A novel pathway of HMGB1-mediated inflammatory cell recruitment that requires Mac-1-integrin
2007; Springer Nature; Volume: 26; Issue: 4 Linguagem: Inglês
10.1038/sj.emboj.7601552
ISSN1460-2075
AutoresValeria V. Orlova, Eun Young Choi, Changping Xie, Emmanouil Chavakis, Angelika Bierhaus, Eveliina Ihanus, Christie M. Ballantyne, Carl G. Gahmberg, Marco E. Bianchi, Peter P. Nawroth, Triantafyllos Chavakis,
Tópico(s)Immune Response and Inflammation
ResumoArticle1 February 2007free access A novel pathway of HMGB1-mediated inflammatory cell recruitment that requires Mac-1-integrin Valeria V Orlova Valeria V Orlova Experimental Immunology Branch, NCI, NIH, Bethesda, MD, USA Search for more papers by this author Eun Young Choi Eun Young Choi Experimental Immunology Branch, NCI, NIH, Bethesda, MD, USA Search for more papers by this author Changping Xie Changping Xie Department of Internal Medicine I, University Heidelberg, Heidelberg, Germany Search for more papers by this author Emmanouil Chavakis Emmanouil Chavakis Molecular Cardiology, Department of Internal Medicine III, University of Frankfurt, Frankfurt, Germany Search for more papers by this author Angelika Bierhaus Angelika Bierhaus Department of Internal Medicine I, University Heidelberg, Heidelberg, Germany Search for more papers by this author Eveliina Ihanus Eveliina Ihanus Division of Biochemistry, Faculty of Biosciences, University of Helsinki, Finland Search for more papers by this author Christie M Ballantyne Christie M Ballantyne Section of Atherosclerosis and Lipoprotein Research, Department of Medicine, Baylor College of Medicine and Center for Cardiovascular Disease Prevention, Methodist DeBakey Heart Center, Houston, TX, USA Search for more papers by this author Carl G Gahmberg Carl G Gahmberg Division of Biochemistry, Faculty of Biosciences, University of Helsinki, Finland Search for more papers by this author Marco E Bianchi Marco E Bianchi Faculty of Medicine, San Raffaele University, Milano, Italy Search for more papers by this author Peter P Nawroth Peter P Nawroth Department of Internal Medicine I, University Heidelberg, Heidelberg, Germany Search for more papers by this author Triantafyllos Chavakis Corresponding Author Triantafyllos Chavakis Experimental Immunology Branch, NCI, NIH, Bethesda, MD, USA Search for more papers by this author Valeria V Orlova Valeria V Orlova Experimental Immunology Branch, NCI, NIH, Bethesda, MD, USA Search for more papers by this author Eun Young Choi Eun Young Choi Experimental Immunology Branch, NCI, NIH, Bethesda, MD, USA Search for more papers by this author Changping Xie Changping Xie Department of Internal Medicine I, University Heidelberg, Heidelberg, Germany Search for more papers by this author Emmanouil Chavakis Emmanouil Chavakis Molecular Cardiology, Department of Internal Medicine III, University of Frankfurt, Frankfurt, Germany Search for more papers by this author Angelika Bierhaus Angelika Bierhaus Department of Internal Medicine I, University Heidelberg, Heidelberg, Germany Search for more papers by this author Eveliina Ihanus Eveliina Ihanus Division of Biochemistry, Faculty of Biosciences, University of Helsinki, Finland Search for more papers by this author Christie M Ballantyne Christie M Ballantyne Section of Atherosclerosis and Lipoprotein Research, Department of Medicine, Baylor College of Medicine and Center for Cardiovascular Disease Prevention, Methodist DeBakey Heart Center, Houston, TX, USA Search for more papers by this author Carl G Gahmberg Carl G Gahmberg Division of Biochemistry, Faculty of Biosciences, University of Helsinki, Finland Search for more papers by this author Marco E Bianchi Marco E Bianchi Faculty of Medicine, San Raffaele University, Milano, Italy