The Effects Of Arginine And GRIM-19 In The Pathogenesis Of Respiratory Diseases

BackgroundAsthma is an inflammatory airway disease characterized by intermittent and variable degrees of airway obstruction and bronchial hyperresponsiveness.There are several studies that point to enhanced arginine catabolism in the airways of asthmatic patients, which may lead to reduced local bioavailability of arginine.Arginine is a non-essential amino acid,acquired by eukaryotic cells by synthesis in the Krebs urea cycle and uptake via the cationic amino acid transporters(CAT).CATs are blocked by polycations like major basic protein(MBP).MBP is present in airways of asthmatics and thus may also lead to reduced arginine availability to cells,even in the presence Of extra cellular arginine.Our previous studies into the regulation of the production of two key pro-inflammatory mediators,interleukin(IL)-6 and IL-8,by airway epithelial cells have shown that reduced levels of the essential amino acid tryptophan,which affects cellular metabolism,led to exaggerated production of the above mediators by post-transcriptional mechanisms.It is unknown whether reduced levels of arginine per se give rise to similar exaggerated mediator responses.ObjectiveWe were interested to assess whether reduced extra cellular arginine levels as well as blocking arginine uptake by cationic protein would influence the epithelial production of the inflammatory mediators IL-6 and IL-8.And to explore the mechanisms underlying the exaggerated IL-6 and IL-8 production by poly-L-arginine.Method1.Cell culture and experimental set upFor mediator release,3x10⁵ cells were plated and cultured overnight in 500μl in 24-well plates.For isolation of mRNA and nuclear extracts,15x10⁵ cells were plated and cultured overnight in 2.5 ml in 6-well plates.Cells were washed and then exposed in fresh medium for 20 hours to doses of recombinant human TNF-α,LPS.When indicated,L-arginine and poly-L-arginine were added.For a number of experiments, we used primary bronchial epithelial cells(NHBE).NHBE cells were cultured and propagated as recommended by the supplier,and cells were used between passage 3 and 5.For cytokine production,0.2x10⁵ cells were plated in 48-well plates,and before exposure to stimuli,cells were washed with PBS and incubated with MEM plus or minus L-arginine.2.Measurement of IL-6 or IL-8 protein and mRNAIL-6 and IL-8 in culture supernatants were measured by sandwich ELISA.Total RNA was extracted with Trizol and IL-6,IL-8 and GAPDH mRNA were determined by dotblotting and hybridization with specific ³²p-labeled probes for IL-6,IL-8 and GAPDH.Blots were quantified using a phosphorimager and variable loading was corrected by expressing mRNA levels relative to that of the housekeeping gene GAPDH.3.L-¹⁴C-arginine uptakeNCI-H292 cells(6x10⁴ cells) were plated in 100μl H292 medium per well in a 96-well plate.After overnight incubation,cells were washed with 100μl Hank's balanced salt solution(HBSS) without L-arginine and then exposed to poly-L-arginine or major basic protein(MBP) in 100μl HBSS.Then,10μl per well of 100μM L-¹⁴C-arginine was added.After 30 minutes,cells were washed 3 times with 100μl HBSS and lysed for 5 minutes in a final 0.1%(v/v) Triton X-100 in PBS.After the addition of 100μl scintillation fluid,radioactivity was counted.4.Degradation of the IL-6 or IL-8 mRNAIL-6 or IL-8 mRNA decay was determined 2 hours after stimulation by co-incubation with 5μg/ml of the transcriptional blocker actinomycin-D.At time zero and after an additional 40 and 80 minutes,remaining IL-6 and IL-8 mRNA were determined,as described above.5.Transcriptional activityNCI-H292 cells were grown to 70%confluence in 6-well