1 BMC Immunology 2011 Vol: 12(1):28. DOI: 10.1186/1471-2172-12-28

Nitric oxide/cGMP pathway signaling actively down-regulates α4β1-integrin affinity: an unexpected mechanism for inducing cell de-adhesion

Integrin activation in response to inside-out signaling serves as the basis for rapid leukocyte arrest on endothelium, migration, and mobilization of immune cells. Integrin-dependent adhesion is controlled by the conformational state of the molecule, which is regulated by seven-transmembrane Guanine nucleotide binding Protein-Coupled Receptors (GPCRs). α4β1-integrin (CD49d/CD29, Very Late Antigen-4, VLA-4) is expressed on leukocytes, hematopoietic progenitors, stem cells, hematopoietic cancer cells, and others. VLA-4 conformation is rapidly up-regulated by inside-out signaling through Gαi-coupled GPCRs and down-regulated by Gαs-coupled GPCRs. However, other signaling pathways, which include nitric oxide-dependent signaling, have been implicated in the regulation of cell adhesion. The goal of the current report was to study the effect of nitric oxide/cGMP signaling pathway on VLA-4 conformational regulation. Using fluorescent ligand binding to evaluate the integrin activation state on live cells in real-time, we show that several small molecules, which specifically modulate nitric oxide/cGMP signaling pathway, as well as a cell permeable cGMP analog, can rapidly down-modulate binding of a VLA-4 specific ligand on cells pre-activated through three Gαi-coupled receptors: wild type CXCR4, CXCR2 (IL-8RB), and a non-desensitizing mutant of formyl peptide receptor (FPR ΔST). Upon signaling, we detected rapid changes in the ligand dissociation rate. The dissociation rate after inside-out integrin de-activation was similar to the rate for resting cells. In a VLA-4/VCAM-1-specific myeloid cell adhesion system, inhibition of the VLA-4 affinity change by nitric oxide had a statistically significant effect on real-time cell aggregation. We conclude that nitric oxide/cGMP signaling pathway can rapidly down-modulate the affinity state of the VLA-4 binding pocket, especially under the condition of sustained Gαi-coupled GPCR signaling, generated by a non-desensitizing receptor mutant. This suggests a fundamental role of this pathway in de-activation of integrin-dependent cell adhesion.

Mentions
Figures
Figure 1: NO/cGMP signaling cascade and small molecules that modulate this pathway, used in the current study. Nitric oxide, generated by nitric oxide synthase, diffuses across the plasma membrane and through the cytoplasm. In leukocytes NO reacts with the active site of guanylyl cyclase (guanylate GC), and stimulates the production of the intracellular mediator cyclic GMP (cGMP). Next, cGMP interacts with the cGMP-dependent protein kinase (PKG), which phosphorylates multiple substrates, and participates in signal propagation. Cyclic nucleotide phosphodiesterases (PDEs, not shown) can rapidly hydrolyze cGMP and terminate signal propagation. The NO/cGMP signaling pathway can be specifically targeted using small molecules. The nitric oxide donor provides an exogenous source of NO. The activator of soluble guanylyl cyclase binds to GC, and induces enzyme activation in the absence of NO. The cell permeable analog of cGMP diffuses across the plasma membrane, and thus, activates cGMP-dependent signaling. Figure 2: Effect of nitric oxide addition on binding and dissociation of the LDV-FITC probe on U937 cells, treated with different Gαi-coupled receptor ligands. LDV-FITC probe binding and dissociation on U937 cells stably transfected with different GPCRs plotted as mean channel fluorescence (MCF) versus time. A, The experiment involved sequential addition of fluorescent LDV-FITC probe (4 nM, below saturation, added 2 min prior to addition of Gαi-coupled receptor ligand, fMLFF, 100 nM), and different concentrations of DEA-NONOate (nitric oxide donor). Dashed line indicates the non-specific binding of the LDV-FITC probe determined using an excess of unlabelled LDV competitor (as shown in D,E). B, The experiment involved sequential addition of fluorescent LDV-FITC probe (4 nM), CXCL12/SDF-1 (12 nM), and DEA-NONOate (250 μM, nitric oxide donor) or vehicle (control). Rapid and reversible binding of the probe reflects the VLA-4 affinity change [14]. C, The experiment involved sequential addition of the fluorescent LDV-FITC probe (4 nM), CXCL8/IL-8 (20 nM), and DEA-NONOate (250 μM, nitric oxide donor) or vehicle (control). D, The experiment involved sequential addition of the DEA-NONOate (250 μM, nitric oxide donor) or vehicle (control) at the 0 time point, and the fluorescent LDV-FITC probe (4 nM), CXCL12/SDF-1 (12 nM). Excess unlabelled competitor LDV (1 μM) is added at the end of the experiment to determine the non-specific binding of the probe (panels D, and E). E, The experiment