RESUMO
Procalcitonin (PCT) is a highly sensitive and specific marker of systemic bacterial infection and sepsis. In contrast to its diagnostic significance, the cellular sources of plasma procalcitonin remain to be clarified. Two forms of PCT mRNAs originate from calcitonin/calcitonin gene-related peptide gene (CALC-I gene) along with mRNA for calcitonin gene-related peptide-I (CGRP-I). Reverse transcription polymerase chain reaction with newly designed primers detecting different PCT mRNAs and CGRP-I mRNA was used to identify tissues that might contribute to PCT production. Our study indicates that a variety of human tissues (13 of the 16 analyzed overall) express PCT-I, PCT-II, and/or CGRP-I mRNAs, with the highest levels detected for liver, testis, lung, prostate, kidney, and small intestine. Various tissues differ in the proportions of PCT-I, PCT-II, and CGRP-I mRNA expression levels. Thus we demonstrate the complexity of tissue-specific regulation of CALC-I gene expression and suppose a variety of tissues as a potential source of CALC-I-encoded peptides.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/genética , Calcitonina/genética , Precursores de Proteínas/genética , RNA Mensageiro/genética , Calcitonina/sangue , Peptídeo Relacionado com Gene de Calcitonina/sangue , Primers do DNA , DNA Complementar , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/fisiologia , Especificidade de Órgãos , Reação em Cadeia da Polimerase , Precursores de Proteínas/sangue , Mapeamento por Restrição , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The search for sensitive and specific markers of systemic infection has shown that procalcitonin levels are increased in sepsis, and, consequently, this plasma protein has come into the focus of clinical research. Human procalcitonin is encoded by the Calc-l gene, which gives rise to two alternatively spliced transcripts. Despite systemic investigation of the Calc-l gene and mechanisms of the tissue-specific regulation and alternative splicing, little is known about the biology of procalcitonin and the cells which express this protein during inflammation. Here we focus on the molecular and biochemical properties of the molecule and summarize the known biological functions of procalcitonin. We report on the structure of the Calc-l gene, the amino acid conservation of procalcitonin in different species, and the consensus sequences of the protein with regard to sites relevant for posttranslational modification, spatial distribution, and homologies to other cytokines. We discuss aspects of intracellular location of procalcitonin and demonstrate that it has the characteristics of a secreted protein.
Assuntos
Calcitonina/genética , Precursores de Proteínas/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Calcitonina/sangue , Calcitonina/química , Peptídeo Relacionado com Gene de Calcitonina , DNA Complementar , Regulação da Expressão Gênica , Humanos , Inflamação/genética , Macrófagos/metabolismo , Dados de Sequência Molecular , Monócitos/metabolismo , Biossíntese de Proteínas , Precursores de Proteínas/sangue , Precursores de Proteínas/química , Processamento de Proteína Pós-Traducional , Homologia de Sequência de AminoácidosRESUMO
Procalcitonin (PCT), the precursor of calcitonin, was recently put forward as a diagnostic marker of systemic bacterial infection and sepsis. The major PCT production site in sepsis still remains unclear. Because of a certain association between increased levels of PCT and leukocyte-derived cytokines during sepsis, we assessed the possible expression of PCT in human peripheral blood mononuclear cells (PBMCs) and the modulation of PCT by lipopolysaccharides (LPS) and various sepsis-related cytokines by reverse transcriptase-polymerase chain reaction (RT-PCR) by using a novel primer set and flow cytometric analysis with intracellular staining with antibodies to the PCT components calcitonin and katacalcin. RT-PCR and flow cytometric analysis demonstrated that PBMCs express PCT both on mRNA and on protein levels. LPS and various proinflammatory cytokines (interleukin-1beta (IL-1beta), IL-6, tumor necrosis factor-alpha (TNF-alpha), IL-2) had pronounced stimulatory effects on the expression of PCT mRNA. Under identical experimental conditions the anti-inflammatory cytokine IL-10 had no effect on the expression of mRNA for PCT. Flow cytometric analysis demonstrated increased intracellular amounts of PCT components after LPS stimulation. Thus we demonstrate for the first time that PCT is expressed in PBMCs. This expression is modulated by bacterial LPS and sepsis-related cytokines. Therefore PBMCs may be among the sources of elevated PCT levels in patients with sepsis.
