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The Journal of Immunology, 2007, 179, 2180 -2186
Copyright © 2007 by The American Association of Immunologists, Inc.
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Acute Rejection of Allografted CTL-Susceptible Leukemia Cells from Perforin/Fas Ligand Double-Deficient Mice1

Hayahito Nomi*,{dagger}, Junko Tashiro-Yamaji*, Yumiko Yamamoto*, Sayako Miura-Takeda*, Masako Miyoshi-Higashino*, Takeshi Takahashi*, Haruhito Azuma{dagger}, Haruhiko Ueda{dagger}, Yoji Katsuoka{dagger}, Takahiro Kubota* and Ryotaro Yoshida2,*

* Department of Physiology and {dagger} Department of Urology, Osaka Medical College, Takatsuki, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The generation of knockout mice demonstrated that CD4+, but not CD8+, T cells were essential for the rejection of allografted skin or heart, presumably because these targets were CTL resistant. In the case of CTL-susceptible targets (e.g., P815 mastocytoma cells and EL-4 or RLmale1 T lymphoma cells), however, it is assumed that the CTL is the effector cell responsible for allograft rejection and that perforin and Fas ligand (FasL) pathways are the killing mechanisms. In the present study, we examined the role of these cytotoxic molecules in the rejection of i.p. allografted CTL-susceptible leukemia cells. Unexpectedly, the allografted leukemia cells were acutely rejected from gld (a mutation of FasL), perforin–/–, or double-deficient mice. The peritoneal exudate cells from gld or normal mice showed T cell-, TCR{alpha}beta-, and perforin-dependent cytotoxic activity against the allograft, whereas the exudate cells from perforin–/– mice exhibited almost full cytotoxic activity in the presence of Fas-Fc. Furthermore, the infiltrates from double-deficient mice showed a high cytotoxic activity against the allografted cells even in the presence of anti-TCR{alpha}beta Ab or in the absence of T cells. The cytotoxic cells appeared to be macrophages, because they were Mac-1+ mononuclear cells with a kidney- or horseshoe-shaped nucleus and because the cytotoxic activity was completely suppressed by the addition of NG-monomethyl-L-arginine, an inhibitor of inducible NO synthase. These results indicate that macrophages are ready and available to kill CTL-susceptible allografts when CTLs lack both perforin and FasL molecules.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
It is generally accepted that allograft rejection does not occur in vivo in the absence of T cells (1, 2). Therefore, most of the previous studies concerning allografted skin, organ (e.g., heart and kidney), or tumor (e.g., P815 mastocytoma cells and EL-4 lymphoma cells) rejection have concentrated on the cytotoxic activity of CD8+ T cells against the allograft (3, 4). However, the generation of knockout mice demonstrated that noncytotoxic CD4+, but not cytotoxic CD8+, T cells were essential for the rejection of allografted skin or heart (5, 6, 7). We reported that a type of activated macrophage in C57BL/6 (B6) (3) mice was the major effector cell responsible for the rejection of allogeneic BALB/c skin (8) or Meth A fibrosarcoma cells (9), a CTL-resistant cell line (10).

In the case of CTL-susceptible targets (e.g., P815 mastocytoma cells, EL-4 T lymphoma cells, and RLmale1 T lymphoma cells), it is assumed that CD8+ T cells are the effector cell responsible for allograft rejection (11) and that perforin and Fas ligand (FasL)3 pathways are the killing mechanisms (12). To know the role of these cytotoxic molecules in the rejection of CTL-susceptible cells, we i.p. transplanted these cells into allogeneic or syngeneic gld (a mutation of FasL), perforin–/–, double-deficient, or normal mice and determined the number of grafted tumor cells and the cytotoxic activity of peritoneal exudate cells (PEC) against the graft. The results showed that gld, perforin–/–, double-deficient, and normal mice acutely rejected the allograft, but not isografts, with similar time courses. Unexpectedly, the PEC from the double-deficient mice exhibited almost full cytotoxic activity even in the absence of T cells. The possible involvement of innate immunity in the rejection of allografted CTL-susceptible leukemia cells is discussed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

