Effect of Hydroxyurea Treatment on In Vitro Adhesion of Sickle Erythrocytes to Sickle Leukocyte Subpopulations.

Abstract Microvascular vaso-occlusion in sickle cell disease is thought to involve adhesive interactions among erythrocytes (RBCs), leukocytes and vascular endothelial cells. Recent studies have demonstrated the presence of a significant inflammatory response in sickle cell disease, including changes in the cell surface adhesion molecules that mediate cell-cell interactions in the microvasculature. In this study, we used a parallel-plate flow chamber assay to determine the subpopulations of leukocytes that are involved in sickle leukocyte-RBC interactions. We also studied the effect of treatment with hydroxyurea (HU) on these adhesive interactions. Populations of monocytes, neutrophils (PMNs) and T cells were isolated by negative selection from the peripheral blood of untreated patients with sickle cell disease (SS), sickle patients receiving HU (SS-HU), and healthy control subjects (AA). Adhesive interactions involving these leukocyte subpopulations, human umbilical vein endothelial cells (HUVECs) pretreated with tumor necrosis factor-α (TNF-α ), and autologous RBCs were measured under a shear stress of 1 dyne/cm2. Compared to the corresponding cell populations from AA individuals, PMNs, monocytes, and T cells from SS individuals were significantly more adherent to TNF-α-treated HUVECs (774±59 vs. 502±27 cells/mm2, p=0.001; 533±66 vs. 348±36 cells/mm2, p=0.024; and 470±75 vs. 227±26 cells/mm2, p=0.009, respectively). HU therapy significantly decreased the adhesion of SS PMNs to HUVECs (774±59 cells/mm2 for SS vs. 604±36 for SS-HU, p=0.025). Compared to adherent AA leukocytes, adherent SS leukocytes exhibited greater participation in adhesive interactions with autologous RBCs (41±3% for SS vs. 27±3% for AA, p=0.002), and HU treatment decreased the fraction of leukocytes that captured autologous RBCs to the control level (29±3% for SS-HU, p=0.006 vs. SS). Compared to adherent PMNs from SS individuals, adherent PMNs from SS-HU individuals showed significantly reduced participation in the capture of RBCs (53±6% for SS vs. 35±5% for SS-HU, p=0.021). Although adherent T cells from SS individuals participated significantly more in RBC capture than adherent T cells from AA individuals (28±5% for SS vs. 10±2% for AA, p=0.007), HU therapy did not have a significant effect on this parameter (21±5% for SS-HU, p=0.373). Compared to AA leukocytes, SS leukocytes captured more RBCs per participating adherent leukocyte (2.8±0.2 vs. 1.9±0.1 RBCs/cell, p=0.001). HU therapy reduced the number of RBCs captured per PMN but not the number captured per T cell. Compared to AA T cells, SS T cells captured adherent RBCs for a significantly longer period of time (51±9 vs. 26±6 seconds, p=0.035). Our data suggest that sickle neutrophils, monocytes and T cells may all be involved in adhesive interactions with sickle RBCs. PMN-RBC and monocyte-RBC interactions appear to be more numerous than T cell-RBC interactions, although T cell-RBC interactions may be stronger. HU therapy appears to target PMN-RBC and monocyte-RBC interactions preferentially. Future studies will focus on the role of particular adhesion molecules in mediating these interactions and on the potential for therapeutic interventions targeting cell-cell adhesion.

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Role of Adhesion Molecules and Vascular Endothelium in the Pathogenesis of Sickle Cell Disease

Hematology ◽

10.1182/asheducation-2007.1.84 ◽

2007 ◽

Vol 2007 (1) ◽

pp. 84-90 ◽

Cited By ~ 27

Author(s):

Marilyn J. Telen

Keyword(s):

Sickle Cell Disease ◽

Red Blood Cells ◽

Adhesion Molecules ◽

Sickle Cell ◽

Vascular Endothelium ◽

Blood Cells ◽

Cell Disease ◽

Adhesive Interactions ◽

Lines Of Evidence

AbstractA number of lines of evidence now support the hypothesis that vaso-occlusion and several of the sequelae of sickle cell disease (SCD) arise, at least in part, from adhesive interactions of sickle red blood cells, leukocytes, and the endothelium. Both experimental and genetic evidence provide support for the importance of these interactions. It is likely that future therapies for SCD might target one or more of these interactions.

