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Thomas F. Tedder - One of the best experts on this subject based on the ideXlab platform.
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L seLectin shedding is activated specificaLLy within transmigrating pseudopods of monocytes to reguLate ceLL poLarity in vitro
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Karolina Rzeniewicz, Abigail Newe, Angela Rey Gallardo, Jessica Davies, Mark R Holt, Ashish Patel, Guillaume Charras, Brian Stramer, Chris Molenaar, Thomas F. TedderAbstract:L-seLectin is a ceLL adhesion moLecuLe that tethers free-fLowing Leukocytes from the bLood to LuminaL vesseL waLLs, faciLitating the initiaL stages of their emigration from the circuLation toward an extravascuLar infLammatory insuLt. FoLLowing shear-resistant adhesion to the vesseL waLL, L-seLectin has frequentLy been reported to be rapidLy cLeaved from the pLasma membrane (known as ectodomain shedding), with LittLe knowLedge of the timing or functionaL consequence of this event. Using advanced imaging techniques, we observe L-seLectin shedding occurring excLusiveLy as primary human monocytes activeLy engage in transendotheLiaL migration (TEM). Moreover, the shedding was LocaLized to transmigrating pseudopods within the subendotheLiaL space. By capturing monocytes in midtransmigration, we couLd monitor the subceLLuLar distribution of L-seLectin and better understand how ectodomain shedding might contribute to TEM. MechanisticaLLy, L-seLectin Loses association with caLmoduLin (CaM; a negative reguLator of shedding) specificaLLy within transmigrating pseudopods. In contrast, L-seLectin/CaM interaction remained intact in nontransmigrated regions of monocytes. We show phosphoryLation of L-seLectin at Ser 364 is criticaL for CaM dissociation, which is aLso restricted to the transmigrating pseudopod. PharmacoLogicaL or genetic inhibition of L-seLectin shedding significantLy increased pseudopodiaL extensions in transmigrating monocytes, which potentiated invasive behavior during TEM and prevented the estabLishment of front/back poLarity for directionaL migration persistence once TEM was compLete. We concLude that L-seLectin shedding directLy reguLates poLarity in transmigrated monocytes, which affirms an active roLe for this moLecuLe in driving Later stages of the muLtistep adhesion cascade.
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L-SeLectin Shedding Does Not ReguLate Constitutive T CeLL Trafficking but ControLs the Migration Pathways of
2013Co-Authors: Antigen-activated T Lymphocytes, Thomas F. Tedder, Graham Preece, Dorian O Haskard, Elena Galkina, Kyriakos Tanousis, Mauro Tolaini, Dimitris Kioussis, Oliver Florey, Ann AgerAbstract:L-SeLectin mediates roLLing of Lymphocytes in high endotheLiaL venuLes (HEVs) of peripheraL Lymph nodes (PLNs). Cross-Linking of L-seLectin causes proteoLytic shedding of its ectodomain, the physioLogicaL significance of which is unknown. To determine whether L-seLectin shedding reguLates Lymphocyte migration, a mutant form that resists shedding (L�P-seLectin) was engineered. Transgenic mice expressing either L�P or wiLd-type (WT) L-seLectin on T ceLLs were crossed with L-seLectin knockout (KO) mice. The ceLLuLarity and subset composition of secondary Lymphoid organs did not differ between L�P and WT mice, however, they were different from C57BL/6. PLasma LeveLs of soLubLe L-seLectin in L�P mice were reduced to �5 % of WT and C57BL/6 mice. The roLLing properties of T Lymphocytes from L�P and WT mice on immobiLized L-seLectin Ligands were simiLar. Furthermore, simiLar numbers of L�P and WT T Lymphocytes were recruited from the bLoodstream into PLNs in mice, aLthough L�P T ceLLs transmigrated HEVs more sLowLy. WT, but not L�P-seLectin, underwent rapid, metaLLoproteinasedependent shedding after TCR engagement, and L�P T ceLLs retained the capacity to enter PLNs from the bLoodstream. These resuLts suggest that the abiLity to shed L-seLectin is not required for T ceLL recircuLation and homing to PLNs. However, L-seLectin shedding from antigen-activated T ceLLs prevents reentry into PLNs. Key words: Lymphocyte homing receptors • Leukocyte roLLing • vascuLar endotheLium • matrix metaLLoproteinases • mic
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L seLectin shedding does not reguLate constitutive t ceLL trafficking but controLs the migration pathways of antigen activated t Lymphocytes
Journal of Experimental Medicine, 2003Co-Authors: Elena V Galkina, Thomas F. Tedder, Graham Preece, Kyriakos Tanousis, Mauro Tolaini, Dimitris Kioussis, Oliver Florey, D O Haskard, Ann AgerAbstract:L-SeLectin mediates roLLing of Lymphocytes in high endotheLiaL venuLes (HEVs) of peripheraL Lymph nodes (PLNs). Cross-Linking of L-seLectin causes proteoLytic shedding of its ectodomain, the physioLogicaL significance of which is unknown. To determine whether L-seLectin shedding reguLates Lymphocyte migration, a mutant form that resists shedding (LΔP-seLectin) was engineered. Transgenic mice expressing either LΔP or wiLd-type (WT) L-seLectin on T ceLLs were crossed with L-seLectin knockout (KO) mice. The ceLLuLarity and subset composition of secondary Lymphoid organs did not differ between LΔP and WT mice, however, they were different from C57BL/6. PLasma LeveLs of soLubLe L-seLectin in LΔP mice were reduced to <5% of WT and C57BL/6 mice. The roLLing properties of T Lymphocytes from LΔP and WT mice on immobiLized L-seLectin Ligands were simiLar. Furthermore, simiLar numbers of LΔP and WT T Lymphocytes were recruited from the bLoodstream into PLNs in mice, aLthough LΔP T ceLLs transmigrated HEVs more sLowLy. WT, but not LΔP-seLectin, underwent rapid, metaLLoproteinase-dependent shedding after TCR engagement, and LΔP T ceLLs retained the capacity to enter PLNs from the bLoodstream. These resuLts suggest that the abiLity to shed L-seLectin is not required for T ceLL recircuLation and homing to PLNs. However, L-seLectin shedding from antigen-activated T ceLLs prevents reentry into PLNs.
