visualized with the DNA dye TO-PRO-3 (blue color) and the fluorescent OPN-coated beads are seen in pink. Cell-associated beads located in the vicinity of the cells are indicated with thick arrows, whereas free beads are indicated with thin arrows. J, Internalization of OPN-coated beads by phagocytosis was con- formed by staining acidic subcellular compartments with LysoTracker Green. Colocalization of the beads (yellow) with LysoTracker Green indicates that the beads have been internalized. Cells with varying numbers of internalized beads are shown. The flow cytometic data are representative of four independent experiments, and the confocal images are representative of two independent experiments. The Journal of Immunology 6949

␣ Fig. 6, E and F, preincubation of the cells with an anti- X Ab cifically binds to and mediates phagocytosis of bacteria. Notice- (14.0%, MFI of 10.3), but not isotype control (29.3%, MFI of ably, we were able to observe phagocytosis in the absence of OPN. 31.0), inhibited the phagocytosis of the OPN-coated beads to a This is expected, since bacteria contain numerous proteins that are level comparable with the uncoated and the BSA-coated beads. involved in this process. Therefore, to ensure that the uptake was Confocal microscopy further demonstrated that untreated cells also mediated by OPN, we confirmed our data using OPN-coated (Fig. 6G) and cells preincubated with the isotype control (Fig. 6I) beads. Whereas CD44 seems to be responsible for macrophage had associated several OPN-coated beads, whereas no or only few migration (16, 49), it does not appear to be a receptor for phago- ␣ OPN-coated beads were associated with the anti- X-treated cells cytosis of OPN-coated beads (26). This suggested that OPN has a (Fig. 6H). To confirm internalization of the OPN-coated beads by hitherto unrecognized receptor for phagocytosis. phagocytosis, the cells were stained with LysoTracker Green, Monocytes, macrophages, and dendritic cells are known to ex- ␣ ␤ ␣ ␤ which accumulates in acidic cellular compartments like phagoly- press high levels of the M 2 and X 2 integrin receptors. Of ␣ ␤ sosomes. The beads colocalized with the LysoTracker, indicating these, the X 2 integrin is known to bind unfolded negatively that the beads were indeed internalized into the cell (Fig. 6J). charges proteins, making it a potential candidate for an OPN receptor. Using an adhesion assay measuring OPN binding to mono- ␣ ␤ Discussion cytes, we identify the X 2 integrin as a novel OPN receptor and ␤ In this study, we demonstrate that OPN binds to both capsulated the first 2 integrin to bind OPN. This interaction was dependent 2ϩ ␤ and noncapsulated bacteria in a dose-dependent manner. A direct on activation of the integrin by either Mn or the 2-activating ␣ ␤ interaction between OPN and bacteria has not been described pre- Ab KIM127. Although OPN is ubiquitously present and the X 2 viously. Importantly, quantification of the binding of OPN to S. integrin is constitutively expressed on monocytes, dendritic cells, Downloaded from aureus indicated that quantitative binding was obtained even at and macrophages, OPN does not bind this receptor unless it be- concentrations Ͻ1 ␮g/ml. Similar observations were made for S. comes activated, as seen during inflammation. This is consistent agalactiae. These are physiologically relevant concentrations, with the notion that OPN-deficient mice appear normal, but display since infant plasma contains 0.3 ␮g/ml OPN and human milk con- impaired responses during infections (5–8). tains as much as 138 ␮g/ml OPN (L. Schack, A. Lange, J. Kelsen, To examine whether the novel OPN receptor was responsible

