RESUMO
In patients with eosinophilic esophagitis (EoE), symptoms often do not correlate with peak eosinophil counts (PEC) determined on histopathological examination of biopsy specimens. This may be because eosinophils degranulate during active disease and lose their morphological identity as intact cells and, therefore, are not enumerated on microscopic examination. Eosinophil granule proteins that are released into tissues with degranulation, including major basic protein 1 (eMBP1), likely contribute to disease pathogenesis and, therefore, may correlate with symptoms better than PEC. We sought to determine whether symptoms in patients with EoE more closely relate to eosinophil granule protein deposition than to eosinophil enumeration, especially in patients with fewer than 15 eosinophils per high power field (HPF). Esophageal biopsy specimens from 34 patients diagnosed with EoE were obtained for histopathological examination and for evaluation of eMBP1 staining by indirect immunofluorescence. PEC by histopathology were compared to extracellular eMBP1 grades by immunostaining. PEC and eMBP1 grades also were analyzed for their relationship to symptoms and clinical course. Biopsy specimens from 19 of the 34 patients had fewer than 15 PEC on histopathological examination, and the other 15 patients had 15 or greater PEC. Positive eMBP1 immunostaining was found in all symptomatic patients. EoE symptoms were related to eMBP1 immunostaining grades (p = 0.0001), but not PEC (P = 0.14). Eosinophil granule protein deposition, specifically eMBP1, is increased in esophageal biopsy specimens from symptomatic patients with EoE and may be a marker of disease activity, including patients with EoE who have 'resolved' disease.
Assuntos
Proteína Básica Maior de Eosinófilos/metabolismo , Esofagite Eosinofílica/metabolismo , Esofagite Eosinofílica/patologia , Eosinófilos/patologia , Proteoglicanas/metabolismo , Adulto , Idoso , Doenças Assintomáticas , Biomarcadores/metabolismo , Biópsia , Mucosa Esofágica/patologia , Feminino , Humanos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Avaliação de Sintomas , Adulto JovemRESUMO
Hereditary angioedema (HAE) is characterized by potentially life-threatening recurrent episodes of oedema. The open-label extension (OLE) phase of the For Angioedema Subcutaneous Treatment (FAST)-1 trial (NCT00097695) evaluated the efficacy and safety of repeated icatibant exposure in adults with multiple HAE attacks. Following completion of the randomized, controlled phase, patients could receive open-label icatibant (30 mg subcutaneously) for subsequent attacks. The primary end-point was time to onset of primary symptom relief, as assessed by visual analogue scale (VAS). Descriptive statistics were reported for cutaneous/abdominal attacks 1-10 treated in the OLE phase and individual laryngeal attacks. Post-hoc analyses were conducted in patients with ≥ 5 attacks across the controlled and OLE phases. Safety was evaluated throughout. During the OLE phase, 72 patients received icatibant for 340 attacks. For cutaneous/abdominal attacks 1-10, the median time to onset of primary symptom relief was 1·0-2·0 h. For laryngeal attacks 1-12, patient-assessed median time to initial symptom improvement was 0·3-1·2 h. Post-hoc analyses showed the time to onset of symptom relief based on composite VAS was consistent across repeated treatments with icatibant. One injection of icatibant was sufficient to treat 88·2% of attacks; rescue medication was required in 5·3% of attacks. No icatibant-related serious adverse events were reported. Icatibant provided consistent efficacy and was well tolerated for repeated treatment of HAE attacks.
