Endothelial Chemokines Destabilize L-selectin-mediated Lymphocyte Rolling without Inducing Selectin Shedding
2002; Elsevier BV; Volume: 277; Issue: 23 Linguagem: Inglês
10.1074/jbc.m201763200
ISSN1083-351X
AutoresValentin Grabovsky, Oren Dwir, R. Alon,
Tópico(s)Platelet Disorders and Treatments
ResumoChemokines presented on specialized endothelial surfaces rapidly up-regulate leukocyte integrin avidity and firm arrest through Gi-protein signaling. Here we describe a novel, G-protein-independent, down-regulatory activity of apical endothelial chemokines in destabilizing L-selectin-mediated leukocyte rolling. Unexpectedly, this anti-adhesive chemokine suppression of rolling does not involve L-selectin shedding. Destabilization of rolling is induced only by immobilized chemokines juxtaposed to L-selectin ligands and is an energy-dependent process. Chemokines are found to interfere with a subsecond stabilization of selectin tethers necessary for persistent rolling. This is a first indication that endothelial chemokines can attenuate in situ L-selectin adhesion to endothelial ligands at subsecond contacts. This negative feedback mechanism may underlie the jerky nature of rolling mediated by L-selectin in vivo. Chemokines presented on specialized endothelial surfaces rapidly up-regulate leukocyte integrin avidity and firm arrest through Gi-protein signaling. Here we describe a novel, G-protein-independent, down-regulatory activity of apical endothelial chemokines in destabilizing L-selectin-mediated leukocyte rolling. Unexpectedly, this anti-adhesive chemokine suppression of rolling does not involve L-selectin shedding. Destabilization of rolling is induced only by immobilized chemokines juxtaposed to L-selectin ligands and is an energy-dependent process. Chemokines are found to interfere with a subsecond stabilization of selectin tethers necessary for persistent rolling. This is a first indication that endothelial chemokines can attenuate in situ L-selectin adhesion to endothelial ligands at subsecond contacts. This negative feedback mechanism may underlie the jerky nature of rolling mediated by L-selectin in vivo. Selectins mediate the reversible capture (tethering) of circulating leukocytes to vascular endothelium at numerous types of inflamed or lymphoid target tissues (1Kansas G.S. Blood. 1996; 88: 3259-3287Crossref PubMed Google Scholar). Leukocyte tethers are short-lived and must be rapidly propagated into rolling adhesions, to allow the recruited leukocyte to survey the endothelial lining for additional stimulatory molecules, predominantly chemokines (2Mackay C.R. Nat. Immunol. 2001; 2: 95-101Crossref PubMed Scopus (707) Google Scholar). Chemokines elicit rapid signals through binding to specific G-protein-coupled receptors (GPCR) 1The abbreviations used are: GPCRG-protein-coupled receptorFCSfetal calf serumGlyCAM-1glycoprotein cell adhesion molecule 1HEVhigh endothelial venuleHSAhuman serum albuminILinterleukinmAbmonoclonal antibodyJAKJanus kinasePNAdperipheral node addressinPBLperipheral blood lymphocytesPBSphosphate-buffered salinePMAphorbol 12-myristate 13-acetatePSGL-1P-selectin glycoprotein ligandPTXpertussis toxinSDF-1αstromal cell-derived factor-1αSLCsecondary lymphoid tissue chemokineSTATsignal transducers and activators of transcriptionVLA-4very late antigen 41The abbreviations used are: GPCRG-protein-coupled receptorFCSfetal calf serumGlyCAM-1glycoprotein cell adhesion molecule 1HEVhigh endothelial venuleHSAhuman serum albuminILinterleukinmAbmonoclonal antibodyJAKJanus kinasePNAdperipheral node addressinPBLperipheral blood lymphocytesPBSphosphate-buffered salinePMAphorbol 12-myristate 13-acetatePSGL-1P-selectin glycoprotein ligandPTXpertussis toxinSDF-1αstromal cell-derived factor-1αSLCsecondary lymphoid tissue chemokineSTATsignal transducers and activators of transcriptionVLA-4very late antigen 4 on tethered leukocytes, which trigger the avidity of leukocyte integrins to endothelial ligands, and thereby stabilize secondary leukocyte adhesion, arrest, and subsequent extravasation (3Campbell J.J. Butcher E.C. Curr. Opin. Immunol. 