Normal Human Polymorphonuclear Neutrophils Research Articles

Inhibition of Human Neutrophil Apoptosis by Paracoccidioides brasiliensis: Role of Interleukin‐8

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidiodes brasiliensis that presents a wide spectrum of clinical manifestations. Because of the great number of neutrophils polymorphonuclear neutrophils (PMN) found in the P. brasiliensis granuloma, studies have been done to evaluate the role of these cells during the development of the infection. This fungus is found intracellularly in PMN and monocytes/macrophages, suggesting that it is capable of evading damage and surviving inside these cells. Thus, in the present study, we investigated whether P. brasiliensis can prolong the lifetime of PMN, and if this process would be related with IL-8 levels. PMN apoptosis and intracellular levels of IL-8 were analysed by flow cytometry and culture supernatants IL-8 levels were evaluated by enzyme-linked immunosorbent assay. We found that coincubation with P. brasiliensis yeast cells results in an inhibition of PMN apoptosis, which was associated with increase in IL-8 production by these cells. Cocultures treatment with monoclonal antibody anti-IL-8 reversed the inhibitory effect of P. brasiliensis on PMN apoptosis, besides to increase spontaneous apoptosis of these cells. These data show that, in contrast to other microbial pathogens that drive phagocytes into apoptosis to escape killing, P. brasiliensis can extend the lifetime of normal human PMN by inducing autocrine IL-8 production.

Activation of polymorphonuclear neutrophils by immune complex: possible involvement in development of transfusion‐related acute lung injury

Transfusion-related acute lung injury (TRALI) is a serious side-effect of transfusion. We presumed that immune complex (IC)-activated polymorphonuclear neutrophils (PMNs) are involved in the development of TRALI. The aim of this study is to examine the various effects of ICs on normal human PMNs. ICs used here were artificially formed by combining soluble human leucocyte antigen (HLA) class II-positive serum and anti-HLA class II antiserum. The abilities of ICs to trigger PMNs and induce the production of soluble mediators and the involvement of the Fc receptor (FcR) in the activation of PMNs by ICs were investigated. Moreover, the ability of the culture supernatant of PMNs incubated with ICs regarded to induce the apoptosis of lung microvascular endothelial (LME) cells was examined. The results proved that PMNs are triggered by ICs resulting in the acceleration of the production of tumour necrosis factor-alpha (TNF-alpha), perforin and Fas ligand, in which FcR on PMNs appears to be involved. Furthermore, the culture supernatants of PMNs cultured with ICs were revealed to induce the apoptosis of LME cells. In conclusion, the ICs used here were proved to induce PMNs to release cytotoxic factors upon activation. These results suggest that ICs are mediators of the development of TRALI.

Parabutoporin—an antibiotic peptide from scorpion venom—can both induce activation and inhibition of granulocyte cell functions

Parabutoporin (PP) affects motility and NADPH oxidase activity in normal human polymorphonuclear neutrophils and in granulocytic HL-60 cells. These PP-induced interactions utilize a Rac activation pathway. PP induces chemotaxis of neutrophils and HL-60 cells via a pertussis toxin-sensitive way, thus using trimeric G-proteins. The enhanced chemotaxis is also apparent in undifferentiated HL-60 cells which lack functional formyl peptide receptors. On the other hand, PP strongly reduces the superoxide production by the NADPH oxidase complex after either PMA or fMLP activation of granulocytes. These combined results strongly suggest a direct activation of G-proteins and subsequent Rac activation as the basis for the observed effects. The unexpected inhibitory effect of PP, despite Rac activation, on superoxide production in granulocytes is explained by the direct interaction of membrane localized PP which prevents the formation of a functional NADPH oxidase complex.

