GTMB 7 - Gene Therapy & Molecular Biology

More documents

Recommendations

Info

Chavakis et al: Leukocyte adhesion and statinsassociated protein (CD47), members of the tetraspaninfamily, syndecans, caveolin-1 or urokinase typeplasminogen activator receptor (uPAR) (CD87), leading tothe formation of transient multireceptor complexes thatfacilitate the dynamic recruitment of signaling moleculesto sites of cellular contacts or focal adhesions (Ossowskiand Aguirre-Ghiso, 2000; Preissner et al, 2000). Besidesits ability to regulate integrin-dependent adhesionphenomena, uPAR can also directly mediate leukocyteadhesion to matrix-associated VN (Wei et al, 1994; Sitrinet al, 1996; May et al, 1998).Recently, attention has been drawn to the role ofmicrodomain structures of the plasma membrane, denotedlipid rafts, in cell adhesion. Lipid rafts are enriched inglycosphingolipids, cholesterol, transmembrane proteinsand signaling molecules. GPI-anchored proteins maybecome sequestered into the microdomains as well, whichhave a lower fluidity than the surrounding membraneallowing the formation of multireceptor adhesioncomplexes. On epithelial cells, caveolin is a unique raftcomponent, that has the intrinsic propensity to oligomerizeand, thereby, contribute to formation of membraneinvaginations termed caveolae (Horejsi et al, 1999;Kurzchalia and Parton, 1999; Smart et al, 1999; Simonsand Toomre, 2000). Although leukocytes lack caveolinexpression, they still contain lipid rafts that may facilitatethe formation of adhesion complexes. The possibility thatlipid rafts might regulate leukocyte adhesion bymodulating integrin avidity has already been suggested(Krauss and Altevogt, 1999).Statins inhibit the key enzyme of cholesterolbiosynthesis 3-hydroxy-3-methylglutaryl coenzyme-Areductase (HMG-CoA reductase). In addition to loweringplasma cholesterol, increasing evidence suggests thatstatins play a pleiotropic role in the vascular system byeffects on nitric oxide synthesis, smooth muscle cellproliferation, fibrinolysis or the immune system (Soma etal, 1993; Aikawa et al, 1998; Essig et al, 1998; Guisarro etal, 1998; Laufs and Liao, 1998; Laufs et al, 1998, 1999;Kwak et al, 2000; Diomede et al, 2001; Kwak and Mach,2001). In particular, statins could inhibit leukocyterecruitment by regulating the expression of monocytechemoattractant protein-1 (Romano et al, 2000) and ofadhesion receptors (Weber et al, 1997; Ganne et al, 2000;Yoschida et al, 2001) or they might modulate integrinaffinity by preventing geranyl-geranylation o f RhoA (Liuet al, 1999). Cholesterol depletion by statins might alsodisrupt lipid rafts and, thereby, affect cell adhesion (Krausand Altevogt, 1999; Simons and Toomre, 2000). Finally, arecent report suggested that different statins selectivelybind to LFA-1, thereby blocking LFA-1 mediatedleukocyte adhesion (Kallen et al, 1999; Weitz-Schmidt etal, 2001).These observations prompted us to investigate inmore detail the role of lovastatin in β2-integrin- anduPAR-mediated leukocyte interactions. Two distinctmechanisms, a HMG-CoA reductase-dependent and an–independent, for inhibition of leukocyte adhesion aredescribed, which further help to understand theantiinflammatory role of statins.II. Materials and methodsA. ReagentsTwo-chain high molecular weight urokinase typeplasminogen activator (uPA) was from American Diagnostica(Bergstrasse, Germany). VN was purified from human plasmaand converted to the multimeric form as previously described(Chavakis et al, 1998). FBG and fibronectin were purchasedfrom Sigma (Munich, Germany). Vitamin