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Human neutrophils generally function adherent to an extracellular matrix. We have previously reported that upon adhesion to laminin- or fibronectin-coated, but not uncoated, plastic there is a depolymerization of actin in neutrophils. This phenomenon was not affected by inhibitors of the more well-studied components of the signal transduction pathway, specifically, pertussis toxin, an inhibitor of G-proteins, H-7 or staurosporine, inhibitors of protein kinase C, or herbimycin A, an inhibitor of nonreceptor tyrosine kinase. We therefore focused our attention on actin-binding proteins and measured the changes in the partitioning of gelsolin between the Triton X-100-soluble and -insoluble cellular fractions which occur upon neutrophil adhesion by means of quantitating anti-gelsolin antibody binding to aliquots of these fractions. It was found that approximately 90% of the total cellular gelsolin was found in the Triton X-100-soluble fraction in suspended cells, but that upon adherence to either fibronectin- or laminin-coated plastic about 40% of the soluble gelsolin could be detected in the insoluble fraction. This effect was not observed in cells adherent to uncoated plastic, wherein more than 90% of the gelsolin was found in the soluble fraction. Results of immunofluorescence microscopy of these cell preparations was consistent with this data. A gelsolin translocation to the insoluble cellular actin network may account for a part of the observed actin depolymerization.

Mannose-binding proteins (MBPs), members of the collectin family, have been implicated as lectin opsonins for various viruses and bacteria. Two distinct but related MBPs, MBP-A and MBP-C, with approximately 55% identity at the amino acid level, have been previously characterized from rodents. In humans, however, only one form of MBP has been characterized. In this paper we report studies elucidating the evolution of primate MBPs. ELISA and Western blot analyses indicated that rhesus and cynomolgus monkeys have two forms of MBP in their sera, while chimpanzees have only one form, similar to humans. Two distinct MBP cDNA clones were isolated and characterized from a rhesus monkey liver cDNA library. Rhesus MBP-A is closely related to the mouse and rat MBP-A, showing 77% and 75% identity at the amino acid level, respectively. Rhesus MBP-A also has three cysteines at the N-terminus, similar to mouse and rat MBP-A and human MBP. Rhesus MBP-C shares 90% identity with the human MBP at the amino acid level and has three cysteines at the N-terminus, in contrast to two cysteine residues found in rodent MBP-C. A stretch of nine amino acids close to the N-terminus, absent in both mouse and rat MBP-A, but present in rodent MBP-C, chicken and human MBPs, is also found in the rhesus MBP-A. The phylogenetic analysis of rhesus and other mammalian MBPs, coupled with the serological data suggest that at least two distinct MBP genes existed prior to mammalian radiation and the hominoid ancestor apparently lost one of these genes or failed to express it.

Surfactant protein-D (SP-D) is a collectin found associated with surfactant in the lung. SP-D has also been functionally characterized as an opsonin for diverse microorganisms and a chemoattractant for phagocytic cells. To determine the structure of mouse SP-D, we isolated and characterized clones from a B6/CBAF1J strain lung cDNA library using a PCR-derived genomic probe. The deduced sequence predicts a 19-amino acid signal sequence, a 25-amino acid long NH2 terminus with two cysteines, followed by an uninterrupted collagen domain with 59 Gly-X-Y repeats. Next, a short "neck" domain of 28 amino acids, with a potential to form trimeric alpha-helical coiled coil is found ending in a COOH-terminal 125-amino acid carbohydrate recognition domain. The mature mouse SP-D protein of 355 amino acids shows strong homology to rat (92% identity), human (76%), and bovine (72%) SP-D amino acid sequences. Northern blot and RT-PCR analysis revealed that the mouse SP-D gene is expressed predominantly in lung and, surprisingly, also in heart, stomach, and kidney but not in brain. In contrast, mouse surfactant protein-A (SP-A) mRNA expression was found to be restricted to lung. Human lung and stomach, but not heart or liver, were found to express SP-D mRNA, as determined by PCR. The mouse SP-D gene (Sftp4) has been localized to chromosome 14 (to a region syntenic to human chromosome 10), closely linked to the genes for other collagenous lectins, mannose-binding protein-A (MbI1), and SP-A (Sftp1).

Two research traditions in immunology, supposedly centered on the same issue of immune identification, have followed different theoretical goals and originated from competing philosophical foundations. These may be labelled modernist and postmodernist, respectively, thereby applying cultural and philosophical categories to immunology in order to articulate potential scientific resonances with the broader culture. To accept that exercise an important caveat is imposed, namely, this translation is most appropriately discussed at the level of metaphor. In other words, I will structure my treatment of these issues as expressed in the metaphorical language of the discipline, and thus the bulk of this discussion will focus on how the language and modeling of the science draws from the culture-at-large. Scientists seek images from their everyday lives to describe phenomena that may be poorly articulated in their technical discourse; such is the utility and importance of metaphors generally, and thus it is not surprising that we might discern echoes of a postmodernist sentiment in the metaphors borrowed from post-World War II culture. I will also discuss, to a more limited extent, how postmodernists have sought support for their own ideological arguments in immunology. This last topic serves only to illustrate the bidirectionality of scientific discourse with the society in which it is embedded.

