Distinct cytotoxic lymphocytes subsets suggest a role for the liver in immune homeostasis

59 WORKING 1 WP4/25

PARTY 4: Liver cell biology

If--Kg

1

PROINK4MM4TORY CWOKINB WMULATE NII’RIC OXIDE PO) PRODUmON AND INDUCIBLE NIIRIC OXIDE WNlXASE

EXFRESIONlNRATCEfOLANGIOClTE3 WOWA. Granato. L. Calb, C. Spirli, NF. LaRusso*, D. Vianello. E. Duner. L. Okolicsanti#, G. Crepaldi, M. Strazabosco. Inst. of Internal Medicine, University of Padova, Italy, *Mayo Clinic, Rochester, Minnesota, U.S.A., #Gastroenterol. Univ. Parma, Italy. In the liver, nitric oxide (NO) plays a role in the regulation of portal haemodinamics and inflammation, is a potent inhibitor of stellate cell contractility and possesses cytotoxic as well as antioxidant properties. Since a Thl predominant pattern of T-cell activation is present in primary biliary cirrhosis, we studied the effects of IL-l, TNFa, IFNy and IL-4 on NO production and iNOS gene expression in a normal rat cholangiocytes cell line (NRC-l) that maintains a polarized distribution of membrane markers and ion carriers. Methods Polarized NRC-I monolayers were cultured semipermeable membrane inserts. IL- l(ZOU/ml), over TNFa(SOOU/ml), IFNy(lOOU/ml), n-4(0.1% v/v), alone or in different combinations, were added to the basolateral side. At different time points, basolateral medium was collected and NO2MO3 concentrations measured by ELISA. At the same points, cellular RNA was extracted and iNOS gene expression determined by comparative RT/PCR. Results When given alone, only IL-1 and IFNr significantly increased NO production with maximal effects between 24 and 48 hours. iNOS mRNA expression increased, peaking at the 12 hours and then slowly decreased. IL-1 and TNFa had sinergistic effect when administered together with IFNy, while IL-4, alone or in combination with Thl cytokines had no effect on NO production. iNOS mRNA expression paralleled these findings. NO production was inhibited by L-Nh4MA and by aminoguanidine, an irreversible inhibitor of iNOS. Conclusions These data indicate that the intrahepatic bile duct epithelium is able to produce NO following stimulation with Thl, but not Th2 cytokines. Effects of Thl cytokines are sinergistic, facilitated by IFNr and mediated through an increase in iNOS gene expression.

DISTINCT CYTOTOXIC LYMPHOCYTES A ROLE FOR THE LIVER IN IMMUNE DG

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SUBSETS SUGGEST HOMEOSTASIS

o erty. S No ‘s. C Co s. G M&tee. 0 Travnor. J Heaartv. ERC 8. tver &it, !tyVincent’s Hospital, Dublin 4, Ireland.

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APPROACHES TO A MURINE MODEL OF AMA POSITIVE NON-SUPPURATIVE DESTRUCTIVE CHOLANGITIS (NSDC) MF Bassendine. JM Palmer. D Decruz*. GW McCauehan*. DH Strickland*. JD Sedgewick’. SJ Yeaman. AD Burt. DJZJ Jones. Centre for Liver Research, University of Newcastle upon Tyne, NE2 4HH, UK and *Centenary Institute, Sydney, NSW 2042, Australia. Previous attempts to induce the autoimmune disease (AD) primary biliary cirrhosis (PBC) in experimental animals by immunising with mitochondrial (M) autoantigens (Ags) have been recombinant unsuccessful. In other AD models there is clear evidence that the genetic background of the animal plays a role in influencing disease incidence and severity. We have therefore attempted to induce features of PBC in mice by immunisation of a variety of inbred strains with M Ags (purified pyruvate dehydrogenase complex[PDC], EUE3BP components which copurify and p163[ 14-mer peptide spanning the lipoyl domain of PDC-E2]) and appropriate adjuvants. Four inbred strains of mice (A[n=30],C[n=45], D[n=30], E [n=lS]) were selected in which AD either occur spontaneously or can be induced, and compared to BALB/C (B[n=15]). Female mice were immunised in groups of 5,aged 4-8 wks with M Ags and adjuvant (FCA ip[n=l lo] or subcut [n=35], + Pertussis toxin N days 0 and 2 [n=25] or LPS IV on day 2[n=30]. Presence of AMA was measured by ELISA and hepatic histology assessed blind at 30-35 weeks. All mice immunised with PDC or PDC-E2/E3BP developed high titre AMA, which inactivated the catalytic function of PDC in vitro (a property of AMA in PBC sera). Development of AMA in mice immunised with ~163 was strain dependent [S/lOA, 0/15C, 0/5D, 4/5E, 5/5B]. 60 mice immunised with M Ags + FCA ip were directly comparable [15 of B,C,D & E]. Portal tract inflammation was seen in 3/15C, 1/15D, u and 1115B but granulomatous NSDC, reminiscent of the classical histological lesion of PBC, was only seen in strain E [7/151 Summary: An inbred strain of mouse has been identified which develops AMA and bile duct damage classical of PBC when immunised with M Ags + FCA ip. This experimental model of NSDC should be of value in understanding the pathophysiology of PBC.