Assuntos
Angioedemas Hereditários/tratamento farmacológico , Bradicinina/análogos & derivados , Adulto , Angioedemas Hereditários/diagnóstico , Bradicinina/administração & dosagem , Bradicinina/efeitos adversos , Bradicinina/uso terapêutico , Antagonistas dos Receptores da Bradicinina , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Retratamento , Resultado do Tratamento , Adulto JovemRESUMO
Several lines of evidence suggest that deficiency of eosinophils is not associated with any characteristic abnormality. Patients lacking eosinophils, in the setting of immunodeficiency or as a consequence of IgG-mediated eosinophil precursor destruction, do not display any distinguishing abnormalities related to eosinophil reduction. The observation that eosinophil-deficient mice do not display any distinctive syndrome or failure of their health is evidence that, under ordinary laboratory conditions, the eosinophil does not play a critical role in the well-being of mammals. Observations that monoclonal antibodies to interleukin-5 (IL-5) are well tolerated appear unsurprising in light of these findings. For example, patients with the hypereosinophilic syndrome have received mepolizumab, an anti-IL-5 monoclonal antibody, for as long as 6 years and have not developed any characteristic set of adverse events. Safety data for reslizumab, another anti-IL-5 monoclonal antibody, and benralizumab, a monoclonal antibody to the IL-5 receptor α-chain, are comparatively limited, especially for benralizumab, although reports of administration of these antibodies to humans suggest that they are well tolerated. Thus, data to the present suggest that reduction of eosinophils appears to have no characteristic ill effects on normal health, and monoclonal antibodies that deplete eosinophils have the potential to be widely employed in the treatment of eosinophil-associated diseases.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Eosinófilos/imunologia , Síndromes de Imunodeficiência/sangue , Síndromes de Imunodeficiência/imunologia , Timoma/imunologia , Neoplasias do Timo/imunologia , Animais , Anticorpos Monoclonais Humanizados/efeitos adversos , Estudos de Coortes , Modelos Animais de Doenças , Eosinófilos/metabolismo , Feminino , Humanos , Síndromes de Imunodeficiência/tratamento farmacológico , Incidência , Interleucina-5/efeitos adversos , Interleucina-5/imunologia , Interleucina-5/uso terapêutico , Contagem de Leucócitos , Masculino , Camundongos , Prognóstico , Medição de Risco , Timoma/sangue , Timoma/epidemiologia , Neoplasias do Timo/sangue , Neoplasias do Timo/epidemiologiaRESUMO
BACKGROUND: Mast cell (MC) - eosinophil (Eos) activating cross-talk might be critical for the severity and chronicity of allergy. Among soluble mediators, eosinophil major basic protein (MBP), a hallmark of allergy, is particularly important because it was shown to activate specific MC subtypes. We previously demonstrated that MBP activates IgE-desensitized rat MC and human lung and cord blood-derived MC (CBMC) after priming with fibroblast membranal stem cell factor. However, a distinct mechanism for this activation was missing. Therefore, we aimed to investigate it. METHODS: Major basic protein-1 activation of CBMC primed with fibroblast-derived membranes (FBM) was measured by ß-hexosaminidase and tryptase release. Chemical cross-linking followed by micrometric flow cytometry probed direct interactions. Antibodies neutralized integrin-ß1 and recognized its active form. Pertussis toxin (Ptx) was used to decrease integrin-ß1 active form expression. Hematopoietic cell kinase (Hck) was identified by immunoprecipitation (IP) and silenced by siRNA. RESULTS: Major basic protein-1-induced CBMC activation is mediated partly by MBP1-integrin-ß1 interaction on the MC surface. FBM prime CBMC via a G protein, as confirmed by Ptx, to shift integrin-ß1 to its active form. Following MBP1 binding, integrin-ß1 binds Hck that further transduces the activation signal. MC priming with FBM leads to up-regulation in Hck protein level. MC integrin-ß1 neutralization inhibits MBP1-induced activation and uptake. Hck silencing results with reduced MBP1-induced activation. CONCLUSIONS: Fibroblast-derived membranes, integrin-ß1, and Hck are involved in MBP1-induced activation of CBMC and therefore represent a distinct mechanism for this activation. This finding might implicate integrin-ß1 and Hck as targets for decreasing MC - Eos activating cross-talk in allergy.
