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Biomedical subjects

V Gruart

Publications and source records attributed to V Gruart.

At least 19 recordsLinked to original sources
PubMed1995

Human eosinophils express a receptor for secretory component. Role in secretory IgA-dependent activation.

The existence of a functional receptor for secretory component (SC) on the eosinophil membrane might explain the preferential degranulation induced by secretory IgA (sIgA) when compared to serum IgA. Indeed, flow cytometry analysis revealed that purified human SC could bind to a subpopulation (4-59%) of blood eosinophils purified from 19 patients with eosinophilia. Binding of radiolabeled human SC could be competitively inhibited using unlabeled SC or secretory IgA but not with serum IgA or IgG. Immunoprecipitation and immunosorbent chromatography using human SC revealed the presence of a major component at 15 kDa in eosinophil extracts as well as in culture supernatants but not in neutrophils. The 15-kDa protein eluted from the human SC immunosorbent was able to bind to SC or to sIgA but not to serum IgA. Eosinophils preincubated with human SC or sIgA released eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) after addition of anti-SC or anti-IgA monoclonal antibody as respective cross-linking reagents. These results indicated that binding of free or complexed SC to human eosinophils could induce eosinophil degranulation. Furthermore, the dose-dependent inhibition by SC of mediator release induced by sIgA but not by serum IgA, suggested that the receptor for SC could be involved in the preferential degranulation mediated by sIgA. These results indicate a novel pathway of eosinophil activation and its potential involvement in mucosal immunity, particularly in inflammatory diseases associated with infiltration of eosinophils and the enhanced production of sIgA.

Binding, Competitive↗
PubMed1993

Human neutrophils express immunoglobulin E (IgE)-binding proteins (Mac-2/epsilon BP) of the S-type lectin family: role in IgE-dependent activation.

It has been suggested that neutrophils may be involved in the late-phase reaction of immunoglobulin E (IgE)-dependent hypersensitivity states. However, the identity of neutrophil-associated molecules inducing the release of mediators remains unclear. In this report, we demonstrate that human neutrophils from normal donors or from patients with inflammatory disorders could bind myeloma IgE proteins, especially after desialylation. Northern blot, immunoprecipitation, and flow cytometry analyses revealed that neutrophils did not express Fc epsilon RII/CD23, but rather Mac-2/epsilon binding protein (BP), belonging to the S-type lectin family. Similarly to IgA used as positive control, myeloma IgE proteins, as well as polyclonal IgE antibodies with or without antibody specificity, were both capable of inducing a neutrophil respiratory burst. Anti-Mac-2 but not anti-CD23 mAb strongly decreased the IgE-dependent activation of neutrophils, induced either by the specific antigen or by anti-IgE antibodies. These findings open new perspectives on the functional role of neutrophils in IgE-associated diseases including allergic states or parasitic infections.

Antigens, Differentiation↗
PubMed1993

IgE-binding molecules (Mac-2/epsilon BP) expressed by human eosinophils. Implication in IgE-dependent eosinophil cytotoxicity.

Macrophage cell-surface protein 2 (Mac-2), a galactose specific S-type lectin identified in inflammatory macrophages, presents a high degree of homology with the rat IgE-binding protein (epsilon BP). In the present study, we show by different experimental approaches that human eosinophils can express Mac-2/epsilon BP. Flow cytometry analysis revealed that a large proportion of eosinophilic patients expressing binding sites for IgE on their eosinophil membrane, were able to bind anti-Mac-2 monoclonal antibody (mAb). Northern blot performed with eosinophil RNA hybridized with the human Mac-2 or epsilon BP cDNA probes revealed that eosinophils presented a unique transcript at 1.2 kb. Immunoprecipitation of eosinophil extracts with anti-Mac-2 mAb revealed the presence of a molecule of 29 kDa corresponding to Mac-2 protein, as well as one additional molecule of 15 kDa, absent from control alveolar macrophages. The function of these molecules was investigated in a radiolabeled IgE binding assay. Anti-Mac-2 mAb as well as galactose and lactose saccharides significantly inhibited the binding of radiolabeled human myeloma IgE protein to eosinophils. Moreover, the dose-dependent inhibition by anti-Mac-2 mAb of IgE-dependent eosinophil-mediated cytotoxicity towards parasite targets indicated the role of these IgE-binding molecules in the function of human eosinophils. These results suggest that in addition to transmembrane receptors, lectin-type molecules can participate in the IgE-dependent effector function of eosinophils.

