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Biomedical subjects

R Moqbel

Publications and source records attributed to R Moqbel.

At least 91 records · Page 5Linked to original sources
PubMed1990

IgG-dependent generation of platelet-activating factor by normal and low density human eosinophils.

We have compared normal and low density human eosinophils for their ability to generate platelet activating factor (PAF) in response to IgG-dependent and nonimmunologic stimulation. After 45 min incubation with IgG-coated Sepharose beads the concentrations of cell-associated PAF recovered from normal density eosinophils were significantly greater than from low-density eosinophils or neutrophils. Moreover, eosinophils stimulated with calcium ionophore A23187 had a considerably greater capacity to generate PAF than had previously been described. Although the quantities of cell-associated PAF recovered from normal and low density eosinophils and neutrophils after A23187 stimulation were similar, the amounts of extracellular PAF recovered from both eosinophil populations were significantly greater than from neutrophils. The amounts of PAF recovered from the low density eosinophils may not reflect the full synthetic capacity of these cells, because PAF-turnover was found to be more rapid than that observed with normal density eosinophils. When exogenous [3H]PAF was added to the two stimulated eosinophil populations subsequent analysis of the [3H]PAF metabolites by DIOL-HPLC revealed that low density eosinophils incorporated PAF into the phosphatidylcholine (PC) pool more rapidly than did normal density eosinophils or neutrophils. Alkaline hydrolysis of the PC fraction from whole cell extracts followed by treatment with acetic anhydride resulted in all the PC-associated radioactivity being converted to [3H]PAF, confirming PAF incorporation to PC via this pathway. These findings suggest that the contribution of eosinophils to inflammatory processes through the generation of PAF may be greater than previously appreciated, and that Ig-mediated stimulation may be important in initiating generation of the mediator. Low density eosinophils, that are presumed to be similar to tissue eosinophils, may have a role in regulating PAF concentrations in tissues through their enhanced rate of metabolism.

Calcimycin↗
PubMed1990

Receptor expression and functional status of cultured human eosinophils derived from umbilical cord blood mononuclear cells.

Selective use of recombinant human cytokines has enabled the culture of large numbers of eosinophils from human cord blood mononuclear cells, raising the possibility of their use as a model of eosinophil function. Cultured eosinophils (CE) were compared with normal-density peripheral blood eosinophils (PBE) in terms of their membrane receptor expression and function. Fc gamma R and CR1 expression of CE and PBE was similar. In contrast, the specific mean fluorescence for LFA-1 alpha, p150,95 alpha, ICAM-1, and HLA-DR was significantly elevated for CE compared with PBE. CE responded in PAF-induced chemotaxis in a similar fashion to PBE. CE gave higher numbers of both resting and platelet activating factor (PAF)-stimulated immunoglobulin G (IgG)- and C3b-dependent rosettes than PBE. CE and PBE had comparable capacity to kill IgG- and C-opsonized schistosomula in terms of both baseline values and PAF-induced enhancement of cytotoxicity. Baseline adherence by CE and PBE to plasma-coated glass was essentially the same, but stimulated adhesion (PAF) of CE was lower. Compared with PBE, CE generated less than half the amounts of extracellular and cell-associated PAF induced by calcium ionophore A23187 stimulation. Unlike PBE, CE did not generate PAF after exposure to IgG-coated Sepharose particles. CE stimulated with IgG-coated beads generated small quantities of LTC4, while A23187 stimulation resulted in approximately half the LTC4 levels observed with PBE. The total cell content of eosinophil peroxidase (EPO) was similar for CE and PBE. These data suggest that although CE and PBE have many phenotypic and functional properties in common there are quantitative differences that may be a consequence of their immaturity and/or the influence of the cytokines used in their culture.

Animals↗
PubMed1990

Effects of inhaled PAF in humans on circulating and bronchoalveolar lavage fluid neutrophils. Relationship to bronchoconstriction and changes in airway responsiveness.