2000; 12: 336-341Crossref PubMed Scopus (555) Google Scholar). Whether chemokine signals transduced to a rolling leukocyte can also modulate the adhesive properties of its selectin or selectin ligands has not been demonstrated. Soluble chemoattractants have been shown, on the other hand, to trigger L-selectin shedding by cell surface endoproteolysis, implicating blood-borne chemokines as potential down-regulators of selectin-mediated rolling (4Smith C.W. Kishimoto T.K. Abbassi O. Hughes B. Rothlein R. McIntire L.V. Butcher E. Anderson D.C. Abbass O. J. Clin. Invest. 1991; 87: 609-618Crossref PubMed Scopus (348) Google Scholar, 5Alexander S.R. Kishimoto T.K. Walcheck B. J. Leukocyte Biol. 2000; 67: 415-422Crossref PubMed Scopus (37) Google Scholar). Selectin rolling is a highly dynamic process that depends on subsecond coupling of tethers successively formed and broken at the cell front and trailing edge under disruptive shear forces (6Kaplanski G. Farnarier C. Tissot O. Pierres A. Benoliel A.M. Alessi M.C. Kaplanski S. Bongrand P. Biophys. J. 1993; 64: 1922-1933Abstract Full Text PDF PubMed Scopus (148) Google Scholar, 7Alon R. Hammer D.A. Springer T.A. Nature. 1995; 374: 539-542Crossref PubMed Scopus (600) Google Scholar). L-selectin rolling adhesions can be mediated by single tethers preferentially formed at microvillar surface projections where L-selectin is preferentially localized (8Picker L.J. Warnock R.A. Burns A.R. Doerschuk C.M. Berg E.L. Butcher E.C. Cell. 1991; 66: 921-933Abstract Full Text PDF PubMed Scopus (482) Google Scholar, 9von Andrian U.H. Hasslen S.R. Nelson R.D. Erlandsen S.L. Butcher E.C. Cell. 1995; 82: 989-999Abstract Full Text PDF PubMed Scopus (329) Google Scholar). Notably, L-selectin-mediated leukocyte rolling in vivo is extremely fast and jerky in nature (10Warnock R.A. Askari S. Butcher E.C. von Andrian U.H. J. Exp. Med. 1998; 187: 205-216Crossref PubMed Scopus (392) Google Scholar), even at endothelial sites expressing high levels of L-selectin ligands such as the peripheral lymph node high endothelial venules (HEV) (10Warnock R.A. Askari S. Butcher E.C. von Andrian U.H. J. Exp. Med. 1998; 187: 205-216Crossref PubMed Scopus (392) Google Scholar, 11Stein J.V. Rot A. Luo Y. Narasimhaswamy M. Nakano H. Gunn M.D. Matsuzawa A. Quackenbush E.J. Dorf M.E. von Andrian U.H. J. Exp. Med. 2000; 191: 61-76Crossref PubMed Scopus (363) Google Scholar). Stabilization of L-selectin-mediated rolling, characterized by smooth rather than jerky motion, is critically dependent on the number of bonds simultaneously formed at each microvillar contact site (12Chen S. Springer T.A. J. Cell Biol. 1999; 144: 185-200Crossref PubMed Scopus (200) Google Scholar). We therefore speculated that the jerky nature of L-selectin-mediated rolling in various in vivo settings might be caused by reduced L-selectin adhesiveness on leukocytes interacting with endothelium-displayed L-selectin ligands. In the present in vitro study, we found that several key chemokines, shown to be displayed on endothelial surfaces in vivo, are capable of strongly destabilizing the rolling activity of L-selectin in different types of leukocytes. Notably, this suppression of rolling was mediated by immobilized rather than soluble chemokines. Leukocyte capture to ligand, although normal, allowed the rapid encounter of immobilized chemokines co-displayed with L-selectin ligands, thereby