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Berkeley Sickle Mice Demonstrate CD8- and NK1.1-Dependent Increased Rejection of Allogeneic Hematopoietic Stem Cell Transplants.

Blood ◽

10.1182/blood.v106.11.2332.2332 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2332-2332

Author(s):

Leslie Kean ◽

Kelly Hamby ◽

Jennifer Perry ◽

Christian Larsen ◽

David Archerq

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Sickle Cell Disease ◽

Hematopoietic Stem Cell ◽

Sickle Cell ◽

Cell Disease ◽

Hematopoietic Stem ◽

Cell Immunity ◽

Increased Risk

Abstract While hematopoietic stem cell transplantation (HSCT) represents the only curative therapy for sickle cell disease, sickle patients undergoing HSCT face many complications, including an increased risk of graft rejection compared to non-sickle patients. We have used the Berkeley sickle mouse model to study the potential mechanisms underlying this increased risk of rejection. Using a CD28/CD40 costimulation-blockade-based non-myeloablative HSCT regimen, we transplanted Berkeley sickle mice with fully allogeneic SJL bone marrow. While the vast majority (>85%, n=25) of control C57BL/6 animals became stably chimeric and immunologically donor-tolerant with this transplant regimen, sickle mice were much more prone to reject the transplant (~20% graft acceptance, n=25). Both CD8+ cells and NK1.1+ cells were found to contribute to this rejection, as depletion of either of these cell populations led to a marked increase in the percent of engrafted mice (>85% graft acceptance, n=15–25), while depletion of CD4+ cells led to the opposite effect, with 0% (n=25) animals engrafted in this depletion cohort. The increased propensity of HSCT rejection in the Berkeley sickle mice may, in part, be explained by the presence of increased numbers of donor-reactive T cells (5–10-fold compared to C57BL/6 controls) in naïve sickle mice, despite their lack of exposure to donor antigens, and their housing in a Specific-Pathogen-Free environment. We speculate that these increased numbers of anti-donor T cells may occur as a result of heightened inflammation in the context of active sickle cell disease, which could lead to increased expansion and persistence of a T cell repertoire containing anti-donor heterologous T cell immunity. This heterologous immunity may have a profound effect on the success of HSCT for sickle cell disease, especially when non-myeloablative regimens are employed.

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Heme Induces Endothelial Tissue Factor Expression: Potential Role in Hemostatic Activation in Patients with Intravascular Hemolysis Including Sickle Cell Disease.

Blood ◽

10.1182/blood.v112.11.1902.1902 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1902-1902

Author(s):

Yamaja Setty ◽

Suhita Gayen Betal ◽

Jie Zhang ◽

Nigel S Key ◽

Marie Stuart

Keyword(s):

Flow Cytometry ◽

Endothelial Cells ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Whole Blood ◽