Animals↗
PubMed1992

Decreased expression of eosinophil peroxidase and major basic protein messenger RNAs during eosinophil maturation.

We evaluated the levels of mRNAs encoding cationic proteins in peripheral blood eosinophils (PBE) purified from patients with eosinophilia and in eosinophils differentiated from cord blood cells (CBC) by culture with recombinant human interleukin-3 (rhIL-3), rhGM-CSF, and rhIL-5. Messenger RNAs encoding eosinophil peroxidase (EPO), major basic protein (MBP), eosinophil-derived neurotoxin (EDN), and eosinophil cationic protein (ECP) were detected by Northern blot hybridization with the respective specific oligonucleotide probes. In mature PBE, MBP mRNA appeared to be absent, whereas EPO mRNA was barely detectable in only 5 of the 19 patients. In contrast, EDN and ECP mRNAs were observed in the PBE of all patients. In CE, EPO, and MBP, mRNAs were abundant in immature eosinophils and their amounts decreased after differentiation toward eosinophils. ECP and EDN mRNAs followed the same patterns, but mRNAs were less abundant at all timepoints studied. Study of mRNA t1/2 during the time course of differentiation indicated that changes in the stability of the different mRNAs were not responsible for the variations observed in the steady-state levels. Together, these results suggest that regulation of expression differs among EPO, MBP, EDN, and ECP mRNAs during the time course of eosinophil differentiation.

Actins↗
PubMed1992

Cloning and expression of a cDNA encoding a non-classical MHC class I antigen (HLA-E) in eosinophils from hypereosinophilic patients.

A cDNA library, constructed from purified blood eosinophils, was screened with the B cell CD23 cDNA probe. A clone designated EO15 has been isolated and found to encode a non-classical HLA class I gene transcript. EO15 was compared with HLA-E and found to be 99.9% similar at the nucleotide level and to extend further in the 3' untranslated region. The presence of an additional polyadenylation signal in the EO15 3' end suggests that EO15 clone represents a copy of the 3.3-kb mRNA species detected in Northern blot analyses. HLA-E transcripts of 1.9 and 3.3 kb have been described in a variety of cell types. The two EO15 mRNA species, similar in size to the previously defined HLA-E mRNA, were present at high levels in blood leukocyte populations and at variable levels in different cell lines. The EO15 transcripts were found at abundant levels in hypodense and normodense eosinophils from hypereosinophilic patients. In situ hybridization confirmed the expression of EO15 mRNA in eosinophils. Neutrophils and lymphocytes from normal donors of from patients with hypereosinophilia also strongly expressed EO15 mRNA. Among the cell lines studied, the highest levels of EO15 transcripts were detected in B and monocytic cell lines, whereas intermediate and lower levels were found in eosinophilic, NK-like, megakaryocytic, and T cell lines, respectively. Similar to its effect on classical HLA class I transcripts, IFN-gamma increased the levels of EO15 mRNA in eosinophils and neutrophils from hypereosinophilic patients. These results suggest that purified blood eosinophils as well as neutrophils express EO15/HLA-E mRNA; however, further experiments are needed to investigate the localization and the function of EO15 protein products.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1992

Eosinophil IgE receptor and CD23.