RPMI 1640 medium was purchased from Nissui Seiyaku. FCS was obtained from ICN Biomedicals and used after heat inactivation. Na2[51Cr]O4 (10.5 GBq/mg) was purchased from PerkinElmer New England Nuclear. Low-Tox-M rabbit complement was a product of Cedarlane Laboratories. Con A was purchased from Sigma-Aldrich and NG-monomethyl-L-arginine (NMMA) from Calbiochem. Abs used were the following: FITC-labeled anti-mouse CD3 (145-2C11), CD11c (HL3), and Ly-6G (1A8) Abs; PE-labeled anti-mouse CD49b (DX5), CD8 (53-6.7), CD4 (RM4-5), CD11b (M1/70), CD95 (Jo2), and CD45R/B220 (RA3-6B2) Abs; and purified anti-mouse TCRbeta (H57-597), TCR{gamma}{delta} (GL3), Thy-1.2 (53-2.1), CD3 (145-2C11), and CD16/CD32 (2.4G2) Abs (BD Pharmingen).

Mice

Specific pathogen-free male (or female) BALB/c, DBA/2, B6, C3H/HeJ, and C3H/HeJ-gld mice were purchased from Japan SLC. B6-gld mice were purchased from The Jackson Laboratory. perforin–/– B6 mice were donated by Dr. H. Hengartner (Department of Pathology, Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland). The perforin/FasL double-deficient mice died early (69 ± 10 days old) of severe pancreatitis; and female mice, in addition, were infertile and suffered from hysterosalphingitis, as described earlier (13, 14). Therefore, to obtain double-deficient (perforin–/–, gld/gld) B6 mice, we bred F1 (perforin+/–, +/gld) B6 mice with gld/gld B6 mice, and genomic DNAs from peripheral blood samples of F2 were screened by PCR to select perforin+/–, gld/gld mice. The F2 mice were then brother-sister mated to obtain double- deficient mice.

The mice were housed in our animal facility under specific pathogen-free conditions in an air-conditioned room maintained at 25 ± 2°C and {approx}50% humidity. These mice were used at 7–10 wk of age for experiments. All experiments were conducted in accordance with the Guidelines on Animal Experiments of Osaka Medical College and the Japanese Government Notification on Feeding and Safekeeping of Animals (Notification no. 6 of the Prime Minister’s Office), and the experimental protocol was approved by the Review Committee for Animal Experiments of Osaka Medical College.

Tumor cells

EL-4 (B6; H-2b) T lymphoma cells and P815 (DBA/2; H-2d) mastocytoma cells were purchased from American Type Culture Collection and RLmale1 (BALB/c; H-2d) T lymphoma cells were donated by the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan).

PEC

To investigate the cellular and cytotoxic mechanism of rejection of allografted tumor cells, we i.p. injected P815, EL-4, or RLmale1 tumor cells (3 x 106 cells) into allogeneic normal, perforin–/–, gld, or double-deficient mice. On days 4–21 after tumor transplantation, peritoneal cells were harvested by peritoneal lavage with PBS. PEC (10–20 µm in diameter) were separated from the allografted tumor cells (>20 µm in diameter) by FACS, as described previously (9, 15, 16, 17, 18, 19, 20, 21, 22, 23).

Elimination of T cells from PEC

T cells among PEC were eliminated by complement-dependent cell lysis with anti-Thy-1.2 Ab, as described previously (9). The PEC (2 x 106 cells) were suspended in 500 µl of fresh RPMI 1640 medium containing 10% FCS and then incubated at 37°C for 45 min with 16 µl of anti-Thy-1.2 Ab and 160 µl of diluted rabbit complement, which had been reconstituted in 1 ml of cold distilled water and then diluted three times with RPMI 1640 medium containing 10% FCS. To confirm that the T cells had been eliminated, we stained the Ab plus complement-treated cells with fluorescein-labeled anti-Thy-1.2 Ab and then examined them for their surface Ag expression by FCM.

