RESUMO
Over expression of hepcidin antimicrobial peptide is a common feature of iron-restricted anemia in humans. We investigated the erythroid response to either erythropoietin or RAP-011, a "murinized" ortholog of sotatercept, in C57BL/6 mice and in hepcidin antimicrobial peptide 1 over expressing mice. Sotatercept, a soluble, activin receptor type IIA ligand trap, is currently being evaluated for the treatment of anemias associated with chronic renal disease, myelodysplastic syndrome, ß-thalassemia, and Diamond Blackfan anemia and acts by inhibiting signaling downstream of activin and other Transforming Growth Factor-ß superfamily members. We found that erythropoietin and RAP-011 increased hemoglobin concentration in C57BL/6 mice and in hepcidin antimicrobial peptide 1 over expressing mice. While erythropoietin treatment depleted splenic iron stores in C57BL/6 mice, RAP-011 treatment did not deplete splenic iron stores in mice of either genotype. Bone marrow erythroid progenitors from erythropoietin-treated mice exhibited iron-restricted erythropoiesis, as indicated by increased median fluorescence intensity of transferrin receptor immunostaining by flow cytometry. In contrast, RAP-011-treated mice did not exhibit the same degree of iron-restricted erythropoiesis. In conclusion, we have demonstrated that RAP-011 can improve hemoglobin concentration in hepcidin antimicrobial peptide 1 transgenic mice. Our data support the hypothesis that RAP-011 has unique biologic effects which prevent or circumvent depletion of mouse splenic iron stores. RAP-011 may, therefore, be an appropriate therapeutic for trials in human anemias characterized by increased expression of hepcidin antimicrobial peptide and iron-restricted erythropoiesis.
Assuntos
Eritropoese/efeitos dos fármacos , Hemoglobinas/análise , Hepcidinas/genética , Ferro/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Receptores de Activinas Tipo II/química , Animais , Transporte Biológico , Contagem de Células Sanguíneas , Células Precursoras Eritroides/efeitos dos fármacos , Eritropoetina/farmacologia , Feminino , Imunoglobulina G/química , Ferro/sangue , Ligantes , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Baço/metabolismoRESUMO
Increased hepcidin antimicrobial peptide correlates with hypoferremia and anemia in various disease states, but its requirement for anemia of inflammation has not been adequately demonstrated. Anemia of inflammation is usually described as normocytic and normochromic, while diseases associated with over expression of hepcidin, alone, are often microcytic and hypochromic. These differences in erythrocyte parameters suggest anemia in many inflammatory states may not be fully explained by hepcidin-mediated iron sequestration. We used turpentine-induced sterile abscesses to model chronic inflammation in mice with targeted disruption of Hepcidin 1 [Hepc1 (-/-)] or its positive regulator, Interleukin-6 [IL-6 (-/-)], to determine whether these genes are required for features characteristic of anemia of inflammation. Although hemoglobin levels did not decline in Hepc1 (-/-) mice with sterile abscesses, erythrocyte numbers were significantly reduced compared to untreated Hepc1 (-/-) mice. In contrast, both hemoglobin concentration and erythrocyte number declined significantly in wild type and IL-6 (-/-) mice with sterile abscesses. Both Hepc1 (-/-) and IL-6 (-/-) mice had increased erythrocyte mean cell volume and mean cell hemoglobin following sterile abscesses, while wild types had no change. Thus, IL-6 (-/-) mice with sterile abscesses exhibit an intermediate phenotype between wild type and Hepc1 (-/-). Our results demonstrate the requirement of Hepc1 for the development of anemia in this rodent model. Simultaneously, our results demonstrate hepcidin-independent effects of inflammation on the suppression of erythropoiesis. Our results suggest chronic anemia associated with inflammation may benefit from interventions protecting erythrocyte number in addition to anti-hepcidin interventions aimed at enhancing iron availability.
