scholarly journals CD44 is a physiological E-selectin ligand on neutrophils

2005 ◽  
Vol 201 (8) ◽  
pp. 1183-1189 ◽  
Author(s):  
Yoshio Katayama ◽  
Andrés Hidalgo ◽  
Jungshan Chang ◽  
Anna Peired ◽  
Paul S. Frenette
Keyword(s):  
Posttranslational Modifications ◽  
Leukocyte Adhesion ◽  
Binding Activity ◽  
Polymorphonuclear Neutrophil ◽  
Selectin Ligands ◽  
Selectin Family ◽  
Selectin Ligand ◽  
Human Pmns ◽  
Deficiency Type

The selectin family of adhesion molecules and their glycoconjugated ligands are essential for blood polymorphonuclear neutrophil (PMN) extravasation into inflammatory and infectious sites. However, E-selectin ligands on PMNs are not well characterized. We show here that CD44 immunopurified from G-CSF–differentiated 32D cells or from peripheral blood PMNs binds specifically to E-selectin. In contrast, CD44 extracted from bone marrow stromal or brain endothelial cell lines does not interact with E-selectin, suggesting cell-specific posttranslational modifications of CD44. PMN-derived CD44 binding activity is mediated by sialylated, α(1,3) fucosylated, N-linked glycans. CD44 enables slow leukocyte rolling on E-selectin expressed on inflamed endothelium in vivo and cooperates with P-selectin glycoprotein ligand–1 to recruit neutrophils into thioglycollate-induced peritonitis and staphylococcal enterotoxin A–injected skin pouch. CD44 extracted from human PMNs also binds to E-selectin. Moreover, we demonstrate that CD44 is hypofucosylated in PMNs from a patient with leukocyte adhesion deficiency type II, suggesting that it contributes to the syndrome. These findings thus suggest broader roles for CD44 in the innate immune response and uncover a potential new target for diseases in which selectins play a prominent role.

ESL-1 Is a Major Physiological Leukocyte Ligand for E-Selectin That Cooperates with PSGL-1 and CD44, and Together Mediate All Binding Activity to Endothelial Selectins In Vivo.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1787-1787
Author(s):  
Andres Hidalgo ◽  
Anna J. Peired ◽  
Paul S. Frenette
Keyword(s):  
Affinity Purification ◽  
Critical Role ◽  
Binding Activity ◽  
Wild Type ◽  
Selectin Ligands ◽  
Tnf Α ◽  
Polarized Cells ◽  
Selectin Ligand ◽  
Soluble E Selectin

