ELSEVIER Immunology Letters 59 (1997) 159%170

Induction of autoimmune-like hepatic and ductal lesions by administration of lipopolysaccharide in mice undergoing graft-versus-host reaction across MHC class I difference

a Animal Center fbr Biomedical Research. Faculty of’ Medicine, Tile University of Tokyo, 7-3-l Hongo, Bunkyo-ku, Tokyo I13, Japan b Department of Internal Medicine (1). The Jikei University School of Medicine, 3-25-8 Nishi-shinbashi. Minato-ku, Tokvo 105, Japan

’ Research Laboratory, Zenyaku-Kogyo, 33-7, Ohizumi-machi 2-chome, Nerima-ku, Tokyo 178, Japan

Received 20 May 1997: accepted 5 August 1997

Abstract

In this paper, we examined the induction of autoimmune-like histologic changes in the liver and other organs of mice undergoing graft-versus-host reaction (GVHR) with MHC class I disparity by the administration of bacterial lipopolysaccharide (LPS), on the assumption that stimulation with LPS could be an exacerbating factor. Spleen cells of C57BL/6 (B6) mice were injected twice into (B6 x bml) Fl recipient mice at an interval of 7 days to induce MHC class I GVHR and then challenged with 1 fig of LPS intravenously on the next day of the cell transfer. The hepatic lesions of the group of MHC class I GVHR mice challenged with LPS showed marked cellular infiltration at the portal area and focal necrosis was observed in the hepatic lobule. The major infiltrating cells were CD8 + , and others including CD4+ cells being of minor populations. In addition, ductal lesions in extrahepatic organs, including the pancreas and salivary glands also showed marked cellular infiltration. Thus, we have demonstrated that LPS induced ductal lesions in mice with MHC class I disparity. CD8+ cells were detected at the destructive hepatic lesions, which might be effector cells. These findings indicate that LPS might be one of the potential factors which augment autoimmune-like lesions. 0 1997 Elsevier Science B.V.

Kqvwords: Graft-versus-host reaction (GVHR); Lipopolysaccharide (LPS); MHC class I; Autoimmune-like disorders

1. Introduction

Graft-versus-host reaction (GVHR) is induced by the

transferred donor T lymphocytes which recognize dif-

ferent histocompatibility antigens of the host. Out-

comes of GVHR are varied depending upon strain

combination, transferred T cell subset or difference in

histocompatibility antigens. Gleichmann et al. [l] re-

ported that there were two different forms of GVHR,

autoimmune (immunoproliferative) diseases and severe

immunodeficiency. We have used MHC class I or class

*Corresponding author. Tel.: + 81 3 34331 Ill; fax: + 81 3

34350569.

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PIf SOl65-2478(97)00120-X

II-mutant mice and observed cellular infiltration

around the interlobular bile ducts in mice with GVHR

due only to the difference in MHC class II [2]. Histo-

logical changes induced by this type of GVHR were

quite similar to those observed in the early stage of

primary biliary chirrhosis (PBC) characterized by

chronic non-suppurative destructive cholangitis (CNSDC) and Sogren’s syndrome. Since PBC has been considered to have autoimmune etiology but remains

largely unknown in pathogenesis, we have extensively

studied the developmental mechanism of PBC by using

this animal model [3-61. In contrast to these well-stud-

ied MHC class II GVHR, little is known concerning

histopathological lesions of MHC class I GVHR

160 A. Hayashi et al. /Immunology Letters 59 (1997) 159-170

Table 1 Occurence of GVHR in (bml x B6) Fl mice

Group”

1 2 3 4

Donor

B6 B6

Recipient

(bml x B6)Fl (bml x B6)Fl (bml x B6)Fl (bml x B6)Fl

Cell transfer

+, x2 +. x2 -

LPS

+. X2

f, X2

Number of mice Spleen indexb Liver indexb

5 11.7 * 7.1* 63.2 + 8.3* 5 6.1 _+ 1.6 48.7 * 5. I 3 6.0* 1.2 43.3 * 2.5 3 6.6k 1.7 45.3 * 3.2

a (bml x B6)Fl mice were sacrificed 2 weeks after the first cell transfer. The occurence of GVHR was assessed by spleen index and liver index. b Spleen index was represented as spleen weight (mg)/body weight (g). Liver index was represented as liver weight (mg)/body weight (g). The data are presented as the mean i SD. * PiO.01 by Wilcoxon test, as compared with the other groups.

mouse. Previously, [7] hepatic involvement was not observed in MHC class I GVHR mice.

