Physicochemical and functional changes in human leukemic cell line HL-60

The recently established human promyelocytic cell line HL-60 was induced to differentiate in the present of DMSO. During this process, physiochemical, and functional changes were detected simultaneously. After exposure to DMSO for more than 1 day, the cell volume decreased and the tendency for hydrophobic interaction increased. Using a hydrophobic two-phase system in counter current distribution fashion, it was then possible to separate more mature metamyelocytes and segmented granulocytes from immature myeloblasts and promyelocytes. Increased functional maturity was reflected by increased chemiluminescence (CL) response and phagocytic activity. Using yeast particles opsonized with IgG as stimulating agent, the CL response increased already after 1 day in DMSO, in parallel with increased phagocytosis of these particles. In contrast, C3b-opsonized yeast and phorbol 12-myristate 13-acetate (PMA) did not enhance the CL response conspiquously until days 3–4. These data suggest that Fc receptor function linked to phagocytosis and the activation of oxidative metabolism develop earlier than that of C3b and PMA. The dissociation between Fc- and PMA-dependent stimulation of the oxidative metabolism may reflect different mechanisms of activation.     

The effect of bombesin-related peptides on the phagocytic function of mouse phagocytes in vitro

Bombesin (BBS) at doses of 0.1, 1.0, 10.0 and 100.0 nM stimulated chemiluminescence (CL) production by phagocytic cells (monocytes, macrophages and polymorphonuclear leucocytes) in mice in the presence of ZAP (opsonized zymosan particles containing luminol). These data suggest that BBS increased the phagocytic function of mouse phagocytes. BBS-related peptides, gastrin-releasing peptides (GRP)-27, GRP-14, GRP-10 and neuromedin B, also induced similar CL responses compared with BBS. The CL response elicited by BBS was depressed dramatically by various concentrations of EGTA (a Ca++ chelator), indicating that a Ca++ pathway may play a key role in the BBS-stimulated CL response.     

Cellular chemiluminescence of activated phagocytes of whole blood

Both, the phagocytic process and the activation of phagocytes with soluble stimuli are accompanied by increased production of reactive oxygen species (ROS). Chemiluminescence (CL) measurement is a simple and sensitive method for the detection of ROS generation. Phagocytes (mainly polymorphonuclear leukocytes, PMNL) were stimulated with soluble stimulus or via phagocytosis in diluted whole blood, and the generation of Luminol-enhanced CL was registered. The time dependence of CL, determined in whole blood, corresponds to the CL from isolated leukocytes. A relationship between peak CL and the number of leukocytes as well as of PMNL was observed. The specific CL, i.e. the CL response related to a defined PMNL number, increases with the age of investigated healthy individuals. No correlations were found between CL and the capacity of PMNL to ingest zymosan particles. Relations between CL and spontaneous platelet aggregation suggest, that reactivity of blood platelets may be a contributing factor to the kinetics of the CL signal in our test system. The inhibition of CL by the sulphydryl reagents diamide and fever few extract indicate the role of cellular sulphydryl groups for phagocyte function. Measurement of CL in whole blood is proved to be a simple assay for assessment of PMNL function and allows measurements in very small blood samples (greater than or equal to 10 ul).     

Exoenzyme Tat-C3 inhibits association of zymosan particles, phagocytosis, adhesion, and complement binding in macrophage cells

Phagocytosis by macrophages is most important in the initial stages of an immune response. Although RhoA regulates cell adhesion, its roles in the integrin-related association of particles with macrophages and in phagocytosis are not clearly understood. We introduced C3 exoenzyme, a specific inhibitor of Rho, into J774A.1 macrophage cells fused with the 9 amino acid (49-57) transduction domain (RKKRRQRRR) of HIV-1 Tat. The presence of this Tat-C3 vector altered RhoA mobility on non-denaturing gels, indicating that Tat-C3 modified RhoA by ADP-ribosylation. Uptake of (FITC)-conjugated serum-opsonized zymosan particles and adhesion to fibrinogen-coated plates were reduced as was the association of serum-opsonized zymosan particles, and complement C3 and C3bi with the transfected cells. These results suggest that Rho regulates the activity of integrins that are involved in the association of particles with macrophages, phagocytosis, adhesion, and binding of complement C3 and C3bi.     