Assuntos
Calcitonina/sangue , Citocinas/farmacologia , Lipopolissacarídeos/farmacologia , Monócitos/efeitos dos fármacos , Precursores de Proteínas/sangue , Sepse/metabolismo , Sequência de Bases , Calcitonina/genética , Peptídeo Relacionado com Gene de Calcitonina , DNA , Primers do DNA , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Técnicas In Vitro , Receptores de Lipopolissacarídeos/análise , Dados de Sequência Molecular , Monócitos/imunologia , Monócitos/metabolismo , Fragmentos de Peptídeos/sangue , Fito-Hemaglutininas/farmacologia , Precursores de Proteínas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Although cytokines are known to be involved in the regulation of a variety of hepatocellular functions, hepatocytes themselves are generally considered only targets but not producers of these important mediators. In order to investigate whether cells of hepatocellular linages are a potential source of various regulatory cytokines we have estimated the multiple cytokine gene expression in the culture of well differentiated human HepG2 hepatoma cells using RT-PCR. Our findings demonstrate that HepG2 cells express mRNAs for interferon gamma (IFN-gamma), tumour necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGF-beta), macrophage colony-stimulating factor (M-CSF), oncostatin-M (OSM), intercellular adhesion molecule (ICAM-1), interleukin 4 (IL-4), IL-5, IL-7, IL-10, IL-11, IL-12 and IL-6 receptor (IL-6R). At the same time the expression of IL-1, IL-2, IL-3, IL-6, CD40 ligand and IL-2R genes was not detected. It was concluded that hepatocytes are potential producers of a variety of cytokines, some of them being able to regulate hepatocellular functions directly, while others are important regulators of leukocyte activity. Thus, on the one hand, hepatocytes may express autoregulatory cytokines and on the other hand, influence the functions of other liver cells like Kupffer, Ito or endothelial cells. Due to their large amount, liver parenchymal cells could be an important source of sytemically acting pro- and anti-inflammatory and other regulatory cytokines.
Assuntos
Citocinas/genética , Citocinas/metabolismo , Expressão Gênica , Fígado/metabolismo , Antígenos CD40/genética , Antígenos CD40/metabolismo , Ligante de CD40 , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Interferon gama/genética , Interferon gama/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Leucócitos Mononucleares/metabolismo , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Oncostatina M , Peptídeos/genética , Peptídeos/metabolismo , RNA Mensageiro/metabolismo , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Immunofluorescence analysis of CD27 expression by CD4 lymphocytes from the peripheral blood of healthy humans or rheumatoid arthritis (RA) patients and from the synovial fluid (SF) of RA patients was carried out, along with the estimation of cytokine gene [interleukin (IL) 2, IL-3, IL-4, IL-5, IL-6, IL-6R, IL-10 and interferon-gamma (IFN-gamma)] expression in these lymphocyte subsets by RT-PCR. Although no differences in CD27-positive and -negative peripheral blood CD4 cell subset distribution were revealed, marked differences in IL-3, IL-4, IL-5 and IFN-gamma mRNA expression were detected between these lymphocyte subsets and between control and disease states. These results showed that phenotyping of different cell subsets in disease cannot provide adequate information about lymphocyte functional status. To estimate differences in cytokine gene expression, CD4 lymphocytes from the peripheral blood and SF of RA patients were compared. In both cases, mRNAs for IL-2, IL-4, IL-10 and IFN-gamma were detected, but CD4 cells from SF failed to express detectable levels of IL-5 mRNA despite our findings of a CD27-cell accumulation within the synovial population of CD4 lymphocytes. These are the first data to demonstrate that expression of the IL-5 gene in RA SF CD27- lymphocytes is down-regulated and that IL-5 disregulation in RA cannot be ruled out.
Assuntos
Artrite Reumatoide/genética , Antígenos CD4/análise , Citocinas/genética , Regulação da Expressão Gênica , Subpopulações de Linfócitos/fisiologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/análise , Artrite Reumatoide/imunologia , Citometria de Fluxo , Imunofluorescência , Humanos , Antígenos Comuns de Leucócito/análise , RNA Mensageiro/análise , Líquido Sinovial/citologia , Líquido Sinovial/metabolismoRESUMO
Normalizing influence of different lymphocyte populations and their soluble factors on L-929 transformed malignant fibroblasts (L cells) has been examined. It was demonstrated that splenic T-lymphocytes caused stable (heritable) normalization of receptor apparatus, biophysical and proliferative characteristics of L cells. Lymphokine with the activity of normalization factor (NF) was purified from 24-hour immunocyte conditioned medium. Stability of the normalization phenomenon was caused by NF induced synthesis of functionally analogous factor in L cells. The results obtained indicate the existence of non-cytotoxic mechanisms of tumour growth immunological control. The isolated lymphokine possessed also the activity of early embryonal cell differentiation factor. It is suggested that lymphokines with the activity of NF are physiological regulators of nonlymphoid cell differentiation.