RESUMO
There are a number of problems associated with the development of standards suitable for use in the most commonly used assays to detect cytokines in biological fluids. These problems include: (i) the failure of some MoAbs used in immunoassays to detect all different <
Assuntos
Citocinas/análise , Substâncias de Crescimento/análise , Imunoensaio/métodos , Animais , Bioensaio , Líquidos Corporais/química , Citocinas/normas , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Substâncias de Crescimento/normas , Humanos , Imunoensaio/normas , Imunoensaio/estatística & dados numéricos , Interferon gama/análise , Interferon gama/normas , Interleucina-1/análise , Interleucina-1/normas , Interleucina-4/análise , Interleucina-4/normas , Camundongos , Padrões de ReferênciaRESUMO
Three ampouled preparations of interleukin-1 alpha (IL-1 alpha) and three ampouled preparations of interleukin-1 beta (IL-1 beta) were evaluated by 13 laboratories in six countries for their suitability to serve as international standards for these materials. The preparation were assayed in in vitro and in vivo bioassays, radioreceptor assays and immunoassays. On the basis of the results reported here, with the agreement of participants in the study and with the authorization of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO), one of the preparations of IL-1 alpha and one of the preparations of IL-1 beta were established as international standards for these materials. Further, since the relative activities of IL-1 alpha and IL-1 beta were dependent upon the assay systems used, it was decided that reference should be made to units of IL-1 alpha activity or IL-1 beta activity rather than to IL-1 activity.
Assuntos
Interleucina-1/normas , Estudos de Avaliação como Assunto , Interleucina-1/análise , Agências Internacionais , Controle de Qualidade , Ensaio Radioligante , Proteínas Recombinantes , Padrões de Referência , Sensibilidade e Especificidade , Organização Mundial da SaúdeRESUMO
We examined the biological effects induced by both human recombinant interleukin-1 alpha (IL-1 alpha) and beta (IL-1 beta) in five different cell types of human, rat and mouse origin. IL-1 alpha and beta preparations were standardized in terms of biological activity in the EL-4/CTLL bioassay and, in parallel, employed to stimulate PGE2 secretion in human fibroblasts, mesangial cells (MC), C57B1/6 mouse MC, DBA/2 mouse macrophages and Sprague Dawley rat MC. In addition, the co-mitogenic effects of IL-1 alpha and beta were determined in freshly prepared Sprague Dawley rat thymocytes. No significant differences in IL-1 alpha and beta concentration dependent PGE2 production were detectable in the different cell types (MC, fibroblasts and macrophages) of human or mouse origin. Incubation of Sprague Dawley rat MC with both IL-1 alpha and beta resulted in a concentration dependent production of PGE2. However, in contrast to mouse or human MC the potency of IL-1 beta to induce PGE2 in Sprague Dawley rat MC was 26-fold higher compared to IL-1 alpha. In addition, the potency of IL-1 beta to enhance co-stimulated proliferation of Sprague Dawley thymocytes was 200-fold higher than that of equal biological activities of IL-1 alpha. When we tested the additive effects on Sprague Dawley cells, increasing IL-1 beta concentrations added to a fixed IL-1 alpha concentration resulted in a cumulative rise in both, PGE2 secretion by MC and thymocyte proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Interleucina-1/farmacologia , Animais , Células Cultivadas , Dinoprostona/biossíntese , Mesângio Glomerular/efeitos dos fármacos , Mesângio Glomerular/metabolismo , Humanos , Indometacina/farmacologia , Interleucina-1/normas , Ativação Linfocitária , Polimixina B/farmacologia , Ratos , Ratos Endogâmicos , Linfócitos T/imunologiaRESUMO
Fresh human alveolar macrophages and blood monocytes were stimulated with LPS and assessed for their ability to produce and release antigenic IL-1 beta. Using a sensitive and specific ELISA for IL-1 beta, monocytes released 13.3 +/- 3.1 ng/10(6) cells compared to 3.5 +/- 0.8 ng/10(6) cells for alveolar macrophages (p less than 0.01). To investigate the reason for this difference in IL-1 beta release, monocytes were compared to alveolar macrophages for total IL-1 beta production (i.e., the amount released plus that detected in the lysates). Monocytes produced a total of 19.0 +/- 3.2 ng/10(6) cells whereas alveolar macrophages produced 24.8 +/- 5.6 ng/10(6) cells (p = 0.37). The relative increase in alveolar macrophage intracellular IL-1 beta was confirmed by Western blot analysis of cell lysates. Thus, the limitation in IL-1 release from alveolar macrophages appears to be due to a decrease in the processing and release of the IL-1 beta precursor. In addition, TNF production studies demonstrated that the limitation in IL-1 release was not a generalized defect. In contrast to the IL-1 beta data, when TNF was measured from monocytes and macrophages, monocytes released only 14.6 +/- 3.4 ng/10(6), whereas macrophages released 101 +/- 30 ng/10(6) (p less than 0.02). In this same context, when fresh monocytes were allowed to mature in vitro they took on monokine production characteristics similar to alveolar macrophages. In vitro matured monocytes had a greater than 20-fold decrease in their ability to release IL-1 beta and a 6- to 8-fold increase in their ability to release TNF. Taken together, these studies suggest that IL-1 beta release is limited in mature mononuclear phagocytes as compared to fresh blood monocytes, and furthermore, that IL-1 beta regulation differs significantly from that of TNF-alpha.
