Granules Of Granulocytes Research Articles

The development of an assay for human neutrophil lipocalin (HNL) — to be used as a specific marker of neutrophil activity in vivo and vitro

Human neutrophil lipocalin (HNL) is a newly discovered protein from human neutrophil secretory granules. A double-antibody radioimmunoassay (RIA) was developed for the measurement of HNL in various body fluids and its high specificity was confirmed by the absence of cross-reaction with other granulocyte granule proteins. The RIA measures HNL within the range of 4–256 μg/l. The intra- and interassay coefficients of variation were less than 6% and 10%, respectively. When HNL was added to serum samples full recovery was obtained. Sera and plasma from 100 apparently healthy individuals revealed a mean level of 78.40 μg/l (range 37.95–190.87 μg/l) in serum and a mean level of 50.65 μg/l (range 30.51–105.8 μg/l) in EDTA-plasma. The distribution of HNL after gel filtration indicated that HNL exists mainly in two major forms, dimer and monomer. This, in addition to the excellent recovery, suggests that these major forms of HNL do not bind to compounds in serum or plasma that would interfere with the assay. The high specificity, sensitivity, reproducibility and accuracy of the present assay should facilitate the measurement of HNL in blood and other body fluids.

Immunocytochemical localization of beta-1,3-glucan recognition protein in the silkworm, Bombyx mori.

A monospecific antibody against beta-1,3-glucan recognition protein (a 62 kDa protein) of the larval silkworm prophenoloxidase activating system was used to study the localization of the protein. Among tissues from 5th instar larvae, only hemocytes and plasma were shown to contain a 62 kDa polypeptide immunoreactive with the antibody. Ultra-thin sections of the hemocytes were stained by an indirect immunogold staining method. Labelling occurred in the granules and cytoplasm of granulocytes and in the spherules and cytoplasm of spherulocytes. It was most conspicuous in granules of granulocytes and uniformly labelled spherules of spherulocyte, whereas no labelling was evident in prohemocytes, plasmatocytes and oenocytoids. The results are discussed in relation to the mode of recognition of fungi as non-self in insect hemocoel.

Fundamental and clinical aspects of anti neutrophil cytoplasmic antibodies (ANCA).

Anti-neutrophil cytoplasmic antibodies (ANCA) are associated with different forms of both primary and secondary vasculitis and glomerulonephritis. These antibodies are directed against different enzymes located in granulocyte granules. In this review ANCA-related antigens and solid phase assays for ANCA detection will be discussed. Furthermore, we will address the clinical relevance of ANCA and will deliberate on their possible pathogenic implications.

Metal content of neutrophil granules is altered in chronic inflammation

The mass fraction of certain elements was measured in isolated granulocytes and isolated granulocyte granule fractions from patients with active inflammatory arthritides (N = 6) and healthy controls (N = 6). The patients had significantly increased amounts of Ca in the granulocytes, in the specific and light azurophil granules, but normal Ca amounts in the dense azurophil granules. Sr was below the detection limit in the granulocytes and granule fraction from controls, but it appeared in high concentrations in the granulocytes and all granule fractions from the patients. The patients had considerably increased granulocyte amounts of Mn but only slightly increased Mn concentrations in the specific granules. Mn was not detectable in azurophil granules from patients and controls. A prominent accumulation of Fe was seen in the granulocytes from the patients, together with an Fe accumulation in the specific granules. Fe was below the detection limit in azurophil granules from patients and controls. The patients had reduced granulocyte Zn and reduced amounts of Zn in the dense and light azurophil granules but normal Zn amounts in the specific granules. The results obtained indicate that the granulocyte accumulation of Ca, Sr, and Fe observed during chronic inflammation is associated with corresponding granule accumulation of these metals; the considerable Mn accumulation in granulocytes during inflammation is not localized in their granules; and the granule subpopulations differ in their capacity to store certain metals.

