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Aims: This study tested inhibitory effects of in vitro Montelukast treatment on nasal airway epithelial cells (AEC) cultured from asthmatic patients treated with Montelukast with and without concomitant allergic rhinitis. We further examined the effect of Montelukast with drawal in these patients on cytokine release from cultured nasal AEC. Methods: Nasal AEC were collected by brushings from subjects with a history of stable (no exacerbations or change in medication for ≥1 month) physician confirmed mild/moderate asthma whose asthma symptoms were documented to benefit from Montelukast treatment (NCT01230437). Release of the following mediators by nasal AEC were measured: IL-8, IL-6, IL-10, GM-CSF, RANTES, eotaxin and IFN-γ. Nasal AEC were cultured before and one week after g withdrawal of their Montelukast treatment. Results: Forty two asthmatics were recruited. Nasal AEC were successfully cultured in 17 at the first assessment, 14 at the second assessment and in 10 individuals at both assessments. Nasal AEC release was no different between asthmatics with or without allergic rhinitis. Montelukast significantly suppressed the release of IL-8 ( p = 0.016), IL-6 (p = 0.006), RANTES (p = 0.002) and IFN-γ (p = 0.046), in a dose dependent manner in unstimulated cultures but not in those stimulated with IL-1/TNF. Withdrawal of Montelukast treatment, was associated with increased IL-8 and RANTES secretion in unstimulated nasal AEC cultured from subjects with asthma and allergic rhinitis but not with asthma alone. Conclusions: Montelukast treatment for asthma symptoms reversibly suppresses nasal AEC release of pro-inflammatory mediators (i.e. IL-8 and RANTES) but only in those cells cultured from subjects with concomitant allergic rhinitis. [ABSTRACT FROM AUTHOR]

Summary Background Eosinophils are now recognized as major effector cells in allergic and asthmatic disease with a potent armoury of mediators whose release makes a major contribution to the inflammation underlying these conditions. Objectives The purpose of this study was to compare cultured eosinophils (CE) with normal-density peripheral blood eosinophils (PBE) in terms of their membrane receptor expression and to analyse the expression and storage of the eosinophil granule proteins major basic protein (MBP) and eosinophil cationic protein (ECP) during eosinophil maturation in vitro . Methods Purified human peripheral blood CD34+ cells were cultured in the presence of recombinant human IL-3, IL-5, rhGM-CSF, SCF, and FLT-3 ligand. PBE were isolated by density gradient centrifugation and negative immunomagnetic selection. Expression of CD11b, CD18, CD45, CD45RA, CD45RB, CD45RO, CD69, CD95, IL-5Rα, IL-9Rα, CCR1, CCR3, and CXCR4 by CE as they matured in culture were assessed by immunostaining and flow cytometry and expression of these receptors compared with freshly isolated PBE. Immunohistochemical staining and labophot-2TM light microscopy determined expression of MBP, ECP, and CD69 during eosinophil maturation. Results Positive immunostaining for MBP and ECP was detectable in a proportion (15–20%) of CE as early as 3 days of culture even though these cells were mononuclear in appearance. The numbers of CE positive for both granule proteins increased in rhIL-3 and rhIL-5 treated cells to a maximum of ∼ 80% by day 28. Maturing eosinophils exhibited positive immunostaining for CD69 after 14, 21 and 28 days of culture. Compared with PBE, CE had lower expression of pan-CD45 and CD45 isoforms, CD95 and CD11b. In contrast, the specific mean fluorescence for CD69, CD18, IL-5Rα, and IL-9Rα was significantly elevated for CE compared with PBE. CCR3 expression by CE and PBE was similar with no expression of CXCR4... [ABSTRACT FROM AUTHOR]