We have compared the effect of inhaled platelet activating factor (PAF) on circulating neutrophils with its ability to induce bronchoconstriction and bronchial hyperresponsiveness in humans. Human volunteers inhaled PAF, given as six successive inhalations 15 min apart, followed by bronchoalveolar lavage (BAL) 4 h later. The mean density and volume of circulating neutrophils were measured by metrizamide gradients and flow cytometry, respectively. PAF caused a decrease in Vp20 of 38.2 +/- 4.5% at 5 min after the first inhalation (p less than 0.001). This was associated with a fall in the peripheral blood neutrophil count from 3.15 +/- 0.3 to 1.1 +/- 0.3 x 10(6) per ml (p less than 0.001), followed by a rebound neutrophilia (p less than 0.01). The mean density of peripheral blood neutrophils fell significantly at 15 min (p less than 0.02), with a return to baseline values despite further PAF inhalations; this was associated with an increase in neutrophil volume (n = 4; p less than 0.05). The numbers of neutrophils (x 10(5] in BAL fluid after PAF were significantly greater than after inhalation of lyso-PAF: 7.1 +/- 1.4 (n = 7) versus 1.3 +/- 0.3 (n = 5, p less than 0.01); eosinophil counts did not change significantly. The PC40 (the concentration of methacholine needed to cause a fall in Vp30) decreased from 17.1 (GSEM 1.40) to 8.7 (1.44) mg/ml (n = 12, p less than 0.02) 3 days after PAF. Inhaled lyso-PAF was inactive in all these respects.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Inhalation↗
PubMed1990

T lymphocytes and eosinophils in allergen-induced late-phase asthmatic reactions in the guinea pig.

The kinetics and phenotype of T lymphocytes infiltrating the airways of guinea pigs undergoing late-phase asthmatic reactions (LAR) were studied with monoclonal antibodies, cytofluorimetry, and immunocytochemistry. Challenge of sensitized animals with aerosolized ovalbumin was followed by early (2 h) and late-phase (17 h) bronchoconstriction. The induction of hypersensitivity, by aerosolized antigen, was associated with an increase in mucosal T cell numbers, which consisted almost entirely of CD8+ T cells. Following allergen challenge of fully sensitized animals, a biphasic rise in total T cell (CD3+) numbers was observed in the bronchial mucosa, peaking at 17 and 48 h. A similar pattern of T cell accumulation was observed in the bronchial adventitia but with an extra early peak at 2 h. In contrast to the T cell influx of the sensitization phase, the postchallenge infiltrate consisted largely of CD3+, CD8- cells. Eosinophil numbers were elevated in both submucosa and adventitia, with a single broad peak between 17 and 48 h. T cell infiltration was compared with eosinophil accumulation; while correlations between T cell and eosinophil numbers varied over the 96 h of the experiment, strong associations were observed between CD8+ numbers and eosinophils in the adventitia at 6 h (r = 0.733, p less than 0.01) and between CD3+ numbers and eosinophils in the submucosa at 72 h (r = 0.88, p less than 0.001). No significant changes were detected in T cell or eosinophil numbers in the lung parenchyma. There was a postchallenge increase in eosinophils (but not T cells) in bronchoalveolar lavage (BAL).(ABSTRACT TRUNCATED AT 250 WORDS)

Airway Resistance↗
PubMed1990

Enhancement of the expression of eosinophil IgE receptor (Fc epsilon R2) and its function by platelet-activating factor.

Eosinophils possess low-affinity Fc immunoglobulin E (IgE) receptors (Fc epsilon R2). We have compared platelet-activating factor (PAF) with lyso-PAF, leukotriene B4 (LTB4) and histamine for their ability to influence the binding of a native myeloma IgE to normal-density human eosinophils, using flow cytometry and a fluorescein-conjugated anti-IgE polyclonal antibody. Preincubation with PAF gave a dose- and time-dependent increase in IgE binding optimal at 10(-7) M (P less than 0.01) and 30 min respectively. A smaller but significant effect was observed with LTB4 and histamine (optimal at 10(-7) M (P less than 0.01) and 10(-5) M (P less than 0.05) respectively). Diluent alone or lyso-PAF had no effect. These results suggest enhanced expression of Fc epsilon R2 by stimulated eosinophils. Further confirmation of enhancement was obtained using two functional assays, i.e., cytotoxicity and mediator (LTC4) generation. IgE-dependent cytotoxicity against schistosomula of Schistosoma mansoni was enhanced by prior incubation of normal eosinophils with PAF (optimal at 10(-7) M), but not lyso-PAF. No LTC4 was detectable following incubation of unstimulated eosinophils with S. mansoni coated with specific IgE. Preincubation of these cells with PAF (10(-7) M) resulted in the generation of 4.4 pmol of LTC4 per 10(6) cells. Our data demonstrate that normal-density eosinophil Fc epsilon R2 expression can be up-regulated in vitro by PAF and LTB4 and that these changes are accompanied by enhanced functional properties.