Neutrophil and eosinophil involvement of the small bowel in patients with celiac disease and Crohn's disease: Studies on the secretion rate and immunohistochemical localization of granulocyte granule constituents

purpose: The concentrations of myeloperoxidase (MPO), a neutrophil granule constituent, and eosinophil cationic protein (ECP), a specific eosinophil granule protein, were measured in jejunal perfusion fluid in an attempt to elucidate the neutrophil and eosinophil involvement of the small bowel in health and disease. patients and methods: The control group consisted of 14 males and two females. Ten patients (seven males and three females) with Crohn's disease and seven patients (two males and five females) with celiac disease were also studied; in addition, one patient with relapsing giardiasis, one patient with giardiasis and complete absence of plasma cells in small intestinal lamina propria, and one patient with selective IgA deficiency and no IgA plasma cells in duodeno-jejunal lamina propria were evaluated. Segmental perfusion of the jejunum was performed according to a previously described method. MPO and ECP were measured by radioimmunoassays. results: In healthy control subjects, the concentrations of both granule proteins were in a narrow range and much higher than would have been anticipated from passive leakage from circulating blood. In patients with celiac disease, the perfusion fluid concentrations of MPO and ECP were on average 3.5 and eight times, respectively, higher than the values seen in the controls. The jejunal segment perfused in patients with Crohn's disease was endoscopically and histologically normal. The perfusion fluid concentrations of MPO and ECP were increased 3.5 and two times, respectively, compared with that in the control subjects. Both patient groups and the control group had similar perfusion fluid concentrations of albumin. Data on MPO and ECP expressed as jejunal secretion rates gave the same differences between patients and controls as just described for the jejunal fluid concentrations. Immunohistochemical studies of jejunal biopsy specimens from another group of patients with celiac disease demonstrated a prominent extracellular deposit of ECP in the lamina propria of the atrophic intestinal mucosa, whereas the release of neutrophil constituents (cathepsin G, MPO) was scarce. In Crohn's disease, an extracellular degranulation ECP and, to a lesser extent, of cathepsin G was observed in relation to ulcerations only. conclusion: Data obtained indicate that the local release of neutrophil and eosinophil granule components is enhanced in the jejunal tissue from patients with celiac sprue and Crohn's disease. The prominent extracellular deposit of eosinophil granule constituents with cytotoxic properties at the site of inflammatory intestinal lesions in celiac sprue might reflect a pathophysiologic mechanism. The deposit of eosinophil and neutrophil granule components in ulcerated areas in Crohn's disease suggests that granulocytes may contribute to the inflammatory lesions also in this disease.

Neutrophil and eosinophil involvement of the small bowel affected by chronic alcoholism.

Excessive ethanol intake may affect the intestinal mucosa functionally and morphologically. The ethanol effect could partly be the result of inflammatory mechanisms, possibly reflected by an enhanced local granulocyte turnover. This study investigated habitual alcoholics by segmental perfusion of the jejunum and analysed the perfusion fluid content of granulocyte granule constituents. The mean jejunal secretion rate of myeloperoxidase (MPO), a neutrophil granule constituent, was 152 (26) (SE) ng/min/40 cm jejunal segment in the controls (n = 16). The MPO secretion rate in non-cirrhotic habitual alcoholics (n = 7) was on average 450 (103) ng/min and significantly increased compared with controls (p less than 0.001). In contrast alcoholics with cirrhosis (n = 6) had normal MPO secretion rate. The mean secretion rate of eosinophil cationic protein (ECP), an eosinophil granule constituent, was in the controls 77 (15) ng/min/40 cm jejunal segment. Corresponding values in non-cirrhotic and cirrhotic alcoholics were 141 (38) and 130 (93) ng/min, respectively (ns). The data suggest an enhanced neutrophil granulocyte turnover in the jejunum in alcoholics, possibly contributing to the ethanol induced affection of the small bowel. The lack of increased neutrophil activity in cirrhotic alcoholics may reflect a role of the liver for granulocyte activity.

Killing of Proteus mirabilis by polymorphonuclear leukocyte granule proteins: evidence for species specificity by antimicrobial proteins.

