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. 2001 Jul;49(1):106–111. doi: 10.1136/gut.49.1.106

Gene expression of interleukin 18 in unstimulated peripheral blood mononuclear cells of patients with alcoholic cirrhosis

C Hanck 1, T Manigold 1, U Bocker 1, M Kurimoto 1, C Kolbel 1, M Singer 1, S Rossol 1
PMCID: PMC1728343  PMID: 11413118

Abstract

BACKGROUND—Most patients with alcohol induced cirrhosis (AC) and chronic endotoxinaemia are not suffering from clinically evident systemic inflammatory reactions. This may be due to altered gene expression of cytokines, possibly related to endotoxin (for example, tolerance and sensitisation). Interleukin 18 (IL-18; interleukin γ inducing factor) modulates local cytokine production in response to endotoxin (lipopolysaccharide (LPS)).
AIM—To investigate the systemic immune response of patients with AC and to see if unstimulated peripheral blood mononuclear cells (PBMC) from patients with AC are activated and contribute to gene expression of IL-18.
METHODS—Plasma levels of endotoxin (LPS) and serum levels of IL-18 were measured by enzyme linked immunoassay and the amoebocyte lysate test in 74 abstinent patients with different stages of AC (Child-Pugh stage A, n=18; B, n=22; C, n=34) and compared with healthy controls (n=43). Gene expression of IL-18 was assessed by semiquantitative reverse transcription-polymerase chain reaction in freshly isolated unstimulated PBMC of a subgroup of 14 patients with AC compared with five healthy controls.
RESULTS—Gene expression of IL-18 specific mRNA in unstimulated PBMC was significantly enhanced in patients with advanced AC (Child-Pugh stage C) and correlated with plasma LPS and serum CD14 levels (Spearman rank correlation factors r=0.76 and r=0.72). Serum concentrations of IL-18 were also elevated compared with healthy controls (p<0.001) but correlation with serum levels of CD14 and plasma levels of LPS was much weaker compared with mRNA data (Spearman rank correlation factors r=0.47 and r=0.26).
CONCLUSIONS—Our in vivo data suggest a presensitisation of "unstimulated" PBMC in the circulation of patients with AC by endotoxin. The term "unstimulated" may be inadequate in patients with AC. Further investigations are needed to define the exact mechanisms and localisation of sensitisation of PBMC in vivo.


Keywords: liver cirrhosis; peripheral blood mononuclear cells; interleukin 18; lipopolysaccharide

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Figure 1  .

Figure 1  

Interleukin 18 (IL-18) serum levels (ELISA) were significantly increased in patients with alcohol induced cirrhosis with Child-Pugh stage C disease (n=34; median 456.15 pg/ml; range 163.1-1643.0) compared with healthy controls (n=43; median 135.5 pg/ml; range 62.9-476.1) and patients with Child-Pugh stage A (n=18; median 195.25 pg/ml; range 59.8-598.9). Moreover, significant differences were found between healthy controls and Child-Pugh stage B (n=22; median 355.15 pg/ml; range 119.8-792.9). Box plots indicate the median and 95% confidence intervals (CI). Individual data points are mean serum levels above or below the 95% CI. ***p<0.001.

Figure 2  .

Figure 2  

CD14 serum levels (ELISA) were significantly increased in patients with alcohol induced cirrhosis with Child-Pugh stage C disease (n=34; median 5.95 µg/ml; range 2.62-7.71) compared with healthy controls (n=43; median 2.98 µg/ml; range 2.19-5.9) and patients with Child-Pugh stage A (n=18; median 4.0 µg/ml; range 1-6.18). Moreover, significant differences were found between healthy controls and Child-Pugh stage B (n=22; median 4.8 µg/ml; range 2.86-6.54). Box plots show median and 95% confidence intervals (CI). Individual data points are mean serum levels above or below the 95% CI. ***p<0.001.

Figure 3  .

Figure 3  

Lipopolysaccharide (LPS) plasma levels were significantly increased in patients with alcohol induced cirrhosis with Child-Pugh stage C disease (n=34; median 1.4 pg/ml; range 0-63) compared with healthy controls (n=43; median 0 pg/ml; range 0-0) and patients with Child-Pugh stage A (n=18; median 0 pg/ml; range 0-0.7). Moreover, significant differences were found between healthy controls and Child-Pugh stage B (n=22; median 0 pg/ml; range 0-6.7). Box plots show median and 95% confidence intervals (CI). Individual data points are mean serum levels above or below the 95% CI. ***p<0.001.

Figure 4  .

Figure 4  

Spearman rank analysis reveals a significant correlation between serum levels of interleukin 18 (IL-18) and CD14 (r= 0.47, p<0.001).

Figure 5  .

Figure 5  

(A) mRNA expression of human interleukin 18 (IL-18) in unstimulated freshly isolated peripheral blood mononuclear cells of patients with alcohol induced cirrhosis (Child's stage A-C; n=14) compared with healthy controls (n=5). One patient with Child-Pugh stage A was retrospectively excluded as this patient did not fulfill the inclusion criteria (for example, a single drink/alcohol intake shortly before liver biopsy and venous puncture; histologically no alcoholic hepatitis; no blood parameters of chronic alcohol consumption either at the time of venous puncture or at previous examinations in the outpatient department for more than two years). GPDH, glyceraldehyde 3-phosphate dehydrogenase. (B) Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) (ratio of mRNA of IL-18/GPDH) revealed that significantly more RT mRNA was expressed in Child's stages B/C than in stage A. Median (range) for Child's stage A, 0.02 (0.01-0.15); for Child's stage B, 0.21 (0.09-0.38); and for Child's stage C, 0.39 (0.32-1.69). Box plots show median and 95% confidence intervals (CI). Individual data points are mean serum levels above or below the 95% CI. *p<0.05.

Figure 6  .

Figure 6  

(A) Spearman rank analysis demonstrated a significant correlation between mRNA expression of interleukin 18 (IL-18) in unstimulated peripheral blood mononuclear cells of patients with alcohol induced cirrhosis and sCD14 (r= 0.72; p < 0.01). (B) Spearman rank analysis demonstrated a significant correlation between mRNA expression of IL-18 and lipopolysaccharide (LPS) (r=0.76; p<0.01). GPDH, glyceraldehyde 3-phosphate dehydrogenase.

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