Search for more papers by this author Peter P Nawroth Peter P Nawroth Department of Internal Medicine I, University Heidelberg, Heidelberg, Germany Search for more papers by this author Triantafyllos Chavakis Corresponding Author Triantafyllos Chavakis Experimental Immunology Branch, NCI, NIH, Bethesda, MD, USA Search for more papers by this author Author Information Valeria V Orlova1, Eun Young Choi1, Changping Xie2, Emmanouil Chavakis3, Angelika Bierhaus2, Eveliina Ihanus4, Christie M Ballantyne5, Carl G Gahmberg4, Marco E Bianchi6, Peter P Nawroth2 and Triantafyllos Chavakis 1 1Experimental Immunology Branch, NCI, NIH, Bethesda, MD, USA 2Department of Internal Medicine I, University Heidelberg, Heidelberg, Germany 3Molecular Cardiology, Department of Internal Medicine III, University of Frankfurt, Frankfurt, Germany 4Division of Biochemistry, Faculty of Biosciences, University of Helsinki, Finland 5Section of Atherosclerosis and Lipoprotein Research, Department of Medicine, Baylor College of Medicine and Center for Cardiovascular Disease Prevention, Methodist DeBakey Heart Center, Houston, TX, USA 6Faculty of Medicine, San Raffaele University, Milano, Italy *Corresponding author. Experimental Immunology Branch, NCI, NIH, 10 Center Drive, Rm 4B17, Bethesda, MD 20892, USA. Tel.: +1 301 451 2104; Fax: +1 301 496 0887; E-mail: [email protected] The EMBO Journal (2007)26:1129-1139https://doi.org/10.1038/sj.emboj.7601552 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info High-mobility group box 1 (HMGB1) is released extracellularly upon cell necrosis acting as a mediator in tissue injury and inflammation. However, the molecular mechanisms for the proinflammatory effect of HMGB1 are poorly understood. Here, we define a novel function of HMGB1 in promoting Mac-1-dependent neutrophil recruitment. HMGB1 administration induced rapid neutrophil recruitment in vivo. HMGB1-mediated recruitment was prevented in mice deficient in the β2-integrin Mac-1 but not in those deficient in LFA-1. As observed by bone marrow chimera experiments, Mac-1-dependent neutrophil recruitment induced by HMGB1 required the presence of receptor for advanced glycation end products (RAGE) on neutrophils but not on endothelial cells. In vitro, HMGB1 enhanced the interaction between Mac-1 and RAGE. Consistently, HMGB1 activated Mac-1 as well as Mac-1-mediated adhesive and migratory functions of neutrophils in a RAGE-dependent manner. Moreover, HMGB1-induced activation of nuclear factor-κB in neutrophils required both Mac-1 and RAGE. Together, a novel HMGB1-dependent pathway for inflammatory cell recruitment and activation that requires the functional interplay between Mac-1 and RAGE is described here. Introduction Leukocyte recruitment as an integral part of inflammatory processes requires multistep adhesive and signaling events including selectin-dependent rolling, chemokine-dependent leukocyte activation, and integrin-mediated firm adhesion and diapedesis (Springer, 1994). During firm endothelial adhesion of leukocytes, leukocyte β2-integrins, LFA-1 (αLβ2, CD11a/CD18), Mac-1 (αMβ2, CD11b/CD18), and p150,95 (αXβ2, CD11c/CD18), as well as β1-integrins interact with endothelial counterligands such as ICAM-1, surface-associated fibrinogen (FBG) or VCAM-1 (Gahmberg, 1997; Plow et al, 2000; Hogg et al, 2003). Among leukocyte integrins, Mac-1 plays an important role in innate immunity, as it may regulate inflammatory cell recruitment as well as pathogen recognition, phagocytosis, and neutrophil survival (Ehlers, 2000; Mayadas and Cullere, 2005). Interestingly, Mac-1 ligation on leukocytes may lead to activation of nuclear factor-κB (NF-κB) (Sitrin et al, 1998) and the activation of the consequent gene expression (Rezzonico et al, 