Animals↗
PubMed1990

Fc gamma and CD11/CD18 receptor expression on normal density and low density human eosinophils.

We have studied the expression of Fc gamma receptors and leucocyte integrins (CD11/CD18 family) on human eosinophils using specific monoclonal antibodies (mAb) and flow cytometric analysis. Peripheral blood eosinophils of normal density and low density were compared with neutrophils and monocytes. Several properties of the human eosinophil were established. These were (i) that the eosinophil expressed Fc gamma RII (CDw32) only (unlike monocytes which bear Fc gamma RI and Fc gamma RII and neutrophils which bear Fc gamma RII and Fc gamma RIII); (ii) that the absence of Fc gamma RIII (CD16) on eosinophils served as a basis for distinguishing eosinophil and neutrophil populations by immunofluorescence; (iii) that the leucocyte adhesion glycoproteins, LFA-1 alpha (CD11a), CR3-alpha (CD11b), p150, 95-alpha (CD11c) and the common beta-chain (CD18), were expressed on the eosinophil as well as the neutrophil; (iv) that CD18 expression was significantly reduced on low-density eosinophils from the hypereosinophilic syndrome. Thus, our findings emphasize the unique phenotype of the human eosinophil in terms of Fc gamma receptor expression, the similarity of the eosinophil and neutrophil with regard to the leucocyte integrins and that eosinophils of low density do not differ greatly from those of normal density in terms of receptor expression.

Antigens, Differentiation↗
PubMed1990

Release of leukotriene C4 (LTC4) from human eosinophils following adherence to IgE- and IgG-coated schistosomula of Schistosoma mansoni.

The release of leukotriene C4 (LTC4) from human low-density eosinophils following adherence to live or formalin-fixed schistosomula of Schistosoma mansoni coated with parasite-specific IgE or IgG obtained from pooled human anti-S. mansoni serum has been studied. IgE-rich fractions were obtained after fractionation of pooled immune sera on fast-protein liquid chromatography (FPLC; polyanion SI-17 column) and were identified by parasite-specific RAST. Contaminating IgG was removed by adsorption on a Staphylococcus aureus-protein A affinity column. IgG-rich FPLC fractions were identified by a specific ELISA assay. IgG-dependent activities were confirmed by protein A adsorption. Low-density eosinophils adhered to live and formalin-fixed schistosomula coated with specific antisera and released 11.7 +/- 2.7 and 16.5 +/- 3.5 pmoles of LTC4/10(6) cells, respectively. LTC4 release induced by A23187 (5 x 10(-6) M) from the same cells was 80 +/- 24 pmoles/10(6) cells and 9.9 +/- 1 pmoles/10(6) cells in the presence of Sepharose particles (CNBr-activated 4B beads) covalently coated with normal human IgG. Fixed schistosomula coated with FPLC-purified IgE and IgG gave 7.6 +/- 0.4 and 6.0 +/- 0.1 pmoles of LTC4 per 10(6) low-density eosinophils, respectively. The same IgE- and IgG-rich fractions induced eosinophil-mediated cytotoxicity of live schistosomula in vitro. Removal of IgE by an anti-IgE affinity column abolished both the IgE-dependent release of LTC4 and the in vitro killing of larvae. Conversely, IgG-dependent activities were abolished by protein A, but not anti-IgE, adsorption. Normal density eosinophils generated undetectable amounts of LTC4 when incubated with IgE-coated schistosomula, whereas with IgG-coated larvae 4.6 pmoles/10(6) cells were obtained. Following preincubation with platelet-activating factor (PAF) (10(-7) M) and leukotriene B4 (LTB4) (10(-7) M), normal density eosinophils released LTC4 when in contact with larvae coated with antigen-specific IgE. Lyso-PAF had no effect in any of the systems tested. The synthetic chemotactic tripeptide formyl-methionyl-leucyl-phenylalanine (FMLP) had no influence on IgE-dependent release of LTC4 from eosinophils. In contrast, FMLP (10(-7) M) enhanced the IgG-dependent LTC4 release, with PAF and LTB4 also showing a small enhancing effect. None of these agents substantially altered the release potential of low-density eosinophils in either IgE- or IgG-dependent events. Thus the results presented here indicate that in an IgE-dependent system, human low-density eosinophils can be induced to adhere to and kill IgE-coated helminthic targets and release biologically relevant amounts of LTC4.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