Autoimmune diseases are thought to be augmented by the infection of bacteria or viruses during the clinical course of the disease. Burroughs et al. [8] reported that bacteriuria frequently followed in patients with PBC and that it may be significantly associated with the exacerbation of the disease. Morreale et al. [9] also reported that patients with PBC and with the bacteri- uria showed higher mortality. Sera from PBC patients showed a higher level of antibodies (IgM class) against lipid A, which is a part of lipopolysaccharide (LPS), suggesting a possibility of involvement of LPS in the disease progress [lO,ll]. From these reports, we as- sumed that endotoxin could be an inducing or exacer- bating factor for the development of autoimmune-like lesions, although there has been a report against spe- cific association between exacerbation of PBC and prevalence of bacteriuria [ 121.

In this article, we report the induction of ductal lesions in mice undergoing MHC class I GVHR by the administration of LPS and discuss the findings with reference to the developmental mechanisms of the au- toimmune-like lesions.

2. Materials and methods

2.1. Mice

C57BL/6 (B6) (haplotype:H-2Kb, I-Ab, H-2Db) mice were purchased from Charles River Japan, Atsugi, Kanagawa. B6.C-H-2b”’ (bml) (haplotype: H-2Kb”‘, I-A, H-2Db) mice which carrys a mutant gene at the H-2K locus of the MHC, obtained from the Jackson Laboratory (Bar Harbor, ME) were used to obtain Fl hybrid mice. These mice were bred and maintained at our own animal facilities. All mice were used at lo-12 weeks of age.

2.2. LPS

LPS of Escherichiu coli (0111: B4, Difco Laborato- ries, Detroit, MO) was resuspended in phosphate- buffered saline (PBS) and stored at - 20°C until used.

2.3. Preparation of spleen T cells

Spleen cells from B6 mice were prepared by mincing spleen and passing cells through stainless-steel mesh in Eagle’s minimum essential medium (MEM, Nissui, Tokyo) buffered with 5 mM Hepes (Sigma, St. Louis, MO) containing 2% fetal calf serum (FCS) (Bockneck, Rexdale, Ont.). Cells were washed with the same medium and treated with 0.83% ammonium chloride- Tris buffer to eliminate red blood cells. After washing serially with MEM-Hepes containing 10 and 2% FCS, the cell suspension was passed through a nylon wool column and the non-adherent fraction was collected as described previously [5].

2.4. Induction of GVHR and LPS challenge

To evoke GVHR, sex-matched B6 spleen cells were inoculated into (B6 x bml ) Fl recipient mice at a dose of 1 x 10’ intravenously twice at weekly intervals (on day 0 and day 7). LPS (1.0 fig/mouse) was injected intravenously on day 1 and day 8. All mice were sacrificed on day 14 for examinations. Experimental groups were divided into four groups according to cell transfer and/or LPS injection as listed in Table 1.

2.5. Assays of the occurrence of GVHR

At sacrifice, about 1 ml of blood was obtained by cardiac puncture from each mouse and the spleen was weighed to calculate spleen index using the following formula:

A. Hayashi et al. / fmmunolog_v Letters 59 (1997) 159- 170 16

(A41

Fig. 1. (Continued)

62 A. Hayashi et al. /Immunology Letters 59 (1997) 159-170

Fig. 1. (Continued)

A. Hayashi et at. / fmmunology Letters 59 (1997) 159- I70

Spleen index = spleen weight (mg)/body weight (g).

The weight of the liver was also measured and the index was calculated using the same formula.

2.6. Histological studies

(B6 x bml) Fl mice were sacrificed at 2 weeks after the first cell transfer. Age-matched control mice were also sacrificed at the same time. The liver was resected individually for histological examinations. The liver specimens were divided into two groups: one for light microscopic study and the other for immunohistochem- ical analyses. The former specimens were fixed in 10% phosphate-buffered formalin, embedded in paraffin, and sections made were stained with hematoxylin and eosin. Five portal areas in each specimen were counted at a magnification of x 200 under a microscope equipped with a micrometer reticule (0.25 mm2, Olym- pus, Tokyo). The lesions were graded according to the average number of infiltrating cells in five or six portal tracts chosen at random as follows: grade 0, none; grade 1, mild ( < 100); grade 2, moderate (100-400); grade 3, marked ( > 400). The latter specimens were immediately frozen in isopentane precooled in acetone and dry ice, and stored at - 80°C until use. Sections 446 pm thickness were cut using a cryostat (Sakura- seiki, Tokyo) and attached to slide glass coated with 0.01% poly-L-lysine.