Low-molecular-weight (Mr, ca. 3,800) polypeptides containing human defensins HNP-1 and HNP-2 (T. Ganz, M. S. Selsted, D. Szlarek, S. L. Harwig, K. Daher, D. F. Bainton, and R. I. Lehrer, J. Clin. Invest. 76:1427-1434, 1985) prepared in our laboratory from acid extracts of human polymorphonuclear granulocyte granules and purified human defensins were found to exert potent bactericidal action against Proteus mirabilis. The antimicrobial action of the extracts of human polymorphonuclear leukocytes granules against P. mirabilis appears to be due to the presence of the defensins. Because P. mirabilis resists the antimicrobial action of other granule proteins, we interpret the present results to mean that the various antimicrobial proteins display species specificity in their microbicidal action.

C. I. Acid Red 52: A New Stain for Cells of Granulocytic Origin

Using the xanthene dye C.I. acid red 52 (C.I. 45100) as a single agent stain applied to coverslip preparations of blood and bone marrow, primary and secondary granules in cells of neutrophilic origin stained brilliant pink. In eosinophils, granules stained dark red. In leukemic myeloblasts that also stained with Sudan black B and demonstrated myeloperoxidase and specific esterase activity, a few bright red staining granules were visualized with acid red 52. In some leukemic promyelocytes, Auer rods stained bright red. In leukemic lymphoblasts, no red granules were seen. Of a wide variety of dyes tested so far, acid red 52 is the most sensitive stain for primary and secondary granules of granulocytes in blood and bone marrow.

Wide-spectrum antibiotic activity of bovine granulocyte polypeptides.

The antibiotic activity of a polypeptide fraction purified from bovine granulocyte granules was tested against Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Bacillus subtilis, Bacillus stearothermophilus, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus faecalis, Streptococcus pyogenes, and clinical isolates of Staphylococcus and Enterobacter spp. All of these bacterial species were susceptible to the antibiotic polypeptide(s), with MICs ranging from 3 to 100 micrograms of protein per ml. The antimicrobial activity was resistant to boiling and abolished by proteinase treatment. Saccharomyces cerevisiae and human fibroblasts grew normally in the presence of 100 and 50 micrograms of antibiotic polypeptide(s) per ml, respectively. [3H]thymidine incorporation into bacterial, but not fibroblast, DNA was efficiently and promptly inhibited by the antimicrobial polypeptide preparation. This suggests that its main target is a component of the system, which catalyzes and regulates the biosynthesis of bacterial DNA.

Human Neutrophil Dysfunction With Giant Granules and Defective Activation of the Respiratory Burst

We describe a patient whose peripheral blood neutrophils and bone marrow precursors (beyond promyelocytes) contained multiple large azurophilic granules. There were also giant granules in eosinophils, basophils, melanocytes, renal tubules, thyroid, and neurones, but not lymphocytes or monocytes. His clinical course included recurrent (ultimately fatal) infections and severe neurologic impairment. Immunofluorescent staining with fluoroscein- and rhodamine-conjugated antisera to primary and secondary granule markers showed virtually all of the granulocyte granules and rare monocyte granules to be fusion products containing both markers. Electron microscopy showed the granules to be large peroxidase-containing lysosomes. Only rare normal primary and secondary granules were present. Superoxide generation in response to opsonized zymosan was 7.3 nmole/min/10(6) cells (control 8.9); but in response to phorbol myristate acetate, only 2.2 (control 9.4). Nitroblue tetrazolium slides showed 3+ dye reduction in response to opsonized zymosan by 90% of granulocytes (control 91%) and to phorbol myristate acetate by 22% (control 99%), with 71% producing only a minimal 1+ response. Cellular contents of myeloperoxidase and beta-glucuronidase were elevated, but the percent release during exocytic degranulation was equivalent to control. Ingestion of complement-opsonized Staphylococcus aureus and zymosan was also normal. Killing of Staphylococcus aureus was 60% at 90-min incubation (control 92%). Granulocyte cyclic adenosine monophosphate (AMP) content was 4 pmole/10(7) cells (control 3.1). In order to determine whether these characteristics derived from the cells' genetic program or their environment, the patient's bone marrow was grown in long-term culture. Granulocytes produced in vitro demonstrated the same morphology, same defect in activation of nitroblue tetrazolium reduction, and same normal cyclic AMP level as those harvested from peripheral blood. These studies describe a new disorder of granulocytes; the structural similarity to, but biochemical differences from, Chediak-Higashi disease indicate the probable heterogeneity of mechanisms for the same morphological abnormality.

