The influence of age and sex on phagocyte chemiluminescence

The process of ageing is associated with increased susceptibility to infection. Phagocytes form the primary defence mechanism against infecting microorganisms, but the influence of ageing on phagocyte function remains controversial. In this study we have applied a microtitre plate phagocyte chemiluminescence (CL) assay suitable for clinical use to compare phagocyte oxidative metabolism in younger healthy subjects (age 20–60 years) and healthy older (60–70 years) subjects. Polymorphonuclear leukocytes (PMNL) and monocytes were stimulated using phorbol myristate acetate (PMA), serum opsonized zymosan (SOZ), and non-opsonized zymosan (ZYM) in the presence of both lucigenin and luminol. Monocytes showed a higher luminolenhanced CL response to PMA in males compared with females in the younger age group. No PMNL differences were observed between the sexes. Although no difference were found in relation to age when cells were stimulated with PMA and SOZ, significantly lower background (unstimulated) CL was obtained from PMNL with luminol. PMNL luminol-enhanced CL responses were also lower in response to ZYM. The findings suggest a reduced response of PMNL from older subjects to minimal stimulation. This could be related to abnormalities in the triggering of the respiratory burst or myeloperoxidase release due to ageing. The influence of age and sex should be taken into account in clinical studies of phagocyte CL.     

Factors affecting the measurement of chemiluminescence in stimulated human polymorphonuclear leucocytes

Optimum conditions were established for the generation and measurement of luminoldependent chemiluminescence (CL) in human polymorphonuclear leucocytes (PMNL) stimulated with a variety of particulate and soluble agents. Several factors had a particular influence on the kinetics of CL stimulated by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Two peaks, both azide-sensitive, were observed at 21°C and 25°C. but these increased in magnitude and merged t o give a single, early peak when the temperature was increased t o 37°C. Pre-exposure of PMNL to a buffer containing calcium was essential for the expression of both phases of fMLP-stimulated CL, while the second peak decreased dramatically if the cells were stored at 4°C for 4 hours before assay. In contrast, storage of PMNL at 4°C for up t o 8 hours in a buffer without divalent cations did not alter the kinetics or magnitude of CL induced by other stimuli, and had the benefit of minimizing the rate of cell aggregation. This study confirms that measurement of luminol-dependent CL in stimulated PMNL is a useful analytical tool, but shows that careful attention t o experimental design is required t o ensure that the observed CL provides a true measure of the parameter under investigation.     

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.     

Luminol-dependent chemiluminescence in antibody-sensitized neutrophils stimulated with protein A-bearing staphylococci

When mouse polymorphonuclear leukocytes (PMNs) sensitized with rabbit antibody to mouse Ehrlich ascites tumor cells were stimulated by Staphylococcus aureus Cowan I cells, a conspicuous luminol-dependent chemiluminescence was observed in the absence of opsonin. The profile of the chemiluminescence (CL) response evoked by staphylococcal cells from antibody-sensitized PMNs had two peaks. An initial peak, observed within 1 min after stimulation, was sharp and high and a second peak, observed about 5 min after stimulation, was low and extended. The CL response of antibody-sensitized PMNs stimulated by S. aureus Cowan I cells was dose-dependently blocked by preincubation with soluble SpA. Cells of a mutant derived from S. aureus Cowan I strain with trace amounts of cell-bound SpA failed to stimulate the antibody-sensitized PMNs to generate the CL response. The antibody-sensitized PMNs were found to phagocytize SpA-bearing S. aureus cells even in the absence of opsonic serum. These results suggest that the observation presented here might provide a useful tool for the investigation of CL response of PMNs.     

Changes in Glutathione Peroxidase Activities and the Oxidative Burst of Leukocytes During Inflammation in the Mouse and Rat

The relationship between glutathione peroxidase (GSH-Px) activity and opsonized zymosan-induced chemiluminescence (CL) has been studied with exudate leukocytes obtained at different times after induction of inflammatory responses in the mouse peritoneal cavity with heat-killed Corynebacterium parvum and in the rat pleural cavity with I-carrageenin. GSH-Px activity in mouse peritoneal exudate cells fell markedly after 2–4h, returning to normal within 1–2 days. The lowered enzyme activity was associated with an increased ability of the cells to generate CL. Rat pleural exudate cells exhibited a slight fall in GSH-Px activity after 6h which increased to supranormal levels within 1–2 days. During this period the ability of the cells to generate CL continually increased. The data indicate that during the early phase of increased generation of reactive oxygen species (ROS) by inflammatory leukocytes, the intracellular protective mechanism, represented by GSH-Px, is compromised. Subsequently, GSH-Px activity increases to or above initial levels possibly due to the presence of mononuclear cells and/or as a response to the increased generation of ROS.     