Abstract Endothelial selectins mediate leukocyte (WBC) rolling on activated endothelium. ESL-1 was isolated by affinity purification more than 10 years ago but whether it is a physiological ligand for E-selectin remains unclear. Recent studies suggest that PSGL-1 and CD44 are physiological E-selectin ligands on neutrophils (PMNs)(J. Exp. Med. 2005 201:1183). To analyze the contribution of ESL-1, PSGL-1 and CD44, we knocked down ESL-1 expression using lentiviral transfer of short hairpin (sh) RNA into wild-type (WT), PSGL-1- or/and CD44-deficient bone marrow lineage-negative (Lin-) cells. Sorted GFP+ cells from mice transplanted with Lin- cells transduced with the shESL-1 exhibited >90% reduction in total ESL-1 levels by Q-PCR and Western blot analyses, and complete absence of ESL-1 on the PMN surface. shESL-1 transduced Lin- cells engrafted recipients mice with the same efficiency as control vector. Downregulation of ESL-1 slightly affected (~33%) the binding of soluble E-selectin to PMNs, but did not alter WBC rolling numbers in TNF-α-stimulated cremasteric venules. Absence of ESL-1 or CD44, but not PSGL-1, resulted in significant increases in mean rolling velocities (4.7 ± 0.4 μm/s for WT; 8.3 ± 0.4 μm/s for shESL-1*; 7.2 ± 0.2 μm/s for CD44−/−* and 5.4 ± 0.4 μm/s for PSGL-1−/−; *p<0.001). Moreover, WBCs lacking ESL-1 exhibited a characteristic “skipping” rolling motion, suggesting that ESL-1 plays a critical role in stabilizing WBC rolling. Remarkably, ESL-1 downregulation in the absence of PSGL-1 led to dramatic reductions in WBC binding to soluble E-selectin (~99%), and rolling (~93%) on TNF-α-stimulated venules. To determine the contribution of these 3 glycoproteins in WBC recruitment, mice transplanted with WT, PSGL-1- and/or CD44-deficient Lin- cells that were transduced with the shESL-1 or control vectors, were injected i.p. with thioglycollate and the number of peritoneal GFP+ PMNs determined. Only the absence of all 3 glycoproteins compromised PMN recruitment to the levels observed in P- and E-selectin double-deficient mice (82% reduction compared to WT; p<0.001), suggesting that ESL-1, PSGL-1 and CD44 comprise all endothelial selectin ligand activity on PMNs in vivo. To assess the role of E-selectin ligands in WBC activation, we monitored in vivo L-selectin clustering on the surface of over 4,000 rolling WBCs using high-speed multichannel fluorescence videomicroscopy. The majority of wild-type rolling WBCs exhibited polarization of L-selectin 150 to 230 min after TNF-α. L-selectin clustering was greatly reduced in E-selectin−/− mice (58 ± 4% polarized cells in WT vs 23.5 ± 1% in E-selectin−/− mice; p<0.001). To determine which E-selectin ligand(s) mediated L-selectin clustering in vivo, we evaluated L-selectin distribution on rolling WBCs deficient in ESL-1, CD44 or PSGL-1. Absence of CD44, but not ESL-1 or PSGL-1, reduced L-selectin redistribution to levels found in E-selectin−/− mice (20 ± 2% polarized cells), indicating that CD44 is the signalling E-selectin ligand required for L-selectin clustering. In addition, E-selectin-mediated CD44 signalling was p38-dependent, as assessed in WT mice treated with the p38 inhibitor SB203580 (77% reduction; p<0.001). Our results indicate that ESL-1, PSGL-1 and CD44 comprise all selectin ligand binding activity on WBCs, and suggest specialized contributions for each ligand where PSGL-1 mediates the initial tethering, ESL-1 is critical for the conversion into steady slow rolling, and CD44 is required for E-selectin-mediated signals contributing to WBC activation.

Discontinuation of fucose therapy in LADII causes rapid loss of selectin ligands and rise of leukocyte counts

Blood ◽  
2001 ◽  
Vol 97 (1) ◽  
pp. 330-332 ◽  
Author(s):  
Kerstin Lühn ◽  
Thorsten Marquardt ◽  
Erik Harms ◽  
Dietmar Vestweber
Keyword(s):  
Leukocyte Adhesion ◽  
Leukocyte Adhesion Deficiency ◽  
Rapid Loss ◽  
Selectin Ligands ◽  
Ligand Expression ◽  
Selectin Ligand ◽  
Carbohydrate Ligands ◽  
Leukocyte Counts ◽  
Deficiency Type ◽  
Peripheral Neutrophil

Abstract Leukocyte adhesion deficiency type II (LADII) is a rare inherited disorder of fucose metabolism. Patients with LADII lack fucosylated glycoconjugates, including the carbohydrate ligands of the selectins, leading to an immunodeficiency caused by the lack of selectin-mediated leukocyte-endothelial interactions. A simple and effective therapy has recently been described for LADII, based on the administration of oral fucose. Parallel to this treatment the lack of E- and P-selectin ligands on neutrophils was corrected, and high peripheral neutrophil counts were reduced to normal levels. This study reports that discontinuation of this therapy leads to the complete loss of E-selectin ligands within 3 days and of P-selectin ligands within 7 days. Peripheral neutrophil counts increased parallel to the decrease of selectin ligands. Selectin ligands reappeared promptly after resumption of the fucose therapy, demonstrating a causal relationship between fucose treatment and selectin ligand expression and peripheral neutrophil counts.

scholarly journals L-Selectin–Dependent Leukocyte Adhesion to Microvascular But Not to Macrovascular Endothelial Cells of the Human Coronary System

Blood ◽  
1997 ◽  
Vol 89 (9) ◽  
pp. 3228-3235 ◽  
Author(s):  
A. Zakrzewicz ◽  
M. Gräfe ◽  
D. Terbeek ◽  
M. Bongrazio ◽  
W. Auch-Schwelk ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Time Course ◽  
Leukocyte Adhesion ◽  
Flow Chamber ◽  
Shear Stresses ◽  
Selectin Ligands ◽  
Tnf Α ◽  
Factor Α

