An in vitro model of human acute ethanol exposure that incorporates CXCR3- and CXCR4-dependent recruitment of immune cells

Abstract

Alcoholic liver disease (ALD) is one of the commonest causes of cirrhosis and liver failure in the developed world. Hepatic inflammation is the critical stage in progression of both ALD and non-ALD, but it remains difficult to study the underlying mechanisms in a human system, and current animal models do not fully recapitulate human liver disease. We developed a human tissue-based system to study lymphocyte recruitment in response to ethanol challenge. Precision-cut liver slices (PCLS) from human livers were incubated in culture, and hepatic function was determined by albumin production, 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium bromide assay, glucose uptake responses, and morphometric assessment. Responses of tissue and lymphocytes to ethanol exposure were determined by PCR, flow cytometry, histology, and lymphocyte infiltration assays. Human PCLS demonstrated appropriate upregulation of CYP2E1, ADH1α, and ADH3 in response to ethanol treatment. Ethanol also induced expression of endothelial VCAM-1 and ICAM-1, production of sICAM-1 and CXCL8, and the chemokine receptors CXCR3 and CXCR4 on CD4 and CD8 lymphocytes. CXCR3- and CXCR4-dependent migration of lymphocytes into the tissue increased significantly in response to treatment with ethanol. We have demonstrated that ethanol increases chemokine receptor expression and lymphocyte recruitment into human liver tissue, suggesting that it may operate directly to promote hepatitis in ALD. The physiological and pathophysiological responses of the PCLS to ethanol in vitro highlight the potential of this assay for dissecting the molecular mechanisms underlying human liver inflammation and as a screening tool for novel therapeutics.

FIG. 1.

(A) Production of albumin by cultured liver slices (six representative slices from three different liver specimens) measured by capture ELISA. Paired t-test showed no significant difference between control and 24h incubations (p = 0.4). (B) MTT reduction assay using between 40 and 56 replicate slices from normal and resection margin tissue only (NL) or all liver (ALL) slices, which included slices from five end-stage cirrhotic specimens. Paired t-test showed no significant difference between signal from normal livers and all livers and a significant reduction in signal at 24h compared with control. Data represent signal normalized per 500mg of tissue ± SEM for triplicate slices from each liver. (C) Image J quantification of images from representative fields of view captured from normal sections stained using bisbenzimide to visualize nuclear integrity. Data represent mean ± SEM% of area stained positive in five HPF analyzed for duplicate slices from four liver samples. Paired t-test revealed significant reduction in nuclear number at 48h. (D) Representative image from stained normal liver after 48h in culture showing loss in nuclear integrity in central area of slice compared with periphery. Bar represents 100 µm. (E) Representative images showing gross morphology of parenchymal areas of sections cut from PCLS collected fresh (Control) or cultured in a well for 24h (24 Hour) stained using hematoxylin and eosin (original magnification ×200, bar represents 50 µm). (F) Uptake of radiolabeled glucose in response to insulin treatment of normal PCLS. Data represent mean ± SEM uptake of six replicate slices (left graph) and periodic acid-Schiff stain of sections from representative PCLS confirming the presence of glycogen granules after 24 and 48h in culture (right figures). Glycogen stains a reddish purple (see samples indicated by arrowheads), and bar represents 50 µm.

FIG. 2.

(A) Semiquantitative PCR data from representative PCLS from three liver specimens: fresh liver (control), samples cultured in media alone (control 24hr), and 100mM ethanol (24hr EtOH) for 24h prior to RNA extraction. Data represent mean ± SEM densitometric signal for ADH1, ADH3, ICAM-1, and E-Selectin expressed as a fold change relative to expression of housekeeping gene. *Paired t-test indicates significant increase in expression relative to control p < 0.05. (B) Representative histological staining from samples of the same treated PCLS (i–iii) or of liver from a patient with ALD (iv) showing localization of positive signal for ADH1 (PT = portal tract, brown staining localized to periportal hepatocytes), ICAM-1 (EC = endothelial cells in portal vessel staining positive), and CYP2E1 (localized brown staining throughout lobule in PCLS and localized to periportal areas in ALD liver, PT = portal tract and C = central area of lobule) and expression of VCAM-1 (EC = endothelial cells in portal vessel) on untreated (v) and treated (100mM, 24h) (vi) PCLS. Samples were stained using standard immunohistochemical methods and DAB chromogen, and isotype controls were negative (not shown). Bars indicate 50µM.

FIG. 3.

(A) Production of proadhesive mediators by human PCLS in response to ethanol exposure. PCLS from two representative donors (one normal and one diseased) were incubated for 24h in the absence (white bars) or presence (black bars) of 100mM ethanol, and secretion of mediators into supernatants was measured using a cytokine profiler kit according to manufacturer’s instructions. Data represent mean + SD relative intensity of spots from four samples.

FIG. 4.

Exposure of lymphocytes to ethanol modulates chemokine receptor expression. (A) Typical flow cytometric analysis of a representative sample of normal PBL which were maintained in culture in media alone (untreated) or indicated concentrations of ethanol (50–100mM) in complete media. Cells were gated on FS/SC (left panels); expression of CD4 and CD8 (central panels); and expression of the chemokine receptors CCR5, CXCR3, and CXCR4 (histograms). Data represent percentage of CD4 and CD8 cells positive for indicated chemokine receptor and expression was determined relative to indicated isotype controls. (B) Pooled data for expression of the chemokine receptors CCR5, CXCR3, and CXCR4 indicated by percentage positive cells and MCF values for CD4 (left graphs) and CD8+ (right graphs) lymphocytes from seven donors. Data represent mean ± SEM, and bars indicate significant differences between control and ethanol-treated lymphocytes.

FIG. 5.

Adhesion of PBL (A) to cryosections cut from PCLS treated with media alone, ethanol (100–200mM), or TNF-α (20ng/ml) for 24 and 4h, respectively. Data represent mean ± SEM adhesion to endothelial structures per HPF. Representative images from a typical control and treated sample are shown (B). Bar represents 50µM, and PBL localize to endothelial cells in sinusoids (SEC).

FIG. 6.

(A) Infiltration of CellTracker Green–labeled lymphocytes into PCLS in the presence of ethanol. Labeled lymphocytes were incubated with PCLS in the absence (control) or presence of indicated concentration of ethanol (50–100mM) for 24h in complete media. Data represent mean ± SEM number of infiltrating cells per HPF of view (n = 15 samples from duplicate slices) Paired t-test confirms that alcohol pretreatment significantly increases lymphocyte infiltration (***p < 0.001). (B) Representative image of the edge of a treated PCLS showing predominant localization of labeled cells to the periphery with occasional cells infiltrating central areas (white arrows). Bar represents 50µm. (C) Adhesion of PBL to cryosections cut from PCLS treated with ethanol (100mM) for 24h. Lymphocytes were untreated or treated with 10 µg/ml of CXCR3 or CXCR4 mab, or a combination of the two (“both”) as indicated. Data represent mean ± SEM adhesion to endothelial structures per HPF in three replicate experiments using three liver donors, and paired t-test indicated significant reduction in adhesion when lymphocytes were treated with both antibodies compared with control (**p < 0.01)