Different effects of peptides and their amino acids on antibody production and phagocytic activity of the neutrophils in mice

The effects of some natural polypeptides fragments and amino acids which were incorporated in them on the thymus-dependent SRBC immune response and on the phagocytosis of Staphylococcus by murine peritoneal neutrophils were compared. It was found that the peptides LGIP and PYIK which consisted of those amino acids that stimulated phagocytosis but did influence the immune response (K, P, Y, L) did not change the immune response as well as phagocytosis. The peptides TKPR, TTKD and LGIPE which included those amino acids that were able to stimulate both immune response and phagocytosis (T, E, D) enhanced both activities. Nevertheless the peptide PYIKV containing valine (that stimulated the antibody production as well as phagocytosis) did not effect the immune response but enhanced the phagocytic index. The existence of two immunoregulatory systems--the peptide system and the amino acid one--was suggested.     

Correlation between chemiluminescence response and rate of zymosan uptake by rat alveolar macrophages. Scanning electron microscope observations

The rate of particle uptake by rat alveolar macrophages (AM) exposed to zymosan (mean number of zymosan particles becoming bound to 100 cells at a fixed time interval) was determined with the aid of the scanning electron microscope (SEM). The intensity of the chemiluminescence (CL) emitted by the AM on addition of zymosan, was measured concomitantly. During the whole course of CL emission, all the particles were found to be either attached or engulfed, but not ingested by the AM. A good correlation was obtained between the time dependence of CL intensity and that of the rate of particle uptake, both reaching a peak value at about 5 min after exposure. It is therefore assumed that the CL emitted by AM exposed to zymosan reflects the attachment and engulfment stages of phagocytosis.     

Hybrid human immunodeficiency virus Gag particles as an antigen carrier system: induction of cytotoxic T-cell and humoral responses by a Gag:V3 fusion.

In attempts to increase the immunogenicity of recombinant antigens, a number of particulate antigen presentation systems have been developed. In this study, we used human immunodeficiency virus Gag particles as carriers for the human immunodeficiency virus envelope V3 region. Gag:V3 fusion proteins were expressed from baculovirus expression vectors; they migrated to the insect cell membrane and budded from the cells as hybrid particles. An immunization study carried out with rats showed that the particles elicited a strong anti-Gag antibody response and a weak antibody response to the V3 region. A strong anti-V3 cytolytic T-cell response was elicited in immunized mice. These data show that retroviral Gag particles can be used as antigen presentation vehicles.     

Altered circadian rhythms of corticosterone, melatonin, and phagocytic activity in response to stress in rats

Corticosterone is thought to be the main glucocorticoid secreted in response to stressful exercise, while melatonin buffers the adverse immunological effects of stress. The present work was aimed to evaluate whether swimming-exercise-induced stress leads to changes in the chronobiology parameters of the circadian rhythms of melatonin and corticosterone, and in the number and phagocytosis of peritoneal macrophages in 3-month-old male Wistar rats. The animals were subjected to a physical activity trial consisting of 2 h of free swimming. Radioimmunoassay was used to determine the plasma levels of melatonin and corticosterone. Phagocytosis was measured by the latex-bead phagocytosis index (PI), i.e., the number of latex beads ingested by 100 macrophages, the phagocytosis percentage (PP), i.e., the percentage of cells that had phagocytosed at least one latex bead, and the phagocytosis efficiency (PE), i.e., the ratio PI: PP which indicates how effectively the phagocytes ingested the particles. Stress significantly decreased the MESOR and amplitude of the melatonin rhythm, and significantly increased the MESOR of the corticosterone rhythm. The control animals' peritoneal macrophage number and PI showed a circadian rhythm with maxima at 02:00 and 03:00, respectively. The stressed group displayed higher values of PI than the controls at most hours of the night, but the number of cells in the peritoneal cavity was practically the same at all hours studied. These data confirm that melatonin and corticosterone act as modulators of the innate immune response, and that the circadian rhythm of the two hormones are altered in situations of stress.     