Abstract To characterize L-selectin–dependent cell adhesion to human vascular endothelium, human cardiac microvascular endothelial cells (HCMEC) and human coronary endothelial cells (HCEC) were isolated from explanted human hearts. The adhesion behavior of human (NALM-6) and mouse (300.19) pre-B cells transfected with cDNA encoding for human L-selectin was compared with that of the respective nontransfected cells in a flow chamber in vitro. More than 80% of the adhesion to tumor necrosis factor-α (TNF-α)–stimulated HCMEC at shear stresses <2 dyne/cm2 was L-selectin dependent and could be equally well blocked by an anti–L-selectin antibody or a L-selectin-IgG-chimera. No L-selectin dependent adhesion to HCEC could be shown. The L-selectin dependent adhesion to HCMEC was insensitive to neuraminidase, but greatly inhibited by addition of NaClO3 , which inhibits posttranslational sulfation and remained elevated for at least 24 hours of stimulation. E-selectin dependent adhesion of HL60 cells to HCMEC was blocked by neuraminidase, but not by NaClO3 and returned to control levels within 18 hours of HCMEC stimulation. It is concluded that microvascular, but not macrovascular endothelial cells express TNF-α–inducible sulfated ligand(s) for L-selectin, which differ from known L-selectin ligands, because sialylation is not required. The prolonged time course of L-selectin dependent adhesion suggests a role in sustained leukocyte recruitment into inflammatory sites in vivo.

Correction of Leukocyte Adhesion Deficiency Type II With Oral Fucose

Blood ◽  
1999 ◽  
Vol 94 (12) ◽  
pp. 3976-3985 ◽  
Author(s):  
Thorsten Marquardt ◽  
Kerstin Lühn ◽  
Geetha Srikrishna ◽  
Hudson H. Freeze ◽  
Erik Harms ◽  
...  
Keyword(s):  
Mental Retardation ◽  
Culture Medium ◽  
Leukocyte Adhesion ◽  
Effective Therapy ◽  
Type Ii ◽  
Leukocyte Adhesion Deficiency ◽  
Oral Supplementation ◽  
Selectin Ligands ◽  
Deficiency Type ◽  
Core Fucosylation

Abstract We describe a simple, noninvasive, and effective therapy for leukocyte adhesion deficiency type II (LAD II), a rare inherited disorder of fucose metabolism. This disorder leads to an immunodeficiency caused by the absence of carbohydrate-based selectin ligands on the surface of neutrophils as well as to severe psychomotor and mental retardation. The fucosylation defect in LAD II fibroblasts can be corrected by addition of L-fucose to the culture medium. This prompted us to initiate dietary fucose therapy on a patient with LAD II. Oral supplementation of fucose in this patient induced the expression of fucosylated selectin ligands on neutrophils and core fucosylation of serum glycoproteins. During 9 months of treatment, infections and fever disappeared, elevated neutrophil counts returned to normal, and psychomotor capabilities improved.

scholarly journals Neutrophil-activating protein 1/interleukin 8 stimulates the binding activity of the leukocyte adhesion receptor CD11b/CD18 on human neutrophils.

1990 ◽  
Vol 171 (4) ◽  
pp. 1155-1162 ◽  
Author(s):  
P A Detmers ◽  
S K Lo ◽  
E Olsen-Egbert ◽  
A Walz ◽  
M Baggiolini ◽  
...  
Keyword(s):  
Leukocyte Adhesion ◽  
Binding Activity ◽  
Receptor Expression ◽  
Human Neutrophils ◽  
Adhesion Receptor ◽  
Biological Responses ◽  
Specific Granules ◽  
Inflammatory Stimuli ◽  
Adhesive Interactions

