Contribution of CD1d-unrestricted hepatic DX5+ NKT cells to liver injury in Plasmodium berghei-parasitized erythrocyte-injected mice

Keishi Adachi1,5, Hiroko Tsutsui1,5, Ekihiro Seki2,5, Hiroki Nakano1,5, Kazuyoshi Takeda3, Ko Okumura3, Luc Van Kaer4 and Kenji Nakanishi1,5

1 Department of Immunology and Medical Zoology and 2 First Department of Surgery, Hyogo College of Medicine, Nishinomiya, Hyogo 663-8501, Japan, 3 Department of Immunology, Juntendo University, Tokyo 113-8431, Japan, 4 Howard Hughes Medical Institute, Department of Microbiology and Immunology, Vanderbilt University, School of Medicine, Nashville, TN 37232, USA 5 Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan

Correspondence to: K. Nakanishi; E-mail: nakaken{at}hyo-med.ac.jp
Transmitting editor: M. Miyasaka


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Inoculation with erythrocytes infected with Plasmodium berghei, a protozoan causing mouse lethal malaria, induces liver injury in mice, although the parasite cannot invade host hepatocytes at this infectious stage. As previously reported, hepatic infiltrates participate in this liver injury by exerting their perforin-dependent killing action. Here, we have investigated the cellular mechanisms underlying P. berghei-induced incidental liver injury. Hepatic lymphocytes from P. berghei-infected mice killed normal hepatocytes, but not ConA-induced lymphoblasts. Furthermore, the hepatic lymphocytes from infected C57BL/6 mice displayed cytotoxicity against hepatocytes from C57BL/6 and BALB/c mice, indicating MHC-unrestricted hepatocytotoxicity by these hepatic lymphocytes. NK cells were not involved in this hepatocytotoxicity. However, DX5+ cells sorted from the liver of infected CD1d-deficient mice killed normal hepatocytes, indicating that CD1d-independent DX5+ T cells are the effector cells. The hepatocytotoxicity of these hepatic DX5+ T cells did not require TCR engagement. These findings indicate that hepatic CD1d-independent DX5+ T cells play a critical role in P. berghei-induced liver injury. Our studies may have general implications for tissue injuries that are caused by ‘bystander killing’ or other poorly defined cell-mediated killing mechanisms.

Keywords: cytotoxicity, malaria, bystander killing, hepatitis


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Lymphocyte-mediated cytotoxicity is critically important for host defense and tumor surveillance as well as homeostasis of the immune system. CTLs and NK cells effectively eliminate microbe-infected cells and delete abnormal and potentially harmful cells in an MHC class I-restricted or -dependent manner, respectively (14). Immunological damage to tissues by cell-mediated cytotoxicity often occurs in the absence of direct infection of the target cells. This phenomenon is often referred to as bystander killing, and its mechanism is poorly understood (5,6). Hepatitis, induced in response to blood stage malaria parasite, represents an example of tissue injury mediated by bystander killing because the protozoan parasitizes RBCs but not hepatocytes in hosts (710).

The immune system of the liver has unique properties (11). The liver contains Kupffer cells and a large number of resident lymphocytes, including NK cells and NKT cells (12). In mice, NKT cells express unique TCR heterodimers and the majority of these cells is selected by an MHC class I-related molecule, CD1d (1315). Unlike conventional T cells, hepatic NKT cells are efficiently activated by IL-12 to exert powerful antitumor action (16). Recent studies have shown that hepatic CD1d-restricted NKT cells can be mobilized against metastatic cancers (16,17) and against malaria infection (18). In contrast, little is known about the pathophysiological roles of a second group of NKT cells that do not require CD1d for their development and have a diverse TCR repertoire (1921).