eliciting the in situ reduction of L-selectin tether avidity to ligand. Surprisingly, destabilization of rolling was not the result of proteolytic L-selectin shedding. Furthermore, chemokine-mediated suppression of selectin rolling, although dependent on metabolic energy, did not involve intracellular signaling through the chemokine receptor. This is a first demonstration that endothelial chemokines may regulate selectin-mediated leukocyte rolling through a nonproteolytic Gi-protein-independent process, prior to and independent of their triggering of integrin adhesiveness.EXPERIMENTAL PROCEDURESAntibodies and ReagentsThe anti-L-selectin mAb, DREG-200 (13Kishimoto T.K. Jutila M.A. Butcher E.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2244-2248Crossref PubMed Scopus (387) Google Scholar), was provided by Dr. T. K. Kishimoto (Boehringer-Ingelheim Pharmaceuticals, Ridgefield, CT). The anti-very late antigen 4 (VLA-4) mAb, HP1/2 (14Hemler M.E. Huang C. Takada Y. Schwarz L. Strominger J.L. Clabby M.L. J. Biol. Chem. 1987; 262: 11478-11485Abstract Full Text PDF PubMed Google Scholar), was a gift from Dr. Roy Lobb (Biogen Inc., Cambridge, MA). The anti-glycoprotein cell adhesion molecule 1 (GlyCAM-1), purified from mouse serum by immunoaffinity chromatography (15Lasky L.A. Singer M.S. Dowbenko D. Imai Y. Henzel W.J. Grimley C. Fennie C. Gillett N. Watson S.R. Rosen S.D. Cell. 1992; 69: 927-938Abstract Full Text PDF PubMed Scopus (581) Google Scholar), was a gift from Dr. S. D. Rosen (University of California, San Francisco, CA). P-selectin glycoprotein ligand 1 (PSGL-1) was affinity-purified from human neutrophil lysates, was a generous gift from Dr. R. P. McEver (University of Oklahoma, Oklahoma City, OK), and was stored frozen in 1%n-octyl-β-d-glucopyranoside/PBS. Peripheral node addressin (PNAd) purified from human tonsil lysates by MECA-79 mAb affinity chromatography (16Berg E.L. Robinson M.K. Warnock R.A. Butcher E.C. J. Cell Biol. 1991; 114: 343-349Crossref PubMed Scopus (266) Google Scholar), a generous gift from Drs. E. L. Berg (Protein Design Laboratories, Mountain View, CA) and J. J. Campbell (Children's Hospital, Boston, MA), was stored in 1% octyl glucoside/PBS solution at 4 °C. Chemokines were obtained from R&D Systems (Minneapolis, MN), except for BCA-1, a gift from Dr. P. Loetscher (University of Bern, Bern, Switzerland). Chemokines were functionally inactivated by brief heat inactivation for 5 min at 100 °C as described previously (17Grabovsky V. Feigelson S. Chen C. Bleijs R. Peled A. Cinamon G. Baleux F. Arenzana-Seisdedos F. Lapidot T. van Kooyk Y. Lobb R. Alon R. J. Exp. Med. 2000; 192: 495-505Crossref PubMed Scopus (287) Google Scholar). Biotin-labeled stromal cell-derived factor-1α (SDF-1α) derivatives (modified either at the COOH or NH2 terminus of the chemokine) were a kind gift from Dr. F. Baleux (Institute Pasteur, Paris, France) and Dr. N. Fujii (Kyoto University, Kyoto, Japan). Both derivatives exhibited similar chemotactic activity toward T cells in Transwell chemotaxis assays. Biotin-labeled PSGL-1-derived sialyl Lewisx(sLex)-decorated glycopeptide and a nonfucosylated control peptide, both corresponding to the 19-residue NH2 terminus of human PSGL-1, and each containing a single biotin group at its COOH terminus (18Somers W.S. Tang J. Shaw G.D. Camphausen R.T. Cell. 2000; 103: 467-479Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar), were a gift from Dr. R. T. Camphausen (Genetics Institute, Cambridge, MA). Neutralite avidin (19Marttila A.T. Laitinen O.H. Airenne K.J. Kulik T. Bayer E.A. Wilchek M. Kulomaa M.S. FEBS Lett. 2000; 467: 31-36Crossref PubMed Scopus (86) Google Scholar) was a gift from Dr. E. A. Bayer, (Weizmann Institute of