Purification of human blood basophils by single step isopycnic banding on Percoll

Human venous blood, anticoagulated with EDTA, was layered onto a discontinuous Percoll gradient, made from solutions of density 1.088, 1.079, and 1.070 gm/ml. After centrifugation at 700g for 15 min at 22 degrees C, the majority of the blood basophils was found in a narrow band at the density 1.070-1.079 interface (Percoll band 2). For 15 normal donors, mean total basophil number recovered from all locations in the gradient was 3.8 +/- 1.2 (SD) X 10(4) basophils per ml of blood applied. Thus, 95% of the values ranged from 1.5 to 6 X 10(4), which compares favorably with the reported range of 1 to 8 X 10(4) basophils per ml for normal subjects. In the basophil-rich Percoll band 2, 2.8 +/- 0.8 X 10(4) basophils were recovered per ml of blood applied. The mean percentage of basophils in Percoll band 2 was 19%, with a range of 5 to 53%. Monocytes and neutrophils were present in very small numbers; the majority of accompanying cells were small lymphocytes.     

The Requirements for Transferrin-Dependent Adherence of Human Granulocytes to Pollen Grains

Human granulocyte/pollen binding protein (GPBP), previously identified as serum transferrin, promoted prolonged firm adherence of neutrophils to Timothy grass pollen. Some characteristics of this adherence reaction are reported. GPBP-induced binding was time-, temperature- and concentration-dependent. Maximal adherence was observed by 2 h and was only slightly decreased at 18 h. The optimal temperature for adherence was 37 degrees C. Concentrations of GPBP as low as 1.25 microgram/ml gave significantly greater binding than the albumin or lactoferrin control. Eosinophils, monocytes and lymphocytes did not appear to participate in GPBP-induced pollen binding reactions at concentrations up to 300 micrograms/ml. In the presence of GPBP, neutrophils adhered to a range of grass, weed and tree pollens. These included timothy, meadow, false oat, rye, giant and short ragweed, plantain, silver birch and ash. GPBP did not facilitate the adherence of granulocytes to inert particles of similar size such as Sephadex beads and agarose. The adherence was Mg++- but not Ca++-dependent and was not inhibited by a monoclonal antibody to the transferrin receptor (OKT9). Transferrin/GPBP did not bind to either neutrophils or pollen grains. A purified commercial transferrin reacted in all respects like GPBP in these pollen binding studies. These observations indicate that GPBP/transferrin-induced adherence of granulocytes to pollen grains is a hitherto unrecognized property of transferrin which appears unrelated to iron transport or the conventional transferrin receptor.     

Neutrophil erythrotoxicity induced by phorbol myristate acetate: mechanisms involved in neutrophil activation

The capacity of phorbol myristate acetate (PMA) to prime neutrophil cytotoxic responses induced by a second stimulus was investigated. Treatment of neutrophils with low concentrations of PMA (0.2-0.5 ng/ml) for 18 hr at 37 degrees C markedly enhanced cytotoxicity triggered by Ca2+ ionophore A23187, N-formyl-methionyl-leucyl-phenylalanine (FMLP) and PMA. Pretreatment with PMA also enabled neutrophils to mediate significant cytotoxicity when triggered by platelet-activating factor (PAF), a stimulus unable to induce untreated cells to display cytotoxicity. Conversely, neutrophil cytotoxicity triggered by immune complexes (IC) was not modified by PMA treatment, whereas cytolytic activity of neutrophils against antibody-sensitized target cells was significantly increased. Treatment with PMA concentrations higher than 1 ng/ml directly triggered neutrophil cytotoxicity. Interestingly, we found that PMA-triggered neutrophils were able to sustain maximal levels of cytotoxicity for at least 8 hr after stimulation. With regard to the mechanisms involved in neutrophil activation by PMA, we found that catalase but not superoxide dismutase (SOD) prevented neutrophil activation measured as 1) induction of cytotoxic responses, 2) increase of neutrophil adhesiveness to cell-free surfaces, and 3) inhibition of chemotactic responses to FMLP. These findings suggest that H2O2 may play a major role in neutrophil activation induced by PMA.     