Chemiluminescence response of phagocytes in E. coli induced experimental ascending pyelonephritis.

The mechanism of tissue injury at the cellular level by following the chemiluminescence response of various phagocytes in E. coli induced experimental pyelonephritis in mice was investigated. There was a marked increase in the capacity of various phagocytic cells viz; renal neutrophils and macrophages peritoneal macrophages, blood monocytes and neutrophils to produce reactive oxygens species through the respiratory burst activity as monitored by the chemiluminescence response. The chemiluminescence response was observed to be increased significantly (p less than 0.001) with increasing days post infection in all phagocytic cells. However, the quantity of total reactive oxygen species produced per million cells was much more in the renal and peritoneal macrophages as compared to blood monocytes and neutrophils. The peak chemiluminescence response time was observed to be decreased from 4 to 2 minutes with the progression of the diseases. The implications of these findings have been discussed.     

Thrombospondin-exposed human monocytes display augmented luminol-enhanced chemiluminescence upon receptor triggering

It was tested whether the exposure to blood platelet thrombospondin (TSP) influences the function of monocytes. TSP-treated monocytes displayed luminol-enhanced chemiluminescence (CL) upon triggering with polyclonal or monoclonal anti-TSP. This response was mediated by an Fc receptor, since F(ab')2 fragments were without effect. Evidence is provided that a CL signal was induced only when antibodies bound to TSP and Fc receptors of the same monocyte. TSP-treated monocytes exerted enhanced CL to aggregated IgG when compared with untreated or albumin-treated cells, suggesting that TSP up-regulated the cells' capacity to mediate Fc receptor-dependent generation of reactive oxygen. A similar enhancement was observed when TSP-treated cells were stimulated with anti-CD36, or with fMLP. Upon stimulation of TSP-pretreated cells with monoclonal anti-fibrinogen (Fg), a much stronger enhancement was noted, which was similar in magnitude to that induced by anti-TSP. The effect of anti-Fg cannot be explained by a trace contamination of TSP with Fg alone. In contrast to receptor-mediated CL, PMA-induced and zymosan-induced CL were influenced little by TSP pretreatment. IgG-mediated phagocytosis was not enhanced in TSP-treated cells. Thus, TSP selectively modulates certain monocyte functions which could be of physiological relevance.     

Change in the chemiluminescence reactivity pattern during in vitro differentiation of human monocytes to macrophages

We determined the luminol-enhanced chemiluminescence (CL) of fresh human monocytes and monocytes cultured for 1-14 days in vitro, within hydrophobic membranes, using a variety of stimuli known to trigger the respiratory burst of phagocytes. It was assured that CL emerged from an adherent subpopulation of mononuclear cells; polymorphonuclear leukocytes (PMN) contaminating mononuclear leukocytes (MNL) contributed little, if anything, to the CL response of MNL. Typical response patterns were established for fresh monocytes triggered by phorbol 12-myristate 13-acetate (PMA), zymosan, the Ca2+ ionophore A 23187, antibody-coated erythrocytes and Sendai virus. Differentiation in vitro into macrophages was associated with a general decrease in magnitude of the CL peak, in an overproportional decrease of the A23187 triggered response and in a complete loss of the response to Sendai virus--a loss which could not be prevented by addition of myeloperoxidase (MPO). In contrast to monocyte CL, macrophage CL was resistant to sodium azide, indicating its MPO-independent origin. Macrophage-type reactivity was obtained at day 4 of culture. Activation of macrophages with recombinant interferon-gamma for the last 2 days of culture was associated with a quantitative (approx. threefold) increase of the CL signal, although qualitatively the same reactivity pattern was obtained as with control macrophages. In contrast to luminol-dependent CL, the lucigenin-dependent CL response of macrophages was greater than that of monocytes, an increase which was particularly prominent for PMA stimulation.     