D3 was from Biomol(Hamburg, Germany), transforming growth factor-β was from R& D Systems (Boston, MA), and interleukin-3 was from PBH(Hannover, Germany). Phorbol 12-myristate 13-acetate (PMA)was from Gibco (Paisley, Scotland,UK). The blockingmonoclonal antibody against human CD18, 60.3, was kindlyprovided by Dr. J. Harlan (University of Washington, Seattle,WA), the blocking monoclonal antibody against human CD11a,L15, was a generous gift from Dr. C. Figdor (University ofNijmegen, The Netherlands) and anti-uPAR monoclonalantibodies R3 and R4 (Chavakis et al, 1999) were given by Dr.G. Hoyer-Hansen (The Finsen Laboratory, Copenhagen,Denmark). Monoclonal antibodies K20 against β1-integrins(CD29), 6.5B5 against ICAM-1, 2LPM19c against CD11b,KB90 against CD11c, MHM24 against CD11a and polyclonalrabbit-anti-FBG were from Dako (Hamburg, Germany). IsolatedMac-1, LFA-1 and ICAM-1 were kindly obtained from Dr. S.Bodary (Genentech, San Francisco, CA). Recombinant solubleuPAR was kindly provided by Dr. D. Cines (University ofPennsylvania, Philadelphia, PA). Lovastatin, mevalonate,farnesyl-pyrophosphate and geranyl-pyrophosphate were fromSigma (Munich, Germany). Peroxidase-conjugated secondaryanti-mouse and anti-rabbit immunoglobulins were from DAKO(Hamburg, Germany).B. Cell cultureMyelomonocytic cells (U937) obtained from AmericanType Culture Collection (ATCC) (Rockville, MD) were culturedin RPMI-1640 medium containing 10% (vol/vol) fetal calfserum. K562 cells transfected with Mac-1 were kindly providedby Dr. M. Robinson (Celltech Ltd, Slough, England) and K562cells transfected with LFA-1 or p150.95 were a generous giftfrom Dr. Y. van Kooyk (University of Nijmegen, TheNetherlands) and were cultivated in a mixture of 75% RPMIcontaining 10% fetal calf serum and 25% ISCOVE´s mediumcontaining 5% fetal calf serum. Expression of the respective β2-integrins was tested by FACS analysis (see below). All culturemedia were from Gibco (Eggenstein, Germany), and the cellculture plastic was from Nunc (Rocksilde, Denmark).C. Cell adhesion assaysCell adhesion to VN, ICAM-1 and FBG coated plates (andto BSA-coated wells as control) was tested according topreviously described protocols (Chavakis et al, 1999, 2000, 2001,2002). Briefly, multiwell plates were coated with 5 µg/ml ICAM-1, FBG or 2 µg/ml VN (dissolved in bicarbonate buffer, pH 9.6),respectively, and blocked with 3% (wt/vol) BSA. U937 cells,which had been differentiated for 24 h with vitamin D3 (100 nM)and transforming growth factor-β (2 ng/ml), or K562 cells werewashed in serum-free RPMI and plated onto the precoated wellsfor 60-90 min at 37°C in the absence or presence of competitorsin serum-free RPMI as indicated in the figure legends. Whereindicated, U937 cells were preincubated for various time periodswithout or together with lovastatin in the absence or presence ofmevalonate, farnesyl-pyrophosphate or geranyl-pyrophosphate.Following the incubation period for the adhesion assay, the wellswere washed and the number of adherent cells was quantified by104