Assuntos
Anemia/sangue , Eritropoese/fisiologia , Hepcidinas/sangue , Inflamação/sangue , Anemia/patologia , Animais , Modelos Animais de Doenças , Feminino , Imunofenotipagem , Inflamação/patologia , Ferro/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Amidst the rise of frailty among a globally aging population, olfactory decline has emerged as a harbinger of frailty and mortality in population-level studies. However, the relationships between frailty and the olfactory subdomains of identification (OI), discrimination (OD), and threshold (OT) remain unexplored. This study prospectively examined the association between olfactory subdomains and the physical frailty phenotype (PFP) to investigate olfactory evaluation as a means of frailty screening. METHODS: A caseâcontrol study of 45 frail and 45 non-frail individuals matched by age and sex. OT, OD, OI (range 0â16), and composite sum (threshold, discrimination, and identification scores [TDI], range 0â48) were measured with Sniffin' Sticks. PFP was defined by presence of three or more criteria: physical inactivity, self-reported exhaustion, muscle weakness, slow gait, and unintentional weight loss. Conditional logistic regression evaluated associations between olfactory subdomains and frailty. RESULTS: Ninety individuals with mean age of 83.1 ± 4.9 years, 60% female (n = 54), and 87.8% white (n = 79) were included. Olfactory scores were significantly lower in the frail group for OI (9.2 vs. 12.1, p < 0.001), OD (8.1 vs. 11.6, p < 0.001), OT (4.4 vs. 8.5, p < 0.001), and TDI (21.7 vs. 32.2, p < 0.001) than in the non-frail group. A single-point decrease in olfactory score was associated with increased odds of frailty in OT (odds ratio [OR]: 2.21, 95% confidence interval: [1.22, 3.98]), OD (OR: 2.19, 95% CI: [1.32, 3.65]), OI (OR: 2.29, 95% CI: [1.19, 4.39]), and TDI (OR: 1.54, 95% CI: [1.14, 2.08]). CONCLUSION: The robust association between olfactory subdomain scores and frailty suggests that olfaction may be an accessible signifier of frailty. Future studies should investigate this relationship longitudinally to assess predictive relationships.
RESUMO
Anemia is common in older adults and associated with adverse health outcomes in epidemiological studies. A thorough understanding of the complex pathophysiological mechanisms driving anemia in the elderly is lacking; but inflammation, iron restriction, and impaired erythroid maturation are thought to influence the phenotype. We hypothesized that interleukin-6 contributes to this anemia, given its pro-inflammatory activities, its ability to induce hepcidin antimicrobial peptide, and its negative impact on several tissues in older adults. We tested this hypothesis by comparing changes in indices of inflammation, iron metabolism and erythropoiesis in aged C57BL/6 mice to aged mice with targeted deletions of interleukin-6 or hepcidin antimicrobial peptide. Circulating neutrophil and monocyte numbers and inflammatory cytokines increased with age. Decline in hemoglobin concentration and red blood cell number indicated that C57BL/6, interleukin-6 knockout mice, and hepcidin antimicrobial peptide knockout mice all demonstrated impaired erythropoiesis by 24 months. However, the interleukin-6 knock out genotype and the hepcidin antimicrobial peptide knock out genotype resulted in improved erythropoiesis in aged mice. Increased erythropoietic activity in the spleen suggested that the erythroid compartment was stressed in aged C57BL/6 mice compared to aged interleukin-6 knockout mice. Our data suggest C57BL/6 mice are an appropriate mammalian model for the study of anemia with age. Furthermore, although interleukin-6 and hepcidin antimicrobial peptide are not required, they can participate in the development of anemia in aging mice, and could be targeted, pre-clinically, with existing interventions to determine the feasibility of such agents for the treatment of anemia in older adults.
Assuntos
Envelhecimento/genética , Envelhecimento/metabolismo , Anemia/sangue , Anemia/genética , Hepcidinas/fisiologia , Interleucina-6/fisiologia , Animais , Feminino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade da EspécieRESUMO
BACKGROUND: We and others have shown previously that over-expression of hepcidin antimicrobial peptide, independently of inflammation, induces several features of anemia of inflammation and chronic disease, including hypoferremia, sequestration of iron stores and iron-restricted erythropoiesis. Because the iron-restricted erythropoiesis evident in hepcidin transgenic mice differs from the normocytic, normochromic anemia most often observed in anemia of inflammation, we tested the hypothesis that chronic inflammation may contribute additional features to anemia of inflammation which continue to impair erythropoiesis following the acute phase of inflammation in which hepcidin is active. DESIGN AND METHODS: We compared erythropoiesis and iron handling in mice with turpentine-induced sterile abscesses with erythropoiesis and iron handling in hepcidin transgenic mice. We compared erythrocyte indices, expression of genes in the hepcidin regulatory pathway, tissue iron distribution, expression of heme and iron transport genes in splenic macrophages, the phenotype of erythroid maturation and chloromethyl dichlorodihydrofluorescein diacetate, acetyl ester fluorescence. RESULTS: Mice with sterile abscesses exhibited an intense, acute inflammatory phase followed by a mild to moderate chronic inflammatory phase. We found that erythrocytes in mice with sterile abscesses were normocytic and normochromic in contrast to those in hepcidin transgenic mice. We also observed that although hypoferremia resolved in the late phases of inflammation, erythropoiesis remained suppressed, with evidence of inefficient maturation of erythroid precursors in the bone marrow of mice with sterile abscesses. Finally, we observed increased oxidative stress in erythroid progenitors and circulating erythrocytes of mice with sterile abscesses which was not evident in hepcidin transgenic mice. CONCLUSIONS: Our results suggest that chronic inflammation inhibits late stages of erythroid production in the turpentine-induced sterile abscess model and induces features of impaired erythropoiesis which are distinct from those in hepcidin transgenic mice.