Nitric oxide production from a macrophage cell line: Interaction with autologous and allogeneic lymphocytes

The indirect stimulation of macrophages to produce nitrite was examined by using the macrophage cell line J774. J774 spontaneously produced nitrite, when cultured at high concentration. J774 cultured in low concentration ( < 10⁴ cells in 100 μl) barely produced nitrite. J774 cultured in low concentration produced a large amount of nitrite by the co-culture of nonadherent spleen cells or nonadherent peritoneal exudate cells, which were stimulated with con A, anti-CD3, or staphylococcal enterotoxin A. J774 (BALB/c derived: H-2^d) cultured with either syngeneic (BALB/c) or allogeneic (B6; H-2^b B10BR; H-2^k) nonadherent lymphocytes, which were stimulated with conA or anti-CD3, produced nitric oxide. However, J774 produced nitric oxide by stimulation with SEA only when co-cultured with SEA-reactive T lymphocytes. Peritoneal exudate cells from mice, which did not proliferate by the stimulation of conA or anti-CD3, proliferated well by the addition of L-arginine homologue, N^G-monomethyl-L-arginine. The proliferation of nonadherent peritoneal exudate cells stimulated with conA or anti-CD3 was suppressed by the addition of peritoneal macrophages. This suppression was abolished by the addition of N^G-monomethyl-L-arginine.     

Activation of human monocytes by interleukin 2: Role of T lymphocytes

The effect of interleukin 2 (IL 2) on the capability of human monocytes to secrete reactive oxygen species triggered via Fc-γ receptor (Fc-γ R) function had been investigated by measurement of chemiluminescence (CL). IL 2 did not activate highly purified (hp) monocytes to respond to Fc-γ R mediated phagocytic stimulation with an enhanced respiratory burst activity unless low numbers of T cells had been co-cultured with hp monocytes. Supernatants from IL 2 treated PBMC contained interferon-γ (IFN-γ) and monocyte activating factor (MAF) activity. The secretion of both cytokine activities was strongly enhanced by cooperative function of monocytes. The correlation of IL 2 induced secretion of IFN-γ and MAF activity was striking, however, monoclonal antibody (mAb) anti-human IFN-γ failed to abrogate IL 2 stimulated and lymphocyte dependent monocyte activation. Although IL 2 had no direct monocyte activating effect, pretreatment of hp monocytes with IL 2 led to monocyte priming: subsequent co-culture with autologous control T cells enhanced the monocyte Fc-γ R mediated CL response. The priming of monocytes by IL 2 was dependent on the interaction of IL 2 with the monocytic IL 2 receptor as shown by inhibition experiments with anti IL 2 R monoclonal antibody. Thus the IL 2 driven monocyte/T-cell interaction leads to an increased Fc-γ R mediated monocytic respiratory burst activity and to the secretion of a soluble MAF activity, but there were no detectable amounts of IFN-γ.     

A distinct "slow" cellular pathway involving soluble mediators for the T cell-instructed induction of monocyte tissue factor activity in an allogeneic immune response

Allogeneic stimulation of human lymphoid cells initiates a collaborative cellular pathway that relatively rapidly induces in monocytes the synthesis and cell surface expression of tissue factor, the initiating cofactor of the extrinsic coagulation pathway. T cells are required for the monocyte procoagulant generation, because the addition of autologous or allogeneic T cells fully reconstitutes the activity in otherwise nonresponding highly purified monocytes. Despite a strict T cell requirement, only low T cell to monocyte ratios are necessary for maximal PCA response. Our results further demonstrate that the collaborative signal from allogeneically stimulated T cells to effector monocytes is transferred by a soluble mediator rather than direct cell to cell contact. Other aspects of the present study include the observation that among normal peripheral blood lymphoid cells, monocytes elicit the strongest allogeneic PCA response. This response is clearly exceeded by that induced upon stimulation with Daudi lymphoblastoid B cells. Our data demonstrate the existence of a second distinct cellular pathway that mediates the lymphoid procoagulant response. This pathway differs from the previously characterized response to bacterial LPS in respect to: a) kinetics of T cell triggering; b) mediation by a soluble product; c) lack of genetic restriction of T cell; monocyte collaboration; and d) deficient capacity for direct T cell induction of the monocyte PCA response.     