2.7. Electron microscopic studies

The mice were perfused through the heart with phys- iologic saline and subsequently with 2.5% glutaralde- hyde in phosphate buffer at pH 7.4. The tissue blocks were postfixed with 1% osmium tetraoxide in 1.8% potassium ferrocyanide, dehydrated, and embedded in Araldite. Once ultrathin sections were stained with uranium acetate and lead citrate, the samples were examined under an electron microscope (Hitachi H- 7000, Hitachi, Ibaraki, Japan).

2.8. Immunohistochemical techniques

The cryostat sections were dried in air and fixed in cold acetone for 10 min. The slides were next washed in PBS, pretreated with Biotin/Avidin solutions (Blocking Kit, Vector Laboratories, Burlingame, CA) for 10 min

163

and then with 20% normal sheep serum diluted in 0.1 M PBS at pH 7.4 for 30 min to block nonspecific binding. After washing with PBS, sections were incu- bated with the biotinylated first antibodies at various dilutions for 90 min at room temperature. The follow- ing biotinylated mAbs were used: anti-CD8 (clone 53- 6.7, IgG2a), anti-CD4 (clone GK1.5, IgG2b), anti-Mac-l (clone M-70.15, IgG2b), anti-B220 (clone RA3-6B2, IgG2a), anti-H-2Kb (clone MIH 030- 11 B, IgG2a) anti-I-Ab,k (clone MIH 040-100, IgG2a) anti- LFA-1 (M17/4, IgG2a), anti-ICAM- (3E2, IgG). Then streptoavidin-horseradish peroxidase complex (Vectas- tatin Elite ABC Kit, Vector Laboratories, Burlingame, CA) was layered on the sections for 45 min at room temperature. The sections were washed with PBS and the reaction products were visualized using H,O,-con- taining 3,3’-diaminobenzidine in 0.05 M Tris buffer. These sections were counterstained with Trypan Blue (Nakarai Tesque, Kyoto) and mounted with mounting media (Mount-Quick, Daido Sangyo, Tokyo) after de- hydration in graded ethanol and xylene.

2.9. FIow cytometry

CD8 and CD4 molecules on spleen cells were exam- ined by flow cytometry. Spleen cell suspension was treated with hemolytic buffer, adjusted to 1 x lo6 cells/ ml in PBS containing 2% bovine serum albumin and 0.02% sodium azide, and incubated with appropriately diluted FITC-conjugated anti-CD8 mAb or an FITC- conjugated anti-CD4 mAb for 30 min on ice and washed twice with PBS. The stained cells were analysed by FACScan (Becton Dickinson, Mountain View, CA) with the Lysis II program.

2.10. Measurement of serum alanine aminotransferase (ALT) level

ALT levels in the serum of each mouse were mea- sured by Reitman-Frankel method using S.T.A. test kit (Wako Pure Chemical Industries, Osaka).

2.11. Statistical analyses

The statistical analysis was performed for each parameter by Wilcoxon’s rank-sum test, except the serum ALT level, to which Student’s t-test was applied.

Fig. 1. Light microscopic findings of the organ lesions (liver, pancreas and salivary gland) of LPS-challenged MHC class I GVHR mice (A-l, B-l and C-l; H.E. x 66). Severe cellular infiltration of the portal area involving CNSDC-like bile duct lesions (D-l; H.E. x 66) piece meal necrosis and also spotty necrosis in the hepatic lobule (D-2; H.E. x 66) were observed. Cellular infiltration into the portal area in the liver and ductal lesions of pancreas and salivary gland were not observed in MHC class I GVHR mice (A-2. B-2 and C-2; H.E. x 66), LPS-challenged (B6 x bml)Fl mice (A-3, B-3 and C-3; H.E. x 66) and (B6 x bml) Fl control mice (A-4, B-4 and C-4; H.E. x 66).