plate and transfected with 5μg of chloramphenicol acetyltransferase(CAT) reporter construct driven by the wild-type IL-8 or IL-6 promoter.Cells were stimulated as indicated,24 h after transfection and 18 h before cell lysis.CAT production was measured by CAT ELISA and all data were normalized for total protein content.6.Statistical analysisAll parameters and comparisons were performed using SPSS 11.5 software.Data were expressed as means±standard error of the mean(SEM).The paired or Independent-Samples t-test and Pearson Correlation were used when appropriate to evaluate statistical significance.For multiple comparisons we used one-way ANOVA, followed by least significance difference when equal variances are assumed or Dunnett's T3 when no equal variances are assumed,and the multi-regression model method.Differences were considered significant at P≤0.05. Result1.Reduced levels of arginine enhanced IL-6 and IL-8 production by airway epithelial cellsThe basal as well as LPS- and TNF-α-stimulated IL-6 and IL-8 production was similar for NCI-H292 cells maintained in either RPMI-1640 medium or MEM-A⁺.The IL-6 and IL-8 production by cells maintained in MEM-A^-,as compared to that of cells in MEM-A⁺ or in RPMI-1640 medium was markedly increased(IL-6:P<0.001,P=0.001 and P<0.001;IL-8:P=0.048,P=0.005 and P=0.037 for non-stimulated,LPS- and TNF-α-stimulated cells,respectively).In fact,between 0 to 1 mM arginine,IL-6 production inversely correlated with the concentration of arginine(r=-0.513;P=0.03). NHBE cells in MEM-A^- for 21 h displayed enhanced production of IL-6 and IL-8 as compared to NHBE cells in MEM-A⁺,in the absence of a stimulus(P=0.001),with LPS(P=0.003).2.Hyperresponsive IL-6 and IL-8 production by reduced levels of L-arginineWhen NCI-H292 cells were exposed to MEM medium with or without L-arginine,we observed a dose dependent IL-6 and IL-8 production on TNF-α(r=0.604,P=0.001; r=0.730,P<0.001,IL-6 and IL-8),but not on LPS(r=0.017,P=0.929;r=0.218,P=0.246. IL-6 and IL-8).3.Cationic protein increased IL-6 and IL-8 production to LPS,dependent of inhibition of arginine uptake by NCI-H292 cellsPoly-L-arginine(≥10μg/ml) markedly reduced the uptake of L-¹⁴C-arginine(P<0.001). Moreover,the uptake of L-¹⁴C-arginine was inversely correlated with the concentration of poly-L-arginine within 80μg/ml(r=-0.715,P<0.001).The combination of poly-L-arginine with LPS significantly potentiated IL-6(P<0.001) and IL-8(except poly-L-arginine is 80μg/ml,the other P<0.001) production with a maximum at the concentration of 5μg/ml of poly-L-arginine.Interestingly,there was a negative correlation between the uptake of L-¹⁴C-arginine and the production of IL-6 to poly-L-arginine by NCI-H292 cells,whatever LPS was co cultured or not(r=-0.662, P=0.001;r=-0.598,P<0.01,for with LPS and without LPS,respectively).However the IL-8 is different to IL-6.The correlation was depended on the consentration of poly-L-arginine.When poly-L-arginine ranged from 0 to 5μg/ml,there was negative correlation between the uptake of L-¹⁴C-arginine by poly-L-arginine and the level of IL-8 after exposure to LPS and poly-L-arginine on H292 cells(r=-0.895,P<0.001).On the contrary,when poly-L-arginine varied from 5 to 80μg/ml,there was positive correlation between the uptake of L-¹⁴C-arginine and the production of IL-8(r=0.896, P<0.001).Major basic protein(MBP) also markedly reduced the uptake of L-¹⁴C-arginine (P<0.001).The uptake of L-¹⁴C-arginine was inversely correlated with the concentration of MBP within 20μg/ml(r=-0.737,P<0.001).MBP lower than 20μg/ml largely increased the LPS-induced IL-6 and IL-8 production(P<0.01 or P<0.001,for IL-6 and IL-8),apparently dependent of blocking uptake of L-¹⁴C-arginine