MFG-E8 inhibits neutrophil migration through αvβ3-integrin-dependent MAP kinase activation

Aziz,Monowar; Yang,Weng‑Lang; Corbo,Lana  M.; Chaung,Wayne  W.; Matsuo,Shingo; Wang,Ping

doi:10.3892/ijmm.2015.2196

MFG-E8 inhibits neutrophil migration through αvβ3-integrin-dependent MAP kinase activation

Authors:
- Monowar Aziz
- Weng‑Lang Yang
- Lana M. Corbo
- Wayne W. Chaung
- Shingo Matsuo
- Ping Wang
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Affiliations: Center for Translational Research, The Feinstein Institute for Medical Research and Department of Surgery, Hofstra North Shore‑LIJ School of Medicine, Manhasset, NY, USA
Published online on: April 23, 2015 https://doi.org/10.3892/ijmm.2015.2196
Pages: 18-28
Copyright: © Aziz et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].

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Abstract

We have previously demonstrated the involvement of milk fat globule-epidermal growth factor-factor 8 (MFG‑E8) in reducing neutrophil infiltration in a murine model of acute lung injury (ALI). In the present study, we aimed to delineate the mechanisms through which MFG‑E8 attenuates neutrophil migration. Recombinant human MFG‑E8 (rhMFG‑E8) was expressed and purified in our facility. The human differentiated neutrophil cell line, dHL‑60, was treated with rhMFG‑E8 and cell migration assay was performed in a Boyden chamber using recombinant interleukin‑8 (IL‑8) as the chemoattractant. Surface CXCR2 and intracellular G protein‑coupled receptor kinase 2 (GRK2) levels were evaluated by flow cytometry or western blot analysis. The levels of mitogen‑activated protein (MAP) kinases were determined by western blot analysis. Treatment with rhMFG‑E8 resulted in a significant inhibition of dHL‑60 cell migration in a dose‑dependent manner. There was a 46% decrease in CXCR2 expression in the rhMFG‑E8‑treated dHL‑60 cells, which was associated with a 32% increase in GRK2 expression. In the dHL‑60 cells, treatment with rhMFG‑E8 promoted the phosphorylation of p38 and extracellular signal-regulated kinase (ERK) within 10‑30 min. The use of SB203580, a p38 inhibitor, and PD98059, an ERK inhibitor, resulted in the restoration of dHL‑60 cell migration which was significantly inhibited treatment with rhMFG‑E8. Furthermore, blocking the MFG‑E8 receptors, αvβ3/αvβ5‑integrins, by anti‑αv‑integrin neutralizing antibody (Ab) inhibited the activation of p38 and ERK, and reversed the rhMFG‑E8‑induced inhibition of dHL‑60 cell migration. Finally, treatment of the dHL‑60 cells with SB203580 and PD98059 neutralized the rhMFG‑E8‑induced downregulation of CXCR2 expression and upregulation of GRK2 expression, as well as the inhibitory effects on cell migration. Our findings reveal a novel mechanism of action of MFG‑E8 through which it inhibits neutrophil migration through αvβ3-integrin-dependent MAP kinase activation.