We have previously demonstrated that requirement of endogenous IL-12 for liver injury induced by the inoculation of mice with Plasmodium berghei-parasitized erythrocytes, and reported that hepatic but not splenic lymphocytes isolated from the inoculated mice kill hepatocytes equally from both infected and uninfected mice (10). SCID mice are resistant to this liver injury, indicating T cell-dependent hepatitis. Furthermore, perforin-deficient mice, while not Fas-deficient mice, were also resistant to this liver injury (10), indicating cell-mediated, perforin/granzyme-dependent hepatocytotoxicity. As P. berghei-elicited hepatic lymphocytes killed uninfected normal hepatocytes, we speculated that the specificity and killing mechanisms utilized by these cells are distinct from conventional CTLs. Here, we have investigated the characteristics of effector lymphocytes that accumulate in the liver of mice after infection with P. berghei. These cells were identified as CD1d-unrestricted DX5+ NKT cells. SCID mice are resistant to liver injury (10) but recombination-activating genes (RAG)3 2-deficient mice that carry transgene for an OVA-specific TCR develop normal liver injury, indicating that hepatocytotoxicity is T cell-dependent but independent of TCR engagement. These findings provide new insights into the mechanism of bystander killing, which causes tissue damage in a variety of pathological conditions.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Female C57BL/6 (B6) mice (6–10 weeks old) and female BALB/c mice (6–10 weeks old) were purchased from SLC (Shizuoka, Japan) and CLEA Japan (Osaka, Japan), respectively. B10.D2 mice, SCID mice and beige mice (C57BL/6 bg/bg) were from The Jackson Laboratory (Bar Harbor, ME). MHC class II-deficient mice on the B6 background were kindly provided by Dr Taniguchi (The University of Tokyo, Tokyo, Japan) and female mice (6–10 weeks old) were used for this study. Female mice deficient in ß2 microglobulin on the B6 background (6–10 weeks old) were kindly provided by Dr Abo at Niigata University (Niigata, Japan). CD1d-deficient mice on the B6 background (6–10 weeks old) have been described (22). RAG2-deficient, OVA-specific TCR-transgenic mice on the B10.D2 background (RAG2–/–DO11.10) were kind gifts from Dr Koyasu at Keio University (Tokyo, Japan). All mice were maintained under specific pathogen-free conditions.

Reagents
Hepatocytes were cultured in William’s medium. Spleen cells were cultured in RPMI-1640. Both culture media were supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µM 2-ME and 2 mM L-glutamine. FITC-conjugated or biotinylated mAb against mouse CD3{epsilon}, PE-conjugated or biotinylated mAb for mouse pan-NK cell marker (DX5), PE-conjugated or biotinylated mAb for NK1.1, streptavidin-CyChrome or streptavidin-allophycocyanin were purchased from PharMingen (San Diego, CA).

P. berghei-induced liver injury
Mice were inoculated i.p. with erythrocytes parasitized with P. berghei (NK65). Inoculation was conducted with several doses (103, 106, 108 cells) and at day 4, serum levels of IL-12 and the liver enzyme alanine aminotransferase (ALT), an indicator of injured liver, and parasitemia were measured (10). ALT levels paralleled with IL-12 and parasitemia (Fig. 1). Since we found that the inoculation with high and low doses of parasitized erythrocytes caused too rapid and too delayed kinetics, respectively, to induce liver injury and immune responses in mice, we employed the inoculation using 106 parasitized erythrocytes for infection of mice (10,2325). ALT levels of all uninfected mice were <50 IU/l, and liver specimens of these mice showed normal histological findings.



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Fig. 1. Positive correlation were observed among parasitemia, IL-12 levels and ALT levels. Mice were inoculated with 103 (square, n = 10), 106 (triangle, n = 10) and 108 (circle, n = 9) parasitized erythrocytes and at day 4 serum levels of IL-12 and ALT and parasitemia were measured. IRBCs, infected red blood cells.

 
Preparation of hepatocytes and liver lymphocytes
Hepatocytes and liver lymphocytes were prepared from inoculated or non-inoculated mice, as previously described (10). In some experiments, hepatic lymphocytes were sorted by autoMACS (Miltenyi Biotec, Gladbach, Germany), negatively with biotinylated anti-CD3{epsilon} mAb plus streptavidin-beads (Miltenyi Biotec) or biotinylated anti-NK1.1 mAb plus streptavidin-beads. DX5-, CD4- or CD8-beads (Miltenyi Biotec GmbH) were used for both negative and positive selection.

Ex vivo assay for hepatocytotoxicity
Hepatocytotoxicity of liver lymphocytes was determined by 4 h [51Cr]-release assays, and percent cytotoxicity was calculated as previously described (10,26). In most experiments, cytotoxicity of hepatic lymphocytes or hepatic lymphocyte subsets against hepatocytes isolated from uninfected mice (2.5 x 103/100 µl/well) was determined at an E/T ratio of 100:1. Relative lytic activity was calculated as follows: relative lytic activity (%) = 100 x hepatocytotoxicity of experimental lymphocyte fraction / hepatocytotoxicity of corresponding control. Spontaneous release of [51Cr] by hepatocytes was <5% of the maximal release. In some experiments, we measured cytotoxicity of P. berghei-elicited hepatic lymphocytes against YAC-1 cells (27,28) or Con A blasts which were prepared by the incubation of syngeneic spleen cells with Con A for 3 days, by 4 h [51Cr]-release assay.