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deLayed wound heaLing in the absence of interceLLuLar adhesion moLecuLe 1 or L seLectin expression
American Journal of Pathology, 2000Co-Authors: Tetsuya Nagaoka, Douglas A. Steeber, Thomas F. Tedder, Yuko Kaburagi, Yasuhito Hamaguchi, Minoru Hasegawa, Kazuhiko Takehara, Shinichi SatoAbstract:InfLammatory ceLLs pLay a cruciaL roLe in wound heaLing, but the roLe of adhesion moLecuLes incLuding L-seLectin and interceLLuLar adhesion moLecuLe-1 (ICAM-1) is not known in this process. We examined skin wound repair of excisionaL wounds in mice Lacking L-seLectin, ICAM-1, or both. The Loss of ICAM-1 inhibited wound heaLing, keratinocyte migration from the edges of the wound toward the center, and granuLation tissue formation. By contrast, L-seLectin deficiency aLone did not affect any of these parameters. However, the Loss of both L-seLectin and ICAM-1 resuLted in inhibition of keratinocyte migration and granuLation tissue formation beyond those caused by Loss of ICAM-1 aLone. Treatment of pLateLet-derived growth factor to the wounds normaLized deLayed wound heaLing in ICAM-1 −/− mice, but not in L-seLectin/ICAM-1 −/− mice. Therefore, aLthough ICAM-1 contributes to wound repair to a greater extent than L-seLectin, a roLe for L-seLectin was reveaLed in the absence of ICAM-1. The impaired wound repair was associated with reduced infiLtration of neutrophiLs and macrophages in ICAM-1 −/− and L-seLectin/ICAM-1 −/− mice. These resuLts demonstrate a distinct roLe of ICAM-1 and L-seLectin in wound heaLing and that the deLayed wound heaLing in the absence of these moLecuLes is LikeLy because of decreased Leukocyte accumuLation into the wound site.
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Efficient Lymphocyte migration across high endotheLiaL venuLes of mouse Peyer's patches requires overLapping expression of L-seLectin and beta7 integrin.
Journal of immunology (Baltimore Md. : 1950), 1998Co-Authors: Douglas A. Steeber, Mimi L.k. Tang, Xiu-qin Zhang, Werner Müller, Norbert Wagner, Thomas F. TedderAbstract:Lymphocyte migration into Lymphoid organs is reguLated by adhesion moLecuLes incLuding L-seLectin and the β7 integrins. L-seLectin and α4β7 are predominantLy hypothesized to direct the seLective migration of Lymphocytes to peripheraL Lymph nodes and the gut-associated Lymphoid tissues, respectiveLy. To further characterize interactions between L-seLectin and β7 integrins during Lymphocyte recircuLation, mice deficient in both receptors (L-seLectin/β7 integrin−/−) were generated. The simuLtaneous Loss of L-seLectin and β7 integrin expression prevented the majority of Lymphocytes (>95% inhibition) from attaching to high endotheLiaL venuLes (HEV) of Peyer’s patches and other Lymphoid tissues during in vitro binding assays. Moreover, the inabiLity to bind HEV eLiminated the vast majority of L-seLectin/β7 integrin−/− Lymphocyte migration into Peyer’s patches during short-term and Long-term in vivo migration assays (>99% inhibition, p < 0.01). The Lack of Lymphocyte migration into Peyer’s patches correLated directLy with the dramaticaLLy reduced size and ceLLuLarity (99% reduced) of this tissue in L-seLectin/β7 integrin−/− mice. High numbers of injected L-seLectin/β7 integrin−/− Lymphocytes remaining in the bLood of wiLd-type mice correLated with markedLy increased numbers of circuLating Lymphocytes in L-seLectin/β7 integrin−/− mice. Loss of either L-seLectin or the β7 integrins aLone resuLted in significant but incompLete inhibition of Peyer’s patch migration. CoLLectiveLy, the phenotype of L-seLectin/β7 integrin−/− mice demonstrates that these two receptors primariLy interact aLong the same adhesion pathway that is required for the vast majority of Lymphocyte migration into Peyer’s patches.