Dependent Manner ◽

Cell Disease ◽

Intravascular Hemolysis ◽

Tnf Α ◽

Endothelial Tissue

Abstract Plasma levels of heme in the 20 to 600 μM range are found in clinical conditions associated with intravascular hemolysis including paroxysmal nocturnal hemoglobinuria and sickle cell disease, conditions also associated with a thrombotic tendency. Objectives: To investigate whether heme, an inflammatory mediator and a product of intravascular hemolysis in patients with hemolytic anemia including sickle cell disease (SCD), could modulate hemostasis by an effect on endothelial tissue factor (TF) expression. Additionally, in SCD patient-related studies, we assessed whether any association existed between whole blood TF activity (WBTF) and levels of surrogate markers of intra-vascular hemolysis including lactate dehydrogenase (LDH) and reticulocyte counts. Methods: Following incubation of human endothelial cells (from umbilical vein and/or lung microvasculature) with heme (1 to 100 μM) for various times (30 minutes to 8 hours), levels of TF protein were assessed using ELISA, flow cytometry and/or Western blotting; and TF mRNA by a semi-quantitative RT-PCR. An assay for TF functional activity was performed using a chromogenic tenase activity kit where specificity of TF activity was tested in antibody-blocking experiments. Three TF-specific antibodies including a rabbit polyclonal and two mouse monoclonal (clones hTF-1 and TF9-10H10) antibodies were used in assays involving TF protein analysis. All experiments were performed in media containing polymyxin B to neutralize any potential endotoxin contamination. In patient-related studies, 81 subjects with SCD (1 to 21 years) were evaluated for levels of WBTF, LDH, and reticulocyte counts and data analyzed for potential relationships. Results: Heme induced TF protein expression on the surface of both macro- and micro-vascular endothelial cells in a concentration-dependent manner with 12- to 50-fold induction noted (ELISA assays) between 1 and 100 μM heme (P<0.05, n=3 to 6). Complementary flow cytometry studies showed that the heme-mediated endothelial TF expression was quantitatively similar to that induced by the cytokine TNF-α. Heme also up-regulated endothelial expression of TF mRNA (8- to 26-fold, peak expression at 2 hours postagonist treatment), protein (20- to 39-fold, peak expression at 4 hours) and procoagulant activity (5- to 13-fold, peak activity at 4 hours post-agonist treatment) in a time-dependent manner. Time-course of heme-mediated TF antigen expression paralleled induction of procoagulant activity with antibody blocking studies demonstrating specificity for TF protein. Potential involvement of endogenously released cytokines including IL-1α and TNF-α in mediating the heme effect was next explored. We found that the latter cytokines are not involved, since antibodies against IL-1α and TNF-α, and an IL-1- receptor antagonist failed to block heme-induced endothelial TF expression. Inhibition of heme-induced TF mRNA expression by sulfasalazine and curcumin suggested that the transcription factor NFκB was involved in mediating heme-induced effect. In patient-related studies, whole blood TF levels in SCD correlated positively with both LDH (r=0.72, p<0.000001), and reticulocyte count (r=0.60, p<0.000001). Conclusions: Our findings demonstrate that heme induces TF expression in endothelial cells, and that the observed effects occurred at patho-physiologically relevant heme concentrations. Our results suggest that heme-induced endothelial TF expression may provide a pathophysiologic link between the intravascular hemolytic milieu and the hemostatic perturbations previously noted in patients with hemolytic anemia including sickle cell disease.

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Hydroxyurea Therapy Increases Expression of BCAM/LU and Adhesion to Laminin in Children with Sickle Cell Disease

Blood ◽

10.1182/blood.v112.11.4806.4806 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4806-4806

Author(s):

Clarissa E Johnson ◽

Marilyn J. Telen

Keyword(s):

Extracellular Matrix ◽

Endothelial Cells ◽

Sickle Cell Disease ◽

Adhesion Molecules ◽

Sickle Cell ◽

Adhesion Molecule ◽

Extracellular Matrix Protein ◽

Published Data ◽

Adhesion Molecule Expression ◽

Cell Disease

Abstract Vaso-occlusion is the major cause of morbidity and mortality in sickle cell disease. The tendency of red blood cells (RBCs) to adhere to extracellular matrix molecules and the vascular endothelium is believed to be a significant contributor to the vaso-occlusive process. Some published studies have shown that hydroxyurea decreases sickle (SS) RBC adhesion to some ligands, although the mechanism by which this occurs is not completely understood. SS RBCs demonstrate increased expression of several adhesion molecules, especially BCAM/LU, and also conserve functional signaling pathways that are associated with upregulation of adhesion. BCAM/LU mediates adhesion to the extracellular matrix protein laminin. We hypothesized that patients responsive to hydroxyurea (HU) therapy would exhibit reduced adhesion to laminin as well as a decrease in adhesion molecule expression. Our subjects included patients with Hb SS between the ages of 5 to 18. They were divided into three groups: children not receiving HU therapy (n = 3); children receiving HU therapy for over 6 months (n = 5), and children initially not receiving HU but who were initiating therapy at the time of study enrollment (n = 5). Adhesion to laminin was examined using a graduated height flow chamber to quantitate the adhesion of SS RBCs. Expression of adhesion molecules was analyzed by western blot and densitometry, using monoclonal antibody to BCAM/LU. We found that HU therapy was associated with significantly increased expression of BCAM/LU (HU: 145.8 ± 14.0 SEM; no HU: 60.8 ± 11.0 SEM densitometry units, p = .0014). This somewhat unexpected finding confirms results published earlier this year by Odievre et al. (2008). Adhesion to laminin was also increased for patients on HU (HU: 9.3 ± 5.9; no HU: .3 ± .3, p=.2), although this increase was not significant, given the variability in adhesion seen among patients and the small number of subjects. Nevertheless, the increase in adhesion corresponded to the increase in BCAM/LU expression. In contrast, adhesion to endothelial cells was decreased, although not significantly, in patients on HU (HU: 38.1 ± 38; no HU: 127.2 ± 122.5, p=.6). Our findings thus confirm earlier published data showing that HU increases the expression of BCAM/LU measured by flow cytometry and further shows that this increased expression is associated with increased adhesion to laminin but not to endothelial cells. Potential mechanisms by which HU affects adhesion molecule expression and activity merit further investigation, as does the physiologic role of these alterations. Comparison of results from patient-matched pre-treatment and post-treatment samples should also help define the effects of HU. Figure Figure