The cytokine NAP-1/IL-8 is produced by a variety of different cells in response to inflammatory stimuli and elicits several biological responses from PMN. Experiments presented here demonstrate that PMN exposed to NAP-1/IL-8 expressed increased amounts of CD11b/CD18, as well as CD11c/CD18 and CR1, on their cell surface, while expression of Fc gamma RIII and HLA-A,B,C remained essentially unchanged. Increased CD11b/CD18 and CD11c/CD18 appears to correspond with the release of specific granules by NAP-1/IL-8. NAP-1/IL-8 was also a potent stimulator of several of the binding activities of CD11b/CD18. Ligation of EC3bi by CD11b/CD18 was rapidly enhanced by NAP-1/IL-8, but phagocytosis of the ligated particles was not induced by the agonist. In addition, enhanced binding of EC3bi was observed in the absence of an increase in receptor expression as shown with PMN cytoplasts. NAP-1/IL-8 promoted additional adhesive interactions between CD11b/CD18 and the biosynthetic precursor of LPS, lipid IVa, fibrinogen, and endothelial cells, suggesting that NAP-1/IL-8 may promote leukocyte adhesion in vivo that could lead to recruitment of PMN to sites of tissue inflammation.

Multiple Myeloma Cells Express Functional E-Selectin Ligands Which Can be Inhibited Both in-Vitro and in-Vivo Leading to Prolongation of Survival in a Murine Transplant Model

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4718-4718 ◽  
Author(s):  
Alessandro Natoni ◽  
Michele Moschetta ◽  
Siobhan Glavey ◽  
Ping Wu ◽  
Gareth J. Morgan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
High Risk ◽  
Research Funding ◽  
Prolonged Exposure ◽  
Residual Disease ◽  
Critical Role ◽  
Cell Trafficking ◽  
Selectin Ligands ◽  
Selectin Ligand

Abstract Introduction Selectins and their ligands play an important role in the adhesion and trafficking of multiple myeloma (MM) cells. While PSGL-1 is strongly expressed by MM cells, E-selectin binding is seen in a small minority only and its contribution to MM cell trafficking is unclear. As E-selectin is constitutively expressed by BM endothelial cells it may play a critical role in the BM homing and retention of MM cells. We wished to determine (1) if MM cells express functional E-selectin ligands (2) whether hypoxia enhances E-selectin ligand positivity (3) the potential of the E-selectin inhibitor GMI-1271 to prolong survival in a murine model of MM minimal residual disease (4) the ability to identify patients most likely to benefit from E-selectin inhibition. Methods To detect E-selectin ligands MM cell lines were stained with an antibody to HECA452, which indicates reactivity with sialofucosylated glycoforms of PSGL-1 and CD44, which can bind E-selectin. To test the effect of hypoxia, MM cells were maintained in either 1% O2 or normoxia at 37¡C for 5 days and then stained with HECA452. MM cells were sorted into E-selectin ligand positive and negative populations based on HECA452 staining. We evaluated rolling of MM cells on E-selectin +/- 10 µM GMI-1271 for 1 h at 37¡C, following which 50 µl of cell suspension were loaded onto a channel of a microfluidic chip (Cellix) pre-coated with 30 µg/ml of recombinant E-Selectin (Peprotech). Cells were perfused into the channel at 0.5 Dyne/cm2 using Mirus Evo Nanopump (Cellix). After 2 min of perfusion, images were acquired at 5 different positions of the channel using an AX10 Vert.A1 microscope (Zeiss) equipped with aQImaging QIClickª digital CCD camera. Thirty frames were acquired at 5 different positions with 0.5 sec between each frame. Each sample was assayed in triplicate. To evaluate in-vivo activity of GMI-1271, SCID-beige mice were injected with luciferase-expressing MM1S cells. Seventy two hours after inoculation, mice were divided into cohorts treated with vehicle, GMI-1271 (40mg/kg bid), bortezomib (0.75 mg/kg IP weekly for 4 weeks), or a combination of GMI-1271 and bortezomib. Tumor burden was determined by bioluminescence imaging and animals were followed for survival. We evaluated gene expression levels of 2 genes involved in selectin ligand expression, the sialyltransferase ST3GAL6 and FUCA1 (fucosidase), which we have previously shown to be important in MM prognosis, in the context of ISS and FISH in the MRCIX dataset. Specifically, we analyzed the interaction of high ST3GAL6 and low FUCA1 with ISS/FISH risk, where patients are considered high risk if ISS=3 along with 17pdel or t(4;14). Results Twelve percent (12%) of MM1S and 15.8% of RPMI8226 cells were HECA452 positive. We consistently observed rolling of sorted HECA452 positive RPMI8226 and MM1S cells on E-selectin with absence of rolling in HECA452 negative fraction. Treatment with GMI-1271 completely inhibited rolling on E-selectin (100% inhibition). Prolonged exposure of RPMI8226 cells to hypoxia resulted in a significant increase in HECA452 positive cells (38.8% versus 25.2%, P=0.0215), consistent with our previous observation that hypoxia upregulates ST3GAL6. The survival of mice treated with either GMI-1271 alone or bortezomib alone was significantly greater than vehicle treated control mice. GMI-1271 in combination with bortezomib treatment however, resulted in the longest survival and significantly longer than treatment with bortezomib alone (p=0.0025). The high ST3GAL6/low FUCA1 signature was overrepresented in high risk compared to low risk [39% (6% of whole population) .v. 13.9% p=0.01] patients and conferred poor prognosis with a median survival of 10 months versus 33 months in the remaining 61% of high-risk patients. On multivariate analysis this pattern was independent of ISS/FISH for OS (p=0.013). Conclusions A small fraction of MM cells express functional E-selectin ligands, which may be higher in the hypoxic BM niche. The binding of these cells to E-selectin is blocked by GMI-1271 and GMI-1271 significantly enhances the activity of bortezomib in vivo. These data indicate the importance of E-selectin ligand expressing MM cells in homing and engraftment and provide a rationale for targeting E-selectin in MM. Furthermore, high ST3GAL6 + low FUCA1 identifies a subgroup of patients with high risk ISS/FISH with particularly poor outcome who may benefit from E-selectin inhibition. Figure 1 Figure 1. Disclosures Magnani: GlycoMimetics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Ghobrial:GlycoMimetics: Consultancy, Research Funding. O'Dwyer:GlycoMimetics: Consultancy, Research Funding.