In the present review, eosinophil Fc epsilon RII was compared to CD23, a differentiation marker of B cells. Biochemical analysis revealed that molecules of similar molecular weight were immunoprecipitated from eosinophils and B cells by an anti-CD23 monoclonal antibody (mAb) or by BB10, and anti-eosinophil Fc epsilon RII. By flow cytometry, a correlation was found between the binding of anti-CD23 mAb and myeloma IgE. However, a low expression of different epitopes of CD23 was observed in various hypereosinophilic patients. Northern blot analysis of eosinophil RNA with the cDNA probe of CD23 revealed a weak message in only 3 of the 6 patients expressing membrane CD23. The inhibition by anti-CD23 mAbs of IgE-mediated cytotoxicity and IgE binding to eosinophils clearly indicated the participation of CD23 or a related molecule in IgE-dependent eosinophil functions. However, the differential effects of anti-CD23 mAbs on eosinophils and B cells suggest major differences in the characteristics of the molecule expressed by eosinophils and by B cells.

Antibodies, Monoclonal↗
PubMed1992

The interleukin 2 receptor in the hypereosinophilic syndrome.

The hypereosinophilic syndrome (HES) has been previously described as a clinicobiological entity characterized by a blood eosinophil count of over 1.5 x 10(9)/L of unknown cause associated with several clinical complications. In reality, HES is a heterogeneous group of diseases with variable and unpredictable progress in visceral lesions, thought to be related to the deleterious effects of tissue eosinophil infiltration. Various criteria for discrimination between benign and severe forms of HES have been described. These previous retrospective clinical investigations, using biological and clinical markers, have defined different stages of HES. It appears more relevant, however, to consider elements of disease activity by studying mechanisms of induction of persistent hypereosinophilia. The T-cell dependence of blood eosinophilia has led us to evaluate various markers of T-cell activation in particular. In the present review, we report previous results and perspectives suggested by the study of the interleukin 2 receptor in HES.

Biomarkers↗
PubMed1991

Elevated serum levels of soluble interleukin-2 receptor: a marker of disease activity in the hypereosinophilic syndrome.

We report here the presence of very high serum levels of the soluble interleukin-2 receptor (sIL-2R) in patients with blood hypereosinophilia with or without detectable markers of malignancy or signs of visceral involvement. The highest sIL-2R levels were observed in 16 eosinophilic patients with T-cell lymphoma (3,440 to 79,500 U/mL). Elevated levels of sIL-2R were also present (1,330 to 22,500 U/mL) in sera from 38 patients with the hypereosinophilic syndrome (HES) without detectable T-cell lymphoma. In this group of patients, the highest levels were noted in the patients with the malignant form of HES. Significantly lower levels were measured in sera of patients with hypereosinophilia associated with parasitic diseases, allergic disorders, or other miscellaneous diseases. Elevated serum sIL-2R levels were not closely paralleled by changes in the number of CD25-positive peripheral blood mononuclear cells as assessed by flow cytometric analysis. However, expression of IL-2R messenger RNA was detected in blood mononuclear cells collected from HES patients. In eight eosinophilic patients with T-cell lymphoma, the serum sIL-2R levels were significantly correlated with the eosinophil counts, and with the total number of blood hypodense eosinophils. alpha-Interferon (alpha-IFN) therapy resulted in both a dramatic clinical improvement and a rapid decrease in sIL-2R levels and blood hypereosinophilia. Similar beneficial effects of alpha-IFN were noted in patients with malignant HES who lacked a detectable T-cell lymphoma. Our data indicate that HES is associated with elevated serum IL-2R levels. The highest levels were observed in the most severe forms of HES with hematologic markers of malignancy or evident visceral involvement. Serum levels of sIL-2R might represent a useful indicator for the management of HES patients. In addition, the respective changes of sIL-2R and blood eosinophilia might reflect distinct processes of mononuclear cell activation affecting the eosinophil lineage.

Adult↗
PubMed1991

Human eosinophils from hypereosinophilic patients spontaneously express the p55 but not the p75 interleukin 2 receptor subunit.