Assuntos
Peptídeos Catiônicos Antimicrobianos/biossíntese , Células Precursoras Eritroides/metabolismo , Eritropoese , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Doença Crônica , Células Precursoras Eritroides/patologia , Hepcidinas , Inflamação/sangue , Inflamação/induzido quimicamente , Inflamação/genética , Mediadores da Inflamação/sangue , Irritantes/efeitos adversos , Irritantes/farmacologia , Camundongos , Camundongos Transgênicos , Terebintina/efeitos adversos , Terebintina/farmacologiaRESUMO
Previously, we demonstrated a negative correlation between histamine release to histamine-releasing factor/translationally controlled tumor protein (HRF/TCTP) and protein levels of SHIP-1 in human basophils. The present study was conducted to investigate whether suppressing SHIP-1 using small interfering (si)RNA technology would alter the releasability of culture-derived mast cells and basophils, as determined by HRF/TCTP histamine release. Frozen CD34+ cells were obtained from the Fred Hutchinson Cancer Research Center (Seattle, WA, USA). Cells were grown in StemPro-34 medium containing cytokines: mast cells with IL-6 and stem cell factor (100 ng/ml each) for 6-8 weeks and basophils with IL-3 (6.7 ng/ml) for 2-3 weeks. siRNA transfections were performed during Week 6 for mast cells and Week 2 for basophils with siRNA for SHIP-1 or a negative control siRNA. Changes in SHIP-1 expression were determined by Western blot. The functional knockdown was measured by HRF/TCTP-induced histamine release. siRNA knockdown of SHIP-1 in mast cells ranged from 31% to 82%, mean 65 +/- 12%, compared with control (n=4). Histamine release to HRF/TCTP was increased only slightly in two experiments. SHIP-1 knockdown in basophils ranged from 34% to 69%, mean 51.8 +/- 7% (n=4). Histamine release to HRF/TCTP in these basophils was dependent on the amount of SHIP knockdown. Mast cells and basophils derived from CD34+ precursor cells represent suitable models for transfection studies. Reducing SHIP-1 protein in cultured mast cells and in cultured basophils increases releasability of the cells.
Assuntos
Basófilos/fisiologia , Biomarcadores Tumorais/farmacologia , Liberação de Histamina/efeitos dos fármacos , Mastócitos/fisiologia , Toxina Pertussis/farmacologia , Monoéster Fosfórico Hidrolases/deficiência , Basófilos/citologia , Basófilos/efeitos dos fármacos , Células Cultivadas , Citometria de Fluxo , Humanos , Imunoglobulina E/farmacologia , Inositol Polifosfato 5-Fosfatases , Mastócitos/citologia , Mastócitos/efeitos dos fármacos , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/genética , RNA Interferente Pequeno/genética , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
BACKGROUND: We have previously defined IgE+ as the IgE on basophils from a subset of highly allergic asthmatic subjects that release histamine after stimulation with histamine-releasing factor (HRF). The mechanism of IgE+ remains an enigma. Recently, there have been reports of monomeric highly cytokinergic IgEs causing mediator release, cytokine release, and phosphorylation events in cultured rodent and human mast cells in the absence of antigen. OBJECTIVE: We investigated whether human IgE+ might exist as highly cytokinergic IgE in the human system. METHODS: IgE+ was defined as causing greater than 10% histamine release by using HRF as a stimulus of human basophils. By definition, IgE- did not support histamine release to HRF. Once defined, serum and various purified human IgEs were used to stimulate purified human basophils or cultured human mast cells. The cells were examined for histamine release, extracellular signal-regulated kinase (ERK) phosphorylation, and IL-13 secretion. RESULTS: We found that neither IgE+ nor IgE- induced ERK phosphorylation, histamine release, and IL-13 release from freshly isolated basophils in the absence of a specific antigen. However, human IgE alone did stimulate ERK phosphorylation in cultured human mast cells and IL-3-primed human basophils. CONCLUSION: Human IgE+ is not an equivalent of the mouse highly cytokinergic IgE. However, human IgE did have effects on cultured mast cells and basophils. The effect of highly cytokinergic IgE on ERK phosphorylation and cytokine secretion might be due to the priming effect of human basophils and mast cells.