Science, Rehovot, Israel). Bovine serum albumin (fraction V), Ca2+- and Mg2+-free Hanks' balanced salt solution, Ficoll-Hypaque 1077, and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma-Aldrich. Human serum albumin (HSA, fraction V), pertussis toxin (PTX), and tyrphostin AG490 were obtained from Calbiochem (La Jolla, CA). The protease inhibitors Ro31-9790 (20Preece G. Murphy G. Ager A. J. Biol. Chem. 1996; 271: 11634-11640Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar) and KD-IX-73-4 (21Walcheck B. Kahn J. Fisher J.M. Wang B.B. Fisk R.S. Payan D.G. Feehan C. Betageri R. Darlak K. Spatola A.F. Kishimoto T.K. Nature. 1996; 380: 720-723Crossref PubMed Scopus (265) Google Scholar) were obtained from Dr. P. Altevogt (German Cancer Research Center, Heidelberg, Germany) and T. K. Kishimoto, respectively.CellsHuman peripheral blood lymphocytes (PBL; obtained from healthy donors) were isolated from citrate-anticoagulated whole blood by dextran sedimentation and density separation over Ficoll-Hypaque. The mononuclear cells thus obtained were washed and further purified on nylon wool and plastic adherence as described (22Carr M.W. Alon R. Springer T.A. Immunity. 1996; 4: 179-187Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). The resulting purified PBL consisted of more than 90% CD3+ T lymphocytes and were cultured in lipopolysaccharide-free RPMI, 10% FCS for 15–18 h before use. Peripheral blood neutrophils were isolated from anticoagulated blood after dextran sedimentation and density separation over Ficoll-Hypaque (23English D. Anderson B.R. J. Immunol. Methods. 1974; 5: 249-252Crossref PubMed Scopus (575) Google Scholar). Murine B lymphocytes were derived from fresh splenocytes by positive immunoselection with mAb B220 followed by magnetic cell sorting purification, as described (24Flaishon L. Hershkoviz R. Lantner F. Lider O. Alon R. Levo Y. Flavell R.A. Shachar I. J. Exp. Med. 2000; 192: 1381-1387Crossref PubMed Scopus (69) Google Scholar). The murine pre-B 300.19 cell line, stably expressing either native human L-selectin or tail-truncated L-selectin, was a generous gift from Dr. G. S. Kansas (Northwestern University, Chicago, IL) (25Kansas G.S. Ley K. Munro J.M. Tedder T.F. J. Exp. Med. 1993; 177: 833-838Crossref PubMed Scopus (185) Google Scholar). Clones were maintained in RPMI 1640, supplemented with antibiotics, 10% FCS, 2 mm glutamine, and 0.1 μm 2-mercaptoethanol. The human umbilical vein endothelial cell-derived line, ECV-304 (LS12), stably transfected with α-1,3-fucosyltransferase and N-acetylglucosamine 6-O-sulfotransferase and expressing functional sulfated L-selectin ligands (26Kimura N. Mitsuoka C. Kanarmori A. Hiraiwa N. Uchimura K. Muramatsu T. Tamatani T. Kansas G.S. R. K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4530-4535Crossref PubMed Scopus (115) Google Scholar) was a kind gift from Dr. R. Kannagi (Aichi Cancer Center, Nagoya, Japan). Cells were maintained in RPMI 1640, 10% FCS, 2 mm glutamine, and antibiotics.Immunofluorescence Flow CytometryIndirect immunofluorescence was performed on washed cells that were suspended in PBS supplemented with 5% FCS and 5 mmEDTA. Cells were incubated at 4 °C either with 10 μg/ml L-selectin mAb DREG-200 or with pre-immune mouse IgG. Cells were washed, stained with fluorescein isothiocyanate-conjugated goat anti-mouse Ig, resuspended in PBS supplemented with 0.05% sodium azide, and immediately analyzed in a FACScan flow cytometer (BD PharMingen, Erembodegem, Belgium). To assess protein kinase C-induced L-selectin shedding, PBL or neutrophils were suspended in cell binding medium (see below) in the presence of protease inhibitors or with control carrier solution for 15 min at 25 °C as described previously (20Preece G. Murphy G. Ager A. J. Biol. Chem. 1996; 271: 11634-11640Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 21Walcheck B. Kahn J. Fisher J.M. Wang B.B. Fisk R.S. Payan D.G. Feehan C. Betageri R. Darlak K. Spatola A.F. Kishimoto T.K. Nature. 