The expression of interleukin 2 receptor (IL2R) on eosinophils was investigated in patients with hypereosinophilia. Hypodense activated eosinophils have been described in various diseases such as parasitic or allergic diseases, hypereosinophilic syndrome (HES) associated in some cases to myeloproliferative markers, and more recently described in patients undergoing recombinant IL2 treatment. The presence of p55 alpha chain of IL2R (CD25) on purified eosinophils collected from blood of hypereosinophilic patients was detected by flow cytometry. In 10 out of 19 cases, more than 10% of eosinophils were CD25+. Cross-linking studies on enriched eosinophils showed one 64-75-kDa band, consisting of IL2 (15 kDa) cross-linked to the IL2R p55 subunit. In Northern blot analysis the two messenger mRNA (3.5 and 1.5 kb) encoding the IL2R p55 subunit were identified after hybridization with a CD25 cDNA probe. In contrast, the presence of the IL2R p75 subunit was not detected. These data provide preliminary evidence for the expression of a low-affinity receptor for IL2 on in vivo activated eosinophils and raise the question of the role played by this cytokine in eosinophil differentiation and activation.

Eosinophilia↗
PubMed1991

Heterogeneous expression of CD23 epitopes by eosinophils from patients. Relationships with IgE-mediated functions.

The receptor for IgE (Fc epsilon RII) on human eosinophils presents some common characteristics with CD23, a differentiation marker of B cells. We have used flow cytometry for evaluating the expression of various epitopes of CD23 on purified eosinophils from patients with eosinophilia. A correlation was found between the binding of myeloma IgE protein and the binding of a monoclonal antibody (mAb 135), directed against the IgE-binding site of B cell CD23. Using two additional anti-CD23 mAb, directed (8-30) or not (3-5) against the IgE-binding site, a low expression of these CD23 epitopes was observed on eosinophils from different eosinophilic patients. Northern blot analysis of eosinophil RNA with the cDNA probe of CD23 revealed a low-abundance transcript in three of the six patients expressing membrane CD23. The inhibition by all anti-CD23 mAb of IgE-mediated cytotoxicity and IgE binding to eosinophils clearly indicated the participation of a CD23-related molecule in IgE-dependent eosinophil functions.

Antibodies, Monoclonal↗
PubMed1991

Release of granule proteins by eosinophils from allergic and nonallergic patients with eosinophilia on immunoglobulin-dependent activation.

The release of eosinophil peroxidase (EPO) and eosinophil cationic protein (ECP) was evaluated after incubation of eosinophils (EOSs) from allergic subjects with the specific allergen or with anti-IgE monoclonal antibodies (MAbs). High levels of EPO could be released after addition of the specific allergen (and not unrelated ones) or anti-IgE MAb. Moreover, EPO release with the two stimuli was significantly correlated both in allergic and in nonallergic patients. In the same supernatants, another granule protein, ECP, could not be detected, suggesting a lack of correlation between EPO and ECP release after IgE-dependent stimulation. However, when EOSs with surface-IgA antibodies were incubated with anti-IgA MAb, both EPO and ECP were released. In contrast, incubation of EOSs with anti-IgG MAb induced mainly the release of ECP and not EPO. These results indicate that pharmacologically active mediators can be released by EOSs from allergic and nonallergic patients on immunoglobulin-dependent activation. The results also confirm the hypothesis of a selective release of the various granule proteins and raise the question of transduction signals delivered by the three Fc receptors (Fc epsilon R, FC alpha R, and FC gamma R) present on human EOSs.

Allergens↗
PubMed1990

Characterization of a receptor for interleukin 5 on human eosinophils: variable expression and induction by granulocyte/macrophage colony-stimulating factor.