Assuntos
Citocinas/biossíntese , Imunoglobulina E/biossíntese , Imunoglobulina E/metabolismo , Anafilaxia/imunologia , Animais , Basófilos/imunologia , Basófilos/metabolismo , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Separação Celular , Células Cultivadas , Citocinas/fisiologia , Humanos , Imunoglobulina E/fisiologia , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
When using a model to study disease, it may be advantageous to identify molecules responsible for biologic functions observed in the model to better understand the disease process being studied. The late phase reaction is used as a model for chronic inflammation, and the histamine releasing activity observed from late phase fluids was thought to be an important factor in the propagation of symptoms that remain in both the late-phase reaction and in chronic inflammation, when the offending antigen is no longer present. Purification from biologic fluids and identification may be helpful in understanding the role of the histamine-releasing factors in inflammation. Once the specific molecule is identified and cloned, techniques such as yeast two-hybrid screens and co-immunoprecipitation experiments can be used to identify binding partners and further elucidate the role of the cloned molecule. The purification and cloning of human recombinant histamine-releasing factor and the subsequent yeast two-hybrid screen and co-immunoprecipitation will be described to illustrate how any functionally defined molecule can be investigated.
Assuntos
Liberação de Histamina , Imunoprecipitação/métodos , Proteínas de Neoplasias/análise , Proteínas Nucleares/análise , Técnicas do Sistema de Duplo-Híbrido , Biomarcadores Tumorais , Linhagem Celular , Clonagem Molecular , Histamina/metabolismo , Humanos , Inflamação/imunologia , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
The human recombinant histamine releasing factor (HrHRF), also known as translationally controlled tumor protein (TCTP), p23 and fortilin, has been described to have both extra- and intracellular functions. To elucidate an extra- or intracellular role for HrHRF, we used the yeast two-hybrid system with HrHRF as the bait and a Jurkat T cell library. We isolated a partial cDNA clone of the human elongation factor-1 delta (EF-1delta) encoding for amino acids 12 to 281. This interaction was confirmed by co-immunoprecipitation experiments. Previously, both HrHRF and EF-1delta have been isolated and identified in association with malignancy in numerous studies. EF-1delta is part of the EF-1 complex responsible for kinetic proofreading in protein synthesis. Additionally, DNA microarray data classifies TCTP (HrHRF) as co-regulated with ribosomal proteins and recent structural analysis of TCTP (HrHRF) relates it to a guanine nucleotide-free chaperone. Our findings of an interaction between HrHRF and EF-1delta taken with some of the recently published information concerning the TCTP (HrHRF) mentioned above suggest a possible intracellular role for TCTP/HrHRF.
Assuntos
Biomarcadores Tumorais/metabolismo , Fator de Iniciação 1 em Eucariotos/metabolismo , Fator 1 de Elongação de Peptídeos/genética , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Clonagem Molecular , DNA Complementar/genética , Fator de Iniciação 1 em Eucariotos/genética , Humanos , Células Jurkat , Proteínas Recombinantes/metabolismo , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
BACKGROUND: Asthma is a disease that affects all ages, races and ethnic groups. Its incidence is increasing both in Westernized countries and underdeveloped countries. It involves inflammation, genetics and environment and therefore, proteins that exacerbate the asthmatic, allergic phenotype are important. Our laboratory purified and cloned a histamine releasing factor (HRF) that was a complete stimulus for histamine and IL-4 secretion from a subpopulation of allergic donors' basophils. Throughout the course of studying HRF, it was uncovered that HRF enhances or primes histamine release and IL-13 production from all anti-IgE antibody stimulated basophils. In order to further delineate the biology of HRF, we generated a mouse model. METHODOLOGY/PRINCIPAL FINDINGS: We constructed an inducible transgenic mouse model with HRF targeted to lung epithelial cells, via the Clara cells. In antigen naïve mice, overproduction of HRF yielded increases in BAL macrophages and statistical increases in mRNA levels for MCP-1 in the HRF transgenic mice compared to littermate controls. In addition to demonstrating intracellular HRF in the lung epithelial cells, we have also been able to document HRF's presence extracellularly in the BAL fluid of these transgenic mice. Furthermore, in the OVA challenged model, we show that HRF exacerbates the allergic, asthmatic responses. We found statistically significant increases in serum and BAL IgE, IL-4 protein and eosinophils in transgenic mice compared to controls. CONCLUSIONS/SIGNIFICANCE: This mouse model demonstrates that HRF expression enhances allergic, asthmatic inflammation and can now be used as a tool to further dissect the biology of HRF.