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1	Nathan C: Neutrophils and immunity: Challenges and opportunities. Nat Rev Immunol. 6:173–182. 2006. View Article : Google Scholar : PubMed/NCBI
2	Rot A and von Andrian UH: Chemokines in innate and adaptive host defense: basic chemokinese grammar for immune cells. Annu Rev Immunol. 22:891–928. 2004. View Article : Google Scholar : PubMed/NCBI
3	Etzioni A and Tonetti M: Leukocyte adhesion deficiency II-from A to almost Z. Immunol Rev. 178:138–147. 2000. View Article : Google Scholar
4	Webb PR, Wang KQ, Scheel-Toellner D, Pongracz J, Salmon M and Lord JM: Regulation of neutrophil apoptosis: a role for protein kinase C and phosphatidylinositol-3-kinase. Apoptosis. 5:451–458. 2000. View Article : Google Scholar
5	Ayala A, Chung CS, Lomas JL, Song GY, Doughty LA, Gregory SH, Cioffi WG, LeBlanc BW, Reichner J, Simms HH and Grutkoski PS: Shock-induced neutrophil mediated priming for acute lung injury in mice: divergent effects of TLR-4 and TLR-4/FasL deficiency. Am J Pathol. 161:2283–2294. 2002. View Article : Google Scholar : PubMed/NCBI
6	Abraham E: Neutrophils and acute lung injury. Crit Care Med. 31(Suppl): S195–S199. 2003. View Article : Google Scholar : PubMed/NCBI
7	Aziz M, Jacob A, Yang W-L, Matsuda A and Wang P: Current trends in inflammatory and immunomodulatory mediators in sepsis. J Leukoc Biol. 93:329–342. 2013. View Article : Google Scholar :
8	Aziz M, Matsuda A, Yang W-L, Jacob A and Wang P: Milk fat globule-epidermal growth factor-factor 8 attenuates neutrophil infiltration in acute lung injury via modulation of CXCR2. J Immunol. 189:393–402. 2012. View Article : Google Scholar : PubMed/NCBI
9	Cui T, Miksa M, Wu R, Komura H, Zhou M, Dong W, Wang Z, Higuchi S, Chaung W, Blau SA, et al: Milk fat globule epidermal growth factor 8 attenuates acute lung injury in mice after intestinal ischemia and reperfusion. Am J Respir Crit Care Med. 181:238–246. 2010. View Article : Google Scholar :
10	Wagner JG and Roth RA: Neutrophil migration mechanisms, with an emphasis on the pulmonary vasculature. Pharmacol Rev. 52:349–374. 2000.PubMed/NCBI
11	Lee WL and Downey GP: Neutrophil activation and acute lung injury. Curr Opin Crit Care. 7:1–7. 2001. View Article : Google Scholar : PubMed/NCBI
12	Zarbock A and Ley K: Mechanisms and consequences of neutrophil interaction with the endothelium. Am J Pathol. 172:1–7. 2008. View Article : Google Scholar :
13	Donnelly SC, Strieter RM, Kunkel SL, Walz A, Robertson CR, Carter DC, Grant IS, Pollok AJ and Haslett C: Interleukin-8 and development of adult respiratory distress syndrome in at-risk patient groups. Lancet. 341:643–647. 1993. View Article : Google Scholar : PubMed/NCBI
14	Goodman RB, Strieter RM, Martin DP, Steinberg KP, Milberg JA, Maunder RJ, Kunkel SL, Walz A, Hudson LD and Martin TR: Inflammatory cytokines in patients with persistence of the acute respiratory distress syndrome. Am J Respir Crit Care Med. 154:602–611. 1996. View Article : Google Scholar : PubMed/NCBI
15	Olson TS and Ley K: Chemokines and chemokine receptors in leukocyte trafficking. Am J Physiol Regul Integr Comp Physiol. 283:R7–R28. 2002. View Article : Google Scholar : PubMed/NCBI
16	Reutershan J, Morris MA, Burcin TL, Smith DF, Chang D, Saprito MS and Ley K: Critical role of endothelial CXCR2 in LPS-induced neutrophil migration into the lung. J Clin Invest. 116:695–702. 2006. View Article : Google Scholar : PubMed/NCBI
17	Murphy PM: Neutrophil receptors for interleukin-8 and related CXC chemokines. Semin Hematol. 34:311–318. 1997.PubMed/NCBI
18	Johnston RA, Mizgerd JP and Shore SA: CXCR2 is essential for maximal neutrophil recruitment and methacholine responsiveness after ozone exposure. Am J Physiol Lung Cell Mol Physiol. 288:L61–L67. 2005. View Article : Google Scholar
19	Vroon A, Heijnen CJ and Kavelaars A: GRKs and arrestins: Regulators of migration and inflammation. J Leukoc Biol. 80:1214–1221. 2006. View Article : Google Scholar : PubMed/NCBI
