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At least 397 records · Page 22Linked to original sources
PubMed1988

A menadione-stimulated pyridine nucleotide oxidase from resting bovine neutrophil membranes. Purification, properties, and immunochemical cross-reactivity with the human neutrophil NADPH oxidase.

A menadione-stimulated, superoxide-generating enzyme was purified 127-fold from resting bovine polymorphonuclear leukocyte (neutrophil) membranes with a yield of 34%. The enzyme was extracted with Triton X-100 and purified by chromatography on DEAE-Sepharose CL-6B, NAD-agarose, and Sephacryl S-200. The purified enzyme contained FAD and had an apparent molecular mass of 93 kDa by sodium dodecyl sulfate gel electrophoresis. In a nondenaturing gel electrophoresis system, the enzyme was multimeric (Mr greater than 400,000). The oxidase showed 3-4-fold higher activity (Vm) with NADH compared with NADPH, but the Km for both pyridine nucleotides was similar (39 and 47 microM, respectively). The enzyme transferred electrons to cytochrome c, dichlorophenolindophenol, and nitro blue tetrazolium. Cytochrome c reduction was stimulated 4-fold by menadione and was inhibited 70% by superoxide dismutase. Cytochrome c reduction was not inhibited by several mitochondrial respiratory chain inhibitors (azide, cyanide, and rotenone) but was sensitive to thiol-reactive agents (p-chloromercuribenzoate and monoiodo acetate). The catalytic properties of this enzyme distinguish it from the NADPH-dependent superoxide-generating respiratory burst oxidase (NADPH-oxidase) of human neutrophils. Nevertheless, antibodies to this enzyme inhibited not only the purified menadione-stimulated oxidase, but also the respiratory burst oxidase in membranes isolated from activated human neutrophils, indicating similar antigenic determinants are shared by these enzymes. Western blots of human neutrophil membranes visualized a plasma membrane protein of molecular mass 67 kDa, corresponding in size to a protein previously reported in preparations of the human respiratory burst oxidase.

Animals↗
PubMed1986

Do human neutrophils make hydroxyl radical? Determination of free radicals generated by human neutrophils activated with a soluble or particulate stimulus using electron paramagnetic resonance spectrometry.

Using electron paramagnetic resonance spectrometry and the spin trap 5,5-dimethyl-1-oxide (DMPO), neutrophil free radical production in response to phorbol myristate acetate and opsonized zymosan was investigated. Using phorbol myristate acetate and zymosan (3 mg/ml), the superoxide spin-trapped adduct 2-2-dimethyl-5-hydroperoxy-1-pyrrolidinyloxyl (DMPO-OOH) and the hydroxyl spin-trapped adduct 2-2-dimethyl-5-hydroxy-1-pyrrolidinyloxyl (DMPO-OH) were detected. Only DMPO-OH was observed with zymosan (0.5 mg/ml). Hydroxyl radical production in the presence of dimethylsulfoxide (Me2SO) and DMPO yields 2,2,5-trimethyl-1-pyrrolidinyloxyl. The only 2,2-trimethyl-1-pyrrolidinyloxyl detected following neutrophil stimulation was that expected from DMPO-OOH degradation. Superoxide dismutase but not catalase inhibited generation of all three spin-trapped adducts. These data indicate that DMPO-OH arose from DMPO-OOH degradation and does not represent hydroxyl radical production. Under certain conditions DMPO-OH is the predominant spin-trapped adduct resulting from neutrophil superoxide production, perhaps due to cellular bioreduction of DMPO-OOH to DMPO-OH. Cytochalasin B, which prevents phagosome closure, inhibited zymosan-stimulated neutrophil oxygen consumption and electron paramagnetic resonance superoxide detection. No hydroxyl radical was detected. Spin trapping with DMPO appears to detect intraphagosomal free-radical formation.

Cell Separation↗
PubMed1988

Purified protein kinase C phosphorylates a 47-kDa protein in control neutrophil cytoplasts but not in neutrophil cytoplasts from patients with the autosomal form of chronic granulomatous disease.