Gene Therapy and Molecular Biology Vol 7, page 105crystal violet staining at 590 nm.D. Analysis of uPAR and integrin expressionby flow cytometryAfter incubation for 18 h in the absence or presence oflovastatin differentiated U937 cells were washed twice withHEPES-buffered saline and were incubated with saturatingconcentrations of primary antibody (10 µg/ml) for 60 min at 4°C.Cells were washed again, resuspended in HEPES buffer andphycoerythrin-conjugated F(ab , )2 fragment of goat anti-rabbit (ormouse) IgG (Dianova, Hamburg, Germany) was added insaturating concentrations for 60 min at 4°C. After washing andresuspension, mean fluorescence of 10,000 cells was measured ina flow cytometer (Beckton Dickinson, Heidelberg, Germany).Nonspecific fluorescence was determined using control speciesandisotype-matched primary antibody.inhibitory effect of lovastatin on ICAM-1 adhesion wasunchanged in the presence of the isoprenoid metabolitesmevalonate, farnesyl-pyrophosphate, or geranylpy ro p h o s p h a t e ( F i gu r e 1B). None of these threemetabolites alone could affect U937 cell adhesion toICAM-1 (not shown).U937 cells engage both Mac-1 and LFA-1 forICAM-1-dependent adhesion; however, the lack ofinhibitory activity of lovastatin on Mac-1-related adhesionto FBG indicated that lovastatin interacts only with LFA-1directly.E. ELISA for ligand-receptor interactionsMaxisorp plates (high binding capacity; Nunc) were coatedwith Mac-1 or LFA-1 (5 µg/ml) dissolved in 20 mM HEPES,150 mM NaCl, 1 mM Mn 2+ , pH 7.2 and then blocked with 3%(wt/vol) bovine serum albumin (BSA) in the same buffer.Binding of FBG (10 µg/ml) or ICAM-1 (10 µg/ml) to theimmobilized integrin was performed in a final volume of 50 µl ofthe same buffer as above together with 0.05% (wt/vol) Tween-20and 0.1 % (wt/vol) BSA in the absence or presence of differentcompetitors as indicated in the figure legends. After incubationfor 2 h at 22°C and a washing step, bound ligands were detectedby the addition of polyclonal rabbit anti-FBG or monoclonalmouse anti-ICAM-1 followed by the addition of 1:1000 dilutedperoxidase-conjugated antibody against rabbit or mouseimmunoglobulins, respectively. The conversion of the substrate2,2-azino-di(3-ethly)benzthiazoline sulphate (Boehringer,Mannheim, Germany) was monitored at 405 nm in a Thermomaxmicrotitre plate reader (Molecular Devices, Menlo Park, CA).Nonspecific binding to BSA-coated wells was used as blank andwas subtracted to calculate the specific binding. The sameprotocol was used when binding of multimeric VN (2 µg/ml) toimmobilized uPAR (5 µg/ml, dissolved in bicarbonate buffer, pH9.6) was tested, except that the binding buffer was TBScontaining 0.05 % (wt/vol) Tween-20 0.1 % (wt/vol) BSA.Bound VN was detected with the anti-VN monoclonal antibodyVN7 and additional steps of quantitation were the same asmentioned above.III. ResultsA. HMG-CoA reductase independentregulation of leukocyte adhesion by lovastatinAs previously established, the adhesion of myelomonocyticU937 cells [differentiated with TGFβ (2 ng/ml)and vitamin D3 (100 nM) for 24 h] to immobilized FBG ispredominantly mediated by Mac-1, whereas both Mac-1and LFA-1 mediate adhesion to immobilized ICAM-1.U937 cell adhesion to FBG and ICAM-1 is enhanced byMn 2+ or phorbol ester (PMA). Moreover, U937 celladhesion to VN is uPAR-dependent; uPA can stimulateadhesion, as it increases the affinity of the uPAR/VNinteraction(Chavakis et al, 2000, 2001 Preissner et al,2000). In the presence of lovastatin, adhesion of U937cells to ICAM-1was markedly reduced, whereas adhesionto FBG or VN was not affected at all (Figure 1A). TheFigure 1. U937 cell adhesion to ICAM-1, FBG and VN. (A)PMA (50 ng/ml)-stimulated U937 cell adhesion to immobilizedICAM-1 (5 µg/ml) and FBG (5 µg/ml) or uPA (50 nM)-stimulated U937 cell adhesion to immobilized VN (2 µg/ml) wasstudied in the absence (open bars) or the presence of lovastatin(100 µM, filled bars) or the following blocking antibodies(hatched bars): anti-CD18 (15 µg/ml) for ICAM-1- and FBGmediatedadhesion, anti-uPAR (10 µg/ml) for VN-dependentadhesion. (B) PMA (50 ng/ml)-stimu