Assuntos
Biomarcadores Tumorais/metabolismo , Modelos Animais de Doenças , Animais , Asma/genética , Asma/metabolismo , Sequência de Bases , Biomarcadores Tumorais/genética , Western Blotting , Líquido da Lavagem Broncoalveolar , Primers do DNA , Proteínas de Fluorescência Verde/genética , Humanos , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Tumoral 1 Controlada por Tradução , Uteroglobina/genéticaRESUMO
We previously identified a negative correlation between histamine release to histamine releasing factor/translationally controlled tumor protein (HRF/TCTP) and protein levels of the Src homology 2 domain-containing inositol 5' phosphatase (SHIP) in basophils. We have also demonstrated that HRF/TCTP primes basophils to release mediators. The purpose of this study was to begin characterization of signal transduction events directly induced by HRF/TCTP and to investigate these events when HRF/TCTP is used as a priming agent for human basophil histamine release. Highly purified human basophils were examined for surface expression of bound HRF/TCTP, changes in calcium, and phosphorylation of Akt, mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated kinase (ERK), Syk, and FcepsilonRIgamma. Results showed that basophils from all donors bound HRF/TCTP. There was a biphasic calcium response to HRF/TCTP, which corresponded to the magnitude of histamine release. Furthermore, those donors who have direct histamine release when exposed to HRF/TCTP (HRF/TCTP responder [HRF/TCTP-R] donors) have phosphorylation of Syk, Akt, MEK, and ERK. Remarkably, basophils from HRF/TCTP-nonresponder (HRF/TCTP-NR) donors do not show phosphorylation of these molecules. This finding is different from IL-3, which also primes basophils for histamine release, but does show phosphorylation of these events. We conclude that priming induced by HRF/TCTP is distinct from that induced by IL-3.
Assuntos
Basófilos/fisiologia , Biomarcadores Tumorais/fisiologia , Basófilos/citologia , Biomarcadores Tumorais/genética , Cálcio/sangue , Liberação de Histamina/fisiologia , Humanos , Interleucina-3/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucotrieno A4/sangue , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Receptores de IgE/sangue , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Quinase Syk , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
In the general population, Syk expression in human basophils is highly variable and correlates well with the IgE-mediated responsiveness of these cells. Previous studies established that IgE-mediated stimulation results in loss of Syk expression. The current studies investigated whether stimulation through other receptors results in loss of Syk. Two classes of stimulation were examined, those that operate through the kinase Syk and those that operate through a GTP-binding protein. These studies demonstrated that aggregation of leukocyte Ig-like receptor LILRA-2 resulted in phosphorylation of Syk and c-Cbl, was inhibited by a third generation Syk inhibitor with an expected IC(50), and induced histamine release in strict proportion to release induced by anti-IgE Ab. Stimulation of LILRA-2 for 18 h resulted in modest loss of Syk that correlated with the more profound loss of Syk induced by anti-IgE Ab. Human recombinant histamine-releasing factor has also recently been shown to induce Syk phosphorylation and in the current studies has also been shown to induce loss of Syk in 18-h cultures. fMLP stimulation for 18 h was also found to induce modest loss of Syk. fMLP induced phosphorylation of c-Cbl that was sustained for at least 45 min. Phosphorylation of c-Cbl was inhibited by a Syk kinase inhibitor but with an IC(50) that was not consistent with Syk activity, suggesting another kinase was responsible for Cbl phosphorylation following fMLP. These studies demonstrate that it is possible to induce the loss of Syk expression in human basophils by a non-IgE-dependent mechanism and even by a mechanism that does directly involve Syk in the reaction complex.