1996; 380: 720-723Crossref PubMed Scopus (265) Google Scholar). Leukocytes were then treated with PMA (100 ng/ml, 2–10 min, 25 °C) and immediately incubated at 4 °C with 10 μg/ml DREG-200, followed by staining with secondary mAb, as described above.Preparation of Ligand-coated SubstratesAliquots of GlyCAM-1, PNAd, or PSGL-1 were diluted in coating medium (PBS, supplemented with 20 mm bicarbonate, pH 8.5) and adsorbed onto polystyrene plates as described previously (27Dwir O. Kansas G.S. Alon R. J. Biol. Chem. 2000; 275: 18682-18691Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Washed substrates were adsorbed with 0.1–4 μg/ml amount of either intact or heat-inactivated chemokines for 3 h at 4 °C. The anti-L-selectin mAb DREG-200 was mixed with either intact or heat-inactivated chemokines in the presence of 2 μg/ml HSA and coated onto polystyrene plates overnight at 4 °C. Neutralite avidin was diluted in PBS, 40 mm bicarbonate, pH 9.0, and adsorbed onto a polystyrene plate overnight at 4 °C, followed by HSA blocking at 4 °C. An equimolar mixture of biotin-labeled PSGL-1-derived selectin-binding peptide (2 × 10−2 nm) and either biotin-labeled SDF-1α or an inactive biotin-labeled control PSGL-1 peptide was diluted in cell binding medium (see below) and adsorbed for 4 h at 4 °C on the avidin-coated plate. Substrates coated with avidin complexed with inactive biotin-labeled glycopeptides lacked any adhesive activity to all L-selectin-expressing leukocytes tested.Laminar Flow AssaysCell Tethering and Rolling MeasurementsThe polystyrene plate, on which purified ligand was adsorbed, was assembled in a parallel plate laminar flow chamber as described previously (28Lawrence M.B. Springer T.A. Cell. 1991; 65: 859-873Abstract Full Text PDF PubMed Scopus (1872) Google Scholar). Various leukocyte populations were washed in H/H medium (Hanks' balanced salt solution, 10 mm HEPES, pH 7.4, supplemented with 2 mg/ml bovine serum albumin) containing 5 mm EDTA, resuspended in cell binding medium (H/H medium supplemented with 2 mm CaCl2) at 2 × 106cells/ml, and perfused at room temperature through the flow chamber at a rate generating wall shear stress of 0.1 dyn/cm2, as described (27Dwir O. Kansas G.S. Alon R. J. Biol. Chem. 2000; 275: 18682-18691Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Once reaching the upstream side of the test adhesive substrate, the flow rate was elevated to generate a shear stress of 0.75, 1, or 1.75 dyn/cm2, and all cellular interactions were visualized at two different fields of view (each one 0.17 mm2 in area) using a 10× objective of an inverted phase contrast microscope (Diaphot 300, Nikon Inc., Tokyo, Japan). An imaging system was used for analysis of instantaneous velocities of leukocytes, WSCAN-Array-3 (Galai, Migdal-Ha'emek, Israel) as described previously (27Dwir O. Kansas G.S. Alon R. J. Biol. Chem. 2000; 275: 18682-18691Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Accumulation of rolling leukocytes on the test fields was determined by computerized cell motion tracking. Adhesive interactions of transiently tethered cells were also manually analyzed as described (27Dwir O. Kansas G.S. Alon R. J. Biol. Chem. 2000; 275: 18682-18691Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The frequency of rolling cells was defined as the number of cells out of the cell flux that initiated persistent rolling on the adhesive substrate lasting at least 3 s after initial tethering. Transient tethers were cells that attached only briefly to the