by MBP when LPS was absent(r=-0.752,P<0.001;r=-0.784,P<0.001,for IL-6 and IL-8, respectively).While LPS was co cultured with MBP,the correlation was disappear (r=0.083,P=0.744;r=-0.080,P=0.752,for IL-6 and IL-8,respectively).4.Poly-L-arginine blocked arginine-uptake and increased IL-6 and IL-8 production to TNF-αby NHBE cells.10μg/ml Poly-L-arginine also block arginine-uptake by NHBE cells(P<0.001). Poly-L-arginine enhanced IL-6 and IL-8 production reduced by LPS and TNF-αon NHBE cells(t=11.49,P<0.0001;t=13.161,P<0.0001;t=8.894,P<0.0001;t=10.045. P<0.0001,for LPS and TNF-α;IL-6 and IL-8,respectively).TNF-α.but not LPS significantly synergized with poly-L-arginine in IL-6 and IL-8 production by NHBE cells,(multi regression model:t=7.264,P<0.0001;t=6.002,P<0.0001;t=-1.693, P=0.098;r=-0.738,P=0.464,for TNF-αand LPS;IL-6 and IL-8,respectively).5.The enhanced expression of IL-6 and IL-8 by poly-L-arginine might be due to increased transcriptional activityThe IL-6 and IL-8 mRNA profiles in NCI-H292 cells over different times were determined.LPS increased IL-8 and IL-6 mRNA 2- to 3-fold at 2 h,followed by a decrease at 4 h.Exposure to LPS and poly-L-arginine led to a significant 7- to 8-fold increase of the IL-6 and IL-8 mRNA level at 2 h.Subsequently,IL-6 and IL-8 mRNA levels decreased and reached a plateau at 6 h,2 to 3 times higher than LPS alone.6.Poly-L-arginine enhanced LPS-induced IL-6 and IL-8 gene transcription by transfected NCI-H292 cells with CAT constructs linked to the IL-6 or IL-8 promoter The mRNA profiles with poly-L-arginine are indicative of an enhanced transcriptional activity,and thus we transfected NCI-H292 cells with 5μg of chloramphenicol acetyltransferase(CAT) reporter constructs driven by the wild-type IL-6 or IL-8 promoter.Poly-L-arginine induced IL-6 and IL-8 gene transcription as deduced by quantification of CAT.Both of IL-6-CAT or IL-8-CAT induced by poly-L-arginine and LPS was increased than that of single poly-L-arginine or LPS.7.The enhanced expression of IL-6 and IL-8 by poly-L-arginine was not due to degradation of mRNAOur experiment indicated that poly-L-arginine had no effect on the degradation of IL-6 and IL-8 mRNA by H292 cells(P>0.05). Conclusion1.L-arginine(≤20μM) enhanced basal and synergized with stimulus-induced epithelial IL-6 and IL-8 production.2.MBP and poly-L-arginine enhanced the stimulus-induced but not basal IL-6 and IL-8 production,however,blocking arginine uptake and the enhanced IL-6 and IL-8 production appeared related.3.Poly-L-arginine enhances LPS-induced IL-6 and IL-8 production by up-regulating the gene transcription and mRNA expression on NCI-H292 cells. BackgroundLung cancer has been a severe threat to human health.In 2000,a report declared by WHO suggested that lung cancer,which has became the first malignant cause of death in man and the second in women,accounts for 19 percent in all malignant cancers.According to incomplete statistic,about 70 percent of lung cancer has been diagnosed in metaphase or advanced stages.They have lost the chance of surgeon.Gene associated with retinoid-interferon mortality-19(GRIM-19) is a new cell death regulatory gene,which was first identified by researcher in Maryland medical college. GRIM-19 is one of the death-related genes induced by retinoid-interferon.Over expression of GRIM-19 could suppress proliferation and promote apoptosis of tumor cells growth,enhance the sensitivity of cells to IFN-RA-induced death.Research has confirmed that GRIM-19 was concerned with human renal cell carcinomas and urinary system rumor and many other malignancies,but its expression in lung cancer and lung cancer development remains unclear.ObjectiveThe expression and distribution