The neutrophil activators phorbol 12-myristate 13-acetate (PMA), formyl-methionyl-leucyl-phenylalanine, serum-treated zymosan, and IgG-coated latex cause an increase in protein phosphorylation in human neutrophil cytoplasts, concomitantly with an increase in oxygen consumption. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, phosphorylation was apparent in many proteins, must abundantly in 42-, 47-, 50-, 60-, and 80-kDa proteins. In neutrophil cytoplasts from autosomal chronic granulomatous disease (CGD) patients that were stimulated with PMA, the phosphorylation of a 47-kDa protein is absent. The localization of this protein in PMA-activated control cytoplasts is mainly in the cytosol and, to a lower and more variable extent, in the membrane. After addition of purified protein kinase C to lysates of nonstimulated control cytoplasts, phosphorylation occurred at the 47-kDa level in both the cytosol and the membrane fraction. With lysates of autosomal CGD cytoplasts, in vitro phosphorylation of the 47-kDa protein was completely absent. After separation of cytoplast proteins on a sodium dodecyl sulfate-polyacrylamide gel and excision of the 47-kDa protein(s), phosphorylation of the isolated 47-kDa band was observed in the presence of purified protein kinase C. This reaction was again absent when autosomal CGD cytoplasts were used as starting material. Our studies have identified the 47-kDa protein in neutrophil cytoplasts as a true substrate for protein kinase C and indicate that the defect in phosphorylation at the 47-kDa level in autosomal CGD cytoplasts is due to a defective protein.

Chromosome Aberrations↗
PubMed1987

Macrophage-neutrophil interactions: contrasting effects of the monokines interleukin-1 and tumour necrosis factor (cachectin) on human neutrophil adherence.

Incubation of human neutrophils with recombinant human interleukin-1 alpha (IL-1 alpha) resulted in the suppression of neutrophil adherence. In contrast, similar treatment with recombinant human tumour necrosis factor-alpha (TNF alpha, cachectin) resulted in the enhancement of neutrophil adherence. These contrasting effects were noted as early as 5 min after incubation, and persisted for at least 60 min. Simultaneous addition of these two monokines resulted in intermediate values between suppression by IL-1 and enhancement by TNF alpha. The stimulatory effects of the chemotactic peptide FMLP and the phorbol ester PMA were ameliorated by IL-1 but augmented by TNF alpha. The effects of these monokines on neutrophil adherence were abolished by heating but not by polymyxin B treatment, showing that their modulatory properties were not mediated by endotoxin.

Dose-Response Relationship, Immunologic↗
PubMed1985

An antibody binding to human neutrophils demonstrates antigenic heterogeneity detected early in myeloid maturation which correlates with functional heterogeneity of mature neutrophils.

An IgG1 mouse monoclonal antibody (31D8) labels a subpopulation of human peripheral neutrophils. Heterogeneous binding to granulocyte precursors is seen in the bone marrow as early as the myelocyte stage of maturation. 31D8 binding is not affected by the in vitro stimulation or incubation of neutrophils. We demonstrate that the cells which bind 31D8 strongly (31D8-bright) are the same cells which depolarize, reduce nitroblue tetrazolium, and migrate chemotactically when stimulated with the chemoattractant N-formylmethionylleucylphenylalanine. Treatment with cytochalasin B modulates functional activity so that all cells depolarize or reduce nitroblue tetrazolium in response to this chemoattractant, but the heterogeneous binding of 31D8 does not change. The stable binding of 31D8 at 37 degrees C and during activation permitted characterization of the functional capacity of the two populations. These results demonstrate neutrophil heterogeneity early in myeloid maturation and correlate antigenic with functional heterogeneity. 31D8 will make it possible to study the significance of neutrophil heterogeneity in a variety of clinical situations.

Antibody Specificity↗
PubMed1985

Neutrophilic nodules in the intestinal walls of Japanese monkeys associated with the neutrophil chemotactic activity of larval extracts and secretions of Oesophagostomum aculeatum.

