Leukocyte Suspensions Research Articles

Influenza A virus enhances allergic histamine release

Histamine release was examined in leukocyte suspensions from patients allergic to house dust mite, grass pollen, birch pollen or cat dander. Influenza A virus was found to enhance the antigen-induced mediator release, but did not cause release of histamine from the cells per se. Also histamine release induced by anti-IgE in cell suspensions from normal individuals was enhanced by virus. The potentiating effect of influenza A virus might be due to neuraminidase on the surface of virus, since a similar effect was caused by a purified neuraminidase obtained from Vibrio cholerae, and the effect of virus as well as the neuraminidase was completely abolished by a potent neuraminidase inhibitor. The synergistic enhancement in IgE-mediated histamine release by virus could be of significance for the conversion from latent to manifest asthma.

Influenza A virus enhances Staphylococcus aureus‐induced basophil histamine release in normal individuals and patients with intrinsic asthma

Influenza A virus was found to enhance Staphylococcus aureus-induced histamine release in human leukocyte suspensions, but did not per se release histamine. This potentiating effect was found to be similar in cells from normal individuals and from patients with intrinsic asthma. The enhancement of mediator release could be due to viral neuraminidase on the surface of the virus, since a similar potentiating effect was caused by a purified neuraminidase preparation obtained from Vibrio cholerae, and the effect of virus as well as of the purified neuraminidase was completely abolished by a potent neuraminidase inhibitor. The potentiating effect of virus on bacteria-induced mediator release might be of importance for the conversion from latent to manifest asthma in upper respiratory tract infections.

Detection of Granulocyte Antibodies Using Flow Cytometric Analysis of Leucocyte Immunofluorescence

Antigranulocyte antibodies are involved in the pathophysiology of a number of clinical disorders, which include: febrile transfusion reactions, severe pulmonary reactions to transfusion, auto-immune neutropenia, drug-induced neutropenia, and iso-immune neonatal neutropenia. Owing to the inherent difficulties of manipulating granulocytes in vitro, many of the serological techniques described for the detection of antigranulocyte antibodies are complex and sometimes difficult to reproduce. We describe the detection of alloreactive granulocyte antibodies using flow cytometric analysis of donor leucocyte suspensions in an indirect immunofluorescent test. The technique provides a semiquantitative detecion of granulocyte antibodies in two groups of patients studied and, by providing as a comparison the reactivity on the corresponding mononuclear leucocytes, allows the distinction between granulocyte- specific antibodies and antibodies directed against the histocompatibility antigens.

Detection of granulocyte antibodies using flow cytometric analysis of leucocyte immunofluorescence.

Antigranulocyte antibodies are involved in the pathophysiology of a number of clinical disorders, which include: febrile transfusion reactions, severe pulmonary reactions to transfusion, auto-immune neutropenia, drug-induced neutropenia, and iso-immune neonatal neutropenia. Owing to the inherent difficulties of manipulating granulocytes in vitro, many of the serological techniques described for the detection of antigranulocyte antibodies are complex and sometimes difficult to reproduce. We describe the detection of alloreactive granulocyte antibodies using flow cytometric analysis of donor leucocyte suspensions in an indirect immunofluorescent test. The technique provides a semiquantitative detection of granulocyte antibodies in two groups of patients studied and, by providing as a comparison the reactivity on the corresponding mononuclear leucocytes, allows the distinction between granulocyte-specific antibodies and antibodies directed against the histocompatibility antigens.

