Supernatant Of Polymorphonuclear Neutrophils Research Articles

Polymorphonuclear neutrophils promote endothelial apoptosis by enhancing adhesion upon stimulation by intermittent hypoxia.

This study explored the interactive effects between polymorphonuclear neutrophils (PMNs) and vascular endothelial cells under intermittent hypoxia (IH) and investigated the mechanisms underlying these effects. Endothelial cells were co-cultured with PMNs isolated from rats exposed to normoxia or IH. The PMN apoptotic rate was determined using flow cytometry. Expression of apoptosis-related proteins in the endothelial cells were evaluated using Western blotting, and the levels of intercellular adhesion molecules in the co-culture supernatants were measured using enzyme-linked immunosorbent assay. The PMN apoptotic rate in the IH-exposed rat group was significantly lower than that of the normoxia control group. There was a positive relationship between the PMN apoptotic rate and IH exposure time. In endothelial cells co-cultured with PMNs isolated from IH-exposed rats, a significant increase in the protein expression levels of Bax, Bcl-2, and caspase-3 and a significant decrease in the Bcl-2/Bax ratio were observed. Furthermore, the intercellular cell adhesion molecule-1(ICAM-1) and E-select element (E-S) levels were elevated significantly in the co-cultured supernatants of endothelial cells and PMNs from IH-exposed rats compared to that from controls. The above IH-induced alterations were partially restored by tempol pretreatment. The apoptotic rate was low in PMNs from IH-exposed rats, which consequently increased the apoptotic signals in endothelial cells in vitro. This may be associated with the increased levels of intercellular adhesion molecules. Further, tempol partially attenuates the PMN-mediated pro-apoptotic effects on endothelial cells under IH.

The Role of TLR2 and TLR4 in Recognition and Uptake of the Apicomplexan Parasite Eimeria bovis and Their Effects on NET Formation.

Background: Bovine polymorphonuclear neutrophils (PMN) constitutively express the Toll-like receptors (TLRs) TLR2 and TLR4 and have been shown to generate Neutrophil extracellular traps (NETs) upon exposure to Eimeria bovis. The present work investigated the role of TLR2 and TLR4 in the recognition and uptake of E. bovis sporozoites, IL-8 production and neutrophil extracellular trap (NET) formation. Methods: TLR expression was performed by flow cytometric analysis on PMN exposed to live carboxyfluorescein succinimidyl ester (CFSE)-stained sporozoites. Supernatants of PMN exposed to different E. bovis sporozoite preparations and antigens in the absence or presence of TLR antibodies were assessed for IL-8 secretion. Cells were exposed to sporozoite preparations and assessed for the activation of transcription factor NF-κB using a luciferase reporter assay. Immunofluorescence analysis was done to investigate TLR2 and TLR4 surface expression and NET formation on bovine PMN exposed to vital sporozoites. Results: we observed significantly increased TLR2 and TLR4 expression with a mean increase in expression that was greater for TLR2 than TLR4. This upregulation neither inhibited nor promoted sporozoite phagocytosis by bovine PMN. Live sporozoites together with anti-TLR2 mAb resulted in a significant enhancement of IL-8 production. NF-κB activation was more strongly induced in TLR2-HEK cells than in TLR4/MD2-HEK cells exposed to heat-killed sporozoites and antigens. Immunofluorescence analysis showed TLR-positive signals on the surface of PMN and concomitant NET formation. Conclusions: This is the first report on E. bovis-induced concomitant TLR2 and TLR4 expression during bovine PMN-derived NETosis.

The role of neutrophil density in neutrophils-mediated inflammatory response induced by monosodium urate crystals