High neutrophil chemotactic activity was detected in the culture medium from Oesophagostomum aculeatum larvae in vitro using blind-well chambers with Millipore filters, and guinea pig leucocytes as indicator cells. Neutrophil chemotactic activity was also detected in the extract from larval worms in a dose dependent fashion. This activity was detected in the low molecular weight fractions adjacent to a sodium chloride marker by gel filtration on Sephadex G200. These results were further confirmed with monkey neutrophils. The possible role of this activity in the formation of granulomatous lesions rich in neutrophils found in O aculeatum infections in the Japanese monkey is discussed.

Animals↗
PubMed1979

The in vitro evaluation of certain neutrophil and lymphocyte functions following the ingestion of 150 mg oral dose of levamisole: assessment of the extent and duration of stimulation of neutrophil chemotaxis, protein iodination and lymphocyte transformation.

Certain functions of human blood neutrophils and lymphocytes were investigated at varying time intervals after the ingestion of a single 150 mg dose of levamisole. The functions tested were neutrophil chemotaxis and post-phagocytic metabolic activity and mitogen-induced DNA and protein synthesis of lymphocytes. It was found that levamisole causes a stimulation of neutrophils motility (cell- and serum-associated) and post-phagocytic hexose monophosphate shunt activity and protein iodination. Increased lymphocyte DNA synthesis, but not protein synthesis, to the mitogen phytohaemagglutinin was observed. The stimulation which was detected almost immediately of these neutrophil and lymphocyte functions was still evident 24 hr later but not at 48 hr, indicating that a single oral dose of levamisole can cause the alteration (stimulation) of leucocyte functions which persists until 24--48 hr after intake of the drug.

Chemotaxis, Leukocyte↗
PubMed1984

Stimulus response coupling in the human neutrophil. Differential requirements for receptor occupancy in neutrophil responses to a chemoattractant.

Activated neutrophils aggregate, generate superoxide (O-2), and degranulate. The role of Ca as "second messenger" in neutrophil activation was examined using as agonist the chemotactic peptide fMet-Leu-Phe and its antagonist t-butoxycarbonyl-Phe-Leu-Phe-Leu-Phe to systematically vary the time of receptor occupancy. Release of enzymes from specific and azurophil granules showed a finite requirement for receptor occupancy; the cells were committed to full degranulation after 10 s of receptor-agonist interaction. In contrast, continuous occupation of the receptor by agonist was required to initiate and maintain O-2 generation and aggregation. Cytosolic Ca ( Quin2 fluorescence) increased immediately in response to fMet-Leu-Phe, requiring less than 2 s of agonist-receptor interaction to initiate an optimal response. Mobilization of membrane-associated Ca (chlorotetracycline fluorescence) also demonstrated a finite time requirement; the cells were fully committed after 10 s of agonist-receptor interaction. Increased Ca permeability (45Ca uptake) was fully launched after 15 s of agonist-receptor interaction. The data indicate that Ca movements ( quin2 , chlorotetracycline fluorescence, 45Ca uptake) are both necessary and sufficient to account for degranulation by neutrophils activated by fMet-Leu-Phe. However, neutrophil aggregation and the generation and release of O-2 in response to the same stimulus require a further unknown factor(s) associated with receptor occupancy to maintain these responses.

Adult↗
PubMed1984

An abnormality of neutrophil adhesion: autosomal recessive inheritance associated with missing neutrophil glycoproteins.

A 3-month-old Japanese female infant, with persistent navel infection due to Pseudomonas aeruginosa since birth and recurrent bacterial skin infections, was found to have a severe abnormality of neutrophil adhesion on a surface, leading to a lack of chemotaxis and a mild impairment of phagocytosis. Neither neutrophil bactericidal activity nor nitroblue tetrazorium reduction was impaired. Sodium dodecyl sulfate polyacrylamide-gel electrophoresis of neutrophil membrane proteins from the patient disclosed the lack of two glycoproteins, one with a molecular weight (mol wt) of 110 K on the cell surface, and the other with mol wt of 115 K, possibly in intracellular membranes. The levels of the two glycoproteins were below normal in neutrophils from both parents, with the 110 K glycoprotein reduced to half the normal level. These findings indicate that the disease was inherited in an autosomal recessive fashion.