IMMUNOTHERAPY 3F HEPATOCELLULAR CARCINOMA (HCC) BY ENGINEERING TUMOR CELLS TO EXPRESS B7-1 ANDIOR IL-12 Y. Sun. C. Oian. D. Pene. J. Prieto. Department

of Internal

Pamplona,

The liver has been postulated to be a site of inactivation and apoptosis of activated T cells, in the maintenance of peripheral immune tolerance and homeostasis. Programmed cell death, or apoptosis, is mediated by CDS+ T cells and natural killer (NK) cells, which are present at high levels in the liver. We have also described a third cytotoxic lymphocyte population, natural T (NT) cells, that appear to be exclusive to the liver. Here we have investigated the functional properties and specificities of hepatic T, NK and NT cells taken from 23 normal liver transplant donors. The relative proportions of T, NK and NT cells and their expression of receptors involved in activation and inhibition of cytotoxicity were determined by three colour flow cytometry of gated CD3+CD56-. CD56+CD3-, and CD3+CD56+ hepatic lymphocytes (HL) respectively. The ability of each fraction to lyse NK-sensitive and T cell-sensitive target cells was tested in chromium-release assays using immunomagnetic bead-separated cell populations as effecters. Expression of Va24-JuQ T cell receptor (TCR), which recognises non-peptide antigens, was analysed by RT-PCR.

One

of

the strategies

vaccinanon

with

as 87-l

have

Investigated

and/or

retroviral

vectors

Material

and

vectors

School

cytokmes

carrymg

of Medicine,

LNCX

University

B7-I

of Navarra,

and/or

gene

IL-12

87-l

Thl

responses.

(BNLMeo)

HCC

kinds

(LNC/B7-I),

cells

87-l

Objective: celis

murine

and

with

animal

model.

retroviral

or both

(LNCIIL-12)

and IL-12

We

(BNL)

were modltied

as a control

or both

by

molecules

of recombinant IL-12

BNL

immunity

costimulatory

in a syngenic

three

cDNAs.

IL-IZ(BNLML-I2),

antitumoral

of transducing

constructed

contammg

increase to express

facihtate

effect

(LNC/B7-I/IL-12)

resistance

I(BNLIB’I-I),

is to

engineered which

We

Method:

from

cancer

cells

the anti-tumor

and IL-12

neomycin

to treat

neoplastic

such

B7-I

Medicine,

Spain

high

to express

level

of

B7-

(BNL/B7-l/IL-12)

by

the cells with the corresponding recombinant retroviruses. Results: Parental BNL cells expressed low level of MHC class I but not MHC

transducnon

of

class II molecules. expressron order

The gene-modified

nor growth

to know

tumongemclty

The mean proportions of NK (31% vs 13%), NT (32% vs 2%) and CD& T cells (72% vs 36%) were significantly raised among HL compared to peripheral blood lymphoc es. Although hepatic NK cells expressed the na’ive CD45RA and CD56 y;’ @“‘phenotypes, they spontaneously lysed K562 and P615 cells. The maiority of T cells (44-63%) expressed activated CD45RO+ CD56dim phenotypes, and were capable of TCR-mediated cytolysis of P615 cells. Up to 95% of NT cells expressed activated phenotypes, they could be induced to kill NK-sensitive K562 cells by stimulation in the presence of IL-2, and they demonstrated TCR mediatedcytolysis of P615 cells. NK but not NT cells expressed the activatory receptor CD16, and cytotoxicity by both NK and NT cells could be inhibited by receptors specific for MHC class I molecules. Vc124-JaQ TCR chain mRNA was expressed by hepatic NT cells, but not by NK cells or T cells.

cells expressmg

Our findings of three distinct populations of cytotoxic HL suggest a role for the liver as a specialised organ in the elimination of cells which may include pathogens, virus-infected and tumour cells as well as activated T cells. Deficient removal of these cells could result in overwhelming infection or autoimmune disease.

Vacclnanon

respecnvely,

cells developed

parental

cells was observed

12

addnlon,

In

found

parental

of these gene-modified

III I I 8%, 80% and 95% of mice

and BNL/B7-I/IL-l2

slgnlficant

cells did not change with

B7- I, IL- I2 or both into syngeneic

was produced or BNL/Neo

BNL

rates as compared

the

InhIbited

on Increase

BNLIB7.l/IL-l2

while

tumor. only

growth in mxe

We found BNL/B7-I,

all mice

inoculated effect

a tumor with

in the production and a marked

IL-2

elevation

established BNWlL-I2

with

against

BNLIIL-I2

molecule

in vifro.

we implanted

mice.

III nnce receiving of

cells,

MHC cells

receiving

A protective

treated

BNL

that no tumor BNLIIL-I2 parental

BNL

rechallenge

with

and BNL/B7-l/IL-

at a distant

place

and BNL/B7-I-IL-12.

by splenocytes in the production

In

BNL

of mice of both

We

injected IL-2

was with

and INF-

received BNLIIL-12. Conclusions: The inJectIon of HCC cells genencally modified to produce 87-l and/or IL-12 protects against tumor gro\rrh III a murlne model. The protective effect observed after tumor gamma

III mxe

transducnon malignancy

which

wnh with

IL-12

cDNA

gene-modified

or prevent

recurrence

involves tumor

cells

the

stimulation

might

after resectton.

of

Thl

be used to eliminate

responses. restdual