Gerard B. Nash

New guidelines for hemorheological laboratory techniques

This document, supported by both the International Society for Clinical Hemorheology and the European Society for Clinical Hemorheology and Microcirculation, proposes new guidelines for hemorheolog ...

Decreased rotational diffusion of band 3 in melanesian ovalocytes from Papua, New Guinea

SummaryMelanesian ovalocytes from Papua New Guinea have an N-terminal extension of the band 3 polypeptide (Jones, G.L., Edmunson. H.M., Wesche, D., Saul, A. 1990.Biochim. Biophys. Acta1096:33–40). The ovalocytes showed a threefold increase in shear elastic modulus as determined by micropipette aspiration measurements of membrane rigidity. Time-resolved phosphorescence anisotropy has been used to study the rotational freedom of band 3 in membranes prepared from ovalocytes. The ovalocytic polymorphism was found to be associated with a marked decrease in the rotational mobility of band 3. This may indicate participation of band 3 in large homoaggregates or in complexes with other proteins at the cytoplasmic surface. There was no morphological clustering of band 3 detectable by immunofluorescence microscopy.

Copy number of FCGR3B, which is associated with systemic lupus erythematosus, correlates with protein expression and immune complex uptake.

Copy number (CN) variation (CNV) has been shown to be common in regions of the genome coding for immune-related genes, and thus impacts upon polygenic autoimmunity. Low CN of FCGR3B has recently been associated with systemic lupus erythematosus (SLE). FcγRIIIb is a glycosylphosphatidylinositol-linked, low affinity receptor for IgG found predominantly on human neutrophils. We present novel data demonstrating that both in a family with FcγRIIIb-deficiency and in the normal population, FCGR3B CNV correlates with protein expression, with neutrophil uptake of and adherence to immune complexes, and with soluble serum FcγRIIIb. Reduced FcγRIIIb expression is thus likely to contribute to the impaired clearance of immune complexes, which is a feature of SLE, explaining the association between low FCGR3B CNV and SLE that we have confirmed in a Caucasian population. In contrast, antineutrophil cytoplasmic antibody–associated systemic vasculitis (AASV), a disease not associated with immune complex deposition, is associated with high FCGR3B CN. Thus, we define a role for FCGR3B CNV in immune complex clearance, a function that may explain why low FCGR3B CNV is associated with SLE, but not AASV. This is the first report of an association between disease-related gene CNV and variation in protein expression and function that may contribute to autoimmune disease susceptibility.

Rolling and stationary cytoadhesion of red blood cells parasitized by Plasmodium falciparum: separate roles for ICAM‐1, CD36 and thrombospondin

Summary. Adhesion of parasitized erythrocytes to microvascular endothelium is a central event in the pathogenesis of severe falciparum malaria. We have characterized the adhesion of flowing parasitized red blood cells to three of the known endothelial receptors coated on plastic surfaces (CD36, intercellular adhesion molecule‐1 (ICAM‐1) and thrombospondin (TSP)), and also to cells bearing these receptors (human umbilical vein endothelial cells (HUVEC) and platelets). All of the surfaces could mediate adhesion at wall shear stress within the physiological range. The great majority of adherent parasitized cells formed rolling rather than static attachments to HUVEC and ICAM‐1, whereas static attachments predominated for platelets, CD36 and TSP. Studies with monoclonal antibodies verified that binding the HUVEC was mainly via ICAM‐1, and to platelets via CD36. Adhesion via ICAM‐1 was least sensitive to increasing wall shear stress, but absolute efficiency of adhesion was greatest for CD36, followed by ICAM‐1, and least for TSP. TSP did not give long‐lasting adhesion under flow, whereas cells remained adherent to CD36 or ICAM‐1. We propose that the different receptors may have complementary roles in modulating adhesion in microvessels. Initial interaction at high wall shear stress may be of a rolling type, mediated by ICAM‐1 or other receptors, with immobilization and stabilization occurring via CD36 and/or TSP.

Identification of a phenotypically and functionally distinct population of long-lived neutrophils in a model of reverse endothelial migration

Recent studies have demonstrated that neutrophils are not a homogenous population of cells. Here, we have identified a subset of human neutrophils with a distinct profile of cell‐surface receptors [CD54high, CXC chemokine receptor 1low (CXCR1low)], which represent cells that have migrated through an endothelial monolayer and then re‐emerged by reverse transmigration (RT). RT neutrophils, when in contact with endothelium, were rescued from apoptosis, demonstrate functional priming, and were rheologically distinct from neutrophils that had not undergone transendothelial migration. In vivo, 1–2% of peripheral blood neutrophils in patients with systemic inflammation exhibit a RT phenotype. A smaller population existed in healthy donors (≈0.25%). RT neutrophils were distinct from naïve circulatory neutrophils (CD54low, CXCR1high) and naïve cells after activation with formyl‐Met‐Leu‐Phe (CD54low, CXCR1low). It is important that the RT phenotype (CD54high, CXCR1low) is also distinct from tissue‐resident neutrophils (CD54low, CXCR1low). Our results demonstrate that neutrophils can migrate in a retrograde direction across endothelial cells and suggest that a population of tissue‐experienced neutrophils with a distinct phenotype and function are present in the peripheral circulation in humans in vivo.

Omega-3 Fatty Acids and Inflammation: Novel Interactions Reveal a New Step in Neutrophil Recruitment

While investigating new mechanisms by which the dietary omega-3 fatty acids regulate inflammation, the authors have identified a new step in the regulation of neutrophil migration across vascular endothelial cells.