Anti-immunoglobulin induced histamine release from naturally abundant basophils in the snapping turtle, Chelydra serpentina

The dose response, temperature sensitivity, time course and calcium dependency of histamine release from snapping turtle basophils treated with rabbit anti-turtle immunoglobulin (RATIg) sera was explored. This investigation indicated that the level of histamine release induced by RATIg was dependent upon the concentration of RATIg with concentrations of 350 mcg/ml inducing optimal release. In addition, release was temperature dependent with release increasing over a temperature range of 10 degrees C to maximal at 27 degrees C. Release was also dependent on the length of exposure to RATIg. Release increased steadily over a time period of 0 to 30 minutes with the higher concentrations of RATIg inducing the most rapid release. Basophil-histamine release was also found to be calcium dependent. This study indicates that the snapping turtle basophil possesses similar characteristics to that of its mammalian counterparts. It is proposed that the very wide temperature range over which turtle basophils release histamine is an important feature in he immune resistance of this ectothermic animal.     

Selective depletion of rat neutrophils by in vivo administration of a monoclonal antibody

A monoclonal antibody (RP-3) that depletes rat neutrophils selectively in vivo was developed by hybridization of mouse myeloma cells (P3-X63.Ag8.653) and spleen cells of BALB/c mice sensitized with peritoneal neutrophils of WKA/Hok rats. RP-3 reacted with rat neutrophils but not with lymphocytes, macrophages, natural killer cells, basophils, eosinophils, or tissues of various organs. The mitogenic responsiveness to concanavalin A (ConA), phytohemagglutinin (PHA), and lipopolysaccharide (LPS) of rats given RP-3 was not significantly different from that of normal rats. Administration of RP-3 into the peritoneal cavity of rats that had been kept under specific pathogen-free (SPF) or clean conditions induced selective depletion of circulating neutrophils to under 100/mm3 (0.5% WBC). The numbers of monocytes, lymphocytes, and platelets were not changed. Administration of 2 ml of RP-3 reduced blood neutrophils to under 100/mm3 for approximately 24 h, and administration of 1 ml caused depletion for approximately 12 h.     

Functional differences of human basophils with different densities

It has been reported that blood basophils comprise two populations of different densities. In this study, we compared the responses of two populations of basophils (band-1 and 2 basophils) to several secretagogues and anti-allergic agents. Twelve patients with bronchial asthma, 3 patients with Japanese cedar pollinosis and 6 healthy subjects were included in this study. Band-2 basophils collected from the interface between Percoll of density 1.078 and 1.068 g/ml released histamine better in response to anti-IgE than band-1 basophils taken from the interface between plasma and Percoll 1.068 (band 1 vs. band 2, 13.2 +/- 2.7 vs. 21.4 +/- 3.6% at 1:10(5), p less than 0.01; 47.1 +/- 5.0 vs. 57.7 +/- 6.1% at 1:10(4), p less than 0.01). Band-2 basophils also responded better to formyl-methionyl-leucyl-phenylalanine than band-1 basophils (34.4 +/- 4.9 vs. 43.2 +/- 5.6% at 10(-6) M, p less than 0.01; 35.6 +/- 5.0 vs. 45.2 +/- 5.7% at 10(-5) M, p less than 0.01). In contrast, band-1 basophils were much more sensitive to the calcium ionophore A23187 stimulation than band-2 basophils (13.0 +/- 3.7 vs. 6.3 +/- 1.8% at 0.05 microgram/ml, p less than 0.05; 52.5 +/- 4.3 vs. 33.8 +/- 3.9% at 0.1 microgram/ml, p less than 0.01; 71.3 +/- 3.3 vs. 51.5 +/- 4.2% at 0.2 microgram/ml, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Venom skin tests in insect-allergic and insect-nonallergic populations