Superoxide anion can modulate vascular smooth muscle tone and potentially affect the growth response in vascular disease. The present studies were undertaken to characterize the source of superoxide in rabbit aorta. Rings of aorta (5 mm) were incubated in physiological salt solution (PSS) for 30 min at 37 degrees C in the presence of 10 mM diethyldithiocarbamate (DDC) with or without inhibitors of superoxide-generating systems. Rings were then placed in PSS containing 250 microM lucigenin at 37 degrees C in the presence or absence of inhibitors, and changes in amounts of superoxide were determined by measuring chemiluminescence (units). The inhibitors of xanthine oxidase, oxypurinol (300 microM), and of mitochondrial NADH dehydrogenase, rotenone (50 microM), had no significant effect on superoxide levels. An inhibitor of NADPH oxidase, iodonium thiophen, caused a concentration-dependent inhibition of superoxide anion (12.49 +/- 1.48 vs 5.27 +/- 1.81 and 2.30 +/- 0.36 units, control vs 7 microM and 70 microM iodonium thiopen, respectively). A structurally related iodonium compound, diphenyleneiodonium (20 microM), caused a 78% reduction in basal and DDC-evoked superoxide levels. In the presence or absence of DDC, exogenous administration of NADPH (10 microM-1 mM), but not NADP (1 mM), elicited a concentration-dependent rise in superoxide levels that was inhibited by iodonium thiophen. Particulate fractions of whole aortic tissue exhibited NADPH-dependent superoxide production that was inhibited by 1 microM diphenyleneiodonium.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial superinfections are the most common cause of mortality during influenza epidemics. Depression of phagocyte functions by influenza A viruses (IAVs) is a likely contributory cause of such infections. We used an in vitro model of viral depression of neutrophil respiratory burst responses to FMLP and PMA to examine the mechanism of IAV-induced phagocyte deactivation. Respiratory burst responses or intracellular calcium mobilization were triggered by the virus itself, but these were not causally related to deactivation. By treating neutrophils with neuraminidase, and by use of purified IAV hemagglutinin (HA) preparations, cross-linking of sialic acid-bearing neutrophil surface components by the IAV HA was shown to be responsible for deactivation. IAV competed for binding to neutrophils with Abs directed against CD43, sialyl-Le(x), CD45, and gangliosides. Deactivation could be reproduced by treating neutrophils with anti-CD43 or -sialyl-Le(x) Abs in the absence of IAV. However, treatment of neutrophils with elastase markedly reduced CD43 expression, without affecting overall IAV binding or the ability of IAV to cause deactivation. Hence, although IAV binding to CD43 can account for deactivation, other IAV-binding proteins exist (e.g., those bearing sialyl-Le(x)) that can independently mediate functional depression.

Human neutrophils have multiple C1q-binding proteins. Direct ligand-binding studies with the globular domain of C1q and two-dimensional Western blot analysis revealed two gC1q-binding proteins (gC1q-R): a 33,000 M(r) protein (pI 4.5) mainly in the neutrophil plasma membrane and an 80,000-90,000 M(r) protein (pI 4.1-4.2) located mainly in the granules. Direct binding studies showed that C1q bound to this higher molecular weight protein under physiological conditions. In contrast, anti-cC1q-R antibody, which recognizes a protein binding to collagenous tails of C1q, detected only a 68,000 M(r) protein in the plasma membrane. Both the 33,000 and 68,000 M(r) receptors appear early on the surface of differentiating HL-60 cells. On mature neutrophils, surface expression of both C1q receptors was evident, but no upregulation was observed upon stimulation. Phorbol myristate acetate treatment of neutrophils downregulated both the receptors from cell surface, and significant amounts of soluble gC1q-R were in cell media supernatants, suggesting receptor shedding or secretion. gC1q-R, unlike cC1q-R, did not bind to other C1q-like ligands, namely mannose binding protein, surfactant protein-A, surfactant protein-D, or conglutinin under normal ionic conditions, suggesting a greater specificity for C1q than the "collectin" type receptor (cC1q-R). Rather, gC1q-R only bound purified C1q, and the binding was enhanced under low ionic conditions and in the absence of calcium. The role of C1q receptor shedding and its biologic consequence remain to be defined, but may contribute to the diversity of C1q-mediated responses observed in many cell types.

Mannose-binding protein (MBP) is a member of a family of collagenous lectins (collectins), which are believed to play an important role in first-line host defense. In this study, the two genes encoding MBP in mice--Mbl1 and Mbl2--have been isolated and their exon-intron structure studied to understand their evolutionary relationship to the single human (MBL) and the two rat MBP genes. Mouse Mbl1 and Mbl2 have five and six exons, respectively. The structure of the mouse Mbl genes is similar to that of the rat and human MBP genes and shows homology to the other collectin genes, with the entire carbohydrate recognition domain being encoded in a single exon and all introns being in phase 1. The MBP encoded by mouse Mbl1 with three cysteines in the first coding exon, like the rat Mbl1 and human MBL, is capable of a higher degree of multimerization and has apparent ability to fix complement in the absence of antibody or C1q. However, the structural features of other exons, that is, the larger size of collagen domain region in the first coding exon (64 bp in Mbl2 vs 46 bp in Mbl1) and the smaller size of the exon encoding the trimerization domain (69 bp in Mbl2 vs 75 bp in Mbl1) reveal that the single human MBL gene is closely related to rodent Mbl2 rather than rodent Mbl1. The findings in this study suggest that in contrast to the evolution of another collectin gene--bovine surfactant protein-D--which duplicated in bovidae after divergence from humans, MBP gene most likely duplicated prior to human-rodent divergence, and that the human homolog to Mbl1 was perhaps lost during evolution.

