Changes In Polymorphonuclear Leukocytes Research Articles

Enhanced expression of aquaporin 9 in activated polymorphonuclear leukocytes in patients with systemic inflammatory response syndrome.

Aquaporins (AQPs) are water channels of cell membranes. All living cells experience osmotic pressure changes in their environment, but the mechanism by which water influx occurs was not known until the discovery of AQPs. AQP9, which is expressed in human polymorphonuclear leukocytes (PMNLs), is reported to relate to morphologic changes of PMNLs in vitro. We examined the expression of AQP9 in PMNLs from patients with systemic inflammatory response syndrome (SIRS) and addressed the role of AQP9 in both morphologic and functional changes of PMNLs in the SIRS condition. Fourteen patients with SIRS were included in our study. Polyclonal antibody was used for the AQP9 assay. F-actin polymerization, oxidative activity, and the expression of AQP9 in PMNLs with and without stimulation by N-formylmethionyl-leucyl-phenylalanine were evaluated by flow cytometry. Expression of AQP9, F-actin polymerization, and oxidative activity in PMNLs were increased significantly in patients with SIRS compared with those in healthy volunteers. The time course of AQP9 fluorescence in PMNLs corresponded to the time course of F-actin polymerization, which showed peak fluorescence at 1 min after N-formylmethionyl-leucyl-phenylalanine stimulation. The expression of AQP9 in PMNLs is increased significantly in SIRS patients. The increased expression of AQP9 in SIRS patients might be associated with F-actin polymerization in PMNLs, which could affect both morphologic and functional changes of PMNLs in the SIRS condition.

Phosphodiesterase type 4 inhibition of activated polymorphonuclear leukocytes in a simulated extracorporeal circulation model

Objectives: Cardiopulmonary bypass is associated with a systemic inflammatory response syndrome and the risk of multiorgan injuries mediated by activated polymorphonuclear leukocytes. Phosphodiesterase type 4 is the predominant phosphodiesterase isozyme in polymorphonuclear leukocytes and plays a key role in the regulation of polymorphonuclear leukocyte activation. The aim of this study was to examine the effect of rolipram, a selective phosphodiesterase type 4 inhibitor, on the functional changes of polymorphonuclear leukocytes by using simulated extracorporeal circulation. Methods: Simulated extracorporeal circulation was established by recirculating heparinized human blood for 120 minutes on a membrane oxygenator with and without 10 μmol/L rolipram. F-actin content and L-selectin and CD11b expression of polymorphonuclear leukocytes were measured by means of flow cytometry. Polymorphonuclear leukocyte deformability was evaluated with a microchannel array flow analyzer that had a similar diameter as the capillaries. Polymorphonuclear leukocyte elastase was measured with an enzyme immunoassay. Results: Rolipram reduced the increase of F-actin content of polymorphonuclear leukocytes and the increase of transit time of 100 μL of blood sample through a microchannel. Rolipram reduced the increase of CD11b expression and the decrease of L-selectin expression of polymorphonuclear leukocytes. Rolipram reduced the release of elastase from polymorphonuclear leukocytes. Conclusion: Rolipram inhibited the deformability change mediated by F-actin assembly, the changes in adhesion molecules, and the release of elastase from activated polymorphonuclear leukocytes in simulated extracorporeal circulation. This study suggests that phosphodiesterase type 4 inhibition could be a feasible therapeutic strategy to prevent the exaggerated inflammatory response related to cardiopulmonary bypass.J Thorac Cardiovasc Surg 2003;125:172-7

Protease inhibitors, the responsible components for the serum-dependent enhancement of Actinobacillus actinomycetemcomitans leukotoxicity.