Immunological release of histamine and lipid mediators is known to occur when basophils, contained in whole blood human leucocytes, are incubated with anti-IgE (reversed anaphylaxis). In the present study we show that IgE-dependent stimulation of basophils was associated with activation of bystander eosinophils and neutrophils., as assessed by enhanced complement (C3b) and IgG (Fc) rosettes, and increased cytotoxicity for complement-coated schistosomula of Schistosoma mansoni. These changes in eosinophil and neutrophil function were totally inhibited in a dose-dependent fashion by prior incubation with disodium cromoglycate (DSCG). In all in vitro systems examined, complete inhibition of enhancement was observed with concentrations as low as 10-7 moles/l. In contrast, DSCG had no effect on histamine release, or the percentage of rosettes or cytotoxicity prior to anti-IgE stimulation. These results suggest that DSCG inhibits activation of inflammatory cells consequent to an IgE-dependent stimulus. [ABSTRACT FROM AUTHOR]

Sheep erythrocytes (E) coated with either human IgG1, IgG2, IgG3 or IgG4 myeloma proteins formed rosettes with human neutrophils and eosinophils. Prior incubation with the chemotactic agents f-met-leu-phe or leukotriene B4 enhanced neutrophil and eosinophil E-IgG1 rosettes in a time- and dose-dependent fashion. There were comparable increases in neutrophil and eosinophil f-met-leu-phe-induced rosettes with either E-IgG1, E-IgG2, E-IgG3 or E-IgG4 whereas this agent did not appear to influence E-IgA1, E-IgD, E-IgM or E-IgE binding to these granulocytes. These studies indicate that human neutrophil and eosinophil IgG (Fc) receptors are enhanced by chemotactic agents in a similar way to that previously described for complement receptors. [ABSTRACT FROM AUTHOR]

A comparative study of membrane expression of immunoglobin (Fc) receptors on human eosinophils and neutrophils has been undertaken using human IgG-1, IgG-2, IgG-3, IgG- 4, IgA-1, IgA-2, IgM, IgD and IgE myeloma proteins. Sheep erythrocytes (E) coated with either human IgG-I, IgG-2, IgG-3 or IgG-4 myeloma proteins formed rosettes with human neutrophils and eosinophils. Proportionally more (1½-2 times) rosettes were observed with neutrophils compared to eosinophils. In contrast, E-IgE bound to eosinophils (but not to neutrophils) to a degree that was comparable to E-IgG-1. Although IgG and IgE rosettes were inhibited by aggregates prepared from their corresponding myeloma protein there was no evidence that eosinophils and neutrophils have distinct receptors for IgG subclasses. Cells from four patients with hypereosinophilia were separated on density (metrizamide) gradients. The percentages of E-IgG-1 and E-IgE rosettes with normal-and light-density eosinophils were similar. Neutrophils, but not eosinophils, also bound significantly more E-IgA-1 and E-IgA-2 than the E-human albumin (E-Alb) control. In contrast, neutrophil and eosinophil rosette formation with E-IgM and E-IgD was not significantly different from E-Alb or E alone. These experiments indicate that (i) human neutrophils and eosinophils bind homologous IgG subclass myeloma proteins, (ii) eosinophils, hut not neutrophils, bind E-lgE with a similar avidity to that observed with E-IgG1, (iii) neutiophils, but not eosinophils, readily express demonstrable receptors for IgA-1 and IgA-2 and (iv) neither neutrophils nor eosinophils form E-IgM or E-IgD rosettes in greater numbers than the E-Alb controls. [ABSTRACT FROM AUTHOR]

We have compared the rosette technique (using C3b-eoatcd red cells) with the uptake of a 125I F(ab')2 anti-complement receptor type I (CR1) monoclonal antibody (Ell) for studying the phenomenon of chemotactic factor (fMLP)-induced complement receptor enhancement (CRE) on human neutrophils. With both methods the dose responses of fMLP were similar with maximal C RE being observed al 10-7 moles 1-1. Conversely, the time course of CRE and the effects of disodium cromoglycate (DSCG) on inhibition of enhancement were discrepant. Maximal CRE with the rosette method was observed al 30 min, whereas increased uptake of anti-CR1 had still not reached a plateau after 1 h. DSCG inhibited fMLP-induced CRE, as assessed by the rosette technique, whereas this agent had no effect on increased binding of radio-labelled anti-CR1 antibody. These results suggest that in CRE the increased adherence of C3b-coated red cells to fMLP-stimulated neutrophils, as measured by the rosette technique, is not dependent exclusively on the increased numbers of CR1. [ABSTRACT FROM AUTHOR]