In vivo depletion of NK cells
To deplete NK cells, anti-NK1.1 mAb (PK136, 10 mg/mouse) was administered i.p. at 2 days prior to, and 0 and 3 days after inoculation. Mice were sacrificed on day 7 of the infection and hepatic lymphocytes and sera were sampled for FACS analysis and measurement of ALT, respectively.

FACS analysis
The surface phenotypes of hepatic lymphocytes were characterized by three-color flow cytometric analysis (29). Briefly, cells were incubated with FITC-conjugated anti-CD3 mAb, PE-conjugated anti-DX5 mAb and a battery of biotinylated anti-NK1.1 mAb and streptavidin-CyChrome. In some experiments, cells were incubated with FITC-conjugated anti-CD3 mAb, PE-conjugated anti-NK1.1 mAb and a battery of biotinylated anti-DX5 mAb and streptavidin-allophycocyanin.

Statistics
All data are shown as the mean value of triplicate samples. Significance between the control group and a treated group was examined with the unpaired Student’s t-test. The correlation coefficient was obtained by Spearman’s rank correlation test. P-values <0.05 were considered significant.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Lymphocytes that accumulate in the liver of P. berghei-infected mice kill hepatocytes in an MHC-unrestricted fashion
We have previously shown that hepatic lymphocytes from P. berghei-infected mice display cytotoxic activity against hepatocytes isolated from both infected and uninfected mice (10). These findings prompted us to investigate the cytotoxicity of the hepatic lymphocytes against other cell types. Consistent with our previous report (10), the hepatic lymphocytes killed hepatocyte in an E/T ratio-dependent manner (Fig. 2A). We performed hepatocytotoxic assay using hepatic lymphocytes prepared from mice on day 0, day 3, day 4, day 5 and day 6 postinfection, and found that the yield and hepatocytotoxicity of the lymphocytes was highest at day 6 (data not shown). In general, mice died at day 7 postinfection. For the following ex vivo experiments, we used hepatic lymphocytes isolated from the mice on day 6 postinfection. As shown in Fig. 2(A), hepatic lymphocytes isolated from P. berghei-infected mice did not kill Con A-induced lymphoblasts. We could not find lytic activity of the hepatic lymphocytes against Con A-blasts by 8 h [51Cr]-release assay (data not shown). At an E/T ratio of 200:1, the hepatic lymphocytes did not kill Con A blasts (data not shown).



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Fig. 2. Hepatocytotoxic activity of hepatic lymphocytes from P. berghei-infected mice is independent of classical MHC. (A) Hepatic lymphocytes were isolated from infected (day 6 postinfection) B6 mice, and their cytotoxicity against hepatocytes (Hepat.) or spleen lymphoblasts (Con A Blasts) prepared from uninfected B6 mice were compared. (B) Cytotoxicity of the hepatic lymphocytes shown in (A) against hepatocytes from B6 or BALB/c mice. (C) At day 6 postinfection, liver specimens of infected ß2 microglobulin-deficient (ß2 MG–/–), MHC class II-deficient (Class II–/–) or wild-type (WT) B6 mice were sampled for histological analysis (hematoxylin & eosin, HE, original magnification, x200). Liver specimens from all uninfected mice examined showed normal findings (data not shown). Hepatic lymphocytes were isolated from ß2 MG–/–, Class II–/– or WT mice at day 0 (Uninf.) or day 6 postinfection (Inf.), and hepatoytotoxicity assay using normal B6 hepatocytes as target cells and FACS analysis were performed. Percentage of DX5+CD3+ population prepared from the infected mice is shown, while that from uninfected mice is shown in parenthesis. (D) Hepatocytotoxicity assay was performed using normal B6 hepatocytes as target cells. As effectors we used unfractionated total P. berghei-elicited hepatic lymphocytes (Unfract.), CD8-enriched (purity >90%, CD8-enrich., left), CD8-depleted (purity >85%, CD8-depl., left), CD4-enriched (purity >95%, CD4-enrich., right) or CD4-depleted (purity >85%, CD4-depl., right) cells. Similar results were obtained in three independent experiments.