substrate (< 0.2 s). Frequency of each category of tethers was expressed in % units; 1% unit measured at 1.75 dyn/cm2 corresponded to tethering rate of 5.25 × 10−3 event × cell−1 mm−1s−1.To block GPCRs on target leukocytes, cells suspended in binding medium were preincubated for 45 min at 37 °C with 0.5 μg/ml soluble chemokines and perfused into the chamber. For blocking Gi-protein signaling, PBL were cultured for 15 h at 37 °C in culture medium alone or in the presence of 100 ng/ml PTX. For blocking JAK/STAT pathway stimulation by chemokines, lymphocytes were preincubated for 2 h at 37 °C with 150 μmJAK inhibitor, tyrphostin AG490, or with control Me2SO solution (0.1%, v/v). To inhibit metabolic energy without interfering with intact L-selectin rolling activity (29Dwir O. Shimron F. Chen C. Singer M. Rosen S.D. Alon R. Cell. Adhes. Commun. 1998; 6: 349-370Crossref PubMed Scopus (28) Google Scholar), lymphocytes were pretreated with 0.05% sodium azide for 2 min at room temperature, and perfused unwashed into the chamber. To adsorb SDF-1α on an endothelial monolayer, SDF-1α (100 ng/ml) was overlaid for 5 min on an L-selectin ligand-expressing ECV-304 cell monolayer assembled on the lower plate of the flow chamber. Unbound chemokine was removed by extensive washing. Overlaid chemokine remained bound to the monolayer throughout the assay, as confirmed in repetitive experiments after extensive washings.Dissociation Kinetics of Individual Transient Tethers and Successive Rolling TethersMicrokinetics of individual cells exhibiting jerky rolling on medium density GlyCAM-1 was analyzed on digitized video segments using the WSCAN-Array-3 software as described previously (27Dwir O. Kansas G.S. Alon R. J. Biol. Chem. 2000; 275: 18682-18691Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Individual cell displacement analysis at 0.02-s intervals monitored changes in instantaneous cell velocities in the flow direction, depicted as successive transient pauses (27Dwir O. Kansas G.S. Alon R. J. Biol. Chem. 2000; 275: 18682-18691Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The natural log of the number of all pauses or tethers formed by a given number of perfused leukocytes that remained bound after a given duration was plotted as a function of time, yielding tether dissociation curves with slopes representing −koff.DISCUSSIONSelectin-mediated tethering and rolling are prerequisite for leukocytes to survey the endothelial lining for proadhesive and promigratory signals, primarily apical chemokines (3Campbell J.J. Butcher E.C. Curr. Opin. Immunol. 2000; 12: 336-341Crossref PubMed Scopus (555) Google Scholar, 10Warnock R.A. Askari S. Butcher E.C. von Andrian U.H. J. Exp. Med. 1998; 187: 205-216Crossref PubMed Scopus (392) Google Scholar). Rolling leukocytes must integrate chemokine signals within subsecond contacts along the direction of flow (17Grabovsky V. Feigelson S. Chen C. Bleijs R. Peled A. Cinamon G. Baleux F. Arenzana-Seisdedos F. Lapidot T. van Kooyk Y. Lobb R. Alon R. J. Exp. Med. 2000; 192: 495-505Crossref PubMed Scopus (287) Google Scholar, 40Campbell J.J. Hedrick J. Zlotnik A. Siani M.A. Thompson D.A. Science. 1998; 279: 381-384Crossref PubMed Scopus (834) Google Scholar, 41Constantin G. Majeed M. Giagulli C. Piccio L. Kim J.Y. Butcher E.C. Laudanna C. Immunity. 