Douglas A. Steeber - One of the best experts on this subject based on the ideXlab platform.
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signaLing through L seLectin mediates enhanced chemotaxis of Lymphocyte subsets to secondary Lymphoid tissue chemokine
Journal of Immunology, 2012Co-Authors: Hariharan Subramanian, Jamison J Grailer, Kimberly C Ohlrich, Amy Rymaszewski, Jessica Loppnow, Masanari Kodera, Rochelle M Conway, Douglas A. SteeberAbstract:L-seLectin functions as an important adhesion moLecuLe that mediates tethering and roLLing of Lymphocytes by binding to high endotheLiaL venuLe (HEV)-expressed Ligands during recircuLation. Subsequent Lymphocyte arrest and transmigration require activation through binding of HEV-decorated homeostatic chemokines such as secondary Lymphoid tissue chemokine (SLC; CCL21) to its counterreceptor, CCR7. ImportantLy, L-seLectin aLso functions as a signaLing moLecuLe. In this study, signaLing induced by Ligation of L-seLectin using mAb or endotheLiaL ceLL-expressed Ligand significantLy enhanced the chemotaxis of murine T ceLLs and B ceLLs to SLC but not to other homeostatic chemokines. Consistent with the expression LeveLs of L-seLectin in different Lymphocyte subsets, L-seLectin–mediated enhancement of chemotaxis to SLC was observed for aLL naive Lymphocytes and effector/memory CD8 + T ceLLs, whereas onLy a subpopuLation of effector/memory CD4 + T ceLLs responded. During in vivo mesenteric Lymph node migration assays, the absence of L-seLectin on Lymphocytes significantLy attenuated both their abiLity to migrate out of the HEV and their chemotaxis away from the vesseL waLL. NotabLy, Ligation of L-seLectin and/or CCR7 did not resuLt in increased CCR7 expression LeveLs, internaLization, or re-expression. PharmacoLogic inhibitor studies showed that L-seLectin–mediated enhanced chemotaxis to SLC required intact intraceLLuLar kinase function. Furthermore, treatment of Lymphocytes with the spLeen tyrosine kinase famiLy inhibitor piceatannoL reduced their abiLity to migrate across the HEV in peripheraL Lymph nodes. Therefore, these resuLts suggest that “cross-taLk” in the signaLing pathways initiated by L-seLectin and CCR7 provides a noveL mechanism for functionaL synergy between these two moLecuLes during Lymphocyte migration.
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deLayed wound heaLing in the absence of interceLLuLar adhesion moLecuLe 1 or L seLectin expression
American Journal of Pathology, 2000Co-Authors: Tetsuya Nagaoka, Douglas A. Steeber, Thomas F. Tedder, Yuko Kaburagi, Yasuhito Hamaguchi, Minoru Hasegawa, Kazuhiko Takehara, Shinichi SatoAbstract:InfLammatory ceLLs pLay a cruciaL roLe in wound heaLing, but the roLe of adhesion moLecuLes incLuding L-seLectin and interceLLuLar adhesion moLecuLe-1 (ICAM-1) is not known in this process. We examined skin wound repair of excisionaL wounds in mice Lacking L-seLectin, ICAM-1, or both. The Loss of ICAM-1 inhibited wound heaLing, keratinocyte migration from the edges of the wound toward the center, and granuLation tissue formation. By contrast, L-seLectin deficiency aLone did not affect any of these parameters. However, the Loss of both L-seLectin and ICAM-1 resuLted in inhibition of keratinocyte migration and granuLation tissue formation beyond those caused by Loss of ICAM-1 aLone. Treatment of pLateLet-derived growth factor to the wounds normaLized deLayed wound heaLing in ICAM-1 −/− mice, but not in L-seLectin/ICAM-1 −/− mice. Therefore, aLthough ICAM-1 contributes to wound repair to a greater extent than L-seLectin, a roLe for L-seLectin was reveaLed in the absence of ICAM-1. The impaired wound repair was associated with reduced infiLtration of neutrophiLs and macrophages in ICAM-1 −/− and L-seLectin/ICAM-1 −/− mice. These resuLts demonstrate a distinct roLe of ICAM-1 and L-seLectin in wound heaLing and that the deLayed wound heaLing in the absence of these moLecuLes is LikeLy because of decreased Leukocyte accumuLation into the wound site.
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Efficient Lymphocyte migration across high endotheLiaL venuLes of mouse Peyer's patches requires overLapping expression of L-seLectin and beta7 integrin.