Contrasting effects of Mycoplasma fermentans and M. felis on the viability and chemiluminescence response of human polymorphonuclear leukocytes

Abstract Trypan blue exclusion was used to estimate the viability of human polymorphonuclear leukocytes (PMNL) in the presence of Mycoplasma felis and two strains of M. fermentans (PG18 and incognitus). The competence of PMNL to mount a respiratory burst when challenged with the mycoplasmas was also monitored by luminol-dependent chemiluminescence (CL). Both un-opsonised and non-immune human serum opsonised M. felis cells had little effect on PMNL viability. In contrast, PMNL viability was reduced markedly by un-opsonised cells of M. fermentans strain incognitus and, to a lesser extent, strain PG18, and opsonisation of these mycoplasmas further enhanced killing. Death of PMNL in the presence of M. fermentans was not associated with the autonomous production of active oxygen species during the respiratory burst as M. felis induced a high CL response from PMNL, whereas that induced by M. fermentans strain incognitus was significantly lower. M. fermentans may invade mammalian cells and it is suggested that the mechanism of PMNL death could be related to the ability of M. fermentans to penetrate host cell membranes.     

Reactive Oxygen Metabolite Production is Inhibited by Histamine and H 1 -antagonist Dithiaden in Human PMN Leukocytes

The study evaluated the distinction between extracellular and intracellular production of reactive oxygen metabolites (ROM) in isolated polymorphonuclear leukocytes (PMNL) stimulated with opsonised zymosan (OZ) and investigated its modulation by the endogenous mediator histamine (0.1-100 &#119 mol/l) and by the H 1 -antagonist dithiaden (1-100 &#119 mol/l). For this observation, a modified luminol and an isoluminol amplified chemiluminescence (CL) technique were used. Our results showed that PMNL activated with OZ responded with a respiratory burst accompanied by both extra- and intracellular generation of ROM. Histamine and dithiaden significantly decreased both the extra- and intracellular component of chemilumiescence stimulated with OZ. While dithiaden decreased both the extra- and intracellular part of CL with the same potency, histamine decreased preferentially the extracellular part of CL. The fact that histamine as well as the H 1 -antagonist dithiaden decreased the respiratory burst indicates that not only histamine receptors but also non-receptor mechanisms could be involved in the reduction of CL. Interaction with enzymes (NADPH-oxidase, myeloperoxidase, phospholipase A 2 ) or interference with PMNL membrane structure may well result in reduction of the chemiluminescence signal.     

Reactive oxygen metabolite production is inhibited by histamine and H1-antagonist dithiaden in human PMN leukocytes

The study evaluated the distinction between extracellular and intracellular production of reactive oxygen metabolites (ROM) in isolated polymorphonuclear leukocytes (PMNL) stimulated with opsonised zymosan (OZ) and investigated its modulation by the endogenous mediator histamine (0.1-100 μmol/l) and by the H 1 -antagonist dithiaden (1-100 μmol/l). For this observation, a modified luminol and an isoluminol amplified chemiluminescence (CL) technique were used. Our results showed that PMNL activated with OZ responded with a respiratory burst accompanied by both extra- and intracellular generation of ROM. Histamine and dithiaden significantly decreased both the extra- and intracellular component of chemilumiescence stimulated with OZ. While dithiaden decreased both the extra- and intracellular part of CL with the same potency, histamine decreased preferentially the extracellular part of CL. The fact that histamine as well as the H 1 -antagonist dithiaden decreased the respiratory burst indicates that not only histamine receptors but also non-receptor mechanisms could be involved in the reduction of CL. Interaction with enzymes (NADPH-oxidase, myeloperoxidase, phospholipase A 2 ) or interference with PMNL membrane structure may well result in reduction of the chemiluminescence signal.     

Lymphocyte recruitment in delayed-type hypersensitivity. The role of IFN-gamma

Lymphocytes are recruited out of the blood into delayed-type hypersensitivity (DTH) reactions, but the factors controlling their migration are poorly understood. Our previous studies have shown that IFN-alpha/beta, its inducers, and T cell lymphokines can induce lymphocyte migration into the skin after intradermal injection. The present studies were designed to determine the effect of rIFN-gamma, IL-1, and anti-IFN-gamma on lymphocyte recruitment into DTH. Small peritoneal exudate lymphocytes, which preferentially migrate to inflammatory sites, were labelled with 111In and injected i.v. into rats. The intradermal injection of IFN-gamma stimulated the migration of these lymphocytes into the skin. IL-1 induced very little migration by itself, but enhanced the effect of IFN-gamma. Kinetic analysis demonstrated that the migration of lymphocytes to IFN-gamma was rapid, with a peak at 6 h, whereas migration into a DTH reaction was minimal for the first 8 h and reached a peak 24 h after intradermal injection. Polyclonal rabbit anti-IFN-gamma anti-serum, and a Mab to IFN-gamma, DB-2, could almost completely block lymphocyte migration induced by IFN-gamma. Furthermore, DB-2 inhibited lymphocyte recruitment into DTH reactions by 50 to 90%. This Mab did not affect migration in response to IFN-alpha/beta, although it partially inhibited the response to polyI:C. The effect of IFN-gamma on lymphocyte recruitment was not specific for small peritoneal exudate lymphocytes, because both spleen T cells and lymph node cells migrated in response to IFN-gamma and DB-2 inhibited the recruitment of splenic T cells to DTH. Thus, IFN-gamma is a potent stimulator of lymphocyte migration into the skin and a major mediator of lymphocyte recruitment into DTH.     