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Activated monocytes in sickle cell disease: potential role in the activation of vascular endothelium and vaso-occlusion

Blood ◽

10.1182/blood.v96.7.2451.h8002451_2451_2459 ◽

2000 ◽

Vol 96 (7) ◽

pp. 2451-2459 ◽

Cited By ~ 1

Author(s):

John D. Belcher ◽

Paul H. Marker ◽

Jill P. Weber ◽

Robert P. Hebbel ◽

Gregory M. Vercellotti

Keyword(s):

Endothelial Cells ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Nuclear Translocation ◽

Mononuclear Leukocytes ◽

Cell Contact ◽

Cell Disease ◽

Tnf Α ◽

Activated Monocytes

Sickle cell anemia is characterized by painful vaso-occlusive crises. It is hypothesized that monocytes are activated in sickle cell disease and can enhance vaso-occlusion by activating endothelium. To test this hypothesis, human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (MVEC) with sickle and normal mononuclear leukocytes were incubated, and endothelial activation was measured. Endothelial cells incubated with sickle mononuclear leukocytes were more activated than those incubated with normal mononuclear leukocytes, as judged by the increased endothelial expression of adhesion molecules and tissue factor and the adhesion of polymorphonuclear leukocytes (PMNL). Monocytes, not lymphocytes or platelets, were the mononuclear cells responsible for activating endothelial cells. Sickle monocytes triggered endothelial nuclear factor-kappa B (NF-κB) nuclear translocation. Cell-to-cell contact of monocytes and endothelium enhanced, but was not required for, activation. Antibodies to tumor necrosis factor-alpha (TNF-α) and interleukin-1-beta (IL-1β) blocked activation of the endothelium by monocytes. Peripheral blood monocytes from patients with sickle cell disease had 34% more IL-1β (P = .002) and 139% more TNF-α (P = .002) per cell than normal monocytes. Sixty percent of sickle monocytes expressed the adhesion molecule ligand CD11b on their surfaces compared with only 20% of normal monocytes (P = .002). Serum C-reactive protein, a marker of systemic inflammation, was increased 12-fold in sickle serum than in normal serum (P = .003). These results demonstrate that sickle monocytes are activated and can, in turn, activate endothelial cells. It is speculated that vascular inflammation, marked by activated monocytes and endothelium, plays a significant role in the pathophysiology of vaso-occlusion in sickle cell anemia.

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Inhibition of cell adhesion by anti–P-selectin aptamer: a new potential therapeutic agent for sickle cell disease

Blood ◽

10.1182/blood-2010-05-285718 ◽

2011 ◽

Vol 117 (2) ◽

pp. 727-735 ◽

Cited By ~ 73

Author(s):

Diana R. Gutsaeva ◽

James B. Parkerson ◽

Shobha D. Yerigenahally ◽

Jeffrey C. Kurz ◽

Robert G. Schaub ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Adhesion ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Therapeutic Agent ◽