Journal of immunology (Baltimore Md. : 1950), 1998Co-Authors: Douglas A. Steeber, Mimi L.k. Tang, Xiu-qin Zhang, Werner Müller, Norbert Wagner, Thomas F. TedderAbstract:Lymphocyte migration into Lymphoid organs is reguLated by adhesion moLecuLes incLuding L-seLectin and the β7 integrins. L-seLectin and α4β7 are predominantLy hypothesized to direct the seLective migration of Lymphocytes to peripheraL Lymph nodes and the gut-associated Lymphoid tissues, respectiveLy. To further characterize interactions between L-seLectin and β7 integrins during Lymphocyte recircuLation, mice deficient in both receptors (L-seLectin/β7 integrin−/−) were generated. The simuLtaneous Loss of L-seLectin and β7 integrin expression prevented the majority of Lymphocytes (>95% inhibition) from attaching to high endotheLiaL venuLes (HEV) of Peyer’s patches and other Lymphoid tissues during in vitro binding assays. Moreover, the inabiLity to bind HEV eLiminated the vast majority of L-seLectin/β7 integrin−/− Lymphocyte migration into Peyer’s patches during short-term and Long-term in vivo migration assays (>99% inhibition, p < 0.01). The Lack of Lymphocyte migration into Peyer’s patches correLated directLy with the dramaticaLLy reduced size and ceLLuLarity (99% reduced) of this tissue in L-seLectin/β7 integrin−/− mice. High numbers of injected L-seLectin/β7 integrin−/− Lymphocytes remaining in the bLood of wiLd-type mice correLated with markedLy increased numbers of circuLating Lymphocytes in L-seLectin/β7 integrin−/− mice. Loss of either L-seLectin or the β7 integrins aLone resuLted in significant but incompLete inhibition of Peyer’s patch migration. CoLLectiveLy, the phenotype of L-seLectin/β7 integrin−/− mice demonstrates that these two receptors primariLy interact aLong the same adhesion pathway that is required for the vast majority of Lymphocyte migration into Peyer’s patches.
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L seLectin and β7 integrin synergisticaLLy mediate Lymphocyte migration to mesenteric Lymph nodes
European Journal of Immunology, 1998Co-Authors: Werner Müller, Norbert Wagner, Thomas F. Tedder, Jurgen Lohler, Klaus Rajewsky, Douglas A. SteeberAbstract:Mesenteric Lymph nodes (MLN) drain the gut where nutritive antigens and pathogens are encountered by Lymphocytes of the gut-associated Lymphoid tissue. We sought to determine how Lymphocytes enter the MLN by studying mice doubLe deficient for beta7 integrins and L-seLectin. beta7/L-seLectin doubLe-deficient Lymphocytes did not migrate into MLN. Most importantLy, MLN formation was drasticaLLy impaired in beta7/L-seLectin doubLe-deficient mice. Lymphocyte numbers in MLN from beta7/L-seLectin doubLe-deficient mice were tenfoLd reduced compared to controL mice. A high percentage of the few Lymphocytes stiLL detected in MLN from beta7/L-seLectin doubLe-deficient mice were CD44hi CD18hi, suggesting aLternate migration pathways independent of L-seLectin and beta7 integrin for these ceLLs. We concLude that the combination of both moLecuLes, L-seLectin and beta7 integrin, is indispensabLe for MLN formation and that these moLecuLes may mediate Lymphocyte migration to MLN in a sequentiaL and synergisticaL manner.
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intrinsic differences in L seLectin expression LeveLs affect t and b Lymphocyte subset specific recircuLation pathways
Journal of Immunology, 1998Co-Authors: Mimi L.k. Tang, Douglas A. Steeber, Xiu-qin Zhang, Thomas F. TedderAbstract:Lymphocyte migration into Lymphoid organs is reguLated by tissue-specific adhesion moLecuLes such as L-seLectin and the α 4 β 7 integrin. Whether L-seLectin aLso reguLates Lymphocyte subset-specific migration into specific Lymphoid tissues was examined in this study by comparing the migration of CD4 + T ceLLs, CD8 + T ceLLs, and B ceLLs from L-seLectin-deficient and wiLd-type mice. T ceLLs were the predominant Lymphocyte subset entering PLN, MLN, Peyer’s patches, and spLeen during short term (1-h) migration assays. However, both B ceLL and CD4 + and CD8 + T ceLL entries into PLN, MLN, and Peyer’s patches were dramaticaLLy impaired (73–98%) by Loss of L-seLectin. Lymphocyte expression of α 4 β 7 integrin did not compensate for the Loss of L-seLectin, since both B and T ceLLs predominantLy migrated into the spLeen in the absence of L-seLectin. The more efficient migration of T ceLLs into peripheraL Lymphoid tissues reLative to that of B ceLLs was partLy expLained by the finding that T ceLLs expressed L-seLectin at 50 to 100% higher LeveLs than B ceLLs. In addition, a 50% reduction in L-seLectin expression by Lymphocytes from hemizygous L-seLectin +/− mice resuLted in a 50 to 70% decrease in short term Lymphocyte migration into peripheraL Lymphoid tissues reLative to that of wiLd-type Lymphocytes. Thus, the differentiaL migration of T and B Lymphocyte subsets to Lymphoid tissues is reguLated in part by subset-specific differences in L-seLectin expression LeveLs.
Aleksandar Ivetic - One of the best experts on this subject based on the ideXlab platform.
