Rahima Zennadi

Carbohydrate Recognition on MUC1-Expressing Targets Enhances Cytotoxicity of a T Cell Subpopulation

The influence of the epithelial mucin MUC1 on T cell‐mediated lysis was analysed using lymph node lymphocytes (LNL) from patients with colorectal carcinoma. LNL were stimulated with allogeneic, MUC1‐transfected B cells and the bulk cultures were cloned. Alloreactive cytotoxic T cell clones were obtained which preferentially lysed MUC1‐expressing targets. The majority was CD4+ and MHC‐class II‐restricted, and a minor group was CD8+ and MHC‐class I‐restricted. All the clones expressed CD3 and TCRαβ, and were CD56−. The capacity to preferentially kill MUC1‐expressing targets was stable in several clones for up to 6 months in culture. The enhancing effect of MUC1 on the lysis was investigated in more detail. It was only seen after inhibition of O‐linked glycosylation in the targets. Furthermore, this effect was completely abrogated by the monoclonal antibody 3C9, directed against the Thomsen–Friedenreich antigen (T‐antigen, Galβ1–3GalNAc bound α1–3 to Ser/Thr) as well as by the soluble disaccharide Galβ1–3GalNAc, but not by other similar disaccharides. The authors conclude that in their system the preferential killing of MUC1‐expressing targets is due to the recognition of an internal carbohydrate epitope accessible on underglycosylated MUC1, possibly T‐antigen, by an auxiliary receptor molecule on T cells.

Sickle red cells induce adhesion of lymphocytes and monocytes to endothelium.

Infusion of epinephrine-activated human sickle erythrocytes (SS RBCs) into nude mice promotes both SS RBC and murine leukocyte adhesion to vascular endothelium in vivo. We hypothesized that interaction of epinephrine-stimulated SS RBCs with leukocytes leads to activation of leukocytes, which then adhere to endothelial cells (ECs). In exploring the underlying molecular mechanisms, we have found that coincubation in vitro of epinephrine-treated SS RBCs with human peripheral blood mononuclear cells (PBMCs) results in robust adhesion of PBMCs to ECs. Sham-treated SS RBCs had a similar but less pronounced effect, whereas neither sham- nor epinephrine-treated normal RBCs activated PBMC adhesion. PBMC activation was induced via at least 2 RBC adhesion receptors, LW and CD44. In response to SS RBCs, leukocyte CD44 and beta2 integrins mediated PBMC adhesion to ECs, a process that involved endothelial E-selectin and fibronectin. SS RBCs activated adhesion of both PBMC populations, lymphocytes and monocytes. Thus, our findings reveal a novel mechanism that may contribute to the pathogenesis of vaso-occlusion in sickle cell disease, in which SS RBCs act via LW and CD44 to stimulate leukocyte adhesion to endothelium, and suggest that RBC LW and CD44 may serve as potential targets for antiadhesive therapy designed to prevent vaso-occlusion.

Impaired adenosine-5'-triphosphate release from red blood cells promotes their adhesion to endothelial cells: a mechanism of hypoxemia after transfusion.