Critical Role ◽

High Specificity ◽

Cell Disease ◽

Adhesive Interactions

Abstract Adhesive interactions between circulating sickle red blood cells (RBCs), leukocytes, and endothelial cells are major pathophysiologic events in sickle cell disease (SCD). To develop new therapeutics that efficiently inhibit adhesive interactions, we generated an anti–P-selectin aptamer and examined its effects on cell adhesion using knockout-transgenic SCD model mice. Aptamers, single-stranded oligonucleotides that bind molecular targets with high affinity and specificity, are emerging as new therapeutics for cardiovascular and hematologic disorders. In vitro studies found that the anti–P-selectin aptamer exhibits high specificity to mouse P-selectin but not other selectins. SCD mice were injected with the anti–P-selectin aptamer, and cell adhesion was observed under hypoxia. The anti–P-selectin aptamer inhibited the adhesion of sickle RBCs and leukocytes to endothelial cells by 90% and 80%, respectively. The anti–P-selectin aptamer also increased microvascular flow velocities and reduced the leukocyte rolling flux. SCD mice treated with the anti–P-selectin aptamer demonstrated a reduced mortality rate associated with the experimental procedures compared with control mice. These results demonstrate that anti–P-selectin aptamer efficiently inhibits the adhesion of both sickle RBCs and leukocytes to endothelial cells in SCD model mice, suggesting a critical role for P-selectin in cell adhesion. Anti–P-selectin aptamer may be useful as a novel therapeutic agent for SCD.

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Proof of Concept for Repair of Sickle Cell Disease By Non-Myeloablative MHC Disparate T Cell Depleted HSCT Combined with Donor-Derived Veto Cells

Blood ◽

10.1182/blood-2019-124594 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1919-1919

Author(s):

Aloukick Kumar Singh ◽

Elias Schetzen ◽

Esther BACHAR Lustig ◽

Wei-Hsin Liu ◽

Raj Kumar Yadav ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Cd8 T Cells ◽

Culture Conditions ◽

Central Memory ◽

Cell Disease ◽

Memory Cd8 T Cells ◽

Veto Cells

Although life extending medical treatments are available for sickle cell disease (SCD), allogenic hematopoietic stem cell transplantation (HSCT) is considered a treatment of choice (Ozdogu et al., Bone Marrow Transplant 2018; 53(7): 880-890). However, HSCT is associated with several limitations, including conditioning-related toxicity and graft-versus-host disease (GvHD), especially when using MHC disparate transplants. Thus, the development of safe transplantation protocols for MHC disparate HSCT in sickle cell disease is vital. While the risk of GvHD and conditioning toxicity can be effectively reduced by the use of T-cell-depleted HSCT (TD-HSCT) under reduced intensity conditioning (RIC), rejection of TD-HSCT remains a challenge. We previously demonstrated in wild type mice that this barrier can be overcome using donor-derived veto cells. Here, we demonstrate the safety and efficacy of this approach in a well-defined murine model for sickle cells disease. Veto activity is based on the ability of certain cells to attack host CTL-precursors (CTLp) which are directed against antigens expressed on the veto cells themselves, sparing cells that are not targeted against the veto cells including T cells needed for defense against pathogens. Central memory CD8 T cells exhibit the most robust veto activity upon transplantation; however, these cells are also endowed with marked GvH activity. We overcame this issue by expanding naïve or memory CD8 T cells against 3rd party MHC or viral antigens, respectively, under culture conditions favoring expression of central memory phenotype. Such anti-3rd party central memoryCD8 T cells (Tcm), which are endowed with marked veto activity, also exhibit reduced risk for GvHD in fully mis-matched recipients (Reviewed in Reisner Y, Or-Geva N. Semin Hematol. 2019; 56(3): 173-182.) To generate Tcm veto cells, splenocytes obtained from Balb/c donors (H2d) were cultured against irradiated third-party splenocytes (FVB; H2q) under cytokine deprivation. The selective expansion of CD8 mouse T cells against 3rd party stimulators leads to selective 'death by neglect' of bystander anti-host T cell clones potentialy mediating GvHD, and these are further diluted out by subsequent expansion of anti-3rd party T clones during continued culture in the presence of IL-15. Apart from selective loss of GvH reactive T cells, these culture conditions induce a central memory phenotype shown to be important for attaining robust veto activity in vivo (Ophir et al., Blood. 2013; 121(7): 1220-8). We first calibrated the optimal irradiation dose for sickle mice (Berkeley model, H2b), comparing 4.5 Gy versus 5 Gy TBI in a conditioning protocol also including short term rapmycin treatment (Scheme 1). Higher levels of engraftment and chimerism were found in the group receiving 5Gy TBI (Fig. 1). All mice of both treatment groups survived >140 days with no evidence of GvHD. To further evaluate this treatment approach, 8 week old sickle mice (N=7) were given bone marrow transplants using the protocol described in Scheme 1, including conditioning with 5 Gy TBI (day -1), rapamycin treatment (day -1 to day 4), and transplantation of NuBM (day 0) plus veto cells (day 7; Scheme 1). Notably, at 44 days post-transplant, 6 out of 7 mice receiving NuBM + TCM + Rapa (85.7%) showed donor chimerism in the peripheral blood, ranging between 77-94% (Fig.2A-B), while no chimerism was detected in mice receiving conditioning alone, or conditioning and transplantation with only NuBM or only veto cells . All mice in all groups survived (N=26) , and no GvHD was detected with a follow up of 77 days, even in the transplanted group which exhibited high donor-derived chimerism. Furthermore, reversal of sickle disease symptoms was observed, including reticulocyte levels (p=0.001;Fig.2C) and expression of wild type hemoglobin ( Fig.2D) in all engrafted mice. Our results offer a proof of concept for the treatment of sickle disease by MHC disparate non-myeloablative T cell depleted HSCT in conjunction with anti-3rd party central memory veto CD8 T cells. A clinical trial testing the safety and efficacy of anti-3rd party veto cells in the context of low toxicity non-myeloablative TD-HSCT in hematological malignancies is currently ongoing at MD Anderson Cancer Center. If successful, our present results support further evaluation of this platform for sickle cell disease. Disclosures Lustig: Yeda Ltd.: Patents & Royalties. Reisner:Cell Source, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