2001), although the underlying mechanisms are poorly understood. The role of Mac-1 in innate immunity is in line with its propensity to be a highly versatile multiligand receptor (Ehlers, 2000) interacting with numerous ligands and counter-receptors. In addition, the functions of Mac-1 may be regulated by interactions in cis, that is, on the same leukocyte surface with other receptors, such as the FcγRIII or the urokinase receptor (Zhou et al, 1993; Tang et al, 1997; Petty et al, 2002; Mayadas and Cullere, 2005). Interestingly, although Mac-1 is notorious for interacting in trans with different cellular counter-receptors or matrix proteins, only a few membrane partners of Mac-1 in cis are identified that may regulate its activity (Ehlers, 2000; Petty et al, 2002). High-mobility group box 1 (HMGB1), also named amphoterin, is a nuclear protein loosely bound to DNA that stabilizes nucleosome formation and regulates transcription (Dumitriu et al, 2005b; Lotze and Tracey, 2005). Emerging evidence has demonstrated an important role for extracellular HMGB1 as a very potent inflammatory mediator (Wang et al, 1999; Scaffidi et al, 2002; Dumitriu et al, 2005b; Lotze and Tracey, 2005). HMGB1 can be secreted into the extracellular space by activated macrophages and mature dendritic cells by an active process that may require the acetylation of the molecule in the nucleus (Bonaldi et al, 2003). Alternatively, HMGB1 is passively released by necrotic, but not apoptotic cells (Scaffidi et al, 2002), thereby representing a signal for tissue damage. Extracellular HMGB1 may interact with toll-like receptors (TLR) and/or RAGE (receptor for advanced glycation end products) (Dumitriu et al, 2005b; Lotze and Tracey, 2005). In particular, an interaction between HMGB1 and TLR-2 or TLR-4 has been demonstrated that may mediate the proinflammatory actions of HMGB1 (Park et al, 2004, 2006). On the other hand, RAGE is a multiligand receptor on vascular cells that plays a key role in inflammatory processes, especially at sites where its ligands accumulate (Schmidt et al, 2001; Chavakis et al, 2004). RAGE ligation may activate a range of signaling pathways including MAP kinases, rho GTPases, as well as activation of NF-κB (Schmidt et al, 2001; Yan et al, 2003). Recently, we established that endothelial RAGE interacts also with Mac-1 on leukocytes (Chavakis et al, 2003). Extracellular HMGB1 evokes a strong inflammatory response; it stimulates the release of multiple proinflammatory cytokines such as tumor necrosis factor (TNF) and interleukins in macrophages and neutrophils (Andersson et al, 2000) and induces the expression of adhesion molecules on endothelial cells, such as VCAM-1 and selectins, as well as it enhances dendritic cell maturation (Dumitriu et al, 2005a, 2005b; Lotze and Tracey, 2005). Robust leukocyte recruitment is a prominent hallmark associated with HMGB1-mediated inflammation (Dumitriu et al, 2005b; Lotze and Tracey, 2005). As observed in studies that entailed HMGB1 blockade in vivo, HMGB1 is important in the pathogenesis of sepsis (Wang et al, 1999; Yang et al, 2000), as well as in arthritis (Yang et al, 2005). Recent studies also indicated that HMGB1 may mediate inflammatory cell recruitment in acute hepatic necrosis (Tsung et al, 2005) and in acute lung injury (Kim et al, 2005; Lin et al, 2005). However, the molecular mechanisms underlying this proinflammatory function of HMGB1 remain to be clarified. In particular, it is not established yet whether HMGB1 affects extravasation-related functions of leukocytes, such as adhesion and migration. Here, we identify a novel pathway for HMGB1-mediated neutrophil recruitment that requires the functional interplay between RAGE and the β2-integrin Mac-1. Results HMGB1-mediated