Serum enhances the leukotoxic activity of Actinobacillus actinomycetemcomitans against human polymorphonuclear leukocytes (PMNL) by a mechanism that still is unknown. Early attempts to identify the serum components responsible for this enhancement gave no conclusive results, but indicated that the lipoprotein-containing fraction of the serum was involved in the interaction. This study aimed to clarify the role of serum lipoproteins in the leukotoxin interaction, and to identify other serum components involved. The main hypothesis examined was that the leukotoxicity enhancement might depend on serum protease inhibitors that block proteolytic cleavage of leukotoxin by enzymes released from the leukocytes. PMNL were isolated from human peripheral blood and incubated with purified leukotoxin in the presence of serum or purified serum components or lipoprotein-deficient serum. Leukotoxin was also incubated with purified elastase and cathepsin G or with enzyme mixtures from degranulated PMNL. The leukotoxic activity in these mixtures was determined as the extracellular release of lactate dehydrogenase from PMNL. Cleavage of the toxin was showed by gel electrophoresis and Western blot. Morphological changes in PMNL from the above mixtures were examined by electron microscopy. Enzymes from degranulated PMNL cleaved leukotoxin to non-cytotoxic fragments. Elastase and cathepsin G were mainly responsible for the cleavage. Inhibition of leukotoxin degradation was found in the presence of whole serum or of the serum protease inhibitors alpha2-macroglobulin and alpha1-proteinase inhibitor. Under these conditions enhanced PMNL lysis was also observed. A similar enhancement of PMNL lysis was found when PMNL degranulation was blocked by EDTA. On the other hand, lipoprotein-deficient serum had no influence on the leukotoxic activity. The results indicate that the increased leukotoxicity of A. actinomycetemcomitans observed in the presence of human serum is caused by the serum protease inhibitors that counteract proteolytic degradation of leukotoxin. The degradation is caused by enzymes from degranulated PMNL triggered by leukotoxin.

Changes in Polymorphonuclear Leukocytes in the Vagina of Patients with Preterm Labor

To evaluate changes in vaginal polymorphonuclear leukocytes (vPMNL) in patients with preterm labor, we obtained vPMNL-rich suspensions from vaginal fluids of 77 women (21 with preterm labor and 56 control women with uncomplicated pregnancy). Total counts of vPMNL and % viability of vPMNL (viable vPMNL:total vPMNL) were significantly higher in patients with preterm labor (37 ± 92 × 10⁵, 23 ± 29%) vs. controls (11 ± 15 × 10⁵, 6.9 ± 8.3%). The concentration of granulocyte elastase in vaginal fluid diluted to 20 ml was also higher in patients with preterm labor than in controls (4,806 ± 3,818 vs. 2,739 ± 2,556 ng/ml). We conclude that PMNL and granulocyte elastase are abundant in the vagina in patients with preterm labor, which suggests that the PMNL may be involved in the pathogenesis of preterm labor.

Changes of polymorphonuclear leukocytes (PMNs) functions under dietary nucleic acid deficiency and effect of administration of nucleotide and nucleosides mixture solution in mice

Changes of polymorphonuclear leukocytes (PMNs) functions under dietary nucleic acid deficiency and effect of administration of nucleotide and nucleosides mixture solution in mice

Biochemical changes in polymorphonuclear leucocytes in patients suffering from diabetes

The microbicidal capacity of polymorphonuclear leucocytes of diabetic and control subjects was evaluated by estimating the release of lysosomal enzymes viz beta-glucuronidase, lysozyme, acid phosphatase, alkaline phosphatase, in response to a particulate stimulus-serum treated zymosan (STZ). The cells untreated and pretreated with cytochalasin B were exposed to STZ The total enzyme activities were estimated after cell lysis.

Inhibition by L-arginine of the endothelin-mediated increase in cytosolic calcium in human neutrophils

The effect of endothelin (ET) on the cytosolic-free calcium ([Ca 2+] i) changes in polymorphonuclear leukocytes (PMN) from normal humans and Wistar rats was investigated. ET induced a dose-related [Ca 2+] i peak. This [Ca 2+] i transient was blunted by TMB-8 (10 −5M) and by Ca 2+-free EGTA medium, therefore suggesting a role of both intracellular Ca 2+ release and Ca 2+ influx in the generation of the [Ca 2+] i peak. Preincubation of PMN with the nitric oxide (NO)-donor L-arginine (L-Arg) markedly blocked the ET-induced [Ca 2+] i transient in an enantiomerically-specific manner. A similar blunting effect of L-Arg on the fMLP (10 −7M)-induced [Ca 2+] i transient was detected. The L-Arg antagonist, N G-monomethyl-L-arginine (L-NMMA), reverted the L-Arg blocking effect on both ET- and fMLP-induced [Ca 2+] i transients. These data suggest that ET has a potential role in activating Ca 2+ mobilization in PMN, an effect that can be inhibited by L-Arg.