164

Table 2

A. Hayashi et al. /Immunology Letters 59 (1997) 159- 170

Grading of cellular infiltrations in liver, pancreas and salivary gland by light microscopy in (bml x B6)Fl mice

Group Cell transfer (l-2 x 10’ B6 T cells) LPS (1 1(g) Cellular infiltration”

Liver Pancreas Salivary gland

1 +, x2 +, x2 2 2 2 2 f, x2 O/l 0 0 3 +, X2 0 0 0 4 0 0 0

a Infiltrating cells were enumerated in five to six portal tracts and graded as follows: grade 0, none; grade 1. mild (< 100); grade 2, moderate (100-400); grade 3, marked (>400). Counting methods were described in detail in Section 2.

3. Results

3. I. Administration of LPS augmented splenomegaly and hepatomegaly of mice with MHC class I GVHR

(B6 x bml) Fl mice were divided into four groups according to cell transfer and/or LPS injection as listed in Table 1. Mild hepatomegaly, though statistically insignificant, was observed in (B6 x bml) Fl recipient mice which were injected with B6 splenic T cells (Group 2), as compared with control mice (Groups 3, 4). Both the spleen index and the liver index of LPS-challenged mice with MHC class I GVHR (Group 1) were remark- ably elevated (P < 0.01).

3.2. LPS administration induced destructive changes on hepatic lesions of mice with MHC class I GVHR

The histological findings of the liver, pancreas and salivary glands were as shown in Fig. 1 and summa- rized in Table 2. The changes of histological findings of the liver which meet the criteria were seen in all mice in each experimental group. The pathological changes of the liver were observed not only in portal areas (Fig. l A-l) but also in hepatic lobules (Fig. lD-2). Cellular infiltration was not observed in non LPS challenged MHC class I GVHR mice (Fig. l A-2) and LPS chal- lenged control mice (Fig. l A-3) and non LPS chal- lenged control mice (Fig. lA-4). CNSDC-like bile duct lesions (Fig. lD-1) in the portal area and focal necrosis in the hepatic lobule (Fig. lD-2) were characteristic features of LPS-challenged MHC class I GVHR mice (Group 1).

Cellular infiltrations were also observed around ducts of the pancreas and salivary glands only in Group 1 mice (Fig. lB-1 and lC-1), but never in control mice (Fig. IB-2, 3, 4 and lC-2, 3, 4).

Electron microscopic analysis of the liver in LPS- challenged MHC class I GVHR mice revealed that liver-infiltrating lymphocytes had crossed over the en- dothelial cells and directly attached to hepatocytes in a broad area. There were some hepatocytes showing signs of necrosis; swelling of mitochondria, decrease in the

number of cristae of mitochondria, vacuolization and splitting of plasma membranes (Fig. 2A), and necrotiz- ing hepatocytes were ingested by macrophages (Fig. 2B). Lymphocytes which have dense granular bodies (arrow heads) infiltrated into the intercellular space of the bile duct epithelial cells (Fig. 2C). The basement membrane and epithelial cells of bile ducts appeared intact. There were no destructive changes in the liver of non-LPS challenged MHC class I GVHR mice or other control mice.

3.3. Immunohistochemical analyses of the hepatic lesions of mice with GVHR

Immunohistochemical studies revealed that the major infiltrating cells in hepatic lesions of mice with MHC class I GVHR challenged by LPS were CD8 + (Fig. 3A). CD4+ cells were scarcely found in the portal area and at the portion of focal necrosis (Fig. 3B). Mac-l + cells were frequently detected at the portion of focal necrosis (Fig. 3C), and B220+ cells existed around the infiltrating lesions (Fig. 3D).

Though, H-2Kb antigen was normally expressed in the liver (Fig. 3E), an aberrant expression of I-Ab antigen was observed on the bile duct epithelial cells (Fig. 3F).

Almost all mononuclear cells infiltrating the liver were LFA-l-positive (Fig. 3G). ICAM-1, which is the ligand of LFA-1, was expressed on endothelial cells of hepatic sinusoids and cytoplasmic membrane of hepato- cytes (Fig. 3H).

3.4. CD8-positive cells expanded in the spleen of LPS challenged MHC class I GVHR mice

Flow cytometric analyses of spleen cells revealed that the number of CD8+ cells had remarkably increased and predominated over CD4+ cells in MHC class I GVHR mice administered with LPS (Fig. 4). There was no significant difference in the percentage of CD8+ cells among non-LPS-challenged mice with MHC class I GVHR, LPS-challenged and untreated mice, in which CD4 + cells predominated.