 
Next, we investigated whether the hepatic lymphocytes kill hepatocytes in an MHC-restricted or -unrestricted manner. As shown in Fig. 2(B), hepatic lymphocytes from infected B6 mice exhibited almost equivalent cytotoxic activity against hepatocytes from MHC-mismatched BALB/c mice as compared with MHC-identical mice. To confirm and extend these findings, we inoculated MHC class I- and MHC class II-deficient mice with P. berghei. Both types of mutant mice showed a comparable increase of serum ALT levels as compared with wild-type mice (data not shown). Furthermore, apoptotic and necrotic cells, fatty degeneration of hepatocytes and dense infiltration of mononuclear cells were observed in the liver of both mutant mice, comparable to wild-type mice (Fig. 2C). Therefore, malaria-elicited hepatic lymphocytes require neither MHC class I nor class II to exert their hepatocytotoxicity. Indeed, hepatic lymphocytes prepared from infected ß2-microglobulin- or MHC class II-deficient mice exhibited comparable and substantial cytotoxicity against hepatocytes, respectively, when compared with wild-type mice (Fig. 2C). MHC class I- and MHC class II-deficient mice lack conventional CD8+ T cells and conventional CD4+ T cells, respectively. This suggests that cells that express neither CD8 nor CD4 might be involved in this liver injury. In fact, CD4+ or CD8+ cell-enriched fraction prepared from the liver lymphocytes of P. berghei-infected wild-type mice exhibited little hepatocytotoxicity (Fig. 2D). Collectively, these results suggest that unconventional T cells and/or NK cells may act as hepatocytotoxic cells in this liver injury.

NK cells are dispensable for P. berghei-induced liver injury
Next, we investigated whether NK cells play a role as effector cells in this liver injury. Lymphocyte fraction isolated from the livers of uninfected or P. berghei-infected SCID mice consisted mainly of NK cells (data not shown). These P. berghei-elicited hepatic lymphocytes attacked against YAC-1 cells, which are authentic target cells for determining NK cell activity, demonstrating normal NK activity of these cells (Fig. 3A). However, these lymphocytes did not kill hepatocytes from normal SCID mice (Fig. 3A), indicating that NK cells solely do not cause liver injury, particularly in the absence of T cells in this model. Consistent with our previous report (10), SCID mice were resistant to the liver injury after P. berghei infection (Fig. 3B). In contrast, beige mice, which have severe and moderate deficiency in NK cell and CTL function, respectively (3033), were susceptible to this liver injury (Fig. 3C and D), suggesting that cell fraction other than NK cells functioned as effector cells in P. berghei-induced liver injury. At the same experimental day, we inoculated wild-type B6 mice as well and found comparable levels of ALT (data not shown). These results strongly indicate that NK cells are not essential for this liver injury.



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Fig. 3. NK cells are not the relevant effector cells in P. berghei-induced hepatocytolysis. (A) At day 6 postinfection, hepatic lymphocytes (Hepatic Lym.) were isolated from SCID mice and their cytotoxicity against hepatocytes (Hepat.) from uninfected SCID mice or YAC-1 cells at an E/T ratio of 25:1 was determined. (B and D) Liver specimens of SCID mice (B) or beige mice (D) were sampled for histological analysis (hematoxylin & eosin, HE, original magnification, x200) prior to or post (Day 6) infection. (C) Before (day 0) and after P. berghei infection (day 6 postinfection), sera of beige mice were sampled for measurement of ALT levels. Data represent the mean ± SD of four mice in each group. (E and F) At day 6 postinfection, lymphocytes and sera were sampled from P. berghei-infected B6 mice intravenously administered PBS or anti-NK1.1 mAb for determining their phenotype and for measuring ALT levels, respectively. Data represent the mean ± SD of three mice in each group. (A–F) Similar results were obtained in three independent experiments. ND, not detected.

 
To investigate whether NK cells are dispensable for this liver injury in wild-type B6 mice, we administered anti-NK1.1 mAb to them prior to P. berghei infection. We simultaneously identified NK cells in the livers of these mice not only by their expression of NK1.1 but also by that of DX5, which is an alternative NK cell marker. As shown in Fig. 2(E), this treatment depleted hepatic NK cells but not T cells including NK1.1+ T cells or DX5+ T cells. NK cell-depleted mice exhibited comparable elevation of ALT serum levels as untreated mice after the infection (Fig. 3F). Administration of control hamster IgG had no effect on the liver injury (data not shown). Collectively, we conclude that NK cells are not required for induction of P. berghei-induced liver injury. Thus, we assume that class I- and class II-unrestricted unconventional T cells are involved in this liver injury.

