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Myeloperoxidase (MPO) from human neutrophils has been purified and found to exist in three isoenzymatic forms, resolved by ion exchange chromatography. In addition to differences in subunit size and cellular compartmentalization of the isoenzymes, differences have been reported in their activity and susceptibility to inhibition. The structural basis of these isoenzymes is unclear; we attempted to further define their functional characteristics and structural identity. First, we measured respective enzymatic activity using a panel of substrates; MPO I was found to have lower activity with some substrates (pyrogallol, guaiacol, potassium iodide [KI]), but similar activity to the other isoenzymes with 4-aminoantipyrine. These studies confirm that MPO I is enzymatically distinct from MPO II and MPO III. Next, we examined the structural basis of these differences by evaluating the iron-containing prosthetic group in each form using electron paramagnetic resonance (EPR) and determination of the pyridine hemochrome. No significant difference between the isoenzymes was noted in these parameters, suggesting that the prosthetic group is the same in each protein. The cause for any difference in enzymatic activity must lie then in variations extrinsic to the heme, and based on previous studies of the gene and protein analysis, the posttranslational modification of MPO must account for these isoenzymatic species.

Phorbol esters are potent stimulants of the respiratory burst of the human neutrophil as assessed by superoxide (O2-) generation in whole cells and by NADPH-oxidase activity in a broken-cell 27,000-g particulate fraction. Phorbol 12-myristate, 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) stimulate production of O2- by human neutrophils with ED50 concentrations of 3.9 +/- 2.1 and 41.7 +/- 7.1 nM, respectively. The relation of biologic activity to receptor occupancy was assessed with binding studies of PMA and PDBu. Phorbol ester binding revealed a single high affinity phorbol ester receptor present at 7.6 x 10(5) sites/cell. The binding affinities for PMA and PDBu, 4.9 nM and 38.4 nM, respectively, agreed quantitatively with that of biologic potencies. Because of the high concentration of phorbol ester receptors (up to 125 nM) and the large amount of nonspecific binding at high cell density, apparent discrepancies between ED50's for NADPH-oxidase and whole cell O2- generation were noted. With the use of low cell concentrations, quantitative agreement between intact cell production of O2-, NADPH-oxidase activity, and receptor binding was found. These results further support the identity of the NADPH-oxidase as the enzymatic source of respiratory burst O2- production in human neutrophils.

Influenza A virus (IAV) activates the human neutrophil, but induces a dysfunctional state as well. Cell activation may contribute to the containment of the virus and/or cause local tissue damage. Certain features of the neutrophil activation response elicited by IAV are distinctive when compared with that triggered by formyl-methyl-leucyl- phenylalanine (FMLP). An atypical respiratory burst response occurs in which hydrogen peroxide, but no superoxide, is formed. This unusual respiratory burst stoichiometry persists despite marked priming of the IAV-induced response. A comprehensive examination of the activation cascade initiated by these stimuli failed to show an explanation for these differences. Both IAV and FMLP comparably stimulate inositol trisphosphate and phosphatidic acid production. The subsequent increase in intracellular calcium (Ca2+i) upon FMLP stimulation was more dependent on extracellular Ca2+ than with IAV activation, but both stimuli induced Ca2+ influx. FMLP and IAV exhibited equal susceptibility to inhibition by protein kinase inhibitors in eliciting the respiratory burst, and actin polymerization occurred in response to each agonist. A possible explanation for the anomalous respiratory burst induced by IAV is that O2- is generated at an intracellular site inaccessible to assay, and/or virus binding to sialic acid constituents of the plasma membrane alters the O2- generating capacity of the respiratory burst oxidase; evidence for each mechanism is offered.