2000; 13: 759-769Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). Here we suggest that apically displayed endothelial chemokines may not merely transmit integrin-activating signals to rolling leukocytes, but in fact directly modulate the rolling process itself, through an in situ reduction of selectin tether stability. Thus, rolling adhesions that allow a captured leukocyte to sample the endothelium for specific chemokines are subjected to a negative feedback mechanism by these very chemokines. Rather than being discrete and sequential events (42Springer T.A. Cell. 1994; 76: 301-314Abstract Full Text PDF PubMed Scopus (6373) Google Scholar), reversible selectin interactions and chemokine receptor occupancy events appear to simultaneously operate at particular adhesive zones bearing immobilized chemokines juxtaposed to L-selectin ligands. As a result, selectin-mediated rolling, which has been predicted to increase encounter of endothelium-displayed chemokines (43Hafezi-Moghadam A. Thomas K.L. Prorock A.J. Huo Y. Ley K. J. Exp. Med. 2001; 193: 863-872Crossref PubMed Scopus (182) Google Scholar), is in fact attenuated by this encounter. Attenuation of rolling is predicted to be more robust at sites of leukocyte interaction with high densities of L-selectin ligand, probably at endothelial regions within lymph node HEV enriched with L-selectin ligands (11Stein J.V. Rot A. Luo Y. Narasimhaswamy M. Nakano H. Gunn M.D. Matsuzawa A. Quackenbush E.J. Dorf M.E. von Andrian U.H. J. Exp. Med. 2000; 191: 61-76Crossref PubMed Scopus (363) Google Scholar). In addition, because the local density of chemokine on endothelial surfaces is heterogeneous (35Middleton J. Neil S. Wintle J. Clark-Lewis I. Moore H. Lam C. Auer M. Rot A. Cell. 1997; 91: 385-395Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar), this attenuation mechanism may result in multiple dynamic outcomes. In regions of low chemokine density, L-selectin-mediated rolling is expected to be accelerated (Fig. 2), whereas L-selectin-mediated rolling on specific regions expressing high density chemokine is expected to be strongly suppressed (Fig. 2). This would cause a rolling leukocyte to detach from such sites, while allowing it to jerk and rebind ligand at an adjacent downstream sites. Furthermore, chemokine distribution on individual endothelial cells is nonuniform, as chemokines can be found in clusters on endothelial microvilli (35Middleton J. Neil S. Wintle J. Clark-Lewis I. Moore H. Lam C. Auer M. Rot A. Cell. 1997; 91: 385-395Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar). These domains could be preferential sites of chemokine destabilization of L-selectin rolling. The jerky nature of L-selectin rolling is not controlled solely by chemokines. Anti-adhesive glycoproteins like CD43 (44Stockton B.M. Cheng G. Manjunath N. Ardman B. von Andrian U.H. Immunity. 1998; 8: 373-381Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), topological heterogeneity of both leukocyte and endothelial surfaces (45Finger E.B. Bruehl R.E. Bainton D.F. Springer T.A. J. Immunol. 1996; 157: 5085-5096PubMed Google Scholar), as well as intrinsic properties of L-selectin bonds (46Alon R. Chen S. Puri K.D. Finger E.B. Springer T.A. J. Cell Biol. 1997; 138: 1169-1180Crossref PubMed Scopus (312) Google Scholar,47Puri K.D. Finger E.B. Springer T.A. J. Immunol. 1997; 158: 405-413PubMed Google Scholar) can each contribute to the jerky nature of L-selectin-mediated rolling of leukocytes along various blood vessels. The existence of such multiple mechanisms for attenuating L-selectin rolling suggests that the jerky nature of L-selectin-mediated rolling is of major physiological significance. One possible outcome of such suppression of rolling could be to attenuate direct integrin activation by L-selectin, a process that depends on L-selectin ligation by ligand and bypasses chemokine regulation of leukocyte arrest on integrin ligands (48Hwang S.T. Singer M.S. Giblin P.A. Yednock T.A. Bacon K.B. Simon S.I. Rosen S.D. J. Exp. Med. 1996; 184: 1343-1348Crossref PubMed Scopus (158) Google Scholar, 49Steeber D.A. Engel P. Miller A.S. Sheetz M.P. Tedder T.F. J. Immunol. 