Hydroxylation at C4′ or C6 is essential for apoptosis-inducing activity of flavanone through activation of the caspase-3 cascade and production of reactive oxygen species

Previous studies demonstrated that hydroxyl groups play important roles in the antioxidative activities of flavonoids; however, the importance of structurally related hydroxylation in their apoptosis-inducing activities is still undefined. In the present study, flavanone with hydroxylation at C4′ and C6 had a significant cytotoxic effect in human leukemia HL-60 cells accompanied by the occurrence of DNA ladders, apoptotic bodies, and hypodiploid cells, characteristics of apoptosis. The replacement of a hydroxyl group (OH) by a methoxyl (OCH 3) group at C4′ or C6 attenuated the apoptotic effect in cells, and there was no significant cytotocity of flavanone or flavanone with OH or OCH 3 in C7-treated HL-60 cells. Induction of enzyme activity of caspase-3 and -9, but not caspase-1 and -8, accompanied by release of cytocrome C from mitochondria to cytosol and the appearance of cleaved of PARP (85 kDa), D4-GDI (23 kDa), and caspase-3 (p17/p15) fragments, was identified in 4′-OH- or 6-OH- flavanone-treated HL-60 cells. Caspase-3 and -9 inhibitors Ac-DEVD-FMK and Ac-LEHD-FMK, but not caspase-1 and -8 inhibitors Ac-YVAD-FMK and Ac-LETD-FMK, attenuated 4′-OH- or 6-OH-flavanone-induced cell death. And, inhibition of capsase-9 activity by Ac-LEHD-FMK suppresses caspase-3 protein procession induced by 4′-OH- and 6-OH-flavanone, indicative of caspase-9 activation locating upstream of caspase-3. A decrease in the antiapoptotic protein Mcl-1 and increases in the pro-apoptotic proteins Bax and Bad were found in 4′-OH- or 6-OH-flavanone-treated HL-60 cells. Induction of endogenous ROS production was detected in 4′-OH- or 6-OH-flavanone-treated HL-60 cells by the DCHF-DA assay. Antioxidants such as N-acetylcysteine (NAC), catalase (CAT), superoxide dismutase (SOD), and allopurinol (ALL), but not pyrrolidine dithiocarbamate (PDTC) or diphenylene iodonium (DPI), significantly inhibited 4′-OH- or 6-OH-flavanone-induced ROS production, with blocking of the apoptosis induced by 4′-OH- or 6-OH-flavanone. The apoptosis-inducing activity of 4′-OH- or 6-OH-flavanone was also observed in another leukemia cell line (Jurkat), but was not found in mature monocytic cells (THP-1) and normal human polymorphonuclear neutrophils (PMNs). This suggests that hydroxylation at C4′ or C6 is important to the apoptosis-inducing activities of flavanone through ROS production, and that activation of the caspase-3 cascade, downstream of caspase-9 activation, is involved.

Store-operated calcium entry in human neutrophils reflects multiple contributions from independently regulated pathways.

Human polymorphonuclear neutrophil (PMN) responses to G protein-coupled chemoattractants are highly dependent upon store-operated Ca(2+) entry (SOCE). Recent research suggests that SOCE currents can be mediated by a variety of related channel proteins of the transient receptor potential superfamily. SOCE has been regarded as a specific response to depletion of cell calcium stores. We hypothesized that net SOCE might reflect the contributions of more than one calcium entry pathway. SOCE was studied in normal human PMN using Ca(2+) and Sr(2+) ions. We found that PMN SOCE depends on at least two divalent cation influx pathways. One of these was nonspecific and Sr(2+) permeable; the other was Ca(2+) specific. The two pathways show different degrees of dependence on store depletion by thapsigargin and ionomycin, and differential sensitivity to inhibition by 2-aminoethyoxydiphenyl borane and gadolinium. The inflammatory G protein-coupled chemoattractants fMLP, platelet-activating factor, and IL-8 elicit unique patterns of Sr(2+) and Ca(2+) influx channel activation, and SOCE responses to these agonists displayed differing degrees of linkage to prior Ca(2+) store depletion. The mechanisms of PMN SOCE responses to G protein-coupled chemoattractants are physiologically diverse. They appear to reflect Ca(2+) transport through a variety of channels that are independently regulated to varying degrees by store depletion and by G protein-coupled receptor activation.