The effect of platelet-activating factor on IgE binding to, and IgE-dependent biological properties of, human eosinophils.

This study investigated the effect of platelet-activating factor (PAF), leukotriene B4 (LTB4) histamine and formyl-methionyl-leucyl-phenylalanine (FMLP) on immunoglobulin E (IgE) binding and IgE-dependent cytotoxicity of human normal density eosinophils. The binding of a native myeloma IgE to normal human eosinophils was measured by flow cytometry using a fluorescein-conjugated polyclonal anti-IgE antibody. Preincubation with PAF (optimal at 10(-7)M), but not lyso-PAF or FMLP, gave dose-dependent increases in IgE binding. PAF and LTB4 gave significant increases in IgE binding after 5 min preincubation (P less than 0.05); the effect was further enhanced at 30 min (P less than 0.01). This was further confirmed using the rosette assay where PAF and LTB4, but not lyso-PAF or FMLP, gave dose- and time-dependent increases in IgE eosinophil rosettes. Eosinophil cytotoxicity for schistosomula of Schistosoma mansoni, incubated with immune serum, was also significantly enhanced (P less than 0.01) by PAF in a dose-dependent fashion (optimal at 10(-8) M). Schistosomula coated with FPLC-purified IgE fractions were susceptible to killing by normal density eosinophils, and this was enhanced with PAF (10(-8)M), LTB4 (10(-7)M) and histamine (10(-5)M) but not with FMLP (10(-7)M) or lyso-PAF. IgE-dependent cytotoxicity was confirmed by the removal of contaminating IgG from IgE-rich fractions, and by the abolishment of IgE-dependent cytotoxicity after IgE adsorption. These results suggest that PAF (and to a lesser extent LTB4 and histamine) increase IgE binding, IgE-dependent adherence and cytotoxicity of normal human eosinophils. Although IgE receptors have not been identified, the data support current concepts that certain biological properties of eosinophils may be IgE associated.

Cells, Cultured↗
PubMed1989

Leukotriene release enhancing factor. Purification, specific allergen induction, and further biologic properties.

Human PBMC cultured with PHA produced a factor which enhanced neutrophil LTB4 production. After progressive purification by gel filtration and chromatofocusing, leukotriene release enhancing factor (LREF) gave a single band on SDS-PAGE corresponding to a Mr of 60 kDa and a isoelectric point of 5.0 to 5.2. Human T lymphocytes were identified as a source of LREF because a long term human CD4+ T cell line, specific for house dust mite (Dermatophagoides farinae) generated leukotriene release enhancing activity (LREA) when stimulated with the inducing Ag together with irradiated accessory cells. Cultures of accessory cell and Ag alone did not induce neutrophil activation. Similarly, control cultures using an irrelevant allergen (grass pollen) failed to produce this activity. Stimulation of PBMC with anti-CD3 mAb also generated LREA. LREA had other neutrophil-associated biologic activities. These included the enhancement of neutrophil adherence to nylon wool, increased cytotoxicity of opsonised schistosomula of Schistosoma mansoni and upregulation of C receptors (CR1 and CR3). LREA had minimal neutrophil chemotactic activity. Therefore, LREF has potential as a mediator of inflammation and provides a link between lipid mediators, neutrophils, and cell-mediated immunity in the context of allergic diseases.

Adjuvants, Immunologic↗
PubMed1989

Inhibition of platelet-activating factor (PAF)-induced chemotaxis and PAF binding to human eosinophils and neutrophils by the specific ginkgolide-derived PAF antagonist, BN 52021.