Objective: To study the role of neutrophil density and molecular mechanism in neutrophils-mediated inflammatory response induced by monosodium urate (MSU) crystals. Methods: Polymorphonuclear neutrophils (PMNs) isolated from healthy human peripheral blood were treated with MSU crystals at different density (5×10(6)/ml, 20×10(6)/ml, 100×10(6)/ml) in vitro. The mean fluorescence intensity (MFI) of PMNs and production of reactive oxygen species (ROS) were detected by flow cytometry. The distribution of MSU crystals was observed by polarized light microscopy. The neutrophil extracellular traps (NETs) formation was detected by immune fluorescence. The cytokines in cell supernatant were measured by beads assay including interleukin 1β (IL-1β) , tumor necrosis factor α (TNFα) , interleukin 8 (IL-8) , interferon inducible protein 10 (IP-10) , macrophage inflammatory protein 1 (MIP-1) , monokine induced by interferon-γ (MIG) , macrophage inflammatory protein 1α (MIP-1α) , macrophage inflammatory protein 1β (MIP-1β) . Results: (1) After MSU crystal intervention, the side scatters (SSC) of neutrophils with medium-cell density (20×10(6)/ml) and high-cell density (100×10(6)/ml) were 128±13 and 93±9 respectively, both significantly lower than 170±19 in low-cell density (5×10(6)/ml) group.(2) Similarly, compared with low-cell density group, the MFI (lucifer yellow) of PMNs with high-cell density was 1.8±0.2, also significantly decreased (P<0.05). When co-treated with oxygenated adenosine triphosphate (oxATP), MFI of PMNs were all enhanced consistently. (3) In MSU crystals stimulated PMNs, after adding 2',7'-dichlorodihydrofluorescein diacetate, the MFI values were 0.85±0.32, 2.49±0.78, 4.54±1.02 in low cell density groups, medium cell density groups, and high cell density groups respectively, indicating that the generation of ROS was positively correlated with the increase of PMN density (P<0.05). After the intervention of oxATP, the ROS production was significantly reduced. (4) MSU crystal induced NETs formation, especially at high cell density. NETs formation promotes MSU crystal aggregation, which could be partially overcome by oxATP pretreatment. (5) The expression of cytokines were all significantly decreased in the supernatant of PMNs at high cell density exposed to MSU crystals compared with PMNs at medium cell density (P<0.05) . Conclusion: The PMN-mediated inflammation induced by MSU crystals is cell density dependent, and ATP may play a role in partially overcoming the process.

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.

Tumour necrosis factor alpha production by polymorphonuclear neutrophils treated with mouse anti-human CD19 monoclonal antibody.

Polymorphonuclear neutrophils (PMNs) play pivotal roles as phagocytic cells in immune defence against bacteria and parasites, exerting their effects by production of reactive oxygen species, several cytokines, chemokines and by phagocytotic reaction. In our investigation of properties of activated PMNs, we discovered that one of the two kinds of mouse anti-human CD19 monoclonal antibodies (mAbs) clone SJ25-C1, weakly binds to freshly prepared PMNs. Moreover, the treatment of freshly prepared PMNs with anti-CD19 mAb (clone SJ25-C1) at 37 degrees C for 6 h induces the production and the secretion of tumour necrosis factor alpha (TNF alpha) by PMNs in vitro which was detectable in culture supernatants by bioassay using mouse cell line L929 cells. The concentration of TNF alpha secreted into the culture supernatant of PMNs cultured in the presence of anti-CD19 mAb (clone SJ25-C1) was higher than those of PMNs treated at 37 degrees C for 6 h with various PMN activators, such as anti-CD24 mAb, granulocytes-macrophage colony stimulation factor (GM-CSF) or interferon gamma (IFN gamma). In contrast, another clone of anti-CD19 mAb, HD37, did not bind to freshly prepared PMNs and failed to produce TNF alpha. To confirm that anti-CD19 mAb (clone SJ25-C1)-treated PMNs definitely produce TNF alpha, we measured the levels of intracellular expression of TNF alpha in PMNs permeabilized by saponin. These cells were treated with fluorescence-conjugated mouse anti-human TNF alpha mAb for detection of intracellular TNF alpha expression. Consequently, large amounts of intracellular TNF alpha were detected in PMNs treated with anti-CD19 mAb (clone SJ25-C1) but not in those treated with anti-CD19 mAb (clone HD37).

ACTIVATION OF POLYMORPHONUCLEAR NEUTROPHILS BY IMMUNE COMPLEX: POSSIBLE INVOLVEMENT IN THE DEVELOPMENT OF TRANSFUSION-RELATED ACUTE LUNG INJURY