Plasma complement C5 protects endothelial cells from polymorphonuclear neutrophil-derived, H2O2-mediated cytotoxicity.

In vitro studies suggest that polymorphonuclear neutrophils (PMN) can damage endothelial cells (EC) by releasing hydrogen peroxide. In vivo this can lead to anasarca secondary to capillary leakage of fluid, protein, and electrolytes. The result is multiple organ dysfunction syndrome, which is associated with high mortality. In vivo, circulating PMN-EC interactions take place in the presence of plasma, and we have shown previously that plasma affords protection to EC from PMN-mediated damage. Human umbilical vein endothelial cells were primed with cytokines, cultured to a confluent monolayer, and coincubated with normal human PMNs. Cytotoxicity was assayed by gamma scintigraphy, plasma C5 was determined by sepharose column elution, and H(2)O(2) was assayed by R-Phycoerythrin fluorescence. Addition of C5, but not C3, to RPMI resulted in EC cytoprotection equivalent to adding whole serum. Removal of C5 from serum using F(ab')(2) rabbit IgG anti-human C5 coupled to CNBr-activated 4 sepharose beads resulted in significant loss of EC cytoprotection against H(2)O(2)-mediated damage, whereas adding back C5 restored the cytoprotection. C5 also reduced H(2)O(2)-mediated destruction of R-Phycoerythrin. The data suggest that the protection of EC against hydrogen peroxide-mediated damage is partly mediated through complement component C5.

Hemofiltration reduces the serum priming activity on neutrophil chemiluminescence in septic patients

Priming of the polymorphonuclear neutrophil (PMN) response has been implicated in the activation of oxidative burst and tissue injury in patients with septic shock and acute renal failure (ARF). This study evaluated whether hemofiltration (HF) removes substances able to enhance the oxidative burst of PMNs. Chemiluminescence (CL) priming activity induced by sera and ultrafiltrates of seven patients with septic shock, multiorgan dysfunction syndrome, and ARF (ARF/HF group) and of 10 uremic stable patients (Control/HF group) was evaluated on normal human PMNs stimulated with bacterial formyl-methionyl-leucyl-phenylalanine (FMLP). Patients submitted to HF were studied by determining blood and ultrafiltrate interleukin-8 (IL-8), platelet-activating factor (PAF), and CL priming activity at the beginning (T0), and after four hours (T4) of treatment. Preincubation of normal human PMNs with sera and ultrafiltrates from septic patients induced a potent priming of CL activity in subsequent FMLP stimulation. In the ARF/HF group, the prefilter blood concentrations of IL-8 and CL PMN-priming activity significantly decreased during the four hours of HF treatment, with a loss of IL-8 in the ultrafiltrate of 6930 (median, range 4292 to 9282) ng per four hours. PAF detected in the ultrafiltrate and associated with the membrane (7.3 ng, range 1.45 to 9.89) was minimal. In the ARF/HF group, a significantly positive correlation between CL PMN-priming activity and IL-8 concentrations was observed. The CL priming activity in blood and ultrafiltrates was reduced to 55 and 46% by preabsorption with monoclonal antibody (mAb) anti-IL-8. In contrast, the PAF receptor antagonist WEB 2170 did not affect CL priming activity. In the control/HF group, the CL PMN-priming activity was significantly lower than in the ARF/HF group and was independent of IL-8. Sera from septic patients demonstrate an enhanced CL priming activity on PMNs. This activity is reduced by ultrafiltration and is due, at least in part, to ultrafiltered IL-8.

Monoclonal anti-double stranded DNA antibody is a leucocyte-binding protein to up-regulate interleukin-8 gene expression and elicit apoptosis of normal human polymorphonuclear neutrophils.