Assuntos
Basófilos/imunologia , Basófilos/metabolismo , Imunoglobulina E/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Receptores de IgE/fisiologia , Basófilos/enzimologia , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/fisiologia , Células Cultivadas , Regulação para Baixo/imunologia , Liberação de Histamina/imunologia , Humanos , Soros Imunes/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/biossíntese , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-cbl/biossíntese , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Proteínas Proto-Oncogênicas c-cbl/fisiologia , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Receptores Imunológicos/fisiologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/imunologia , Quinase Syk , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
Human recombinant histamine-releasing factor (HrHRF) preincubation enhances the secretion of histamine, IL-4, and IL-13 from FcepsilonRI-stimulated human basophils. In GM-CSF-primed human eosinophils, HrHRF increases IL-8 production. Our recent experiments were designed to evaluate the effects of HrHRF on human T cell cytokine production. Purified T cells were preincubated with GST-tagged HrHRF, followed by stimulation with PMA and A23187 overnight. A partial inhibition of IL-2 and IL-13 production (30 and 75%, respectively) was detected compared with that in cells treated with PMA/A23187 alone. However, the production of IFN-gamma was similar in PMA/A23187 stimulated cells with or without HrHRF. The inhibition of cytokine protein production was dose dependent and specific to the HrHRF portion of GST-HrHRF. The inhibition was not due to endotoxin, since preincubation with polymyxin B and HrHRF gave similar results to that with HrHRF alone. The same pattern and specificity of cytokine regulation were replicated in the Jurkat T cell line as for primary T cells. The PMA/A23187-stimulated activity of a proximal promoter IL-13, IL-4, or IL-2 luciferase construct transfected into Jurkat cells was partially inhibited (60, 32, or 70%, respectively) upon GST-HrHRF preincubation, suggesting that HrHRF functions to inhibit cytokine production in Jurkat cells by preventing gene transcription. The inhibition of IL-2 promoter activation was specific to the HrHRF portion of GST-HrHRF. We conclude that HrHRF, in addition to functioning as a histamine-releasing factor, can differentially modulate the secretion of cytokines from human basophils, eosinophils, T cells, and murine B cells, suggesting that it may induce a complex array of responses at sites of allergic inflammation.
Assuntos
Biomarcadores Tumorais/fisiologia , Citocinas/antagonistas & inibidores , Citocinas/genética , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Transcrição Gênica/imunologia , Apoptose/imunologia , Biomarcadores Tumorais/farmacologia , Antígenos CD28/fisiologia , Calcimicina/farmacologia , Divisão Celular/imunologia , Separação Celular , Citocinas/metabolismo , Relação Dose-Resposta Imunológica , Sinergismo Farmacológico , Regulação da Expressão Gênica/imunologia , Liberação de Histamina/genética , Liberação de Histamina/imunologia , Humanos , Interleucina-13/antagonistas & inibidores , Interleucina-13/biossíntese , Interleucina-13/genética , Interleucina-13/metabolismo , Interleucina-2/antagonistas & inibidores , Interleucina-2/genética , Interleucina-2/metabolismo , Interleucina-4/antagonistas & inibidores , Interleucina-4/genética , Interleucina-4/metabolismo , Células Jurkat , Regiões Promotoras Genéticas/imunologia , Receptores de Antígenos de Linfócitos T/fisiologia , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/fisiologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Tempo , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
BACKGROUND: Activity of immunoglobulin (Ig)E-dependent histamine-releasing factor (HRF) is dependent on the IgE molecules bound to the surface of basophils. Sera capable of passively sensitizing basophils to release histamine to HRF were designated IgE+ sera. IgE+ and HRF have been suggested to play a role in late allergic reaction (LAR). OBJECTIVE: The working hypothesis was tested that IgE+ induces a LAR. Further, activity of HRF produced by mononuclear cells (HRF(mn)) was compared with that of recombinant HRF p23. METHODS: Atopic patients (n = 82) were bronchially provoked with Dermatophagoides pteronyssinus extract and the change in forced expiratory volume in 1 second was monitored. A LAR was defined as forced expiratory volume in 1 second as percentage of baseline < 80% 4 to 10 hours after allergen challenge. The presence of HRF-responsive IgE in serum was determined using basophils sensitized in vitro by serum. RESULTS: The presence of HRF(mn)-responsive IgE (IgE(mn+)) in serum was shown not be essential for a LAR: 63% of the patients with a LAR had no IgE(mn+) in their serum. Further, 71% of patients with IgE(mn+) did not have a LAR. HRF(mn) and recombinant HRF p23 were not equivalent in the bioassay: serum of 38 of 82 atopic patients sensitized basophils to release histamine to HRF(mn), whereas this was found with serum of 1 of 82 patients to HRF p23. CONCLUSIONS: The results do not support the hypothesis that IgE(mn+) induces a LAR, but do not exclude the alternative hypothesis that HRFs are released during a LAR and contribute to asthma severity.