We have investigated the effects of BN 52021 (a specific PAF antagonist derived from Ginkgo biloba) on PAF-induced human eosinophil and neutrophil chemotaxis. In response to an optimal concentration of PAF (10(-6) mol/L), the drug was significantly more potent (p less than 0.001) in inhibiting eosinophil as compared to neutrophil locomotion. These inhibitory effects were observed in a dose-dependent manner with a concentration of drug required to produce 50% inhibition of 7.0 (+/- 2.2) X 10(-6) mol/L and 2.3 (+/- 0.2) X 10(-5) mol/L for eosinophils and neutrophils, respectively. Sodium cromoglycate, nedocromil sodium, salbutamol, and dexamethasone (preincubated with cells up to 6 hours) had no effect over a wide dose range (10(-3) to 10(-9) mol/L). BN 52021 was significantly more effective in inhibiting chemotaxis when the cells were preincubated with the compound for up to 1 hour before commencement of the locomotion assay, whereas washing the cells completely abolished this effect. Inhibition by BN 52021 was specific for PAF in that it had no effect on chemotaxis induced by either leukotriene B4, N-formyl-methionyl-leucyl-phenylalanine, or a purified human mononuclear cell-derived neutrophil chemotactic factor. BN 52021 also inhibited the specific binding of [3H]-PAF (10(-8) mol/L) to eosinophils and neutrophils in a concentration-dependent fashion with a concentration of drug required to produce 50% inhibition of 1.5 (+/- 0.3) X 10(-6) mol/L and 9.1 (+/- 2.5) X 10(-7) mol/L, respectively. These results suggest that BN 52021 has potential as an anti-inflammatory agent in conditions associated with PAF-induced accumulation of neutrophils and eosinophils.

Albuterol↗
PubMed1989

The effect of nedocromil sodium on human eosinophil activation.

Nedocromil sodium inhibited the increase in eosinophil activation (measured by IgG- and complement-dependent cytotoxicity assays) induced by platelet activating factor (PAF). Inhibition of the upregulation in eosinophil effector function by nedocromil sodium was dose-dependent (optimal at 10(-7) mol/L) and paralleled that produced by a specific PAF antagonist, BN 52021. In addition, preliminary data suggest that nedocromil sodium can inhibit the increase in IgG-dependent release of LTC4 from human eosinophils after stimulation with the synthetic tripeptide, formyl-methionyl-leucyl-phenylalanine (fMLP). These data support our previous hypothesis that part of the mode of action of nedocromil sodium may be due to its ability to directly block the chemotactic factor-induced enhancement of inflammatory cell activity.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1989

Action of nedocromil sodium and sodium cromoglycate on cloned human allergen-specific CD4+ T lymphocytes.

Nedocromil sodium (and sodium cromoglycate) are topical, prophylactic, anti-inflammatory agents used in reversible obstructive airways disease. To investigate their potential mode of action, the effects of these agents on specific allergen- or IL-2-induced proliferation of a human CD4+ house dust mite-specific T-cell clone isolated from an individual with perennial rhinitis was examined. Although strong proliferation was observed in control cultures, no inhibitory effect was observed with any of the drug concentrations tested. In contrast, dexamethasone was able to inhibit the T-cell proliferation. These observations suggest that the clinically observed suppression of the late-phase asthmatic response may not be due to an effect on CD4+ T lymphocytes.

Allergens↗
PubMed1989

An in vitro model of allergen-dependent IgE synthesis by human B lymphocytes: comparison of the response of an atopic and a non-atopic individual to Dermatophagoides spp. (house dust mite).

An allergen-dependent in vitro model of immunoglobulin E (IgE) synthesis by human B cells is reported. Using this model, it is demonstrated that polyclonal T cells and CD4+ Dermatophagoides spp. (house dust mite)-specific T-cell clones derived from an atopic, house dust mite (HDM)-allergic individual are able to support IgE synthesis by autologous B cells. The helper activity was interleukin-4 (IL-4) dependent as only cloned T cells expressing detectable mRNA for IL-4 were able to induce IgE synthesis without the addition of exogenous IL-4. Peripheral and cloned T cells reactive with HDM could also be identified from a non-atopic individual but neither population was able to support IgE production even in the presence of exogenous IL-4.