neutrophil recruitment in vivo requires Mac-1 We first studied whether HMGB1 administration can elicit rapid inflammatory cell recruitment in vivo. Interestingly, intraperitoneally (i.p.) injection of HMGB1 resulted in a rapid (4 h) recruitment of leukocytes (mostly neutrophils) into the peritoneum (Figure 1A). As a comparison, we studied thioglycollate-induced peritonitis (Figure 1B) (Chavakis et al, 2002, 2003). The HMGB1-mediated effect on neutrophil recruitment was reduced in RAGE-deficient mice (Figure 1A). In addition, HMGB1-induced neutrophil emigration to the peritoneum was blocked by systemic pretreatment of wild-type mice with soluble RAGE 1 h before HMGB1 injection (data not shown). Whereas thioglycollate-induced neutrophil infiltration was blocked by blocking monoclonal antibody (mAb) against LFA-1 and, to a less extent, by blocking mAb against Mac-1, HMGB1-mediated neutrophil emigration to the peritoneum was only blocked by blocking mAb to Mac-1, but not affected by antibody against LFA-1 (Figure 1B). To define further the underlying mechanisms of HMGB1-mediated neutrophil extravasation, we engaged mice deficient in Mac-1 or LFA-1. Consistent with the antibody inhibition studies, HMGB1-induced neutrophil emigration was decreased in Mac-1-deficient mice but not in LFA-1-deficient mice (Figure 1C). In contrast, thioglycollate-induced peritonitis was prevented in LFA-1-deficient mice (Figure 1D), consistent with previous reports showing an important role of LFA-1 in thioglycollate-induced peritonitis (Coxon et al, 1996; Lu et al, 1997; Berlin-Rufenach et al, 1999; Ding et al, 1999). Together, these findings suggest that HMGB1 stimulates neutrophil recruitment in vivo and that this process requires Mac-1 as well as RAGE. Figure 1.HMGB1-mediated inflammatory cell recruitment in vivo. (A) The number of neutrophils in wild-type (open bars) or RAGE−/− (filled bars) mice is shown 4 h after the i.p. injection of buffer (−) or HMGB1 (10 μg). (B) Sixty minutes before thioglycollate (open bars) or HMGB1 (filled bars) administration, wild-type mice were treated with isotype control mAb, with a blocking mAb against LFA-1 or with a blocking mAb against Mac-1 (each 100 μg). (C) HMGB1 induced peritonitis in wild-type, Mac-1−/−, and LFA-1−/− mice. (D) Thioglycollate induced peritonitis in wild-type, Mac-1−/−, and LFA-1−/− mice. (E) HMGB1 induced peritonitis in sublethally irradiated wild-type mice reconstituted with bone marrow cells from wild-type mice (wt → wt), sublethally irradiated wild-type mice reconstituted with bone marrow cells from RAGE−/− mice (RAGE−/− → wt) and sublethally irradiated RAGE−/−mice reconstituted with bone marrow cells from wild type (wt → RAGE−/−). Data are expressed as absolute numbers of emigrated neutrophils into the peritoneum. *P<0.01; #P<0.05; ns: not significant. Data are mean±s.d. (n=3–6 mice/group). Download figure Download PowerPoint RAGE is expressed on both endothelial cells and hematopoietic cells including neutrophils (Collison et al, 2002; Yan et al, 2003). Endothelial RAGE can interact with leukocyte Mac-1 in trans (Supplementary Figure 1, and Chavakis et al, 2003). To differentiate whether endothelial- or neutrophil-associated RAGE was required for the activity of HMGB1 to stimulate inflammatory cell recruitment in vivo, we performed bone marrow transplantation experiments. In particular, wild-type mice received irradiation and were then reconstituted with bone marrow from either wild-type or RAGE-deficient mice (wt → wt and RAGE−/− → wt, respectively). In the reverse experiment, irradiated RAGE−/− mice were reconstituted with bone marrow from wild-type mice (wt → RAGE−/−). Interestingly, the decrease in HMGB1-induced neutrophil extravasation observed in