Intradermal skin tests with varying concentrations of honeybee, yellow jacket, white-faced hornet, yellow hornet, and Polistes venoms were done on 85 patients with histories of insect-sting anaphylaxis and on 56 insect-nonallergic subjects. Positive skin tests (wheal greater than or equal to 5 to 10 mm and flare greater than or equal to 11 to 20 mm) were present in 67 insect-allergic patients at venom concentrations ranging from 0.001 microgram/ml to 0.1 microgram/ml. Seven additional allergic patients had positive skin tests with the 1.0 microgram/ml venom concentration. Twenty-six nonallergic subjects had positive skin tests at the venom concentration of 1.0 microgram/ml, and two patients had positive skin tests at the lower venom concentrations (0.001 to 0.1 microgram/ml). These results confirm venom skin tests as a highly sensitive method of detecting venom-specific IgE in the evaluation of patients with stinging-insect hypersensitivity. Since a large percentage of insect-nonallergic subjects reacted to the 1.0 microgram/ml concentration, clinical judgment and further in vitro testing should be considered in the evaluation of patients who react only at this venom concentration.     

Effect of Cyclosporin A on the Generation of Cytotoxic T Lymphocytes in Mouse Mixed Lymphocyte Culture

Cyclosporin A inhibited at equal concentrations both the proliferative response and the generation of cytotoxic T lymphocytes in one-way mouse spleen cell mixed lymphocyte culture. The 50% inhibitory concentration was in all experiments 10(-2) to 10(-1) microgram/ml. The inhibition was directly proportional to how early the drug was added to the culture: a complete inhibition of both responses was obtained if the drug was added on day 3, and a partial inhibition if added on day 4 of culture. Cytological analysis of the cultured cells demonstrated that resting lymphocytes were not damaged at 100-1000-fold concentrations of the drug giving complete inhibition of the blastogenic response. The results suggest that cyclosporin A is most effective if present throughout the induction phase of the immune response.     

Inhibitory effect of cyclosporin A on peripheral blood and bone marrow T lymphocyte colony formation

The effect of cyclosporin A on the formation of T lymphocyte colonies from human peripheral blood and bone marrow was tested using a double-layer technique. A moderate inhibition (27%) was observed on peripheral blood lymphocytes with concentrations of 0.1 microgram/ml of the drug; this increased to 57% with 1 microgram/ml and to almost 70% with 10 microgram/ml. Bone marrow cells were less sensitive to cyclosporin A. This was more evident at the lowest concentration of the drug (0.1 microgram/ml), with which a 14% inhibition was found. Higher concentrations (1--10 microgram/ml) produced 37 and 56% inhibition respectively. Overnight incubation with the drug followed by repeated washing of the cells did not influence colony growth. E-rosette formation was also not affected by cyclosporin A. The apparent greater sensitivity to the drug of peripheral blood than bone marrow T lymphocytes, possibly related to a different distribution of T colony-forming cells or of T lymphocyte subsets, may have some bearing on the clinical application of cyclosporin A in the prevention and treatment of graft-versus-host disease in man.     

Evidence for the role of superoxide radicals in neutrophil-mediated cytotoxicity.

Human peripheral neutrophils became cytotoxic to chicken red blood cells (CRBC) in the presence of lectins as assessed by release of 51chromium from labelled target cells. Phytohaemagglutinin (PHA) and concanavalin A (Con A), which caused time-dependent and dose-dependent cytotoxicity over a concentration range of 25--400 microgram/ml, also caused significant generation of superoxide radicals as measured by ferricytochrome C reduction. Pokeweed mitogen, which does not induce cytotoxicity over the same concentration range, was unable to promote superoxide generation by neutrophils. PHA-induced generation of superoxide paralleled and appeared to precede PHA-dependent cytotoxicity. Superoxide dismutase (SOD), which enzymatically destroys superoxide, caused moderate inhibition of PHA-dependent cytotoxicity over the concentration range of 100--500 microgram/ml whereas catalytically inactive enzyme had no effect. Incubation under oxygen-depleted conditions caused a marked decrease in both PHA-induced superoxide generation and cytotoxicity relative to that obtained with neutrophils incubated aerobically. These findings suggest a central role for superoxide radicals in causing target cell damage in this model of neutrophil-mediated cytotoxicity.     

