Urinary trypsin inhibitor (Ulinastatin®) was generously gifted from Halim pharmaceutical (Seoul, Korea). Escherichia coli 055 : B5 endotoxin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Immunoreactive TNF-α and IL-6 were quantified using commercially available enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA). RPMI 1,640/25 mM HEPES/L-glutamine was obtained from Mediatech (Herndon, VA), while FBS and penicillin/streptomycin were purchased from Mediatech. Bicinchoninic acid (BCA) protein assay reagent was obtained from Pierce (Rockford, IL, USA). Antibodies specific for phosphorylated (p)-ERK1/2, JNK and p38, as well as total ERK, JNK and p38 were purchased from Cell Signaling Technologies (Beverly, MA, USA).

For isolation of human neutrophils, peripheral blood was obtained from healthy volunteers under a protocol approved by the institutional review board. None of the volunteers had a history of infection or allergy; none of them were smokers or undergoing immunosuppressive therapy. Neutrophils were isolated by plasma-Percoll gradients after dextran sedimentation of erythrocytes, as described previously [19]. Dextran was added to a final concentration of 6%, and erythrocytes were sedimentated under gravity for 45 minutes at room temperature. The erythrocyte-depleted supernatant containing leukocytes was centrifuged at 1,100 rpm for 6 minutes. The resulting leukocyte-enriched pellet was resuspended in platelet poor plasma (PPP). The leukocytes were then layered over a discontinuous gradient of percoll (2 ml of 42% and 2 ml of 51%) in a 15 ml polypropylene centrifugation tube. Percoll densities were created from stock percoll diluted with PPP. The percoll densities and layered cells were then centrifuged at 1,100 rpm for 10 minutes. Neutrophils were found at the 51-42% percoll layer interface. Moreover, when a few red blood cells (RBCs) stayed with neutrophils, we were able to use RBC lysing buffer (SIGMA, UK) for the lysis of RBCs. Neutrophils were removed from the new tube and washed twice with phosphate buffered saline (PBS). Cells were counted with a hemocytometer (Marienfeld, Germany) and resuspended at 5x106cells/ml with RPMI-1640 containing 5% of serum. Purity (Wright's stain) and viability (trypan blue exclusion) of the isolated cells were kept more than 97% and 95%, respectively.

Neutrophils (5 × 10⁶/ml) were incubated for 4 hours with varying concentrations of UTI (1, 10, 100, 1,000 and 10,000 U/ml) or LPS (100 ng/ml) alone or LPS plus UTI. Unstimulated neutrophils, exposed to neither UTI nor LPS, were also incubated as a control group. Concentrations of TNF-α and IL-6 in the culture media were determined using ELISA kits (R&D System, Minneapolis, MN, USA), according to the manufacturer's instructions and as described previously [20].

Neutrophils were incubated for 30 minutes with or without UTI (10,000 U/ml), or LPS (100 ng/ml) alone or with LPS plus UTI. Western blots were performed to detect the levels of phosphorylated and total p38, ERK1/2 and JNK, as previously described [20,21]. Neutrophils were lysed in ice-cold lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM Na₂ EDTA, 1% Triton X-100, 1 mM EGTA, 1 mM Na₃ vanadate, 2.5 mM Na pyrophosphate, 1 mM β-glycerophosphate, 1 mM PMSF, 1 µg/ml leupeptin, pH 7.5), and then resuspended and sonicated for 30 seconds. Debris from the lysed cell was pelleted by centrifugation at 14,000 rpm for 20 minutes. The supernatant was then removed and stored at -86℃. Protein concentration of each sample was assayed using the BCA protein assay kit (Pierce Chemical, Rockford, IL) standardized to BSA, according to the manufacturer's protocol. For the Western blot, 50 µg of protein was loaded and run on a 10% Tris-HCl SDS polyacrylamide gel. Protein was electrotransferred to a nitrocellulose membrane and then blocked with 5% non-fat dry milk, 20 mM TBS with 0.1% Tween. After blocking, the membrane was incubated overnight at 4℃ with a rabbit polyclonal specific primary antibody against p-p38, p-ERK1/2 and p-JNK, or at a dilution ratio of 1/1,000 in 1% BSA, followed by anti-rabbit or anti-rat Ig HRP-coupled secondary antibody at a dilution of 1/2,000 in 5% nonfat dry milk. After washing five times, bands were detected using ECL Western blotting detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ). The membranes were then stripped using stripping buffer (63 mM Tris-HCl, pH 6.8, 2% SDS, 100 mM 2-ME; from Bio-Rad, Hercules, CA), and reprobed with Abs specific for total ERK, JNK, or p38. Densitometry was performed by the chemiluminescence system and analysis software (Bio-Rad) to determine the ratio between phosphorylated and total kinases.