Whole blood luminol-enhanced chemiluminescence: Statistical analysis of the responses of different subjects

Whole blood luminol-enhanced chemiluminescence (CL) was studied using N-formyl-methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan particles as stimuli. The peak and the integral responses of CL were recorded. In the FMLP-induced CL the initial activation (1-minute values) was also studied, because it coincides with the extracellular production of oxy radicals. Correction factors based on neutrophil count and on haemoglobin concentration were found to decrease dispersion and shape the distributions of the CL responses close to normal in a study of 50 healthy adults. One-minute values were significantly lower in women than in men but there were no significant differences for peak or integral values between sexes. Depressed reaction is in accordance with the previous findings that phagocytic oxy radical production is depressed in female plasma. Thus, our results suggest that 1-minute value is a variable more sensitive than peak or integral value of the CL response.     

The chemiluminescence reactions of the blood neutrophils and of peritoneal exudate cells in Syrian hamsters to the intraperitoneal administration of cellular and microbial materials]

The luminol-dependent chemiluminescence (CL) activity of peritoneal exudate cells and blood neutrophils of Syrian hamsters inoculated intraperitoneally with heat-inactivated microbial particles of Candida albicans, (C. albicans), heated irradiated normal cells and native or heated irradiated malignant tumor cells was studied. The inoculation with particles of C. albicans and heated normal cells induced significant activation of CL of peritoneal exudate cells, but did not influence the CL reaction of blood neutrophils. The inoculation of animals with nonheated irradiated tumor cells led to increase of CL response of both peritoneal exudate cells and blood neutrophils. The inoculation with heated irradiated tumor cells did not activate CL of peritoneal exudate cells and led to slight, but long-lasting decrease of CL response of blood neutrophils.     

Functional integrity of human neutrophils following 24 hour incubation with hydroxyapatite and fluoride

We have previously shown that hydroxyapatite (HA) priming of human neutrophils to a second stimulus of formyl-methionyl-leucyl-phenylalanine (fMPL) is influenced by a bisphosphonate and fluoride. The purpose of this study was to investigate the long-term effects of low concentrations of NaF (10⁻³–10⁻¹¹ mol/L F) on HA-mediated neutrophil chemiluminescence (CL) as a measure of oxidative function. CL assays were conducted following extended time periods of incubation (30 min, 3 h, 18 h and 24 h). Results were calculated as integrals of total energy output and expressed as the difference between the experimental (NaF/HA) and control (cells alone) assays. Transmission electron microscopy (TEM) was used to estimate cellular integrity and confirm HA phagocytosis. CL inhibition was observed at all fluoride concentrations at 30 min incubation. No significant difference compared to the control was observed in the CL output at 3 or 18 h. However, at 24 h the response showed a significant increase in activity at all NaF concentrations. The TEM results confirmed the functional integrity of the neutrophils, particularly those phagocytosing HA particles up to 24 h. Based on these results we demonstrate that human peripheral blood neutrophils can be maintained in a fully functional state with respect to the respiratory burst and morphology for at least 24 h. © 1997 John Wiley & Sons, Ltd.     

Selective down-regulation of alveolar macrophage oxidative response to opsonin-independent phagocytosis