of GRIM-19 was investigated in lung cancer tissue and lung inflammation tissue.We have explored the correlations with histology,clinical stage, primary lesion,lymph node or distant metastasis of lung cancer.To further explore the value of early diagnosis and clinical significance in Lung Cancer.Methods1.The expression of GRIM-19 in lung cancer and lung inflammation was examined by immunohistochemistyThe immunohistochemical expression of GRIM-19 was investigated in 30 lung cancers(13 squamous cell carcinomas;11 adenocarcinomas and 8 SCLC) and 10 lung inflammation tissues.2.Analyze the expression of GRIM-19 in lung cancer and lung inflammationThe expression of GRIM-19 was semi-quantitatively measured by optical density (O.D.) from JD 801 morphology image analysis system.3.Analyze the relationship between GRIM-19 and clinical-pathologic features in lung cancerGroup GRIM-19 expressions according to sex,age,smoking,primary lesion,lymph node or distant metastasis and clinical staging of NSCLC(non small cell lung carcinoma,NSCLC) patients,then analyze the relationship between GRIM-19 and clinical-pathologic features in NSCLC.4.The cellular distribution of GRIM-19 in lung cancer and lung tissuesThe cellular distribution of GRIM-19 in lung cancer and lung tissues was observed by laser scanning confocal microscope.5.Statistical AnalysisAll parameters and comparisons were performed using SPSS 11.5 software.Data were expressed as the means±SD.The paired or Independent-Samples t-test and Pearson Correlation were used when appropriate to evaluate statistical significance. For multiple comparisons we used one-way ANOVA,followed by least significance difference when equal variances are assumed or Dunnett's T3 when no equal variances are assumed,and the multi-regression model method.Differences were considered significant at P<0.05.Result1.The compare of GRIM-19 in lung cancer and lung inflammationThe expression of GRIM-19 had significant difference in NSCLC,SCLC and lung inflammation(P<0.001).The expressions of GRIM-19 in squamous cell carcinomas, adenocarcinomas and SCLC were lower than that of lung inflammation(P<0.05, P<0.05,P<0.001,respectively).Compared with SCLC,the GRIM-19 of squamous cell carcinomas,adenocarcinomas enhanced significantly(P<0.01,P<0.01).There was no difference between squamous cell carcinomas and adenocarcinomas (P>0.O5).2.The relationship between the expression of GRIM-19 and clinical-pathologic features in lung cancerThe expression of GRIM-19 in the stageâ… -â…¡of NSCLC was significantly higher than that of stageâ…¢-â…£(P<0.01).There was negative correlation between the expression of GRIM-19 and primary lesion(P<0.05).This study also discovered that the expression of GRIM-19 is no correlation with lymph node or distant metastasis,sex, age and smoking of NSCLC patients(P>0.05).3.The cellular distribution of GRIM-19 in lung cancer and lung tissuesGRIM-19 was dominantly located in the cytoplasm of normal lung cells and lung inflammation.However,it was enriched in the nuclei of lung cancer cells besides the cytoplasm. Conclusion1.GRIM-19 was expressed in normal lung tissue.Its expression in lung inflammation significantly increased compared to lung cancer.It had relationship with the tumors histological type of lung cancer.2.GRIM-19 expression is down-regulated along the primary lesion and clinical development of lung cancer,and no correlation with lymph node or distant metastasis. The findings indicate that the reduction of GRIM-19 may play an important role in lung cancer carcinogenesis.GRIM-19 nuclear expression is an early and important phenomenon in the pathogenesis of lung cancer.It maybe a potential suitable target for early diagnosis.