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R Moqbel

Publications and source records attributed to R Moqbel.

At least 73 records · Page 4Linked to original sources
PubMed1993

Interleukin-8 is a chemo-attractant for eosinophils purified from subjects with a blood eosinophilia but not from normal healthy subjects.

Interleukin-8 (IL-8), a pro-inflammatory cytokine with potent neutrophil chemotactic activity, was studied for its effect on eosinophil migration responses, in vitro. Normal density eosinophils were isolated from healthy, non-atopic subjects (<0.35 x 10(9) eosinophils/l) and individuals with various diseases associated with a blood eosinophilia (range 0.56 x 10(9)-12.2 x 10(9) eosinophils/l). IL-8 produced a dose-dependent migrational response for eosinophils from subjects with an eosinophilia, optimal at 10(-8) M (P < 0.01) and the major component of the migrational response was chemokinesis. On a molar basis, IL-8 (EC50 approximately 10(10) M) was 100-fold more potent than platelet activating factor (PAF), although a comparison of the migrational responses showed that at optimal concentrations IL-8 (10(-8) M) produced only 30% maximal responses stimulated by PAF (10(-6) M). In contrast, IL-8 tested over a wide concentration range had a negligible effect on eosinophils from normal subjects. A direct correlation between the total blood eosinophil counts for all subjects and the absolute magnitude of the migrational response to IL-8 (r = 0.727, P < 0.01 at 10(-8) M), PAF (r = 0.551, P < 0.03 at 10(-6) M) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) (r = 0.689, P < 0.02 at 10(-8) M), suggested that heightened eosinophil migrational responses to inflammatory mediators may arise as a consequence of in vivo priming mechanism(s) associated with the development of an eosinophilia. In this regard, eosinophils derived from human cord blood mononuclear cells cultured in the presence of eosinophilopoietic cytokines IL-3 and IL-5, produced migrational responses to IL-8 and PAF, that were comparable with that of eosinophils from eosinophilic subjects. Furthermore, incubation of eosinophils from normal donors with IL-5 (optimal concentration 10(-9) M), significantly enhanced the subsequent migrational responses to both IL-8 (10(-8) M, P < 0.01) and PAF (10(-8) M, P < 0.05). Therefore, the increased responsiveness of eosinophils from eosinophilic subjects may reflect in vivo priming by IL-5 and this phenomenon may contribute partly to the mechanism(s) by which eosinophils preferentially accumulate at sites of allergic inflammation.

Cells, Cultured↗
PubMed1993

T cells are the principal source of interleukin-5 mRNA in allergen-induced rhinitis.

We have investigated the phenotype of interleukin-5 (IL-5) mRNA+ cells in the nasal mucosa of subjects with allergic rhinitis. Serial cryostat sections were cut from paraformaldehyde-fixed snap-frozen nasal biopsies from six patients, before and 24 h after local allergen provocation with grass pollen. Immunocytochemistry (APAAP) was followed by in situ hybridization on the same sections. For immunocytochemistry, antibodies against CD3, tryptase, and major basic protein (MBP) were used to identify T cells, mast cells, and eosinophils, respectively. Hybridization studies were performed using a Digoxigenin-labeled IL-5 riboprobe. Nitroblue tetrazolium (NBT) and X-phosphate-5-bromo-4-chloro-3- indoly phosphate (BCIP) served as chromogens to detect hybridized IL-5 mRNA signals. The majority of IL-5 mRNA+ cells were CD3+ (83.2%), whereas the remainder were either tryptase+ (11.3%) or MBP+ (5.4%). In contrast, only a few IL-5 mRNA+ cells were observed in nasal biopsies before challenge, all of which were co-localized to CD3+ cells. These results indicate that CD3+ cells are the principal cellular source of IL-5 transcripts in the nasal mucosa 24 h after allergen-induced late-phase nasal responses.

Adult↗
PubMed1993

Eosinophil accumulation induced by human interleukin-8 in the guinea-pig in vivo.