Blood Bactericidal Activity↗
PubMed1978

Postmitotic marrow neutrophils and neutrophil mobilization in man: role of the spleen.

The relationship between postmitotic marrow neutrophils (PMMN) and neutrophil increment in blood after an intravenous dose of 3 mg hydrocortisone/kg was studied in patients with normal-sized spleens and compared with splenectomized and splenomegalic patients. PMMN were quantified from the ferrokinetic measurement of the normoblast number and the PMMN/normoblast ratio in bone marrow biopsy sections. In 12 control patients with normal PMMN the increment was 3.50 +/- 1.13 X 10(9) neutrophils/liter. An excellent correlation was found between the number of PMMN and the maximal neutrophil increment (y = 826x - 1021, r = 0.93, p less than 0.001) among 24 patients with normal spleen size and a wide range of marrow cellularity. Significantly higher responses were observed in 10 splenectomized patients (y = 872x + 1429, r = 0.92, p less than 0.001). The two regression lines were shown to be parallel, indicating a diminution of the response by 2.5 X 10(9) neutrophils/liter in the presence of a normal spleen. In 11 hypersplenic patients the responses were further reduced and more variable. Peak neutrophilia occurred after median values of 2, 3, and 4 hr in the hypersplenic, the control, and the splenectomized group, respectively. These studies indicate that allowing for the different response curves neutrophil increments may be used as an index of PMMN in patients with normal spleen size and in splenectomized patients. They further suggest sequestration of the prematurely released cells by the spleen.

Bone Marrow Cells↗
PubMed1993

Neutrophils and neutrophil-endothelial cell adhesion in adult respiratory distress syndrome.

The neutrophil has been implicated as a pivotal player in the pathogenesis of adult respiratory distress syndrome (ARDS) and multiple organ failure. An important first step in the process of neutrophil-mediated organ injury involves the binding of neutrophils to endothelial cells. This process is largely regulated by complementary adhesion molecules, some of which are present constitutively on the cell surface and others that can be up-regulated in response to chemotactic and proinflammatory stimuli. Several different adhesion molecules have been described. The leukocyte integrins consist of a common beta 2 chain (CD18) covalently linked to one of three different alpha chains (CD11a, CD11b, CD11c). CD11a/CD18 is expressed on all leukocytes, whereas CD11b/CD18 and CD11c/CD18 are restricted to cells of myeloid origin. CD11b/CD18 is involved in transendothelial migration and adherence-dependent formation of reactive oxygen species. Recently, a relationship between CD11b/CD18 expression, as an indication of neutrophil activation, and the development of ARDS has been suggested. The potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in a large number of experimental models. Protective effects with anti-CD18 antibodies have been observed in a wide variety of inflammatory, immune, and ischemia-reperfusion injuries. Blockage of CD18, however, would affect all leukocytes, as would antibodies to CD11a/CD18. Targeting CD11b/CD18 would affect cells of the myeloid lineage only, which could prove to be beneficial. Anti-CD11b treatment has been used effectively to reduce tissue injury initiated by ischemia-reperfusion, complement activation, and endotoxemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

The CXC-chemokine neutrophil-activating peptide-2 induces two distinct optima of neutrophil chemotaxis by differential interaction with interleukin-8 receptors CXCR-1 and CXCR-2.

The CXC-chemokines interleukin-8 (IL-8), neutrophil-activating peptide-2 (NAP-2), and melanoma growth-stimulatory activity (MGSA) are chemoattractants with high selectivity for neutrophils. Although IL-8 has been shown to act as an extremely potent mediator, reports on NAP-2 and MGSA are still contradictory. Here we show for the first time that NAP-2 and MGSA induce two distinct optima of neutrophil chemotaxis. A first optimum is elicited within a concentration range as low as it is characteristic for IL-8. However, a second optimum appears at more than 200-fold higher stimulus concentrations, at which IL-8 is inactive. Investigating the involvement of the two chemokine receptors CXCR-1 and CXCR-2 in NAP-2-mediated chemotaxis, we observe that the cells become desensitized to the first optimum of the chemokine after selective downregulation of CXCR-2, while both optima disappear upon simultaneous downregulation of both receptors. Blocking monoclonal antibodies (MoAbs) specific for CXCR-2 or CXCR-1 either suppress the first optimum of NAP-2-induced chemotaxis or drastically reduce the second one, respectively. These results provide evidence that both receptors are involved in NAP-2-induced neutrophil chemotaxis, with CXCR-2 rendering the cells responsive to low dosages of the chemokine, and with CXCR-1 extending their responsiveness to NAP-2 dosages higher by several orders of magnitude.