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Activated monocytes in sickle cell disease: potential role in the activation of vascular endothelium and vaso-occlusion

Blood ◽

10.1182/blood.v96.7.2451 ◽

2000 ◽

Vol 96 (7) ◽

pp. 2451-2459 ◽

Cited By ~ 214

Author(s):

John D. Belcher ◽

Paul H. Marker ◽

Jill P. Weber ◽

Robert P. Hebbel ◽

Gregory M. Vercellotti

Keyword(s):

Endothelial Cells ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Nuclear Translocation ◽

Mononuclear Leukocytes ◽

Cell Contact ◽

Cell Disease ◽

Tnf Α ◽

Activated Monocytes

Abstract Sickle cell anemia is characterized by painful vaso-occlusive crises. It is hypothesized that monocytes are activated in sickle cell disease and can enhance vaso-occlusion by activating endothelium. To test this hypothesis, human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (MVEC) with sickle and normal mononuclear leukocytes were incubated, and endothelial activation was measured. Endothelial cells incubated with sickle mononuclear leukocytes were more activated than those incubated with normal mononuclear leukocytes, as judged by the increased endothelial expression of adhesion molecules and tissue factor and the adhesion of polymorphonuclear leukocytes (PMNL). Monocytes, not lymphocytes or platelets, were the mononuclear cells responsible for activating endothelial cells. Sickle monocytes triggered endothelial nuclear factor-kappa B (NF-κB) nuclear translocation. Cell-to-cell contact of monocytes and endothelium enhanced, but was not required for, activation. Antibodies to tumor necrosis factor-alpha (TNF-α) and interleukin-1-beta (IL-1β) blocked activation of the endothelium by monocytes. Peripheral blood monocytes from patients with sickle cell disease had 34% more IL-1β (P = .002) and 139% more TNF-α (P = .002) per cell than normal monocytes. Sixty percent of sickle monocytes expressed the adhesion molecule ligand CD11b on their surfaces compared with only 20% of normal monocytes (P = .002). Serum C-reactive protein, a marker of systemic inflammation, was increased 12-fold in sickle serum than in normal serum (P = .003). These results demonstrate that sickle monocytes are activated and can, in turn, activate endothelial cells. It is speculated that vascular inflammation, marked by activated monocytes and endothelium, plays a significant role in the pathophysiology of vaso-occlusion in sickle cell anemia.

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The Adhesion of Sickle Cell Disease Neutrophils to Endothelial Layers, In Vitro, Is Mediated by the Mac-1, LFA-1 and VLA-4 Integrins.