Cell number and viability

The cell number in the suspensions was determined with a hemocytometer after dilution of the cells in Turk’s solution. The viability of cells was assessed by the trypan blue exclusion method.

Isolation of DNA and PCR amplification

Mouse peripheral blood ({approx}0.1 ml) was obtained from a tail vein, and the genomic DNA was isolated from it by using a Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer’s instructions. For detection of the gld point mutation, the genomic DNA was amplified with one miss-match PCR primer set, 5'-CAATTTTGAGGAATCTAAGGCC-3' and 5'-TAAGGACCACTCCATGGACC-3'. PCR was conducted by 5-min denaturation at 94°C and 30-s annealing at 62°C, followed by 45 cycles of 30 s at 94°C, 30 s at 62°C, and 30 s at 72°C, with a final extension of 7 min at 72°C in a PCR thermal cycler (GeneAmp PCR System 9700; PerkinElmer). A mouse perforin primer set (5'-AAGCTACACCAGAGCAGTTCTCAACCT-3' and 5'-CACAGATGTTCTGCCCGGAAATTGCT-3') was used to amplify a 328-bp fragment. PCR was conducted by a 5-min denaturation at 94°C and a 30-s annealing at 70°C, followed by 35 cycles of 30 s at 94°C, 30 s at 70°C, and 60 s at 72°C, with a final extension of 7 min at 72°C. The PCR products were electrophoresed on 1.5% agarose gels.

FCM analysis

Cells (5 x 105 cells) suspended in 25 µl of cold PBS containing 2% FCS in the presence (for staining) or absence (for staining and sorting) of 0.1% sodium azide were stained with a fluorescein-labeled Ab at 4°C for 20 min, washed, and analyzed by FCM. Dead cells and erythrocytes were gated out on the basis of light scattering as the data were being collected.

Cytotoxic assays

The cytotoxic activity against 51Cr-labeled EL-4, RLmale,1 or P815 tumor cells was determined as described before (9). Effector cells (1.25 x 104–2 x 105 cells) in 0.2 ml of RPMI 1640 medium supplemented with 10% FCS were mixed with 51Cr-labeled targets (5 x 103 cells) in 20 µl of the medium in Costar 3799 U-shaped microtiter wells. The Fas-Fc was provided by Dr. S. Nagata (Department of Genetics, Osaka University, Osaka, Japan). After a 4- or 12-h incubation, an aliquot of the supernatant was removed from each well and assayed for released 51Cr in a gamma counter. Lysis of 51Cr-labeled targets in each well was quantified as percent specific lysis: percent specific lysis = [(cpmexperiment – cpmspontaneous)/(cpmmax – cpmspontaneous)] x 100.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Growth of EL-4, RLmale1, and P815 cells i.p. injected into syngeneic or allogeneic strains of mice and cytotoxic activity of PEC against these cells in vitro

Fig. 1, a–c, shows the growth of EL-4, RLmale1, and P815 leukemia cells i.p. injected into syngeneic strains of mice. The tumor cells grew time-dependently in the peritoneal cavity until around day 11 after transplantation and then gradually migrated into the neighboring lymph nodes. The animals died 18–20 days after the injection of the tumor cells. At all time intervals after the injection, there was very low or no cytotoxic activity of PEC taken from the transplantation site against 51Cr-labeled leukemia cell targets.