Allergens↗
PubMed1989

IgE-mediated release of rat mast cell protease II, beta-hexosaminidase and leukotriene C4 from cultured bone marrow-derived rat mast cells.

Functional characteristics of cultured bone marrow-derived rat mast cells (BMMC) were studied. BMMC were shown to release in a time- and dose-dependent fashion the mucosal mast cell (MMC)-specific enzyme, rat mast cell protease II (RMCPII), following IgE-mediated activation in vitro. RMCPII release was temporally associated with that of the mast cell granule-derived enzyme, beta-hexosaminidase (beta-hex). Release of the pre-formed granule constituents, RMCPII and beta-hex, was associated with the generation of the membrane-derived lipid mediator, leukotriene C4 (LTC4) and, in older cultures, substantial amounts were generated (25.2 ng/10(6) BMMC). Absolute amounts of RMCPII, beta-hex and LTC4 released were dependent upon the age of the BMMC. These results extend our previous observations on the staining properties and protease content of rat BMMC and provide evidence that these cells are functionally, as well as histochemically, analogous to the MMC subset, which is so prominent during intestinal nematode infections in rats.

Animals↗
PubMed1989

The activated eosinophil in allergy and asthma.

Our studies indicate that human eosinophils can be activated in terms of up-regulation of surface receptors, increased membrane lipid generation, and enhanced cytotoxicity by a variety of pharmacological mediators, through IgE-, IgG-, and complement-dependent pathways. PAF is an impressive activator of eosinophil function and is the most potent chemotactic mediator for eosinophils so far described. It is not selective in that neutrophils are also attracted by PAF. In our hands, IL-1, IL-2, granulocyte/macrophage-colony stimulating factor, tumor necrosis factor, interferon-gamma, and IL-5 tested over a wide concentration range gave very weak chemotaxis for eosinophils in vitro. Nevertheless, in vivo infiltration of eosinophils into the site of allergic tissue reactions is closely associated with the local accumulation of activated (CD4+, IL-2 rec+) T cells. We suggest, therefore, that the selective eosinophil infiltration seen in allergic inflammation may result from the initial recruitment and activation of granulocytes by lipid mediators and the subsequent selective activation of eosinophils by cytokines.

Asthma↗
PubMed1988

Leukotriene C4 generation from human eosinophils stimulated with IgG-Aspergillus fumigatus antigen immune complexes.

Sepharose beads coated with IgG stimulate eosinophils to produce leukotriene C4 (LTC4). This observation has been extended with specific immobilized IgG/antigen immune complexes to elicit mediator generation. An extract of Aspergillus fumigatus was covalently coupled to Sepharose beads and incubated with the IgG fraction of immune serum from patients with allergic bronchopulmonary aspergillosis. These beads elicited generation of 7.72 +/- 1.7 pmol of LTC4 immunoreactive material (n = 5) from 1 X 10(6) normal eosinophils of greater than 86% purity, and significantly less LTC4 (0.73 +/- 0.19 pmol per 10(6) cells; n = 3) was produced by eosinophils after incubation with beads treated with IgG from normal nonimmune serum. The maximum antibody-dependent release achieved represented approximately 20% of that induced by the calcium ionophore (A23187). LTC4 was measured by radioimmunoassay and validated by reverse-phase high-performance liquid chromatography. The amount of LTC4 generated was dependent on the concentration of A. fumigatus-specific IgG, and mediator release was completely abolished by prior adsorption of the IgG fraction onto Sepharose-protein A (Staphylococcus aureus). Grass pollen-specific IgG antibody/antigen complexes, in combination with Sepharose beads, also triggered generation of LTC4 immunoreactive material. There was no evidence to suggest that IgE/A. fumigatus immune complexes triggered LTC4 generation, although IgE myeloma protein, in association with Sepharose beads, was a weak stimulus. The efficacy of the IgG immune complex-dependent stimulation of eosinophils suggests a possible physiologic mechanism whereby these cells could participate in the inflammatory changes associated with allergic bronchopulmonary aspergillosis and similar allergic disorders.

Antigen-Antibody Complex↗