Penglin Ma，¹ Xiaolin Cui，Shuibang Wang，Jianhua Zhang，Ervant V. Nishanian，² Weihan Wang，Robert A. Wesley，and Robert L. Danner³<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />

Critical Care Medicine Department，Warren Grant Magnuson Clinical Center，National Institutes of Health，Bethesda，Maryland

　　Nitric oxide （NO） contributes to vascular collapse in septic shock and regulates inflammation. Here，we demonstrate in lipopolysaccharide （LPS）－stimulated human THP－1 cells and monocytes that NO regulates interleukin （IL）－8 and tumor necrosis factor а （TNF－а） by distinct mechanisms. Dibutyryl－cyclic guanosine 5’－monophosphate （cGMP） failed to simulate NO－induced increases in TNF－а or IL－8 production. In contrast，dibutyryl－cyclic adenosine monophosphate blocked NO－induced production of TNF－а（P＝0.009） but not IL－8. NO increased IL－8 （5.7－fold at 4 h；P＝0.04） and TNF－а mRNA levels （2.2－fold at 4 h；P＝0.037）. However，nuclear run－on assays demonstrated that IL－8 transcription was slightly decreased by NO （P＝0.08），and TNF－аwas increased （P＝0.012）. Likewise，NO had no effect on IL－8 promoter activity （P＝0.84）as measured by reporter gene assay. In THP－1 cells and human primary monocytes treated with actinomycin D，NO had no effect on TNF－а mRNA stability （P>0.3 for both cell types） but significantly stabilized IL－8 mRNA （P＝0.001 for both cell types）. Because of its role in mRNA stabilization，the p38 mitogen－activated protein kinase （MAPK） pathway was examined and found to be activated by NO in LPS－treated THP－1 cells and human monocytes. Further，SB202190，a p38 MAPK inhibitor，was shown to block NO－induced stabilization of IL－8 mRNA （P<0.02 for both cell types）. Thus，NO regulates IL－8 but not TNF－а post－transcriptionally. IL－8 mRNA stabilization by NO is independent of cGMP and at least partially dependent on p38 MAPK activation. J. Leukoc. Biol. 76：278－287；2004.

　　Key Words：cytokines protein kinases gene regulation mRNA cAMP cGMP.

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　　In the current investigation，we sought to comprehensively examine NO regulation of IL－8 in LPS－stimulated THP－1 cells. These cells were chosen as a reproducible，LPS－responsive，human cellular model that has been evaluated extensively in studies of post－transcription regulation ^[49]. Further，we wanted to approach this question in a system that was different from our previous studies in phorbol ester－differentiated U937 cells ^[35～38]. Several key experiments were also performed in human primary monoctyes to investigate the biological relevance of results obtained with THP－1 cells. Specific goals were to directly compare the cyclic nucleotide dependence of IL－8 and TNF－а gene regulation；test effects of NO on IL－8 and TNF－а mRNA kinetics，transcription，and stability；investigate the IL－8 promoter for NO－induced changes in activity and transcription factor binding；and finally，examine the role，if any，of p38 MAPK in the regulation of IL－8 by NO.

　　MATERIALS AND METHODS

　　Reagents and cell cultures

　　Salmonella minnesota Re595 LPS was obtained from List Biologic （Campbell，CA）. Dibutyryl－cGMP （Bt₂cGMP） and ibutyryl－cAMP （Bt₂cAMP） were purchased from Seikagaku America （Rockville，MD）. Actinomycin D and glutathione （GSH） were obtained from Sigma－Aldrich （St. Louis，MO）. S－nitrosoglutathione （SNOG） and p38 MAPK inhibitor SB202190 were obtained from Calbiochem （San Diego，CA）.