To determine whether anti-double stranded DNA (anti-dsDNA) autoantibody could bind and affect the functions of normal human polymorphonuclear neutrophils (PMN). Normal human PMN were incubated with different concentrations of a monoclonal mouse anti-dsDNA antibody (12B3) or mouse isotype-matched IgG2a. The binding of anti-dsDNA and PMN was measured by flow cytometry and interleukin-8 (IL-8) gene expression in PMN was detected by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). PMN apoptosis was justified by morphological changes. The cognate antigen(s) of anti-dsDNA on the PMN surface was identified by membrane biotinylation, immunoprecipitation and Western blot. The binding of PMN with anti-dsDNA was much higher than with non-specific mouse IgG2a (70.8 vs 2.0%). Anti-dsDNA at concentrations higher than 12.5 ng/ml significantly enhanced the production and mRNA expression of IL-8 by PMN. However, anti-dsDNA facilitated PMN apoptosis after 3 h incubation. Western blot analysis of biotinylated PMN cell lysates demonstrated that a 50-52 kDa membrane molecule is the cognate antigen of anti-dsDNA. Anti-dsDNA autoantibody up-regulates IL-8 gene expression and elicits activation-induced cell death (AICD) of human PMN via binding to a 50-52 kDa membrane-expressed molecule.

Polymorphonuclear leukocyte adhesion to articular cartilage is inhibited by cartilage surface macromolecules.

The present studies deal with polymorphonuclear neutrophil (PMN) adhesion inhibitory properties of cartilage surface proteoglycans. Normal human PMN were used in adhesion experiments with bovine cartilage surfaces exposed to neutrophil elastase and reconstituted with fibronectin (Fn) or on plastic-bound Fn. An extract of cartilage surface small proteoglycans (SE) and purified fibromodulin (FM), decorin (DCN), biglycan (BGN), and aggrecan (AGN) on the surface of normal cartilage were used to test for inhibition of Fn-dependent cell adhesion. The PMN did not adhere to intact articular cartilage surfaces, whereas significant adhesion was measured using cartilage explants digested with elastase and reconstituted with Fn. Incubation of elastase-treated, Fn-reconstituted cartilage with 45 microg/ml SE inhibited PMN adhesion by 50.7 +/- 5.8% (P < 0.0001). Addition of 50 microg/ml purified FM to the reconstituted articular surfaces inhibited cell adhesion by 71.2 +/- 13.9% (P < 0.0001). Inhibition of PMN adhesion to plastic-bound Fn was seen with 1.7 microg/ml SE (20.4 +/- 8.0%). Maximal inhibition of 67.4 +/- 14.8% (P < 0.01) was obtained with 17.0 microg/ml SE. With FM, concentrations of 4.3 microg/ml resulted in 34.7 25.2 inhibition (P < 0.001), and maximal inhibition of 66.3 16.2% (P < 0.01) was obtained with 43.0 microg/ml. Similar results were obtained with purified bovine DCN and BGN. The main component of cartilage matrix, AGN, failed to inhibit cell adhesion significantly. The results indicate that macromolecules normally present on articular cartilage surfaces act as a barrier to PMN adhesion. Since cartilage surface proteins are susceptible to breakdown by proteases from synovial fluid inflammatory cells, we postulate that the degradation of this barrier may be responsible for increasing PMN adhesion and subsequent cartilage damage in inflammatory arthritis.

Oncostatin M Production by Blood and Alveolar Neutrophils during Acute Lung Injury

Polymorphonuclear neutrophils (PMN) are involved in the pathogenesis of acute lung injury (ALI), secreting numerous mediators such as proteases, reactive oxygen species, and cytokines. Because we had recently observed the ability of normal human PMN to degranulate and synthesize oncostatin M (OSM), an IL-6–family cytokine, we quantified OSM production ex vivo by highly purified blood and alveolar PMN from 24 ventilated patients with ALI, including some patients with severe pneumonia. Most of the patients had no detectable OSM in plasma, and OSM production by cultured blood PMN was similar to that of healthy controls. However, OSM was present in bronchoalveolar lavage (BAL) fluid supernatant, with significantly higher levels during pneumonia. In addition, alveolar OSM levels correlated with the number of PMN obtained by BAL, suggesting that PMN are an important source of OSM within the alveoli. Indeed, purified alveolar PMN from all of the patients, especially those with pneumonia, strongly produced OSM. Interestingly, in the latter patients, alveolar PMN always produced more OSM than autologous blood PMN. These results document the functional duality of PMN in ALI by showing the participation of PMN in the modulation of lung inflammation.

