The molecular basis of antibody protection against West Nile virus.

West Nile virus (WNV) infection of mosquitoes, birds, and vertebrates continues to spread in the Western Hemisphere. In humans, WNV infects the central nervous system and causes severe disease, primarily in the immunocompromised and elderly. In this review we discuss the mechanisms by which antibody controls WNV infection. Recent virologic, immunologic, and structural experiments have enhanced our understanding on how antibodies neutralize WNV and protect against disease. These advances have significant implications for the development of novel antibody-based therapies and targeted vaccines.

A subpopulation of Mac-1 (CD11b/CD18) molecules mediates neutrophil adhesion to ICAM-1 and fibrinogen.

We report that a subpopulation (10%) of the Mac-1 (CD1 1b/CD18) molecules on activated neutrophils mediates adhesion to ICAM-1 and fibrinogen. We describe a novel mAb (CBRM1/5) that binds to an activation-specific neoepitope on a subset of Mac-1 molecules on neutrophils and monocytes after stimulation with chemoattractants or phorobol esters but does not recognize Mac-1 on resting myeloid cells. CBRM1/5 immunoprecipitates a subpopulation of Mac-1 molecules from detergent lysates of neutrophils, binds to immunoaffinity-purified Mac-1, and localizes to the I domain on the alpha chain of Mac-1. Because CBRM1/5 recognizes a fraction of Mac-1 on activated neutrophils, but still blocks Mac-1-dependent adhesion to fibrinogen and ICAM-1, we suggest that only a small subset of Mac-1 molecules is competent to mediate adhesion.

Leishmania promastigotes require opsonic complement to bind to the human leukocyte integrin Mac-1 (CD11b/CD18).

Previous reports have suggested that Leishmania spp. interact with macrophages by binding to Mac-1 (CD1 1b/CD18), a member of the leukocyte integrin family. To better define this interaction, we tested the ability of leishmania promastigotes to bind to purified leukocyte integrins and to cloned integrins expressed in COS cells. We show that leishmania promastigotes bind to cellular or purified Mac-1 but not lymphocyte function-associated antigen-1 in a specific, dose-dependent manner that requires the presence of serum. Binding is inhibited with specific monoclonal antibodies to Mac-1. In the absence of complement opsonization, three different species of leishmania tested fail to bind directly to any of the three leukocyte integrins. We show that binding to Mac-1 requires the third component of complement (C3). Organisms incubated in heat-inactivated serum or serum that has been immunologically depleted of C3 fail to bind to Mac-1. Because the addition of purified C3 to C3-depleted serum restores leishmania binding to Mac-1, we suggest that parasites gain entry into macrophages by fixing complement and subverting a well-characterized adhesive interaction in the immune system between Mac-1 and iC3b.

Heparin is an adhesive ligand for the leukocyte integrin Mac-1 (CD11b/CD1).

Previous studies have demonstrated that the leukocyte integrin Mac-1 adheres to several cell surface and soluble ligands including intercellular adhesion molecule-1, fibrinogen, iC3b, and factor X. However, experiments with Mac-1-expressing transfectants, purified Mac-1, and mAbs to Mac-1 indicate the existence of additional ligands. In this paper, we demonstrate a direct interaction between Mac-1 and heparan sulfate glycans. Heparin affinity resins immunoprecipitate Mac-1, and neutrophils and transfectant cells that express Mac-1 bind to heparin and heparan sulfate, but not to other sulfated glycosaminoglycans. Inhibition studies with mAbs and chemically modified forms of heparin suggest the I domain as a recognition site on Mac-1 for heparin, and suggest that either N- or O-sulfation is sufficient for heparin to bind efficiently to Mac-1. Under conditions of continuous flow in which heparins and E-selectin are cosubstrates, neutrophils tether to E-selectin and form firm adhesions through a Mac-1-heparin interaction.

The I domain is a major recognition site on the leukocyte integrin Mac-1 (CD11b/CD18) for four distinct adhesion ligands.