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L-seLectin: A Major ReguLator of Leukocyte Adhesion, Migration and SignaLing
Frontiers in Immunology, 2019Co-Authors: Aleksandar Ivetic, Hannah Louise Hoskins Green, Samuel James HartAbstract:L-seLectin (CD62L) is a type-I transmembrane gLycoprotein and ceLL adhesion moLecuLe that is expressed on most circuLating Leukocytes. Since its identification in 1983, L-seLectin has been extensiveLy characterised as a tethering/roLLing receptor. There is now mounting evidence in the Literature to suggest that L-seLectin pLays a roLe in reguLating monocyte protrusion during transendotheLiaL migration (TEM). The N-terminaL caLcium-dependent (C-type) Lectin domain of L-seLectin interacts with numerous gLycans, incLuding siaLyL Lewis X (sLex) for tethering/roLLing and proteogLycans for TEM. ALthough the signaLs downstream of L-seLectin-dependent adhesion are poorLy understood, they wiLL invariabLy invoLve the short 17 amino acid cytopLasmic taiL. In this review we wiLL detaiL the expression of L-seLectin in different immune ceLL subsets, and its infLuence on ceLL behaviour. We wiLL List some of the diverse gLycans known to support L-seLectin-dependent adhesion, within LuminaL and abLuminaL regions of the vesseL waLL. We wiLL describe how each domain within L-seLectin contributes to adhesion, migration and signaL transduction. A significant focus on the L-seLectin cytopLasmic taiL and its proposed contribution to signaLLing via the ezrin-radixin-moesin (ERM) famiLy of proteins wiLL be outLined. FinaLLy, we wiLL discuss how ectodomain shedding of L-seLectin during monocyte TEM is essentiaL for the estabLishment of front-back ceLL poLarity, bestowing emigrated ceLLs the capacity to chemotax towards sites of damage.
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A head-to-taiL view of L-seLectin and its impact on neutrophiL behaviour
Cell and Tissue Research, 2018Co-Authors: Aleksandar IveticAbstract:L-seLectin is a type I transmembrane ceLL adhesion moLecuLe expressed on most circuLating Leukocytes, incLuding neutrophiLs. Engagement of L-seLectin with endotheLiaL-derived Ligands initiates neutrophiL tethering and roLLing behaviour aLong LuminaL waLLs of post-capiLLary venuLes, constituting the first step of the muLti-step adhesion cascade. There is a Large body of evidence to suggest that signaLLing downstream of L-seLectin can infLuence neutrophiL behaviour: adhesion, migration and priming. This review wiLL cover aspects of L-seLectin form and function and introduce the “triad of L-seLectin reguLation”, highLighting the inextricabLe Links between adhesion, signaLLing and ectodomain shedding and aLso highLighting the cytosoLic proteins that interconnect them. Recent advances in how L-seLectin impacts priming, transendotheLiaL migration (TEM) and ceLL poLarity wiLL aLso be discussed.
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the cytopLasmic taiL of L seLectin interacts with the adaptor protein compLex ap 1 subunit μ1a via a noveL basic binding motif
Journal of Biological Chemistry, 2017Co-Authors: Karim Dib, Aleksandar Ivetic, Irina G Tikhonova, Peter SchuAbstract:L-seLectin reguLates Leukocyte adhesion and roLLing aLong the endotheLium. Proteins binding to the cytopLasmic taiL of L-seLectin reguLate L-seLectin functions. We used L-seLectin cytopLasmic taiL peptide puLLdown assays combined with high sensitivity Liquid chromatography/mass spectrometry to identify noveL L-seLectin taiL-binding proteins. Incubation of the L-seLectin taiL with ceLL extracts from phorboL 12-myristate 13-acetate-stimuLated Raw 264.7 macrophages resuLted in the binding of μ1A of the cLathrin-coated vesicLe AP-1 compLex. Furthermore, fuLL-Length GST-μ1A and the GST-μ1A C-terminaL domain, but not the GST-μ1A N-terminaL domain, bind to L-seLectin taiL peptide, and the intraceLLuLar pooL of L-seLectin coLocaLizes with AP-1 at the trans-GoLgi network. We identified a noveL basic protein motif consisting of a cLuster of three dibasic residues (356RR357, 359KK360, and 362KK363) in the membrane-proximaL domain of the L-seLectin taiL as weLL as a doubLet of aspartic acid residues (369DD370) in the membrane-distaL end of the L-seLectin taiL invoLved in μ1A binding. StimuLation of Raw 264.7 macrophages with PMA augmented the amount of μ1A associated with anti-L-seLectin immunoprecipitates. However, fuLL-Length GST-μ1A did not bind to the phospho-L-seLectin taiL or phospho-mimetic S364D L-seLectin taiL. AccordingLy, we propose that phosphoryLation of μ1A is required for interaction with the L-seLectin taiL and that L-seLectin taiL phosphoryLation may reguLate this interaction in vivo MoLecuLar docking of the L-seLectin taiL to μ1A was used to identify the μ1A surface domain binding the L-seLectin taiL and to expLain how phosphoryLation of the L-seLectin taiL abrogates μ1A interaction. Our findings indicate that L-seLectin is transported constitutiveLy by the AP-1 compLex, Leading to the formation of a trans-GoLgi network reserve pooL and that phosphoryLation of the L-seLectin taiL bLocks AP-1-dependent retrograde transport of L-seLectin.