20	Chuang TT, Iacovelli L, Sallese M and De Blasi A: G protein- coupled receptors: Heterologous regulation of homologous desensitization and its implications. Trends Pharmacol Sci. 17:416–421. 1996. View Article : Google Scholar : PubMed/NCBI
21	Aragay AM, Ruiz-Gómez A, Penela P, Sarnago S, Elorza A, Jiménez-Sainz MC and Mayor F Jr: G protein-coupled receptor kinase 2 (GRK2): mechanisms of regulation and physiological functions. FEBS Lett. 430:37–40. 1998. View Article : Google Scholar : PubMed/NCBI
22	Penn RB, Pronin AN and Benovic JL: Regulation of G protein-coupled receptor kinases. Trends Cardiovasc Med. 10:81–89. 2000. View Article : Google Scholar
23	Penela P, Ribas C and Mayor F Jr: Mechanisms of regulation of the expression and function of G protein-coupled receptor kinases. Cell Signal. 15:973–981. 2003. View Article : Google Scholar : PubMed/NCBI
24	Pitcher JA, Tesmer JJ, Freeman JL, Capel WD, Stone WC and Lefkowitz RJ: Feedback inhibition of G protein-coupled receptor kinase 2 (GRK2) activity by extracellular signal-regulated kinases. J Biol Chem. 274:34531–34534. 1999. View Article : Google Scholar : PubMed/NCBI
25	Liu X, Ma B, Malik AB, Tang H, Yang T, Sun B, Wang G, Minshall RD, Li Y, Zhao Y, et al: Bidirectional regulation of neutrophil migration by mitogen-activated protein kinases. Nat Immunol. 13:457–464. 2012. View Article : Google Scholar : PubMed/NCBI
26	Hanayama R, Tanaka M, Miwa K, Shinohara A, Iwamatsu A and Nagata S: Identification of a factor that links apoptotic cells to phagocytes. Nature. 417:182–187. 2002. View Article : Google Scholar : PubMed/NCBI
27	Hanayama R, Tanaka M, Miyasaka K, Aozasa K, Koike M, Uchiyama Y and Nagata S: Autoimmune disease and impaired uptake of apoptotic cells in MFG-E8-deficient mice. Science. 304:1147–1150. 2004. View Article : Google Scholar : PubMed/NCBI
28	Ensslin MA and Shur BD: Identification of mouse sperm SED1, a bimotif EGF repeat and discoidin-domain protein involved in sperm-egg binding. Cell. 114:405–417. 2003. View Article : Google Scholar : PubMed/NCBI
29	Hanayama R and Nagata S: Impaired involution of mammary glands in the absence of milk fat globule EGF factor 8. Proc Natl Acad Sci USA. 102:16886–16891. 2005. View Article : Google Scholar : PubMed/NCBI
30	Ensslin MA and Shur BD: The EGF repeat and discoidin domain protein, SED1/MFG-E8, is required for mammary gland branching morphogenesis. Proc Natl Acad Sci USA. 104:2715–2720. 2007. View Article : Google Scholar : PubMed/NCBI
31	Aziz M, Jacob A, Matsuda A, Wu R, Zhou M, Dong W, Yang W-L and Wang P: Pre-treatment of recombinant mouse MFG-E8 downregulates LPS-induced TNF-α production in macrophages via STAT3-mediated SOCS3 activation. PLoS One. 6:e276852011. View Article : Google Scholar
32	Brissette MJ, Lepage S, Lamonde AS, Sirois I, Groleau J, Laurin LP and Cailhier JF: MFG-E8 released by apoptotic endothelial cells triggers anti-inflammatory macrophage reprogramming. PLoS One. 7:e363682012. View Article : Google Scholar : PubMed/NCBI
33	Matsuda A, Jacob A, Wu R, Zhou M, Nicastro JM, Coppa GF and Wang P: Milk fat globule-EGF factor VIII in sepsis and ischemia-reperfusion injury. Mol Med. 17:126–133. 2011. View Article : Google Scholar :
34	Nuzzi PA, Lokuta MA and Huttenlocher A: Analysis of neutrophil chemotaxis. Methods Mol Biol. 370:23–36. 2007. View Article : Google Scholar : PubMed/NCBI
35	Qiang X, Li J, Wu R, Ji Y, Chaung W, Dong W and Wang P: Expression and characterization of recombinant human milk fat globule-EGF factor VIII. Int J Mol Med. 28:1071–1076. 2011.PubMed/NCBI
36	Schneider CA, Rasband WS and Eliceiri KW: NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 9:671–675. 2012. View Article : Google Scholar : PubMed/NCBI
37	Belperio JA, Keane MP, Burdick MD, Londhe V, Xue YY, Li K, Phillips RJ and Strieter RM: Critical role for CXCR2 and CXCR2 ligands during the pathogenesis of ventilator-induced lung injury. J Clin Invest. 110:1703–1716. 2002. View Article : Google Scholar : PubMed/NCBI