Assuntos
Membrana Celular/imunologia , Proteína Básica Maior de Eosinófilos/imunologia , Eosinófilos/imunologia , Eosinófilos/metabolismo , Fibroblastos/imunologia , Integrina beta1/imunologia , Mastócitos/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Comunicação Celular/imunologia , Membrana Celular/metabolismo , Proteína Básica Maior de Eosinófilos/metabolismo , Fibroblastos/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Inativação Gênica , Humanos , Integrina beta1/metabolismo , Camundongos , Ligação Proteica , Proteínas Proto-Oncogênicas c-hck/genética , Proteínas Proto-Oncogênicas c-hck/metabolismoRESUMO
BACKGROUND: Acid-sensing ion channels (ASIC) are a family of acid-activated ligand-gated cation channels. As tissue acidosis is a feature of inflammatory conditions, such as allergic rhinitis (AR), we investigated the expression and function of these channels in AR. OBJECTIVES: The aim of the study was to assess expression and function of ASIC channels in the nasal mucosa of control and AR subjects. METHODS: Immunohistochemical localization of ASIC receptors and functional responses to lactic acid application were investigated. In vitro studies on cultured epithelial cells were performed to assess underlying mechanisms of ASIC function. RESULTS: Lactic acid at pH 7.03 induced a significant rise in nasal fluid secretion that was inhibited by pre-treatment with the ASIC inhibitor amiloride in AR subjects (n = 19). Quantitative PCR on cDNA isolated from nasal biopsies from control and AR subjects demonstrated that ASIC-1 was equally expressed in both populations, but ASIC-3 was significantly more highly expressed in AR (P < 0.02). Immunohistochemistry confirmed significantly higher ASIC-3 protein expression on nasal epithelial cells in AR patients than controls (P < 0.01). Immunoreactivity for EPO+ eosinophils in both nasal epithelium and submucosa was more prominent in AR compared with controls. A mechanism of induction of ASIC-3 expression relevant to AR was suggested by the finding that eosinophil peroxidase (EPO), acting via ERK1/2, induced the expression of ASIC-3 in epithelial cells. Furthermore, using a quantitative functional measure of epithelial cell secretory function in vitro, EPO increased the air-surface liquid depth via an ASIC-dependent chloride secretory pathway. CONCLUSIONS: This data suggests a possible mechanism for the observed association of eosinophils and rhinorrhoea in AR and is manifested through enhanced ASIC-3 expression.
Assuntos
Peroxidase de Eosinófilo/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Mucosa Nasal/metabolismo , Rinite Alérgica Sazonal/metabolismo , Canais de Sódio/biossíntese , Canais Iônicos Sensíveis a Ácido , Adolescente , Adulto , Biópsia , Células Cultivadas , Células Epiteliais/patologia , Feminino , Humanos , Ácido Láctico/farmacologia , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mucosa Nasal/patologia , Reação em Cadeia da Polimerase , Rinite Alérgica Sazonal/patologiaRESUMO
Eosinophil granule major basic protein (MBP), a potent toxin for helminths and various cell types, is a 13.8-kD single polypeptide rich in arginine with a calculated isoelectric point (pI) of 10.9. A cDNA for human MBP was isolated from a gamma GT10 HL-60 cDNA library. The nucleotide sequence of the MBP cDNA indicates that MBP is translated as a 25.2-kD preproprotein. The 9.9-kD pro-portion of proMBP is rich in glutamic and aspartic acids and has a calculated pI of 3.9, while proMBP itself has a calculated pI of 6.2. We suggest that MBP is translated as a nontoxic precursor that protects the eosinophil from damage while the protein is processed through the endoplasmic reticulum to its sequestered site in the granule core toxic MBP, and we present results from the literature suggesting that other cationic toxins, which damage cell membranes, may also be processed from nontoxic precursors containing distinct anionic and cationic regions.
Assuntos
Proteínas Sanguíneas/genética , DNA/genética , Eosinófilos , Precursores de Proteínas/genética , Ribonucleases , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Proteínas Granulares de Eosinófilos , Humanos , Dados de Sequência Molecular , Biossíntese de Proteínas , RNA Mensageiro/genéticaRESUMO
Elucidation of the functions of the eosinophil might be accomplished by analysis of the granule constituents. We have purified eosinophils (93% or greater) from the peritoneal cavity of the guinea pig and have investigated a variety of methods to disrupt cells and liberate intact granules. Lysis in 0.34 M sucrose gave the best yield of granules and these had the characteristic morphology of eosinophil granules when examined by electron microscopy. Granules were solubilized by a variety of treatments and the solutions analyzed by polyacrylamide electrophoresis at pH 3 in 6 M urea. Comparison of the electrophoretic patterns of solubilized eosinophil and neutrophil granules revealed a difference: a major portion (53+/-3%; x +/-1 SE) of the protein from the eosinophil granule migrated as a single component. This major band protein has a molecular weight between 6,000 and 12,000 daltons and a pI of 10 or greater. Analysis of eosinophil granule constituents on Sephadex G-50 revealed two main peaks; peak 1 possessed peroxidase activity and peak 2 contained the major band protein. These studies indicate that eosinophil granules contain a cationic protein of low molecular weight which lacks peroxidase activity and which accounts for greater than 50% of granule protein.