The in vitro immunomodulatory effects of sulfasalazine on human polymorphonuclear leukocytes, mononuclear cells, and cultured glomerular mesangial cells

Sulfasalazine (SSA) was investigated for its effects on phagocytic activity of normal human polymorphonuclear neutrophils (PMN), proliferation of mononuclear cells (MNC) and cultured glomerular mesangial cells. At concentrations from 25 to 100 μM, it inhibited phagocytic activity of PMN and the 3H-thymidine incorporation of phytohemagglutinin (PHA)-stimulated human MNC in a dose-dependent manner. At comparable concentrations, sulfapyridine and 5-aminosalicylic acid, two of its major metabolites, did not show similar effects. SSA exhibited an inhibitory effect on both mouse and rat mesangial cells but at rather higher concentrations (0.5 mM). Excretion of interleukin (IL)-8 by lipopolysaccharide (LPS)-stimulated PMN was also markedly deterred in a dose-dependent manner but excretion of IL-8 by LPS-stimulated MNC was not interfered by SSA. Production of tumor necrosis factor (TNF)-α and IL-1β by mouse mesangial cells was not blocked by SSA but production of IL-4 by these cells was inhibited by it (>0.1 mM). Inhibition of MNC was not due directly to cytotoxic effect of SSA on these cells as shown by fluorescein diacetate stain. Collectively, SSA inhibits phagocytosis and IL-8 excretion by PMN as well as mitogen-stimulated MNC reaction. On the other hand, at high concentrations, it inhibits glomerular mesangial cells and their IL-4 excretion but not TNF-α and IL-1β excretion. These results can account for minimal nephrotoxic characteristic of SSA and suggest that it may be helpful in the treatment of immune-mediated glomerulonephritis.

Differential lytic and agglutinating activity of the anti-Lewis(x) monoclonal antibody FC-2.15 on human polymorphonuclear neutrophils and MCF-7 breast tumor cells. In vitro and ex vivo studies.

The Lewis(x) (Le(x)) trisaccharide (CD15) linked to proteins and glycolipids is highly expressed on the surface of normal human polymorphonuclear neutrophils (PMN) and several human neoplasias, such as breast and gastrointestinal carcinomas and chronic myeloid leukemias. FC-2.15 is an IgM murine mAb that specifically recognizes Le(x) and has been previously shown to mediate the in vitro lysis of Le(x)(+) cells by human complement. In a phase I clinical trial of FC-2.15, a temporary neutropenia was the main toxicity, and antitumor responses were observed. In order to characterize FC-2.15 further and determine the physiological relevance of Le(x) binding, the reactivity of FC-2.15 on PMN was investigated under several conditions. Flow cytometry revealed a strong reactivity of FC-2.15 with almost 100% of PMN, and Scatchard analysis demonstrated an affinity constant of 5.14 x 10(9) M(-1) and 1.11 x 10(6) antigen sites/cell. In vitro, the binding of Le(x) epitopes by FC-2.15 induced PMN homotypic aggregation, only 28.4 +/- 4.1% remaining as single cells. When PMN and the Le(x)(+) MCF-7 breast cancer cells were co-incubated, FC-2.15 induced heterotypic aggregation. In 51Cr-release assays employing human complement, FC-2.15 lysed 93.4 +/- 7.9% of PMN and 87.8 +/- 10.7% of MCF-7 cells. However, when the effect of FC-2.15 was tested in ex vivo circulating blood, no lytic activity against PMN was detected, whereas MCF-7 cells were still lysed. Blood smears demonstrated that FC-2.15 induced PMN agglutination and heterotypic aggregates when MCF-7 cells were present. A pretreatment of PMN with colchicine impaired PMN agglutination both in vitro (single PMN = 81.15 +/- 4.35%) and in ex vivo circulating blood. In the latter condition, FC-2.15-lytic activity was restored, suggesting that PMN homotypic aggregation by FC-2.15, but not lysis, is dependent on microtubule integrity and that PMN agglutination hinders their lysis. Moreover, when 51Cr-release assays were performed following agglutination, FC-2.15 cytotoxicity was restricted to isolated PMN. It is suggested that crosslinking of Le(x) epitopes by FC-2.15 induces PMN to form homotypic aggregates. It is suggested that the neutropenia observed in FC-2.15-treated patients would be due to PMN agglutination and margination, rather than lysis. In addition, FC-2.15 appears to be able to lyse Le(x)(+) tumor cells in circulation.