Differential morphological changes in human polymorphonuclear leucocytes induced by culture products of Porphyromonas (Bacteroides) gingivalis W50 and its colonial variants with reduced virulence

The colonial variants of W50, derived after growth in a chemostat and previously designated W50/BP1, W50/BR1 and W50/BE1, produced black-, brown- and beige-pigmented colonies, respectively, and showed reductions in mouse virulence and proteolytic activity correlating with their reduced pigmentation. Incubation of glass-adherent polymorphonuclear leucocytes with culture supernatants from the virulent, highly proteolytic, black-pigmented strain produced significant changes in morphology when compared with changes among sterile bacterial growth medium treated, control polymorphonuclear leucocytes. Large, non-polar cells (⩾ 18 μm dia) increased by 130% ( p < 0.01) while polarized and small non-polar cells (< 18 μm dia) decreased by 48 and 30% ( p < 0.05), respectively. Changes in percentages of the different morphological forms of polymorphonuclear leucocyte after exposure to the culture supernatant from the less virulent W50/BR1 variant showed similar, though less marked trends, while the avirulent W50/BE1 variant failed to produce significant changes in any morphological category. The specific activities of trypsin-like enzyme in the culture supernatants of the different variants were greater in black-pigmented than brown-pigmented and least in the beige-pigmented variant, thus correlating with the morphological changes in polymorphonuclear leucocytes. Morphological changes similar to those seen with culture supernatants from black-pigmented strains were reproduced by exposure of polymorphonuclear leucocytes to commercially available trypsin type II and prior heat treatment of these culture supernatants abolished the shape changes in polymorphonuclear leucocytes. Thus a bacterial, trypsin-like enzyme may play a role in the morphological changes observed.

Phagocytosis of Bacteroides in suspension and on a glass surface determined by a modified fluorochrome assay

Phagocytosis of Bacteroides fragilis and Bacteroides thetaiotaomicron by human polymorphonuclear leukocytes (PMNL) was studied using a modified fluorochrome assay. Bacteria were grown overnight, washed and opsonized in normal, human, pooled serum. Preopsonized bacteria, either in suspension or preadhered onto a glass cover slip, were then incubated with PMNL. Afer appropriate incubation, the mixtures were centrifuged onto the cover glasses. The cover glasses were stained with acridine orange, while duplicate cover glasses were also stained with Giemsa solution. The total number and distribution of bacteria and PMNL, as well as morphological changes in PMNL, were observed with the Giemsa stain. The acridine orange stained only ingested bacteria which provided an accurate indication of phagocytosis. Bacteroides cells adhered to a glass surface were phagocytized significantly more efficiently than Bacteroides in suspension.

4] Purification and crystallization of staphylococcal leukocidin

This chapter describes the purification and crystallization of Staphylococcal Leukocidin. Staphylococcal Leukocidin, known to be important in the pathogenicity of certain staphylococcal diseases, consists of two protein components (S and F) that act synergistically to induce cytotoxic changes in human and rabbit polymorphonuclear leukocytes. These components when tested individually are not cytotoxic. The F and S components crystallize in the form of plates and needles, respectively. The amino-terminal residues of both components are alanine. Leukocidin activity is determined also by assaying for 86 Rb release from 86 Rb-labeled polymorphonuclear leukocytes. Similar data are obtained by examining morphological changes of polymorphonuclear leukocytes. The terminal event in leukocidin action is cytoplasmic degranulation, rupture of nuclei, and complete cell lysis. The purified F and S components of leukocidin stored at –80° in 0.1 M phosphate-buffered saline (pH 7.2) containing 0.5% gelatin are stable for at least 3 months.

Leukotoxic activity in Actinobacillus (Haemophilus) actinomycetemcomitans isolated from periodontal disease patients.