The Ca2+ storage protein calreticulin is associated with the endoplasmic reticulum and shares a high degree of amino acid homology with the surface receptor C1q-R. In this study, flow cytometric analysis detected calreticulin on the neutrophil surface, which decreased during stimulation probably as a consequence of shedding, as calreticulin was found by ELISA in the cell supernatants of stimulated cells. Antibodies raised against C1q-R and calreticulin demonstrated a high degree of immunological cross-reactivity for purified calreticulin as determined by dot blot analysis. Western blots of neutrophil subcellular fractions located calreticulin in both the cytosol and cell membrane fractions; C1q-R was largely confined to the cell membrane. Calreticulin and C1q-R both bind to C1q and mannan-binding protein. Therefore, calreticulin may be shed on cell activation and may be associated with the cell membrane, where it can potentially interact with C1q and serum lectins. The implications of this are discussed.

We tested the hypothesis that pulmonary surfactant-associated lectins--surfactant proteins A and D (SP-A, and -D)--contribute to initial protective mechanisms against influenza A viruses (IAVs). SP-D potently inhibited hemagglutination activity of several strains of IAV as well as causing viral aggregation. SP-D enhanced neutrophil binding of IAV and neutrophil respiratory burst responses to the virus. Neutrophil dysfunction resulting from IAV exposure was diminished when the virus was pre-incubated with SP-D. Each of these effects was mediated by the calcium-dependent carbohydrate-binding property of SP-D. Native SP-D preparations of both human and rat origin, as well as recombinant rat SP-D, had similar activity. SP-A also inhibited IAV hemagglutination activity. We have previously reported that related mammalian serum lectins (mannose-binding lectin [MBL] and conglutinin) have similar effects. SP-D was at least 10-fold more potent at causing hemagglutination inhibition than were SP-A or MBL. SP-D was shown to contribute to potent anti-IAV activity of human bronchoalveolar lavage fluid. These results suggest that SP-D--alone, and in conjunction with SP-A and phagocytic cells--constitutes an important component of the natural immune response to IAV infection within the respiratory tract.

Bovine conglutinin (BC), a member of the mammalian C-type collectin subfamily, is a serum protein synthesized in liver that is believed to play a role in natural host defense. Previously, we have characterized a full length BC cDNA and we now describe the partial characterization of a genomic clone that encodes for the BC gene (CGN1). BC is encoded by nine exons spanning > 11 kb and has been localized previously to band 18 of bovine (Bos taurus) chromosome 28. Genomic sequencing demonstrated that the signal peptide/amino-terminal domain, the carbohydrate recognition domain, and the linking peptide, a domain between the collagenous region and the carbohydrate recognition domain, are each encoded by a single exon. The collagenous domain is split into five exons, with the 5' most region being located within the exon that also encodes the signal peptide/amino terminus. The remaining four collagenous domain exons are tandemly arranged with lengths of 117, 108, 108, and 117 bp, respectively. Overall, the BC genomic organization is very similar to that of the human surfactant protein-D gene, SFTP4. On the basis of identical collagen domain structures, we suggest that conglutinin and bovine surfactant protein-D evolved from a gene duplication event occurring in Bovidae after divergence from other mammals.

We have studied in detail the in vitro interactions of influenza A viruses (IAVs) with human neutrophils to clarify why these cells become dysfunctional during IAV infection. Unosponized IAV elicited a respiratory burst response in neutrophils which, like that triggered by formylmethionyl-leucyl-phenylalanine (fMLP), involved mediation of signal-transducing GTP-binding proteins and tyrosine kinase activation. The IAV-induced response differed from that provoked by fMLP in that H2O2 was produced without concomitant O2- release. IAV also did not cause extracellular release of granule enzymes in cytochalasin B-treated neutrophils. Using chemiluminescence assays, the respiratory burst response to IAV was found to occur at an intracellular location. These findings may, in part, explain the anomalous nature of the respiratory burst response elicited by IAV and suggest strategies for determining the mechanism of IAV-induced neutrophil deactivation.

Bovine conglutinin (BC) is a C-type lectin isolated from bovine serum that appears to play a role in first-line host defense. The BC cDNA was cloned from a bovine liver library and the nucleotide (nt) sequence of 1519 bp was determined. The longest open reading frame encoded a 20-amino-acid (aa) signal sequence and a mature protein of 351 aa. Analysis of the nt and deduced aa sequences revealed 87 and 78% identity, respectively, with the sequences of another vertebrate lectin: bovine surfactant protein-D (SP-D). Of interest, the expression of the BC mRNA, as determined by RNase protection assay, is restricted to liver, unlike bovine SP-D, a lung-surfactant protein.

Immunology is one of the unique products of the darwinian age--born in the controversies of that fresh announcement that all species, including ourselves, were not static entities, but subject to change as a result of the vicissitudes of time and circumstance. Darwinism postulated an everchanging species defined by evolutionary necessity. In this scheme, the organism is not given, but evolves. Always adapting, it is always changing. Thus, this raises the core issue of organismal identity as a problem. Here, Alfred Tauber explores the concept of self and traces the development of the term from Metchnikoff's theory that immunity resides in the active pursuit of identity.