l a t e d U 937 cell adhesion toimmobilized ICAM-1 (5 µg/ml) was studied in the absence (-) orpresence of a blocking anti-CD18 antibody (15 µg/ml), ablocking anti-LFA-1 (CD11a) antibody (15 µg/ml), lovastatinalone (100 µM), or in combination with mevalonate (100 µM,MEV), farnesyl-pyrophosphate (100 µM, FP), or geranylpyrophosphate(100 µM, GP). Cell adhesion is expressed aspercent of control, which is represented by the adhesion in thepresence of PMA (or uPA, where adhesion to VN is shown) andin the absence of any competitor. Data are mean ± SEM (n=3) ofa typical experiment; similar results were obtained in at leastthree separate experiments.105
Page 7 and 8:
Instructions to authors:Gene Therap
Page 9:
Please submit an electronic version
Page 12:
103-111 ResearchArticle113-133 Revi
Page 17 and 18:
Gene Therapy and Molecular Biology
Page 19 and 20:
Page 21 and 22:
Page 23 and 24:
Page 25 and 26:
Page 27 and 28:
Page 29 and 30:
Page 31 and 32:
Page 33 and 34:
Page 35 and 36:
Page 37 and 38:
Page 39 and 40:
Page 41 and 42:
Page 43 and 44:
Page 45 and 46:
Page 47 and 48:
Page 49 and 50:
Page 51 and 52:
Page 53 and 54:
Page 55 and 56:
Page 57 and 58:
Page 59 and 60:
Page 61 and 62:
Page 63 and 64:
Page 65 and 66:
Page 67 and 68: Gene Therapy and Molecular Biology
Page 77: Gene Therapy and Molecular Biology
Page 80 and 81: Epperly et al: Late injection of Mn
Page 82 and 83: Epperly et al: Late injection of Mn
Page 84 and 85: Goldberg-Cohen et al: Regulation of
Page 90 and 91: Gascón-Irún et al: Gene therapy a
Page 106 and 107: Suzuki et al: Regulation of the Sp/
Page 114 and 115: Li et al: MET amplification in live
Page 116 and 117: Li et al: MET amplification in live
Page 120 and 121: Chavakis et al: Leukocyte adhesion
Page 128 and 129: Sanlioglu et al: Adenovirus mediate
Page 150 and 151: George et al: Gene therapy for vasc
Page 168 and 169:
Zhang et al: Angiogenic Gene Therap
Page 170 and 171:
Page 172 and 173:
Page 174 and 175:
Page 176 and 177:
Page 178 and 179:
Page 180 and 181:
Page 182 and 183:
Xu et al: G-CSF receptor-mediated S
Page 184 and 185:
Page 186 and 187:
Page 188 and 189:
Burek et al: Calcium induced cell d
Page 190 and 191:
Page 192 and 193:
Page 194 and 195:
Page 196 and 197:
David et al: Current status and fut
Page 198 and 199:
Page 200 and 201:
Page 202 and 203:
Page 204 and 205:
Page 206 and 207:
Page 208 and 209:
Page 210 and 211:
Page 212 and 213:
Page 214 and 215:
Page 216 and 217:
Page 218 and 219:
Page 220 and 221:
Page 222 and 223:
Page 224 and 225:
Page 226 and 227:
Stoll et al: The role of EBV and ge
Page 228 and 229:
Page 230 and 231:
Page 232 and 233:
Page 234 and 235:
Page 236 and 237:
Maruyama et al: Kidney-targeted pla
Page 238 and 239:
Page 240 and 241:
Page 242 and 243:
Page 244 and 245:
Kren et al: Hepatocyte-targeted del
Page 246 and 247:
Page 248 and 249:
Page 250 and 251:
Page 252 and 253:
Page 254 and 255:
Zeng: PRL-3 as a target for cancer
Page 256 and 257:
Page 258 and 259:
Page 260 and 261:
Latchman: Protective effect of heat
Page 262 and 263:
Page 264 and 265:
Page 266 and 267:
Page 268 and 269:
Page 270 and 271:
Cai et al: Lung cancer gene therapy
Page 272 and 273:
Page 274 and 275:
Page 276 and 277:
Page 278 and 279:
Page 280:
Page 283 and 284:
Page 285 and 286:
Page 287 and 288:
Page 289 and 290:
Page 291 and 292:
Page 293 and 294:
Page 295 and 296:
Page 297 and 298:
Page 299 and 300:
Page 301 and 302:
Page 303 and 304:
Page 305 and 306:
Page 307 and 308:
Page 309:
show all

GTMB 7 - Gene Therapy & Molecular Biology

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?