Objective:Transfusion of red blood cells has been linked to disappointing clinical outcomes in the critically ill, but specific mechanisms of organ dysfunction after transfusion remain poorly understood. We tested the hypothesis that red blood cell storage impairs the ability of red blood cells to release adenosine-5′-triphosphate and that impaired adenosine-5′-triphosphate release was injurious in vivo, in part through increased red blood cell adhesion. Design:Prospective, controlled, mechanistic study. Setting:University research laboratory. Subjects:Human and mouse blood donors; nude mouse transfusion recipients. Interventions:Manipulation of adenosine-5′-triphosphate release, supplemental adenosine-5′-triphosphate, and antibodies to red blood cell and endothelial adhesion receptors were used in vitro and in vivo to probe the roles of released adenosine-5′-triphosphate and adhesion in responses to (transfused) red blood cells. Measurements and Main Results:The ability of stored red blood cells to release adenosine-5′-triphosphate declined markedly within 14 days after collection despite relatively stable levels of adenosine-5′-triphosphate within the red blood cells. Inhibiting adenosine-5′-triphosphate release promoted the adhesion of stored red blood cells to endothelial cells in vitro and red blood cell sequestration in the lungs of transfused mice in vivo. Unlike transfusion of fresh human red blood cells, stored red blood cell transfusion in mice decreased blood oxygenation and increased extravasation of red blood cells into the lungs alveolar air spaces. Similar findings were seen with transfusion of fresh red blood cells treated with the adenosine-5′-triphosphate release inhibitors glibenclamide and carbenoxolone. These findings were prevented by either coinfusion of an adenosine-5′-triphosphate analog or pretransfusion incubation of the red blood cells with an antibody against the erythrocyte adhesion receptor Landsteiner-Wiener (intercellular adhesion molecule-4). Conclusions:The normal flow of red blood cells in pulmonary microvessels depends in part on the release of antiadhesive adenosine-5′-triphosphate from red blood cells, and storage-induced deficiency in adenosine-5′-triphosphate release from transfused red blood cells may promote or exacerbate microvascular pathophysiology in the lung, in part through increased red blood cell adhesion.

Erythrocyte plasma membrane–bound ERK1/2 activation promotes ICAM-4–mediated sickle red cell adhesion to endothelium

The core pathology of sickle cell disease (SCD) starts with the erythrocyte (RBC). Aberration in MAPK/ERK1/2 signaling, which can regulate cell adhesion, occurs in diverse pathologies. Because RBCs contain abundant ERK1/2, we predicted that ERK1/2 is functional in sickle (SS) RBCs and promotes adherence, a hallmark of SCD. ERK1/2 remained active in SS but not normal RBCs. β(2)-adrenergic receptor stimulation by epinephrine can enhance ERK1/2 activity only in SS RBCs via PKA- and tyrosine kinase p72(syk)-dependent pathways. ERK signaling is implicated in RBC ICAM-4 phosphorylation, promoting SS RBC adhesion to the endothelium. SS RBC adhesion and phosphorylation of both ERK and ICAM-4 all decreased with continued cell exposure to epinephrine, implying that activation of ICAM-4-mediated SS RBC adhesion is temporally associated with ERK1/2 activation. Furthermore, recombinant ERK2 phosphorylated α- and β-adducins and dematin at the ERK consensus motif. Cytoskeletal protein 4.1 also showed dynamic phosphorylation but not at the ERK consensus motif. These results demonstrate that ERK activation induces phosphorylation of cytoskeletal proteins and the adhesion molecule ICAM-4, promoting SS RBC adhesion to the endothelium. Thus, blocking RBC ERK1/2 activation, such as that promoted by catecholamine stress hormones, could ameliorate SCD pathophysiology.

Proteomic analysis of ERK1/2-mediated human sickle red blood cell membrane protein phosphorylation

BackgroundIn sickle cell disease (SCD), the mitogen-activated protein kinase (MAPK) ERK1/2 is constitutively active and can be inducible by agonist-stimulation only in sickle but not in normal human red blood cells (RBCs). ERK1/2 is involved in activation of ICAM-4-mediated sickle RBC adhesion to the endothelium. However, other effects of the ERK1/2 activation in sickle RBCs leading to the complex SCD pathophysiology, such as alteration of RBC hemorheology are unknown.ResultsTo further characterize global ERK1/2-induced changes in membrane protein phosphorylation within human RBCs, a label-free quantitative phosphoproteomic analysis was applied to sickle and normal RBC membrane ghosts pre-treated with U0126, a specific inhibitor of MEK1/2, the upstream kinase of ERK1/2, in the presence or absence of recombinant active ERK2. Across eight unique treatment groups, 375 phosphopeptides from 155 phosphoproteins were quantified with an average technical coefficient of variation in peak intensity of 19.8%. Sickle RBC treatment with U0126 decreased thirty-six phosphopeptides from twenty-one phosphoproteins involved in regulation of not only RBC shape, flexibility, cell morphology maintenance and adhesion, but also glucose and glutamate transport, cAMP production, degradation of misfolded proteins and receptor ubiquitination. Glycophorin A was the most affected protein in sickle RBCs by this ERK1/2 pathway, which contained 12 unique phosphorylated peptides, suggesting that in addition to its effect on sickle RBC adhesion, increased glycophorin A phosphorylation via the ERK1/2 pathway may also affect glycophorin A interactions with band 3, which could result in decreases in both anion transport by band 3 and band 3 trafficking. The abundance of twelve of the thirty-six phosphopeptides were subsequently increased in normal RBCs co-incubated with recombinant ERK2 and therefore represent specific MEK1/2 phospho-inhibitory targets mediated via ERK2.ConclusionsThese findings expand upon the current model for the involvement of ERK1/2 signaling in RBCs. These findings also identify additional protein targets of this pathway other than the RBC adhesion molecule ICAM-4 and enhance the understanding of the mechanism of small molecule inhibitors of MEK/1/2/ERK1/2, which could be effective in ameliorating RBC hemorheology and adhesion, the hallmarks of SCD.