Since the 1940's, non-Ig inhibitors of influenza A virus (IAV) hemagglutination activity and infectivity have been recognized in mammalian sera. Recently, the heat labile (beta) inhibitor of this type was identified by indirect methods as the lectin, conglutinin. In support of this hypothesis, we found that purified conglutinin strongly inhibited hemagglutination activity and infectivity of IAV. By using IAV strains with specific variations in glycosylation of the hemagglutinin molecule, we showed these effects to be mediated by binding of conglutinin to high mannose carbohydrate attachments on the viral hemagglutinin. Through the same mechanism conglutinin caused aggregation of IAV particles. Human neutrophils produce hydrogen peroxide upon exposure to IAV. Also, after a brief period of exposure to IAV, neutrophils exhibit depressed responsiveness (deactivation) upon exposure to other stimuli (e.g., chemotactic peptides). These phenomena may be related to the in vivo inflammatory response during IAV infection, and to the propensity of IAV-infected subjects to suffer bacterial superinfection. Pre-incubation of IAV with conglutinin markedly potentiated human neutrophil hydrogen peroxide production in response to the virus. This effect correlated with the ability of conglutinin to aggregate the virus. IAV treated with conglutinin also caused significantly less neutrophil deactivation than did the unopsonized virus. These enhancements of neutrophil respiratory burst responses by conglutinin were again mediated by binding of the lectin to viral carbohydrates. The mammalian C-type lectin family includes conglutinin, mannose-binding protein, and surfactant proteins A and D. These lectins may be important constituents of the initial host response to IAV, by inhibiting IAV infectivity directly, causing viral aggregation, and acting as opsonins to enhance phagocyte responses to the virus.

The interactions of annexin I with specific granules isolated from human neutrophils were investigated. Unfractionated cytosol induced Ca(2+)-dependent granule self-aggregation and fusion of granules with model phospholipid vesicles. High Ca2+ concentrations were required for these processes (500-600 microM for the half-maximal rate of granule self-aggregation; 100-200 microM for the half-maximal rate of fusion with phospholipid vesicles). These activities were inhibited by a monoclonal antibody specific for annexin I and immunodepletion of cytosol by this antibody greatly reduced activity, implicating annexin I as the major mediator of these processes in neutrophil cytosol. The fact that the Ca2+ concentration dependences differed for different membranes suggests that specificity may be controlled by the type of intracellular membrane involved and the local Ca2+ concentration. Trypsin treatment of granules enhanced the rate of fusion of phospholipid vesicles with granules, suggesting that access to phospholipids in the granule membrane may be modulated by granule proteins or that a fusogenic protein factor in the granule membrane is activated by trypsin treatment. Coaggregation of specific granules with plasma membrane vesicles mediated by Ca2+ and annexin I was suggested by the fact that granules preincubated with Ca2+, cytosol and plasma membrane vesicles blocked the fusion of subsequently added phospholipid vesicles with the plasma membrane vesicles. These data suggest a role for annexin I as part of a multiprotein system involved in membrane-membrane contact necessary for exocytosis of specific granules in human neutrophils.

Influenza A viruses (IAVs) cause substantial morbidity and mortality in yearly epidemics, which result from the ability of the virus to alter the antigenicity of its envelope proteins. Despite the rapid replication of this virus and its ability to infect a wide variety of cell types, viremia is rare and the infection is generally limited to the upper respiratory tract. The preimmune host defense response against IAV is generally, therefore, successful. We have previously provided (and summarized) evidence that neutrophils contribute to defense against IAV, although neutrophil dysfunction and local tissue damage may be less salutory byproducts of this response. Here we provide evidence that the serum lectin mannose-binding protein directly inhibits hemagglutinin activity and infectivity of several strains of IAV. In addition mannose-binding protein acts as an opsonin, enhancing neutrophil reactivity against IAV. Opsonization of IAV by mannose-binding protein also protects the neutrophil from IAV-induced dysfunction. These effects are observed with physiologically relevant concentrations of mannose-binding protein. Two different allelic forms of recombinant mannose-binding protein are found to have similar effects. We believe, on the basis of these data, that mannose-binding protein alone and in conjunction with phagocytic cells is an important constituent of natural immunity (i.e., preimmune defense) against IAV.