Interleukin-8 (IL-8) is a neutrophil chemoattractant cytokine. Initially IL-8 appeared to exhibit specificity for neutrophils over other cells of the immune system. However, several recent studies have shown that this mediator can also activate other leucocyte types in vitro. In this study we have used an in vivo model of local [111In]leucocyte accumulation in the guinea-pig and an in vitro assay of leucocyte activation (changes in cytosolic-free Ca2+) to investigate the eosinophil chemoattractant activity of IL-8. The intradermal injection of recombinant human (rh)IL-8 induced a dose-dependent accumulation of intravenously administered [111In]eosinophils into the skin sites over 4 hr. Time-course experiments revealed that this cell infiltration was delayed in onset, occurring between 1 and 2 hr after injection of IL-8. The delay may indicate that IL-8 operates via an indirect mechanism. In contrast, eosinophil accumulation induced by the complement fragment C5a occurred within the first hour following injection. Other human cytokines, IL-1, IL-3, IL-5, tumour necrosis factor (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), were not eosinophil chemoattractants in this in vivo test system. Direct activation of eosinophils by IL-8 was demonstrated in vitro by a transient elevation in cytoplasmic-free Ca2+ levels where it was less potent than either rhC5a or leukotriene B4 (LTB4). Experiments using [111In]neutrophils in vivo indicated that rhIL-8 and rhC5a were similar in potency in inducing local neutrophil infiltration into guinea-pig skin. The demonstration of the eosinophil chemoattractant activity of IL-8 in vivo raises the possibility that this cytokine, or a structurally related molecule, contributes towards eosinophil infiltration in a number of inflammatory conditions such as asthma, helminthic infections and adult respiratory distress syndrome.

Animals↗
PubMed1992

Human eosinophils synthesize and secrete interleukin-6, in vitro.

Using the technique of in situ hybridization, we have shown that resting, unstimulated, human peripheral blood eosinophils, obtained from subjects with greater than 8% eosinophilia, transcribe and translate messenger RNA (mRNA) for interleukin-6 (IL-6). After incubation for 24 hours in culture medium alone, approximately 19% of eosinophils were positive for IL-6 mRNA. This may be a reflection of their in vivo activation, but also may suggest that the gene for this cytokine is constitutively expressed in eosinophils. After stimulation with interferon gamma (IFN gamma) (500 U/mL), the percentage of IL-6-mRNA+ cells increased to 51.3%. This was accompanied by an enhancement of intensity of the hybridization signals. The specificity of the IL-6 probe and the hybridization signals was confirmed by the use of an IL-6 sense probe and RNase pretreatment of cell preparations. Evidence for the translation of IL-6 mRNA was obtained by immunocytochemical staining. Normal and activated eosinophils gave IL-6-specific immunoreactivity with a polyclonal antihuman IL-6 antibody. A higher percentage of positive cells was detected among activated eosinophils than those treated with medium alone. Using a specific immunoenzymetric assay, we detected 190.15 +/- 18.1 and 403.32 +/- 213.6 pg/mL of IL-6 in supernatants of unstimulated and IFN gamma-treated (24 and 48 hours) eosinophils, respectively. These data indicate that eosinophils are an important cellular source of IL-6.

Antibodies↗
PubMed1992

Preferential messenger RNA expression of Th1-type cells (IFN-gamma+, IL-2+) in classical delayed-type (tuberculin) hypersensitivity reactions in human skin.

We recently established that the allergen-induced late-phase cutaneous reaction in atopic subjects was associated with high mRNA expression for the cytokine gene cluster IL-3, IL-4, IL-5, and granulocyte/macrophage-CSF (GM-CSF), compared with IFN-gamma and IL-2, suggesting that allergic skin reactions contained the equivalent of murine Th2 cells. We now show that, in humans, classical delayed-type hypersensitivity is associated with cells preferentially expressing a Th1-type cytokine profile. Cryostat sections from skin biopsies from 24-h tuberculin reactions in 10 nonatopic subjects were hybridized with 35S-labeled RNA probes and processed by using in situ hybridization. On the whole, tuberculin biopsies showed preferential expression of mRNA encoding IFN-gamma and IL-2, although in some cases mRNA expression for IL-3, IL-4, IL-5, and GM-CSF was also observed. Biopsies from diluent control sites gave only occasional signals. The difference in the number of cells expressing mRNA in the diluent compared with tuberculin sites was statistically significant for IL-2 and IFN-gamma (p less than 0.01) but not for IL-3, IL-4, IL-5, and GM-CSF. These results suggest that cells infiltrating the site of the 24-h tuberculin reaction preferentially transcribe mRNA encoding IFN-gamma and IL-2, supporting the hypothesis that delayed-type hypersensitivity is associated with preferential activation of cells having a cytokine profile similar to the murine Th1 subset.