Hepatic DX5+ NKT cells are required for hepatocytotoxicity induced by P. berghei
Our present results (Figs 2 and 3) exclude major contribution of conventional CTLs, conventional CD4+ T cells and NK cells to P. berghei-induced liver injury as the hepatocytolytic function. Indeed, CD4+ or CD8+ cell-enriched fraction prepared from the liver of P. berghei-infected wild-type mice exhibited little hepatocytotoxicity (Fig. 2D). In addition, CD4- or CD8-depleted cells can equally kill hepatocytes to the unfractionated parental cells (Fig. 2D). Thus, we focused on the identification of the relevant effector cells. Because of the abundance of NKT cells in normal liver we investigated their potential roles as effector cells. NKT cells increased in numbers in the liver after P. berghei infection (Fig. 4A). Depletion of CD3+ cells from the effector lymphocyte population reduced but did not completely abrogate the hepatocytotoxicity (Fig. 4B). This may be explained by contamination of residual CD3dull cells which compose the NKT cell population (Fig. 4B). Importantly, depletion of NK1.1+ cells entirely abrogated the cytotoxicity (Fig. 4B). Based on the negligible role of NK cells in this liver injury (Fig. 3), these results indicate the importance of NKT cells in this liver injury. We next employed DX5 for cell sorting, because anti-NK1.1 Ab has the potential to transduce an activation signal through NK1.1 molecules on NKT cells. DX5+ T cells, like NK1.1+ T cells, increased in the liver after P. berghei infection (Fig. 4A). Compared with unfractionated hepatic lymphocytes, DX5+ cell-enriched lymphocytes showed augmented hepatocytotoxicity. Like NK1.1+ cell-depleted fraction, DX5+ cell-depleted cells showed little hepatocytotoxicity (Fig. 4C). DX5+ cell-enriched lymphocytes from uninfected mice showed little hepatocytotoxicity (Fig. 4C), indicating development of the DX5+ T cells into hepatocytotoxic cells during P. berghei infection. Similar results were obtained through the experiments using hepatocytes prepared from the infected mice as targets (data not shown). As both NK1.1+ cell- and DX5+ cell-depleted fraction revealed obvious deterioration of the hepatocytotoxicity (Fig. 4B and C), the relevant effector cells in this hepatitis may be T cells expressing both NK1.1 and DX5. In fact, FACS analysis revealed that the proportion of DX5-expressing cells was a fifth of NK1.1+CD3+ cells and that this overlapping population increased moderately after infection (data not shown). It is notable that the absolute number of these DX5+ NKT cells was extensively facilitated. As cell yields of hepatic lymphocytes increased to at least 10-fold after infection (10), hepatic DX5+NK1.1+CD3+ cell number grew to more than 30-fold (Fig. 4A and D). However, we did not examine hepatocytotoxic activity of this population. To date, we have no appropriate approach to sort these DX5+ NKT cells without activating them, because commercially available antibodies recognizing T cell phenotype always activate T cells, including NKT cells.



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Fig. 4. DX5+ hepatic NKT cells are required for P. berghei-induced liver injury. (A) The proportion of NK1.1+CD3+ or DX5+CD3+ cells among hepatic lymphocytes (Hepatic Lym.) of B6 mice was measured before (Uninfected) or after (day 6 postinfection) infection. (B and C) To identify the effector cell population, we fractionated the hepatic lymphocytes in vitro, based on their surface phenotypes by autoMACS and measured hepatocytotoxicity of each fractionated cell population. Hepatocytotoxicity assay was performed using normal B6 hepatocytes as targets. As effector cells we used total P. berghei-elicited hepatic lymphocytes (Unfract., B and C), CD3-depleted cells (inset, CD3-depl.), NK1.1-depleted cells (inset, NK1.1-depl.) (B), DX5-enriched (purity >80%, DX5-enrich.) or DX5-depleted (purity >80%, DX5-depl.) cells (C), or DX5-enriched hepatic lymphocytes from uninfected (Uninf.) B6 mice (C). (B) The average percent hepatocytotoxicity of unfractionated cells was 15%. The relative lytic activity of hepatocytotoxicity of each fraction to that of unfractionated cells was calculated. The proportion of residual CD3+ (left) or NK1.1+ cells (right) in CD3-depleted and NK1.1-depleted fraction, respectively, is shown in insets of (B). (B) Dotted line represents staining with isotype control. (D) The cell yields of DX5+NK1.1+CD3+ cells isolated from a P. berghei-uninfected (Uninfect.) or -infected (day 6 postinfection, Day6) mouse liver are represented. Data represent the mean ± SD of four mice in each group. ND, not detected.

 
Next, we examined whether the proportion of hepatic DX5+ NKT cells increased in the absence of MHC class I or MHC class II. After P. berghei infection, both mutant mice showed proportional and numerical increase of this cell population comparable to wild-type mice (Fig. 2C and data not shown).

Taken together, all the results strongly indicate that DX5+ NKT cells play a critical role in P. berghei-induced liver injury as effector cells.