Background: Human polymorphonuclear neutrophils (PMNs) are suspected as a causative factor in the development of febrile nonhemolytic transfusion reactions (FNHTRs), such as transfusion-related acute lung injury (TRALI), although the mechanisms underlying FNHTRs have not yet been precisely clarified. The aim of this study was to evaluate the potency of immune complexes (ICs) to activate PMNs and to identify soluble factors produced by PMNs. Methods We: investigated the production of tumor necrosis factor α (TNFα) and perform by PMNs on stimulation with artificial oligomerized ICs by enzymelinked immnosorbent assay (ELISA). Furthermore, the potency of culture supernatants of PMNs that were stimulated with ICs was investigated with regard to the growth inhibitory activity against lung-tissue-derived primary cell culture (LT cells) and cytotoxic activity against LT cells by 3H-thymidine uptake experiment and 4-hour 51Cr release assay, respectively. Findings: PMNs activated by ICs were proved to produce TNFα and perform. The growth of LT cells was inhibited in the presence of cell-free culture supernatants of PMNs cultivated with ICs. Moreover, cell-free culture supernatants of PMNs cultivated with ICs appeared to have cytotoxic activity against LT cells. Conclusion: These results suggest that IC-stimulated PMNs produce inflammatory and cytotoxic mediators, which are potent inducers of FNHTRs such as TRALI.

VEGF in the culture of PMN and the serum in oral cavity cancer patients

Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that plays a pivotal role in angiogenesis in vivo. In the present study we examined the ability of polymorphonuclear neutrophils (PMN) to secrete VEGF confronted with the serum levels in oral cavity cancer patients. To investigate whether VEGF may have a prognostic importance, its value in the serum and the culture supernatants was related to the clinical course of patients. The levels of VEGF in the culture supernatant of PMN from patients were significantly higher than those from control. Increased VEGF production by PMN according to clinical progression disease, observed in the present study, seems to suggest a stimulating role of tumour cells in VEGF production by PMN. Additionally, a decrease in the ability of PMN to VEGF release after surgery may be caused by a removal of the tumour mass and then the lack the effects of tumour cells on PMN function. Results obtained appear to suggest that PMN can contribute significantly to the initiation and amplification of tumour angiogenesis and metastasis in oral cavity cancer patients. Increased values of VEGF with progression of disease and decreased values after surgery treatment clearly suggest that VEGF can play a role as a tumour marker in oral cavity cancer patients.

Supernatants of stored polymorphonuclear neutrophils exhibit growth-promoting activity in several cell lines: perforin expression in polymorphonuclear neutrophils and its role in down-regulation of growth-promoting activity.

Polymorphonuclear neutrophils (PMNs) play important roles in the host immune defence. This study was performed to identify roles of PMNs other than those already known. PMNs were separated from the peripheral blood of healthy individuals and stored in vitro for 24 h in the presence or absence of an anti-human Fc receptor (FcR) gamma III antibody, namely, anti-CD16 monoclonal antibody (mAb). Stored supernatants were harvested and incubated with several leukaemia and transformed cell lines for 48 h. The increase in growth rate was assessed by the increase in the amount of 3H-thymidine incorporated into these cells. Expression of perforin on PMNs, which is thought to decrease cell viability, was elucidated by flow cytometry (FCM) analysis. The presence of perforin in the stored supernatants was determined using an enzyme-linked immunosorbent assay (ELISA). A serine protease inhibitor, which is known to block the effect of perforin, was added to the cultures of several leukaemia and transformed cell lines to confirm the effect of perforin in reducing cell viability. Growth promotion of some cell lines cultured with the stored supernatants of PMNs was observed both in the presence and absence of anti-CD16 mAb, which was used as a trigger molecule of PMNs. This was particularly notable in the case of Raji and Daudi (both Burkitt lymphoma) cell lines and Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines (B-LCLs) derived from healthy individuals. Perforin expression was observed in both freshly prepared and stored PMNs, regardless of the presence or absence of anti-CD16 mAb. ELISA also detected perforin in the stored supernatants in both the presence and absence of anti-CD16 mAb. The increase in growth rate was induced in the presence of not only a serine protease inhibitor but also an anti-perforin mAb. Stored supernatants of PMNs exhibit up-regulation of cell growth in several cell lines; this up-regulation is particularly prominent in B-lineage cell lines. Furthermore, perforin appeared to be expressed on PMNs constitutively and secreted into the extracellular fluid. Results of this study strongly suggest that the growth-promoting activity in supernatants of stored PMNs is partially inhibited by perforin, which is thought to be produced by PMNs themselves.