Immunoglobulin E-mediated asthma and hypersensitivity pneumonitis with precipitating anti-hapten antibodies due to diphenylmethane diisocyanate (MDI) exposure

Two workers are presented who were exposed to diphenylmethane diisocyanate (MDI) while coating pipes with a polyurethane foam. After a latent period of exposure, worker A developed immediate-onset asthma and worker B developed a clinical picture of hypersensitivity pneumonitis for which he was hospitalized. The antibody response of these workers to a conjugate of MDI with human serum albumin (MDI-HSA) was measured by gel precipitation, total antibody binding of 125I MDI-HSA, and specific IgG and IgE antibody by polystyrene-tube radioimmunoassay (PTRAI). Worker B had precipitating antibody to MDI-HSA by double immunodiffusion in gel. Both workers had high levels of IgG antibody specific for MDI-HSA which had some cross-reactivity with a conjugate or toluene diisocyanate and HSA. Total serum antibody binding of 125I MDI-HSA was 15 microgram/ml in worker A and 900 microgram/ml in worker B. Both workers had serum IgE antibody specific for MDI-HSA as measured by two PTRIA techniques. These results indicate that a marked immunologic response to MDI is possible in exposed workers and that hypersensitivity pneumonitis can occur subsequent to the inhalation of a low-molecular-weight chemical in the industrail setting.     

Evaluation of Fractional Analysis of Bronchoalveolar Lavage Combined with Cellular Morphological Features

Background. The value of bronchoalveolar lavage (BAL) still remains controversial, prompting a need for further improvement. The purpose of this study was to develop and evaluate a sequential analysis of cell content in fractional BAL (FBAL) from the airways and alveolar sacs with incorporation of the cellular morphologic features. Methods. Initially, 30 ml saline was infused into a subsegmental lobe of the lung and the recovered fluid was assigned as FBAL-I being mainly originated from whole airways. The second and third lavages (FBAL-II and FBAL-III) each were performed using 50 ml saline being from more distal portions of airways and alveolar sacs respectively in the same lobe. Total cell number/ml and percentages of macrophages, lymphocytes, neutrophils, and eosinophils in each fraction together with their morphological alterations and mast cells, basophils and Masson bodies were assessed. Results. In the 12 controls, percentage of neutrophils was high and lymphocytes and macrophages were low in FBAL-I while in FBAL-III, neutrophils decreased to nearly nil and lymphocytes and macrophages were increased. Analysis of FBAL from 76 patients with sarcoidosis and 14 with hypersensitivity pneumonitis (HP) revealed that a predominance of small, round and well-differentiated lymphocytes with relative absence of neutrophils, basophils and Masson bodies correlated best with sarcoidosis. In contrast, neutrophil predominance and presence of lymphocytes having deep nuclear indentations and abundant cytoplasm with a process resembling a “hand-mirror” correlated well with HP. Conclusions. Evaluation of FBAL together with cellular morphological features especially characteristics of lymphocytes provides valuable information for establishing the diagnosis in interstitial lung diseases.     

Quantification of antibodies to the C3d subcomponent of human C3.

Purified soluble C3d has been employed to measure the concentration of anti-C3d antibodies in immune rabbit sera. Multiple batches of C3d, prepared from C3-C3b substrate by treatment with C3b-inactivator (KAF), after labelling with 125I, retained 80% immunoreactivity, and were stable on storage at -50 degrees and +4 degrees. Concentrations of anti-C3d were determined by Scatchard analysis of equilibrium concentrations of bound and free C3d in a mixture of 125I-labelled C3d and anti-C3d. Separation of bound from free C3d was by G-75 Sephadex filtration. Assuming a 1:1 molar ratio in the antibody-C3d complex, anti-C3d antibody concentrations for four rabbit whole antisera and four IgG preparations fell in the range 288-2433 microgram/ml, with Ko values of 6-2 X 10(8)-2-9 X 10(9) litres/mol. One commercial antiglobulin-serum contained 3-6 microgram anti C3d/ml and had a Ko value of 1-7 X 10(8) litres/mol. Values for anti-C3d concentrations measured independently by an indirect method employing 125I-labelled sheep anti-rabbit IgG averaged 20% lower than those obtained with 125I-labelled C3d. Antibody concentrations were correlated with antiglobulin agglutination titres against C3d-coated red cells; a titre of 1 was given by an anti-C3d concentration of 0-5 microgram/ml.     