␣ http://www.jimmunol.org/ J. Agnholt, B. Christensen, T. E. Petersen, and E. S. Sørensen, for the observed phagocytosis, we blocked the X integrin subunit submitted for publication). using azide-free Abs. This inhibited phagocytosis of OPN-coated Since OPN was able to bind bacteria with and without a poly- beads to the level of uncoated beads. This inhibition was confirmed saccharide capsule, it suggests that the binding occurs through using confocal microscopy. We cannot rule out that other recep- common structures on bacterial surfaces. The binding of OPN was tors, such as CD44, could contribute to phagocytosis of bacteria, dependent on the presence of calcium ions, but it is unclear although we definitively demonstrate that OPN-induced phagocy- ␣ ␤ whether this was a requirement for the binding per se or for OPN tosis requires the novel OPN receptor, the X 2 integrin. to adopt a structure that allows binding. OPN is highly phosphor- In conclusion, we have demonstrated that OPN binds directly to ylated with phosphate groups arranged in clusters that could favor activated monocytes and induces their chemotactic migration. We its interaction with calcium (11, 12). We speculate that the chela- speculate that this may be important for recruiting monocytes to by guest on September 26, 2021 tion of divalent cations by OPN through the multiple phosphory- sites of infection. This hypothesis is supported by our finding that lations and acidic side chains of aspartate and glutamate residues OPN opsonizes all known serotypes of S. aureus and S. agalactiae could allow for a molecular pattern that recognizes bacteria. For through a direct calcium-dependent interaction. Importantly, we ␣ ␤ example, the group Ag in both S. agalactiae and S. aureus is com- identify the X 2 integrin on monocytes as a novel receptor for posed of teichoic acid, which contains repeating oligosaccharide OPN and demonstrate that this receptor is responsible for OPN- subunits connected by phosphate diester bonds. We suggest that mediated phagocytosis. Thus, our data reveal a novel innate im- these phosphate groups are responsible for binding OPN through mune function of OPN. calcium chelation, thus forming a molecular pattern based on elec- trostatic interactions. Acknowledgments During inflammation, monocytes extravasate from blood into We thank Dr. Timothy A. Springer (Harvard Medical School, Boston, MA) ␣ ␤ the tissue. This condition also favors up-regulation of OPN ex- for the generous gift of K562 cells transfected with either the X 2 or the ␣ ␤ pression. Several studies have shown that OPN do not induce mi- M 2 integrin. gration of unstimulated monocytes (21, 24). This prompted us to investigate whether OPN is only chemotactic for activated mono- Disclosures cytes. Our data demonstrate that activation of human monocytes is The present study was supported in part by grants from Arla Foods, a crucial for OPN to function as a chemotactic agent, as unstimu- company that produces milk osteopontin and has filed patent applications on the use of osteopontin in infant formula and oral hygiene. lated monocytes only exhibited a low level of migration toward L.S. and E.S.S. are among the inventors on these applications. R.S., B.C., OPN. In contrast, OPN induced IL-1-activated monocytes to mi- E.K.-O., U.B.S.S., T.V.-J., and P.H. declare no competing financial grate approximately half as efficiently as did the potent chemokine interests. MCAF/MCP-1. This strongly suggests that OPN-induced monocyte migration is physiologically relevant during inflammation in References humans. As OPN is able to target bacteria and to induce migration 1. Sodek, J., B. Ganss, and M. D. McKee. 2000. Osteopontin. Crit. Rev. Oral Biol. of monocytes, it is likely that OPN forms a chemotactic gradient Med. 11: 279–303. 