RAGE−/− mice as compared to wild-type mice (Figure 1A) could be reversed in the wt → RAGE−/− group, that is, by restoring the expression of RAGE on neutrophils (Figure 1E). In contrast, HMGB1-induced neutrophil recruitment into the peritoneum was prevented in the RAGE−/− → wt group as compared to the wt → wt group (Figure 1E). The degree of reduction in HMGB1-induced neutrophil recruitment into the peritoneum owing to the hematopoietic-specific absence of RAGE was comparable to the degree of decrease of HMGB1-induced neutrophil emigration in RAGE-deficient mice (Figure 1A). Taken together, these results indicate that Mac-1 and neutrophil RAGE but not endothelial RAGE are required for the HMGB1-induced recruitment of neutrophils in vivo. HMGB1 stimulates Mac-1-dependent leukocyte adhesion As these observations suggested a role for HMGB1 in Mac-1-dependent leukocyte extravasation, we studied whether HMGB1 can affect neutrophil adhesion. Interestingly, Mac-1-dependent neutrophil adhesion to FBG was stimulated three-fold by HMGB1. Whereas adhesion of RAGE-deficient neutrophils to FBG was comparable to the adhesion of wild-type neutrophils, the stimulatory effect of HMGB1 on Mac-1-dependent neutrophil adhesion to FBG was abolished in the absence of RAGE (Figure 2A). Additionally, HMGB1 induced spreading of wild-type but not RAGE-deficient neutrophils on FBG (Figure 2B). In contrast, PMA-induced adhesion and spreading of neutrophils to FBG was not affected by RAGE deficiency (not shown). Moreover, Mac-1−/− neutrophils failed to adhere to FBG and HMGB1 did not stimulate the adhesion of Mac-1−/− neutrophils to FBG, whereas HMGB1 enhanced the FBG adhesion of LFA-1−/− neutrophils (Figure 2C). Thus, HMGB1 stimulates the Mac-1-dependent adhesion of neutrophils to FBG in a RAGE-dependent manner. Figure 2.HMGB1-stimulated adhesion of mouse neutrophils. (A) Adhesion of wild-type or RAGE−/− neutrophils to immobilized FBG in the absence (open bars) or presence of HMGB1 (filled bars, 100 ng/ml) is shown without (−) or with mAb to LFA-1 or mAb to Mac-1 (each at 20 μg/ml). Cell adhesion is represented as number of adherent cells. (B) Spreading of wild-type or RAGE−/− neutrophils on immobilized FBG in the absence (open bars) or presence of HMGB1 (filled bars, 100 ng/ml). Data are represented as % spread cells. (C) Adhesion of wild-type, Mac-1−/−, or LFA-1−/− neutrophils to immobilized FBG in the absence (open bars) or presence of HMGB1 (filled bars, 100 ng/ml) is shown. Cell adhesion is represented as number of adherent cells. In (A), (B), and (C), *P<0.01; ns: not significant. (D) Adhesion of wild-type, Mac-1−/−, or LFA-1−/− neutrophils to immobilized ICAM-1 is shown in the absence (open bars) or presence of HMGB1 (100 ng/ml, filled bars) without (−) or with mAb to Mac-1, mAb to LFA-1, or soluble RAGE (each at 20 μg/ml). Cell adhesion is represented as number of adherent cells. In (D), *P<0.01; #P<0.05; ns: not significant; +P<0.05 as compared to adhesion in the absence of HMGB1 (open bars) and in the absence of competitors (−); ns1: not significant as compared to adhesion in the absence of HMGB1 (open bars) and in the absence of competitors (−); &P<0.01 as compared to adhesion in the presence of HMGB1 (filled bars) and in the absence of competitors (−); ns2: not significant as compared to adhesion in the presence of HMGB1 (filled bars) and in the absence of competitors (−). Data are mean±s.d. of three independent experiments each performed in triplicate. Download figure Download PowerPoint Next, the effect of HMGB1 on the adhesion of neutrophils to immobilized ICAM-1, the major endothelial counter-receptor of leukocyte β2-integrins, was studied. Neutrophil adhesion to ICAM-1 is mediated by both Mac-1 and LFA-1 (Gahmberg, 