involved sequential addition of DEA-NONOate (250 μM, nitric oxide donor) or vehicle (control) at the 0 time point, and the fluorescent LDV-FITC probe (4 nM), CXCL8/IL-8 (20 nM) (arrows). According to the unpaired t test, the means are significantly different (p<0.05) at the peak of activation (marked on panels D and E as “*”), and at the steady state (marked on panels B-E as “**”). Experiments shown in the different panels were performed using different instruments, and therefore MCF values are not identical. Figure 3: Effect of guanylyl cyclase activator on binding and dissociation of the LDV-FITC probe on U937 cells, treated with different Gαi-coupled receptor ligands. LDV-FITC probe binding and dissociation on U937 cells stably transfected with different GPCRs plotted as mean channel fluorescence (MCF) versus time. A, The experiment involved sequential addition of the fluorescent LDV-FITC probe (4 nM, below saturation, added 2 min prior to addition of the Gαi-coupled receptor ligand, fMLFF, 100 nM), and different concentrations of BAY 41-2272 (guanylyl cyclase activator).  B, The experiment involved sequential addition of the BAY 41-2272 (100 μM, guanylyl cyclase activator) or vehicle (control) at the 0 time point, and the fluorescent LDV-FITC probe (4 nM), and CXCL12/SDF-1 (12 nM). C, The experiment involved sequential addition of BAY 41-2272 (100 μM, guanylyl cyclase activator) or vehicle (control) at the 0 time point, and the fluorescent LDV-FITC probe (4 nM), and CXCL8/IL-8 (20 nM). The means are significantly different (p<0.05) at the peak of activation (marked on panels B and C as “*”), and at the steady state (marked in panels B and C as “**”). D, Kinetic analysis of binding and dissociation of the LDV-FITC probe. Cells were sequentially treated with the LDV-FITC probe (25 nM, near saturation), the Gαi-coupled receptor ligand (fMLFF, 100 nM), BAY 41-2272 (guanylyl cyclase activator, 50 μM). At time points indicated by arrows, cells were treated with excess unlabeled LDV containing small molecule (2 μM), and the dissociation of the fluorescent molecule was followed. Dissociation rate constants (koff) were obtained by fitting dissociation curves to a single exponential decay equation. E, Dissociation rate values, obtained in experiments analogous to panel D, summarized as a bar graph showing mean and SEM (n=4). Colors of the dissociation curves in panel D and bars on panel E are matching. The difference between koffs for “resting” and “fMLFF activated”, and between “fMLFF activated” and “fMLFF activated and treated with BAY 41-2272” is statistically significant (P < 0.05) as calculated by one-way ANOVA. Figure 4: Effect of the cell permeable analog of cGMP on binding and dissociation of the LDV-FITC probe on U937 cells stably transfected with the non-desensitizing mutant of FPR. LDV-FITC probe binding and dissociation on U937 cells stably transfected with the non-desensitizing mutant of FPR plotted as mean channel fluorescence (MCF) versus time. The experiment involved sequential addition of the fluorescent LDV-FITC probe (4 nM, below saturation, added 2 min prior to addition of the Gαi-coupled receptor ligand, fMLFF, 100 nM), and different concentrations of dibutyrylguanosine 3',5'-cyclic monophosphate (cell permeable cGMP analog) (arrows). Control cells were treated with vehicle. The MCF value corresponding to cell autofluorescence is indicated by the horizontal arrow. Dashed line indicates the non-specific binding of the LDV-FITC probe determined using excess unlabelled LDV competitor (as shown on B). Rapid and reversible binding of the probe reflects the VLA-4 affinity change 14. Curves are means out of two independent determinations calculated on a point-by-point basis (n = 2). Figure 5: Changes in cell adhesion between U937 FPR (ΔST) and VCAM-1-transfected B78H1 cells in the resting state and in response to receptor stimulation. Real-time aggregation experiments were conducted as described under "Methods". U937/ΔST FPR stably transfected cells, which constitutively express VLA-4, were labeled with red fluorescent dye, and B78H1/VCAM-1 transfectants were stained with green fluorescent dye. Labeled cells were preincubated for 10 min at 37°C with fMLFF only (100 nM, activated control), DMSO (vehicle, resting cells control), or with fMLFF and DEA-NONOate (250 μM, nitric oxide donor) in a manner analogous to the experiment showed in A. Next, cells were mixed and real-time cell aggregation (red and green double positive events) was followed. To determine the level of VLA-4 dependent cell aggregation, 6 min after cell mixing, excess unlabelled VLA-4 specific ligand was added (arrow, LDV block, 2 μM). This induced rapid cellular disaggregation to the level of non-specific binding. A representative experiment out of three experiments is shown.