Expansion of DX5+ hepatocytotoxic NKT cells in the absence of CD1d expression
NKT cells including DX5+ T cells can be divided into two subpopulations: CD1d-restricted and CD1d-unrestricted cells (1921). Consistent with the above results of mice deficient in ß2-microglobulin (Fig. 2C), which is an essential structural component of both MHC class I and CD1d (13), CD1d-deficient mice are sensitive to P. berghei-induced liver injury (10) (Fig. 2C). These suggest that CD1d-restricted NKT cells are excluded as effector cells. Next we examined the hepatocytotoxicity of CD1d-unrestricted NKT cells. First, we tested whether CD3+NK1.1+ cells and/or CD3+DX5+ cells increase in the liver of CD1d-deficient mice after P. berghei infection. As shown in Fig. 5A, both CD3+NK1.1+ cells and CD3+DX5+ cells expanded in CD1d-deficient mice after the infection. Next, we conducted ex vivo hepatocytotoxicity assays using hepatic lymphocytes from P. berghei-infected CD1d-deficient mice as effectors. The hepatic lymphocytes from infected CD1d-deficient mice exhibited robust hepatocytotoxic activity (Fig. 5B). Furthermore, DX5+ cells enriched from the hepatic lymphocytes displayed enhanced hepatocytotoxicity (Fig. 5B). In sharp contrast, depletion of DX5+ lymphocytes abrogated the hepatocytotoxicity (Fig. 5B). Hepatic lymphocytes from uninfected CD1d-deficient mice failed to kill hepatocytes (data not shown). These results strongly indicated that DX5+ CD1d-unrestricted NKT cells play a crucial role in this liver injury. To confirm that hepatocytotoxicity occurs in a CD1d-unrestricted manner, we compared cytotoxic activity of the hepatic lymphocytes from the infected wild-type mice against CD1d-deficient hepatocytes to that against wild-type hepatocytes, and found no difference (data not shown). All of these results strongly indicate that CD1d-unrestricted hepatic DX5+ NKT cells are effector cells in P. berghei-induced liver injury.



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Fig. 5. P. berghei infection increases the number of hepatocytotoxic NKT cells in the liver of CD1d-deficient mice. (A) Hepatic lymphocytes (Hepatic Lym.) were collected from uninfected or infected (day 6 postinfection) CD1d-deficient mice, and the percentage of NK1.1+CD3+ and DX5+CD3+ cells was measured. (B) To identify the effector cell population, we fractionated the hepatic lymphocytes in vitro, based on their surface phenotypes by autoMACS and measured hepatocytotoxicity of each fractionated cell population. B6 hepatocytes were used as targets. Hepatic lymphocytes were isolated from infected (day 6 postinfection) CD1d-deficient mice, and unfractionated cells (Unfract.), DX5-enriched (purity >85%, DX5-enrich.) or DX5-depleted (purity >85%, DX5-depl.) cells were used as effectors. The average percent hepatocytotoxicity was 22% at an E/T ratio of 50:1. Percent hepatocytolysis of each effector at an E/T ratio of 50:1 was compared to that of unfractionated cells (Relative Lytic Activity). Similar results were obtained in three independent experiments.

 
TCR engagement is not required for hepatocytotoxicity of hepatic NKT cells
Finally, we analyzed the molecular basis for the hepatocytotoxicity of the CD1d-independent hepatic DX5+ NKT cells. All the results obtained (Figs 25) led us to hypothesize that these CD1d-unrestricted hepatic DX5+ NKT cells kill hepatocytes independently of conventional TCR engagement. In order to verify this hypothesis, we inoculated RAG2–/–DO11.10 mice, which have only T and NKT cells that express OVA-specific TCR (3436), with P. berghei-infected erythrocytes. Intriguingly, while wild-type mice expressed typical malarial symptoms, such as wasting and poor exercise, and significant elevation of serum ALT around day 6 postinfection, RAG2–/–DO11.10 mice displayed neither of them at that time (Fig. 6A). The mutant mice displayed the symptoms and ALT elevation after day 10 postinfection (Fig. 6A), and the pathological changes of the liver were almost similar to wild-type mice (Fig. 6B). Next, to investigate the involvement of the hepatic DX5+ NKT cells in this liver injury, we compared the proportional changes of this cell population between wild-type mice and the mutant mice when the pathological changes were induced, at day 6 postinfection in wild-type mice and at day 12 postinfection in the mutant mice, respectively. FACS analysis revealed that DX5+ NKT cells increased in the liver of RAG2–/–DO11.10 mice as well as in wild-type mice (Fig. 6C). In order to confirm that the hepatic lymphocytes from RAG2–/–DO11.10 mice acquire the hepatocytotoxic activity after P. berghei infection, we compared hepatocytotoxic activity of hepatic lymphocytes from infected RAG2–/–DO11.10 mice with that from infected wild-type mice. As shown in Fig. 6D, hepatic lymphocytes from the mutant and wild-type mice exhibited comparable hepatocytotoxicity. Taken together, these results strongly indicate that the DX5+ CD1d-unrestricted hepatic NKT cells expanded in the liver of P. berghei-infected mice kill hepatocytes without conventional TCR engagement.