1997; Hogg et al, 2003), and in the absence of HMGB1 neutrophil adhesion to ICAM-1 was blocked by inhibitory mAb to either Mac-1 or LFA-1 (Figure 2D). Both Mac-1- and LFA-1-deficient neutrophils displayed decreased adhesion to immobilized ICAM-1 (Figure 2C). HMGB1 increased adhesion of wild-type neutrophils to ICAM-1 and the HMGB1-induced effect was absent in RAGE−/− neutrophils (not shown). Moreover, the HMGB1-induced upregulation of ICAM-1 adhesion was mediated by Mac-1 but not LFA-1, as evidenced by the following observations: (i) ICAM-1 adhesion of wild-type neutrophils in the presence of HMGB1 was prevented by inhibitory mAb to Mac-1 but was not affected by inhibitory mAb to LFA-1. (ii) The HMGB1-induced stimulation of neutrophil adhesion to ICAM-1 was absent in Mac-1-deficient neutrophils. In contrast, HMGB1 increased adhesion of LFA-1-deficient neutrophils to ICAM-1. This HMGB1-induced increase in ICAM-1 adhesion of LFA-1-deficient neutrophils was prevented by inhibitory mAb to Mac-1 but not mAb to LFA-1 (Figure 2D). Moreover, although soluble RAGE did not affect the adhesion of neutrophils to ICAM-1 in the absence of HMGB1, it blocked the HMGB1-induced upregulation of wild-type or LFA-1-deficient neutrophils (Figure 2D). Furthermore, the effect of HMGB1 to stimulate Mac-1-dependent adhesion of neutrophils to FBG or ICAM-1 was dose-dependent (1–250 ng/ml; data not shown). HMGB1 was active in stimulating Mac-1-dependent adhesion independent of whether it was pre-incubated with the neutrophils and then washed away before the adhesion assay or whether it was co-incubated with the neutrophils during the course of the adhesion assay, indicating that HMGB1 primary acts on the neutrophils. Finally, adhesion of wild-type neutrophils to immobilized fibronectin (FN), which is mediated by VLA-4, was not stimulated by HMGB1 (data not shown). Our data indicate that HMGB1 activates Mac-1 in a RAGE-dependent manner. Studies on Mac-1 activation are more feasible to perform with human Mac-1 owing to the availability of both purified human Mac-1 as well as of well-characterized antibodies against human Mac-1. First, we investigated whether HMGB1 also stimulates Mac-1-dependent adhesion of human leukocytes. Consistent with the data obtained from mouse neutrophils, HMGB1 stimulated the Mac-1-dependent adhesion of myelomonocytic THP-1 cells to immobilized FBG or ICAM-1 by three-fold (Figure 3A and B). The effect of HMGB1 was also dose-dependent (1–250 ng/ml; data not shown). HMGB1-induced Mac-1-dependent adhesion of THP-1 cells to FBG or ICAM-1 was prevented in the presence of antibody to HMGB1 or soluble RAGE. In addition, the HMGB1-induced increase of ICAM-1 adhesion of THP-1 was prevented by inhibitory mAb to Mac-1 but was not affected by inhibitory mAb to LFA-1 (Figure 3B). In contrast, PMA or monocyte chemoattractant protein-1 (MCP-1)- stimulated adhesion of THP1 cells to ICAM-1 was blocked by both antibodies to Mac-1 and LFA-1 (data not shown). Adhesion of THP-1 cells to FN was predominantly mediated by VLA-4 and was not affected by the presence of HMGB1, soluble RAGE or antibody to HMGB1 (Figure 3C). Similar results were also obtained in experiments performed with human neutrophils isolated from peripheral blood (Supplementary Figure 2). These experiments indicate that HMGB1 stimulates Mac-1-dependent adhesive events in human leukocytes in a RAGE-dependent manner. Figure 3.HMGB1-mediated adhesion of human leukocytes. (A, B) Adhesion of THP1 cells to immobilized FBG (A) or immobilized ICAM-1 (B) is shown in the absence (open bars) or presence of HMGB1 (100 ng/ml, filled bars), without (−) or with mAb to CD29, mAb to Mac-1, mAb to LFA-1, antibody to HMGB1 or soluble RAGE (each at 20 μg/ml). (C) Adhesion of