The partial characterization and expression of the C1q receptor (C1q-R) in relation to other complement receptors present on the surface of neutrophils has been examined, as well as the effects of free C1q on cell function. A polyclonal anti-C1q-R antibody recognizes a 68-kD neutrophil surface protein. C1q-R expression was not upregulated upon warming, priming, or exposure to FMLP, but decreased after exposure to phorbol myristate acetate (PMA), because of shedding of the receptor into the extracellular medium, as detected by enzyme-linked immunosorbent assay. CR3 and CR1 expression was upregulated from intracellular pools after cell stimulation by PMA. No evidence of intracellular pools of C1q-R was found, as assessed by immunoblotting of subcellular fractions. But C1q-R appeared to be expressed early in cell differentiation, was detected on undifferentiated HL-60 cells, and like CR3 expression, increased upon 5 days differentiation towards a neutrophil lineage. However, C1q-R expression decreased upon additional culture, whereas CR3 expression continued to increase. A large variation in the percentage of peripheral cells expressing C1q receptors in donors was observed, ranging from 13% to 100%, contrasting with CR3 receptors that exhibited less variability. Interactions between free monomeric C1q and neutrophils were also studied. Incubation of stimulated neutrophils with 10 to 100 micrograms/mL C1q resulted in a further increase in CR3 expression and adherence to albumin-coated surfaces. Staphylococci opsonized with low quantities of C1q (0.1 to 1 microgram/mL) mediated a moderate and sustained respiratory burst in neutrophils, whereas a burst of similar magnitude was generated only with free C1q at concentrations 10- to 100-fold higher. Stimulation was only partially inhibited if cells were first treated with anti-C1q-R antibody, suggesting other C1q binding proteins may be present on the cell surface. In summary, neutrophil C1q receptor is approximately 68-kD, exhibits varying expression on different subjects, and is not upregulated from intracellular stores on exposure to soluble stimuli. Stimulated, but not resting, neutrophils selectively respond to raised levels of free C1q, resulting in altered cell function and enhanced CR3 receptor expression. These studies thus suggest complex roles for C1q in neutrophil function.

Although neutrophils are not viewed as a principal defense against influenza A virus (IAV) infection, their interactions are both complex and clinically relevant. Activation of the neutrophil is distinctive from that described for chemoattractants. To more fully characterize the pathway by which IAV stimulates the human neutrophil, we have examined its binding characteristics. First, inhibition studies with various sialic acid-containing and sialic-free sugars showed that IAV binds to sialic acid residues and activates receptors distinct from those used by Concanavalin-A (Con-A) and formyl-methionyl-leucyl-phenylalanine (FMLP) and that overlap those bound by wheat germ agglutinin (WGA). That viral hemagglutinin (HA) mediates viral binding and activation was shown by preincubating neutrophils with purified monovalent bromelain-released HA (BHA) and showing that IAV-induced membrane depolarization and hydrogen peroxide (H2O2) production were inhibited approximately 95%. However, binding inhibition required significantly higher concentrations of purified HA, suggesting that binding and cell activation have different interactive requirements. Desialation of the neutrophil surface membrane by neuraminidase treatment resulted in a 90.6% +/- 4.4% and 53.1% +/- 8.7% inhibition of IAV activation of neutrophils and viral binding, respectively. Resialation with ganglioside GT1b totally restored viral binding, but did not reverse the inhibition of activation. Thus, although HA was shown to mediate binding and neutrophil activation, viral binding per se was insufficient to stimulate the cell. Having demonstrated the functional role of HA, we sought to establish the mechanism of stimulation. HA in three different forms (BHA, HA-rosettes, and HA-liposomes) failed to activate the cell, although H2O2 production evoked by IAV stimulation was reduced in competitive inhibition studies with each preparation. Upon cross-linking with a monoclonal antibody to HA, activation comparable to that of intact virus was observed. The requirement for cross-linking of functional receptors, as opposed to activation through the neutrophil Fc receptor, was confirmed in experiments using staphylococcal A protein. These studies have shown the chemical specificity of IAV binding to the human neutrophil, the character of the receptor(s) stimulated to activate the IAV-evoked response, and the activation requirement for cross-linking those receptors responsible for stimulating functional responses.