We have extended our previous studies of adherent neutrophils and compared actin depolymerization and intracellular calcium changes induced by adherence to laminin and fibronectin. In order to accurately assess cellular actin changes, F-actin depolymerization in the cell lysates must be inhibited. We found that phalloidin or 3.7% formaldehyde treatment effectively inhibited the depolymerization of F-actin fragments following cell lysis. Formaldehyde and phalloidin treatment reduced G-actin levels 75-80% in suspended cells, 35-73% in cells adherent for 1 min, and about 50% for cells adherent for 3 min. When the actin was fixed, there were highly significant differences in G-actin levels between the suspended and adherent cells as compared with unfixed cells. Adhesion to both laminin and fibronectin initiated a rapid rise in G-actin with a corresponding decrease in F-actin. However, the changes were more pronounced in cells adherent to laminin. The peak of depolymerization occurred by 1 min and, thereafter, G-actin decreased and F-actin increased reaching a steady state at 5 min. Adhesion to both laminin- and fibronectin-coated surfaces was accompanied by an increase of [Ca2+]i with a peak at 3 min, followed by a decrease from 3-5 min and a steady state attained between 5 and 10 min. The rise of [Ca2+]i in laminin-adherent cells was about twice that in fibronectin-adherent cells at 3 min (P < 0.02). Pertussis toxin, H-7, and staurosporin treatments did not alter the dynamic changes of actin in adherent cells, suggesting that these metabolic events are transduced by a G-protein and Protein Kinase C independent mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Human neutrophils adherent to a polystyrene plastic surface are vigorously activated, whereas those adherent to fibronectin manifest only a priming response. The basis of these metabolic differences was further characterized; polystyrene-adherent cells, which were shown to spread quickly upon adhesion, exhibited an increase of cytoskeleton-associated actin (F-actin) (measured by a nitrobenzoxadiazole-phallacidin fluorescent staining assay) and a decrease of monomeric G-actin concentration (measured by a DNase inhibition assay); in contrast, fibronectin-adherent cells exhibited little spreading and decreased their F-actin, after 1.5 min of adhesion, to 33.49 +/- 6.9% (mean +/- SD, n = 5) of initial levels found in suspended cells before plating. Actin depolymerization in fibronectin-adherent cells was confirmed by measuring G-actin, which sharply increased during the first minute of adhesion, rising from 0.065 +/- 0.007 to 0.20 +/- 0.035 microgram/microgram of protein (mean +/- SEM, p less than 0.05), and then remained elevated during 5 min of observation. In contrast, soluble fibronectin induced a decrease of G-actin in suspended cells. Cells pretreated with 1 microM cytochalasin D and allowed to adhere to a plastic surface did not spread, failed to generate O2-, and exhibited elevated concentrations of G-actin (0.1 to 0.2 microgram/microgram of protein) during the 5 min of observation. Actin changes, as well as respiratory burst, in adherent cells were shown to proceed through a pertussis toxin-insensitive pathway. Fluo-3 measurements of intracellular Ca2+ concentrations ([Ca2+]i) showed a fourfold and twofold [Ca2+]i increase in polystyrene- and fibronectin-adherent cells, respectively, after 2 min. The small rise in [Ca2+]i in fibronectin-adherent cells corresponds to a primed response of these cells to subsequent activation with FMLP. Ionomycin (1 microM) added to neutrophils just before adhesion on fibronectin induced full activation, i.e., O2- production and actin polymerization. The metabolic events controlling metabolic priming and actin depolymerization are as yet uncharacterized, but fibronectin receptor-linked responses beyond the mediation of cell adhesion have now been identified, suggesting complex metabolic functions of integrin receptors.

Whole cytosol isolated from human neutrophils was found to accelerate the Ca(2+)-dependent fusion of phospholipid vesicles with neutrophil plasma membranes as measured by several fluorescence resonance energy transfer lipid dilution assays or by the fate of an encapsulated aqueous soluble fluorophore. The Ca2+ (threshold of 2-10 microM) and protein concentration dependencies for fusion mediated by purified human neutrophil annexin I (lipocortin I), recombinant annexin I and des(1-9)annexin I showed behavior similar to that of whole cytosol. A monoclonal antibody against the N-terminal region of annexin I strongly inhibited the action of isolated annexins as well as whole cytosol, indicating that annexin I is the major activity of this type in whole neutrophil cytosol and that it functions even in this complex mixture of proteins. Residual Ca(2+)-dependent fusion activity in the absence of cytosol or annexin I was not inhibited by several antibodies against annexin I, implicating an as yet unknown protein. Kinetic analysis of liposomal fusion showed that annexin I, as in the case of synexin, accelerates aggregation of vesicles but not the actual fusion event per se. The disposition of annexin I in liposomal aggregates was studied by monitoring binding of the protein with a pyrene-phospholipid and by simultaneously monitoring vesicular aggregation by turbidity. An antibody to the N-terminus of annexin I inhibited vesicular aggregation but not binding, suggesting that initial binding of annexin I is similar to that of annexin V. A relatively small proportion of the bound annexin was involved in intervesicular linkage, and no exchange of bound annexin to subsequently added vesicles was observed. The lack of extensive contact between lipids of aggregated vesicles was supported by a lack of energy transfer between phospholipid probes on separate aggregating vesicles. Covalent linkage of maleimidyl or photoaffinity phospholipid derivatives with annexin I in vesicular aggregates did not allow complete disaggregation of vesicles by EDTA, suggesting that monomers of annexin I can contact two membranes simultaneously at the point of intervesicular linkage. These data are discussed in terms of possible models for the structure of this site.

The subcellular localization of a heparan sulfate degrading endoglycosidase, heparanase, was studied in human neutrophils. Unstimulated cells were disrupted by nitrogen cavitation and fractionated on a Percoll density gradient into three components, separating the plasma membranes, specific granules, and azurophilic granules. Heparanase activity was measured by gel filtration analysis of 35S-labeled degradation fragments released from subendothelial extracellular matrix (ECM) or produced during incubation with soluble, ECM-derived, heparan sulfate proteoglycans. Heparanase activity was found mainly in fractions containing the specific granules; this activity was inhibited by heparin. Freezing and thawing was not needed for recovery of the enzyme from the subcellular fraction, confirming previous data about its ready release. The mechanism of the ready release of heparanase from the specific granules requires further investigation.