Antibodies, Monoclonal↗
PubMed1998

IFN-gamma is produced by polymorphonuclear neutrophils in human uterine endometrium and by cultured peripheral blood polymorphonuclear neutrophils.

Cytokines present in the human uterus play an important role both in modulating immune responses to infectious challenge and in the establishment and maintenance of pregnancy. In particular, successful implantation and pregnancy is thought to require the establishment of a Th2 environment, while Th1 cytokines are associated with pregnancy loss and infertility. On the other hand, a Th1 response appears to be required for the resolution of acute infection. Using novel confocal microscopic analysis of fresh sections of human tissue, we have investigated the production of IFN-gamma, a Th1 cytokine, in human endometria. Extracellular IFN-gamma, mostly associated with matrix components, was located immediately beneath the luminal epithelium and along the glandular epithelium proximal to the lumen. As evidenced by intracellular staining, IFN-gamma is produced by both stromal cells and intraepithelial lymphocytes through all stages of the menstrual cycle. Surprisingly, the stromal cell containing intracellular IFN-gamma was identified as a polymorphonuclear neutrophil on the basis of its reactivity with a panel of mAbs and its nuclear morphology. We further found that polymorphonuclear neutrophils isolated from normal donors produce IFN-gamma in response to stimulation with LPS, IL-12, and TNF-alpha. Taken together, these findings suggest that polymorphonuclear neutrophils are capable of producing IFN-gamma both in vitro and in vivo, indicating that their role in shaping immune responses may be more extensive than previously thought. Furthermore, these studies strongly suggest that polymorphonuclear neutrophils play an important role in determining immune responsiveness within the female reproductive tract.

Adult↗
PubMed1998

Delayed neutrophil apoptosis after total body irradiation in mice. The role of granulocyte-macrophage colony-stimulating factor in neutrophil function.

We performed an in vitro study of the long-term effects of a sublethal dose (5 Gy) of x-irradiation on the survival and function of neutrophils in adult mice. For this purpose, we incubated control neutrophils harvested from long-term bone marrow cultures (LTBMCs) with supernatant withdrawn from cultures obtained in adult mice 6 or 9 months postirradiation. We noted a significant increase in superoxide anion production, NADPH, and protein levels in these cells after 3, 6, and 15 hours of incubation compared with the same cells incubated with supernatant from control LTBMCs. We also observed a delay in apoptosis that was correlated with maintenance of adenosine triphosphate levels and survival. Similar differences were found when control LTBMC neutrophils were incubated with granulocyte-macrophage colony-stimulating factor (GM-CSF) (1.3 nM). Indeed, enzyme-linked immunosorbent assay revealed a significant overproduction of this cytokine, together with higher interleukin (IL)-6 and IL-3 levels, in the supernatant from cultures of irradiated mice. Our results suggest that GM-CSF is one of the cytokines responsible for promoting the survival and activation of neutrophil function after total body irradiation.

Animals↗
PubMed2026

ER proteostasis failure in HYOU1 deficiency alters B cells, neutrophils, and interferon signalling.