We have compared the oxidative response of alveolar macrophages (AM) during opsonin-dependent and independent phagocytosis by using multiparameter flow cytometry. The respiratory burst of AM during phagocytosis was quantitated by the intracellular oxidation of the nonfluorescent precursors dichlorofluorescin diacetate (DCFH) or hydroethidine (HE, a reduced precursor of ethidium) to their fluorescent (oxidized) counterparts. After loading freshly isolated normal hamster AM with DCFH or HE, red or green fluorescent beads, respectively, were added to the shaking cell suspensions. Ingestion of opsonized particles by AM caused a marked increase in oxidation of both DCFH and HE proportional to the number of beads ingested. In contrast, uptake of one to three unopsonized particles per cell led to inhibition of oxidative activity compared to control cells incubated without particles. AM ingesting four or more unopsonized particles showed some increase in oxidative metabolism, but far less than that with identical numbers of particles in opsonin-dependent ingestion. Similar results were obtained using fluorescent labeled staphylococcal bacteria. Using three-color flow cytometry to study cells ingesting both types of particles, cells first ingesting unopsonized beads were also found to have an inhibited oxidative response to subsequently ingested opsonized particles. The mitochondrial poison antimycin inhibited most of the intracellular oxidative response to either type of phagocytosis. The remaining antimycin-insensitive, membrane derived respiratory burst of AM was also substantially diminished after phagocytosis of unopsonized particles vs similar numbers of opsonized particles. The greatly increased mitochondrial respiration in AM during phagocytosis of opsonized particles may be related to bactericidal mechanisms. Killing of ingested Staphylococcus by AM was markedly impaired in the presence of antimycin. The results suggest that AM may ingest the numerous, unopsonized inert particles that are inhaled without generation of potentially toxic oxygen metabolites, while retaining the capacity to undergo a respiratory burst after ingesting opsonized particles and bacteria. The mechanism(s) for this distinct response may include generation of an inhibitor of intracellular oxidative metabolism.     

Molecular cloning of rock bream (Oplegnathus fasciatus) tumor necrosis factor-α and its effect on the respiratory burst activity of phagocytes

Rock bream (Oplegnathus fasciatus) tumor necrosis factor-α (rbTNF-α) gene was cloned, recombinantly produced, and the effect of the recombinant rbTNF-α on the respiratory burst activity of rock bream phagocytes was analyzed. Structurally, genomic DNA of rbTNF-α was comprised with four exons and three introns, and deduced amino acid sequence of its cDNA possessed the TNF family signature, a transmembrane domain, a protease cleavage site, and two cysteine residues, which are the typical characteristics of TNF-α gene in mammals and fish. The chemiluminescent (CL) response of rock bream phagocytes was significantly enhanced by pre-incubation with recombinant rbTNF-α, when opsonized zymosan was used as a stimulant of the respiratory burst. However, CL enhancing effect of the recombinant rbTNF-α was very weak when the respiratory burst activity of phagocytes was triggered with phorbol-12-myristate-13-acetate (PMA) instead of zymosan. These results suggest that rock bream TNF-α might have an ability to prime the respiratory burst activity of phagocytes against receptor-mediated phagocytosis inducing stimulants, such as zymosan, but have little ability against stimulants not accompanying receptor-mediated phagocytosis.     

Cell mediated immunity in American cutaneous and mucosal leishmaniasis

Cellular immune responses were studied in 35 Brazilian patients with either active cutaneous leishmaniasis (CL), active mucosal leishmaniasis (ML), or healed cutaneous leishmaniasis. The mean age and duration of illness in the two groups were as follows: 14 CL patients, age 28 +/- 13 yr, disease 5 +/- mo; and 16 ML patients, age 34 +/- 15 yr, disease 86 +/- 117 mo. Patients with CL and ML responded well to leishmania antigen in blastogenesis assays. However, the response of ML patients was over three times greater than the response of CL patients. There was a significant correlation between the magnitude of the lymphoproliferative response and the duration of disease activity. There were no significant differences between CL and ML patients in terms of the following parameters: lymphoproliferative responsiveness to mitogens (phytohemagglutinin, concanavalin A, and pokeweed mitogen) and peripheral blood lymphocyte subpopulations (T and B cells, oKT8+ and OKT4+ cells, OKT4:OKT8 ratio). Peripheral blood mononuclear cells from ML patients also generated interferon-gamma containing lymphokine in response to stimulation with leishmania antigen. This lymphokine was capable of inducing macrophages from ML patients to inhibit the intracellular multiplication of leishmania in vitro. These studies have determined that the parameters of lymphocyte and macrophage functions evaluated in ML and CL patients are comparable, except for an enhanced lymphoproliferative response, with leishmania antigen in ML patients. This later finding may be a function of the long duration of active disease in this population and unrelated to the pathogenesis of their mucosal lesions.     