Influenza A virus (IAV) causes both activation and deactivation of the human neutrophil, which may, respectively, contribute to host defense against the virus and enhanced susceptibility to bacterial superinfection. We have shown that certain features of neutrophil activation by IAV are distinctive compared with activation by chemoattractants in terms of both the stoichiometry of the respiratory burst response and the signal transduction events that precede it. We here demonstrate that related myxoviruses as well as sialic acid-binding lectins elicit a respiratory burst response similar to that induced by IAV, in which hydrogen peroxide is formed with minimal accompanying superoxide generation. Brief preincubation of neutrophils with these agents fully inhibits subsequent activation by IAV, implying that they are binding to the same surface membrane components as IAV. Preincubation with Limax flavus agglutinin (LFA) does, in fact, substantially reduce binding of radiolabeled IAV to the neutrophil. This lectin, like IAV, both activates and deactivates the neutrophil. As in the case of IAV, LFA-induced activation (1) is mediated via stimulation of phospholipase C, (2) is pertussis toxin insensitive, and (3) entails a lesser contribution of calcium influx than is the case for chemoattractants.

During the period of 1895-1910, immunology was preoccupied with defining the cellular (Elie Metchnikoff's phagocytosis theory) as opposed to the humoral basis of bactericidal defense. Although initial discovery of immunopathologic phenomena had been made (e.g., relating to transplantation, autoimmunity, allergy), focus on microbicidal therapy and diagnosis of infectious diseases remained the major stimuli of inquiry. The debate concerning the relative roles of phagocytes, complement, amboceptors (sensibilizing factors, antibody, antitoxins), various lysins (e.g., bacteriolysins, spermatolysins, hemolysins), agglutinins, stimulines, and then Almoth Wright's opsonins reflects the ambiguity of a scientific language being created in an era still struggling with a poorly defined experimental system, for the language, both its vocabulary (newly studied phenomena) and grammar (operational mechanisms) was yet to be codified. The joint award of the Nobel Prize to Metchnikoff and Paul Ehrlich in 1908 for their respective contributions to the "theory of immunity" appeared to proclaim a consensus, but the secret Nobel Committee reports that evaluated Metchnikoff's contributions reveal only a grudging acceptance of his position, and the award was clearly made on the basis of an apparent complementarity between the theoretical views of the humoralists and those elements of the phagocytosis theory that fit the then current discussion of immunity. In this regard, opsonins played an especially important role as both an experimental and conceptual bridge between the competing schools. What was no longer under consideration (and in fact never was explicitly debated) concerned the intellectual foundation of Metchnikoff's original concept of immunity as those activities that defined organismal identity, (developed from Metchnikoff's research in developmental biology) and which regarded host defense mechanisms as only subordinate to this primary function. Immunology in the first half of the 20th century pursued issues pertinent to chemically characterizing immune specificity and only later returned to the Metchnikovian question of how the immune identity was established. This latter venture has achieved molecular sophistication, but even such a formulation may be an inadequate answer to the Metchnikovian postulate. The theoretical discussion between cellularists and humoralists continues in new guises, for the essential debate remains unresolved.

The haptoglobin-related gene product (HGRP) having greater than 90% sequence homology with plasma haptoglobin, has recently been implicated as a tumor maker of recurrent breast cancer. Therefore, availability of a monoclonal antibody which is specific to the HGRP and ensuing ELISAs to quantitate the HGRP without cross-reactivity with plasma haptoglobin will allow determination of the clinical value of the HGRP in human plasma under various pathophysiological conditions. A peptide representing the first 34 residues of the N-terminal portion of the HRGP was synthesized and conjugated to keyhole limpet hemocyanin (KLH) to immunize Balb/c mice. Hybrid clones that produced specific antibodies to HRGP were screened with the same peptide but conjugated to bovine serum albumin (HRGP-BSA). From 12 hybridoma clones that recognized HGRP-peptide, we obtained one IgM producing clone (27.5) by limiting dilution technique that selectively reacted with HGRP-peptide with minimal cross-reactivity with haptoglobin. Using a separate monoclonal antibody, clone 21.7, which is directed to the alpha subunit of haptoglobin, we developed an enzyme-linked immunosorbent assay (ELISA) to determine the quantities of the HGRP in human plasma, which will allow assessment of HGRP as a clinical marker of malignancy.

Membrane fusion was studied using human neutrophil plasma membrane preparations and phospholipid vesicles approximately 0.15 microns in diameter and composed of phosphatidylserine and phosphatidylethanolamine in a ratio of 1 to 3. Liposomes were labeled with N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl (NBD) and lissamine rhodamine B derivatives of phospholipids. Apparent fusion was detected as an increase in fluorescence of the resonance energy transfer donor, NBD, after dilution of the probes into unlabeled membranes. 0.5 mM Ca2+ alone was sufficient to cause substantial fusion of liposomes with a plasma membrane preparation but not with other liposomes. Both annexin I and des(1-9)annexin I caused a substantial increase in the rate of fusion under these conditions while annexin V inhibited fusion. Fusion mediated by des(1-9)annexin I was observed at Ca2+ concentrations as low as approximately 5 microM, suggesting that the truncated form of this protein may be active at physiologically low Ca2+ concentrations. Trypsin treated plasma membranes were incapable of fusion with liposomes, suggesting that plasma membrane proteins may mediate fusion. Liposomes did not fuse with whole cells at any Ca2+ concentration, indicating that the cytoplasmic side of the membrane is involved. These results suggest that annexin I and unidentified plasma membrane proteins may play a role in Ca(2+)-dependent degranulation of human neutrophils.