Despite the identification and characterization of several distinct ligands for the leukocyte integrin (CD11/CD18) family of adhesion receptors, little is known about the structural regions on these molecules that mediate ligand recognition. In this report, we use alpha subunit chimeras of Mac-1 (CD11b/CD18) and p150,95 (CD11c/CD18), and an extended panel of newly generated and previously characterized mAbs specific to the alpha chain of Mac-1 to map the binding sites for four distinct ligands for Mac-1: iC3b, fibrinogen, ICAM-1, and the as-yet uncharacterized counter-receptor responsible for neutrophil homotypic adhesion. Epitopes of mAbs that blocked ligand binding were mapped with the chimeras and used to localize the ligand recognition sites because the data obtained from functional assays with the Mac-1/p150,95 chimeras were not easily interpreted. Results show that the I domain on the alpha chain of Mac-1 is an important recognition site for all four ligands, and that the NH2-terminal and perhaps divalent cation binding regions but not the COOH-terminal segment may contribute. The recognition sites in the I domain appear overlapping but not identical as individual Mac-1-ligand interactions are distinguished by the discrete patterns of inhibitory mAbs. Additionally, we find that the alpha subunit NH2-terminal region and divalent cation binding region, despite being separated by over 200 amino acids of the I domain, appear structurally apposed because three mAbs require the presence of both of these regions for antigenic reactivity, and chimeras that contain the NH2 terminus of p150,95 require the divalent cation binding region of p150,95 to associate firmly with the beta subunit.

ICAM-1 (CD54): a counter-receptor for Mac-1 (CD11b/CD18).

While the leukocyte integrin lymphocyte function-associated antigen (LFA)-1 has been demonstrated to bind intercellular adhesion molecule (ICAM)-1, results with the related Mac-1 molecule have been controversial. We have used multiple cell binding assays, purified Mac-1 and ICAM-1, and cell lines transfected with Mac-1 and ICAM-1 cDNAs to examine the interaction of ICAM-1 with Mac-1. Stimulated human umbilical vein endothelial cells (HUVECs), which express a high surface density of ICAM-1, bind to immunoaffinity-purified Mac-1 adsorbed to artificial substrates in a manner that is inhibited by mAbs to Mac-1 and ICAM-1. Transfected murine L cells or monkey COS cells expressing human ICAM-1 bind to purified Mac-1 in a specific and dose-dependent manner; the attachment to Mac-1 is more temperature sensitive, lower in avidity, and blocked by a different series of ICAM-1 mAbs when compared to LFA-1. In a reciprocal assay, COS cells cotransfected with the alpha and beta chain cDNAs of Mac-1 or LFA-1 attach to immunoaffinity-purified ICAM-1 substrates; this adhesion is blocked by mAbs to ICAM-1 and Mac-1 or LFA-1. Two color fluorescence cell conjugate experiments show that neutrophils stimulated with fMLP bind to HUVEC stimulated with lipopolysaccharide for 24 h in an ICAM-1-, Mac-1-, and LFA-1-dependent fashion. Because cellular and purified Mac-1 interact with cellular and purified ICAM-1, we conclude that ICAM-1 is a counter receptor for Mac-1 and that this receptor pair is responsible, in part, for the adhesion between stimulated neutrophils and stimulated endothelial cells.

GloPID-R report on Chikungunya, O'nyong-nyong and Mayaro virus, part I: Biological diagnostics.

The GloPID-R (Global Research Collaboration for Infectious Disease Preparedness) Chikungunya (CHIKV), O'nyong-nyong (ONNV) and Mayaro virus (MAYV) Working Group is investigating the natural history, epidemiology and medical management of infection by these viruses, to identify knowledge gaps and to propose recommendations for direct future investigations and rectification measures. Here, we present the first report dedicated to diagnostic aspects of CHIKV, ONNV and MAYV. Regarding diagnosis of the disease at the acute phase, molecular assays previously described for the three viruses require further evaluation, standardized protocols and the availability of international standards representing the genetic diversity of the viruses. Detection of specific IgM would benefit from further investigations to clarify the extent of cross-reactivity among the three viruses, the sensitivity of the assays, and the possible interfering role of cryoglobulinaemia. Implementation of reference panels and external quality assessments for both molecular and serological assays is necessary. Regarding sero-epidemiological studies, there is no reported high-throughput assay that can distinguish among these different viruses in areas of potential co-circulation. New specific tools and/or improved standardized protocols are needed to enable large-scale epidemiological studies of public health relevance to be performed. Considering the high risk of future CHIKV, MAYV and ONNV outbreaks, the Working Group recommends that a major investigation should be initiated to fill the existing diagnostic gaps.