Background: Eosinophil-derived inflammatory mediators including cytokines are considered to be important in the pathogenesis of allergic inflammation. Fibronectin (Fn) has been shown to be a physiological trigger of autocrine cytokine production by human eosinophils. Fn is encoded by a single gene, but alternate splicing of the primary RNA transcript results in polypeptide diversity in a cell type-specific fashion. Thus, tissue Fn contains approximately 50% more of the CS-1 cell binding region recognized by the integrin α4/β1 compared with plasma Fn. Objective Since eosinophils are predominantly tissue-dwelling cells we compared the effect of tissue and plasma Fn on eosinophil survival in culture. Methods The viability and cytokine generation of eosinophils (>99.9% pure) cultured for up to 4 days in 96 well plates coated with tissue Fn, plasma Fn or BSA was compared. Results There was a significant difference in the ability of tissue Fn to support eosinophil survival compared with plasma Fn (P < 0.01). Optimal survival with tissue Fn was seen at 25 μg/well (70% ± 2.0% viability at 3 days vs 7% ± 2.2% viability on BSA). Significant (P < 0.001) cell viability on tissue Fn was observed for up to 4 days in culture (54% ± 6.0%) compared with BSA coated wells. Addition of autologous mononuclear cells (final concentration 0.5%, 1% or 2%) resulted in plasma Fn-dependent eosinophil survival comparable to that of 99.9% pure eosinophils adherent to tissue Fn. Tissue Fn-dependent survival was significantly inhibited by anti-interleukin-3, anti-granulocyte macrophage colony stimulating factor (GM-CSF) and anti-IL-5 monoclonal antibodies. Picogram quantities of these three cytokines were detected in supernatants from eosinophils cultured for 3 days on tissue Fn using specific enzyme-linked immunosorbent assays (ELISAs). Eosinophil survival on tissue Fn was significantly inhibited by anti-β1 and α4/β1 monoclonal antibody (MoAb) and also by a MoAb specific for the CS-1 motif in the IIICS region of Fn. Conclusion These observations show preferential survival of eosinophils cultured on tissue Fn as a result of α4/β1-dependent interaction with the CS-1 region of tissue Fn triggering autocrine cytokine synthesis and release, thereby promoting their survival and persistence within the tissues. [ABSTRACT FROM AUTHOR]

Human peripheral blood eosinophils adhered specifically to microtitre plates coated with plasma fibronectin (Fn) in a dose- and time-dependent fashion. Adhesion was optimal at 60 mm at a concentration of 100 μg/ml. Adherence to Fn was up-regulated by platelet-activating factor (PAF; optimum concentration of 10- 6 M) and was significantly inhibited by a polyclonal anti-Fn antibody (P < 0.05). The following evidence suggested that eosinophil adhesion to Fn was mediated by α4β1: (1) eosinophil adherence to Fn was not inhibited by an Arg-GIy-Asp-Ser (RGDS) synthetic peptide; (2) there was a dose-dependent adherence of eosinophils to microtitre plates coated with the 40,000 MW proteolytic fragment of Fn that contains the CS-1 α4β1, binding region, whereas adherence to the 120,000 MW chymotryptic fragment of Fn, which contains the ROD-dependent binding site, was weak and only observed at high concentrations (>250 μg/mI); (3) significant inhibition of eosinophil adherence to Fn was achieved by monoclonal antibodies (mAb) against the u chain of VLA-4 but not by a mAb against CD45 or a mouse myeloma antibody as negative controls. After adhesion to Fn, eosinophils were investigated for their capacity to release leukotriene C4 in response to stimulation with a suboptimal concentration of calcium ionophore (2 × 10-6M). Significant enhancement of release was detected with Fn-coated plates but not with the control bovine serum albumin (BSA) (P < 0.01). Furthermore, this enhancement was significantly inhibited by the α4β1 mAb HP2/1 (P < 0.05) but not by an anti- CD45 mAb. From these studies we conclude that (1) α4β1 (VLA-4) integrin is a major receptor for Fn on human eosinophils and (2) adhesion to Fn may prime eosinophils for mediator release during allergic inflammation. [ABSTRACT FROM AUTHOR]