Effect of misoprostol on the secretion of histamine from basophils of whole blood.

BACKGROUND: Misoprostol (MSP), the synthetic prostaglandin E1 (PGE1) analog, possesses multifunctional features, including modulating some inflammatory aspects of immune and allergic disorders. OBJECTIVES: To investigate the effect of MSP on histamine release (HR) from basophils of whole blood using anti-IgE, specific allergens, and calcium ionophore. METHODS: The study was performed using the automated glass fiber-based whole blood leukocyte histamine release test (LHRT). RESULTS: Very low concentrations of MSP produced a marked inhibition of HR induced with anti-IgE. Maximum inhibition was observed at 10-9 M. It was also shown that the levels of HR inhibition with MSP varied at different incubation times. The greatest inhibition of HR was noted at 1 to 2 hours of incubation at MSP concentrations of 10-8 and 10-9 M, respectively. Incubation of blood from allergic patients at the optimal MSP concentration and optimal elapsed time (2 hours) resulted in significant reductions of allergen-specific HR induced by both Timothy pollen grass allergen and D.pteronissinus. Incubation of blood with varying concentrations of MSP and subsequent stimulation with calcium ionophore A23187 also inhibited HR from basophils. In the latter case, the most effective concentrations of MSP ranged from 10-8 to 10-6 M. CONCLUSIONS: This study demonstrated that MSP can inhibit basophil HR indicating a potentially beneficial role of PGE1 analogs as pharmacotherapy for allergic diseases.     

Antigen-induced histamine release from guinea pig basophils

Guinea pig blood was found to contain an average of 106 +/- 21 ng/ml of histamine. Of this total, approximately 85-90% was of platelet origin and the rest from basophils. Basophils contain about 0.72 pg of histamine/cell. Concanavalin A (1-5 microgram/ml) induced the release of approximately 65% of the basophilic histamine. When basophils were isolated from animals sensitized to ovalbumin or keyhole limpet hemocyanin, addition of the appropriate antigen induced histamine release at concentrations of 0.01 microgram/ml or lower. Individual animals were studied over time by repetitive bleeding. The circulating basophils remained sensitized for at least 17 weeks postsensitization. However, release did not occur if animals had been sensitized less than 7 days earlier. This assay facilitates the investigation of basophil sensitization since animals can be studied on several occasions following immunization. The mechanisms, timing and role of basophil sensitization in various types of immune and hypersensitivity reactions can now be evaluated.     

Comparative study of eosinophil and neutrophil chemotaxis and enzyme release.

It has been well documented that both natural and synthetic chemotactic peptides can induce lysosomal enzyme release from neutrophils treated with cytochalasin B. These same peptides are also potent inducers of unidirectional movement, as demonstrated by the chemotactic response in Boyden chambers. In this study, the ability of another family of leukocytes, eosinophils, to release lysosomal enzymes and exhibit a chemotactic response to both natural and synthetic chemotactic peptides was examined. A striking fundamental difference between neutrophil and eosinophil chemotaxis and enzyme release was shown using C5a, formyl met-leu-phe (FMLP), and ala-gly-ser-glu (AGSG) peptides. The 50% effective doses (ED50) for chemotactic responses to C5a, FMLP, or AGSG by neutrophils and eosinophils were 0.05 microgram/ml and 1.0 microgram/ml, 10(-12) M and 10(-10) M, and 10(-7) M and 10(-7) M, respectively. At the same concentrations, these peptides (C5a, f met-leu-phe, and ala-gly-ser-glu) induced the following release of glucosaminidase from neutrophils and eosinophils, respectively: 42% and 2%, 42% and 2%, and 29% and 2%. In striking contrast, immune complexes and opsonized zymosan particles induced the release of 39% and 42% of the total glucosaminidase from neutrophils, while eosinophils released 32% and 43% of the total glucosaminidase from immune complexes and opsonized zymosan particles, respectively. These data indicate fundamental differences between neutrophils and eosinophils in unidirectional movement induced by chemotactic factors and enzyme release mechanism(s).     