Adult↗
PubMed1992

Immunohistology of the nasal mucosa in seasonal allergic rhinitis: increases in activated eosinophils and epithelial mast cells.

The immunohistology of the nasal mucosa was examined in 13 grass pollen-sensitive patients and in seven normal nonatopic control subjects before and during the pollen season. Cryostat sections (6 microns) of biopsy specimens from the inferior turbinate were immunostained with the alkaline-phosphatase antialkaline-phosphatase method and a panel of monoclonal antibodies. Mast cell subtypes were measured with a double sequential immunostaining method. Within the submucosa, seasonal increases in total (MBP+, p less than 0.01) and "activated" (EG2+, p less than 0.01) eosinophils were observed for the patients, which were significant when these counts were compared with counts for those of control subjects (MBP+ p less than 0.01; EG2+ p less than 0.001). Within the nasal epithelium, seasonal increases in total (p less than 0.05) and "activated" (p less than 0.02) eosinophils were also observed. Mast cell counts revealed seasonal increases in tryptase-only positive mast cell (MCT) (p less than 0.02) but not chymase plus tryptase-positive mast cells (MCTC) within the epithelium that were significant when counts were compared with those of control subjects (p less than 0.03). No significant changes were observed within the submucosa or epithelium for total leukocytes (CD45+ cells) or T-lymphocytes (CD3+, CD4+, CD8+, and CD 25+ cells) for either group. Similarly, no significant changes were observed for neutrophils (antielastase), macrophages (CD68+), nor HLA-DR+ cells. In the subjects with rhinitis, seasonal submucosal CD3+ counts correlated with MBP+ eosinophils (r = 0.56; p less than 0.05) and MCTS (r = 0.65; p less than 0.02). Similarly, seasonal epithelial EG2+ eosinophil counts correlated with MCTs (r = 0.56; p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Characterisation of ovine mast cells derived from in vitro culture of haemopoietic tissue.

Ovine mast cells generated in vitro from bone marrow (BMMC) were compared with mucosal mast cells (MMC) isolated from parasitised abomasum. Ultrastructurally, the granules of BMMC were partially developed and immature. Both cells types contained beta-hexosaminidase, arylsulfatase, histamine, dopamine and sheep mast cell proteinase (SMCP). Greater amounts of beta-hexosaminidase, but less SMCP, histamine and arylsulfatase were present in BMMC. Stimulation with calcium ionophore A23187 caused the secretion of granule constituents and generation of leukotriene C4 by BMMC in a dose-dependent manner. An additional [3H]diisopropylfluorophosphate-binding 31,500 mol. wt. serine esterase, antigenically related to SMCP (27,000 mol. wt.) was present in cultures of BMMC but was not detected in isolated MMC. Both enzymes were detected in BMMC by Day 7 of culture and were secreted concomitantly following stimulation of BMMC with ionophore.

Abomasum↗
PubMed1992

Application of monoclonal antibodies against major basic protein (BMK-13) and eosinophil cationic protein (EG1 and EG2) for quantifying eosinophils in bronchial biopsies from atopic asthma.

A monoclonal antibody prepared against the eosinophil major basis protein (MBP) was compared with the anti-eosinophil cationic protein (ECP) antibodies (EG1 and EG2) in immunostaining of bronchial biopsies from atopic asthma and controls. Anti-MBP (designated BMK-13) did not cross-react with other eosinophil basic proteins (i.e. ECP, eosinophil peroxidase [EPO] or eosinophil-derived neurotoxin [EDN]) and stained more than 98% of peripheral blood eosinophils irrespective of their degree of activation. EG2 stained 15% of resting and 75% of activated eosinophils; EG1 recognized 74% and 78% of resting and activated cells, respectively. The numbers of BMK-13, EG1 or EG2-positive staining cells in bronchial biopsies from asthma were significantly greater than atopic non-asthmatics (P less than 0.02, P less than 0.01 and P less than 0.05, respectively) and normal non-atopic controls (P less than 0.001). For each of the various groups studied, the rank order for the number of eosinophils stained was BMK-13 greater than EG1 greater than EG2. BMK-13 stained significantly more cells from bronchial biopsies of atopic asthma and atopic non asthma when compared to EG2 (P less than 0.001 and P less than 0.05, respectively). Since only a proportion of BMK-13+ cells were EG2+, these results suggest that not all tissue eosinophils are actively secreting. Thus, BMK-13 can serve as a useful pan-eosinophil marker in tissue sections since it appears to stain most eosinophils.