Assuntos
Proteínas Sanguíneas/análise , Eosinófilos/análise , Animais , Bioensaio , Proteínas Sanguíneas/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Eosinófilos/enzimologia , Liofilização , Cobaias , Corpos de Inclusão , Microscopia Eletrônica , PeroxidasesRESUMO
Charcot-Leyden crystals (CLC) are currently believed to be unique to the eosinophil and a hallmark of active eosinophilic inflammation or proliferation. The distinctiveness of the CLC to the eosinophil was questioned in 1965 by Archer and Blackwood (9), but their demonstration of CLC formation in basophils was ignored and later dismissed (1) as being the result of eosinophil contamination of basophil-enriched cell suspensions. We reexamined this question and showed that basophils obtained from the peripheral blood of normal individuals form CLC and that basophils contain a protein that is immunochemically indistinguishable from eosinophil CLC protein. These conclusions are based upon the findings that (a) crystal formation in basophils was demonstrated by specific histochemical staining of crystal-containing cells in highly enriched basophil suspensions prepared by fluorescence-activated cell sorter (FACS) purification of surface IgE-positive cells, (b) that enrichment for surface IgE-positive cells (primarily basophils) by the FACS also enriched for cells staining positively by immunofluorescence for eosinophil CLC protein, and (c) that CLC protein was measured by radioimmunoassay in cell extracts prepared from purified basophil suspensions containing 97-99% basophils and absolutely no contaminating eosinophils. These basophil extracts contained a protein immunochemically indistinguishable from eosinophil CLC protein. Based upon these findings, the CLC or the protein comprising the crystal (lysophospholipase) can no longer be considered as distinctive to the eosinophil. We must now consider the possibility that the presence of CLC in tissues, sputum, or stool may also represent basophil involvement in disease processes.
Assuntos
Basófilos/análise , Glicoproteínas/biossíntese , Animais , Basófilos/metabolismo , Separação Celular , Eosinófilos/análise , Citometria de Fluxo , Imunofluorescência , Congelamento , Glicoproteínas/análise , Humanos , Lisofosfolipase , CoelhosRESUMO
Two of the four principal cationic proteins of the eosinophil granule, major basic protein (MBP) and eosinophil peroxidase (EPO), were shown to be platelet agonists. Both MBP and EPO evoked a dose-dependent nonlytic secretion of platelet 5-hydroxytryptamine in unstirred platelet suspensions even in the presence of 10 microM indomethacin. MBP also evoked secretion of platelet alpha granule and lysosome components. Secretion by MBP and EPO was inhibited by 1 microM PGE1, but the nature of the inhibition differed from that observed with thrombin. Thus, MBP and EPO can be classified as strong platelet agonists with a distinct mechanism of activation.
Assuntos
Proteínas Sanguíneas/farmacologia , Eosinófilos/fisiologia , Ativação Plaquetária/efeitos dos fármacos , Ribonucleases , Alprostadil/farmacologia , Proteínas Granulares de Eosinófilos , Eosinófilos/enzimologia , Humanos , Técnicas In Vitro , Peroxidases/farmacologia , Serotonina/metabolismo , Trombose/etiologia , Tromboxano A2/biossínteseRESUMO
A protein immunochemically related to the eosinophil granule major basic protein (gMBP) is found in increased concentration in the plasma of pregnant women and has been localized to placental trophoblasts by immunofluorescence. Pregnancy MBP (pMBP) is indistinguishable from gMBP in its reactivity with polyclonal antisera and a panel of 14 mouse mAbs. We report the purification of pMBP from human placenta by: (a) affinity chromatography over mAb immobilized on Sepharose, (b) gel filtration in 6 M guanidine.HCl buffer, and (c) reversed-phase HPLC. Purified pMBP and gMBP are biochemically indistinguishable in that both: (a) bind to DNA, (b) polymerize and bind to carrier proteins via disulfide linkages, (c) have a molecular weight of 14,000, (d) have isoelectric points greater than 10.6, (e) comigrate in two-dimensional gels, (f) coelute during reversed-phase HPLC on C18 columns, (g) have identical peptide maps after three different digestions, and (h) have partial amino acid sequence identity. This physicochemical identity has important implications as to the role of pMBP in human placentation.