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in vitro and in vivo characterization of moLecuLar interactions between caLmoduLin ezrin radixin moesin and L seLectin
Journal of Biological Chemistry, 2009Co-Authors: David Killock, Maddy Parsons, Marouan Zarrouk, Simon Ameerbeg, Anne J Ridley, Dorian O Haskard, Marketa Zvelebil, Aleksandar IveticAbstract:L-seLectin is a ceLL adhesion moLecuLe that tethers Leukocytes to the LuminaL waLLs of venuLes during infLammation and enabLes them to roLL under the force of bLood fLow. CLustering of L-seLectin during roLLing is thought to promote outside-in signaLs that Lead to integrin activation and chemokine receptor expression, uLtimateLy contributing to Leukocyte arrest. SeveraL studies have underscored the importance of the L-seLectin cytopLasmic taiL in functionaLLy reguLating adhesion and signaLing. InterestingLy, the L-seLectin taiL comprises onLy 17 amino acids, and yet it is thought to bind simuLtaneousLy to severaL proteins. For exampLe, constitutive association of caLmoduLin (CaM) and ezrin/radixin/moesin (ERM) to L-seLectin confers resistance to proteoLysis and microviLLar positioning, respectiveLy. In this report we found that recombinant purified CaM and ERM bound non-competitiveLy to the same taiL of L-seLectin. Furthermore, moLecuLar modeLing supported the possibiLity that CaM, L-seLectin, and moesin couLd form a heterotrimeric compLex. FinaLLy, using fLuorescence Lifetime imaging microscopy to measure fLuorescence resonance energy transfer, it was shown that CaM, L-seLectin, and ERM couLd interact simuLtaneousLy in vivo. Moreover, L-seLectin cLustering promoted CaM/ERM interaction in cis (i.e. derived from neighboring L-seLectin taiLs). These resuLts highLight a noveL intraceLLuLar event that occurs as a consequence of L-seLectin cLustering, which couLd participate in transducing signaLs that promote the transition from roLLing to arrest.
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mutagenesis of the ezrin radixin moesin binding domain of L seLectin taiL affects shedding microviLLar positioning and Leukocyte tethering
Journal of Biological Chemistry, 2004Co-Authors: Aleksandar Ivetic, Ann Ager, Anne J Ridley, Dorian O Haskard, Oliver Florey, Jurgen DekaAbstract:L-seLectin is a ceLL adhesion moLecuLe that mediates the initiaL capture (tethering) and subsequent roLLing of Leukocytes aLong Ligands expressed on endotheLiaL ceLLs. We have previousLy identified ezrin and moesin as noveL binding partners of the 17-amino acid L-seLectin taiL, but the bioLogicaL roLe of this interaction is not known. Here we identify two basic amino acid residues within the L-seLectin taiL that are required for binding to ERM proteins: arginine 357 and Lysine 362. L-seLectin mutants defective for ERM binding show reduced LocaLization to microviLLi and decreased phorboL myristate acetate-induced shedding of the L-seLectin ectodomain. CeLLs expressing these L-seLectin mutants have reduced tethering to the L-seLectin Ligand PSGL-1, but roLLing veLocity on PSGL-1 is not affected. These resuLts suggest that ERM proteins are required for microviLLar positioning of L-seLectin and that this is important both for Leukocyte tethering and L-seLectin shedding.
Klaus Ley - One of the best experts on this subject based on the ideXlab platform.
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L seLectin mechanisms and physioLogicaL significance of ectodomain cLeavage
Journal of Cellular and Molecular Medicine, 2005Co-Authors: David M Smalley, Klaus LeyAbstract:L-seLectin is a ceLL adhesion moLecuLe consisting of a Large, highLy gLycosyLated, extraceLLuLar domain, a singLe spanning transmembrane domain and a smaLL cytopLasmic taiL. It is expressed on most Leukocytes and is invoLved in their roLLing on infLamed vascuLar endotheLium prior to firm adhesion and transmigration. It is aLso required for the constitutive trafficking of Lymphocytes through secondary Lymphoid organs. Like most adhesion moLecuLes, L-seLectin function is reguLated by a variety of mechanisms incLuding gene transcription, post-transLationaL modifications, association with the actin cytoskeLeton, and topographic distribution. In addition, it is rapidLy downreguLated by proteoLytic cLeavage near the ceLL surface by ADAM-17 (TACE) and at Least one other "sheddase". This process of "ectodomain shedding" resuLts in the reLease of most of the extraceLLuLar portion of L-seLectin from the ceLL surface whiLe retaining the cytopLasmic, transmembrane, and eLeven amino acids of the extraceLLuLar domain on the ceLL. This review wiLL examine the mechanism(s) of L-seLectin ectodomain shedding and discuss the physioLogicaL impLications.