Leukotoxic activity in Actinobacillus (Haemophilus) actinomycetemcomitans isolated from patients with rapidly progressive periodontitis (RP), gingivitis (G), and juvenile periodontitis (JP), and several oral bacteria, was determined by observation of morphological changes in polymorphonuclear leukocytes (PMNs). Many A. actinomycetemcomitans isolates yielded both rough-surfaced and umbonate-shaped colonies (A-type), and smooth-surfaced and convex-shaped colonies (B-type), when stock cultures were streaked on agar medium. Both types of cells were identical in terms of Gram stain, cell morphology, sugar fermentation profile, nitrate reduction and cellular fatty acid composition. Sonic extracts were prepared from 32 A. actinomycetemcomitans strains isolated from patients and from 3 American Type Culture Collection (ATCC) strains. Sonic extracts from 8 isolates and 2 ATCC strains induced sphering of PMNs during a 45-50 min period of incubation at 37 C. Extracts from the other oral bacteria had no effects on PMN morphology. The sphered PMNs were found by their fluorochromatic-negative reactions to be damaged cells. The leukotoxic substance was heat-sensitive (56 C, 30 min), trypsin-sensitive and did not induce sphering of PMNs at 4 C. There was no clear correlation between colony type and leukotoxicity. Among 8 leukotoxic strains, 5 were isolates from an RP patient.

Cell shape of polymorphonuclear leukocytes is influenced by opioids

The effects of β-endorphin(β-End), an endogenous opioid, were tested in vitro on shape changes in polymorphonuclear leukocytes (PMNs). Cell shape changes indicate alterations of the functional status of the cells. Within 2 min, β-End but not the opioid alkaloid levorphanol or the antagonist, diprenorphine, induced a cell spreading. Subsequently, β-End and levorphanol (10 −8 M), but not the dextrorotatory isomer, stimulated an elongation of the cells. Both effects of β-End could be antagonized by diprenorphine in an equimolar concentration. Thus, the effects were stereo specific and antagonizable. In this test system, the morphological changes evoked by β-End were equal to the effects of FMLP, a chemotactic substance, used as a reference. Our findings indicate that endogenous opioids might play a role in modulating the initial phase of the PMNs' offensive behaviour, presumably cell adherence and motility.

Rapid changes in light scattering from human polymorphonuclear leukocytes exposed to chemoattractants. Discrete responses correlated with chemotactic and secretory functions.

A platelet aggregometer was adapted for the simultaneous measurement of perpendicular light scattering in addition to light transmission. The addition of chemoattractants to polymorphonuclear leukocyte suspensions evoked a single wave of increased light transmission, whereas the perpendicular scattering measurement demonstrated a previously unrecognized biphasic response. The first perpendicular scattering response had no detectable latency and peaked at 10 +/- 1 s, then decayed rapidly. The second response peaked at 40 +/- 5 s, and decayed over several minutes. The dose-response curve of chemoattractants for inducing the rapid (10 +/- 1 s) perpendicular scattering peak corresponded to that which initiated chemotaxis. Initiation of the slow (40 +/- 5 s) peak required 10-fold higher amounts of chemoattractants, and the dose-response curve correlated with the induction of lysosomal enzyme secretion and superoxide anion production. Low doses of aliphatic alcohols, which have been shown to enhance chemotaxis but to inhibit secretion and superoxide anion production, abolished the slow perpendicular light-scattering response but left the fast response intact. Stimulants of secretion induced only slow and prolonged responses that were best observed in transmission measurements. In an attempt to resolve the origin of the light-scattering responses, the morphological changes of polymorphonuclear leukocytes were examined microscopically. Neither aggregation nor morphological whole cell polarization could be correlated with changes in light transmission or perpendicular scattering, which suggested that the source of scattering is of subcellular dimensions. The rapid perpendicular light-scattering response of polymorphonuclear leukocytes to chemoattractants appears to record an initial event in the stimulus-response coupling, and its measurement should provide a useful new tool for the study of leukocyte function. The biphasic nature of the light-scattering responses to chemoattractants, moreover, correlates with the dual regulation of the chemotactic and secretory responses of leukocytes.