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References
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    • . . . Previously, we described and characterized in detail a model ligand an LDV-FITC containing small molecule ( 14 42 43 44 , and references therein) for the detection of VLA-4 conformational regulation . . .
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    • . . . Previously, we described and characterized in detail a model ligand an LDV-FITC containing small molecule ( 14 42 43 44 , and references therein) for the detection of VLA-4 conformational regulation . . .
  45. K Lin; HS Ateeq; SH Hsiung; LT Chong; CN Zimmerman; A Castro; WC Lee; CE Hammond; S Kalkunte; LL Chen Selective, tight-binding inhibitors of integrin alpha4beta1 that inhibit allergic airway responses J Med Chem 42, 920-934 (1999) .
    • . . . This VLA-4 specific fluorescent probe was based on a highly specific α4β1-integrin inhibitor BIO1211, which contains the Leu-Asp-Val (LDV) ligand binding motif from the alternatively spliced connecting segment-1 (CS-1) peptide of cellular fibronectin 17 45 . . .
  46. A Chigaev; G Zwartz; SW Graves; DC Dwyer; H Tsuji; TD Foutz; BS Edwards; ER Prossnitz; RS Larson; LA Sklar Alpha4beta1 integrin affinity changes govern cell adhesion J Biol Chem 278, 38174-38182 (2003) .
    • . . . We established that integrin affinity changes, detected using this probe, vary in parallel with the natural VLA-4 ligand, human VCAM-1 46 . . .
    • . . . The slower koff corresponds to higher ligand binding affinity 14 17 46 . . .
    • . . . This model system has been described and characterized previously 42 46 54 55 . . . .
    • . . . The overall extent of activated cell aggregation was similar to previously published data 46 . . .
    • . . . The VLA-4 specific ligand 14 46 47 4-((N'-2-methylphenyl)ureido)-phenylacetyl-L-leucyl-L-aspartyl-L-valyl-L-prolyl-L-alanyl-L-alanyl-L-lysine (LDV containing small molecule), and its FITC-conjugated analog (LDV-FITC) were synthesized at Commonwealth Biotechnologies . . .
    • . . . Kinetic analysis of the binding and dissociation of the LDV-FITC probe was described previously 14 46 . . .
    • . . . The cell suspension adhesion assay has been described previously 46 55 . . .
  47. A Chigaev; T Buranda; DC Dwyer; ER Prossnitz; LA Sklar FRET detection of cellular alpha4-integrin conformational activation Biophys J 85, 3951-3962 (2003) .
    • . . . In the absence of receptor desensitization, the effect of nitric oxide was more evident in cells transfected with a non-desensitizing mutant of FPR (vehicle, Figure 2A) 47 48 . . .
    • . . . The VLA-4 specific ligand 14 46 47 4-((N'-2-methylphenyl)ureido)-phenylacetyl-L-leucyl-L-aspartyl-L-valyl-L-prolyl-L-alanyl-L-alanyl-L-lysine (LDV containing small molecule), and its FITC-conjugated analog (LDV-FITC) were synthesized at Commonwealth Biotechnologies . . .
  48. ER Prossnitz Desensitization of N-formylpeptide receptor-mediated activation is dependent upon receptor phosphorylation J Biol Chem 272, 15213-15219 (1997) .
    • . . . In the absence of receptor desensitization, the effect of nitric oxide was more evident in cells transfected with a non-desensitizing mutant of FPR (vehicle, Figure 2A) 47 48 . . .
