Volume 115, Issue 1, Pages 147-156 (July 1998) Altered biosynthesis of leukotrienes and lipoxins and host defense disorders in patients with cirrhosis and ascites  Joan Clària*, Esther Titos*, Wladimiro Jiménez‡, Josefa Ros‡, Pere Ginès§, Vicente Arroyo§, Francisca Rivera‡, Joan Rodés§  Gastroenterology  Volume 115, Issue 1, Pages 147-156 (July 1998) DOI: 10.1016/S0016-5085(98)70376-2 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 (A) Detection of 5-LO and 15-LO mRNA by RT-PCR and (B) analysis of 5-LO expression by Northern blot in PMN isolated from patients with cirrhosis and ascites and from healthy subjects. RNA was obtained by the guanidinium isothiocyanate–cesium chloride method, and cDNA was produced by reverse transcription. PCR amplification was performed using specific oligonucleotide sequences of 5- and 15-LO and GAPDH. For RNA blot analysis, RNA (6 μg) was electrophoresed in denaturing 1% agarose gels, transferred onto nylon filters, and hybridized with 32P-labeled cDNA probes specific for human 5-LO and GAPDH. Representative results obtained from 3–5 separate donors. Gastroenterology 1998 115, 147-156DOI: (10.1016/S0016-5085(98)70376-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Formation of (A) LXA4 and (B) cysteinyl-containing LT by PMN-PLT coincubations. PMN and PLT were obtained from 6 healthy subjects (HS) and 4 patients with cirrhosis and ascites (CH). PMN suspensions (32–80 × 106 PMN/mL) were exposed to FMLP (100 nmol/L, 5 minutes, 37°C) and subsequently combined with thrombin-activated (1 U/mL, 5 minutes, 37°C) PLT suspensions with the cell ratio adjusted to 1:10 (PMN:PLT). The incubations were terminated after 20 minutes at 37°C by the addition of cold MeOH (2 volumes), and LO products were determined by ELISA. Results are given as mean ± SEM. Gastroenterology 1998 115, 147-156DOI: (10.1016/S0016-5085(98)70376-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 (A) Plasma angiotensin II levels in healthy and cirrhotic subjects. Blood samples were obtained from 6 healthy subjects and 8 patients with cirrhosis (CH) and ascites, and plasma angiotensin II levels were measured by radioimmunoassay as described in Materials and Methods. (B) Modulation of LXA4 biosynthesis by angiotensin II. Coincubations of PMN and PLT were performed as in Figure 2, except that cells were previously exposed to either vehicle or angiotensin II (10 nmol/L) for 20 minutes at 37°C. Results are expressed as mean ± SEM of five experiments performed in cells obtained from 3 different healthy donors. Gastroenterology 1998 115, 147-156DOI: (10.1016/S0016-5085(98)70376-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Individual values of plasma LXA4 levels in cirrhotic patients. Blood samples were obtained from 6 patients with compensated cirrhosis (Compensated CH) and 24 patients with cirrhosis and ascites (CH + ascites). Horizontal lines denote the mean value for each group of patients. Statistical analysis revealed a P value of <0.001 for cirrhosis vs. cirrhosis plus ascites. Inserts show the relationships between plasma LXA4 concentration and urinary LTE4 excretion and between plasma LXA4 levels and PRA in a group of cirrhotic patients included in the study. Gastroenterology 1998 115, 147-156DOI: (10.1016/S0016-5085(98)70376-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Representative RP-HPLC profile of ascites obtained from patients with cirrhosis. Ascites fluid from 4 patients with cirrhosis was pooled, extracted with ethyl acetate, and injected into RP-HPLC. The column, a Kromasil C18 (5 μm, 4.6 × 250 mm), was eluted with MeOH/H2O/acetic acid (65:35:0.01; vol/vol/vol) as phase one (t0, −20 minutes) and a linear gradient with MeOH/acetic acid (99.9:0.1, vol/vol) as phase two (20–45 minutes) at a flow rate of 1.0 mL/min, and the detector was set to record signals at 300 nm. Insert shows UV spectra of material eluting beneath the LX region. Gastroenterology 1998 115, 147-156DOI: (10.1016/S0016-5085(98)70376-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Adhesion of control (▨) and cirrhotic (■) PMN after incubation with either LTB4 or angiotensin II. BCECF-AM–labeled PMN were incubated with 100 nmol/L LTB4 or 100 nmol/L angiotensin II in laminin-coated 96-well plates for 20 minutes at 37°C. Adherent cells were solubilized with 0.1% SDS in 25 mmol/L NaOH, and the remaining fluorescence was quantitated. Insert shows the effect of LXA4 on LTB4- and angiotensin II–induced adhesion. Fluorescent-labeled PMN were treated with LXA4 (100 nmol/L) for 15 minutes at 37°C and incubated with LTB4 or angiotensin II as above. Results represent the mean ± SEM of five different experiments performed in duplicate. Gastroenterology 1998 115, 147-156DOI: (10.1016/S0016-5085(98)70376-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Migration of PMN from control subjects (▨) and patients with cirrhosis and ascites (■) after incubation with either vehicle or LTB4. BCECF-AM–labeled PMN were tested for their ability to migrate across a polycarbonate filter in response to vehicle (0.1% EtOH) or LTB4 (100 nmol/L) as detailed in Materials and Methods. Results are mean ± SEM of 3–4 different experiments. Gastroenterology 1998 115, 147-156DOI: (10.1016/S0016-5085(98)70376-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 Adhesion of control (▨) and cirrhotic (■) monocytes after incubation with either LTB4 or LXA4. BCECF-AM–labeled monocytes were incubated with 100 nmol/L LTB4 or 100 nmol/L LXA4 in laminin-coated 96-well plates for 20 minutes at 37°C. Data are mean ± SEM of four different experiments performed in duplicate. Gastroenterology 1998 115, 147-156DOI: (10.1016/S0016-5085(98)70376-2) Copyright © 1998 American Gastroenterological Association Terms and Conditions