The dynamic regulation of integrin adhesiveness.

The integrins are a family of transmembrane heterodimeric adhesion molecules that play important roles in wound healing, immune system function and organ development. Recent studies indicate that adhesion of integrins to their ligands is not constitutive but is dynamically regulated by intracellular signal transduction pathways.

Subcellular localization and dynamics of Mac-1 (alpha m beta 2) in human neutrophils.

The subcellular localization of Mac-1 was determined in resting and stimulated human neutrophils after disruption by nitrogen cavitation and fractionation on two-layer Percoll density gradients. Light membranes were further separated by high voltage free flow electrophoresis. Mac-1 was determined by an ELISA with monoclonal antibodies that were specific for the alpha-chain (CD11b). In unstimulated neutrophils, 75% of Mac-1 colocalized with specific granules including gelatinase granules, 20% with secretory vesicles and the rest with plasma membranes. Stimulation with nanomolar concentrations of FMLP resulted in the translocation of Mac-1 from secretory vesicles to the plasma membrane, and only minimal translocation from specific granules and gelatinase granules. Stimulation with PMA or Ionomycin resulted in full translocation of Mac-1 from secretory vesicles and gelatinase granules to the plasma membrane, and partial translocation of Mac-1 from specific granules. These findings were corroborated by flow cytometry, which demonstrated a 6-10-fold increase in the surface membrane content of Mac-1 in response to stimulation with FMLP, granulocyte-macrophage colony stimulating factor, IL-8, leukotriene B4, platelet-activating factor, TNF-alpha, and zymosan-activated serum, and a 25-fold increase in response to Ionomycin. Thus, secretory vesicles constitute the most important reservoir of Mac-1 that is incorporated into the plasma membrane during stimulation with inflammatory mediators.

The thin filament of vertebrate skeletal muscle co-operatively activates as a unit.

We find that extraction of as little as one troponin C molecule per troponin-tropomyosin strand on a thin filament reduces the slope of the pCa/tension relation. We interpret this to mean that the regulatory units along a thin filament of rabbit psoas fibers are linked co-operatively so that a thin filament activates as a unit. The presence of extended co-operativity explains why the pCa/tension relation in skinned fibers has a slope much higher than predicted by binding of Ca2+ to one regulatory unit. Replacement of the extracted troponin C with purified troponin C fully reverses the effect of extraction and shows it to be the essential Ca2+ binding protein responsible for the steep slope of the pCa/tension relation.

Effect of different troponin T-tropomyosin combinations on thin filament activation.

The response of permeabilized rabbit fast skeletal muscle fibers to calcium is determined by the troponin T (TnT) and tropomyosin (Tm) isoforms they express. Fibers expressing primarily TnT2f and alpha 2 Tm exhibit steeper pCa/tension relations than those in which either TnT1f or TnT3f and alpha beta Tm predominate. Troponin C extraction studies show that lower slopes do not result from a less concerted transition on the thin filament: the Tn-Tm regulatory strand activates as a unit in all fast fibers. Because the TnT variants differ in their N-terminal segments, and this region overlaps adjacent Tms on the regulatory strand, we propose that both the end-to-end overlap of Tm and the effect of TnT on that interaction are the basis of the concerted transition of the regulatory strand to the active state that occurs in the presence of calcium. Moreover, the effect of different Tn-Tm combinations on the ratio of the affinities of TnC for calcium in the relaxed and active states appears to be a significant determinant of the contractile properties of fast fibers in vivo.