2. Patarca, R., G. J. Freeman, R. P. Singh, F. Y. Wei, T. Durfee, F. Blattner, leading monocytes in the direction of OPN-coated bacteria. D. C. Regnier, C. A. Kozak, B. A. Mock, H. C. Morse, 3rd, et al. 1989. Structural Although cells from OPN-deficient mice have impaired phago- and functional studies of the early T lymphocyte activation 1 (Eta-1) gene: def- cytic capacity, which can be rescued by addition of recombinant inition of a novel T cell-dependent response associated with genetic resistance to bacterial infection. J. Exp. Med. 170: 145–161. OPN (48), it is unknown whether OPN may function as an opsonin 3. Kawamura, K., K. Iyonaga, H. Ichiyasu, J. Nagano, M. Suga, and Y. Sasaki. for bacteria. However, this possibility has been suggested using 2005. Differentiation, maturation, and survival of dendritic cells by osteopontin regulation. Clin. Diagn. Lab. Immunol. 12: 206–212. OPN-coated latex beads, which are internalized more efficiently 4. Sodek, J., A. P. Batista Da Silva, and R. Zohar. 2006. Osteopontin and mucosal than are BSA-coated beads (26). We demonstrate that OPN spe- protection. J. Dent. Res. 85: 404–415. ␣ ␤ 6950 OPN ENHANCES PHAGOCYTOSIS VIA THE X 2 INTEGRIN

5. Rittling, S. R., H. N. Matsumoto, M. D. McKee, A. Nanci, X. R. An, 27. Barry, S. T., S. B. Ludbrook, E. Murrison, and C. M. Horgan. 2000. A regulated ␣ ␤ K. E. Novick, A. J. Kowalski, M. Noda, and D. T. Denhardt. 1998. Mice lacking interaction between 5 1 integrin and osteopontin. Biochem. Biophys. Res. Com- osteopontin show normal development and bone structure but display altered mun. 267: 764–769. osteoclast formation in vitro. J. Bone Miner. Res. 13: 1101–1111. 28. Denda, S., L. F. Reichardt, and U. Muller. 1998. Identification of osteopontin as ␣ ␤ 6. Nau, G. J., L. Liaw, G. L. Chupp, J. S. Berman, B. L. Hogan, and R. A. Young. a novel ligand for the integrin 8 1 and potential roles for this integrin-ligand 1999. Attenuated host resistance against Mycobacterium bovis BCG infection in interaction in kidney morphogenesis. Mol. Biol. Cell. 9: 1425–1435. mice lacking osteopontin. Infect. Immun. 67: 4223–4230. 29. Liaw, L., M. P. Skinner, E. W. Raines, R. Ross, D. A. Cheresh, S. M. Schwartz, 7. Maeno, Y., S. Nakazawa, N. Yamamoto, M. Shinzato, S. Nagashima, K. Tanaka, and C. M. Giachelli. 1995. The adhesive and migratory effects of osteopontin are J. Sasaki, S. R. Rittling, D. T. Denhardt, T. Uede, and K. Taniguchi. 2006. ␣ ␤ mediated via distinct cell surface integrins: role of v 3 in smooth muscle cell Osteopontin participates in Th1-mediated host resistance against nonlethal ma- migration to osteopontin in vitro. J. Clin. Invest. 95: 713–724. laria parasite Plasmodium chabaudi chabaudi infection in mice. Infect. Immun. 30. Yokosaki, Y., K. Tanaka, F. Higashikawa, K. Yamashita, and A. Eboshida. 2005. 74: 2423–2427. ␣ ␤ ␣ ␤ ␣ ␤ Distinct structural requirements for binding of the integrins v 6, v 3, v 5, 8. Ashkar, S., G. F. Weber, V. Panoutsakopoulou, M. E. Sanchirico, M. Jansson, ␣ ␤ ␣ ␤ 5 1 and 9 1 to osteopontin. Matrix Biol. 24: 418–427. S. Zawaideh, S. R. Rittling, D. T. Denhardt, M. J. Glimcher, and H. Cantor. 2000. ␣ ␤ 31. Bayless, K. J., and G. E. Davis. 2001. Identification of dual 4 1 integrin binding Eta-1 (osteopontin): an early component of type-1 (cell-mediated) immunity. Sci- sites within a 38 amino acid domain in the N-terminal thrombin fragment of ence 287: 860–864. human osteopontin. J. Biol. Chem. 276: 13483–13489. 9. Nau, G. J., P. Guilfoile, G. L. Chupp, J. S. Berman, S. J. Kim, H. Kornfeld, and 32. Green, P. M., S. B. Ludbrook, D. D. Miller, C. M. Horgan, and S. T. Barry. 2001. R. A. Young. 1997. A chemoattractant cytokine associated with granulomas in Structural elements of the osteopontin SVVYGLR motif important for the inter- tuberculosis and silicosis. Proc. Natl. Acad. Sci. USA 94: 6414–6419. action with ␣ integrins. FEBS Lett. 503: 75–79. 10. O’Regan, A. W., G. L. Chupp, J. A. Lowry, M. Goetschkes, N. Mulligan, and 4 33. Yokosaki, Y., N. Matsuura, T. Sasaki, I. Murakami, H. Schneider, J. S. Berman. 1999. Osteopontin is associated with T cells in sarcoid granulomas S. Higashiyama, Y. Saitoh, M. Yamakido, Y. Taooka, and D. Sheppard. 1999. and has T cell adhesive and cytokine-like properties in vitro. J. Immunol. 162: The integrin ␣ ␤ binds to a novel recognition sequence (SVVYGLR) in the 1024–1031. 