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 potyclonal 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 rain preincubation (P < 0·05); the effect was further enhanced at 30 min (P < 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 < 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. [ABSTRACT FROM AUTHOR]

We have studied chemotactic factor-induced 'complement receptor enhancement' to determine whether changes in receptor expression and complement-dependent cytotoxicity were associated with alterations in cell density. Ficoll-Paque separated normal human neutrophils (>90%), when further fractionated on discontinuous metrizamide (MTZ)gradients(18, 19, 20, 21, 22. 23% MTZ). consistently gave two major bands at the 20/21% and 21/22% interfaces. Incubation with the synthetic chemotactic peptide, N- formyl-methionyl-leucyl-phenylalanine (fMLP (10-8 M)), converted virtually all neutrophils to low density cells sedimenting on MTZ at the 18/19% and 19/20% interfaces. There was a time-dependent change of density after fMLP-stimulation which was maximal at 30 min, with cells reverting towards normal density by 60 min. Control unstimulated cells did not alter their density at any of the time points examined. Activated, low density neutrophils had increased expression of CR1 and CR3 (as shown by flow cytometry and the uptake of -125-F(ab')2 monoclonal anti-CR1 antibody (E11)). These cells also showed enhanced cytotoxic capacity in vitro for helminthic targets (schistosomula of Schistosoma mansoni) opsonized with autologous complement. There were highly significant correlations between cell density and anti-CRI uptake (P<0.001), and between schistosomular killing and change in density (P<0.001). Increased CR1 expression also correlated with enhanced helminthieidal capacity of neutrophils (P<0.001). Complement dependent cytotoxicity was partially reduced after treatment of cells with anti-human CR1 and :or CR3 antibodies, but only in the presence of a second antibody. These findings indicate that chemotactic factor-induced complement receptor enhancement of human neutrophils is associated with a decrease in cell density and increased complement-dependent cytotoxicity (CTX). [ABSTRACT FROM AUTHOR]

The ability of nedocromil sodium, a new anti-inflammatory agent for the treatment of asthma, to inhibit activation of human eosinophils and neutrophils in vitro, has been studied using an adherence reaction (the "rosette" technique) as well as a cytotoxicity assay. We have also investigated the capacity of nedocromil sodium to inhibit IgE-dependent histamine release from human lung mast cells. The drug was a potent inhibitor (IC50 approx 5 × 10-9M) of fMLP-induced enhancement of eosinophil and neutrophil complement (C3b) and IgG (Fc) rosettes. There was also a comparable inhibition of enhancement, by fMLP, of eosinophil and neutrophil cytotoxicity (for complement-coated schistosomula of Schistosoma mansoni). Although nedocromil sodium also inhibited histamine release from human lung mast cells in a dose-dependent fashion its activity was relatively weak (IC30 5 × 10-6M) compared to its effect on granulocytes. These experiments support the view that the principal mode of action of nedocromil sodium is its capacity to inhibit the activation of inflammatory cells. [ABSTRACT FROM AUTHOR]

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°C. Concentrations of GPBP as low as 1.25 μg/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 μg/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. [ABSTRACT FROM AUTHOR]