  49. SE Martinez; AY Wu; NA Glavas; XB Tang; S Turley; WG Hol; JA Beavo The two GAF domains in phosphodiesterase 2A have distinct roles in dimerization and in cGMP binding Proc Natl Acad Sci USA 99, 13260-13265 (2002) .
    • . . . Intracellular cGMP can directly stimulate the catalytic activity of several cyclic nucleotide phosphodiesterases (PDEs) that hydrolyze cGMP 49 50 51 . . .
    • . . . Activation of PDEs directly by cGMP binding, or indirectly after being phosphorylated by a cGMP dependent kinase (PKG), has been previously reported 49 50 51 52 53 . . . .
  50. SD Rybalkin; IG Rybalkina; M Shimizu-Albergine; XB Tang; JA Beavo PDE5 is converted to an activated state upon cGMP binding to the GAF A domain EMBO J 22, 469-478 (2003) .
    • . . . Intracellular cGMP can directly stimulate the catalytic activity of several cyclic nucleotide phosphodiesterases (PDEs) that hydrolyze cGMP 49 50 51 . . .
    • . . . Another possibility is activation of PDEs through phosphorylation by cGMP-dependent protein kinase (PKG) (Figure 1) 50 51 52 53 . . . .
    • . . . Activation of PDEs directly by cGMP binding, or indirectly after being phosphorylated by a cGMP dependent kinase (PKG), has been previously reported 49 50 51 52 53 . . . .
  51. F Mullershausen; A Friebe; R Feil; WJ Thompson; F Hofmann; D Koesling Direct activation of PDE5 by cGMP: long-term effects within NO/cGMP signaling J Cell Biol 160, 719-727 (2003) .
    • . . . Intracellular cGMP can directly stimulate the catalytic activity of several cyclic nucleotide phosphodiesterases (PDEs) that hydrolyze cGMP 49 50 51 . . .
    • . . . Another possibility is activation of PDEs through phosphorylation by cGMP-dependent protein kinase (PKG) (Figure 1) 50 51 52 53 . . . .
    • . . . Activation of PDEs directly by cGMP binding, or indirectly after being phosphorylated by a cGMP dependent kinase (PKG), has been previously reported 49 50 51 52 53 . . . .
  52. KS Murthy Activation of phosphodiesterase 5 and inhibition of guanylate cyclase by cGMP-dependent protein kinase in smooth muscle Biochem J 360, 199-208 (2001) .
    • . . . Another possibility is activation of PDEs through phosphorylation by cGMP-dependent protein kinase (PKG) (Figure 1) 50 51 52 53 . . . .
    • . . . Activation of PDEs directly by cGMP binding, or indirectly after being phosphorylated by a cGMP dependent kinase (PKG), has been previously reported 49 50 51 52 53 . . . .
  53. KS Murthy Modulation of soluble guanylate cyclase activity by phosphorylation Neurochem Int 45, 845-851 (2004) .
    • . . . Another possibility is activation of PDEs through phosphorylation by cGMP-dependent protein kinase (PKG) (Figure 1) 50 51 52 53 . . . .
    • . . . Activation of PDEs directly by cGMP binding, or indirectly after being phosphorylated by a cGMP dependent kinase (PKG), has been previously reported 49 50 51 52 53 . . . .
  54. BS Edwards; FW Kuckuck; ER Prossnitz; A Okun; JT Ransom; LA Sklar Plug flow cytometry extends analytical capabilities in cell adhesion and receptor pharmacology Cytometry 43, 211-216 (2001) .
    • . . . This model system has been described and characterized previously 42 46 54 55 . . . .
  55. G Zwartz; A Chigaev; T Foutz; RS Larson; R Posner; LA Sklar Relationship between Molecular and Cellular Dissociation Rates for VLA-4/VCAM-1 Interaction in the Absence of Shear Stress Biophys J 86, 1243-1252 (2004) .
    • . . . This model system has been described and characterized previously 42 46 54 55 . . . .
    • . . . Next, the cell populations were mixed, and the appearance of double positive events, representing cellular aggregates, was followed in real-time by flow cytometry (see Figure 1, 2, 3 in 55 for method details) . . .
    • . . . The cell suspension adhesion assay has been described previously 46 55 . . .
  56. C Laudanna; R Alon Right on the spot. Chemokine triggering of integrin-mediated arrest of rolling leukocytes Thromb Haemost 95, 5-11 (2006) .