38	Matsuda A, Wu R, Jacob A, Komura H, Zhou M, Wang Z, Aziz MM and Wang P: Protective effect of milk fat globule-epidermal growth factor-factor VIII after renal ischemia-reperfusion injury in mice. Crit Care Med. 39:2039–2047. 2011. View Article : Google Scholar : PubMed/NCBI
39	Brooks PC, Clark RA and Cheresh DA: Requirement of vascular integrin alpha v beta 3 for angiogenesis. Science. 264:569–571. 1994. View Article : Google Scholar : PubMed/NCBI
40	Cheyuo C, Jacob A, Wu R, Zhou M, Qi L, Dong W, Ji Y, Chaung WW, Wang H, Nicastro J, et al: Recombinant human MFG-E8 attenuates cerebral ischemic injury: Its role in anti-inflammation and anti-apoptosis. Neuropharmacology. 62:890–900. 2012. View Article : Google Scholar :
41	Aziz MM, Ishihara S, Mishima Y, Oshima N, Moriyama I, Yuki T, Kadowaki Y, Rumi MA, Amano Y and Kinoshita Y: MFG-E8 attenuates intestinal inflammation in murine experimental colitis by modulating osteopontin-dependent alphavbeta3 integrin signaling. J Immunol. 182:7222–7232. 2009. View Article : Google Scholar : PubMed/NCBI
42	Bu HF, Zuo XL, Wang X, Ensslin MA, Koti V, Hsueh W, Raymond AS, Shur BD and Tan XD: Milk fat globule-EGF factor 8/lactadherin plays a crucial role in maintenance and repair of murine intestinal epithelium. J Clin Invest. 117:3673–3683. 2007.PubMed/NCBI
43	Jinushi M, Nakazaki Y, Carrasco DR, Draganov D, Souders N, Johnson M, Mihm MC and Dranoff G: Milk fat globule EGF-8 promotes melanoma progression through coordinated Akt and twist signaling in the tumor microenvironment. Cancer Res. 68:8889–8898. 2008. View Article : Google Scholar : PubMed/NCBI
44	Raymond A, Ensslin MA and Shur BD: SED1/MFG-E8: A bi-motif protein that orchestrates diverse cellular interactions. J Cell Biochem. 106:957–966. 2009. View Article : Google Scholar : PubMed/NCBI
45	Alves-Filho JC, Sônego F, Souto FO, Freitas A, Verri WA Jr, Auxiliadora-Martins M, Basile-Filho A, McKenzie AN, Xu D, Cunha FQ, et al: Interleukin-33 attenuates sepsis by enhancing neutrophil influx to the site of infection. Nat Med. 16:708–712. 2010. View Article : Google Scholar : PubMed/NCBI
46	Oda K, Matsuoka Y, Funahashi A and Kitano H: A comprehensive pathway map of epidermal growth factor receptor signaling. Mol Syst Biol. 1:00102005. View Article : Google Scholar
47	Reynolds CM, Eguchi S, Frank GD and Motley ED: Signaling mechanisms of heparin-binding epidermal growth factor-like growth factor in vascular smooth muscle cells. Hypertension. 39:525–529. 2002. View Article : Google Scholar : PubMed/NCBI
48	Liu ZJ, Xiao M, Balint K, Smalley KS, Brafford P, Qiu R, Pinnix CC, Li X and Herlyn M: Notch1 signaling promotes primary melanoma progression by activating mitogen-activated protein kinase/phosphatidylinositol 3-kinase-Akt pathways and up-regulating N-cadherin expression. Cancer Res. 66:4182–4190. 2006. View Article : Google Scholar : PubMed/NCBI
49	Goruppi S, Ruaro E and Schneider C: Gas6, the ligand of Axl tyrosine kinase receptor, has mitogenic and survival activities for serum starved NIH3T3 fibroblasts. Oncogene. 12:471–480. 1996.PubMed/NCBI
50	Hidai C, Zupancic T, Penta K, Mikhail A, Kawana M, Quertermous EE, Aoka Y, Fukagawa M, Matsui Y, Platika D, et al: Cloning and characterization of developmental endothelial locus-1: an embryonic endothelial cell protein that binds the alphavbeta3 integrin receptor. Genes Dev. 12:21–33. 1998. View Article : Google Scholar : PubMed/NCBI
51	Hanayama R, Tanaka M, Miwa K and Nagata S: Expression of developmental endothelial locus-1 in a subset of macrophages for engulfment of apoptotic cells. J Immunol. 172:3876–3882. 2004. View Article : Google Scholar : PubMed/NCBI
52	Choi EY, Chavakis E, Czabanka MA, Langer HF, Fraemohs L, Economopoulou M, Kundu RK, Orlandi A, Zheng YY, Prieto DA, et al: Del-1, an endogenous leukocyte-endothelial adhesion inhibitor, limits inflammatory cell recruitment. Science. 322:1101–1104. 2008. View Article : Google Scholar : PubMed/NCBI