Antineutrophil Cytoplasmic Antibodies Stabilize Adhesion and Promote Migration of Flowing Neutrophils on Endothelial Cells

OBJECTIVE Recruitment of neutrophils to sites of inflammation requires coordinated regulation of their capture, activation, and migration on vascular endothelium. This study examines whether exposure of neutrophils to antineutrophil cytoplasmic antibodies (ANCAs) can disrupt this sequence of events. METHODS Isolated human neutrophils were perfused in the presence or absence of ANCA-positive IgG over endothelial cells that had been activated with either 2 units/ml or 100 units/ml of tumor necrosis factor alpha (TNFalpha) for 4 hours. RESULTS When endothelial cells were activated with 100 units/ml of TNFalpha, neutrophils were captured from flow, a small proportion of adherent cells rolled, and the majority transmigrated through the endothelial cell monolayer. When neutrophils were treated with ANCA IgG immediately before, 5 minutes before, or 15 minutes before perfusion, none rolled on contact with the endothelium, but the majority still transmigrated. When endothelial cells were activated with 2 units/ml of TNFalpha, the majority of untreated adherent neutrophils rolled, a few transmigrated, and the number that attached decreased with time during washout. In contrast, when neutrophils were pretreated with ANCA IgG just before perfusion, adhesion was stabilized, and the number of neutrophils that transmigrated was increased 10-fold. Priming of the neutrophils with TNFalpha before the addition of ANCA further increased the stability of neutrophil binding, but did not significantly increase transmigration. CONCLUSION Rather than frustrating the transmigration process, ANCAs promoted the migration of neutrophils through the endothelium. That the effect was evident at a relatively low level of endothelial activation suggests that ANCAs may potentiate the early vasculitic lesion and promote tissue damage and recruitment of other proinflammatory cells.

CLEC-2 and Syk in the megakaryocytic/platelet lineage are essential for development.

The C-type lectin receptor CLEC-2 signals through a pathway that is critically dependent on the tyrosine kinase Syk. We show that homozygous loss of either protein results in defects in brain vascular and lymphatic development, lung inflation, and perinatal lethality. Furthermore, we find that conditional deletion of Syk in the hematopoietic lineage, or conditional deletion of CLEC-2 or Syk in the megakaryocyte/platelet lineage, also causes defects in brain vascular and lymphatic development, although the mice are viable. In contrast, conditional deletion of Syk in other hematopoietic lineages had no effect on viability or brain vasculature and lymphatic development. We show that platelets, but not platelet releasate, modulate the migration and intercellular adhesion of lymphatic endothelial cells through a pathway that depends on CLEC-2 and Syk. These studies found that megakaryocyte/platelet expression of CLEC-2 and Syk is required for normal brain vasculature and lymphatic development and that platelet CLEC-2 and Syk directly modulate lymphatic endothelial cell behavior in vitro.

ANCA induces β2 integrin and CXC chemokine-dependent neutrophil-endothelial cell interactions that mimic those of highly cytokine-activated endothelium

Antineutrophil cytoplasm antibodies (ANCA) activate neutrophils to undergo a series of coordinated interactions, leading to transendothelial migration, eventually culminating in vascular destruction. The molecular events underlying neutrophil recruitment in ANCA‐associated vasculitis need to be defined to enable effective therapeutic manipulation. A flow‐based adhesion ssay was used to investigate the role of β2 integrins (CD11a/CD18 and CD11b/CD18) and chemokine receptors [CXC chemokine receptor (CXCR)1 and CXCR2] in neutrophil migration through the endothelium. Two endothelial models were used: a highly activated model stimulated with 100 U/ml tumor necrosis factor α (TNF‐α) and a minimally activated model stimulated with 2 U/ml TNF‐α and in which ANCA was present as a secondary neutrophil stimulus. CD11a/CD18, CD11b/CD18, and CXCR2 contributed to adhesion and transendothelial migration in both models. However, when the endothelium was minimally activated with TNF‐α, CD11b/CD18 played an important role in stabilizing adhesion induced by ANCA immunoglobulin G (IgG). Analysis of β2 integrins and chemokine receptors demonstrated that ANCA IgG had no effect on expression levels at the neutrophil surface but enabled an active conformational change of CD11b/CD18. Similar molecular mechanisms control neutrophil adhesion and migration through highly or minimally TNF‐α‐activated endothelium. However, the direct ANCA‐mediated neutrophil stimulation is needed to drive migration through minimally activated endothelium, and CD11b/CD18 is recruited for greater stability of adhesion during this process and can undergo an activatory, conformational change in response to ANCA IgG.

Basis of unique red cell membrane properties in hereditary ovalocytosis

Hereditary ovalocytes from a Mauritian subject are extremely rigid, with a shear elastic modulus about three times that of normal cells, and have increased resistance to invasion by the malaria parasite Plasmodium falciparum in vitro. The genetic anomaly resides in band 3; the protein gives rise to chymotryptic fragments with reduced mobility in SDS/polyacrylamide gel electrophoresis, but this is a result of anomalous binding of SDS and not a higher molecular weight. Analysis of the band 3 gene reveals (1) a point mutation (Lys56----Glu), which also occurs in a common asymptomatic band 3 (Memphis) variant and governs the electrophoretic properties, and (2) a deletion of nine amino acid residues, including a proline residue, encompassing the interface between the membrane-associated and the N-terminal cytoplasmic domains. The interaction of the mutant band 3 with ankyrin appears unperturbed. The fraction of band 3 capable of undergoing translation diffusion in the membrane is greatly reduced in the ovalocytes. Cells containing the asymptomatic band 3 variant were normal with respect to all the properties that we have studied. Possible mechanisms by which a structural change in band 3 at the membrane interface could regulate rigidity are examined.