9 1 thrombin-cleaved amino-terminal fragment of osteopontin. J. Biol. Chem. 274: 11. Sørensen, E. S., P. Højrup, and T. E. Petersen. 1995. Posttranslational modifi- 36328–36334. cations of bovine osteopontin: identification of twenty-eight phosphorylation and three O-glycosylation sites. Protein Sci. 4: 2040–2049. 34. Katagiri, Y. U., J. Sleeman, H. Fujii, P. Herrlich, H. Hotta, K. Tanaka, Downloaded from 12. Christensen, B., M. S. Nielsen, K. F. Haselmann, T. E. Petersen, and S. Chikuma, H. Yagita, K. Okumura, M. Murakami, et al. 1999. CD44 variants ␤ E. S. Sørensen. 2005. Posttranslationally modified residues of native human os- but not CD44s cooperate with 1-containing integrins to permit cells to bind to teopontin are located in clusters: identification of thirty-six phosphorylation and osteopontin independently of arginine-glycine-aspartic acid, thereby stimulating five O-glycosylation sites and their biological implications. Biochem. J. 390: cell motility and chemotaxis. Cancer Res. 59: 219–226. 285–292. 35. Weber, G. F., S. Ashkar, M. J. Glimcher, and H. Cantor. 1996. Receptor-ligand 13. Christensen, B., C. C. Kazanecki, T. E. Petersen, S. R. Rittling, D. T. Denhardt, interaction between CD44 and osteopontin (Eta-1). Science 271: 509–512. and E. S. Sørensen. 2007. Cell type-specific post-translational modifications of 36. Mi, Z., T. Oliver, H. Guo, C. Gao, and P. C. Kuo. 2007. Thrombin-cleaved COOHϪ terminal osteopontin peptide binds with cyclophilin C to CD147 in mouse osteopontin are associated with different adhesive properties. J. Biol. http://www.jimmunol.org/ Chem. 282: 19463–19472. murine breast cancer cells. Cancer Res. 67: 4088–4097. ␤ 14. Weiss, J. M., A. C. Renkl, C. S. Maier, M. Kimmig, L. Liaw, T. Ahrens, S. Kon, 37. Mayadas, T. N., and X. Cullere. 2005. Neutrophil 2 integrins: moderators of life M. Maeda, H. Hotta, T. Uede, and J. C. Simon. 2001. Osteopontin is involved in or death decisions. Trends Immunol. 26: 388–395. the initiation of cutaneous contact hypersensitivity by inducing Langerhans and 38. Elward, K., and P. Gasque. 2003. “Eat me” and “don’t eat me” signals govern the dendritic cell migration to lymph nodes. J. Exp. Med. 194: 1219–1229. innate immune response and tissue repair in the CNS: emphasis on the critical 15. Koh, A., A. P. da Silva, A. K. Bansal, M. Bansal, C. Sun, H. Lee, M. Glogauer, role of the complement system. Mol. Immunol. 40: 85–94. J. Sodek, and R. Zohar. 2007. Role of osteopontin in neutrophil function. Immu- 39. Stapulionis, R., C. L. Oliveira, M. C. Gjelstrup, J. S. Pedersen, M. E. Hokland, nology 122: 466–475. S. V. Hoffmann, K. Poulsen, C. Jacobsen, and T. Vorup-Jensen. 2008. Structural ␣ ␤ 16. Weber, G. F., S. Zawaideh, S. Hikita, V. A. Kumar, H. Cantor, and S. Ashkar. insight into the function of myelin basic protein as a ligand for integrin M 2. 2002. Phosphorylation-dependent interaction of osteopontin with its receptors J. Immunol. 180: 3946–3956. regulates macrophage migration and activation. J. Leukocyte Biol. 72: 752–761. 40. Vorup-Jensen, T., C. V. Carman, M. Shimaoka, P. Schuck, J. Svitel, and

17. Singh, R. P., R. Patarca, J. Schwartz, P. Singh, and H. Cantor. 1990. Definition T. A. Springer. 2005. Exposure of acidic residues as a danger signal for recog- by guest on September 26, 2021 of a specific interaction between the early T lymphocyte activation 1 (Eta-1) ␣ ␤ nition of fibrinogen and other macromolecules by integrin X 2. Proc. Natl. protein and murine macrophages in vitro and its effect upon macrophages in vivo. Acad. Sci. USA 102: 1614–1619. J. Exp. Med. 171: 1931–1942. 41. Kazanecki, C. C., D. J. Uzwiak, and D. T. Denhardt. 2007. Control of osteopontin 18. Giachelli, C. M., D. Lombardi, R. J. Johnson, C. E. Murry, and M. Almeida. signaling and function by post-translational phosphorylation and protein folding. 1998. Evidence for a role of osteopontin in macrophage infiltration in response to J. Cell. Biochem. 