Figure 1
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  		FIGURE 1. Growth of i.p. transplanted EL-4, RLmale1, or P815 cells in the peritoneal cavity ({circ}) or the neighboring lymph nodes (•) and the cytotoxic activity of PEC ({triangleup}) against the above cells used as targets in a 51Cr release assay. At appropriate time intervals, peritoneal cells were recovered and the numbers of tumor cells were determined. Each value for the numbers of tumor cells (x107 cells/mouse) in the peritoneal cavities and the volumes of tumor mass (x cm3/mouse) in the lymph nodes represents the mean ± SD and the mean of four different experiments, respectively. The cytotoxic activity of PEC against 51Cr-labeled leukemia cells was determined at an E:T ratio of 20 in a 12-h assay. The y-axis values relate to two (d–f) or three (a–c) variables. Each value represents the mean ± SD of 16 cultures from four different experiments. a, B6 mice plus EL-4 cells; b, BALB/c mice plus RLmale1 cells; c, DBA/2 mice plus P815 cells; d, BALB/c mice plus EL-4 cells; e, B6 mice plus RLmale1 cells; and f, B6 mice plus P815 cells.

 
The leukemia cells i.p. injected into the peritoneal cavity of the allogeneic strains of mice grew transiently until day 7 after the injection. The growth rate decreased with increasing cytotoxic activity of PEC against the allograft, as detected by the 51Cr release assay, and the tumor cells were rejected around day 12 (Fig. 1, d–f). In contrast, the spleen cells were inactive toward the allograft (4.4 ± 3.0% specific lysis (E:T ratio of 20 in a 12-h assay; n = 7)).

Effects of Ab or Ab plus complement on the cytotoxic activity of PEC against i.p. allografted EL-4, RLmale1, or P815 cells

Most of the cytotoxic activity of PEC against allogeneic P815, RLmale1, or EL-4 cells disappeared after specific removal of the T cells by complement-dependent cell lysis with anti-Thy-1.2 Ab, and the cytotoxic activity of PEC was largely suppressed by the addition of anti-CD3 or anti-TCR{alpha}beta, but not by that of anti-TCR{gamma}{delta} Ab (Fig. 2). These results indicate that Thy-1.2+CD3+TCR{alpha}beta+ T cells were the major effector cells cytotoxic against allogeneic leukemia cells.


Figure 2
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  		FIGURE 2. Effects of Ab or Ab plus complement on the cytotoxic activity of PEC against allogeneic P815, RLmale1, or EL-4 leukemia cells. PEC were recovered on day 10 after transplantation, and an aliquot of PEC was incubated with anti-Thy-1.2 Ab plus complement for 45 min at 37°C to remove the T cells. The cytotoxic activity of PEC against the allograft was determined in the presence or absence of anti-CD3, anti-TCR{alpha}beta, or anti-TCR{gamma}{delta} Ab at an E:T ratio of 10 in a 12-h assay. Each value represents the means ± SD of eight cultures from two different experiments. a, B6 + P815 cells; b, B6 + RLmale1 cells; and c, BALB/c + EL-4 cells.

 
Acute rejection of allografted leukemia cells by perforin–/– or gld mice

It has been suggested that most of the lytic T cell cytotoxicity is mediated through perforin and FasL pathways (12, 24), and gld, a spontaneous loss-of-function mutation, is a mutation of FasL (25). To ascertain that FasL or perforin was involved in the CTL-mediated cytotoxicity against allografted leukemia cells, we injected EL-4 (B6; H-2b) cells i.p. into C3H/HeJ-gld or C3H/HeJ (H-2k) mice. The allografts were acutely rejected from both C3H/HeJ-gld and their control (C3H/HeJ) mice around 13 days after transplantation (Fig. 3a), and almost the same cytotoxic activities of PEC against EL-4 cells were induced in the rejection site of both gld and control mice in a 4-h (data not shown) or 12-h (Fig. 3d) assay. Similarly, when we i.p. injected P815 (DBA/2; H-2d) mastocytoma cells or RLmale1 (BALB/c; H-2d) T lymphoma cells into perforin–/– and control B6 (H-2b) mice, the allografted leukemia cells were acutely rejected from both perforin–/– and the control mice (Fig. 3, b and c); almost the same cytotoxic activities of PEC against the allograft were induced in the rejection site of perforin–/– as well as perforin+/+ mice, as judged from the results of a 12-h 51Cr release assay (Fig. 3, e and f). In a 4-h incubation, however, the cytotoxic activities were largely impaired in perforin–/– mice (data not shown), suggesting the involvement of perforin in the early phase of cytotoxic activity. There was significant expression of Fas Ag on the cells of these three cell lines (EL-4: peak values shifted from 95 (–Ab) to 751 (+Ab); RLmale1: 67–316; and P815: 139–961).