The ionic basis of phorbol 12-myristate 13-acetate (PMA)-stimulated membrane depolarization in the human neutrophil has not previously been established. Alterations in cation permeability are probably not directly responsible for the depolarization response, since the rate or Rb+ influx or efflux is unchanged upon PMA stimulation, and although Na+ fluxes are increased, depolarization is not changed by either the addition of ouabain or reduction of extracellular Na+ from 140 to 0 meq. Furthermore, the enhanced Na+ influx in stimulated cells is blocked by amiloride at 10(-3) M, but not by 10(-5) M, suggesting Na+ influx proceeds through the electroneutral Na(+)-H+ antiporter and is therefore not responsible for depolarization. Upon stimulation, Cl- content of PMA-stimulated neutrophils decreases without change in Na+ or K+ content, as determined by electron probe analysis. In addition, acute reduction in extracellular Cl- enhances the rate and extent of depolarization induced by PMA. This change in intracellular Cl- and effect of reduction in extracellular Cl- concentration on depolarization can best be accounted for by an enhanced efflux via an electrogenic mechanism. Thus enhanced conductive Cl- efflux can account for the observed depolarization. That Ca2+ regulates depolarization is evidenced by the dependence of depolarization on external Ca2+ (Cao2+). Depolarization is absent in Ca2(+)-depleted cells [internal Ca2+ (Cai2+) less than 15 nM] and is restored with titration of extracellular Ca2+, exhibiting a 50% effective dose (ED50) of 100 mM. Thus PMA-initiated depolarization is regulated by Ca2+, either from intra- or extracellular sources, but the Ca2(+)-dependent activity responsible for control of Cl- efflux is as yet uncharacterized.

The subcellular distribution of GTP binding proteins in human neutrophils and their functional coupling to the N-formylmethionylleucylphenylalanine (FMLP) receptor was characterized to provide insight into mechanisms of cellular activation. Human neutrophils were nitrogen cavitated and fractionated on discontinuous Percoll gradients. Four subcellular fractions were obtained: cytosol, light membranes enriched for plasma membranes, specific granules and azurophilic granules. ADP-ribosylation catalyzed by pertussis toxin (PT) revealed a major substrate of 40 kDa only in plasma membrane and cytosol, and antiserum specific for Gi alpha confirmed the presence of neutrophil Gi alpha in plasma membrane and cytosol and its absence from specific granules. The cytosolic PT substrate was shown to be mostly in monomeric form by molecular sieve chromatography. The rate of the ribosyltransferase reaction was several-fold lower in cytosol compared to plasma membranes, and the extent of ADP-ribosylation was greatly augmented by supplementation with beta gamma subunits in cytosol. ADP-ribosylation catalyzed by cholera toxin (CT) revealed substrates of 52, 43 and 40 kDa in plasma membrane alone. FMLP receptors in plasma membrane were shown to be coupled to the 40 kDa substrate for CT by ligand-modulation of ADP-ribosylation, while FMLP added to specific granules did not induce ribosylation of this substrate even though FMLP receptors were found in high density in this compartment. Both 24 and 26 kDa [32P]GTP binding proteins were found to codistribute with FMLP receptors in specific granules and plasma membranes. Functional evidence for the coupling of GTP binding proteins to the FMLP receptor in specific granules was obtained by modulating [3H]FMLP binding with GTP gamma S, and by accelerating [35S]GTP gamma S binding with FMLP.

Nisoldipine inhibits calcium (Ca++) influx in human neutrophils: Preincubation with the dihydropyridine, nisoldipine (1.5 microM) resulted in a 30% decrease in [45]Ca++ influx during formyl-methionine-leucine-phenylalanine (FMLP) stimulation in primed as well as resting cells. Although the drug does not inhibit Ca++ dependent effector functions elicited by FMLP, e.g. superoxide (O2-) production, it inhibits FMLP priming, a phenomenon that is independent of extracellular Ca++. Nisoldipine exhibited a narrow dose response with an ED50 of ca. 1 microM and total inhibition of primed O2- response at 1.5 microM. Nisoldipine (1.5 microM) also abolished the incremental rise of Ca++i in primed neutrophils stimulated with FMLP. The dissociation of nisoldipine inhibitory effects on cell effector function and Ca++ transport were corroborated in studies with neutrophils stimulated with influenza virus and phorbol myristate acetate (PMA), stimuli which do not exhibit an extracellular Ca(++)-dependence in their elicited responses. Unlike in FMLP-stimulated cells, nisoldipine reduced influenza virus and PMA initiated respiratory burst, indicating that this drug has inhibitory effects on neutrophil function independent of its effect on Ca++ metabolism. Possible sites of action are postulated at phospholipase A2 or calmodulin-regulated activities. Caution is thus required in interpreting the effects of dihydropyridine on cell function, when the drug is used at micromolar concentration.