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Fig. 6. Conventional TCR engagement-independent liver injury upon P. berghei infection. (A–C) Before (open column; A, Uninfected; C) and after inoculation (closed column; A, Day6 or Day12; B and C), sera, liver specimens and hepatic lymphocytes (Hepatic Lym.) of wild-type mice (WT) and RAG2–/–DO11.10 mice at day 6 or 12 postinfection, respectively, were sampled for the measurement of ALT levels (A), the histological analysis (B, hematoxylin & eosin, HE, original magnification, x200) and the measurement of the percentage of DX5+CD3+ cells (C). Data represent the mean ± SD of five mice in each group (A). (D) Ex vivo hepatocytotoxic activity assay was conducted with hepatocytes from uninfected wild-type B10.D2 mice as targets. Hepatic lymphocytes were isolated from infected WT (day 6 postinfection) or RAG2–/–DO11.10 (day 12 postinfection) mice, and their hepatocytotoxicity was measured. The average percent hepatocytotoxicity of wild-type mice was 6.5% at an E/T ratio of 50:1. Relative lytic activity of the mutant cells to wild-type cells was calculated. (A–D) Similar results were obtained in three independent experiments.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Malaria protozoa have a complicated life cycle composed of lives in mosquitoes as terminal host and in human and mice as intermediate hosts. In intermediate hosts, the protozoan development is divided into two stages on the basis of their cytotropism: liver stage and blood stage. At the each developmental stage, the parasites induce and/or trigger various unique and complex immunological responses in the hosts, as demonstrated by the pioneering works (7,3739). Upon bite of the protozoa-infected mosquitoes, Plasmodium sporozoits can cause liver injury via their selective infection of host liver cells. This is liver stage infection. After leaving host liver, the protozoa eventually lose the liver tropism and gain the erythrocyte tropism, and do not transform back to the liver stage parasite. However, the blood stage protozoa can still provoke liver injury in human or mice, as previously reported (810,40,41).

We have learned a lot from LPS-induced liver injury (11,26,42,43). LPS, a major constituent of the outer membrane of Gram-negative bacteria, provokes liver injury in host via local activation of proinflammatory cytokines, although it does not possess liver tropism (11). In LPS-induced liver injury, the causative pro-inflammtory cytokine levels correlate with the levels of LPS, and the lethality parallels well with the doses of the cytokines (11). In order to address the roles of parasitemia in P. berghei-induced liver injury, we compared levels of parasitemia, IL-12, which is a relevant pro-inflammatory cytokine to this liver injury (9, 10), and ALT among hosts having received various numbers of the parasitized erythrocytes (Fig. 1). Because the serum levels of IL-12 peaked at day 4 postinfection and fell down to pre-infected level at day 6 postinfection (10), and because the elevation of ALT levels was initiated at day 4 postinfection, we compared the values of each parameter at day 4 postinfection. Expectedly, the levels of parasitemia paralleled well with the doses of injected parasitized erythrocytes (Fig. 1). In addition, there were positive correlations between any pair of the three parameters (Fig. 1). However, mice with defect in IL-12 production or mice administered neutralizing Ab against IL-12 evade the liver injury with comparable levels of parasitemia (9,10). Therefore, like LPS-induced liver injury (11), parasitized erythrocytes dose-dependently induce IL-12 in mice, and the IL-12 in turn dose-dependently induces liver injury. As mentioned above, IL-12-deficient mice did not manifest liver injury even though they exhibited high levels of parasitemia. Hence, it is not the protozoa burden itself but the host immune responses triggered by the infection that cause this liver injury. In fact, perforin-deficient mice were free from P. berghei-induced liver injury (10), suggesting that cell-mediated immunity would be critical for this liver injury. In addition, the immune responses, including the production of pro-inflammatory cytokines, would trigger the sequential changes in hemodynamics within the liver of host, which might exacerbate the liver injury (4447). Furthermore, Kupffer cells isolated from P. berghei-infected mice secrete IL-12 and IL-18 in response to parasitized erythrocytes (our unpublished data), indicating that liver is an immunologically active organ at erythrocyte stage as well as liver stage of the infection. As perforin-deficient mice are free from this liver injury (10), host-derived perforin-expressing lymphocytes play essential roles in this liver injury. Based on these findings, we assumed that P. berghei-infected erythrocytes might activate host immune cells to secrete IL-12, which aberrantly stimulates IL-12-responsive effector cells densely recruited in the liver to kill hepatocytes, leading to the liver injury (10). In this study, we focused on the identification of the hepatocytotoxic effector cells and observed that DX5+ NKT cells generated in a CD1d-unrestricted manner are hepatocytotoxic effector cells of P. berghei-induced liver injury.