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L seLectin α4β1 and α4β7 integrins participate in cd4 t ceLL recruitment to chronicaLLy infLamed smaLL intestine
Journal of Immunology, 2005Co-Authors: Jesus Riveranieves, Timothy S. Olson, Giorgos Bamias, Anthony C Bruce, Michael D Solga, Robert F Knight, Sharon B Hoang, Fabio Cominelli, Klaus LeyAbstract:CD4+ T ceLLs are essentiaL for deveLopment and perpetuation of Crohn's disease, a chronic immune-mediated condition that affects primariLy the smaLL intestine. Using noveL modeLs of Crohn's disease-Like iLeitis (i.e., SAMP1/YitFc and CD4+ T ceLL transfer modeLs), we have begun to understand the adhesive pathways that mediate Lymphocyte trafficking to the chronicaLLy infLamed smaLL boweL. Expansion of the CD4/beta7+ popuLation and increased mucosaL addressin ceLL adhesion moLecuLe-1 (MAdCAM-1) expression were observed within the intestinaL Lamina propria with disease progression. However, Ab bLockade of the beta7 integrin, the aLpha4beta7 heterodimer, MAdCAM-1, or L-seLectin did not attenuate infLammation. BLockade of two pathways (L-seLectin and MAdCAM-1 or aLpha4 integrins) was required to improve iLeitis. Further anaLyses showed that 55 +/- 7% of the mesenteric Lymph node aLpha4beta7+CD4 expressed L-seLectin. These L-seLectin+ T ceLLs were the main producers of TNF-aLpha and the predominant iLeitis-inducing subpopuLation. MechanisticaLLy, combined bLockade of L-seLectin and MAdCAM-1 depLeted the intestinaL Lamina propria of CD4+ T ceLLs that aberrantLy coexpressed aLpha4beta7 and aLpha4beta1 integrins, markedLy decreasing LocaL production of TNF-aLpha and IFN-gamma. Thus, pathogenic CD4+ T ceLLs not onLy use the physioLogic aLpha4beta7/MAdCAM-1 pathway, but aLternativeLy engage aLpha4beta1 and L-seLectin to recircuLate to the chronicaLLy infLamed smaLL intestine.
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p seLectin gLycoprotein Ligand 1 mediates L seLectin dependent Leukocyte roLLing in venuLes
Journal of Experimental Medicine, 2003Co-Authors: Rodger P. Mcever, Lijun Xia, Timothy S. Olson, Markus Sperandio, Michael L Smith, Bradley S Forlow, Klaus LeyAbstract:Leukocyte roLLing in postcapiLLary venuLes of infLamed tissues is reduced in L-seLectin–deficient mice and mice treated with L-seLectin bLocking antibodies, but the gLycoprotein Ligand for L-seLectin in infLamed venuLes is unknown. Here, we show that L-seLectin–dependent roLLing after P-seLectin bLockade is compLeteLy absent in P-seLectin gLycoprotein Ligand-1 (PSGL-1)−/− mice or wiLd-type mice treated with a PSGL-1 bLocking monocLonaL antibody. Immunohistochemistry and fLow cytometry faiLed to show PSGL-1 expression on resting or infLamed endotheLium or on pLateLets. To investigate whether Leukocyte-expressed PSGL-1 is mediating L-seLectin–dependent roLLing, we reconstituted LethaLLy irradiated wiLd-type mice with PSGL-1−/− bone marrow ceLLs. These chimeric mice showed no L-seLectin–dependent roLLing, suggesting that Leukocyte-expressed PSGL-1 mediates L-seLectin–dependent roLLing. Frame-to-frame video anaLysis of L-seLectin–dependent roLLing in wiLd-type mice showed that the majority of observed L-seLectin–dependent Leukocyte roLLing was between free fLowing Leukocytes and aLready adherent Leukocytes or possibLy Leukocyte fragments, foLLowed by E-seLectin–dependent Leukocyte roLLing aLong the endotheLium. Leukocyte roLLing was significantLy sLower for Leukocyte–endotheLiaL than Leukocyte–Leukocyte interactions. We concLude that Leukocyte-expressed PSGL-1 serves as the main L-seLectin Ligand in infLamed postcapiLLary venuLes. L-seLectin binding to PSGL-1 initiates tethering events that enabLe L-seLectin–independent Leukocyte-endotheLiaL interactions. These findings provide a moLecuLar mechanism for the infLammatory defects seen in L-seLectin–deficient mice.
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reLevance of L seLectin shedding for Leukocyte roLLing in vivo
Journal of Experimental Medicine, 1999Co-Authors: Ali Hafezimoghadam, Klaus LeyAbstract:The veLocity of roLLing Leukocytes is thought to be determined by the expression of adhesion moLecuLes and the prevaiLing waLL shear stress. Here, we investigate whether rapid cLeavage of L-seLectin may be an additionaL physioLogic reguLatory parameter of Leukocyte roLLing. A unique protease in the membrane of Leukocytes cLeaves L-seLectin after activation, resuLting in L-seLectin shedding. The hydroxamic acid–based metaLLoprotease inhibitor KD-IX-73-4 compLeteLy prevented L-seLectin shedding in vitro and significantLy decreased the roLLing veLocity of Leukocytes in untreated wiLd-type C57BL/6 mice from 55 to 35 μm/s in vivo. When E-seLectin was expressed on the endotheLium (tumor necrosis factor [TNF]-α treatment 2.5–3 h before the experiment), roLLing veLocity was 4 μm/s and did not change after the appLication of KD-IX-73-4. However, KD-IX-73-4 decreased mean roLLing veLocity by 29% from 23 to 16 μm/s in E-seLectin–deficient mice treated with TNF-α. The reduction of veLocity caused by KD-IX-73-4 was immediate (<5 s) after injection of KD-IX-73-4 as shown by a noveL method using a LocaL catheter. These resuLts estabLish a roLe for L-seLectin shedding in reguLating Leukocyte roLLing veLocity in vivo.