We have previously demonstrated that influenza A virus (IAV) stimulates the human neutrophil through phospholipase C activation. With the use of the fluorescent indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF), cytoplasmic acidification and subsequent alkalinization are shown to accompany this activation. These responses are not inhibited by pertussis toxin (PT). The alkalinization is mediated largely *but not entirely) by the Na(+)-H+ antiporter and is not initiated, or modulated, by the IAV-induced cytosolic Ca2+ (Cai2+) rise. Rather, protein kinase C (PKC) is likely the mediator of cell alkalinization, based on studies using the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). The acidification can be dissociated from the alkalinization response, which is also independent of Cai2+ fluxes and of PKC. Both pHi responses can be dissociated from the respiratory burst. Cytosolic alkalinization and acidification seem to reflect two independently mediated responses of the activated neutrophil, the former resulting ultimately from phospholipase activation and the latter from other activities that are not yet fully characterized.

Haptoglobin, an acute phase reactant protein, has been shown to modulate various facets of immune responses. In this paper we examined the effect of haptoglobin on human neutrophils at the molecular level. First, we found that native haptoglobin binds at two distinct sites on neutrophils. We then examined the effects of this binding at normal and pathophysiological concentrations of haptoglobin found in human serum. Of the various functional parameters assessed, neutrophil respiratory burst activity, as assessed by superoxide (O2-) production, was inhibited by native haptoglobin when the cells were stimulated with formylmethionyl-leucylphenylalanine (FMLP), arachidonic acid (AA), and opsonized zymosan. The rise in intracellular calcium induced by FMLP stimulation was also inhibited by native haptoglobin. Since the generation of O2- was unaffected by native haptoglobin in phorbol myristate acetate (PMA)-stimulated neutrophils, the likely site of haptoglobin inhibition on neutrophil function is at a point of receptor-ligand interaction in the activation cascade. The role of haptoglobin as a modifier of the immune response has here been extended to altered neutrophil function stimulated by diverse agonists.

Natural immune reactions are mediated by lymphocytes, macrophages/monocytes, and neutrophils. The latter have been implicated in a variety of self-surveillance models, i.e., activity against malignant host cells, participation in wound repair, and infliction of damage in postischemic perfusion injury. Better characterized are the interactions with unopsonized pathogens through lectinophagocytosis mechanisms, where the lectin resides either on the phagocyte or on the microorganism. This review examines the infection by influenza A virus (IAV) of the human neutrophil, which results in the vigorous metabolic response of the cell to generate toxic oxygen species. This response is not necessarily characteristic of response to unopsonized particles, as the neutrophil exhibits no such activity to unopsonized zymosan or chlamydia. The virus elicits calcium mobilization from intracellular stores through a pertussis toxin-insensitive mechanism, and in its particulars the activation cascade is unique in comparison to any other characterized agonist. The putative receptor for the IAV binding protein, hemagglutinin (HA), contains the sialic acid residues; identification of specifically linked protein receptors will allow characterization of this stimulation pathway and will define the molecular biology of this activation sequence. Insight into this particular pathway may allow definition of a primitive recognition system that represents a fundamental basis for discernment of self and nonself entities.

Human neutrophils stimulated with either latex particles or opsonized zymosan exhibited equivalent rates of net oxygen consumption as well as hydrogen peroxide release. The quantity of superoxide (O2-) detected in latex-stimulated neutrophils was less than 2% of that seen with opsonized zymosan stimulation, and only several-fold greater than that of resting cells. The failure to detect O2- in the latex-stimulated neutrophils was due neither to latex acting as a O2- scavenger nor to its interference with the O2- -forming system of the neutrophil. An intracellular site of O2- generation could not be demonstrated. NADPH oxidase activity in cells exposed to latex particles was only 10% of that seen in cells comparably activated with opsonized zymosan. Latex particles have the unusual property of stimulating the respiratory burst of the human neutrophil without the extracellular release of O2-. The potential physiologic importance of this finding is discussed.

The myeloperoxidase (MPO)-hydrogen peroxide (H2O2)-halide systems were found to produce chemiluminescence at 1,268 nm, a characteristic emission band for singlet oxygen (1O2). The emission was enhanced by a factor of 29 +/- 5 in deuterium oxide and was inhibited by the 1O2 quenchers, histidine and azide ion. Inactivation of MPO with heat or with cyanide ion prevented light production. The combined weight of all data strongly supported the production of 1O2 by these enzyme systems. The amount of 1O2 produced was sensitive to the conditions employed. Under optimal conditions at pH 5, the MPO-H2O2-bromide (Br-) system produced 0.42 +/- 0.03 mol 1O2/mol H2O2 consumed, close to the theoretical value of 0.5 that was predicted by the reaction stoichiometry. In contrast, the MPO-H2O2-chloride (Cl-) system was much less efficient. The maximum yield of 1O2 was 0.09 +/- 0.02 mol/mol H2O2 consumed and required pH 4 and 5 mM H2O2. At higher pH, the 1O2 production rapidly decreased. The yield at pH 7 was 0.0004 +/- 0.0002 mol/mol H2O2 consumed. Enzyme inactivation was a major factor limiting the yield of 1O2 with both Cl- and Br-. While the MPO-H2O2-halide systems can efficiently produce 1O2, the conditions required are not physiologic, which suggests that the chemiluminescence of the stimulated neutrophil does not derive from 1O2 generated by a MPO mechanism.