102: 912–924. pathological stimuli in vivo. Am. J. Pathol. 152: 353–358. 42. Sørensen, E. S., and T. E. Petersen. 1993. Purification and characterization of 19. Zhu, B., K. Suzuki, H. A. Goldberg, S. R. Rittling, D. T. Denhardt, three proteins isolated from the proteose peptone fraction of bovine milk. C. A. McCulloch, and J. Sodek. 2004. Osteopontin modulates CD44-dependent J. Dairy Res. 60: 189–197. chemotaxis of peritoneal macrophages through G-protein-coupled receptors: ev- 43. Lu, C., M. Ferzly, J. Takagi, and T. A. Springer. 2001. Epitope mapping of idence of a role for an intracellular form of osteopontin. J. Cell. Physiol. 198: antibodies to the C-terminal region of the integrin ␤ subunit reveals regions that 155–167. 2 become exposed upon receptor activation. J. Immunol. 166: 5629–5637. 20. Nystro¨m, T., P. Dune´r, and A. Hultgårdh-Nilsson. 2007. A constitutive endoge- 44. Petruzzelli, L., J. Luk, and T.A. Springer. 1995. Adhesion structure subpanel 5, nous osteopontin production is important for macrophage function and differen- leukocyte integrins: CD11a, CD11b, CD11c, CD18. In Leucocyte Typing V: tiation. Exp. Cell Res. 313: 1149–1160. White Cell Differentiation Antigens. S. F. Schlossman, L. Boumsell, W. Gilks, 21. Yamamoto, N., F. Sakai, S. Kon, J. Morimoto, C. Kimura, H. Yamazaki, J. Harlan, T. Kishimoto, T. Morimoto, J. Ritz, S. Shaw, R. Silverstein, I. Okazaki, N. Seki, T. Fujii, and T. Uede. 2003. Essential role of the cryptic T. Springer, et al., eds. Oxford Univ. Press, New York, pp. 1581–1585. epitope SLAYGLR within osteopontin in a murine model of rheumatoid arthritis. J. Clin. Invest. 112: 181–188. 45. Weetall, M., R. Hugo, C. Friedman, S. Maida, S. West, S. Wattanasin, R. Bouhel, 22. Panzer, U., F. Thaiss, G. Zahner, P. Barth, M. Reszka, R. R. Reinking, G. Wolf, G. Weitz-Schmidt, and P. Lake. 2001. A homogeneous fluorometric assay for U. Helmchen, and R. A. Stahl. 2001. Monocyte chemoattractant protein-1 and measuring cell adhesion to immobilized ligand using V-well microtiter plates. osteopontin differentially regulate monocytes recruitment in experimental glo- Anal. Biochem. 293: 277–287. merulonephritis. Kidney Int. 59: 1762–1769. 46. Robinson, M. K., D. Andrew, H. Rosen, D. Brown, S. Ortlepp, P. Stephens, and ␤ 23. Okada, H., K. Moriwaki, R. Kalluri, T. Takenaka, H. Imai, S. Ban, M. Takahama, E. C. Butcher. 1992. Antibody against the Leu-CAM -chain (CD18) promotes and H. Suzuki. 2000. Osteopontin expressed by renal tubular epithelium mediates both LFA-1- and CR3-dependent adhesion events. J. Immunol. 148: 1080–1085. interstitial monocyte infiltration in rats. Am. J. Physiol. 278: F110–F121. 47. Beletskii, A., M. Cooper, P. Sriraman, C. Chiriac, L. Zhao, S. Abbot, and L. Yu. 24. Burdo, T. H., M. R. Wood, and H. S. Fox. 2007. Osteopontin prevents monocyte 2005. High-throughput phagocytosis assay utilizing a pH-sensitive fluorescent recirculation and apoptosis. J. Leukocyte Biol. 81: 1504–1511. dye. Biotechniques 39: 894–897. 25. Liaw, L., D. E. Birk, C. B. Ballas, J. S. Whitsitt, J. M. Davidson, and B. L. Hogan. 48. Heilmann, K., U. Hoffmann, E. Witte, C. Loddenkemper, C. Sina, S. Schreiber, 1998. Altered wound healing in mice lacking a functional osteopontin gene C. Hayford, P. Holzlohner, K. Wolk, E. Tchatchou, et al. 2008. Osteopontin as (spp1). J. Clin. Invest. 101: 1468–1478. two-sided mediator of intestinal inflammation. J Cell Mol. Med. Epub ahead of 26. Pedraza, C. E., L. G. Nikolcheva, M. T. Kaartinen, J. E. Barralet, and print on July 8; DOI: 10.1111/j.1582-4934.2008.00428.x. M. D. McKee. 2008. Osteopontin functions as an opsonin and facilitates phago- 49. Marcondes, M. C., M. Poling, D. D. Watry, D. Hall, and H. S. Fox. 2008. In vivo cytosis by macrophages of hydroxyapatite-coated microspheres: implications for osteopontin-induced macrophage accumulation is dependent on CD44 expres- bone wound healing. Bone 43: 708–716. sion. Cell. Immunol. 254: 56–62.