Properties of human basophils isolated by fluorescence-activated cell sorting

The aim of the current study was twofold: (1) to consider the applicability of fluorescence-activated cell sorting (FACS) to basophil isolation from small blood volumes and (2) to compare basophils obtained from children with asthma to basophils from healthy children. With FACS, basophil suspensions were prepared with a purity of 84% (range, 75% to 95%) and a recovery of 20% (range, 15% to 30%). The purified basophils had a total histamine content of 1.6 ± 0.12 pg per cell, not differing significantly from total histamine content observed in “total” leukocyte suspensions (1.4 ± 0.07 pg per basophil). The same was true for IgE receptor-mediated histamine release (29 ± 4% versus 27 ± 4%) and for ionophore A23187-induced histamine release (41 ± 6% versus 51 ± 9%). Sorted basophils from subjects with asthma released more histamine after IgE receptor activation (0.67 ± 0.09 pg per cell) than basophils from healthy children (0.40 ± 0.04 pg per cell; p < 0.02). Expressed as percent release, no significant difference was observed (37 ± 3.2% versus 30 ± 2.7%). Ionophore A23187-induced histamine release did not differ significantly between subjects with asthma and control subjects, neither expressed as picograms per cell (1.21 ± 0.17 pg per cell versus 1.02 ± 0.11 pg per cell) nor expressed as percent release (66 ± 4.4% versus 74 ± 3.2%). The IgE receptor-mediated change in membrane potential induced by Ca ++ exposure and by ionophore A23187 exposure demonstrated no significant differences between subjects with asthma and healthy children: 12 ± 1.6 mV versus 14 ± 1.0 mV after Ca ++ exposure and 11 ± 1.8 mV versus 12 ± 1.4 mV in the presence of ionophore A23187. FACS is well suited for the preparation of high-purity basophil suspensions without significant selection bias or cell damage. In response to IgE stimulation, purified basophils from subjects with asthma release more histamine than basophils from healthy children. No such difference could be demonstrated in ionophore A23187-induced histamine release.

Dissociation of the res and immune components in the transient splenic response of embryos and neonatal chicks to immunization

Immunization with mouse erythrocytes (MRBC) elicited transient splenic enlargement as well as PFC formation in immunocompetent 7, 10, and 14 day chicks. An early enlargement within two days was followed by a decline and a second enlargement by day six. Only the early enlargement was observed when MRBC or Percoll was administered to neonatal chicks and late embryos. No significant splenomegaly was observed following cell transfer of bone marrow, spleen, leucocyte, thymocyte or bursal cell suspensions from B haplotype-compatible donors. During the adoptive immune response in embryo hosts, a minor early peak was observed that was followed by an elevated or progressive increase in spleen size. The data suggest that the early splenic response reflects primarily RES-vascular activity and the later enlargement is the consequence of immune proliferation.

Monoclonal antibody detection of carcinoma cells in bone marrow biopsy specimens from breast cancer patients.

The possibility of using immunologic methods for detecting metastatic cells in bone marrow samples from breast cancer patients was investigated. The MBr1 monoclonal antibody, which recognizes a membrane antigen on breast carcinoma cells and is unreactive on normal bone marrow cells, seemed to be an adequate reagent for this kind of approach. When human leukocyte suspensions artificially contaminated with mammary tumor cells were tested by MBr1 immunofluorescence, it was demonstrated that the added tumor cells could be specifically discriminated from normal cells and that as little as one tumor cell in 200,000 could be detected. With the same methodology we screened bone marrow biopsies from breast cancer patients with apparently uninvolved lymph nodes at the moment of surgery. Immunoreactive tumor cells were detected by the MBr1 antibody in 17% of N- patients. None of the bone marrow samples showed any evidence of tumor involvement by conventional histologic analysis.

Altered leukocyte rheology in patients with chronic cerebrovascular disease.

Erythrocyte and leukocyte suspensions were prepared from 28 patients with chronic cerebrovascular disease and 30 age- and concomitant disease-matched control subjects. Cell filterability was studied with the new St. George's filtrometer, which can discriminate between initial filtration rate as an erythrocyte parameter and filter clogging as a leukocyte parameter. Compared with control subjects, a significant increase of filter clogging was found in the patients, suggesting decreased deformability or increased adhesiveness of leukocytes or both in chronic cerebrovascular disease.