A. Hayashi et al. /Immunology Letters 59 (i 997) 159- 170 165

Fig. 2. Electron micrography of the liver of MHC class I GVHR mice challenged with LPS. A: Liver-infiltrating lymphocytes (arrow head) were attached to hepatocytes that had crossed over the endothelial cells and directly attached to hepatocytes. The hepatocytes showed irregularity of nuclear membrane and mild dilatation of endoplasmic reticulum. Some of the hepatocytes (arrow) showed signs of necrosis; i.e. swelling of mitochondria. decrease of cristae of mitochondria, vacuolization and splitting of plasma membranes ( x 7600). B: Macrophage (arrow head) phagocytosed the necrotized hepatocytes. Phagosomes were observed in the cytoplasm of a macrophage (arrow) ( x 9500). C: Bile duct of MHC class I GVHR mice that were challenged by LPS. The bile duct epithelial cells were infiltrated by three lymphocytes that have dense granular bodies (arrow heads). The basement membrane of bile ducts showed no signs of destruction and remained almost normal ( x 7600).

3.5. Serum ALT levels were elevated in MHC class 1 GVHR mice following administration of LPS

In order to substantiate the destruction of the liver biochemically, we examined the serum level of ALT. (B6 x bml )Fl mice injected with cells in which LPS showed a marked elevation of the ALT level (P < 0.05, Fig. 5). On the other hand, the ALT level of Fl mice injected with cells or LPS only was not elevated.

4. Discussion

In this study, we have shown that hepatic and ductal lesions were induced by the administration of LPS following cell transfer in the recipients with GVHR due to MHC class I difference. Histologic changes with lymphoid cell infiltration were observed in the portal area of the liver, pancreas and salivary glands but neither in the skin nor bowel. These findings are quite similar to the feature of class II GVHR [3]. In addition, characteristic destructive changes in the hepatic lobule appeared in these mice.

Immunohistochemical analyses revealed that the number of CD8 + cells had markedly increased in the hepatic lesions of mice with MHC class I GVHR

following the administration of LPS. The increase of the number of CD8+ cells was confirmed by flow cytometric analysis of spleen cells of the mice. It is supposed that transferred CD8 + cells reacting with host MHC class I (H-2Kbm1) antigen were strongly activated by the LPS administration, differentiated into cytotoxic T cells and injured the host bile duct epithelial cells and hepatic cells. In fact, electron microscopic studies revealed that lymphocytes attached to hepato- cytes showed signs of necrotic process. This finding may indicate that destructive changes of hepatocytes are initiated by contact with lymphocytes. Since such destructive changes were never observed in GVHR mice without LPS injection, LPS seems to have a crucial role in the formation of the characteristic destructive lesions as shown in this paper.

The immunomodulatory effect of LPS has been stud- ied for the last three decades, especially as a stimulator of macrophages [13-151. LPS is a potent inducer of monokines such as TNF and IL-l that have been shown to mediate a pathological process. In GVHR, Nestle et al. [16] reported that macrophages during allogeneic reaction secreted a large amount of TNF-a resulting in death following administration of a sub- lethal dose of LPS. Mizoguchi et a1.[17] used a fulmi- nant hepatitis mouse model induced by P. acnes

166 A. Hayashi et al. /Immunology Letters 59 (1997) 159-170

Fig. 2. (Continued)

followed by LPS administration in mice, and reported that hepatic adherent cells primed by P. acnes might be responsible for the induction of liver damage. Recently, the role of Fas/Fas ligand in P. acnes/LPS induced hepatic damage has been clarified. Okamura et al. reported that IFN-1/ inducing factor (IL-l@ from P. acnes/LPS stimulated macrophages/Kupffer cells in- duced IFN-y production [18] which renders Fas molecules functional [ 191. IL-l 8 increases the expression of Fas ligand on NK/cytotoxic T cells [20] that en-

hances cytotoxic activity. Recently, it has been reported that the mechanism of hepatic cell death in this model is conducted through the Fas/Fas ligand system.

In our experiment, an increased number of macrophages at the region of focal necrosis was ob- served following the administration of LPS. CD8 + cells seemed to be effecters for tissue destruction because these cells are major infiltrating cells. Taken together, it is speculated that LPS-activated macrophages/Kupffer cells might be a trigger for the accumulation of CD8 +

A. Hayashi et al. /tmmunolog~ Letters 59 11997) I59- I70 167

(D)

Fig. 3. Immunohistochemical studies of hepatic lesions of MHC class I GVHR mice challenged with LPS. A. CD8 ( x 33); B. CD4 ( x 66); C, Mac-l ( x 33): D, B220 ( x 33); E, H-2Kb ( x 33): F, I-Ab ( x 33); G, LFA-1 ( x 16.5); H. ICAM- ( x 16.5). The nuclei were counterstained with Trypan Blue and immunohistochemical procedures are described in Section 2.

cytotoxic T cells. In addition, IL-18 secreted from activated macrophages/Kupffer cells may induce hep- atic cell death through the Fas/Fas ligand system.