Interleukin-13 increases prostaglandin E2 (PGE2) production by normal human polymorphonuclear neutrophils by enhancing cyclooxygenase 2 (COX-2) gene expression.

To investigate whether interleukin-13 (IL-13) can affect arachidonic acid metabolism and phagocytic activity of normal human polymorphonuclear neutrophils (PMN). Normal human PMN (1 x 10(6) cells/ml) were incubated with different concentrations of IL-13 (0.1-10 ng/ml) for a variety of times (30-120 min). Phagocytosis and intracellular cyclooxygenase-2 (COX-2) were detected by flow cytometry. The expression of COX-1 and COX-2 mRNA was detected by RT-PCR. The concentration of PGE2 in the PMN cultured supernatants was determined by EIA. We found that IL-13 at an optimal concentration of 1 ng/ml significantly enhanced COX-2 gene expression and PGE2 production (121.57 +/- 22.17 pg/ml in IL-13 stimulation vs. 73.16 +/- 11.72 pg/ml in controls) by PMN. In addition, IL-13 stimulated PMN phagocytosis via increased complement receptor type 1 (CR1) and type 3 (CR3), but not IgG Fcgamma receptor type 3 (FcgammaRIII). The cytoplasmic neutral esterase activity of PMN was also enhanced by IL-13 stimulation for 24 h. These results suggest that IL-13 can stimulate PMN and modulates the inflammatory reactions via the cyclooxygenase pathway.

INTERCELLULAR ADHESION MOLECULE-1 (ICAM-1) IS EXPRESSED ON HUMAN NEUTROPHILS AND IS ESSENTIAL FOR NEUTROPHIL ADHERENCE AND AGGREGATION

This study investigated the expression and regulation of intercellular adhesion molecule-1 (ICAM-1) on human polymorphonuclear neutrophils (PMNs), and its potential role in PMN-PMN adherence and aggregation as observed during systemic inflammatory response syndrome. Normal human PMNs were found to express ICAM-1 with 90% positive population, and this expression was augmented by endotoxin (lipopolysaccharide, LPS) and tumor necrosis factor-alpha (TNF-alpha) stimulation. The presence of ICAM-1 mRNA in human PMNs was further detected by reverse transcription-polymerase chain reaction before and after LPS and TNF-alpha treatment. Furthermore, incubation of PMNs with LPS and TNF-alpha resulted in significant increases in PMN-PMN adherence and aggregation, while addition of either anti ICAM-1 mAb or anti CD11b/CD18 mAb significantly inhibited LPS and TNF-alpha-mediated PMN-PMN adherence and aggregation. These novel findings demonstrate that ICAM-1 is expressed on human PMNs and responsible for PMN aggregation, and suggest that the interaction between ICAM-1 and CD11b/CD18 may be the molecular basis for PMN aggregation and clumping in the microcirculation during systemic inflammatory response syndrome.