Interleukin 5 (IL-5) acts on eosinophil differentiation and activation, suggesting the existence of a membrane receptor for IL-5 on eosinophils. Here, we report that 125I-labeled recombinant human IL-5 bound, at 4 degrees C, to high affinity receptors on human eosinophils. The association constant was higher for hypodense eosinophils (1.93 x 10(9) M-1) than for normodense cells (0.39 x 10(9) M-1), with a closely related number of receptor sites per cell. No specific binding occurred on neutrophils. The specific binding of IL-5 was induced by overnight incubation at 37 degrees C of human eosinophils with granulocyte/macrophage (GM)-CSF. The levels of increase were significantly higher for normodense than for hypodense eosinophils, suggesting a previous in vivo activation of the later subpopulation by GM-CSF. IL-3 was ineffective by itself but synergistically enhanced the effect of GM-CSF. Specificity studies showed that the binding of 125I-labeled IL-5 was inhibited by IL-5, but not by other cytokines, on human eosinophils. These results show the existence of a specific binding site for IL-5 on human eosinophils with a variable affinity on eosinophil hypodense or normodense subpopulations, as previously reported for other membrane receptors.

Cells, Cultured↗
PubMed1990

Presence of antibodies against endothelial cells in the sera of patients with episodic angioedema and hypereosinophilia.

We reported three additional cases of a newly described syndrome called episodic angioedema with hypereosinophilia. In order to investigate its pathophysiological mechanisms, four parameters were concurrently investigated, including blood eosinophil density, serum chemoattractant activity, serum major basic protein (MBP) levels and the presence of anti-endothelial cell antibodies. Distribution of eosinophils through a metrizamide density gradient showed a preferential sedimentation of blood eosinophils in intermediate layers, clearly different from the hypodense cells (low-density layers) identified in a group of seven patients with idiopathic hypereosinophilic syndrome (HES). In two of the three patients with cyclic angioedema, a chemotactic activity towards eosinophils was detected in the serum (30 +/- 6 and 42 +/- 12 eosinophils per high-power field; P less than 0.05 compared with a control group). Serum MBP levels were at 1524, 619 and 1200 pg/ml. All three patients had circulating anti-endothelial cell antibodies, predominantly of the IgG isotype, in contrast to controls (P less than 0.01) or to patients with HES (P less than 0.01). Specificity of the antibody for endothelial cells was demonstrated in the three patients studied by the absence of binding to various blood cells, including monocytes, lymphocytes, eosinophils and platelets. In one case (patient 2), the levels of anti-endothelial cell antibodies, as well as the serum chemoattractant activity to eosinophils varied according to the successive acute phases of the disease. Although further investigations are needed to clarify the exact pathophysiology of this syndrome, and especially the possible participation of the anti-endothelial cell antibodies in the cutaneous lesions, these data suggest that angioedema observed in this syndrome could result from the combined effects of activated eosinophils and of immunologically induced endothelial lesions.

Adult↗
PubMed1989

IgE receptor on human eosinophils (FcERII). Comparison with B cell CD23 and association with an adhesion molecule.

IgE FcR (FcERII) on human eosinophils was characterized and compared with FcERII present on B cells (CD23). Two mAb, BB10 (anti-eosinophil FcERII) and 135 (anti-CD23), bound to the major component of FcERII at 45,000 to 50,000 Mr, both on purified hypodense eosinophils and on a B cell line (WIL-2WT). The specific ligand, human myeloma IgE, was able to bind to the molecules immunoprecipitated by BB10. A cross-reactivity between BB10 and a mAb anti-Leishmania gp63, which is a "fibronectin (Fn)-like" molecule, containing the L-arginine-L-glycyl-L-aspartyl (RGD) cell attachment domain indicated the presence of such a sequence in the common structure present on eosinophil and B cell FcERII. The synthetic tetrapeptide RGDS as well as its inverted sequence (SDGR) reduced the binding of BB10 and anti-Fn mAb to eosinophils and B cells. Flow microfluorometry analysis revealed a variable binding of BB10 and anti-Fn mAb to eosinophils purified from different patients, results compatible with recent findings on the inducibility of FcERIIb. The significant inhibition of IgE-dependent cytotoxicity against parasite targets by preincubation of eosinophils with BB10, anti-Fn and anti-CD23 mAb, with anti-RGDS polyclonal antibodies or with the SDGR peptide suggested the requirement of this cell adhesion sequence for the function of low affinity FcERII. The presence of such a sequence in the C-terminal domain of B cell FcERII raised the possibility of its role in B cell adhesion or B cell growth.