Effect of Propranolol as Antiadhesive Therapy in Sickle Cell Disease

Sickle red blood cells (SSRBCs) adhere to both endothelial cells (ECs) and the extracellular matrix. Epinephrine elevates cyclic adenosine monophosphate in SSRBCs and increases adhesion of SSRBCs to ECs in a β‐adrenergic receptor and protein kinase A‐dependent manner. Studies in vitro as well as in vivo have suggested that adrenergic stimuli like epinephrine may contribute to vaso‐occlusion associated with physiologic stress. We conducted both animal studies and a Phase I dose‐escalation study in sickle cell disease (SCD) patients to investigate whether systemically administered propranolol inhibits SSRBC adhesion and to document the safety of propranolol in SCD. Systemically administered propranolol prevented SSRBC adhesion and associated vaso‐occlusion in a mouse model. In patients receiving a single oral dose of 10, 20, or 40 mg propranolol, SSRBC adhesion to ECs was studied before and after propranolol, with and without stimulation with epinephrine. Propranolol administration significantly reduced epinephrine‐stimulated SSRBC adhesion in a dose dependent manner (p = 0.03), with maximum inhibition achieved at 40 mg. Adverse events were not severe, did not show dose dependence, and were likely unrelated to drug. No significant heart rate changes occurred. These results imply that β‐blockers may have a role as antiadhesive therapy for SCD. Clin Trans Sci 2012; Volume 5: 437–444

Role and regulation of sickle red cell interactions with other cells: ICAM-4 and other adhesion receptors

Erythrocytes containing primarily hemoglobin S (SS RBCs) are abnormally adherent. We now know that SS RBCs express numerous adhesion molecules, and that many of these can undergo activation. SS RBCs exposed briefly to epinephrine show markedly increased adhesion to both laminin and endothelial cells. In vivo, infusion of epinephrine-activated but not unstimulated SS RBCs causes RBC adhesion, vaso-occlusion, organ trapping, and shortened RBC survival in the circulation. Epinephrine treatment of SS RBCs before infusion also induces adhesion of murine leukocytes to vascular walls. Indeed, in vitro, SS RBCs can activate leukocyte adhesion and cytokine production. We now have demonstrated both in vitro and in vivo evidence for the importance of RBC signaling and have also shown that SS RBC adhesion is determined by genetic polymorphisms in the signaling pathway that activates adhesion. These advances will hopefully lead to new therapeutic modalities for sickle cell disease.

Sickle Erythrocytes Target Cytotoxics to Hypoxic Tumor Microvessels and Potentiate a Tumoricidal Response