Advances in alfalfa mosaic virus-mediated expression of anthrax antigen in planta

Plant viruses show great potential for production of pharmaceuticals in plants. Such viruses can harbor a small antigenic peptide(s) as a part of their coat proteins (CP) and elicit an antigen-specific immune response. Here, we report the high yield and consistency in production of recombinant alfalfa mosaic virus (AlMV) particles for specific presentation of the small loop 15 amino acid epitope from domain-4 of the Bacillus anthracis protective antigen (PA-D4s). The epitope was inserted immediately after the first 25 N-terminal amino acids of AlMV CP to retain genome activation and binding of CP to viral RNAs. Recombinant AlMV particles were efficiently produced in tobacco, easily purified for immunological analysis, and exhibited extended stability and systemic proliferation in planta. Intraperitional injections of mice with recombinant plant virus particles harboring the PA-D4s epitope elicited a distinct immune response. Western blotting and ELISA analysis showed that sera from immunized mice recognized both native PA antigen and the AlMV CP.     

Human neutrophil response to recombinant neisserial Opa proteins

Interactions of human neutrophils with recombinant Escherichia coli expressing gonococcal outer membrane Opa proteins were examined using chemiluminescent and biological assays. Seven opa loci from Neisseria gonorrhoeae MS11 4.8 were expressed as beta-lactamase-Opa fusion proteins that contained all but the mature N-terminal amino acid of the full-length Opa protein fused to three N-terminal amino acids derived from the mature beta-lactamase. The Opa fusion proteins were exported and assembled in the outer membrane of E. coli in a manner similar to that of Opa in N. gonorrhoeae, as evaluated by antibody binding and in situ proteolytic cleavage. All fusion proteins exhibited the characteristic heat-modifiable migration in SDS-polyacrylamide gel electrophoresis that typifies Opa proteins of neisseriae. Opa fusion proteins conferred on E. coli the ability to stimulate a chemiluminescent response from human neutrophils in the absence of antibody or complement. The nature of the response in terms of chemiluminescence, phagocytosis, and killing was in all cases analogous to that seen using N. gonorrhoeae expressing the equivalent Opa protein. Neither E. coli nor gonococci expressing OpaA elicited a response from neutrophils. Use of E. coli expressing Opa fusions should be useful in defining their biological activities and pathogenic roles.     

Surfactant protein A regulates IgG-mediated phagocytosis in inflammatory neutrophils

Surfactant proteins (SP)-A and SP-D have been shown to affect the functions of a variety of innate immune cells and to interact with various immune proteins such as complement and immunoglobulins. The goal of the current study is to test the hypothesis that SP-A regulates IgG-mediated phagocytosis by neutrophils, which are major effector cells of the innate immune response that remove invading pathogens by phagocytosis and by extracellular killing mediated by reactive oxygen and nitrogen. We have previously shown that SP-A stimulates chemotaxis by inflammatory, but not peripheral, neutrophils. To evaluate the ability of SP-A to modulate IgG-mediated phagocytosis, polystyrene beads were coated with BSA and treated with anti-BSA IgG. SP-A significantly and specifically enhanced IgG-mediated phagocytosis by inflammatory neutrophils, but it had no effect on beads not treated with IgG. SP-A bound to IgG-coated beads and enhanced their uptake via direct interactions with the beads as well as direct interactions with the neutrophils. SP-A did not affect reactive oxygen production or binding of IgG to neutrophils and had modest effects on polymerization of actin. These data suggest that SP-A plays an important role in mediating the phagocytic response of neutrophils to IgG-opsonized particles.     