The Expression of Genes Modulating Programmed Cell Death in Normal Human Polymorphonuclear Neutrophils

Normal human polymorphonuclear neutrophils (PMN) have a short life and die in progression via apoptosis. In order to understand the molecular basis of PMN apoptosis, the expression of apoptosis-related (Fas, Fas-ligand, p53, and c-myc) and survival-related (bcl-2) genes was detected by flow cytometry, Western blot and reverse transcription-assisted polymerase chain reaction (RT-PCR). We found that Fas and Fas-ligand (FasL) were expressed on the surface of most of the cells. However, the disappearance of FasL was much faster than Fas after 24h incubation. p53 and bcl-2 were also expressed in the cytoplasm of most of the cells. In contrast, the expression of c-myc was negligible in PMN. The addition of monoclonal anti-human Fas antibody (25 μg/ml) to PMN suspension enhanced whereas anti-FasL antibody (25 μg/ml) suppressed PMN apoptosis in 48h incubation. These results suggest that the activation of Fas pathway induced by Fas-FasL interaction among PMNs is one of the mechanisms for spontaneous PMN apoptosis. Lack of proto-oncoprotein c-myc expression in PMN is responsible for their non-proliferative property and may aggravate the spontaneous apoptosis of the cells.

Expression of recombinant myeloperoxidase using a baculovirus expression system

Myeloperoxidase (MPO) is a glycosylated heme-containing enzyme present in the azurophilic granules of normal human polymorphonuclear neutrophils. This enzyme plays a major role in the microbicidal activity of the host defense system by catalyzing the formation of the potent oxidant, hypochlorous acid. Although the amino acid sequence of MPO has been deduced from the cDNA, the structural basis for the observed heterogeneity of this enzyme is not known. Furthermore, the nature of the prosthetic group and its mode of linkage to the apoprotein has not been determined. To address questions regarding the structural features of MPO, which arise during the complex posttranslational processing of this enzyme, we utilized a baculovirus system to express MPO in Sf9 insect cells. Two glycosylated, single-chain precursor species of MPO were observed: an 84 kDa species that was secreted and a 74 kDa species that was cell-associated. This is the first report of an expression system in which a cell-associated MPO precursor undergoes posttranslational proteolytic processing.

Pholasin ®: A new chemiluminescent probe for the detection of chloramines derived from human phagocytes

We have previously reported that normal human polymorphonuclear neutrophils (PMN) relaese taurine-chloramine, a long-lived oxidant, in response to stimulation by phorbol myristate acetate (PMA) or opsonized zymosan in the presence of exogenous taurine. We now describe a new, simple, and highly sesitive method for the detection of chloramines, including taurine-chloramine, using the chemiluminescent probe Pholasin ®, the luciferin of the mollusc Pholas dactylus. Taurine-chloramine ( N-chlorotaurine) detection was assessed with both a colometric method (based on the oxidation of potassium iodide) and with the Pholasin-dependent chemiluminescence (CL) method. The taurine-chloramine concentration in PMN supernatants determined using the potassium iodide (KI) method correlated closely with Pholasin-dependent CL. This CL was also assessed in nonoxidative conditions. No taurine-chloramine was detected in supernatants of lymphocytes and PMN from patients with an oxidative burst defect (chronic granulomatous disease, CGD) with the KI method, but Pholasin-dependent CL was consistently observed. The use of methionine, a specific chloramine scavenger in our incubation conditions, allowed us to define a methionine-inhibitable fraction of Pholasin-dependent CL (i.e., chloramine-induced CL).

Interleukin 1 production by human polymorphonuclear neutrophils.

The purpose of this study was to determine whether human polymorphonuclear neutrophils (PMN), which share a common cell lineage with macrophages, could produce factors such as IL 1. Other properties which these two cell types share are their phagocytic nature and the common receptor and antigens on their cell surfaces. IL 1, in many of its physical, biochemical, and functional characteristics, is found to resemble endogenous pyrogen (EP). PMN have been cited as a possible cell source of EP, but there have also been reports in which the capacity of PMN to produce EP has been questioned. This study shows that normal human PMN can be stimulated by particulate agents such as zymosan and soluble agents such as phorbol myristic acetate to produce a factor(s) which induces proliferation of mouse thymocytes, i.e., PMN IL 1. This PMN IL 1 was released from PMN in a dose- and time-dependent fashion. PMN IL 1 was nondialyzable, was heat-labile, and was inactivated at pH below 5 and above 8. PMN IL 1 stimulated the proliferation of normal human synovial fibroblasts and caused release of a neutral protease (plasminogen activator) from synovial cells. The synovial and thymocyte-proliferating capacity of PMN IL 1 was not affected by the protease inhibitor aprotinin or by soybean trypsin inhibitor. Gel filtration studies estimate the m.w. of PMN IL 1 to be approximately 13,000 to 17,000.