Techentium-labelled leucocytes using diethyldithiocarbamate

Diethyldithiocarbamate (DDC) forms a lipid soluble complex with 99Tcm-pertechnetate which is rapidly incorporated into leucocytes in vitro. In seven healthy volunteers, a 'crude' leucocyte suspension was obtained by sedimnetaion and centrifugation and incubated with 99Tcm-DDC complex. Labelling efficiency was found to be 73 +/- 14% (SD). The mean activity remaining on the cells after hourly incubations with plasma was 77 +/- 5%. Percoll-saline differential cell analysis showed that 69 +/- 3% of the activity was associated with the granulocytes, 12 +/- 4% with the lymphocytes/monocytes, 11 +/- 4% with the platelets and 8 +/- 1% with contaminating erythrocytes (n = 3). The mean labelling efficiency decreased by 14% when the leucocyte count was reduced from 42 X 10(6) to 21 X 10(6) cells. Over the range 21 X 10(6) to 5.2 X 10(6) cells there was no decrease in labelling efficiency.

Clumping of labeled leukocyte suspension. A simple measure for avoiding it

Leukocytes in “mixed” suspensions can clump together, resulting in cell clusters which are responsible for false positive hot spots in lungs of patients, in the case of abscess localization studies using 111In labeled leukocytes. Addition of extra ACD (acid-citrate-dextrose) in those labeled leukocyte suspensions prevented cell clumping and avoided occurrence of focal radioactivity accumulation in lungs. The acidification did not interfere in leukocyte migration under agar.

Experimentelle Untersuchungen zur in vitro‐IFN‐Induktion durch verschiedene Polynucleotide in humanen Zellkultur‐Systemen

Several polynucleotides were tested for interferon induction in comparison with the standard Poly (IC) in human diploid fibroblasts and in human leukocyte suspensions. We received high IFN-titre following superinduction of the polynucleotides in human fibroblasts. These results show that under superinduction conditions partly 3 times more interferon is induced in comparison with the standard inductor Poly (IC). The tested concentration of 10 micrograms/ml polynucleotides did not result in any IFN yields or only in very low ones in human leukocytes, which were only within the range of detectability.

Lipoxin A augments release of thromboxane from human polymorphonuclear leukocyte suspensions

Lipoxin A (LXA) is a novel eicosanoid, generated by the interactions of lipoxygenases, which has a variety of biological actions. When added to human polymorphonuclear leukocytes, LXA stimulated thromboxane formation which was monitored as TxB2 by radioimmunoassay. The compound augmented the formation of TxA2 stimulated by the ionophore of divalent cations (A23187). Formation of thromboxane was inhibited by two non-steroidal anti-inflammatory drugs (i.e. indomethacin and proglumetacin). Results of the present study indicate that LXA can provoke the release and transformation of endogenous arachidonic acid to thromboxane. Moreover, they suggest a relationship between lipoxin A and the formation of cyclooxygenase pathway products.

Bovine gut-associated lymphoid tissue — Morphologic and functional studies: I. Isolation and characterization of leukocytes from the epithelium and lamina propria of bovine small intestine

A successful technique for the isolation of highly pure suspensions of viable leukocytes from the small intestine of cattle is described. Procedures ranging from mechanical mincing to enzymatic digestion of tissues were compared. The most reliable and reproducible procedure was the sequential treatment of tissues with dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA) in calcium-magnesium-free salt solutions, and collagenase. Two populations of mucosal leukocytes were obtained from the small intestine. One population was derived from within the epithelium (intraepithelial leukocytes, IEL), the second from within the lamina propria (lamina propria leukocytes, LPL). At least 2 × 10 6 viable leukocytes were obtainable from each square centimeter of the intestinal mucosa from either the epithelium or lamina propria. Erythrocyte rosetting and immunofluorescence characterization with conventional antisera and monoclonal antibodies (MAB) demonstrated that IEL were predominantly T cells (60%), with relatively few B cells present (10%), while LPL contained relatively high numbers of B cells (28%) and a reasonable percentage of T cells (45%). Both cell populations proliferate in resposne to stimulation with T and B cell mitogens. Addition of the thiol compound, 2-mercaptoethanol (2-ME) strongly augmented the mitogenic response of both cell isolates. Human recombinant interleukin-2 (hr-IL-2) in the presence or absence of additional stimuli was found to be able to induce the proliferation of both cell types. These results demonstrate that functinal leukocytes can be isolated from the small intestine of cattle, and that they can maintain their responsiveness to both T and B cell mitogens and to exogenous cloned IL-2.