HUMAN POLYMORPHONUCLEAR LEUKOCYTES OF THE BONE MARROW, CIRCULATION AND MARGINATED POOL: FUNCTION AND GRANULE PROTEIN CONTENT

Polymorphonuclear leukocytes (PMNLs) demonstrate altered function during acute infections and after administration of corticosteroids. We questioned whether such changes are due to population shifts from functionally different compartments of the granulocyte pool. Ten volunteers were given epinephrine 0.3 ml s.c. to induce demargination. PMNLs obtained before and after epinephrine injection were compared for adherence to nylon wool fiber, chemotaxis under agarose, luminol enhanced chemiluminescence, and total content and release of the granule proteins lactoferrin (LF) and β-glucuronidase (β-glu). Similar experiments were carried out using hydrocortisone (HC) to induce PMNL egress from the bone marrow. Epinephrine induced a significant neutrophilia of mature PMNLs, but there was no change in function or granule protein content. HC induced a neutrophilia with significant number of immature PMNLs (bands). These PMNLs demonstrated less adherence, increased chemiluminescence to zymosan stimulation and increased β-glu and LF release to stimulation with phorbol myristate acetate. Total LF and β-glu content was unchanged. There was no correlation between change in function and magnitude of HC induced neutrophilia or the left shift. We conclude that the functional changes of PMNLs after HC infusion are due to an effect of HC upon PMNLs and not a result of a population shift from bone marrow PMNLs.

Physiochemical properties of polymorphonuclear leukocyte surface structures associated with the f-Met-Leu-Phe receptor.

Surface property changes of polymorphonuclear leukocytes (PMNL) as a result of development of functional receptors to f-Met-Leu-Phe and as a result of f-Met-Leu-Phe binding have been studied by aqueous biphasic partitioning of the cells in systems of dextran and polyethylene glycol (PEG) with part of the PEG exchanged for positively charged or hydrophobic PEG. The Phe was associated with increased exposure of hydrophobic surface structures as well as of negatively charged groups on the PMNL surface. Binding of f-Met-Leu-Phe to the PMNL surface receptors caused hiding of hydrophobic and charged structures, indicating that these properties are of importance in the interaction between PMNL and the chemotactic peptide.

Membrane fluidity changes accompanying phagocytosis in normal and in chronic granulomatous disease polymorphonuclear leukocytes

We have studied membrane fluidity changes in polymorphonuclear leukocytes (PMN) during phagocytosis. Membrane fluidity was assessed by electron spin resonance (ESR) using a nitroxide-substituted stearic acid analog (5DS) as a spin probe. PMN from normal subjects and from 3 CGD patients (2 males, 1 female) were incubated in Kreb's Ringers phosphate with or without opsonized zymosan. ESR spectra were obtained and the order parameter (S), which is inversely related to membrane fluidity, was calculated. Without zymosan addition, S for normal (0.638) and for CGD (0.635) were not significantly different (p less than 0.35). The S values indicate that under resting conditions the molecular environment of the CGD membrane is similar to that of normal PMN membranes. However, with addition of opsonized zymosan, the normal, but not the CGD, PMN showed a significant increase (CGD, S = 0.638; normal, S = 0.647; p less than 0.001). This change in S for the normals is consistent with a more restricted movement of 5DS. Treatment of normal PMN with a mixture of scavengers specific for H2O2 (catalase, 1600 U/ml), O2-.(superoxide dismutase, 100 micrograms/ml), and for HO., (sodium benzoate, 1mM) during zymosan stimulation gave S values similar to those of resting cells. Catalase alone also lowered S value, suggesting that H2O2 was instrumental in causing the initial S value increase. This idea was supported by studies in which CGD cells were incubated with zymosan in the presence of glucose oxidase, an enzyme that catalyzes glucose oxidation resulting in the direct reduction of molecular oxygen to H2O2. Our results indicate that reduced O2 by- products, particularly H2O2, can cause altered biophysical properties of PMN membrane during phagocytosis.

Membrane Fluidity Changes Accompanying Phagocytosis in Normal and in Chronic Granulomatous Disease Polymorphonuclear Leukocytes