Amino Acid Sequence↗
PubMed1989

Variations in protein expression related to human eosinophil heterogeneity.

In hypereosinophilic patients, eosinophil heterogeneity has been assessed mainly according to morphologic and biologic criteria. In order to investigate the molecular basis of such heterogeneity, biochemical analysis was performed on various eosinophil subpopulations fractionated on metrizamide gradients. Whole cell extracts from purified eosinophils disrupted with a nonionic (NP-40) detergent were successively analyzed by SDS-PAGE and two-dimensional electrophoresis (isoelectric focusing or nonequilibrium pH gradient electrophoresis in the first dimension). Hypodense eosinophils that sediment in the lightest density gradients (18 to 22% metrizamide solution) differed from other purified eosinophils (intermediate and normodense eosinophils respectively collected in 22 to 23% and 23 to 25% metrizamide solutions). Comparative analysis of protein patterns on both monodimensional and bidimensional electrophoresis showed that a basic protein of Mr 51 kDa, present on normodense or intermediate eosinophils, was poorly detected in the case of hypodense eosinophils. In contrast, two other proteins with apparent Mr of about 23 kDa and 41 kDa were exclusively or predominantly identified in these latter cell fractions. Immunochemical analysis with polyclonal antibodies against eosinophil basic proteins and enzymatic assays revealed that the 51-kDa polypeptide could be related to an eosinophil peroxidase-like molecule. In addition, the two proteins detected only in hypodense eosinophils might be related to proteins newly synthesized by in vivo activated eosinophils. Our results suggest that variations in protein expression might represent a good marker of in vivo activation.

Blood Proteins↗
PubMed1989

Heterogeneity of human eosinophil glucocorticoid receptor expression in hypereosinophilic patients: absence of detectable receptor correlates with resistance to corticotherapy.

Assessment of steroid receptor content in human neoplastic lymphoid cells or mammary tumour cells has been previously used to predict steroid sensitivity in various types of cancers. In the present study, we have evaluated the relationship between glucocorticoid receptor content and the glucocorticoid sensitivity of human eosinophils, since hypereosinophilic patients do not always respond favourably to glucocorticoid, particularly in the hypereosinophilic syndrome (HES). Blood or alveolar eosinophils obtained from seven patients (four with HES without leukaemic markers; two with parasitic diseases; and one with eosinophilic pneumonia) displayed the same specific glucocorticoid receptor content as normal eosinophils (7.58 +/- 1.31 x 10(3) versus 7.76 +/- 0.74 x 10(3) sites/cell). In contrast, glucocorticoid-binding sites were undetectable in purified eosinophils collected from seven HES patients with (n = 3) or without (n = 4) leukaemic markers, whilst their mononuclear cells and/or neutrophils bound glucocorticoid. In one HES patient, kinetic studies showed that blood eosinophils initially positive in glucocorticoid binding assays became negative with the subsequent appearance of leukaemic markers. The absence of specific glucocorticoid binding sites was correlated with the absence of glucocorticoid receptor proteins by the use of a specific anti-glucocorticoid receptor monoclonal antibody. Eosinophil sensitivity to glucocorticoid was investigated by the evaluation of glucocorticoid inhibition of eosinophil chemotaxis and by the clinical outcome of in vivo glucocorticoid therapy. Our data provide evidence of the heterogeneity of eosinophil glucocorticoid receptor expression. In addition, the presence of glucocorticoid receptors is a prerequisite for glucocorticoid activity, in vitro and in vivo, on cells of the eosinophil lineage.

Drug Resistance↗