Figure 3
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  		FIGURE 3. Growth of i.p. allografted leukemia cells in perforin–/– or gld mice and the cytotoxic activity of PEC against 51Cr-labeled target cells of the allograft type. On day 4.5, 7, 10, 14, or 18 after i.p. transplantation of allogeneic leukemia cells, the numbers of tumor cells were determined. Each value represents the mean ± SD of four different experiments. The cytotoxic activities of PEC against the allograft-type cell targets were determined as described in the legend of Fig. 1. Each value represents the mean ± SD of 16 cultures from four different experiments. a and d, C3H/HeJ ({circ}) or C3H/HeJ-gld mice (•) plus EL-4 cells; b and e, perforin+/+ ({circ}) or –/– (•) B6 mice plus P815 cells; and c and f, perforin+/+ ({circ}) or –/– (•) B6 mice plus RLmale1 cells.

 
Effects of Fas-Fc on the cytotoxic activity of PEC against allografts in perforin–/– mice

The above experimental results suggest two possibilities. One is that the cytotoxic activity in perforin–/– (or gld) mice was compensated by FasL (or perforin). The other is that neither perforin nor FasL was essential. To test these possibilities, we examined the effects of Fas-Fc on the cytotoxic activity of PEC induced by the allograft in perforin–/– mice (Fig. 4).


Figure 4
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  		FIGURE 4. Effects of Fas-Fc on the cytotoxic activity of PEC from gld or perforin–/– mice toward 51Cr-labeled targets of the allograft type. On day 7 after i.p. allografting into gld or perforin–/– mice, PEC were obtained. The cells accumulating in the lymph node of MRL-lpr/lpr mice (15 wk old) were used as a positive control. The cytotoxic activities of effector cells (i.e., PEC or lymph node cells) against their target cells were determined in the presence or absence of Fas-Fc (10 µg/ml) at an E:T ratio of 20 in a 12-h incubation. Each value represents the mean ± SD of eight cultures from two different experiments. KO, Knockout.

 
The cells accumulating in the lymph nodes of MRL-lpr/lpr mice (10 wk old or older) are mainly abnormal T cells (CD4–CD8–Thy-1.2+B220+) expressing activation Ags, including FasL (26). In fact, the abnormal T cells were cytotoxic against Fas-expressing Con A blasts, whereas the cytotoxic activity was completely suppressed by the addition of Fas-Fc, indicating Fas/FasL-dependent cytotoxicity (Fig. 4). Another mouse spontaneous loss-of-function mutation, gld, is a mutation of FasL (25). After i.p. transplantation of allogeneic EL-4 cells into C3H/HeJ-gld/gld mice, the cytotoxic activity against the allograft was induced in the bulk PEC from the mutant mice, as described in Fig. 3d, and expectedly, the cytotoxic activity was almost unchanged in the presence of Fas-Fc (Fig. 4). Unexpectedly, however, most ({approx}80%) of the cytotoxic activity that was induced by allograft in perforin–/– mice was retained in the presence of Fas-Fc (Fig. 4).

Acute rejection of allografted P815 cells from perforin/FasL double-deficient mice

The data in Fig. 4 imply that most of the cytotoxic activity of PEC against the allograft in perforin–/– mice was Fas/FasL independent. To confirm this, we ablated both cytotoxic pathways, perforin and FasL, by cross-breeding perforin-deficient mice with FasL-deficient ones. After i.p. transplantation of allogeneic P815 mastocytoma cells (3 x 106 cells/mouse) into the double-deficient or wild-type B6 mice, peritoneal cells were obtained on days 4.5, 10, 14, and 21. Unexpectedly, the allografted P815 cells were acutely rejected from the double-deficient mice with a peak on day 10, and the time course was essentially the same as that for the control mice (Fig. 5).