Antibodies, Monoclonal↗
PubMed1991

Expression of mRNA and immunoreactivity for the granulocyte/macrophage colony-stimulating factor in activated human eosinophils.

Using in situ hybridization, we have shown that activated human peripheral blood eosinophils express mRNA for granulocyte/macrophage colony-stimulating factor (GM-CSF). Between 15 and 27% of eosinophils gave positive hybridization signals for GM-CSF mRNA after stimulation with the calcium ionophore A23187 or interferon gamma, and 4 and 6% after incubation with interleukin 3 (IL-3) or IL-5. Activated eosinophils also gave specific immunoreactivity with an anti-GM-CSF polyclonal antibody, suggesting translation of the mRNA. These data indicate that eosinophils may be an important source of GM-CSF at sites of allergic inflammation. Furthermore, the identification of GM-CSF production by human eosinophils suggests that the pro-inflammatory potential of this cell type may be substantially greater than hitherto recognized.

Calcimycin↗
PubMed1991

Messenger RNA expression of the cytokine gene cluster, interleukin 3 (IL-3), IL-4, IL-5, and granulocyte/macrophage colony-stimulating factor, in allergen-induced late-phase cutaneous reactions in atopic subjects.

Cryostat sections from skin biopsies from 24-h allergen-induced late-phase cutaneous reactions (LPR) in 14 human atopic subjects were hybridized with 35S-labeled RNA probes for a number of cytokines. mRNA was detected for interleukin 3 (IL-3) (8/14), IL-4 (10/14), IL-5 (11/14), and granulocyte/macrophage colony-stimulating factor (GM-CSF) (13/14). Only 5 of 14 gave hybridization signals for IL-2, and 0 of 14 for interferon gamma. Biopsies from diluent controls gave only occasional weak signals. These results suggest that cells infiltrating the site of the 24-h LPR transcribe mRNA for the IL-3, IL-4, IL-5, and GM-CSF gene cluster and support the hypothesis that atopy is associated with preferential activation of cells having a similar cytokine profile to the murine T helper type 2 subset.

Adolescent↗
PubMed1991

TNF alpha mRNA expression in allergic inflammation.

Using the technique of in situ hybridization, we have attempted to identify messenger RNA for tumour necrosis factor-alpha (TNF alpha) in cells infiltrating allergen-induced late phase reaction (LPR) of the skin and the nose of atopic subjects. We have also compared the number of TNF alpha mRNA positive cells in bronchoalveolar lavage (BAL) from atopic asthmatics and normal controls. Twenty-four hours after local allergen challenge, 12/14 skin biopsies and 9/10 nasal biopsies had positive hybridization signals for TNF alpha mRNA whereas only 4/14 and 2/10 biopsies were positive in the relevant diluent controls. Compared with diluent sites significantly increased numbers of cells expressing mRNA for TNF alpha were observed in the LPR of skin (P less than 0.004) and nose (P less than 0.006). All BAL from asthmatics (n = 10) and from normal volunteers (n = 10) had cells showing positive hybridization signals for TNF alpha mRNA but these were at increased frequency in asthmatics (P less than 0.001). These results suggest that TNF alpha may be an important cytokine in atopic allergic inflammation.

Adolescent↗
PubMed1991

Interleukin-5 mRNA in mucosal bronchial biopsies from asthmatic subjects.

Using in situ hybridization, we have investigated the expression of interleukin-5 (IL-5) mRNA in bronchial biopsies from asthmatics (n = 10) and controls (n = 9). The number of IL-5-nRNA-positive cells were compared with the number of CD25+ and EG2+ cells and total eosinophil counts. Specific hybridization signals for IL-5 mRNA were demonstrated in 6 out of the 10 asthmatic subjects but in none of the controls. The 6 IL-5-mRNA-positive asthmatics tended to have more severe disease and showed a significant increase in the degree of infiltration of the bronchial mucosa by activated T lymphocytes and eosinophils.

