Tumour necrosis factor alpha impairs function of liver derived T lymphocytes and natural killer cells in patients with primary sclerosing cholangitis

Abstract

Background: Primary sclerosing cholangitis (PSC) is considered to be a chronic autoimmune disease where infiltrating T lymphocytes have been implicated in the destruction of bile ducts. Altered function of these T cells may reflect abnormalities in the immune response leading to tissue damage.

Aim: We investigated the proliferative and functional capacity of freshly isolated liver derived T lymphocytes (LDLs) and natural killer (NK) cells from PSC patients.

Methods: The proliferative responses to common mitogens such as phytohaemagglutinin (PHA), concanavalin A (Con A), and lipopolysaccharide (LPS) were studied, and the cytotoxic function of T lymphocytes was measured using allogeneic target cells. NK (CD56(+)/16(+)) cytotoxic function was measured using the two cell lines K562 (NK sensitive) and Raji lymphoma cells (NK resistant).

Results: Compared with patients with primary biliary cirrhosis (PBC), autoimmune hepatitis (AIH), and normal controls (without liver disease), in PSC: (1) LDLs contained a low percentage of T cells; (2) there was significantly decreased expression of interleukin (IL)-2 receptor (p<0.001) on activated T cells (HLA-DR(+)); (3) LDLs but not peripheral blood lymphocytes had significantly impaired proliferative responses to mitogens such as PHA, Con A, and LPS (p< 0.001); (4) no cytotoxic activity of PSC liver T and NK cells was recorded; (5) significantly higher levels of tumour necrosis factor alpha (TNF-alpha) and IL-1beta but lower levels of IL-2, IL-10, and interferon gamma were found in the supernatants of mitogen stimulated LDL cultures (p<0.001); (6) higher percentages of freshly isolated PSC LDLs contained intracytoplasmic TNF-alpha and IL-1beta; and (7) pretreatment of PSC LDLs in vitro with neutralising TNF antibodies significantly enhanced proliferative responses and allowed IL-2 receptor expression following stimulation. In addition, the impaired cytolytic activity of both NK and T cells was partially restored. Impaired proliferative or functional capacity of liver derived T cells was not observed in either PBC or AIH patients.

Conclusions: We suggest that reduced T cell reactivity in liver infiltrating cells obtained from patients with PSC is due to high local production of TNF-alpha. Our findings indicate that the use of anti-TNF antibodies as an alternative treatment for PSC patients should be evaluated.

Figure 1

Biliary epithelial cells (BECs, second passage) isolated from two healthy and three primary sclerosing cholangitis (PSC) livers were tested for expression of CD95 and the two tumour necrosis factor (TNF) receptors to study the role of BECs as targets for lymphocyte mediated or direct cytokine mediated damage. TNF-α and IL-1 stimulated PSC BECs demonstrated increased expression of the TNF RI and RII receptors but low expression of CD95. Normal BECs expressed CD95 and TNF RII but had lower expression of TNF RI.

Figure 2

In general, liver derived lymphocytes (LDLs) isolated from controls (n=8) on stimulation with mitogens such as concanavalin A (Con A) and phytohaemagglutinin (PHA) for 72 hours showed lower proliferation responses than those observed in peripheral blood lymphocytes from the same on stimulation. No difference in response was observed after pretreatment with anti-tumour necrosis factor (TNF) monoclonal antibodies. Significantly diminished proliferation of LDLs from the seven primary sclerosing cholangitis (PSC) patients was observed on stimulation which was restored to almost normal by pretreatment with anti-TNF monoclonal antibodies (p<0.001). No significant differences in proliferative responses in mitogen stimulated LDLs from primary biliary cirrhosis (PBC) or autoimmune hepatitis (AIH) patients (either before or after anti-TNF monoclonal antibody pretreatment) was observed compared with controls (NS). Data represent mean counts per minute (cpm) of triplicate cultures. LPS, lipopolysaccharide.

Figure 3

Peripheral blood lymphocytes (PBLs) isolated from controls (n=8), primary sclerosing cholangitis (PSC) (n=7), primary biliary cirrhosis (PBC) (n=4), and autoimmune hepatitis (AIH) (n=3) patients showed normal proliferative responses on stimulation with mitogens such as concanavalin A (Con A) and phytohaemagglutinin (PHA) for 72 hours. No difference in responses was observed after pretreatment with anti-tumour necrosis factor (TNF) monoclonal antibodies. Data represent mean counts per minute (cpm) of triplicate cultures. LPS, lipopolysaccharide.

Figure 4

Cytotoxic liver derived lymphocytes (LDLs) (T cells) generated by stimulation against normal allogeneic LDLs from control, primary biliary cirrhosis (PBC), and autoimmune hepatitis (AIH) livers showed high cytotoxic function against allogeneic target cells. In the case of controls, only the average per cent lysis (from eight experiments in normals, four in PBC, and three in AIH) is shown. Cytotoxic LDLs (T cells ) isolated from primary sclerosing cholangitis (PSC) livers (n=7) on the other hand did not show any cytotoxic activity against allogeneic target cells. The cytotoxic activity of T cells was thus completely abolished in the livers of PSC patients (p<0.001). Natural killer (NK) cells (CD56+16⁺) isolated from control, PBC, and AIH livers showed normal cytotoxic activity against K562 (NK cell sensitive cell line) but not against Raji's (NK resistant cell line) (only mean per cent lysis is shown). NK cells isolated from PSC livers (n=7) on the other hand did not show any cytotoxic activity against K562. NK cell cytotoxic activity was thus completely abolished in the livers of PSC patients (p<0.001). However, overnight treatment of cytotoxic LDLs (T cells) isolated from PSC livers with tumour necrosis factor (TNF) antibodies (abs) (10 mg/ml) showed a moderate increase in cytotoxic activity against allogeneic target cells. Similarly, NK cells (CD56⁺/16⁺) showed enhanced cytotoxic activity against K562 (NK cell sensitive cell line).

Figure 5

Increased immunofluorescent staining of intracellular cytokines tumour necrosis factor α (TNF-α) and interleukin 1β (IL-1β) was seen in freshly isolated liver derived lymphocytes from all seven primary sclerosing cholangitis (PSC) patients but not from controls. A representative picture of this finding from one PSC patient and one normal control is shown. Cells were initially fixed, permeabilised, and stained with fluorescein isothiocynate conjugated anti-TNF/anti-IL-1β antibodies. Unlabelled anti-cytokine monoclonal antibody was used to decrease high background staining.

Figure 6

Effect of anti-cytokine antibodies on restoration of diminished proliferative responses to mitogen stimulation by concanavalin A (Con A) and phytohaemagglutinin (PHA). Liver derived lymphocytes (LDLs) from primary sclerosing cholangitis (PSC) livers remained untreated or pretreated with anti-tumour necrosis factor (TNF) monoclonal antibodies (10 µg/ml) and/or interleukin 1 (IL-1) monoclonal antibodies (10 µg/ml), or control monoclonal antibodies (10 µg/ml) overnight. Con A or PHA was added and ³H thymidine incorporation was detected after 72 hours. Anti-TNF monoclonal antibodies alone restored the diminished proliferative responses in the LDLs from PSC patients.