Assuntos
Proteínas Sanguíneas/isolamento & purificação , Eosinófilos/análise , Placenta/análise , Proteínas da Gravidez/análise , Ribonucleases , Sequência de Aminoácidos , Proteínas Sanguíneas/análise , Cromatografia de Afinidade , Proteínas Granulares de Eosinófilos , Feminino , Humanos , Ponto Isoelétrico , Peso Molecular , Mapeamento de Peptídeos , GravidezRESUMO
Human eosinophil peroxidase (EPO) was purified from eosinophil granules derived from the peripheral blood of patients with eosinophilia. The molecular mass of the H and L subunits was determined by gel filtration to be 57,000 and 11,000 daltons, respectively. The partial amino acid sequences of both subunits were used to construct oligonucleotides for the screening of several cDNA libraries, including one derived from human-induced umbilical cord mononuclear cells. A cDNA clone was isolated corresponding to EPO. The nucleotide sequence revealed an open reading frame of 2,106 bp, corresponding to a prosequence, L chain, and H chain, in this order. Comparison of the EPO nucleotide sequence with other peroxidases, such as myeloperoxidase, suggests the existence of a multigene family.
Assuntos
Clonagem Molecular , Eosinófilos/enzimologia , Família Multigênica , Peroxidase/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Cromatografia em Gel , DNA/genética , Sangue Fetal/citologia , Humanos , Leucócitos Mononucleares/enzimologia , Dados de Sequência Molecular , Peso Molecular , Hibridização de Ácido Nucleico , Peroxidase/isolamento & purificação , Homologia de Sequência do Ácido NucleicoRESUMO
Human peripheral blood eosinophils released eosinophil survival-enhancing activity when stimulated with the calcium ionophore, ionomycin. The release of activity was detected as early as 3 h after stimulation and was inhibited by an immunomodulating agent, cyclosporin A. The survival-enhancing activity was completely abolished by treatment with anti-interleukin 3 (IL-3) and anti-granulocyte/macrophage colony-stimulating factor (GM-CSF) monoclonal antibodies. Moreover, IL-3 and GM-CSF were measurable in ionomycin-stimulated eosinophil supernatants by immunoassay. Eosinophils produced approximately one-half as much IL-3 and one-fifth as much GM-CSF as ionomycin-stimulated mononuclear cells. Neutrophils also produced IL-3 and GM-CSF, but the amounts were less than those produced by eosinophils. These observations suggest a novel role for eosinophils in pathophysiology of allergic inflammation and host defense mechanisms.
Assuntos
Eosinófilos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Interleucina-3/metabolismo , Neutrófilos/metabolismo , Ribonucleases , Sobrevivência Celular/efeitos dos fármacos , Neurotoxina Derivada de Eosinófilo , Eosinófilos/citologia , Humanos , Técnicas In Vitro , Ionomicina/farmacologia , Neurotoxinas/metabolismo , Neutrófilos/citologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Asthma exacerbations, many of which are virus induced, are associated with airway eosinophilia. This may reflect altered inflammatory response to viruses in atopic individuals. Inhibitory M(2) muscarinic receptors (M(2)Rs) on the airway parasympathetic nerves limit acetylcholine release. Both viral infection and inhalational antigen challenge cause M(2)R dysfunction, leading to airway hyperresponsiveness. In antigen-challenged, but not virus-infected guinea pigs, M(2)R dysfunction is due to blockade of the receptors by the endogenous antagonist eosinophil major basic protein (MBP). We hypothesized that sensitization to a nonviral antigen before viral infection alters the inflammatory response to viral infection, so that M(2)R dysfunction and hyperreactivity are eosinophil mediated. Guinea pigs were sensitized to ovalbumin intraperitoneally, and 3 wk later were infected with parainfluenza. In sensitized, but not in nonsensitized animals, virus-induced hyperresponsiveness and M(2)R dysfunction were blocked by depletion of eosinophils with antibody to interleukin (IL)-5 or treatment with antibody to MBP. An additional and unexpected finding was that sensitization to ovalbumin caused a marked (80%) reduction in the viral content of the lungs. This was reversed by the antibody to IL-5, implicating a role for eosinophils in viral immunity.
