Archive for the ‘Pleural Fluid’ Category

Human Peripheral Blood and Pleural Fluid Eosinophils: Eosinophils as Effector Cells in Inflammatory and Immune Reactions

Human Peripheral Blood and Pleural Fluid Eosinophils: Eosinophils as Effector Cells in Inflammatory and Immune ReactionsEosinophils as Effector Cells in Inflammatory and Immune Reactions
The list of human disorders associated with an excess of eosinophils in the blood, tissues and secretions includes diseases characterized by IgE-mediated hypersensitivity, such as allergen-induced bronchial asthma, allergic rhinitis and allergic bronchopulmonary aspergillosis, infections with metazoan parasites, certain drug reactions, a number of connective tissue diseases as well as various solid tumors and lympho-proliferative and lymphoreticular disorders. Although it is universally accepted that eosinophils have a specialized role in host defense against parasites, just as neutrophils destroy and kill various bacteria, their functions in the inflammatory reactions associated with malignant or immune diseases are not completely understood. In this context, a wide variety of solid and hematologic cancers are occasionally accompanied by eosinophilia, but the mechanisms underlying this relationship remain obscure. Read the rest of this entry »

Human Peripheral Blood and Pleural Fluid Eosinophils: Discussion

Although eosinophils are common components of a variety of inflammatory and immune-mediated responses, which role they may play in many pulmonary and pleural diseases is unclear. However, since these diseases often are characterized by injury to parenchymal cells and since eosinophils, when activated, can secrete a variety of cytotoxic products, it is reasonable to hypothesize that eosinophils may be involved in processes leading to the derangement of the normal tissue components. The present study demonstrates the following: (a) Human peripheral blood eosinophils, like peripheral blood neutrophils, possess an intense oxidative metabolic burst which can be induced by soluble membrane activators, such as PMA. (b) In addition to PMA, physiologic stimuli such as IgG and, to a lesser extent, IgE immune complexes, can activate human eosinophils to release considerable amounts of H202. Read the rest of this entry »

Human Peripheral Blood and Pleural Fluid Eosinophils: Ability of Catalase to Inhibit Eosinophil and Neutrophil Cytotoxic Activity

Human Peripheral Blood and Pleural Fluid Eosinophils: Ability of Catalase to Inhibit Eosinophil and Neutrophil Cytotoxic ActivityAbility of Catalase to Inhibit Eosinophil and Neutrophil Cytotoxic Activity
To demonstrate that the cytotoxic damage induced on Cr-labeled Raji cells by stimulated eosinophils and neutrophils was mostly related to the generation of H202, various numbers of peripheral blood or pleural fluid leukocytes were cocultured with the target cells in the presence of PMA, IgG IC and Agg IgE and with or without the H202 specific scavenger. When peripheral blood eosinophils were stimulated with PMA, a cytotoxicity index of 59 ± 9 percent at an effector-to-target ratio of 30:1 was generated while the addition of catalase virtually completely inhibited PMA-induced cytotoxicity (11 ±3 percent; p<0.001) (Fig 4,A). Read the rest of this entry »

Human Peripheral Blood and Pleural Fluid Eosinophils: Cytotoxic Activity of Peripheral Blood Eosinophils and Neutrophils from Control Subjects

Cytotoxic Activity of Peripheral Blood Eosinophils and Neutrophils from Control Subjects
Peripheral blood eosinophils and neutrophils from control subjects exhibited a very low spontaneous cellular cytotoxicity for Cr-labeled Raji cells after 4 h incubation (Fig 2). In contrast, stimulation of the cells with PMA induced a detectable eosinophil- and neutrophil-mediated killing which increased directly, for both eosinophils and neutrophils, with increasing effector-to-target cell ratio. Significant cytotoxicity was present for eosinophils at an effector-to-target ratio of 4:1 and for neutrophils at an effector-to-target ratio of 7.5:1. The cytotoxic activity of PMA-stimulated eosinophils was higher than the cytotoxic activity of PMA-stimulated neutrophils at an effector-to-target cell ratio of 15:1 (p<0.05) and of 30:1 and 60:1 (p<0.01). Read the rest of this entry »