Figure 5
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  		FIGURE 5. Acute rejection of allografted P815 cells by control ({circ}) and perforin/FasL double-deficient (•) B6 mice. On day 4.5, 10, 14, or 21 after i.p. transplantation of allogeneic leukemia cells, peritoneal cells were recovered, and the tumor cell numbers were determined. Each value represents the mean ± SD of four different experiments.

 
Effector cells cytotoxic against the allograft in double-deficient mice

To identify the effector cells responsible for this rejection, we obtained peritoneal cells of the double-deficient mice by peritoneal lavage with PBS on day 10 after the i.p. transplantation of allogeneic P815 cells. At this time, the peritoneal cells mainly consisted of four populations, i.e., allografted P815 cells (>20 µm in diameter) and PEC (10–20 µm in diameter), including lymphocytes, granulocytes, and macrophages. However, their flow cytometric pattern was quite different between the two groups of mice (Table I): for the control mice, Mac-1+, CD8+, CD11c+, Ly6G+, DX5+, B220+, CD3+, and CD4+ cells represented 50.7, 6.1, 2.9, 9.1, 3.1, 9.5, 12.8, and 5.2%, respectively, of the total PEC; whereas in the double-deficient mice, Mac-1+ cells (75.1%) were much more evident as the major population of infiltrating cells, followed by CD3+ cells (7.5%), CD4+ cells (6.2%), and B220 cells (3.4%). Spielman et al. (13) also reported that perforin/FasL double deficiency was associated with macrophage expansion and severe pancreatitis.


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  		Table I. Types of cells infiltrating into the rejection site after P815 cell transplantation into the peritoneal cavity of allogeneic normal or perforin/FasL double-deficient micea

 
The PEC obtained on day 10 after i.p. transplantation of allogeneic P815 cells into double-deficient mice exhibited a high cytotoxic activity against the 51Cr-labeled allograft cell target. The cytotoxic activity, however, did not change significantly either in the presence of anti-TCRbeta Ab or after specific removal of T cells by complement-dependent cell lysis with anti-Thy-1.2 Ab (Fig. 6).


Figure 6
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  		FIGURE 6. Non-T cells were the effector cells responsible for the rejection of allografted P815 mastocytoma cells by perforin/FasL double-deficient B6 mice. On day 10 after i.p. transplantation of P815 cells into the double-deficient mice, PEC were obtained, and the cytotoxic activity was then assessed as described in the legend of Fig. 2. Each value represents the mean ± SD of eight cultures from two different experiments.

 
Macrophage as a candidate of the effector cell responsible for the rejection of allografted P815 mastocytoma cells by perforin/Fas double-deficient mice

The results mentioned above suggest that some cell type(s) other than T cells of the perforin/FasL double-deficient B6 mice might have been the major effector cell responsible for the lysis of the CTL-susceptible P815 cells. When Mac-1+ and Mac-1– cells were isolated from the PEC, the cytotoxic activity was found exclusively in the Mac-1+ cell fraction, whereas the Mac-1– cells had very low, if any, cytotoxic activity (Fig. 7). Furthermore, Fig. 8A reveals that the major population of PEC taken from the double-deficient mice was mononuclear cells with a kidney- or horse shoe-shaped nucleus, suggesting that macrophage might be the effector cell responsible for the rejection of allografted P815 mastocytoma cells.


Figure 7
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  		FIGURE 7. Mac-1+ cells as a candidate of the effector cell responsible for the rejection of allografted P815 mastocytoma cells by perforin/FasL double-deficient mice. On day 10 after i.p. transplantation of P815 cells into the double-deficient mice, PEC were obtained. After staining of PEC with fluorescein-labeled anti-mouse Mac-1 Ab, Mac-1+ and Mac-1– cells were isolated from PEC by FCM. The cytotoxic activity of Mac-1+ or Mac-1– cells against 51Cr-labeled P815 cells was determined at an E:T ratio of 10 in a 12-h assay. Each value represents the mean ± SD of eight cultures from two different experiments.