Assuntos
Hiper-Reatividade Brônquica/etiologia , Eosinófilos/fisiologia , Inflamação/etiologia , Ovalbumina/imunologia , Infecções por Paramyxoviridae/imunologia , Receptores Muscarínicos/fisiologia , Animais , Pressão Sanguínea , Feminino , Cobaias , Frequência Cardíaca , Imunização , Interferon gama/biossíntese , Interleucina-5/fisiologia , Óxido Nítrico/fisiologia , Receptor Muscarínico M2 , Nervo Vago/fisiologiaRESUMO
Guinea pig eosinophil granules are characterized by the presence of a basic protein of low molecular weight which accounts for greater than 50% of granule protein. This protein, termed the major basic protein (MBP), readily aggregates and becomes insoluble, and the formation of aggregates is dependent on the establishment of disulfide bonds. Analysis of concentrated preparations of MBP often revealed a series of bands which were multiples of a monomeric unit with a mol wt of approximately 11,000. Analysis of reduced and alkylated MBP on a 10% agarose column equilibrated with 6 M guanidinium chloride revealed a single polypeptide chain with a mol wt of 10,800. Amino acid analysis of the protein revealed the presence of 13% arginine, consistent with the basic character of the molecule. Four residues of tryptophan, were present, indicating that MBP is not a histone. The MBP did not increase vascular permeability when injected into the skin of guinea pigs, nor did it antagonize the effect of histamine and bradykinin in the skin. MBP also did not contract the isolated guinea pig ileum and when mixed with histamine or bradykinin did not inhibit their activity on the gut. MBP had only weak, if any, antihistaminic activity. MBP possessed weak bactericidal activity when compared to histone and then only with one strain of E. coli. MBP precipitated DNA, neutralized heparin, and activated papain. On a molar basis MBP was more active than cysteine in activating papain. These results do not point to any unique biological activity associated with MBP other than those expected of a protein as basic as it is and one which possesses reactive sulfhydryl groups. Possible functions of eosinophils based on the properties of the MBP are discussed.
Assuntos
Proteínas Sanguíneas , Eosinófilos , Aminoácidos/análise , Animais , Bactérias/crescimento & desenvolvimento , Eletroforese das Proteínas Sanguíneas , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/isolamento & purificação , Vasos Sanguíneos , Bradicinina/farmacologia , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Eosinófilos/análise , Cobaias , Heparina , Histamina/farmacologia , Histonas/farmacologia , Íleo , Peso Molecular , Contração Muscular , Neutrófilos , Papaína/metabolismo , Permeabilidade , Pele/efeitos dos fármacos , Compostos de Sulfidrila/análiseRESUMO
In experiments using an immunofluorescent method to localize human eosinophil granule major basic protein (MBP) in cells and tissues, a small number of cells stained for MBP that subsequently could not be identified as eosinophils. Because the Charcot-Leyden crystal protein, another eosinophil protein, was recently identified in basophils, we tested whether MBP might also be a constituent of blood basophils. Highly purified, eosinophil-free basophil suspensions were prepared using the fluorescence-activated cell sorter (FACS) to sort basophil-containing mononuclear cell preparations stained with fluorescein-conjugated sheep IgG anti-human IgE antibody. Using these FACS-purified basophils, we found that: (a) enrichment for surface IgE-positive cells (greater than 95% basophils) by FACS also enriched for cells staining for MBP by immunofluorescence; (b) MBP appeared to be localized in the histamine-, heparin-containing granules; (c) significant quantities of MBP were measurable by radioimmunoassay (RIA) in freeze-thaw detergent extracts of purified basophils; and (d) RIA dose-response curves for extracts of purified eosinophils and basophils had identical slopes. The MBP content of basophils from normal individuals averaged 140 ng/10(6) cells, whereas purified eosinophils from normal donors and patients with the hyper-eosinophilic syndrome averaged 4,979 and 824 ng/10(6) cells, respectively. MBP was also detected by immunofluorescence and RIA in cells obtained from a patient with basophil leukemia, although the MBP content for basophil leukemia cells was lower than that for normal basophils. We conclude that basophils contain a protein that is immunochemically indistinguishable from eosinophil granule MBP.