Bacterial superinfections are a major cause of morbidity and mortality during influenza A virus (IAV) epidemics. Depression of phagocyte functions resulting from attachment of the IAV hemagglutinin (HA) to cell surface sialo-glycoproteins is a likely contributory cause of these infections. We have proposed that the group of collagenous lectins (termed collectins) present in blood and pulmonary surfactant play a role in initial host defense against IAV. We used here several recombinant human surfactant protein D (RhSP-D) preparations to determine the mechanism through which opsonization of IAV with collectins protects neutrophils against the deactivating effects of IAV on cellular respiratory burst responses in vitro. RhSP-D was markedly more potent than antibodies that inhibited viral hemagglutination activity (anti-HA antibodies) at protecting neutrophils in this assay. Unlike the anti-HA antibodies, RhSP-D was protective at concentrations that minimally inhibited viral hemagglutination activity. Two related features of SP-D--the degree of multimerization and the ability to cause aggregation of IAV particles--were critical determinants of the ability of SP-D to protect neutrophils against deactivation. Similarly SP-D-induced viral aggregate formation resulted in enhanced IAV binding to neutrophils and potentiated the ability of the virus itself to trigger neutrophil respiratory burst responses. In contrast to the case of IAV-antibody complexes, SP-D-IAV complexes attached to and activated neutrophils through a neuraminidase-sensitive mechanism (ie, similar to unopsonized IAV). These results indicate that collectin-mediated viral aggregation per se may be an important host defense mechanism not only by virtue of reducing the number of infectious viral particles, but also by promoting phagocyte responsiveness.

An NADH-ferricyanide reductase activity of ca. 170 nmole ferricyanide reduced/min/10(7) cells is present in the membrane fraction of human neutrophils. This membrane-bound activity constitutes ca. 85% of the total NADH-ferricyanide reductase activity that is present in these cells. The enzyme(s) readily utilize(s) purified cytochrome-b5 from beef liver as an electron acceptor. No other physiologic electron acceptors tested (e.g., ubiquinone-30, menadione) were active. The specificities of electron donors (e.g., NADH congruent to deamino-NADH much greater than NADPH) and acceptors (e.g., Fe(CN)6–3 greater than 2,6-dichlorophenol-indophenol much greater than O2) for the enzyme(s) in unfractionated membranes, along with action of inhibitors (e.g., ADP, p-chloromercuribenzoate) and the pH optimum, indicate that virtually all of the membrane-bound ferricyanide reductase activity in these cells is NADH-cytochrome-b5 reductase. This reductase, however, is only slightly solubilized (ca. 10%) by a phosphate buffer extraction procedure that is effective with the liver enzyme.

The mechanism by which unstimulated human neutrophils initiate a respiratory burst on adherence to a surface has been examined. When neutrophils adhere to a plastic surface, they immediately generate a sustained burst of superoxide (O2-). However, this respiratory burst is not initiated by adherence alone, since neutrophils attached to fibronectin fail to mount a response. Adhesion to plastic is calcium (Ca2+) independent, but O2- production requires Ca2(+)-containing buffer in the initiation phase, that is, during adhesion and the early phase of O2- production. The Ca2(+)-dependent step was shown to involve protein kinase C (PK-C) in that the O2- production, but not adherence, was blocked with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), and PK-C was found to translocate from the cytosol to the membrane on adhesion. Furthermore, it may be inferred that this translocation results in the generation of a Ca2+ independent form of PK-C, PK-M, since leupeptin, which inhibits the generation of PK-M, also blocked O2- production. This finding was corroborated by showing that after 5 minutes in a Ca2(+)-containing buffer, enough time to initiate O2- production and PK-C translocation, Ca2+ is no longer required for sustained O2- release. These results, in aggregate, demonstrate that neutrophils are activated by adhesion to plastic to generate O2-, a PK- C-dependent process that appears to involve a Ca2(+)-independent form of the kinase, PK-M. Why adherent neutrophils generate a respiratory burst on plastic and not fibronectin surfaces probably reflects activation of distinct receptors, whose nature must still be defined. Another issue to address is the priming effect of adhesion, since cells adherent to plastic- or fibronectin-coated surfaces have an enhanced O2- response to formylmethionyl-leucine-phenylalanine (FMLP) compared with neutrophils stimulated in suspension. This may relate to increased Ca2+ mobilization, an important mediator of priming for FMLP responses. Thus, adhesion as a priming event does not necessarily initiate cell effector function, and the further elucidation of the plastic and fibronectin models suggests a means of characterizing the crucial event that control neutrophil activation.