The modulation of phagocyte oxygen metabolism by recombinant cytokines and a complex of natural cytokines

The data on the modulating function of cytokines on the oxygen-producing function of peritoneal exudate cells of rats are presented. As priming agents, recombinant cytokines IL1 beta and TFR beta 1, as well as the natural complex of cytokines, were used. The priming action of cytokines was studied by changing in the production of active forms of oxygen by peritoneal exudate cells of rats, stimulated with opsonized zymosan, by the method of luminol-dependent chemiluminescence. The study revealed that IL1 beta and the natural complex of cytokines primed peritoneal exudate cells for the production of active forms of oxygen. The maximum value of the prestimulation index was 1.9 +/- 0.1 and 2.95 +/- 0.27 respectively. The preincubation of peritoneal exudate cells of rats with TFR beta 1 led to the pronounced inhibition of the intensity of the chemiluminescent response of cells. The prestimulation index did not exceed 1.06 +/- 0.1. Moreover, as revealed with the use of the probe Fura-2/AM, in the process the prestimulation of phagocytes with the natural complex of cytokines the intracellular concentration of calcium increased from 0.86 +/- 0.15 to 1.86 +/- 0.2 microM/ml. The mechanism of the prestimulation of peritoneal exudate cells of rats cytokines seems to be calcium-dependent.     

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).     

Staphylococcal enterotoxin B induced release of macrophage migration inhibition factor from normal lymphocytes

Staphylococcal enterotoxin B (SEB), a potent lymphocyte mitogen, inhibits migration of peritoneal exudate cells from most guinea pigs but does not inhibit migration of purified macrophages. Experiments were designed to test the ability of highly purified SEB to induce normal lymphocytes to release migration inhibition factor (MIF). Supernatants of lymph node lymphocytes cultured with SEB inhibited the migration of purified macrophages, indicating the release of a migration inhibition factor. Mitomycin-C blocked the SEB-induced release of MIF. SEB-induced MIF localized in the albumin fraction on Sephadex G-200 chromatography. Antibody to SEB specifically blocked the inhibitory effect of SEB on migration of normal guinea pig peritoneal exudate cells.     

Tumor-activated NK cells trigger monocyte oxidative metabolism

We have examined the hypothesis that tumor cells can stimulate a respiratory burst by human natural killer (NK) cells in vitro as measured by luminol-dependent chemiluminescence (CL). Percoll-purified NK cells, containing 40% HNK-1+ cells and less than 1 to 4% esterase-positive contaminating monocytes, can generate a strong CL response after stimulation with the NK-susceptible K562 tumor but not with the NK-resistant P815 tumor cells. Although the response was NK dependent, as shown by depletion with NK-directed monoclonal antibodies (HNK-1, OKT-11, and OKM-1), the cell generating the CL response was not the NK cell. On the basis of several independent experimental approaches the CL response always required the presence of monocytes in the NK preparation. a) Treatment with a monocyte-specific monoclonal antibody (MO2) and complement completely abolished CL. b) The cells producing the CL response were strongly adherent to nylon wool columns (NWC), and large granular lymphocyte preparations containing less than 0.1% esterase-positive cells were inactive. c) NK cells cultured in IL 2-containing medium and tested over several days did not generate CL. d) Optimal numbers of monocytes (less than 1 to 2%) added to a non-CL NWC-purified NK population restored CL, whereas larger or smaller amounts were ineffective. Neither these procedures nor the addition of superoxide dismutase (which completely blocked CL) had any effect on NK lytic activity. We subsequently demonstrated that a factor present in supernatants obtained from NK/K562 incubations, but not from NK or tumor cells alone, could stimulate monocyte CL. We therefore propose that the CL response measured in NK-enriched Percoll fractions originated from contaminating monocytes that were triggered by factor(s) released from tumor-activated NK cells, and that superoxide anion was not required for NK lysis.     

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.