Assuntos
Basófilos/análise , Proteínas Sanguíneas/análise , Ribonucleases , Basófilos/imunologia , Sítios de Ligação de Anticorpos , Ligação Competitiva , Proteínas Sanguíneas/imunologia , Separação Celular , Proteínas Granulares de Eosinófilos , Eosinófilos/análise , Citometria de Fluxo , Imunofluorescência , Humanos , Imunoglobulina E/análise , Leucemia/análise , Leucemia/imunologia , RadioimunoensaioRESUMO
Major basic protein (MBP) is a primary constituent of eosinophil granules. In this report, we demonstrate that MBP from human eosinophil granules initiates a nonlytic histamine release from human leukocytes. A direct effect of MBP on basophils was confirmed using purified human basophils. The kinetics of release were similar to those reported for poly-L-arginine, although MBP was less potent than poly-L-arginine of similar molecular weight. Reduction and alkylation of MBP diminished both the potency and efficacy of the molecule. Native MBP also stimulated histamine secretion from purified rat peritoneal mast cells in a manner characteristic of other polycations. These results emphasize the bidirectional nature of the basophil/mast cell-eosinophil regulatory axis.
Assuntos
Basófilos/metabolismo , Proteínas Sanguíneas/farmacologia , Eosinófilos/fisiologia , Histamina/metabolismo , Mastócitos/metabolismo , Ribonucleases , Animais , Basófilos/efeitos dos fármacos , Grânulos Citoplasmáticos/fisiologia , Proteínas Granulares de Eosinófilos , Humanos , Cinética , Masculino , Mastócitos/efeitos dos fármacos , Peptídeos/farmacologia , Ratos , Ratos Endogâmicos , Relação Estrutura-AtividadeRESUMO
We have shown that serum levels of a molecule immunochemically similar to eosinophil granule major basic protein (MBP) are elevated in pregnant women throughout gestation. MBP levels increase during gestation and plateau at approximately 7,500 ng/ml by the 20th wk (greater than 10-fold above normal). Levels return to normal after delivery, with a T1/2 of 13.7 d. The MBP in pregnancy serum is remarkably similar to the eosinophil granule MBP in that: (a) pregnancy MBP fully inhibits the binding of radiolabeled MBP standard in a double antibody radioimmunoassay; (b) this inhibition reaction is specific for human MBP because pregnancy serum produces no inhibition of the binding of radiolabeled guinea pig MBP in the guinea pig MBP radioimmunoassay; (c) in a two-site immunoradiometric assay for MBP, slopes of dose-response curves for pregnancy serum, purified MBP, and serum from a patient with hypereosinophilic syndrome are identical, and maximal binding is comparable; (d) reduction and alkylation of pregnancy sera increases measured MBP 100-fold, as previously shown for eosinophil granule MBP in serum; and (e) the MBP in pregnancy serum demonstrates the same pattern of heat lability as has been previously reported for MBP. Four observations have raised the possibility that the eosinophil is not the source of the MBP in pregnancy serum: (a) no correlation between serum MBP level and peripheral blood eosinophil count exists in pregnant women, in contrast to previous studies of patients with eosinophilia; (b) levels of three other eosinophil-associated proteins are normal or low in pregnancy sera, whereas the serum levels of these proteins are elevated in patients with eosinophilia; (c) the slopes of dose-response curves for pregnancy sera and MBP standards differ in the double antibody radioimmunoassay; and (d) the molecule in pregnancy serum elutes from Sephadex G-50 columns at the void volume, while eosinophil granule MBP and the MBP in serum of patients with eosinophilia elute at a volume consistent with the previously established molecular weight of 9,300. These findings suggest that the MBP in pregnancy serum is derived from a source other than the eosinophil.