We have studied membrane fluidity changes in polymorphonuclear leukocytes (PMN) during phagocytosis. Membrane fluidity was assessed by electron spin resonance (ESR) using a nitroxide-substituted stearic acid analog (5DS) as a spin probe. PMN from normal subjects and from 3 CGD patients (2 males, 1 female) were incubated in Kreb's Ringers phosphate with or without opsonized zymosan. ESR spectra were obtained and the order parameter (S), which is inversely related to membrane fluidity, was calculated. Without zymosan addition, S for normal (0.638) and for CGD (0.635) were not significantly different (p less than 0.35). The S values indicate that under resting conditions the molecular environment of the CGD membrane is similar to that of normal PMN membranes. However, with addition of opsonized zymosan, the normal, but not the CGD, PMN showed a significant increase (CGD, S = 0.638; normal, S = 0.647; p less than 0.001). This change in S for the normals is consistent with a more restricted movement of 5DS. Treatment of normal PMN with a mixture of scavengers specific for H2O2 (catalase, 1600 U/ml), O2-.(superoxide dismutase, 100 micrograms/ml), and for HO., (sodium benzoate, 1mM) during zymosan stimulation gave S values similar to those of resting cells. Catalase alone also lowered S value, suggesting that H2O2 was instrumental in causing the initial S value increase. This idea was supported by studies in which CGD cells were incubated with zymosan in the presence of glucose oxidase, an enzyme that catalyzes glucose oxidation resulting in the direct reduction of molecular oxygen to H2O2. Our results indicate that reduced O2 by-products, particularly H2O2, can cause altered biophysical properties of PMN membrane during phagocytosis.

Membrane changes in polymorphonuclear leukocytes during Ionophore (A23187)-induced lysosomal release

Micromolar concentrations of the divalent ionophore A23187 and millimolar concentrations of extracellular calcium caused the rapid exocytosis of lysosomes from rabbit polymorphonuclear leukocytes. In control experiments the larger granules remained within the leukocytes while another class of smaller granules fused with the plasma membrane. Whenever membrane fusion and exocytosis occurred intramembranous particle-free regions developed within the plasma membrane at the sites of fusion. Neither a massive aggregation of intramembranous particles (IMPs), nor the formation of highly symmetrical rosette patterns of IMPs was seen in either experimental or control preparations.

Membrane Changes in Polymorphonuclear Leukocytes During Ionophore (A23187) - Induced Lysosomal Release

It is thought that calcium and/or magnesium may play important roles in polymorphonuclear (PMN) leukocyte functions such as chemotaxis, adhesion and phagocytosis. Yet, a clear understanding of the biological roles of these ions has awaited the development of techniques which permit a selective alteration of intracellular ion concentrations. Recently, treatment of cells with the ionophore A23187 has been used to alter intracellular divalent cation concentrations. This ionophore is a lipid soluble antibiotic produced by Streptomyces chartreusensis that complexes with both calcium and magnesium (3) and is believed to carry these ions across biological membranes (4). Biochemical investigations of human PMN leukocytes demonstrate that cells treated with A23187 and extracellular calcium release their lysosomal enzymes into the extracellular medium without rupturing and releasing their soluble cytoplasmic enzymes (5,6). The aim of the present study and and a companion report (7) was to investigate the structural changes that occur in leukocytes during ionophore-induced lysosomal enzyme release.

Changes in polymorphonuclear leukocytes as a manifestation of malignant neoplasia.

Malignancy associated changes in polymorphonuclear leukocytes were studied in routine Wright-Giemsa and Feulgen stained smears of blood from 254 patients (131) patients w/o tumors, 70 w/carcinoma and 53 w/multiple myeloma, lymphosarcoma and various leukemias. The cells were examined at the highest magnification of the light microscope by use of a x100 oil immersion objective and x16 wide field oculars and by closed circuit television microscopy at x16,000. In patients without tumor a prominent chromocenter and straight chromatin bands in each of the leukocyte lobules were usually present. In patients with malignant tumors the nuclear structure in the leukocytes consisted either of the orderly MAC with numerous pale circular areas surrounded by conspicuously evident chromatin bands or of the disorderly arranged malignancy associated structure with deeply stained-chromocenters and chromatin bands of varying size. In addition a variety of ancillary changes were noted. These were decreased or abnormal segmentation, nuclear excrescences such as hair-like appendages, short and long rods and clubs and the target formation. These changes, though more frequently found in patients with malignant tumors, were not considered significant for the interpretation of MAC. The presence of MAC in blood leukocytes was noted in 81.4% of patients with carcinoma, in 70.0% of those with various leukemias, in 62.5% of those with multiple myeloma and in 70.6% of those with lymphosarcoma. In patients without evidence of tumor MAC were found in 14.5% of the cases. The presence of MAC in the high percentage of patients with malignant tumors is suggestive of a systemic involvement of the host. The reasons for the occurrence of MAC in cases without evidence of tumor remains undetermined until further information is available from continued observation of these patients.