Effects of immune complexes, zymosan preparations and ionophore A 23187 on leukotriene formation in human leukocytes

Incubation of human leukocytes with opzonized zymosan or IgG immune complexes led to a time dependent release of leukotrienes (LT) B 4 and C 4. After 3–4 min, the levels of LTB 4 and LTC 4 were 93 and 35 pmol/3 ★10 7 cells, respectively. These amounts were 2–4 times lower than those released by leukocytes stimulated with the calcium ionophore A 23187. The levels of LTC 4 were 8 and 20 times lower than those of LTB 4 after incubation with opsonized zymosan or immune complexes, respectively. Heat-inactivation of the serum prior to zymosan coating decreased the effect of opsonized zymosan. Uncoated zymosan was an even weaker stimulus of leukotriene formation. These results suggest that both complement factors and immunoglobulins play a pivotal role in activating leukotriene synthesis in a mixed suspension of human leukocytes.

Lysophosphatidylserine-dependent interaction between rat leukocytes and mast cells

The production of lysophosphatidylserine has been studied in a population of rat peritoneal cells; 67% polymorphonuclear and 33% mononuclear leukocytes. Pulse-chase experiments with l-[U- 14C]serine reveal a net lysophosphatidylserine production of 0.33 nmol/mg protein in 2 h of incubation. The source of lysophosphatidylserine is probably the phosphatidylserine of cells damaged during the incubation, since plasma membrane fragments obtained from the leukocytes yield higher lysophosphatidylserine production (1.9 nmol/mg protein in 1 h of incubation). Both leukocytes and plasma membranes show phosphatidylserine splitting activity when tested with vesicles of this phospholipid. In the presence of albumin a fraction of produced lysophosphatidylserine is recovered in the incubation medium. Under these conditions efficient incorporation of lysoderivative into surrounding leukocytes and conversion to phosphatidylserine requires cell activation by tetradecanoylphorbol acetate. In agreement with radiochemical data it is found that a suspension of leukocytes elicits histamine release when rat peritoneal mast cells and nerve growth factor are subsequently added. This typical, lysophosphatidylserine-dependent mast cell response is retained when leukocyte plasma membranes substitute the whole cells. These results suggest that leukocyte lysis at sites of tissue injury results in the production of a sufficient amount of lysophosphatidylserine to reach and activate surrounding mast cells.

Bacteria and their products peptidoglycan and teichoic acid potentiate antigen-induced histamine release in allergic patients

Histamine release was examined in leukocyte suspensions from patients allergic to grass pollen, mite or cat dander or to bacteria (antigen). When the cells were challenged with specific antigen plus bacteria to which the person was not sensitized, these bacteria were found to potentiate the allergic histamine release. The potentiating effect by bacteria might be due to the bacterial cell wall components, peptidoglycan and teichoic acid, which mimic the effect of bacteria.

Transformation of 15-hydroperoxyeicosapentaenoic acid to lipoxin A5 and B5, mono- and dihydroxyeicosapentaenoic acids by porcine leukocytes.

15-Hydroperoxy[1-14C]eicosapentaenoic acid derived from eicosapentaenoic acid (EPA) was incubated with suspensions of porcine leukocytes. Incubation with porcine leukocytes resulted in the formation of seven dihydroxy compounds, one monohydroxy and one hydroxyepoxy compound. After separation by reverse-phase and straight-phase HPLC, GC/MS analysis revealed that these metabolites were four isomers of 8,15-diHEPEs, two isomers of 14,15-diHEPEs, one isomer of 5,15-diHEPE, 15-HEPE and an epoxyalcohol: 13-hydroxy-14,15-epoxyeicosatetraenoic acid. In addition to the above metabolites, two trihydroxytetraene derivatives were also isolated. GC/MS and ultraviolet spectroscopy identified the two trihydroxypentaene derivatives as 5,6,15-trihydroxy-7,9,11,13,17-eicosapentaenoic acid (lipoxin A5) and 5,14,15-trihydroxy-6,8,10,12,17-eicosapentaenoic acid (lipoxin B5). This study demonstrated that the 15-hydroperoxide of EPA can be actively converted to various hydroxylated products via the 5-, 12- and 15-lipoxygenase as well as epoxyisomerase pathways in the porcine leukocytes.