Co-operative interactions between troponin-tropomyosin units extend the length of the thin filament in skeletal muscle.

Ca2+ binding to troponin C (TnC), a subunit of the thin filament regulatory strand, activates vertebrate skeletal muscle contraction. Tension, however, increases with Ca2+ too abruptly to be the result of binding to sites on individual TnCs. Because extraction of one TnC on average per regulatory strand dramatically reduces the slope of the tension/Ca2+ relationship, we proposed that all 26 troponin-tropomyosin complexes of the regulatory strand form a co-operative system. This study of permeabilized (chemically skinned) rabbit psoas fibers analyzes the extraction time-course, the distribution of extraction sites on regulatory strands and the effects of extraction on the co-operativity of the tension/Ca2+ relationship. Two components of TnC are resolved in the time-course of extraction: a "rapidly extracting" component that can be selectively removed without affecting tension or co-operativity, and a "slow extracting" component whose loss reduces tension and co-operativity. Extraction of [14C]TnC shows that the slowly extracting component is lost randomly, so that, after removal of 5% of the TnC, most extracted strands have lost one TnC. Extraction interrupts the transmission of co-operativity by dividing the regulatory strand into smaller, independent co-operative systems; it reduces tension by preventing Ca2+ activation of TnC-depleted regulatory units. Co-operativity of the tension/Ca2+ relationship is modeled with the concerted-transition formalism for intact systems of 26 regulatory units, and for the smaller systems in extracted fibers.

Changes in subcellular localization and surface expression of L-selectin, alkaline phosphatase, and Mac-1 in human neutrophils during stimulation with inflammatory mediators.

The localization of the adhesion protein L-selectin in human neutrophils was determined by subcellular fractionation and immunoelectron microscopy and compared with the localization of Mac-1 (alpha m beta 2) and alkaline phosphatase, the marker for secretory vesicles. L-selectin was found to be localized exclusively on the plasma membrane of unstimulated cells and also of stimulated cells, although markedly diminished. This was in contrast to Mac-1, which was also localized in secretory vesicles and in specific/gelatinase granules as shown previously [Sengeløv, H., et al. J. Clin. Invest. (1993) 92, 1467-1476]. Stimulation of neutrophils with inflammatory mediators such as tumor necrosis factor (TNF), platelet-activating factor (PAF), or f-Met-Leu-Phe (fMLP), induced parallel up-regulation of the surface membrane content of alkaline phosphatase and Mac-1 and down-regulation of L-selectin, as evidenced by flow cytometry. Preimbedding immunoelectron microscopy confirmed that L-selectin was present mainly on tips of microvilli in unstimulated cells and showed that alkaline phosphatase and Mac-1 were randomly distributed on the surface membrane of fMLP-stimulated cells. These studies indicate that the transition of neutrophils from L-selectin-presenting cells to Mac-1-presenting cells induced by inflammatory mediators is mediated by incorporation of secretory vesicle membrane, rich in Mac-1 and devoid of L-selectin, into the plasma membrane.

Biochemical properties of macrophage fractions and their relation to the mechanism of superoxide production.

Guinea pig alveolar macrophages are separable by density gradient centrifugation into three subpopulations whose capacity for biological activity (e.g. O2- production and chemotaxis) varies directly with buoyant density [(1983) J. Reticuloendothel. Soc. 33, 157-164]. This study demonstrates that the activity per cell of various other enzymes remains constant among the subpopulations. When normalized for cell volume, enzyme activity diminishes with decreasing buoyant density. Intracellular calcium mobilization, linked to formyl peptide and concanavalin A-stimulated O2- production, similarly diminishes. Formyl peptide receptor distribution and affinity remain constant. Decreased responsiveness of lower density cells is probably due to lower concentration of enzyme(s) involved in the transduction of signal distal to ligand recognition (or binding).

Interferon inhibits dengue virus infection by preventing translation of viral RNA through a PKR-independent mechanism.