Normal human neutrophils are a source of a specific interleukin 1 inhibitor.

In the course of our study on neutrophil production of an interleukin 1 (IL-1)-like factor, we found that the addition of polymorphonuclear neutrophils (PMN) to monocytes cultured in the presence of zymosan resulted in decreased IL 1 activity of the resultant supernatant, suggesting that PMN may contain an inhibitor of IL 1. The objective of this investigation was to study this IL 1 inhibitor which normal human PMN contain. The inhibitor is constitutively present in the PMN because 0 hr PMN lysates and unstimulated PMN supernatants also show inhibitory activity. The PMN inhibitor inhibits IL 1 (crude and partially purified) in a dose-response manner and does not affect basal [3H]thymidine incorporation in the presence or absence of PHA-P. The PMN inhibitor does not have any effect on interleukin 2 (IL 2)-induced proliferation of the IL 2-dependent CTLL cells. The inhibitor can be generated in the absence of serum and is not produced as a result of proteolytic activity from PMN enzymes. The inhibitor is heat-labile and is most stable at neutral pH. Gel filtration studies on Sephadex G-200 indicate that the inhibitor is heterogeneous in size. Two inhibitory peaks, at 45,000 to 70,000 m.w. and at greater than 160,000 m.w., were observed. When zymosan-stimulated PMN supernatant was chromatographed, there was separation of inhibitory factor from a 17,000 m.w. proliferating factor. Presence of this PMN inhibitor may be important in negative regulation of IL 1.

Interaction of human leukocytes and Entamoeba histolytica. Killing of virulent amebae by the activated macrophage.

Capable effector mechanisms in the human immune response against the cytolytic, protozoan parasite Entamoeba histolytica have not been described. To identify a competent human effector cell, we studied the in vitro interactions of normal human polymorphonuclear neutrophils, peripheral blood mononuclear cells (PBMC), monocytes (MC), and MC-derived macrophages with virulent axenic amebae (strain HMI-IMSS). Amebae killed neutrophils, PBMC, MC, and MC-derived macrophages (P less than 0.001), without loss of parasite viability. The addition of heat-inactivated immune serum did not enable leukocytes to kill amebae, nor did it protect these host cells from amebae. MC-derived macrophages, activated with lymphokine elicited by the mitogens conconavalin A, phytohemagglutinin, or an amebic soluble protein preparation (strain HK9), killed 55% of amebae by 3 h in a trypan blue exclusion assay (P less than 0.001); during this time, 40% of the activated macrophages died. Lysis of amebae was confirmed using 111Indium oxine radiolabeled parasites and was antibody independent. Macrophage death appeared to be due to the deleterious effect of lysed amebae rather than the contact-dependent effector mechanisms of E. histolytica. Adherence between activated macrophages and amebae was greater than that between other leukocytes and amebae (P less than 0.001). Microscopic observations, kinetic analysis of the killing of amebae by activated macrophages, and suspension of amebae with adherent activated macrophages in a 10% dextran solution indicated that contact by activated macrophages was necessary to initiate the killing of amebae. Catalase but not superoxide dismutase inhibited the amebicidal capacity of activated macrophages (P less than 0.001). However, activated macrophages from an individual with chronic granulomatous disease were able to kill amebae, but not as effectively as normal cells (P less than 0.01). In summary, activated MC-derived macrophages killed virulent E. histolytica trophozoites through a contact-dependent, antibody-independent mechanism involving oxidative-dependent and -independent processes.