　　THP－1 cells，a human monocytic line，were obtained from the American Type Culture Collection （Manassas，VA）. For all experiments，cells were cultured at 37°C in a 5％CO₂ atmosphere using RPMI－1640 complete medium （BioWhittaker，Walkersville，MD） containing 10％ fetal calf serum （FCS；Cellgro，Herndon，VA），100 U/ml penicillin，100 μg/ml streptomycin，25 μg/ml amphotericin B （all from Cellgro），and 50 μM β－mercaptoethanol （Sigma－Aldrich）.

　　Human primary monocytes obtained from healthy donors by standard leukapheresis were purified by counterflow centrifugation （elutriation），which yields cells that are 93－95％ pure and ?98％ viable （Department of Transfusion Medicine，Section of Blood Services and Cell Processing，Clinical Center，National Institutes of Health，Bethesda，MD）. Monocytes （1×10⁷） were cultured at 37°C with 5％ CO₂ in 10 ml RPMI－1640 complete medium in which 10％ autoplasma was substituted for 10％ FCS. For Bt₂cGMP，Bt₂cAMP，LPS，SNOG，GSH，and SB202190，the maximum concentrations examined in THP－1 cells or human monocytes did not alter cell viability as determined by trypan blue exclusion.

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　　Cytokine assay

　　THP－1 cells （5×10⁵/ml/well） were placed in 24－well plates containing RPMI－ 1640 complete medium with Bt₂cGMP （0－50 μM） or Bt₂cAMP （0－50 μM） in combination with LPS （1 μg/ml） and SNOG （0－500 μM）. GSH，the precursor of SNOG，was used to adjust combined SNOG and GSH concentrations to 500 мM in all wells. SNOG （400 μM） in RPMI－1640 complete medium produced peak NO concentrations of ～150 мM at 1 h，which decayed rapidly to levels of ～50 μM （NO Analyzer 280，Sievers，Watertown，MA）. After 20 h incubation，culture supernatants were collected. IL－8 and TNF－а production was measured using an enzyme－linked immunosorbent assay （ELISA） kit （R&D Systems，Minneapolis，MN） according to the manufacturer’s instructions. To examine the effect of p38 MAPK on IL－8 production，THP－1 cells were pretreated with the specific p38 MAPK inhibitor SB202190 （0－0.1 μM） for 1 h and then incubated in complete medium for an additional 8 h with LPS （1 μg/ml） and phosphate－buffered saline （PBS） alone，GSH （400 μM），or SNOG （400 μM）. IL－8 concentrations in supernatant were then determined as described above.

　　Quantitation of cytokine mRNA levels and stability

　　Total RNA from 1 × 10⁷ THP－1 cells or primary monocytes was isolated using RNeasy kits （Qiagen，Valencia，CA） after incubation in medium with various treatments and time－periods as specified in the corresponding figures. IL－8，TNF－а，and glyceraldehyde 3－phosphate dehydrogenase （GAPDH） or β－actin mRNA levels were measured from 1 to 2 мg total RNA using Quantikine^? colorimetric mRNA quantitation kits and specific Quantikine^? mRNA probes （R&D Systems） following the manufacturer’s instructions. All data were normalized to the housekeeping genes GAPDH or β－actin as indicated and expressed in amol/мg total RNA using a standard curve.

　　In one experiment，IL－8 mRNA was measured in THP－1 cells that were pretreated with the p38 MAPK inhibitor SB202190 （0－0.1 μM） for 1 h followed by LPS （1 μg/ml） stimulation and exposure to PBS，GSH （400 μM），or SNOG （400 μM） for 4 h. For measurement of mRNA stability，THP－1 cells or primary monocytes （1×10⁷） were stimulated with LPS （1 μg/ml） for 4 h，followed by 30 min pretreatment with actinomycin D （2.5 μg/ml）. In two experiments measuring mRNA stability，as indicated，pretreatment also included exposure of some cells to SB202190 （0.1 μM）. Then PBS，GSH （400 μM），or SNOG （400 μM） was added，followed by further incubation for 0，1，2，or 4 h. Total RNA was prepared，and mRNA was measured as above.