Asthma↗
PubMed1991

Effects of cetirizine on human eosinophil and neutrophil activation in vitro.

The ability of cetirizine, a novel antihistamine agent, to inhibit the in vivo activation of human eosinophils, neutrophils and monocytes has been investigated using C3b- and IgG-dependent rosette formation, cytotoxicity against opsonised parasitic larvae and adherence to plasma-coated glass (PCG). The drug inhibited platelet-activating factor (PAF)-induced enhancement of eosinophil and neutrophil IgG (Fc) and complement (C3b) rosettes with an IC50 of 2 x 10(-5) M. There was also comparable inhibition of PAF-dependent enhancement of eosinophil cytotoxicity (for complement-coated schistosomula of Schistosoma mansoni). Cetirizine inhibited PAF-induced eosinophil, but not neutrophil, hyperadherence to PCG. These data support the view that cetirizine may exert some of its anti-allergic effects by inhibiting the activation of human granulocytes and that it may also selectively inhibit PAF-induced eosinophil hyperadherence.

Animals↗
PubMed1991

Expression of mRNA for interleukin-5 in mucosal bronchial biopsies from asthma.

We have attempted to identify mRNA for IL-5 in endobronchial mucosal biopsies from asthmatics and controls, using the technique of in situ hybridization. Bronchial biopsies were obtained from 10 asthmatics and 9 nonatopic normal controls. A radio-labeled cRNA probe was prepared from an IL-5 cDNA and hybridized to permeabilized sections. These were washed extensively before processing for autoradiography. An IL-5-producing T cell clone derived from a patient with the hyper-IgE syndrome was used as a positive control. As a negative control, sections were also treated with a "sense" IL-5 probe. Specific hybridization signals for IL-5 mRNA were demonstrated within the bronchial mucosa in 6 out of the 10 asthmatic subjects. Cells exhibiting hybridization signals were located beneath the epithelial basement membrane. In contrast, there was no hybridization in the control group. No hybridization was observed with the sense probe. The six IL-5 mRNA-positive asthmatics tended to have more severe disease than the negative asthmatics, as assessed by symptoms and lung function, and showed a significant increase in the degree of infiltration of the bronchial mucosa by secreting (EG2+) eosinophils and activated (CD25+) T lymphocytes. Within the subjects who showed positive IL-5 mRNA, there was a correlation between IL-5 mRNA expression and the number of CD25+ and EG2+ cells and total eosinophil count. This study provides evidence for the cellular localization of IL-5 mRNA in the bronchial mucosa of asthmatics and supports the concept that this cytokine regulates eosinophil function in bronchial asthma.

Adult↗
PubMed1991

The accumulation of 111In-eosinophils induced by inflammatory mediators, in vivo.

Eosinophils are implicated in the pathogenesis of a variety of allergic inflammatory diseases such as asthma. Several substances have been shown to be chemotactic for eosinophils in vitro, but the inflammatory mediators involved in the accumulation of eosinophils in vivo are as yet unidentified. In this study we have developed a system to measure the accumulation of 111In-eosinophils in guinea-pig skin in vivo. Horse serum-induced guinea-pig peritoneal eosinophils were radiolabelled with 111In and injected intravenously into recipient animals. 125I-albumin was also injected intravenously in order to measure local oedema formation simultaneously. A range of putative mediators was injected intradermally and responses measured for up to 2 hr. Of the mediators tested, guinea-pig C5a des Arg in zymosan-activated plasma was the most active. Recombinant human C5a (rHC5a) was also highly active, but less than the guinea-pig material. C5a des Arg in maximally activated plasma induced a 1500% increase in eosinophil accumulation, while rHC5a (10(-10) mol dose) induced a 600% increase. Platelet-activating factor (PAF) and leukotriene B4 (LTB4) were also tested for comparison. With respect to 111In-eosinophil accumulation, the order of potency of the mediators tested was as follows: guinea-pig C5a des Arg greater than LTB4 greater than PAF. In contrast, the order of potency of the mediators with respect to oedema formation was: PAF greater than guinea-pig C5a des Arg greater than LTB4. The techniques described will facilitate analysis of the mechanisms involved in eosinophil accumulation in defined inflammatory reactions.

Albumins↗