Assuntos
Proteínas Sanguíneas/análise , Proteínas da Gravidez/análise , Ribonucleases , Animais , Ligação Competitiva , Proteínas Sanguíneas/imunologia , Proteínas Sanguíneas/fisiologia , Relação Dose-Resposta Imunológica , Proteínas Granulares de Eosinófilos , Eosinofilia/diagnóstico , Eosinofilia/imunologia , Feminino , Sangue Fetal/química , Cobaias , Humanos , Recém-Nascido , Período Pós-Parto , Gravidez , Proteínas da Gravidez/imunologia , Proteínas da Gravidez/fisiologiaRESUMO
Previous work (1)(1) has shown that normal human eosinophils show a preferential capacity, in comparison with neutrophils, to bind to antibody- coated schistosomula of Schistosoma mansoni. This effect is attributable to a temperature-dependent function of the eosinophil which renders its binding stable and irreversible by aggregated gamma globulin or Staphylococcus aureus protein A. In contrast, the binding of neutrophils is readily reversible by these agents. It has now been shown that the differences observed between eosinophils and neutrophils is a property of their interaction with living schistosomula. When dead or artificially damaged schistosomula were tested, neutrophils showed a markedly enhanced capacity to adhere, in both the presence and absence of anti-chistosomular serum. Subsequent experiments were designed to test the hypothesis that the strong, stable binding of eosinophils was attributable to degranulation, with release of granule contents which would then serve as ligands to bind the cell to the organism. First, an enhanced adherence both of eosinophils and of neutrophils could be demonstrated in the presence of eosinophil major basic protein (MBP) or of protamine, a high molecular weight cation. Second, the binding of eosinophils induced by concanavalin A (Con A) was found to differ markedly from that induced by antischistosomular serum. Con A-mediated binding of eosinophils was fully reversible by alpha-methyl-mannoside, was not associated with damage to the organism, and did not lead to degranulation of the cell, as estimated by measuring the release of MBP into the culture supernate. However, induction of degranulation of concanavalin A-bound eosinophils, but not of neutrophils, with the calcium ionophore A23187 converted the reaction into one which was no longer reversible by alpha- methylmannoside and in which damage to the organism now did occur. These findings support the hypothesis that the stable binding of eosinophils is associated with degranulation, a process which may contribute to the preferential capacity of this cell to mediate antibody-dependent damage to schistosomula.
Assuntos
Eosinófilos/imunologia , Imunidade Celular , Esquistossomose/imunologia , Anticorpos , Calcimicina/farmacologia , Adesão Celular/efeitos dos fármacos , Concanavalina A/farmacologia , Grânulos Citoplasmáticos/metabolismo , Humanos , Imunidade Celular/efeitos dos fármacos , Metilmanosídeos/farmacologia , Neutrófilos/imunologia , Schistosoma mansoni/imunologiaRESUMO
We have recently reported that human pregnancy is characterized by a 10- to 20-fold elevation of eosinophil major basic protein (MBP) immunoreactivity in maternal blood. Here we show, by immunofluorescence, that placental tissue specifically binds antibody to MBP in and around the placental X cells and placental-site giant cells and, using thin plastic sections, that placenta has no infiltrating eosinophils. The X cells line the inner aspects of placental septal cysts, and the cyst fluid, obtained by aspiration, contains immunoreactive MBP at concentrations of 100 micrograms/ml, a sixfold greater concentration than the highest levels measured in maternal blood. The soluble MBP immunoreactivities in placental homogenates and in maternal serum chromatograph identically on Sephadex G-50, and both these gestational MBP molecules migrate as though substantially larger than the MBP found in serum from patients with hypereosinophilic syndrome or purified from the eosinophil granule. Our inability to demonstrate eosinophils in maternal blood or placental tissue, coupled with the large quantities of immunoreactive MBP highly localized in placental cysts and the chromatographic behavior of this molecule, suggest that the MBP detected in human gestation is produced by placenta.