Blood ◽

10.1182/blood.v110.11.2264.2264 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2264-2264 ◽

Cited By ~ 1

Author(s):

Andreia A. Canalli ◽

Renata F. Proenca ◽

Sara T.O. Saad ◽

Nicola Conran ◽

Fernando F. Costa

Keyword(s):

Endothelial Cells ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Umbilical Vein ◽

In Vivo Studies ◽

Integrin Subunit ◽

Cell Disease ◽

Inflammatory Conditions ◽

Tnf Α

Abstract Leukocytes may have a propagating and, possibly, initiating role in sickle cell disease (SCD) vaso-occlusion. In vivo studies suggest that adherent leukocytes capture sickle erythrocytes in the microcirculation and in vitro studies demonstrate an increased ability of SCD neutrophils (neu) to adhere to fibronectin, endothelial cells and endothelial proteins. Previous studies suggest that the expressions of the major neu integrins, CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1) may only be upregulated on the surface of SCD neu following their stimulation, indicating that alterations in integrin function (affinity or avidity) contribute to alter SCD neu adhesion. The objective of this study was to identify the integrins responsible for altered SCD neu adhesion. Neus were isolated from the peripheral blood of healthy controls and SCD individuals in steady state over ficoll-paque gradients. Cell adhesion (2×106cells/ml in RPMI) to cultured human umbilical vein endothelial cells (HUVEC) at confluence was assessed using static adhesion assays (30min, 37°C, 5%CO2). Neus from SCD patients demonstrated a significantly greater adhesion to HUVEC than control neus (20.2±2.8% compared to 11.2±1.0%; n≥7; p<0.03; Mann Whitney test). Subsequently, cells were co-incubated with adhesion molecule-blocking monoclonal antibodies (mAbs) during assays. Control neu adhesion to HUVEC was significantly inhibited by the anti-CD11b mAb (6.7±1.5%;n=6; P<0.05, paired t test), but not by mAbs against CD11a, the VLA-4-integrin subunit, CD49d, or a non-specific negative control mAb (neg control) (data not shown). In contrast, the adhesion of SCD neus to HUVEC was significantly inhibited by both the anti-CD11a and the anti-CD11b mAbs (20.2±2.8% reduced to 11.4±1.2% and 9.1±1.5%; n=9; P<0.01 and P<0.001, respect.). Interestingly, a mAb against CD49d was also found to significantly decrease SCD neu adhesion to HUVEC (10.4±1.1%; n=9; P<0.01), while the neg control mAb did not significantly affect SCD neu adhesion (data not shown). Following the stimulation of HUVEC with TNF-α (10 ng/ml) (3h, 37°C, 5%CO2) to simulate an endothelial layer under inflammatory conditions, the adhesions of control and SCD neus were increased but statistically similar (38.4±2.9% and 34.4±5.0%; n≥4, respect.). Under these conditions anti-CD11a and CD11b mAbs significantly inhibited control neu adhesion to HUVEC (reduced to 28.8±2.9% and 19.6±4.6%; n=4; P<0.01 and P<0.05, respect.). In contrast, SCD neu adhesion to HUVEC was significantly inhibited by mAbs for CD11a (19.5±2.6%; n=6; p<0.01) and CD11b (15.2±2.0%; n=6; p<0.001). The anti-CD49d, but not the neg control mAb, also significantly decreased SCD neu adhesion to TNF-α-stimulated HUVEC (19.5±3.7%; n=6; p<0.05). In conclusion, data indicate that control neu adhesion to endothelial cells appears mainly to be mediated by the Mac-1 (CD11b/18) integrin with a contribution from the LFA-1 integrin (CD11a/18) under inflammatory conditions. In contrast, SCD neu adhesion to endothelium (under both basal and stimulated conditions), at least in vitro, appears to be mediated by the Mac-1 and LFA-1 integrins and, interestingly, by VLA-4 (CD49d/CD29), an integrin found expressed at low levels on neus during certain inflammatory conditions. We speculate that alterations in the affinity/ avidity of these molecules contribute to SCD neu adhesion. Approaches to inhibit the adhesion of all three integrins may be important for preventing leukocyte adhesion to the vascular endothelium and, in turn, vaso-occlusion.

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