    • . . . A current paradigm of the inside-out activation of integrins implies an instantaneous triggering of integrin conformational changes, where a chemokine signal appears to be closely apposed to the integrin 56 . . .
  57. K Ley; C Laudanna; MI Cybulsky; S Nourshargh Getting to the site of inflammation: the leukocyte adhesion cascade updated Nat Rev Immunol 7, 678-689 (2007) .
    • . . . An "updated" adhesion cascade includes several steps in addition to the traditional tethering, rolling, and arrest 57 . . .
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    • . . . While integrin adhesion research is largely focused on activating pathways, the inhibitory Gαs-coupled GPCR/cAMP-dependent signaling pathways is acknowledged for platelet regulation 58 . . .
  59. D Cox; M Brennan; N Moran Integrins as therapeutic targets: lessons and opportunities Nat Rev Drug Discov 9, 804-820 (2010) .
    • . . . The relative lack of interest in the integrin deactivation pathways is potentially compensated by the identification of antagonists that competitively block adhesive interactions, and thus, provide a desirable therapeutic effect 59 . . . .
  60. LA Sklar; PA Hyslop; ZG Oades; GM Omann; AJ Jesaitis; RG Painter; CG Cochrane Signal transduction and ligand-receptor dynamics in the human neutrophil. Transient responses and occupancy-response relations at the formyl peptide receptor J Biol Chem 260, 11461-11467 (1985) .
    • . . . In some cases, this fraction may be less than 0.1% of the total number of receptors 60 . . .
    • . . . A plausible scenario would be to take advantage of natural regulatory pathways to counteract unwanted signaling, especially because antagonistic pathways potentially have similar amplification capacity 60 61 . . . .
  61. LA Sklar; GM Omann Kinetics and amplification in neutrophil activation and adaptation Semin Cell Biol 1, 115-123 (1990) .
    • . . . This is dependent on significant signal amplification for both stimulatory and inhibitory pathways 61 . . .
    • . . . A plausible scenario would be to take advantage of natural regulatory pathways to counteract unwanted signaling, especially because antagonistic pathways potentially have similar amplification capacity 60 61 . . . .
  62. A Chigaev; Y Wu; DB Williams; Y Smagley; LA Sklar Discovery of very late antigen-4 (VLA-4, {alpha}4{beta}1 integrin) allosteric antagonists J Biol Chem 286, 5455-5463 (2011) .
    • . . . Blocking of the VLA-4/VCAM-1 interaction using anti-VLA-4 antibodies, small molecule competitive as well as allosteric VLA-4 antagonists, results in the mobilization of progenitors into the peripheral blood 28 29 30 31 32 62 . . .
  63. J Ervens; R Seifert Differential modulation by N4, 2'-O-dibutyryl cytidine 3':5'-cyclic monophosphate of neutrophil activation Biochem Biophys Res Commun 174, 258-267 (1991) .
    • . . . For example, cyclic cytidine 3',5'-monophosphate (cCMP) was shown to modulate leukocyte activation 63 , to inhibit platelet activation, and to signal through cGMP-dependent protein kinase (PKG) 64 . . .
  64. M Desch; E Schinner; F Kees; F Hofmann; R Seifert; J Schlossmann Cyclic cytidine 3',5'-monophosphate (cCMP) signals via cGMP kinase I FEBS Lett 584, 3979-3984 (2010) .
    • . . . For example, cyclic cytidine 3',5'-monophosphate (cCMP) was shown to modulate leukocyte activation 63 , to inhibit platelet activation, and to signal through cGMP-dependent protein kinase (PKG) 64 . . .
  65. RM Gaion; G Krishna Cytidylate cyclase: possible artifacts in the methodology Science 203, 672-673 (1979) .
    • . . . Although, the existence and metabolism of cCMP in mammals is uncertain 65 , recent report suggests that the adenylyl cyclase toxin edema factor and adenylate cyclase-hemolysin (CyaA), produced by Bacillus anthracis and Bordetella pertussis, can catalyze the formation of cCMP, and other cyclic nucleotides 66 . . .
  66. M Gottle; S Dove; F Kees; J Schlossmann; J Geduhn; B Konig; Y Shen; WJ Tang; V Kaever; R Seifert Cytidylyl and uridylyl cyclase activity of bacillus anthracis edema factor and Bordetella pertussis CyaA Biochemistry 49, 5494-5503 (2010) .