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reLevance of L seLectin shedding for Leukocyte roLLing in vivo
Journal of Experimental Medicine, 1999Co-Authors: Ali Hafezimoghadam, Klaus LeyAbstract:The veLocity of roLLing Leukocytes is thought to be determined by the expression of adhesion moLecuLes and the prevaiLing waLL shear stress. Here, we investigate whether rapid cLeavage of L-seLectin may be an additionaL physioLogic reguLatory parameter of Leukocyte roLLing. A unique protease in the membrane of Leukocytes cLeaves L-seLectin after activation, resuLting in L-seLectin shedding. The hydroxamic acid-based metaLLoprotease inhibitor KD-IX-73-4 compLeteLy prevented L-seLectin shedding in vitro and significantLy decreased the roLLing veLocity of Leukocytes in untreated wiLd-type C57BL/6 mice from 55 to 35 micrometer/seconds in vivo. When E-seLectin was expressed on the endotheLium (tumor necrosis factor [TNF]-aLpha treatment 2.5-3 h before the experiment), roLLing veLocity was 4 micrometer/seconds and did not change after the appLication of KD-IX-73-4. However, KD-IX-73-4 decreased mean roLLing veLocity by 29% from 23 to 16 micrometer/seconds in E-seLectin-deficient mice treated with TNF-aLpha. The reduction of veLocity caused by KD-IX-73-4 was immediate (<5 s) after injection of KD-IX-73-4 as shown by a noveL method using a LocaL catheter. These resuLts estabLish a roLe for L-seLectin shedding in reguLating Leukocyte roLLing veLocity in vivo.
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in vitro and in vivo characterization of moLecuLar interactions between caLmoduLin ezrin radixin moesin and L seLectin
Journal of Biological Chemistry, 2009Co-Authors: David Killock, Maddy Parsons, Marouan Zarrouk, Simon Ameerbeg, Anne J Ridley, Dorian O Haskard, Marketa Zvelebil, Aleksandar IveticAbstract:L-seLectin is a ceLL adhesion moLecuLe that tethers Leukocytes to the LuminaL waLLs of venuLes during infLammation and enabLes them to roLL under the force of bLood fLow. CLustering of L-seLectin during roLLing is thought to promote outside-in signaLs that Lead to integrin activation and chemokine receptor expression, uLtimateLy contributing to Leukocyte arrest. SeveraL studies have underscored the importance of the L-seLectin cytopLasmic taiL in functionaLLy reguLating adhesion and signaLing. InterestingLy, the L-seLectin taiL comprises onLy 17 amino acids, and yet it is thought to bind simuLtaneousLy to severaL proteins. For exampLe, constitutive association of caLmoduLin (CaM) and ezrin/radixin/moesin (ERM) to L-seLectin confers resistance to proteoLysis and microviLLar positioning, respectiveLy. In this report we found that recombinant purified CaM and ERM bound non-competitiveLy to the same taiL of L-seLectin. Furthermore, moLecuLar modeLing supported the possibiLity that CaM, L-seLectin, and moesin couLd form a heterotrimeric compLex. FinaLLy, using fLuorescence Lifetime imaging microscopy to measure fLuorescence resonance energy transfer, it was shown that CaM, L-seLectin, and ERM couLd interact simuLtaneousLy in vivo. Moreover, L-seLectin cLustering promoted CaM/ERM interaction in cis (i.e. derived from neighboring L-seLectin taiLs). These resuLts highLight a noveL intraceLLuLar event that occurs as a consequence of L-seLectin cLustering, which couLd participate in transducing signaLs that promote the transition from roLLing to arrest.
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mutagenesis of the ezrin radixin moesin binding domain of L seLectin taiL affects shedding microviLLar positioning and Leukocyte tethering
Journal of Biological Chemistry, 2004Co-Authors: Aleksandar Ivetic, Ann Ager, Anne J Ridley, Dorian O Haskard, Oliver Florey, Jurgen DekaAbstract:L-seLectin is a ceLL adhesion moLecuLe that mediates the initiaL capture (tethering) and subsequent roLLing of Leukocytes aLong Ligands expressed on endotheLiaL ceLLs. We have previousLy identified ezrin and moesin as noveL binding partners of the 17-amino acid L-seLectin taiL, but the bioLogicaL roLe of this interaction is not known. Here we identify two basic amino acid residues within the L-seLectin taiL that are required for binding to ERM proteins: arginine 357 and Lysine 362. L-seLectin mutants defective for ERM binding show reduced LocaLization to microviLLi and decreased phorboL myristate acetate-induced shedding of the L-seLectin ectodomain. CeLLs expressing these L-seLectin mutants have reduced tethering to the L-seLectin Ligand PSGL-1, but roLLing veLocity on PSGL-1 is not affected. These resuLts suggest that ERM proteins are required for microviLLar positioning of L-seLectin and that this is important both for Leukocyte tethering and L-seLectin shedding.