Neutrophils were incubated for 1 hour with or without UTI (10,000 U/ml), or LPS (100 ng/ml) alone or with LPS plus UTI. EMSAs were performed, as previously described [20]. To obtain nuclear extracts from neutrophils, cells were resuspended in buffer A containing 10 mM HEPES (pH 7.9), 1.5 mM MgCl₂, 10 mM KCl, and 0.5 mM DTT, and the samples were incubated on ice for 20 minutes. After removing the cytoplasm from the nuclei by 15 passages through a 25-gauge needle, the nuclei were collected by centrifugation at 600 × g for 6 minutes at 4℃. The pellets were suspended in buffer C containing 20 mM HEPES, 1.5 mM MgCl₂, 420 mM NaCl, 0.2 mM EDTA, 25% glycerol, and 0.5 mM PMSF. After 30 minutes of incubation on ice, the suspension was centrifuged at 14,000 × g for 20 minutes at 4℃, and the supernatant was collected. Protein concentration in the supernatants was determined using BCA protein assay kits (Pierce Chemical, Rockford, IL). The nuclear extracts (5 µg) were incubated at room temperature for 20 minutes in 20 µl of reaction buffer containing 10 mM Tris-HCl (pH 7.5), 1 mM MgCl₂, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, and 4% glycerol with a ³²P end-labeled, double-stranded oligonucleotide probe specific for the κB site, 5'-AGTTGAGGGGACTTTCCCAGGC-3' (Geneka, Burlington, VT), and 1 µg of poly(dI-dC)·poly(dI-dC). In some experiments, unlabeled NF-κB or CREB oligonucleotide (Promega, Madison, WI) was added to the samples at 200-fold excess before adding the labeled probe. The sample was then incubated for 15 minutes on ice. The complexes were resolved on 5% polyacrylamide gels in Tris-HCl (pH 8.0)-borate-EDTA buffer at 10 V/cm. The dried gels were exposed to Kodak Biomax MS film (Eastman-Kodak, Rochester, NY) for 1-24 hours at -70℃.

Statistical analyses of the data were performed by two-way analysis of variance with repeated measures. The Scheffé test was used for multiple pair-wise comparisons when a significant difference was indicated through analysis of variance. The Kruskal-Wallis H test was used for group comparisons. A value of P < 0.05 was considered significant.

To assess the possible interactions between UTI and LPS on neutrophil activation, neutrophils were incubated with varying concentrations of UTI (1, 10, 100, 1,000 and 10,000 U/ml) plus LPS (100 ng/ml) or LPS alone, and protein levels for IL-6 and TNF-α were determined 4 h later. In these experiments, expression of both cytokines increased after stimulation of LPS. There was no effect of UTI alone over the range of concentrations examined. This LPS induced-expression in both cytokines was attenuated in the presence of UTI (10,000 U/ml) (Fig. 1).

To examine the effects of UTI on LPS-induced activation of these kinase pathways and nuclear translocation of NF-κB, LPS-stimulated neutrophils were incubated with or without UTI (10,000 U/ml), and then the levels of active, phosphorylated forms of p38, ERK1/2, JNK, and nuclear levels of the transcriptional factor NF-κB were determined. Exposure of resting neutrophils to UTI alone had no effect on the activation of p38, ERK and JNK. As shown in Fig. 2, a culture of neutrophils with LPS resulted in the activation of p38, ERK1/2 and JNK. Addition of UTI to the neutrophils stimulated with LPS did not affect p38 and ERK1/2 activation, but did result in the attenuation of JNK phosphorylation. Activation of NF-κB induced by LPS was not affected by UTI (Fig. 3).