GENE EXPRESSION OF ENDOTHELIN-1 (ET-1) AND RELEASE OF MATURE PEPTIDE BY ACTIVATED HUMAN NEUTROPHILS

Polymorphonuclear neutrophils (PMNs) are only regarded as being involved in the cleavage of exogenous big endothelin-1 (ET-1) to the active peptide. The aims of the present study were to investigate whether PMNs may themselves express mRNA for prepro-ET-1 (pp-ET-1, a long precursor of 212 amino acids) and to determine the capacity of several PMN stimulants to modulate mRNA expression and the release of ET-1 in culture medium. PMNs, isolated from seven healthy adult volunteers, were stimulated with lipopolysaccharide (LPS, 0.25–10μg/ml), or LPS (1μg/ml)+phorbol myristate acetate (PMA, 10ng/ml) or N-formyl-L-methionyl-L-leucyl-L-phenylalanine (f-MLP, 10−5M) or tumour necrosis factor-α (TNF-α, 50IU/ml). They were found to express pp-ET-1 mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Enzyme immunoassay (EIA) revealed low levels of ET-1 in the culture supernatants of PMNs stimulated for 3h with LPS (10μg/ml) and with LPS+PMA. Control unstimulated PMNs did not express pp-ET-1 mRNA. The local production of ET-1 by PMNs in vivo has significant implications in inflammatory diseases.

Oxidative metabolism and release of myeloperoxidase from polymorphonuclear leukocytes obtained from blood sedimentation in a ficoll‐hypaque gradient

Polymorphonuclear neutrophils (PMN) have an important role in the host defence response to infection. These cells produce large amounts of reactive oxygen species (O2·−, H2O2 and ONOO−) with microbicidal activity. PMN are commonly isolated from peripheral blood by sedimentation through a gradient of density (Ficoll-Hypaque gradient and dextran), yielding a highly homogeneous cellular population. However, some cellular activation due to membrane perturbation is also expected. We studied how the production of reactive oxygen species and release of myeloperoxidase (MPO) from blood PMN are affected by the use of the Ficoll-Hypaque density gradient. PMN isolated by spontaneous sedimentation and total blood were used for comparisons. Lucigenin- and luminol-enhanced chemiluminescence was used to estimate the production of reactive oxygen from intact cells and shown to be higher for cells isolated by density gradient both in the absence and presence of added stimuli. The release of MPO, estimated by the chemiluminescence of the luminol/H2O2 reaction in the supernatant of PMN incubated in the absence and presence of stimuli and absence and presence of cytochalasin B, was also higher for PMN isolated by a density gradient. In conclusion, it was shown that the PMN isolation procedure affects reactive oxygen species production and MPO release and in some cases may cause a misinterpretation of results. Copyright © 2000 John Wiley & Sons, Ltd.

Oxidative metabolism and release of myeloperoxidase from polymorphonuclear leukocytes obtained from blood sedimentation in a Ficoll-Hypaque gradient.

Polymorphonuclear neutrophils (PMN) have an important role in the host defence response to infection. These cells produce large amounts of reactive oxygen species (O(2).(-), H(2)O(2) and ONOO(-)) with microbicidal activity. PMN are commonly isolated from peripheral blood by sedimentation through a gradient of density (Ficoll-Hypaque gradient and dextran), yielding a highly homogeneous cellular population. However, some cellular activation due to membrane perturbation is also expected. We studied how the production of reactive oxygen species and release of myeloperoxidase (MPO) from blood PMN are affected by the use of the Ficoll-Hypaque density gradient. PMN isolated by spontaneous sedimentation and total blood were used for comparisons. Lucigenin- and luminol-enhanced chemiluminescence was used to estimate the production of reactive oxygen from intact cells and shown to be higher for cells isolated by density gradient both in the absence and presence of added stimuli. The release of MPO, estimated by the chemiluminescence of the luminol/H(2)O(2) reaction in the supernatant of PMN incubated in the absence and presence of stimuli and absence and presence of cytochalasin B, was also higher for PMN isolated by a density gradient. In conclusion, it was shown that the PMN isolation procedure affects reactive oxygen species production and MPO release and in some cases may cause a misinterpretation of results.