E-Selectin Ligand Expression Increases with Progression of Myeloma and Induces Drug Resistance in a Murine Transplant Model, Which Is Overcome By the Glycomimetic E-Selectin Antagonist, GMI-1271

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1805-1805 ◽  
Author(s):  
Alessandro Natoni ◽  
Theodore A.G. Smith ◽  
Niamh Keane ◽  
Silvia C. Locatelli-Hoops ◽  
Isabela Oliva ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Disease Progression ◽  
Cell Lines ◽  
Saline Control ◽  
Selectin Ligands ◽  
Ligand Expression ◽  
Selectin Ligand ◽  
Transplant Model

Abstract There is increasing evidence that E-selectin and its ligands play an important role in the progression of multiple myeloma (MM) and drug resistance. We reported that the sialyltransferase ST3GAL6 influences homing and survival in MM, and postulated that it may function in the synthesis of E-selectin ligands (Glavey et al Blood, 2014). We also found that a small subpopulation of cells (~ 5%) from MM cell lines express functional E-selectin ligands, which could be expanded under hypoxic conditions typical of the bone marrow (BM) microenvironment. These cells were identified by reactivity with an antibody (HECA452), which binds the same carbohydrate epitope required for binding to E-selectin. Rolling of MM1S cells on E-selectin was blocked by a small molecule glycomimetic antagonist to E-selectin (GMI-1271). Moreover, GMI-1271 significantly enhanced the anti-myeloma activity of bortezomib (BTZ) in an in vivo murine transplant model (Natoni et al Blood, 2014). We now extend these observations to obtain a more complete understanding of the role E-selectin plays in MM biology and chemotherapy resistance for its potential clinical relevance. The parental, heterogeneous MM cell lines MM1S and RPMI8226 (MM1Spar, RPMI8226par, respectively) were sequentially sorted to obtain cell lines highly enriched (>85% and 80%) for the expression of cell surface carbohydrates bound by HECA452, and designated MM1SHECA452 and RPMI8226HECA452. The cell lines could be passaged in vitro and were stable for enriched E-selectin ligand expression. In contrast to parental cells, both MM1SHECA452 and RPMI8226HECA452 showed strong binding to E-selectin in static adhesion assays. Both MM1SHECA452 and RPMI8226HECA452 exhibited strong rolling on E-selectin under shear stress. MM1Spar or RPMI8226par failed to roll well on E-selectin. The addition of GMI-1271 during culture conditions led to a marked reduction in adhesion of MM1SHECA452 and profoundly inhibited rolling on E-selectin of both HECA452 enriched MM cell lines. The significance of these in vitro findings was studied in vivo. MM1Spar or MM1SHECA452 cells were injected i.v. into SCID beige mice. Beginning 5 days post tumor injection, the survival impact of treatment with saline control, GMI-1271, BTZ or a combination of both was determined. Mice transplanted with MM1SHECA452 had more aggressive disease with significantly shorter survival compared to those transplanted with MM1Spar. In contrast to MM1Spar cells, mice engrafted with MM1SHECA452 demonstrated a marked resistance to BTZ treatment. Whereas GMI-1271 treatment alone had no impact on survival, the combination of GMI-1271 and BTZ led to a highly significant improvement in survival of MM1Spar engrafted mice (P=0.0363), and more importantly broke the resistance and restored the anti-myeloma activity of BTZ in MM1SHECA452 engrafted mice (P=0.0123) (figure 1). The number of peripheral blood (PB) human CD138+ cells was increased in MM1SHECA452-engrafted mice within 60 min following a single injection of GMI-1271, and persisted for at least 24 hours (2.37% v. 0.03%, p <0.001). This effect was consistent with GMI-1271 disrupting the tumor microenvironment and mobilizing MM1SHECA-452 cells from the BM niche. Given these findings we wished to see if samples from patients with MM express E-selectin ligands and whether higher levels are seen with disease progression. BM and/or PB were obtained following informed consent from patients with MM and plasma cells (CD38+/CD138+) were analyzed for E-selectin ligand expression by flow cytometry using the HECA452 antibody. To date all primary MM samples (n=25) contained HECA452-reactive cell populations (median 22%). A consistently higher proportion of circulating MM cells express HECA452 when compared with paired BM samples (n=14), with a median difference of 33% (Wilcoxon signed rank test, p=0.02). HECA452 expression of MM in PB was significantly higher (on average 40% higher) in samples taken at relapse vs. diagnosis, (unpaired t test, p = 0.0008) These data provide compelling evidence that E-selectin ligand bearing cells play an important role in disease progression and drug resistance in MM, and a strong rationale for clinical strategies incorporating GMI-1271 to improve patient outcome. Disclosures Smith: GlycoMimetics, Inc.: Employment. Locatelli-Hoops:GlycoMimetics, Inc.: Employment. Oliva:GlycoMimetics, Inc.: Employment. Fogler:GlycoMimetics, Inc.: Employment. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. O'Dwyer:Celgene: Honoraria, Research Funding.

scholarly journals ICAM-1–activated Src and eNOS signaling increase endothelial cell surface PECAM-1 adhesivity and neutrophil transmigration

Blood ◽  
2012 ◽  
Vol 120 (9) ◽  
pp. 1942-1952 ◽  
Author(s):  
Guoquan Liu ◽  
Aaron T. Place ◽  
Zhenlong Chen ◽  
Viktor M. Brovkovych ◽  
Stephen M. Vogel ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Adhesion Molecule ◽  
Vascular Inflammation ◽  
Transendothelial Migration ◽  
Binding Activity ◽  
Polymorphonuclear Neutrophil ◽  
Intercellular Adhesion Molecule ◽  
No Production ◽  
Intercellular Adhesion Molecule 1

Abstract Polymorphonuclear neutrophil (PMN) extravasation requires selectin-mediated tethering, intercellular adhesion molecule-1 (ICAM-1)–dependent firm adhesion, and platelet/endothelial cell adhesion molecule 1 (PECAM-1)–mediated transendothelial migration. An important unanswered question is whether ICAM-1–activated signaling contributes to PMN transmigration mediated by PECAM-1. We tested this concept and the roles of endothelial nitric oxide synthase (eNOS) and Src activated by PMN ligation of ICAM-1 in mediating PECAM-1–dependent PMN transmigration. We observed that lung PMN infiltration in vivo induced in carrageenan-injected WT mice was significantly reduced in ICAM-1−/− and eNOS−/− mice. Crosslinking WT mouse ICAM-1 expressed in human endothelial cells (ECs), but not the phospho-defective Tyr518Phe ICAM-1 mutant, induced SHP-2–dependent Src Tyr530 dephosphorylation that resulted in Src activation. ICAM-1 activation also stimulated phosphorylation of Akt (p-Ser473) and eNOS (p-Ser1177), thereby increasing NO production. PMN migration across EC monolayers was abolished in cells expressing the Tyr518Phe ICAM-1 mutant or by pretreatment with either the Src inhibitor PP2 or eNOS inhibitor L-NAME. Importantly, phospho–ICAM-1 induction of Src signaling induced PECAM-1 Tyr686 phosphorylation and increased EC surface anti–PECAM-1 mAb-binding activity. These results collectively show that ICAM-1–activated Src and eNOS signaling sequentially induce PECAM-1–mediated PMN transendothelial migration. Both Src and eNOS inhibition may be important therapeutic targets to prevent or limit vascular inflammation.