 

Figure 8
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  		FIGURE 8. Effector mechanism of macrophages responsible for the rejection of allografted P815 mastocytoma cells by perforin/FasL double-deficient B6 mice. A, Morphology after Giemsa staining of peritoneal cells. Scale bar, 10 µm. B, The cytotoxic activity of PEC against 51Cr-labeled target cells of the allograft type was determined in the presence or absence of anti-TNF-{alpha} Ab (1 µg/ml) or NMMA (0–2.5 mM) at an E:T ratio of 20 in a 12-h assay. Each value represents the mean ± SD of eight cultures from two different experiments.

 
Surprisingly, the cytotoxic activity of PEC against P815 cells was dose-dependently suppressed by the addition of NMMA, an inhibitor of inducible NO synthase (Fig. 8B). In contrast, the inhibitor had no effect on the cytotoxic activity against P815 cells of CTLs that had been induced in P815 cell-allografted normal B6 mice: the cytotoxic activities in the presence of 0, 0.25, and 0.5 mM NMMA were 63.1 ± 2.2, 63.2 ± 4.6, and 68.3 ± 6.8% specific lysis (E:T ratio of 20 in a 12-h assay; n = 4), respectively. Furthermore, anti-TNF-{alpha} Ab had no effect on the activity (Fig. 8B), although this Ab was earlier shown to largely inhibit the cytotoxic activity of LPS/IFN-{gamma}-stimulated casein-macrophages against TNF-{alpha}-sensitive L929 cells (15).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The presence of allograft-induced macrophages (H-2b) results in the apoptotic death of H-2d allografts (e.g., Meth A cells and BALB/c skin) with MHC haplotype (H-2d) specificity, which occurs in a Ca2+- and cell-cell contact-dependent, but Fas-, perforin-, and soluble factor-independent manner (9, 15, 16, 17, 18, 19, 20, 21, 23). The macrophages are inactive toward donor-type Con A blasts (8, 15). In contrast, allograft-induced T cells are highly cytotoxic against donor-type Con A blasts, mastocytoma (e.g., P815) cells, lymphoid (e.g., P388D1 and L1210) tumor cells, and some fibroblastic (e.g., BALB/3T3 and Colon26) cells. Unexpectedly, however, the anti-H-2d CTLs are inactive toward epithelial (e.g., BALB/c skin component) cells, squamous cell carcinoma (e.g., KLN205) cells, and fibrosarcoma (e.g., Meth A) cells (20). Noguchi et al. (10) also reported that Meth A cells were a CTL-resistant cell line. These results suggest that CTLs and macrophages may be the effector cells responsible for the rejection of allografted CTL-susceptible cells and CTL-resistant cells, respectively. In the present study, however, allografted CTL-susceptible P815 mastocytoma cells were acutely rejected from perforin/FasL double-deficient mice, and the effector cell appeared to be a macrophage, not a T cell. Similarly, Spielman et al. (13) reported that allospecific CD8+ CTL lines (H-2b; anti-H-2d), which were generated from spleens of perforin/FasL double-deficient mice, were completely unable to lyse CTL-susceptible P815 (H-2d) targets. Thus, these results indicate that CTLs are not essential for the rejection of CTL-susceptible targets when they lack their two cytotoxic molecules (i.e., perforin and FasL).