Triflavin potentiates the antiplatelet activity of platelet activating factor receptor antagonist on activated neutrophil-induced platelet aggregation

In this study, specific platelet activating factor (PAF) receptor antagonist ginkgolide B (BN52021) was tested for its antiplatelet activity in zymosan activated polymorphonuclear neutrophil-induced platelet aggregation. Triflavin was also tested for its antiplatelet activity compared with PAF receptor antagonist. Triflavin, an Arg–Gly–Asp-containing disintegrin purified from venom peptide inhibited platelet aggregation by interfering with the interaction of fibrinogen with the glycoprotein IIb/IIIa complex. Furthermore, we also report an efficient high resolution method for quantitative analysis of PAF using high-performance capillary electrophoresis (HPCE). The supernatant of polymorphonuclear neutrophils after their activation by opsonized zymosan induces the aggregation of washed rabbit platelets. In rabbit platelets, BN52021 (100–1000 μM) only partially inhibited activated polymorphonuclear neutrophil-induced platelet aggregation, and its maximal inhibition was estimated to be about 79%. Triflavin also partially inhibited platelet aggregation about 82% induced by activated polymorphonuclear neutrophils. Furthermore, after treatment with a combination of triflavin (0.26 μM) with various concentrations of BN52021 (4–1000 μM), the inhibitory effect of platelet aggregation was almost completely. This inhibition was greater than that produced by the individual drugs alone. These results indicate that a combination of glycoprotein IIb/IIIa complex and PAF receptor antagonist could completely inhibit activated polymorphonuclear neutrophil-induced platelet aggregation. In addition, the amount of PAF released from zymosan (6 mg/ml)-activated polymorphonuclear neutrophils was accurately calculated about 11.8±1.5 ng/10 6 cells, and did not further increase even at a high concentration of zymosan (10 mg/ml). These results suggest that PAF play a major role in the interaction between platelets and polymorphonuclear neutrophils. This interaction may be important in the pathogenesis of thrombosis and inflammatory diseases. Our present findings support the hypothesis that combination therapy with glycoprotein IIb/IIIa complex antagonists and PAF receptor antagonists may represent a new approach to the treatment of ischemic disorders.

Mechanisms of the platelet aggregation induced by activated neutrophils and inhibitory effect of specific PAF receptor antagonists

The supernatant of polymorphonuclear neutrophils after their activation by opsonized zymosan induces the aggregation of washed platelets in human. It potentiates platelet aggregation induced by agonists in platelet rich plasma as well as in whole blood. This activation involves the phosphoinositide metabolism. Specific PAF receptor antagonists gingkolides (BN 50726, BN 52021, BN 54068, BN 54062, BN 50730, BN 50749, BN 50744) and benzodiazepine Web2086 antagonize this neutrophil-induced platelet aggregation. BN 50 730, BN 50 749 and Web 2086 can fully inhibit this aggregation at the final concentration of 10 −6 M. Preincubation of platelets with synthetic PAF also inhibits this activation through a desensitization of the receptor. These data suggest the major involvement in our model of PAF acether in the platelet-neutrophil interactions.

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).

Circulating human peripheral blood granulocytes synthesize and secrete tumor necrosis factor alpha.

Circulating peripheral blood polymorphonuclear neutrophils (PMNs) have long been considered terminally differentiated cells that do not synthesize or secrete protein. However, work of others and ourselves has shown that PMNs can secrete the cytokine interleukin 1. In the present study we investigated whether circulating PMNs are capable of synthesizing and secreting another cytokine, tumor necrosis factor alpha (TNF-alpha). Highly purified (greater than 99% granulocytes) PMNs were isolated from normal human volunteer blood and cultured with or without bacterial lipopolysaccharide (LPS) for up to 24 hr. Cell culture supernatants were collected and tested for TNF-alpha, and total RNA was isolated from cells at various times after stimulation and assessed for TNF-alpha mRNA by Northern blot techniques. The results showed that message for TNF-alpha was produced after 60 min of in vitro stimulation with LPS and was maximal at about 4 hr. TNF-alpha was secreted into the supernatant of unstimulated PMNs from two different donors during 24 hr of culture (35-50 pg/ml), but significantly more (160-190 pg/ml) was secreted by PMNs when stimulated with LPS. PMNs from six other normal volunteers showed significant LPS-stimulated secretion of TNF at 60-180 min of culture. The secreted product also had biological activity against the TNF-sensitive L-M cell line, confirming that PMNs can make and secrete immunologically and biologically active TNF. Since it is also possible for monocytes to synthesize and secrete TNF, the amount of TNF secreted by a monocyte population equal to 20% of the PMNs cultured was measured. The results showed that monocytes at a concentration 20 times that potentially contaminating the PMN populations cultured could not produce as much TNF (unstimulated, 26-65 pg/ml; stimulated, 32-87 pg/ml). The PMN must now be considered a cell capable of altering the acute inflammatory response and modulating the immune response through the synthesis and release of cytokines.