1997; 159: 952-963PubMed Google Scholar, 50Simon S.I. Cherapanov V. Nadra I. Waddell T.K. Seo S.M. Wang Q. Doerschuk C.M. Downey G.P. J. Immunol. 1999; 163: 2891-2901PubMed Google Scholar).Spontaneous and chemoattractant-induced proteolytic shedding of L-selectin was traditionally proposed as a major negative feedback mechanism of L-selectin-mediated leukocyte rolling (21Walcheck B. Kahn J. Fisher J.M. Wang B.B. Fisk R.S. Payan D.G. Feehan C. Betageri R. Darlak K. Spatola A.F. Kishimoto T.K. Nature. 1996; 380: 720-723Crossref PubMed Scopus (265) Google Scholar, 51Kishimoto T.K. Jutila M.A. Berg E.L. Butcher E.C. Science. 1989; 245: 1238-1241Crossref PubMed Scopus (905) Google Scholar). However, the Gi-protein-independent chemokine destabilization of L-selectin rolling studied here did not involve L-selectin shedding, previously shown to involve activation of GPCR signaling (4Smith C.W. Kishimoto T.K. Abbassi O. Hughes B. Rothlein R. McIntire L.V. Butcher E. Anderson D.C. Abbass O. J. Clin. Invest. 1991; 87: 609-618Crossref PubMed Scopus (348) Google Scholar, 51Kishimoto T.K. Jutila M.A. Berg E.L. Butcher E.C. Science. 1989; 245: 1238-1241Crossref PubMed Scopus (905) Google Scholar). The insensitivity of chemokine suppression of rolling to PTX blockage of Gi signaling, demonstrated here, also rules out the possibility that chemokines suppress L-selectin rolling through Gi-protein-dependent phosphorylation of the L-selectin cytoplasmic tail (52Haribabu B. Steeber D.A. Ali H. Richardson R.M. Snyderman R. Tedder T.F. J. Biol. Chem. 1997; 272: 13961-13965Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Indeed, even lymphocytes expressing tail-truncated L-selectin were sensitive to chemokine suppression of rolling. Similar to our finding of an accelerated L-selectin rolling induced by low level chemokine (Fig. 2), Campbell and co-authors (40Campbell J.J. Hedrick J. Zlotnik A. Siani M.A. Thompson D.A. Science. 1998; 279: 381-384Crossref PubMed Scopus (834) Google Scholar) reported 2-fold faster L-selectin-dependent rolling of murine lymphocytes on PNAd co-immobilized with chemokines. The study attributed the accelerated rolling to chemokine blockage of L-selectin binding carbohydrates on the substrate. Our evidence that chemokines suppress L-selectin binding to mAb, an L-selectin-binding protein that lacks any selectin-binding carbohydrates, rules out the possibility of L-selectin ligand masking by chemokine. Furthermore, our finding that chemokines fail to suppress L-selectin adhesion to low density ligand (Fig. 7B) also rules out a direct blockage of ligand activity by chemokine. Instead, our data strongly suggest that chemokines induce rapid redistribution of both chemokine receptors and L-selectin at adhesive contact sites, possibly through extracellular or membranal associations of their receptors with juxtaposed L-selectin molecules. Recent electron microscopic analysis of the chemokine receptors for SDF-1α and RANTES (regulated on activation normal T cell expressed and secreted), CXCR4 and CCR5, respectively, in PBL has demonstrated that these GPCRs localize to lymphocyte microvilli (53Singer I.I. Scott S. Kawka D.W. Chin J. Daugherty B.L. DeMartino J.A. DiSalvo J. Gould S.L. Lineberger J.E. Malkowitz L. Miller M.D. Mitnaul L. Siciliano S.J. Staruch M.J. Williams H.R. Zweerink H.J. Springer M.S. J. Virol. 2001; 75: 3779-3790Crossref PubMed Scopus (138) Google Scholar), where L-selectin as well as α4 integrins are preferentially co-expressed (8Picker L.J. Warnock R.A. Burns A.R. Doerschuk C.M. Berg E.L. Butcher E.C. Cell. 1991; 66: 921-933Abstract Full Text PDF PubMed Scopus (482) Google Scholar, 9von Andrian U.H. Hasslen S.R. Nelson R.D. Erlandsen S.L. Butcher E.C. Cell. 1995; 82: 989-999Abstract Fu
Referência(s)