Human neutrophil dysfunction with giant granules and defective activation of the respiratory burst

We describe a patient whose peripheral blood neutrophils and bone marrow precursors (beyond promyelocytes) contained multiple large azurophilic granules. There were also giant granules in eosinophils, basophils, melanocytes, renal tubules, thyroid, and neurones, but not lymphocytes or monocytes. His clinical course included recurrent (ultimately fatal) infections and severe neurologic impairment. Immunofluorescent staining with fluoroscein- and rhodamine-conjugated antisera to primary and secondary granule markers showed virtually all of the granulocyte granules and rare monocyte granules to be fusion products containing both markers. Electron microscopy showed the granules to be large peroxidase-containing lysosomes. Only rare normal primary and secondary granules were present. Superoxide generation in response to opsonized zymosan was 7.3 nmole/min/10(6) cells (control 8.9); but in response to phorbol myristate acetate, only 2.2 (control 9.4). Nitroblue tetrazolium slides showed 3+ dye reduction in response to opsonized zymosan by 90% of granulocytes (control 91%) and to phorbol myristate acetate by 22% (control 99%), with 71% producing only a minimal 1+ response. Cellular contents of myeloperoxidase and beta- glucuronidase were elevated, but the percent release during exocytic degranulation was equivalent to control. Ingestion of complement- opsonized Staphylococcus aureus and zymosan was also normal. Killing of Staphylococcus aureus was 60% at 90-min incubation (control 92%). Granulocyte cyclic adenosine monophosphate (AMP) content was 4 pmole/10(7) cells (control 3.1). In order to determine whether these characteristics derived from the cells' genetic program or their environment, the patient's bone marrow was grown in long-term culture. Granulocytes produced in vitro demonstrated the same morphology, same defect in activation of nitroblue tetrazolium reduction, and same normal cyclic AMP level as those harvested from peripheral blood. These studies describe a new disorder of granulocytes; the structural similarity to, but biochemical differences from, Chediak-Higashi disease indicate the probable heterogeneity of mechanisms for the same morphological abnormality.

Quantitative determination of hemoglobin and cytochemical staining for peroxidase using 3,3′,5,5′-tetramethylbenzidine dihydrochloride, a safe substitute for benzidine

The quantitative determination of hemoglobin employing 3,3′,5,5′-tetramethylbenzidine dihydrochloride (TMB-d) is described. This agent can also be used for staining myeloperoxidase-containing granules in granulocytes. TMB-d is at least as sensitive a reagent as benzidine but, unlike benzidine and diaminobenzidine, it is not a carcinogen.

Experimentally induced elevations in acid phosphatase activity in hemolymph of Biomphalaria glabrata (Mollusca)

The levels of acid phosphatase activity in the hemocytes and serum of the following four groups of the pulmonate gastropod Biomphalaria glabrata were ascertained: (1) those injected with heat-killed Bacillus megaterium, (2) those challenged with sterile distilled water, (3) those shaminjected, and (4) those left untampered. It was determined that challenge with heat-killed bacteria sesulted in significant elevations in acid phosphatase activity in both cells and serum at 1, 2, and 4 hr postinjection, with the level being highest at 2 hr. Injection with water resulted in a significant elevation in cellular enzyme level at 1 hr postinjection but not at 2 and 4 hr; however, there were significant elevations at 2 and 4 hr in serum. This is interpreted to indicate that the elevated intracellular enzyme was subsequently released into serum. Sham injection resulted in significant elevations in acid phosphatase levels in hemocytes at 2 and 4 hr postinjection but no increase of enzyme activity in serum during the course of the experiment. This is interpreted to mean that this hydrolase was not released as a result of sham injection. The source of acid phosphatase was apparently the cytoplasmic granules of granulocytes, which are true lysosomes.