Evaluation of Opsonophagocytic Dysfunctions in Severely Burned Patients by Luminol-Dependent Chemiluminescence

We compared the luminol-dependent chemiluminescence (CL) response of peripheral blood from severely burned patients with that from normal controls to evaluate the primary defense level against bacterial infection in the patients. The CL was measured upon addition to diluted whole blood of a soluble stimulus, phorbol myristate acetate (PMA) or particulate stimuli such as bacteria or zymosan without special opsonization. In the early post-burn days, the initial rate of whole blood CL induced with the particulate stimulus was much lower than that in the normal controls, whereas the rate was higher when PMA was used as a stimulus. The number of granulocytes in the patients' blood had increased and isolated polymorphonuclear leukocytes (PMNs) from the patients exhibited higher CL responses to the particulate or soluble stimulus as compared with those of normal controls. The results suggest that the PMNs in burn patients were activated and normally mobilized in the early post-burn period but the opsonizing capacity in the blood decreased. In fact, the serum levels of complement, immunoglobulins and fibronectin were found to be lower in the blood from the patients than those from normal controls and a supplement of freshly frozen plasma of human immunoglobulin preparations restored the initial rate of the whole blood CL upon phagocytosis. The prognosis is still poor when severe infection occurs in the patients with decreased CL response of whole blood. Recombinant human granulocyte colony-stimulating factor (rhG-CSF) enhanced the CL response of PMNs from burn patients. The administration of rhG-CSF may be useful for decreasing the morbidity of severe infection following burn injury in the near future.     

The Effect of Virus Particle Size on Chemiluminescence Induction by Influenza and Sendai Viruses in Mouse Spleen Cells

Suspensions of orthomyxo- and paramyxoviruses are composed of pleomorphic particles ranging from large filaments to small spheres. Influenza and Sendai viruses were separated according to size by gel filtration and the induction of luminol-dependent chemiluminescence (CL) by particles of similar size was studied in suspensions of mouse spleen cells known to contain phagocytes. CL reflects the generation by the cells of reactive oxygen species. CL induction decreased with particle size for both viruses. Compared with small spheres, large influenza filaments were approximately 10 times as efficient in activating cellular light emission while the ratio between large and small Sendai viruses was 3:1. Small Sendai virus particles were also less efficient in lysing red cells and had lower neuraminidase activity. By contrast, with influenza virus, only neuraminidase and not the hemolytic activity decreased with the virus size. When influenza virus filaments were broken into smaller particles by sonication, the capacity to induce chemiluminescence dropped markedly while the hemolytic and hemagglutinating activities increased and neuraminidase activity remained unaltered. These results suggest that the presentation of influenza virus hemagglutinin and neuraminidase glycoproteins in a large particle, leading to extensive receptor crosslinking, may be an important factor in the efficient activation of CL by filamentous influenza virus. We suggest that radical generation as reflected in cellular CL may relate to the toxic in vivo effects that contribute to the pathogenesis of influenza and infections with paramyxoviruses.     

The Effect of Virus Particle Size on Chemiluminescence Induction by Influenza and Sendai Viruses in Mouse Spleen Cells

Suspensions of orthomyxo- and paramyxoviruses are composed of pleomorphic particles ranging from large filaments to small spheres. Influenza and Sendai viruses were separated according to size by gel filtration and the induction of luminol-dependent chemiluminescence (CL) by particles of similar size was studied in suspensions of mouse spleen cells known to contain phagocytes. CL reflects the generation by the cells of reactive oxygen species. CL induction decreased with particle size for both viruses. Compared with small spheres, large influenza filaments were approximately 10 times as efficient in activating cellular light emission while the ratio between large and small Sendai viruses was 3:1. Small Sendai virus particles were also less efficient in lysing red cells and had lower neuraminidase activity. By contrast, with influenza virus, only neuraminidase and not the hemolytic activity decreased with the virus size. When influenza virus filaments were broken into smaller particles by sonication, the capacity to induce chemiluminescence dropped markedly while the hemolytic and hemagglutinating activities increased and neuraminidase activity remained unaltered. These results suggest that the presentation of influenza virus hemagglutinin and neuraminidase glycoproteins in a large particle, leading to extensive receptor crosslinking, may be an important factor in the efficient activation of CL by filamentous influenza virus. We suggest that radical generation as reflected in cellular CL may relate to the toxic in vivo effects that contribute to the pathogenesis of influenza and infections with paramyxoviruses.