Neutrophil dysfunction consequent to influenza A virus infection has been described in vivo and in vitro and may contribute to the serious bacterial sequelae which occur in influenza-infected hosts. On the premise that such dysfunction may represent a form of “deactivation,” we sought to characterize neutrophil activation by the virus in comparison with other agonists. The virus induces a respiratory burst in which H2O2 (but not O2-) are formed. Preceding the respiratory burst, a rise in intracellular calcium (Ca2+i) is noted, but both responses are nearly independent of extracellular Ca2+, unlike those elicited by the other well-characterized Ca2+-dependent agonists, formyl-methyl-leucyl-phenylalanine (FMLP), or Concanavalin-A (Con-A). The Ca2+ increase is paralleled by IP3 generation, implying that it is the result of phospholipase C (PLC) activation. The virus also elicits neutrophil membrane depolarization, which is independently mediated from the Ca2+ increase and respiratory burst and may reflect protein kinase C (PK-C) activation. Virus-induced responses are insensitive to pertussis toxin (PT); cholera toxin does inhibit these responses but in a nonspecific manner. Thus, although influenza virus activates PLC in neutrophils, it does so in a PT-insensitive manner and does not elicit or require a discernible Ca2+ influx to generate a respiratory burst response. In aggregate, the data indicate that influenza A virus activates neutrophils in a manner distinct from that of other well- described neutrophil agonists. These results illustrate the diversity of neutrophil activation mechanisms and support the notion that further characterization of this pathway may facilitate understanding of neutrophil dysfunction induced by the virus.

Resting neutrophils may be “primed” to augmented effector function, eg, superoxide (O2-) production in the respiratory burst, upon a second stimulation with a variety of soluble agonists including formylated methionyl-leucyl-phenylalanine (FMLP) and phorbol myristate acetate (PMA). At priming concentrations of FMLP (5 x 10(-9) mol/L) that did not initiate O2- generation, two metabolic activities were noted: (1) approximately a threefold increase in the baseline intracellular calcium (Ca++i) level, that was not dependent on extracellular Ca++, and (2) a rapid rise in intracellular pH that was blocked by 5-(N,N- dimethyl) amiloride (DA), that had no effect on the Ca++i response to priming. Furthermore, there were no significant increases in inositol metabolites in cells primed and stimulated with FMLP compared with cells receiving the stimulating dose of FMLP alone and pretreatment with pertussis toxin (PT) (before the addition of the priming -5 x 10(- 9) mol/L dose of FMLP), whereas abolishing the response to FMLP during the second stage of stimulation, had (1) no effect on FMLP-primed cells subsequently stimulated with PMA, and (2) only partially ablated the rise in Ca++i initiated with FMLP. That FMLP priming involved distinctive processes to those of the well characterized FMLP-coupled Ca++-dependent activation cascade was shown by the full priming effect attained in a Ca++-free buffer, which did not sustain an O2- response to a second-stage FMLP stimulation, but sustained a primed response to PMA. These data demonstrate that FMLP primes human neutrophils by a Ca++-independent and PT-insensitive pathway, offering a functional model for studying heterogeneous FMLP receptor-coupled reactions.

The present studies document the cellular and biochemical processes involved in granulocyte O2- production in three patients from two kindreds with variant chronic granulomatous disease (CGD). Rates of O2- production were 9% to 30% of normal, depending on the individual tested and the stimulus; the two brothers from one family responded to each stimulus with rates very similar to each other. Kinetic analysis of NADPH-dependent O2- production in subcellular fractions revealed all three to have NADPH oxidases with both diminished substrate affinity for NADPH (high Kmapp) and decreased maximal velocities of O2- production. Their granulocytes had normal lag times for activation of the respiratory burst but abnormal rates of stimulus-induced membrane depolarization. Cytochrome b was not found in granulocytes or subcellular fractions despite the use of a spectrophotometric assay sensitive enough to detect the cytochrome if its content were proportional to the residual rate of O2- generation. A striking finding in one patient from each kindred was a threefold to tenfold decrease in the rate of O2- production accompanying serious infection. The residual O2(-)-generating activity of CGD variants helps to explain their relative freedom from the recurrent infections of the classic disease. However, the marked decrease described in the present study indicates the potential for a vicious cycle in which an infection, once established, leads to increasing impairment of host defense.