Hypoxia upregulated 1 (HYOU1) is a stress-inducible ER chaperone. We investigated 2 unrelated patients carrying biallelic HYOU1 variants and presenting with primary immunodeficiency. Patient 1, homozygous for p.Pro444His, displayed failure to thrive, hypoglycemia, B cell lymphopenia, and neutropenia. Patient 2, compound heterozygous for p.Arg262Gln and p.Pro757_Glu758insAla, exhibited recurrent infections, enteropathy, and hypogammaglobulinemia. In Patient 1, while HYOU1 transcription was preserved, the protein was severely reduced. Tunicamycin treatment of dermal fibroblasts showed a blunted unfolded protein response and defective induction of ER stress-responsive genes. Immunophenotyping showed near-absence of circulating B cells, and single-cell RNA sequencing of bone marrow identified an arrest at the pro-B cell stage. Neutrophils displayed hypogranulation and dysregulated IFN- and apoptosis-associated transcriptional signatures, unresponsive to G-CSF. HYOU1 deficiency hence results in ER stress-induced proteostasis failure that simultaneously impairs adaptive immunity through B cell developmental arrest and innate immunity through neutrophil dysfunction and IFN pathway imbalance. This work expands the spectrum of HYOU1 deficiency and further identifies ER proteostasis as a central determinant of immune homeostasis.

Journal Article↗
PubMed2001

Immunobiology of CD28 expression on human neutrophils. I. CD28 regulates neutrophil migration by modulating CXCR-1 expression.

CD28, described as a T cell costimulatory molecule so far, is expressed on human peripheral blood neutrophils, as shown by cell surface staining and immunoprecipitation with anti-CD28 monoclonal antibody, and by reverse transcription PCR. The phorbol 12-myristate 13-acetate-augmented expression of CD28 on these cells can be blocked by actinomycin D, an RNA transcription inhibitor, and staurosporin, a protein kinase inhibitor. Cross-linking of CD28 results in an early increase in IL-8 receptor A (IL-8RA or CXCR-1) expression and a concurrent increase in IL-8-induced chemotaxis. The expression of CXCR-1 is down-regulated by receptor internalization 3 h after CD28 cross-linking with concurrent decrease in IL-8-induced chemotactic migration. Thus, our results demonstrate for the first time that CD28 is expressed on human peripheral blood neutrophils and that CD28 may play an important role in the regulation of IL-8RA expression and migration of neutrophils in response to IL-8.

Adult↗
PubMed1994

Activation of neutrophils by a chemically separated but optically coupled neutrophil population undergoing respiratory burst.

Neutrophils from pig blood were used as a model system to investigate the optical communication between cells. It was found that neutrophils stimulated to undergo respiratory burst can activate a second, chemically separated, but optically coupled population of neutrophils. The response of the latter was visualized as a temporary rising of their low-level chemiluminescence and an enhanced generation of superoxide radicals detected by both the reduction of ferricytochrome c and spin trapping. The results provide evidence that a long-range optical coupling of biological significance between living cells exists.

Animals↗
PubMed1999

Cloning and expression of bovine neutrophil beta-defensins. Biosynthetic profile during neutrophilic maturation and localization of mature peptide to novel cytoplasmic dense granules.

beta-Defensins are microbicidal peptides implicated in host defense functions of phagocytic leukocytes and certain surface epithelial cells. Here we investigated the genetic structures and cellular expression of BNBD-4, -12, and -13, three prototypic bovine neutrophil beta-defensins. Characterization of the corresponding cDNAs indicated that BNBD-4 (41 residues) derives from a 63-amino acid prepropeptide and that BNBD-12 (38 residues) and BNBD-13 (42 residues) derive from a common 60-amino acid precursor (BNBD-12/13). The peptides were found to be encoded by two-exon genes that are closely related to bovine epithelial beta-defensin genes. BNBD-4 and BNBD-12/13 mRNAs were most abundant in bone marrow, but were expressed differentially in certain non-myeloid tissues. In situ hybridization and immunohistochemical studies demonstrated that BNBD-4 synthesis is completed early in myelopoiesis. BNBD-12 was localized exclusively to the novel dense granules, organelles that also contain precursors of cathelicidins, antimicrobial peptides that undergo proteolytic processing during phagocytosis. In contrast to cathelicidins, Western blot analyses revealed that mature beta-defensins are the predominant organellar form in myeloid cells. Stimulation of neutrophils with phorbol myristate acetate induced secretion of BNBD-12, indicating that it is co-secreted with pro-cathelicidins. The exocytosis of BNBD-12 by activated neutrophils reveals different mobilization pathways for myeloid alpha- and beta-defensins.

Amino Acid Sequence↗