全血中でのＰＧＩ２生成に関する検討

The factors affecting PGI2 synthesis following the platelet aggregation induced by collagen (1μg/ml) in whole blood were examined.Trapidil, possessing the anti-platelet aggregating activity and the enhancing effect of PGI2 synthesis in endothelial cells, markedly promoted PGI2 synthesis in whole blood, while aspirin showed no effect. The increment of PGI2 synthesis ran parallel to the concentration of trapidil, whereas TxA2 synthesis was decreased. OKY-046, the inhibitor of TxA2 synthetase, also increased PGI2 level and decreased TxA2 level in whole blood in the same manner of trapidil. The level of PGI2 in the suspension of leukocytes added trapidil (1mM), arachidonic acid (0.3M) and collagen (1μg/ml) was signficantly higher than that in the suspension added 1mM of aspirin in place of trapidil.These results suggest that trapidil has the inhibitory activity of TxA2 synthetase, resulting in the increment of PGI2 in whole blood. This synthesis of PGI2 was found in the leukocyte suspension in the presence of arachidonic acid.

Benzene-specific increase in leukocyte alkaline phosphatase activity in rats exposed to vapors of various organic solvents.

Female Wistar rats were exposed to various solvent vapors 8 h/d for 7 d. The leukocyte suspension and serum were prepared from peripheral blood and utilized for the determination of alkaline phosphatase (AP) activity with disodium phenyl phosphate as a substrate [leukocyte AP (LAP) and serum AP (SAP) assay]. While the exposure to benzene at 20 or 50 ppm did not cause significant changes in LAP activity, the exposure at 100 and 300 ppm resulted in a dose-dependent increase of LAP activity up to more than 100% over the control. No further increase was observed at 1000 or 3000 ppm. Similar exposure at 300 ppm to either toluene, m-xylene, n-hexane, trichloroethylene, methyl ethyl ketone, ethyl acetate, or methyl alcohol did not induce any changes in LAP activity. Thus, the increase in LAP activity was considered to be specific to benzene exposure. When the animals were exposed to toluene (300 ppm) in combination with benzene (300 ppm), not only was the benzene-induced leukopenia alleviated as previously reported, but the benzene-induced increase in LAP activity was no longer observed. The parallel inhibitory effects of toluene on benzene-induced increase in LAP and leukopenia suggest that a relation may exist between increase in LAP activity and leukopenia. No changes in SAP activities were observed in the rats under the exposure conditions examined.

Enhanced IgE-dependent basophil histamine release and airway reactivity in asthma.

IgE-dependent basophil histamine release does not necessarily correlate with the amount of cell-bound IgE, thus it has been suggested that basophil "releasability" is an important, but yet undefined, factor in this secretory process. Because mast cell, and possibly basophilic leukocyte, mediator release contributes to airway reactivity, any enhancement of this secretory process would favor asthma provocation. To evaluate IgE-dependent basophil histamine releasability in asthma, suspensions of leukocytes were isolated from patients with an allergic and nonallergic component to their airway disease and stimulated with concanavalin A (Con A) (0.03 to 10.0 micrograms/ml) and anti-IgE (10 to 1,000 ng/ml). Basophil histamine release to Con A and anti-IgE was significantly greater in both allergic and nonallergic asthmatic patients when compared with normal subjects. In contrast, basophil histamine release to the calcium ionophore A23187 was similar in leukocytes from normal subjects and asthmatic patients, suggesting the observed abnormality in secretion may be limited to an IgE-dependent process. To further determine if basophil histamine releasability in asthma correlated to measures of airway reactivity, bronchial provocation with histamine was performed. An inverse correlation was found between the provocative dose of inhaled histamine required to produce a 20% decrease, PD20, in the FEV1 and the leukocyte histamine release to Con A (p less than 0.05) and anti-IgE (p less than 0.05). Thus, we have new evidence that enhanced IgE-dependent release of leukocyte histamine correlates with airway reactivity in asthma. The mechanism of basophil releasability and its relationship to the pathogenesis of airway reactivity in asthma have yet to be established.