NADH-cytochrome b5 reductase is the predominant NADH-diaphorase found in the human neutrophil (Blood 62:152, 1983). Although this reductase segregates with the light membranes of nitrogen-cavitated neutrophils separated on Percoll gradients (which include the plasma membrane markers alkaline phosphatase and NADPH-oxidase), it is approximately 95% excluded from plasma membrane-enriched phagocytic vacuoles. The reductase constitutes approximately 5% of the light membrane fraction FAD-flavoprotein (14.8 +/- 5.5 pmol/mg protein) and was found in equimolar concentration with a high potential b cytochrome also present in this light membrane fraction and tentatively identified as cytochrome b5. Isolation of the reductase from human neutrophils was accomplished by Triton X-114 solubilization of the light Percoll gradient membranes, followed by temperature-dependent phase separation and then affinity chromatography on AMP-Sepharose. The active preparation contained 1.3 mol FAD/mol protein, migrated on sodium dodecyl sulfate-polyacrylamide gels as a single band corresponding to an apparent mol wt of 45,000 daltons, exhibited a pl of 5.7 on chromatofocusing and was obtained in greater than 70% yield, with an overall purification of almost 900-fold. The purified enzyme was characterized by a high specificity for NADH as electron donor (Km = 6.4 mumol/L v Km greater than 1.6 mmol/L for NADPH) and exhibited a maximal turnover of ca. 30,000 min-1 at 22 degrees C with either ferricyanide or cytochrome b5 (Km = 10 nmol/L) as electron acceptor. Although the physical characterization and biochemical properties described here demonstrate that this neutrophil NADH b5 reductase is similar to the corresponding liver and erythrocyte enzymes, its unique function in the neutrophil has yet to be determined.

We describe a patient whose peripheral blood neutrophils and bone marrow precursors (beyond promyelocytes) contained multiple large azurophilic granules. There were also giant granules in eosinophils, basophils, melanocytes, renal tubules, thyroid, and neurones, but not lymphocytes or monocytes. His clinical course included recurrent (ultimately fatal) infections and severe neurologic impairment. Immunofluorescent staining with fluoroscein- and rhodamine-conjugated antisera to primary and secondary granule markers showed virtually all of the granulocyte granules and rare monocyte granules to be fusion products containing both markers. Electron microscopy showed the granules to be large peroxidase-containing lysosomes. Only rare normal primary and secondary granules were present. Superoxide generation in response to opsonized zymosan was 7.3 nmole/min/10(6) cells (control 8.9); but in response to phorbol myristate acetate, only 2.2 (control 9.4). Nitroblue tetrazolium slides showed 3+ dye reduction in response to opsonized zymosan by 90% of granulocytes (control 91%) and to phorbol myristate acetate by 22% (control 99%), with 71% producing only a minimal 1+ response. Cellular contents of myeloperoxidase and beta- glucuronidase were elevated, but the percent release during exocytic degranulation was equivalent to control. Ingestion of complement- opsonized Staphylococcus aureus and zymosan was also normal. Killing of Staphylococcus aureus was 60% at 90-min incubation (control 92%). Granulocyte cyclic adenosine monophosphate (AMP) content was 4 pmole/10(7) cells (control 3.1). In order to determine whether these characteristics derived from the cells' genetic program or their environment, the patient's bone marrow was grown in long-term culture. Granulocytes produced in vitro demonstrated the same morphology, same defect in activation of nitroblue tetrazolium reduction, and same normal cyclic AMP level as those harvested from peripheral blood. These studies describe a new disorder of granulocytes; the structural similarity to, but biochemical differences from, Chediak-Higashi disease indicate the probable heterogeneity of mechanisms for the same morphological abnormality.

The particulate O-2-forming system from human neutrophils was found to oxidize methional and 2-keto-4-methylthiobutyric acid (KMB) to ethylene, indicating the formation by this system of strongly oxidizing radicals. Conforming this interpretation was the observation that ethylene production was inhibited by the radical scavengers benzoate, ethanol, and mannitol. Ethylene production was also sharply reduced by superoxide dismutase, implicatin O-2 as a precursor of oxidizing radicals. In our system catalase only partially inhibited ethylene generation from either methional or KMB, suggesting that oxidizing radicals are generated at least in part by the reacton of O-2 with compounds other than H2O2. We propose that in neutrophils oxidizing radicals are formed in a reaction between O-2 and a peroxide according to the following equation: O-2 + ROOH leads to RO . + OH- + O2, in which ROOH may be hydrogen peroxide, an alkyl peroxide, or an acyl peroxide (i.e., a peroxy acid).