Somatic revertant mosaicism in a patient with leukocyte adhesion deficiency type 1

Blood ◽  
2006 ◽  
Vol 109 (3) ◽  
pp. 1182-1184 ◽  
Author(s):  
Yumi Tone ◽  
Taizo Wada ◽  
Fumie Shibata ◽  
Tomoko Toma ◽  
Yoko Hashida ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Leukocyte Adhesion ◽  
Somatic Mosaicism ◽  
Autosomal Recessive Disorder ◽  
Cell Receptor ◽  
Leukocyte Adhesion Deficiency ◽  
Deficiency Type

Abstract Leukocyte adhesion deficiency type 1 (LAD-1) is an autosomal recessive disorder caused by mutations in the ITGB2 (CD18) gene and characterized by recurrent severe infections, impaired pus formation, and defective wound healing. We describe an unusual case of severe phenotypic LAD-1 presenting with somatic mosaicism. The patient is a compound heterozygote bearing 2 different frameshift mutations that abrogate protein expression. However, CD18 expression was detected in a small proportion of T cells but was undetectable in granulocytes, monocytes, B cells, and natural killer (NK) cells. The T cells were not of maternal origin, lacked the paternal mutation, and showed a selective advantage in vivo. Molecular analysis using sorted CD18+ cells revealed them to be derived from a single CD8+ T cell carrying T-cell receptor VB22. These findings suggest that spontaneous in vivo reversion was responsible for the somatic mosaicism in our patient.

Sialyltransferase specificity in selectin ligand formation

Blood ◽  
2002 ◽  
Vol 100 (10) ◽  
pp. 3618-3625 ◽  
Author(s):  
Lesley G. Ellies ◽  
Markus Sperandio ◽  
Gregory H. Underhill ◽  
James Yousif ◽  
Michael Smith ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Dependent Manner ◽  
Neuraminidase Treatment ◽  
Ovary Cells ◽  
Rolling Velocity ◽  
Selectin Ligands ◽  
Selectin Ligand

Selectin ligands are glycan structures that participate in leukocyte trafficking and inflammation. At least 6 ST3Gal sialyltransferases (I-VI) have been identified that may contribute to selectin ligand formation. However, it is not known which of these sialyltransferases are involved in vivo and whether they may differentially regulate selectin function. We have produced and characterized mice genetically deficient in ST3Gal-I, ST3Gal-II, ST3Gal-III, and ST3Gal-IV. Unlike mice bearing severe defects in selectin ligand formation, there was no finding of leukocytosis with these single ST3Gal deficiencies. Among neutrophils, only ST3Gal-IV was found to play a role in the synthesis of selectin ligands. In vitro rolling of marrow-derived neutrophils on E- or P-selectins presented by Chinese hamster ovary cells was reduced in the absence of ST3Gal-IV. However, in a tumor necrosis factor α (TNF-α)–induced inflammation model in vivo, no defect among P-selectin ligands was observed. Nevertheless, the number of leukocytes rolling on postcapillary venules in an E-selectin–dependent manner was decreased while E-selectin–dependent rolling velocity was increased. We propose that multiple ST3Gal sialyltransferases contribute to selectin ligand formation, as none of these ST3Gal deficiencies recapitulated the degree of E- and P-selectin ligand deficit observed on neuraminidase treatment of intact neutrophils. Our findings indicate a high degree of functional specificity among sialyltransferases and a substantial role for ST3Gal-IV in selectin ligand formation.

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