Both lpr/lpr mice and gld/gld mice, which possess loss of function mutation in Fas and Fas ligand, respectively, were susceptible to P. berghei-induced liver injury (10), while perforin-deficinet mice were resistant to this liver injury. Moreover, in the ex vivo hepatocytotoxic assay, the treatment with perforin inhibitor, but not with anti-Fas ligand mAb, abolished the hepatocytolytic activity of the hepatic lymphocytes isolated from P. berghei-infected mice (10). Therefore, the cytolytic action of this NKT cell population was speculated to depend upon perforin/granzyme system, but not upon Fas/Fas ligand system. In fact, DX5+ CD1d-unrestricted NKT cells expressed undetectable levels of Fas ligand after P. berghei infection, whereas hepatocytes, either from non-infected or infected mice, and Con A blasts expressed Fas (our unpublished data).

In vivo treatment with anti-NK1.1 mAb selectively depleted NK cells but not NK1.1+ T cells in the liver (Fig. 3E), while in vitro treatment with the same mAb eliminated both NK cells and NK1.1+ T cells (Fig. 4B). These dissociated phenomena might be explained by the differences of the intensity of NK1.1 expression between these two types of cells. As shown in Fig. 3(E), NK cells express high and solid levels of NK1.1, while hepatic NKT cells express lower and broadly scattered levels of it. In vivo treatment with anti-NK1.1 mAb might selectively eliminate high level NK1.1-expressing cells, such as NK cells and a small subpopulation of NKT cells, via activation of complement system and/or induction of opsonization. Or, the differences of the susceptibility of the cells to this treatment, whose mechanism remains unknown, might be involved. This is also the case for spleen cells (48). Anti-NK1.1 mAb was insufficient for depletion of NKT cells in in vivo treatment (Fig. 3E). However, microbeads conjugating the antibody seem to sufficiently bind to NKT cells as well as NK cells in vitro to be depleted by negative selection with MACS (Fig. 4B).

Mice that have only T cells and NKT cells expressing OVA-specific TCR underwent the liver injury but with some delay when compared with wild-type mice (Fig. 6A and B). Although the cause of this delay on RAG2–/–DO11.10 mice remains to be elucidated, this might be ascribed to the lack of acquired immunity in the mutant mice.

This is the first report demonstrating the profound involvement of CD1d-unrestricted NKT cells in organ-specific injury induced by bystander killing. Since the majority of resident NKT cells in the liver are CD1d-restricted (19,20), it is possible that the CD1d-unrestricted NKT cells are recruited into the liver from other sites, rather than proliferating inside the liver. Alternatively, certain T cells lacking NK1.1 and/or DX5 might express these molecules after P. berghei infection, because it is reported that NK1.1 is an activation marker (49). These DX5+ NKT cells, whatever their origin, explode in number and may be aberrantly activated by IL-12 as shown previously (10). In this context, it is interesting to note that P. berghei-activated Kupffer cells secrete IL-12 in response to parasitized RBCs in vitro (our unpublished data). Recent reports have demonstrated the heterogeneity of NKT cells in terms of their phenotypes, development manners and biological actions (1921,5052). It remains unclear whether our CD1d-independent hepatocytotoxic DX5+ NKT cells consist of a single or more than two subpopulations of NKT cells. And it also remains unsolved how these CD1d-unrestricted DX5+ NKT cells discriminate between hepatocytes and lymphoblasts. Hepatocytes may express unknown natural ligands that selectively activate the DX5+ NKT cells independently of their TCR in certain pathological situations. Further study will provide us with new tools to prevent and/or to treat organ-specific injury induced by bystander killing.


    Acknowledgements
 
We thank Dr Tomoaki Hoshino (Kurume University, Japan) for kindly providing us with anti-NK1.1 antibody and Dr Shigeo Koyasu at Keio University for enthusiastic discussion. We also thank Ms Shizue Yumikura-Futatsugi for excellent technical assistance. This work was supported in part by Grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), a Hitec Research Center Grant from MEXT and a Grant from Hyogo College of Medicine.


    Abbreviations
 
ALT—alanine aminotransferase

B6—C57BL/6

RAG—recombination-activating genes

RAG2-/-DO11.10—RAG2-deficient, OVA-specific TCR transgenic


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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