RESUMO
BACKGROUND: Lipopolysaccharide (LPS) is a potent activator of human monocytic cells. We have determined that LPS stimulation of the human monocytic cell line, THP-1, results in an increased apoptotic rate. We hypothesized that cDNA expression array analysis could be used to identify target genes involved in the regulation of this process. METHODS: THP-1 cells (1 x 10(6)/mL) were stimulated with LPS (1 microg/mL) or vehicle control. Apoptosis was measured at 0, 24, 48, 72 and 96 h using propidium iodide staining and flow cytometry to determine the percentage of cells with hypodiploid DNA. At 16 h, the Atlas Human cDNA expression array system, containing probes for 205 genes related to apoptosis, was used to survey and quantify transcript expression. The experiment was performed in duplicate and the membranes were normalized to cytoplasmic beta-actin. Standard Western blotting was performed on the conditioned medium to correlate secreted protein expression with RNA expression. Pretreatment with insulin-like growth factor I (IGF-I) was performed to determine whether the effects of insulin-like growth factor binding protein-3 (IGFBP-3) on apoptosis were IGF-dependent. RESULTS: LPS stimulation of THP-1 cells resulted in a greater than 2-fold increase in the rate of apoptosis when compared to vehicle control. When the cDNA expression arrays were compared, there was a 500-fold increase in the expression of the IGFBP-3 transcript in the LPS-stimulated cells. Western blotting of culture medium verified an approximately 2-fold increase in secreted IGFBP-3. Pretreatment with IGF-I did not prevent the increase in apoptosis seen with LPS stimulation. CONCLUSIONS: THP-1 cell apoptosis is increased in response to LPS stimulation and is associated with a significant induction of IGFBP-3 mRNA and protein. IGFBP-3, which reportedly promotes apoptosis and modulates the bioavailability of the pro-survival insulin-like growth factor 1, may serve to regulate apoptosis in monocytic cells in an IGF-independent manner. These data further support the investigation of the role of the IGF axis in programmed cell death of immune cells.
Assuntos
Apoptose/fisiologia , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/biossíntese , Regulação para Cima/fisiologia , Apoptose/efeitos dos fármacos , Linhagem Celular Transformada , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Fator de Crescimento Insulin-Like I/farmacologia , Lipopolissacarídeos , Ativação Linfocitária , Monócitos , Análise de Sequência com Séries de Oligonucleotídeos , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) is a well-documented central inflammatory mediator in sepsis. Specific polymorphisms of the TNF-alpha and TNF-beta genes (TNF2 and LTA + 250, respectively) have been suggested to correlate with higher mortality in septic shock. This study sought to determine whether these polymorphisms of the TNF-alpha and -beta genes are associated with an increased risk of infection in an at-risk surgical intensive care population. MATERIALS AND METHODS: Forty-four consecutive patients with systemic inflammatory response syndrome were enrolled prospectively in the study. Genomic DNA was isolated from whole blood samples using standard phenol/chloroform extraction techniques. Specific fragments including the polymorphic sites of each gene were amplified by polymerase chain reaction, and restriction enzyme digestions were performed. Genotypes were determined by gel electrophoresis and confirmed by direct sequencing. RESULTS: Eighty-six percent of the patients were TNF1 homozygotes (G:G at -308 of the TNF-alpha promoter region), whereas 9% of the patients were homozygous for TNF2 (A:A). There was no difference in the incidence of sepsis, septic shock, or mortality between patients bearing the various alleles. Only 13.6% of the patients exhibited the G:G alleles for TNF-beta, whereas the homozygous A:A was present in 45.4% of the patients. CONCLUSION: The presence of the A allele at these polymorphic sites did not predispose critically ill surgical patients to either infection or septic shock.
Assuntos
Linfotoxina-alfa/genética , Polimorfismo Genético/genética , Sepse/genética , Fator de Necrose Tumoral alfa/genética , Idoso , Sequência de Bases , Cuidados Críticos , Estado Terminal , Infecção Hospitalar/complicações , Infecção Hospitalar/genética , Infecção Hospitalar/imunologia , Feminino , Predisposição Genética para Doença/genética , Humanos , Masculino , Pessoa de Meia-Idade , Risco , Sepse/etiologia , Sepse/imunologia , Choque Séptico/genética , Choque Séptico/imunologia , Síndrome de Resposta Inflamatória Sistêmica/genética , Síndrome de Resposta Inflamatória Sistêmica/imunologia , Resultado do TratamentoRESUMO
Human toll-like receptor 4 (hTLR4) and CD14 are known to be components of the lipopolysaccharide receptor complex. Our study investigated the association between TLR4 mutations (Asp299Gly and Thr399Ile) and CD14 polymorphism(s) with outcome in an intensive care unit (ICU) population at risk for sepsis. By use of a polymerase chain reaction-based restriction fragment-length polymorphism analysis technique, the hTLR4 gene was altered in 14 (18%) of 77 ICU patients (all positive for systemic inflammatory response syndrome) and in 5 (13%) of 39 volunteers. There was a significantly higher incidence of gram-negative infection among patients with the mutations (11 [79%] of 14), compared with that in the wild-type population (11 [17%] of 63; P=.004). No association between CD14 polymorphism(s) and the incidence of infection or outcome was observed. These findings indicate that hTLR4 mutations are associated with an increased incidence of gram-negative infections in critically ill patients in a surgical setting.