Assuntos
Interleucina-1/metabolismo , Macrófagos/análise , Monócitos/análise , Alvéolos Pulmonares/citologia , Adulto , Western Blotting , Diferenciação Celular , Sistema Livre de Células , Citosol/metabolismo , Endotoxinas/farmacologia , Ensaio de Imunoadsorção Enzimática/normas , Feminino , Humanos , Interleucina-1/normas , Ativação de Macrófagos , Macrófagos/imunologia , Masculino , Monócitos/imunologia , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Using three different monoclonal antibodies (McAb no. 374, no. 964 and no. 1190) to human interleukin-1 alpha (rHu-IL-1 alpha), we have established two sandwich enzyme immunoassays (EIA) to differentiate rHu-IL-1 alpha and its deamidated derivative (rHu-Asp36-IL-1 alpha) where the asparagine at position 36 (counting from the N-terminus) of rHu-IL-1 alpha is converted to Asp. The McAb no. 1190 reacts specifically with rHu-IL-alpha and not with the rHu-Asp36-IL-1 alpha whereas both no. 374 and no. 964 can react with the two different forms of rHu-IL-1 alpha. The first EIA (S-EIA I) which uses the McAb no. 964 labelled with horse-radish peroxidase and the McAb no. 1190 fixed to the microtiter plate, only measure rHu-IL-1 alpha. The second EIA (S-EIA II) which uses enzyme labelled no. 964 and no. 374 fixed to the plate, can detect both rHu-IL-1 alpha and rHu-Asp36-IL-1 alpha and this assay of total rHu-IL-1 alpha is comparable to a competitive EIA using an enzyme-labelled rHu-IL-1 alpha and an anti-rHu-IL-1 alpha polyclonal antibody. Thus, the level of rHu-Asp36-IL-1 alpha in the samples containing the two IL-1 alpha s can be calculated by subtracting the level measured by S-EIA I from that measured by S-EIA II. The two EIA systems with an assay range of 1.5-100 ng/ml do not recognize IL-1 beta, IL-2, rHu-TNF alpha, IFN-alpha and IFN-gamma of human origin.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Anticorpos Monoclonais , Soros Imunes , Técnicas Imunoenzimáticas , Interleucina-1/análise , Proteínas Recombinantes/análise , Animais , Anticorpos Monoclonais/classificação , Ácido Aspártico/metabolismo , Ligação Competitiva , Estabilidade de Medicamentos , Feminino , Humanos , Técnicas Imunoenzimáticas/normas , Interleucina-1/análogos & derivados , Interleucina-1/farmacocinética , Interleucina-1/normas , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Controle de Qualidade , Coelhos , Proteínas Recombinantes/análogos & derivados , Proteínas Recombinantes/normasRESUMO
Numerous studies have reported altered levels of in vitro production of the cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF) from blood leukocytes in various human disease states. Most of these studies have used bioassays which are vulnerable to inhibitors produced by these cells. Furthermore in vitro cytokine production is often assessed on a single occasion. The present study was designed to standardize stimulation conditions for in vitro IL-1 beta production and to employ a competitive radioimmunoassay (RIA) to demonstrate reproducibility and long-term variation of in vitro cytokine production in a cohort of healthy human subjects. We also examined relative amounts of immunoreactive IL-1 beta, IL-1 alpha, and TNF induced by the stimuli endotoxin, phytohemagglutinin, or Staphylococcus epidermidis. We show that the RIA can reliably detect IL-1 beta produced from mononuclear cells in concentrations as low as 115 pg/ml. Lysing cells by repeated freeze-thawing yields maximal recovery of total (i.e., secreted plus cell-associated) immunoreactive IL-1 beta, when compared to extraction with the detergent CHAPS or addition of protease inhibitors. Repeated measurement of in vitro cytokine production on different days within 1 week shows good reproducibility for a given individual and a given stimulus (variation coefficient 20 to 30%). Over a long time period (6 months) in vitro cytokine production is stable in some individuals but changes considerably in others. The soluble stimulus endotoxin induces twofold more IL-1 alpha than IL-1 beta or TNF; in contrast the phagocytic stimulus heat-killed S. epidermidis induces fourfold more IL-1 beta and TNF than IL-1 alpha. This distinct pattern of cytokine response indicates differential stimulation of the mononuclear cells by different stimuli. The results form the basis for studying in vitro cytokine production in different human disease states.
Assuntos
Interleucina-1/biossíntese , Leucócitos Mononucleares/análise , Fator de Necrose Tumoral alfa/biossíntese , Animais , Aprotinina , Congelamento , Humanos , Interleucina-1/isolamento & purificação , Interleucina-1/normas , Leucócitos Mononucleares/metabolismo , Fluoreto de Fenilmetilsulfonil , Coelhos , Valores de Referência , Solubilidade , Fator de Necrose Tumoral alfa/isolamento & purificação , Fator de Necrose Tumoral alfa/normasRESUMO
Two separate convenient sandwich enzyme immunoassay methods were developed for measuring the production of the monokines interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta) from peripheral blood mononuclear cells. Polyclonal antisera raised against the recombinant proteins and selected on the basis of their ability to neutralize IL-1-induced IL-2 secretion were used for coating microtiter plates or preparing peroxidase-Fab' conjugates. Both techniques were able to accurately and specifically detect monokines from various sources in the sub-picomolar range and were not influenced by compounds currently used for cell activation. A high molecular weight form of IL-1 beta was demonstrated under certain conditions and the two enzyme immunoassays were successfully applied to the detection of IL-1 alpha and IL-1 beta present in cell supernatants following stimulation with mitogenic or chemical agents.