Human Peripheral Blood and Pleural Fluid Eosinophils: Results

Human Peripheral Blood and Pleural Fluid Eosinophils: ResultsAbility of Peripheral Blood Eosinophils and Neutrophils from Control Subjects to Generate H2O2
The production of H202 by human peripheral blood eosinophils or neutrophils was studied under both resting and stimulated conditions. Although neither peripheral blood eosinophils nor neutrophils produced spontaneously detectable amounts of H202, the addition of PMA to the culture media was able to generate an intense respiratory burst which was greater for eosinophils than for neutrophils (eosinophils: 7.5±0.8 nmol H202; neutrophils: 4.2 ± 0.5 nmol H202; p<0.05) (Fig 1). Similar results were obtained when cells were stimulated with IgG IC, which induced a more intense H202 generation by eosinophils than by neutrophils (eosinophils: 4.1 ±0.6 nmol H202; neutrophils: 1.1 ±0.3 nmol H202; p<0.01), although the levels of H202 measured were lower when compared with the levels observed after PMA stimulation, both for eosinophils and for neutrophils (p<0.05, each comparison). Interestingly, the presence of Agg IgE was able to induce a detectable H202 release only by eosinophils and not by neutrophils (eosinophils: 0.9 ±0.3 nmol H202; neutrophils: undetectable amounts of although the respiratory burst induced in eosinophils by Agg IgE was lower than that observed after activation with PMA or IgG IC (p<0.001, each comparison). No detectable H202 production was observed when peripheral blood eosinophils or neutrophils were incubated with IgG alone or IgE alone (data not shown). !–more–>alta white teeth whiteningAbility of Pleural Fluid and Peripheral Blood Eosinophils from Patients with Eosinophilic Malignant Pleural Effusions to Generate H202
As observed for circulating eosinophils from control subjects, neither pleural fluid eosinophils nor peripheral blood eosinophils from patients with eosinophilic malignant pleural effusions spontaneously released H202 (Table 1). All stimuli used in this study were able to induce a detectable respiratory burst in pleural fluid and peripheral blood eosinophils isolated from cancer patients, but the generation of H202 was higher for peripheral blood eosinophils than for pleural fluid eosinophils either with PMA (peripheral blood eosinophils: 7.7 ±0.9 nmol H202; pleural fluid eosinophils: 4.9 ±0.7 nmol H202; p<0.05) or with IgG IC (peripheral blood eosinophils: 4.6 ±0.6 nmol H2Oz; pleural fluid eosinophils: 2.4±0.2 nmol H202; p<0.05), or with Agg IgE (peripheral blood eosinophils: 2.1 ±0.2 nmol H202; pleural fluid eosinophils: 0.4 ±0.1 nmol H202; p<0.01). As described for peripheral blood eosinophils from control subjects, H202 release was higher after stimulation with PMA than after stimulation with IgG IC or Agg IgE both for peripheral blood and for pleural fluid eosinophils (p<0.05, each comparison) and more elevated after IgG IC stimulation than after Agg IgE stimulation (p<0.05, each comparison). In addition, in the experimental conditions tested in this study, no significant difference in the H202 release was observed between peripheral blood eosinophils from normal control subjects and peripheral blood eosinophils from patients with malignant pleural effusions (p>0.2, all comparisons).

Figure 1. Hydrogen peroxide production by human peripheral blood eosinophils (open bars) and neutrophils (solid bars) from normal subjects in resting conditions (RESTING) and after stimulation with phorbol myristate acetate (PMAX IgG immune complexes (ZgG IC) or human heat-aggregated myeloma (Agg IgE). Hydrogen peroxide was quantified after 30 minutes’ incubation by recording the oxidation of phenol red in the presence of horseradish peroxidase. The data represent the mean ± SEM of six experiments.

Figure 1. Hydrogen peroxide production by human peripheral blood eosinophils (open bars) and neutrophils (solid bars) from normal subjects in resting conditions (RESTING) and after stimulation with phorbol myristate acetate (PMAX IgG immune complexes (ZgG IC) or human heat-aggregated myeloma (Agg IgE). Hydrogen peroxide was quantified after 30 minutes’ incubation by recording the oxidation of phenol red in the presence of horseradish peroxidase. The data represent the mean ± SEM of six experiments.

Table 1—H2O2 Release by Peripheral Blood E osinophils from Control Subjects and from Peripheral Blood and Pleural Fluid Eosinophils from Patients with Eosinophilic Malignant Pleural Effusions

StudyPopulations Source of Eosinophils Spontaneous H202 Release (nmol/min/106 Cells) H202 Release after Eosinophil Activation
PMA IgG IC (nmol/min/108 Cells) Agg IgE
Controlsubjects Peripheralblood Not detectable 7.5±0.8 4.1 ±0.6 0.9±0.3
Patientswith

eosinophilic

malignant

pleural

effusions

Peripheralblood Not detectable 7.7±0.9 4.6±0.6 2.1 ±0.2
Pleuralfluid Not detectable 4.9±0.7 2.4 ±0.2 0.4 ±0.1

Human Peripheral Blood and Pleural Fluid Eosinophils: Analysis of Data

Spontaneous and PMA or IgG IC or Agg IgE-lnduced H202 Production by Peripheral Blood and Pleural Fluid Eosinophils and by Peripheral Blood Neutrophils
Peripheral blood and pleural fluid eosinophils and peripheral blood neutrophils (the number of pleural fluid neutrophils was too low to perform the functional studies) were resuspended in HBSS at a final concentration of 1 x 10® cells/ml. Leukocytes (5 x 10) were added to 15 x 75-mm plastic test tubes containing 0.28 mM phenol red, 8.5 U/ml horseradish peroxidase (type 2, Sigma Chemical Co, St Louis, MO), superoxide dismutase, 5 p-g/ml (Sigma Chemical Co), as previously described. When indicated, phorbol myristate acetate (PMA), 75 ng/ml (PMA, Sigma Chemical Co), or various amounts of IgG alone or IgE alone or of IgG IC or of Agg IgE (50, 100, 200 and 300 fiL) were added to the test tubes containing neutrophils or eosinophils. Read the rest of this entry »

Human Peripheral Blood and Pleural Fluid Eosinophils: Isolation of Peripheral Blood and Pleural Fluid Granulocytes

Human Peripheral Blood and Pleural Fluid Eosinophils: Isolation of Peripheral Blood and Pleural Fluid GranulocytesIsolation of Peripheral Blood and Pleural Fluid Granulocytes
The blood and pleural fluid samples were diluted with an equal volume of 4.5 percent dextran T-500 (Pharmacia Fine Chemicals, Piscataway, N.J.) and allowed to sediment at 37°C for 40 min. The leukocyte-rich suspensions were removed, centrifuged three times at 200 g to remove platelets and the cells were then pelleted at 500 g for 15 min. The leukocytes were then washed in Hanks’ balanced salt solution (IiBSS) and layered on a 12-ml discontinuous metri-zamide gradient (Nyegaard and Co, Oslo, Norway), 20 to 25 percent weight per volume ratio (w + /vol) in HBSS containing 0.1 percent (w/v) gelatin and 15 p.g*ml_1 DNase I (DN-CL, Sigma Chemical Co), pH 6.8, by centrifugation at 400 g for 45 min, according to the technique described by Vadas et al modified by Oliver et al. Read the rest of this entry »

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