Furthermore, infiltrating CD8 + cells responding to MHC class I antigen might produce cytokines such as IL-2. Mizuochi et al. [21,22] reported that CD8 + helper T cells had an ability to produce IL-2. In our model, the presence of CD8+ helper T cells, if any, seems to have a marginal effect, because a minimal accumulation of CD8+ cells was found in MHC class I GVHR. However, it should be noted that CD8 + cells had markedly increased after administration of LPS, which might indicate the accumulation of IL-2-producing CD8 + cells that enhance subsequent allogeneic reaction and tissue destruction in the liver.

In contrast, we have obtained preliminary data show- ing that an increase occurs in CD4+ cells in the portal area but not in CD8 + cells after the administration of LPS in MHC class II GVHR, and that demonstrable tissue destructive changes in the hepatic lobule were not observed [23].

Vogel et al. [24] reported that LPS can directly stimulate the proliferation of murine cytotoxic T cell line. In addition, they found that highly purified splenic T lymphocyte populations contained a small subpopu- lation (about 3%) which was proliferated by LPS. While, Mattern et al. [25] reported that monocytes were required in LPS induced proliferative response of hu- man CD4+ or CD8 + T cells. Thus, in MHC class I/class II GVHR mice, CD8 + /CD4 + cells were stimu- lated by LPS directly or indirectly with the aid of macrophages.

In MHC class II GVHR using (bm 12 x B6) Fl as recipients, donor CD8 + cells could not respond to host MHC class I antigen [26]. Therefore, even if CD8 + cells were stimulated by LPS, donor CD8+ cells could not develop to become cytotoxic killer cells. On the contrary, donor CD8 + cells could respond to allo- geneic class I antigen in (B6 x bml) Fl recipient mice but could induce only slight organ lesions. Mild histo- logical activity of the organ lesions might be due to lack of help from CD4 + or CD8 + helper T cells.

168 A. Hayashi et al. /Immunolog_y Letters 59 (1997) 159-170

(G)

Fig. 3. (Continued)

Bismuth et a1.[27] reported that LPS augmented anti- gen specific proliferation of both CD4+ and CD8 + helper T cell lines, and hence LPS might activate trans- ferred donor CD8 + helper T cells in MHC class I GVHR. From these observations, it is indicated that once these donor CD8 + cells were stimulated by LPS, they could be developed to cytotoxic T cells and induce tissue destruction.

LPS is also known to affect the expression of MHC antigens on several tissues. Jephthah-Ochola et al. [28] reported that LPS augmented MHC expression on cells in the liver and several organs of mice and they as- sumed that LPS stimulated macrophages to produce cytokines that induced the expression of MHC anti- gen(s). In our experiments, expression of MHC class I antigen and an aberrant expression of MHC class II antigen were observed in LPS-stimulated mice with MHC class I GVHR. Because the outcome of GVHR may be affected by the degree of allogeneic MHC antigen(s) expression, LPS can also enhance class I GVHR in this manner.

Finally, LPS, a well known B cell mitogen, has been analyzed in the context of humoral immunity. In this study, LPS given at a dose of 1 ,~gg/mouse might have

induced mild activation of B cells which had little effect on the development of the hepatic lesions. Further evidence of B cell activation in our experiment will be required in the future.

In conclusion, we have shown that LPS can act as an inducing factor for the tissue destruction in MHC class I GVHR. These findings seem to be in accord with several human diseases such as primary biliary cirrho- sis, primary sclerosing cholangitis, acute hepatitis and others in which CD8 + cells and macrophages might be involved in tissue destruction. These results suggest that bacterial infections could be an exacerbating factor of tissue destruction in human liver diseases through the effect of LPS.

Acknowledgements

This work was supported in part by grants from the Ministry of Education, Culture, and Science, and the Ministry of Health and Welfare, of the goverment of Japan. We thank Mrs Sumiko Akiyama for technical assistance.

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