THP-1 cells to immobilized FN is shown in the absence (open bars) or presence of HMGB1 (100 ng/ml, filled bars) without (−) or with mAb to CD29, mAb to Mac-1, antibody to HMGB1 or soluble RAGE (each at 20 μg/ml). Cell adhesion is represented as number of adherent cells. *P<0.01; ns: not significant; +P<0.05 as compared to adhesion in the absence of HMGB1 (open bars) and in the absence of competitors (−); ns1: not significant as compared to adhesion in the absence of HMGB1 (open bars) and in the absence of competitors (−); &P<0.01 as compared to adhesion in the presence of HMGB1 (filled bars) and in the absence of competitors (−); ns2: not significant as compared to adhesion in the presence of HMGB1 (filled bars) and in the absence of competitors (−). Data are mean±s.d. of three independent experiments each performed in triplicate. Download figure Download PowerPoint Chemotactic activity of HMGB1 on neutrophils To assess further HMGB1 as a pro-adhesive/pro-chemotactic factor, we studied whether HMGB1 stimulates lamellipodium formation. Similar to MCP-1 (not shown and Cambien et al, 2001), HMGB1 induced the polarization of THP-1 cells adhering onto FBG. This shape change corresponded with the enrichment of F-actin at the leading edge, indicating that HMGB1-induced lamellipodium formation in these cells. Moreover, consistent with previous reports (Mocsai et al, 2002; Schymeinsky et al, 2005), the non-receptor protein tyrosine kinase syk was also redistributed at the site of lamellipodium formation and colocalized with F-actin upon stimulation with HMGB1 (Figure 4A). Thus, HMGB1 resembles chemotactic factors in that it induces lamellipodium formation. Figure 4.HMGB1-mediated chemotaxis and transendothelial migration of leukocytes. (A) Immunofluorescence for Syk (green) or F-actin (red) was performed followed by confocal microscopy. Representative immunofluorescence of THP-1 cells that were incubated in the absence (−) or presence of HMGB1 (50 ng/ml) for 30 min is shown. Double-stained images were merged. HMGB1 induced the enrichment of F-actin and Syk staining at the leading edge of the cell. (B) Chemotaxis of wild-type mouse neutrophils towards no chemoattractant (open bar), 50 ng/ml MIP-2 (gray bar) or 50 ng/ml HMGB1 (filled bar) is shown. (C) Chemotaxis of wild-type, LFA-1−/−, Mac-1−/−, and RAGE−/− mouse neutrophils towards no chemoattractant (open bars), or 50 ng/ml HMGB1 (filled bars) is shown. (D) Chemotaxis of human neutrophils towards no chemoattractant (open bar), 50 ng/ml IL-8 (gray bar) or 50 ng/ml HMGB1 (filled bars) is shown in the absence (−) or presence of blocking mAb to Mac-1, mAb to LFA-1, soluble RAGE, or antibody to HMGB1 (each at 20 μg/ml). In (B), (C), and (D), chemotaxis data are shown as percent of control. In (B) and (C), chemotaxis of wild-type mouse neutrophils in the absence of stimuli or competitors represents the 100% control; in (D) chemotaxis of human neutrophils in the absence of stimuli or competitors represents the 100% control. *P<0.02; ns: not significant. (E) The transmigration of human neutrophils towards 50 ng/ml IL-8 (gray bars) or 50 ng/ml HMGB1 (filled bars) across HUVEC is shown in the absence (−) or presence of blocking mAb to Mac-1, mAb to LFA-1, soluble RAGE, or antibody to HMGB1 (each at 20 μg/ml). Transmigration is represented as percent of control. Transmigration through HUVEC in the absence of stimuli or competitors represents the 100% control. +P<0.01 as compared to transmigration towards IL-8 (gray bars) and in the absence of competitors (−); ns1: not significant as compared to transmigration towards IL-8 (gray bars) and in the absence of competitors (−); &P<0.01 as compared to transmigration towards HMGB1 (filled bars) and in the absence of competitors (−); ns2: not significant as compared to transmigration towards HMGB1 (filled bars) and in the absence of competitors (−). Data are mean±s.d. of three independent experiments each performed in triplicate. Download figure Download PowerPoint In addition, HMGB1 induced chemotaxis of mouse and human neutrophils. The chemotactic effect of HMGB1 on mouse and human neutrophils was comparable to the effects of MIP-2 and IL-8 on mouse and human neutrophils, respectively (Figure 4B and D). The chemotactic effect of HMGB1 on neutrophils required both Mac-1 and RAGE, as HMGB1 failed to induce chemotaxis of Mac-1−/− and RAGE−/− neutrophils (Figure 4C). Consistently, HMGB1-induced chemotaxis of human neutrophils was blocked by soluble RAGE and an inhibitory mAb to Mac-1 but not by inhibitory mAb to LFA-1 (Figure 4D). As β2-integrins and Mac-1 also play an important role in neutrophil transendothelial migration, we then investigated whether HMGB1 might affect this process. In a transwell assay, HMGB1 significantly stimulated the transmigration of human neutrophils through a monolayer of human umbilical vein endothelial cells (HUVEC) and this effect was blocked by mAb against Mac-1 but not by mAb against LFA-1. In addition, HMGB1-induced transendothelial migration of neutrophils was inhibited by soluble RAGE or antibody to HMGB1. In contrast, IL-8-stimulated transmigration through cultured endothelial cells was blocked by mAb to Mac-1 as well as mAb to LFA-1, and was not affected by soluble RAGE or antibody to HMGB1 (Figure 4E). Similar results were obtained with THP-1 cells (not shown). Thus, HMGB1 exerts a chemotactic activity on neutrophils, inducing Mac-1-mediated chemotaxis and transendothelial migration in a RAGE-dependent manner. HMGB1 increases Mac-1 activity in a RAGE-dependent manner Our data so far suggested that HMGB1 stimulates Mac-1-mediated adhesiveness in a RAGE-dependent manner. Activation of integrin-mediated adhesiveness takes place at the level of avidity or valency (receptor density on the adhesive surface) as well as at the level of affinity for the individual ligand (Carman and Springer, 2003). We found that HMGB1-induced adhesion and spreading of THP-1 cells onto FBG-coated slides was associated with the polarization of Mac-1 to the leading edge of the THP-1 cells, as opposed to the diffuse staining on the cell surface in non-stimulated cells. In addition, we observed a strong colocalization of RAGE with Mac-1 at the leading edge of spreading THP-1 cells upon HMGB1 stimulation (Figure 5A). Figure 5.HMGB1-dependent activation of Mac-1. (A) Immunofluorescence for Mac-1 and RAGE was performed followed by confocal microscopy. Representative immunofluorescence of THP-1 cells that were incubated in the absence (−) or presence of HMGB1 (100 ng/ml) on FBG for 30 min is shown. Double-stained images were merged. (B) Human neutrophils were incubated in the absence (black curves) or presence of HMGB1 (100 ng/ml, red curves) for 20 min. Surface expression of an activation-dependent epitope (CBRM1/5) on Mac-1 was quantitated by FACS analysis. For comparison, quantitative cell surface expression of Mac-1 was analyzed using an antibody, which recognizes an epitope irrespective of the activation state of the integrin. Nonspecific fluorescence was determined using isotype-matched mouse-IgG (dotted thin curves). Download figure Download PowerPoint Increases in the affinity of integrins are associated with conformational changes leading to increased exposure of activation-dependent epitopes on the integrin (Carman and Springer, 2003; Hogg et al, 2003). Activation of Mac-1 on the cell surface can be measured by the binding of the mAb CBRM1/5 that recogniz
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