In the case of CTL-susceptible targets (e.g., P815 mastocytoma cells, EL-4 T lymphoma cells, and RLmale1 T lymphoma cells), it is assumed that CD8+ T cells are the effector cells responsible for allograft rejection (11) and that perforin and FasL pathways are the killing mechanisms (12). We confirmed these findings in the present study (Figs. 1 and 2). Although CTL-resistant allogeneic tissue (e.g., skin) or tumor cells (e.g., Meth A fibrosarcoma cells) were rejected with kinetics of clearance similar to those of allogeneic CTL-susceptible targets, allograft-induced macrophages (AIM) were the major effector cells responsible for the rejection (8, 9). The infiltration of AIM preceded the infiltration of CTLs by several days during the course of allograft rejection (16). Furthermore, we recently demonstrated that macrophage MHC receptor 1 and macrophage MHC receptor 2 on allograft (H-2d)-induced macrophages in B6 (H-2b) mice recognized H-2Dd and H-2Kd Ags, respectively, on the allograft with very low Kd values (10–9 M) (27, 28). These results indicate that two distinct populations of unique cytotoxic cells (i.e., CTLs and AIM) are induced in the allograft rejection site and that the infiltration of AIM responsible for rejection precedes that of the CTLs perforin- or FasL-dependently cytotoxic against cells expressing donor-related alloantigens. Therefore, it is reasonable that in the absence of perforin and FasL, in vivo rejection of some tumors would be mediated by other mechanisms including the NO-mediated cytotoxicity by macrophages.

Perforin/FasL double-deficient mice die early of severe pancreatitis. Female mice, in addition, are infertile and suffer from hysterosalpingitis (13, 14). The occurrence and severity of autoimmune disease in these mice point to an essential function of these two cytolytic effector pathways in regulating cell-mediated tissue destruction. Because neither perforin deficiency alone nor FasL deficiency alone produces this particular syndrome (12), it is clear that each effector pathway alone is capable of preventing this form of tissue destruction and that the combined absence of both effector pathways is necessary for disease expression. It has been reported that the tissue destruction is accompanied by infiltration by Mac-1+ monocyte/macrophages and Mac-1+ T cells and expansion of CD8+ T cells. It is also known that in vivo inactivation of monocyte/macrophages by carrageenan reverses the disease progression and restores fertility to these double-mutant female mice (13). Carageenan affects only macrophages (29), suggesting that infiltration and expansion of T cells may be secondary to monocyte/macrophage presence in the tissue undergoing destruction. Also in the current study, allograft (i.e., P815 cells)-induced T cells in the perforin/FasL double-deficient B6 mice were inactive toward allografts, and the infiltrates including CTLs had no cytotoxic activity against syngeneic (B6) Con A blasts (data not shown), implying that non-T cells may mediate the tissue (e.g., pancreas and uterus) destruction. Spielman et al. (13) also speculated that the failure of activated T cells to lyse Ag-presenting monocytes/macrophages may be responsible for this syndrome.

Komatsu et al. (30) demonstrated that host cells can effectively resist MHC-matched minor histocompatibility complex-mismatched donor progenitor cells via an alternative effector pathway(s) independent of perforin-, FasL-, TNFR1-, and TRAIL-mediated cytotoxicity (30). Our present results demonstrate that the effector cell appears to be a type of macrophage, not a T cell. Therefore, a non-perforin-, FasL-, TNFR1-, and TRAIL-dependent host macrophage pathway(s) must be capable of effecting significant barrier activity following allografting. In this respect, it is of interest that the macrophages killed CTL-susceptible P815 cells through a NO-dependent pathway.


   Acknowledgment
 
We thank T. Ueno for skillful technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by the Mori and Magai Memorial Research Funds from Osaka Medical College and by a research grant from Kissei Pharmaceutical Company. Back

2 Address correspondence and reprint requests to Dr. Ryotaro Yoshida, Department of Physiology, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki, Japan. E-mail address: ryoshida{at}art.osaka-med.ac.jp Back

3 Abbreviations used in this paper: FasL, Fas ligand; AIM, allograft-induced macrophage; NMMA, NG-monomethyl-L-arginine; PEC, peritoneal exudate cell; FCM, flow cytometry. Back

Received for publication March 1, 2006. Accepted for publication May 29, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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