Previously, we demonstrated that pretreatment of cells with interferon (IFN) alpha + gamma or beta + gamma inhibited dengue virus (DV) replication. In this study, experiments were performed to better define the mechanism by which IFN blocks the accumulation of dengue virus (DV) RNA. Pretreatment of human hepatoma cells with IFN beta + gamma did not significantly alter virus attachment, viral entry, or nucleocapsid penetration into the cytoplasm. The inhibitory effect of IFN was retained even when naked DV RNA was transfected directly into cells, confirming that steps associated with viral entry were not the primary target of IFN action. Biosynthetic labeling experiments revealed that IFN abolished the translation of infectious viral RNA that occurred prior to RNA replication. Subcellular fractionation experiments demonstrated that IFN did not significantly alter the ability of viral RNA to attach to ribosomes. The antiviral effect of IFN appeared independent of the IFN-induced, double-stranded RNA-activated protein kinase (PKR) and RNase L, as genetically deficient PKR- RNase L- cells that were infected by DV retained sensitivity to inhibition by IFN. We conclude that IFN prevents DV infection by inhibiting translation of the infectious viral RNA through a novel, PKR-independent mechanism.

The leukocyte integrin p150,95 (CD11c/CD18) as a receptor for iC3b. Activation by a heterologous beta subunit and localization of a ligand recognition site to the I domain.

p150,95 is a member of the leukocyte integrin family of adhesion proteins. Compared with LFA-1 and Mac-1, p150,95 is less well functionally characterized. Although p150,95 has complement receptor activity for iC3b and has been designated complement receptor type 4, transfected cells expressing p150,95 do not bind iC3b-sensitized cells. We report that cells cotransfected with a human p150,95 alpha subunit and a chicken, but not human, beta subunit bind IgM-iC3b-coated erythrocytes, suggesting that interactions between the alpha and beta subunits can regulate p150,95 adhesiveness. Furthermore, purified human p150,95 binds to cell-bound iC3b-coated erythrocytes. Because binding to iC3b by cellular and purified p150,95 is specifically abolished by mAbs that localize to the I domain of p150,95, we suggest that the I domain of the p150,95 alpha subunit is an important ligand recognition site for iC3b.

Separation of bronchoalveolar cells from the guinea pig on continuous gradients of Percoll: functional properties of fractionated lung macrophages.

Lung macrophages from normal guinea pig lungs were separated from bronchoalveolar cells into three fractions according to buoyant density by centrifugation on continuous iso-osmotic gradients of Percoll [3]. A reproducible pattern of functional activity distinguished these three macrophage fractions. With increasing density and decreasing cell size, the respective fractions exhibited increased stimulated migration, superoxide anion release and pinocytosis, and increased protein concentration of the cells. These differences, coupled with previous observations that these fractions also exhibited morphological and cytochemical differences [3], support the notion that these fractions of macrophages may represent different stages of maturation (or differentiation) of alveolar macrophages in the lungs of normal guinea pigs.

A switch region inversion contributes to the aberrant rearrangement of a mu immunoglobulin heavy chain gene in MPC-11 cells.

We describe the unique features of an aberrantly rearranged mu immunoglobulin heavy chain gene isolated from MPC-11 cells (a gamma 2b producing Balb/c plasmacytoma). A novel rearrangement has occurred 1.5 Kb 5' of the MPC-11 mu gene (denoted 18b mu) resulting in the deletion of the majority of the repetitive switch region (S mu) and 5' flanking DNA including the Joining (JH) sequences. The remainder (275 bp) of the S mu repeat has undergone a complete sequence inversion. DNA sequences 5' of the inverted S mu sequence do not resemble Variable (VH), Diversity (D), JH or their conserved flanking sequences. A DNA sequence localized 5' of the inverted S mu sequence, (p18b mu-1.4) detects a small family of homologous sequences in Balb/c DNA. The 18b mu-1.4 like sequences lack homology to S mu, exhibit flanking sequence polymorphisms in 5 out of 6 inbred mouse strains and undergo partial or complete deletion in 5 out of 10 plasmacytomas tested. Two 18b mu-1.4 homologous sequences display a higher copy number in C57Bl/6, AL/N and CAL9 mouse strains.