Macrophages from patients with cirrhotic ascites showed function alteration of host defense receptor

Abstract

Background: Patients with cirrhotic ascites (PCA) are susceptible to spontaneous bacterial peritonitis (SBP) which has increased morbidity and mortality. Since some host defense aspects of peritoneal macrophages (PMф) from PCA are altered this study examined factors related to receptor-mediated phagocytosis.

Methods: Twelve PCA were studied. PMɸ were isolated from ascitic fluid (AF) samples removed from these patients. Uptake of mannose receptor (MR)-specific ligand, fluorescein isothiocyanate-mannosylated-bovine serum albumin (FITC-man-BSA), by patients' PMɸ and controls, a human monocytic cell line, was measured pre- and post-IL-4 treatment. Phagocytosis of FITC-labeled yeast particles by patients' PMɸ was measured pre- and post-IL-4 treatment. Fluorescence values were obtained using a spectrofuorometer. MRC1 gene was analyzed in blood samples from PCA and controls, healthy donors, using standard polymerase chain reaction (PCR) technique.

Results: Past SBP episode(s) were reported in 58.3% of patients. Mean AF volume analyzed per patient was 1.3L. PMɸ ratio in cell yield was 53.73% (SD 18.1). Mean uptake absorbance of patients' PMф was 0.0841 (SD 0.077) compared to 0.338 (SD 0.34) of controls, P = 0.023. Following IL-4 treatment absorbance increased to 0.297 (SD 0.28) in patients' PMф (P = 0.018 on paired sample t-test), and to 0.532 (SD 0.398 in controls (P = 0.053 on independent sample t-test). Mean phagocytosis absorbance of patients' PMф was 0.1250 (SD 0.032) before IL-4 treatment compared to 0.2300 (SD 0.104) after (P = 0.026). PCR analysis for MRC1 gene was negative in all PCA samples compared to positive results in all controls.

Conclusion: Since decreased phagocytosis and MR uptake were enhanced post-IL-4 treatment MR downregulation pre-treatment is plausible. Negative PCR results for MRC1 might suggest an anomaly, but this awaits further ellucidation. These altered host defense findings are relevant to infection pathophysiology, and their relevance to SBP susceptibility in PCA is worth verifying.

Keywords: AF, ascitic fluid; FBS, foetal bovine serum; FITC, fluorescein isothiocyanate; IL-4, interleukin-4; MR, mannose receptor; MRC1 gene; MRC1, gene encoding human MR; PCA, patients with cirrhotic ascites; PCR, polymerase chain reaction; PMф, peritoneal macrophages; RPMI and DMEM, cell culture media; cirrhosis; macrophage host defense; man-BSA, mannosylated bovine serum albumin; mannose receptor; spontaneous bacterial peritonitis.

Figure 1

Photographs of stained sections of patients' PMф at high magnifications (A & B) and phase contrast views of tissue culture plates (C & D). Slide A & B were stained using May Grunwald Giemsa stain. Observe the triking morphological changes following overnight incubation (D). No stimulus was added to these cells.

Figure 2

Effect of IL-4 on phagocytosis in patients' macrophages. The bottom curve (dark diamond symbols) represents phagocytosis measurements before IL-4 treatment and the top curve (square symbols) represents measurements after IL-4 treatment. Letters in horizontal axis denote samples of patients' PMɸ. Values in vertical axis represent absorbance.

Figure 3

Mannose receptor (MR) specific uptake measurement of PMɸ from patients and controls. The panel on the left represents uptake measurements in patients' PMɸ (dark bar) and controls (light bar) before IL-4 treatment P = 0.023 on independent sample t-test. The panel on the right represents uptake measurements after IL-4 treatment in both groups P = 0.053 on independent sample t-test and P = 0.018 on paired sample t-test. The figures on the vertical axis represent absorbance.

Figure 4

a. PCR Results of MRC1 gene in patients with cirrhosis. In lanes 1, 2, 3 and 5 no bands for MRC1 gene were visualized (negative PCR results) in four out of twelve samples from PCA. In lane 4 a clear band is visualized (positive PCR result) for MRC1 gene in a healthy donor's sample, serving as a normal reference. In the remaining eight patients' samples the results were also negative (film not shown). 550 bp on 1.5% agarose gel M: 50 bp DNA size marker. b. PCR Results of MRC1 gene in normal subjects. In lanes 1–7 clear bands were visualized for MRC1 gene (positive PCR results). No patients' samples were included here. M: 50 bp DNA size marker. 550 bp on 1.5% agarose gel.