    • . . . Although, the existence and metabolism of cCMP in mammals is uncertain 65 , recent report suggests that the adenylyl cyclase toxin edema factor and adenylate cyclase-hemolysin (CyaA), produced by Bacillus anthracis and Bordetella pertussis, can catalyze the formation of cCMP, and other cyclic nucleotides 66 . . .
  67. UR Schwarz; U Walter; M Eigenthaler Taming platelets with cyclic nucleotides Biochem Pharmacol 62, 1153-1161 (2001) .
    • . . . Integrin-dependent platelet aggregation and adhesion are known to be actively regulated by cyclic nucleotides 67 . . .
  68. ML Dustin; TA Springer T-cell receptor cross-linking transiently stimulates adhesiveness through LFA-1 Nature 341, 619-624 (1989) .
    • . . . Cyclic AMP is known to rapidly decrease LFA-1 dependent cell adhesion 68 , and binding of the ligand to activated LFA-1 can be rapidly and reversible down-modulated in a manner identical to VLA-4 21 69 . . .
  69. A Chigaev; Y Smagley; Y Zhang; A Waller; MK Haynes; O Amit; W Wang; RS Larson; LA Sklar Real-time analysis of the inside-out regulation of lymphocyte function-associated antigen-1 revealed similarities and differences with very late antigen-4 J Biol Chem , (2011) .
    • . . . Cyclic AMP is known to rapidly decrease LFA-1 dependent cell adhesion 68 , and binding of the ligand to activated LFA-1 can be rapidly and reversible down-modulated in a manner identical to VLA-4 21 69 . . .
  70. D Spina PDE4 inhibitors: current status Br J Pharmacol 155, 308-315 (2008) .
    • . . . Currently, a large number of preclinical in vivo studies on PDE inhibitors exhibit decreased cell recruitment, activation of inflammatory cells and physiological changes in lung function in asthma, chronic obstructive pulmonary disease, and others 70 71 . cAMP phosphodiesterase-4 inhibitor showed anti-inflammatory activity in vitro and in a model of psoriasis 72 . . .
  71. Z Diamant; D Spina PDE4-inhibitors: A novel, targeted therapy for obstructive airways disease Pulm Pharmacol Ther , (2011) .
    • . . . Currently, a large number of preclinical in vivo studies on PDE inhibitors exhibit decreased cell recruitment, activation of inflammatory cells and physiological changes in lung function in asthma, chronic obstructive pulmonary disease, and others 70 71 . cAMP phosphodiesterase-4 inhibitor showed anti-inflammatory activity in vitro and in a model of psoriasis 72 . . .
  72. PH Schafer; A Parton; AK Gandhi; L Capone; M Adams; L Wu; JB Bartlett; MA Loveland; A Gilhar; YF Cheung Apremilast, a cAMP phosphodiesterase-4 inhibitor, demonstrates anti-inflammatory activity in vitro and in a model of psoriasis Br J Pharmacol 159, 842-855 (2010) .
    • . . . Currently, a large number of preclinical in vivo studies on PDE inhibitors exhibit decreased cell recruitment, activation of inflammatory cells and physiological changes in lung function in asthma, chronic obstructive pulmonary disease, and others 70 71 . cAMP phosphodiesterase-4 inhibitor showed anti-inflammatory activity in vitro and in a model of psoriasis 72 . . .
  73. RR Kew; T Peng; SJ DiMartino; D Madhavan; SJ Weinman; D Cheng; ER Prossnitz Undifferentiated U937 cells transfected with chemoattractant receptors: a model system to investigate chemotactic mechanisms and receptor structure/function relationships J Leukoc Biol 61, 329-337 (1997) .
    • . . . Wild type CXCR4 (CD184) receptor, and CXCR2, IL-8RB, (CD128b, CD182) stably transfected U937 cells, and site-directed mutants of the FPR (non-desensitizing mutant of FPR ΔST) in U937 cells were prepared as described 73 and were a gift of Dr . . .
  74. L Osborn; C Hession; R Tizard; C Vassallo; S Luhowskyj; G Chi-Rosso; R Lobb Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes Cell 59, 1203-1211 (1989) .
    • . . . The original construct 74 was subcloned into the pTRACER vector (Invitrogen) . . .
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