Resistance of hypoxic solid tumor niches to chemotherapy and radiotherapy remains a major scientific challenge that calls for conceptually new approaches. Here we exploit a hitherto unrecognized ability of sickled erythrocytes (SSRBCs) but not normal RBCs (NLRBCs) to selectively target hypoxic tumor vascular microenviroment and induce diffuse vaso-occlusion. Within minutes after injection SSRBCs, but not NLRBCs, home and adhere to hypoxic 4T1 tumor vasculature with hemoglobin saturation levels at or below 10% that are distributed over 70% of the tumor space. The bound SSRBCs thereupon form microaggregates that obstruct/occlude up to 88% of tumor microvessels. Importantly, SSRBCs, but not normal RBCs, combined with exogenous prooxidant zinc protoporphyrin (ZnPP) induce a potent tumoricidal response via a mutual potentiating mechanism. In a clonogenic tumor cell survival assay, SSRBC surrogate hemin, along with H2O2 and ZnPP demonstrate a similar mutual potentiation and tumoricidal effect. In contrast to existing treatments directed only to the hypoxic tumor cell, the present approach targets the hypoxic tumor vascular environment and induces injury to both tumor microvessels and tumor cells using intrinsic SSRBC-derived oxidants and locally generated ROS. Thus, the SSRBC appears to be a potent new tool for treatment of hypoxic solid tumors, which are notable for their resistance to existing cancer treatments.

Sevuparin binds to multiple adhesive ligands and reduces sickle red blood cell-induced vaso-occlusion

Sevuparin is a novel drug candidate in phase II development as a treatment for vaso‐occlusive crises (VOC) in patients with sickle cell disease (SCD). As a heparin‐derived polysaccharide, sevuparin has been designed to retain anti‐adhesive properties, while the antithrombin‐binding domains have been eliminated, substantially diminishing its anticoagulant activity. Here, we demonstrate that sevuparin inhibits the adhesion of human sickle red blood cells (SS‐RBCs) to stimulated cultured endothelial cells in vitro. Importantly, sevuparin prevents vaso‐occlusion and normalizes blood flow in an in vivo mouse model of SCD vaso‐occlusion. Analyses by surface plasmon resonance (SPR) and fluorescence correlation spectroscopy (FCS) demonstrate that sevuparin binds to P‐ and L‐selectins, thrombospondin, fibronectin and von Willebrand factor, all of which are thought to contribute to vaso‐occlusion in SCD. Despite low anticoagulation activity, sevuparin has anti‐adhesive efficacy similar to the low molecular weight heparin tinzaparin both in vitro and in vivo. These results suggest that the anti‐adhesive properties rather than the anticoagulant effects of heparinoids are critical for the treatment of vaso‐occlusion in SCD. Therefore, sevuparin is now being evaluated in SCD patients hospitalized for treatment of VOC.

MEK Inhibitors, Novel Anti-Adhesive Molecules, Reduce Sickle Red Blood Cell Adhesion In Vitro and In Vivo, and Vasoocclusion In Vivo

In sickle cell disease, sickle erythrocyte (SSRBC) interacts with endothelial cells, leukocytes, and platelets, and activates coagulation and inflammation, promoting vessel obstruction, which leads to serious life-threatening complications, including acute painful crises and irreversible damage to multiple organs. The mitogen-activated protein kinase, ERK1/2, is abnormally activated in SSRBCs. However, the therapeutic potential of SSRBC ERK1/2 inactivation has never been investigated. I tested four different inhibitors of MEK1/2 (MEK), the kinase that activates ERK1/2, in a model of human SSRBC adhesion to TNFα-activated endothelial cells (ECs). SSRBC MEK inhibition abrogated adhesion to non-activated and TNFα-activated ECs to levels below baseline SSRBC adhesion to non-activated ECs in vitro. SSRBC MEK inhibition also prevented SSRBCs from activating naïve neutrophils to adhere to endothelium. To determine the effect of MEK inhibitors on SSRBC adherence in vivo, sham-treated or MEK inhibitor-treated SSRBCs were infused to nude mice previously treated with TNFα. Sham-treated SSRBCs displayed marked adhesion and occlusion of enflamed vessels, both small and large. However, SSRBC treatment with MEK inhibitors ex vivo showed poor SSRBC adhesion to enflamed vessels with no visible vasoocclusion in vivo. In addition, MEK inhibitor treatment of SSRBCs reduced SSRBC organ trapping and increased the number of SSRBCs circulating in bloodstream. Thus, these data suggest that SSRBC ERK1/2 plays potentially a critical role in sickle pathogenesis, and that MEK inhibitors may represent a valuable intervention for acute sickle cell crises.