The blood leukocytes of Bufo alvarius: a light, phase-contrast, and histochemical study

Blood leukocytes of Bufo alvarius were studied by light and phase-contrast microscopy and histochemical techniques for the localization of glycogen and several hydrolytic enzymes, i.e., acid and alkaline phosphatases, nonspecific esterase, beta-glucuronidase, aryl-sulfatase, and myeloperoxidase (peroxidase). Neutrophils were the only leukocytes to demonstrate alkaline phosphatase activity, while beta-glucuronidase and aryl-sulfatase were not observed in any leukocytes. Periodic acid – Schiff (PAS) positive granules and granules containing hydrolytic enzymes occurred in varying amounts in leukocytes. In eosinophils, most glycogen was associated with smaller granules, while the larger refractile granules were PAS negative. Small lymphocytes were myeloperoxidase (peroxidase) negative. The present study agrees with previous investigations in mammals which indicate that specific granules in granulocytes may be PAS positive as well as contain one or more hydrolytic enzymes. In small lymphocytes of B. alvarius, PAS positive and acid phosphatase positive granules correspond to neutral red granules seen in supravital films. Furthermore, the appearance and histochemical reactivity of acid phosphatase granules in mature neutrophils, metamyelocytes, and late myelocytes correspond closely with the appearance and number of specific neutrophilic granules seen in Wright–Giemsa preparations and with PAS positive granules.

Cell fractionation and isolation of plasma membrane enriched fractions of normal granulocytes and leukemic myeloblasts

Cell fractionation studies were done on normal human granulocytes and leukemic myeloblasts. Methods were developed to prepare plasma membrane enriched fractions from these cells. Homogenization could be performed with a Potter Elvehjem tube or a Dounce homogenizer in hypotonic sucrose. Short sonication in isotomic sucrose also gave good disruption. More reproducible fractionation results were obtained after sonication. As the classical plasma membrane marker enzymes (5′-nucleotidase and alkaline phosphatase) cannot be used for human granulocytes and no plasma membrane marker was known for leukemic myeloblasts we introduced a radioactive marker. 125I-labeled Fab, prepared from an antiserum against human leucocytes was found to be a reliable plasma membrane marker both for normal granulocytes and leukemic myeloblasts. Using sonication for disruption of cells and [ 125I] Fab as plasma membrane marker, plasma membrane enriched fractions could be prepared with high yield (60%) and 10 fold enrichment. It was concluded from fractionation studies that enzyme +5 like β-glucuronidase, peroxidase and lysozyme that are confined to granules in granulocytes have other localization in leukemic myeloblasts.

Degranulation and Other Changes of Molluscan Granulocytes Associated with Phagocytosis

The loss of cytoplasmic granules, which have been shown to be lysosomes, from granulocytes of Mercenaria mercenaria during the in vitro phagocytosis of Bacillus megaterium has been demonstrated semiquantitatively. This process, designated as degranulation, represents the morphological basis of the release of lysosomal enzymes from granulocytes into serum as- sociated with phagocytosis. In addition to degranulation associated with phagocytosis, there is rearrangement of lysosomes, the appearance of large vacuolated areas in the cytoplasm, con- traction of cell margins, and the appearance of atypically large granules in granulocytes of M. mercenaria.

Données sur la localisation ultrastructurale d'une activité phénoloxydasique dans les hémocytes circulants d' Antheraea pernyi au dernier âge larvaire

Phenoloxidase activity has been demonstrated (with Dopa as a substrate) only in oenocytoids and in intermediate forms between plasmatocytes and oenocytoids (pro-oenocytoids) among five haemocyte types identified in the last larval stage of silkworm Antheraea pernyi. This activity is localized within the cytosol and also in the dense granules. No phenoloxidase reaction product has been observed within premelanosome-like granules of granulocytes and pro-oenocytoids. Control and experiment incubations suggest that the localization of reaction product correspond to two forms of phenoloxidase: one bound to cytoplasmic bodies an the other soluble in the cytosol.

骨髄体外組織培養に於ける顆粒球の固有顆粒運動の生態観察

骨髄体外組織培養に於ける顆粒球の固有顆粒運動の生態観察

骨髄体外組織培養に於ける顆粒球の固有顆粒運動の生態観察

骨髄体外組織培養に於ける顆粒球の固有顆粒運動の生態観察