Mast cell histamine-releasing activity from stimulated rat neutrophils

Mast cell histamine-releasing activity (HRA) has been observed in medium derived from stimulated rat neutrophils pretreated with cytochalasin B. This HRA did not require cell-cell contact between mast cells and neutrophils, and its concentration was increased by increasing the number of neutrophils or by raising the concentration of chemotactic peptide used to stimulate the neutrophils. The HRA survived boiling for 5 min and storage overnight at -20 degrees C. In the absence of neutrophil stimulation, no HRA was observed.     

Heart rate and oxygen uptake response to angiotensin in the squirrel monkey at 10 degrees C.

Unanesthetized squirrel monkeys exposed to an ambient temperature of 10 degrees C showed elevations in total body oxygen consumption (VO2), systemic arterial blood pressure (BP), and heart rate (hr) above values recorded at 28 degrees C. Further elevation of BP in the cold by intravenous infusion of phenylephrine (5-50 microgram/kg-min) was accompanied by reduction in both VO2 and HR, and the changes in VO2 were proportional to those in HR. When BP was raised by intravenous infusion of angiotensin (0.05-1.0 microgram/kg-min), large elevations in BP were again accompanied by reductions in HR and VO2. However, for equivalent elevations in BP, the depressions in both HR and VO2 were much smaller with angiotensin than they were with phenylephrine. Previous studies in this laboratory have demonstrated that in response to experimental elevation of BP, reflexes originating at the sinoaortic baroreceptors depress not only HR but also VO2. The present results suggest that angiotensin modulates baro-reflexive responses to elevation in BP. The reductions in HR and VO2 that ordinarily occur in response to baroreceptor stimulation may be modified by an action of angiotensin on the central nervous system.     

The release of lysosomal enzymes from human polymorphonuclear leukocytes by human C3e

C3e is a leukocytosis-inducing peptide of degraded C3 that has the electrophoretic behavior of prealbumin and a MW of 10-12,000. Using a homogeneous preparation, the ability of C3e to promote lysosomal enzyme release from human polymorphonuclear leukocytes (PMNs) was examined by incubation of various concentrations of C3e with cytochalasin B-treated (5 micrograms/ml) human PMNs for 60 min at 37 degrees C. Amounts of extracellular beta-glucuronidase, myeloperoxidase, and lysozyme were determined in the cell-free supernatants and it was found that all three enzymes were released in significant amounts without concomitant release of the cytoplasmic enzyme lactate dehydrogenase (LDH). At a concentration of 25 micrograms/ml of C3e, 25 +/- 1.1% (SD) of lysozyme, 20 +/- 0.9% beta-glucuronidase, and 24.3 +/- 0.9% myeloperoxidase were released into the supernatant while the release of LDH remained within the 4-7% range throughout these studies. Furthermore the supernatants were found to contain a substance which was capable of a generating chemotactic fragment from isolated C5. The same range of C3e concentrations (5-25 micrograms/ml) was, however, incapable of effecting histamine release from basophils. These results suggest that generation of C3e in vivo may serve as a potent stimulus for the generation of the C5-cleaving enzyme from PMNs which in turn may function to recruit more neutrophils by a positive feedback mechanism.     

Characteristics of Pseudomonas aeruginosa in relation to laboratory-induced resistance to gentamicin.

Pseudomonas aeruginosa strain C2 was habituated to gentamicin by serial passage in broth containing increasing concentrations of the antibiotic and up to 250 microgram/ml. The resistant progenies differed from the parent strain in antibiotic susceptibility to two other aminoglycosides, colonial morphology, lytic phage patterns, phage adsorption, and agglutination with the seven Fisher's antisera. All the progenies failed to grow at 42 degrees C and oxidised glucose in O/F tubes after incubation at 37 degrees C for three days but were catalase- and oxidase-positive. Reversion to the original properties of the parent strain was demonstrated in all cases after 10 serial subcultures in antibiotic-free broth.