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The Journal of Immunology, 2000, 165: 5048-5053.
Copyright © 2000 by The American Association of Immunologists

Functional Defects of NK Cells Treated with Chloroquine Mimic the Lytic Defects Observed in Perforin-Deficient Mice1

Mesha Austin Taylor2,*,{dagger}, Michael Bennett{dagger}, Vinay Kumar{ddagger} and John D. Schatzle{dagger}

* Graduate Program in Immunology, {dagger} Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390; and {ddagger} Department of Pathology, University of Chicago, Chicago, IL 60637


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
NK cells are the primary effectors mediating acute rejection of incompatible bone marrow cell grafts. To reduce rejection, we evaluated the ability of chloroquine (CHQ) to prevent perforin-dependent NK cell activity. Perforin is a key cytotoxic component released from the lytic granules of activated NK cells. Generation of functional perforin requires an acidic protease activity that occurs in the secretory, lytic lysosomes. Our hypothesis was that CHQ, a lysosomotropic reagent, would raise the pH of the acidic compartment in which perforin is processed and thereby block perforin maturation and cytotoxicity. We have measured NK cytotoxicity in vivo by clearance of YAC-1 tumor cells from the lungs and by rejection of incompatible bone marrow transplants and in vitro by cytolysis of YAC-1 and Jurkat cells. The engraftment of bone marrow cells was monitored by recolonization of the spleen with hemopoietic cells from transplants of MHC class I-deficient bone marrow cells into lethally irradiated recipient mice. Transplant rejection was compared in two inbred strains of mice: 129, which apparently use perforin-dependent cytotoxicity, and C57BL/6, in which rejection can be perforin-independent. CHQ treatment reduced NK cell activity in 129 mice in which perforin is important for mediating rejection. CHQ affected the fraction of NK cell cytolysis that was Fas independent. In addition, we found that CHQ prevents perforin processing by LAK cells in vitro. These data indicate that CHQ may impair rejection of incompatible bone marrow transplants and other functions mediated by NK and cytotoxic T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Natural killer cells are large granular lymphocytes that can spontaneously lyse tumor and virally infected cells without prior sensitization (1). Unlike solid tissue graft rejection, which is mainly T cell mediated, NK cells are the primary effector cells mediating the acute rejection of bone marrow cell (BMC)3 grafts (2). As proposed by the missing self hypothesis, NK cells are able to efficiently lyse allogeneic targets or MHC class I-deficient targets because the target cell lacks self MHC class I expression (3).

At least three mechanisms of NK cell-mediated lysis have been defined extensively in recent years. These include calcium-dependent release of perforin and granzymes, interaction of ligands with different death receptors, and contact with TNF. The function of perforin in concert with granzymes appears to be a primary cytotoxic mechanism of NK cells. Conjugate formation occurs between the effector and target cell resulting in degranulation of perforin, which forms pores in the target cell membrane and allows for the entrance of granzymes into the target cell for the induction of DNA fragmentation (4). Perforin as a key mediator of NK cell-mediated cytolysis was demonstrated by the lack of killing of Fas+ YAC-1 lymphoma cells, NK cell-sensitive target cells, by NK cells derived from perforin-deficient mice (PKO) (5). Thus, the perforin deficiency results in effectors with decreased ability to reject tumors and clear viral infections (6). Another cytotoxic mechanism is the engagement of Fas ligand (FasL) on an effector cell with the death receptor, Fas (CD95), on the target cell, triggering apoptosis in the target (7, 8). Although Fas was the first member of the TNF receptor superfamily to be described in terms of its apoptotic functions (9), additional members of this family can also transduce apoptotic signals upon binding to their soluble ligands, such as the death receptors, DR4 and DR5, which bind to TNF-related apoptosis-inducing ligand (TRAIL) (10). Although to a smaller magnitude, membrane-bound or secreted TNF also plays a role in NK cell-mediated cytotoxicity. In an 18-h cytotoxicity assay, perforin- and FasL-deficient lymphokine-activated killer cells (LAKs) demonstrate TNF-mediated cytotoxicity against TNF-sensitive targets (11). Although these pathways represent alternative mechanisms of NK cell-mediated cytotoxicity, much remains to be studied in terms of their effects in acute bone marrow rejection.

The importance of perforin in NK cell cytotoxicity is becoming more evident. Recently, the perforin deficiency has been shown to play a role in the pathogenesis of familial hemophagocytic lymphohistiocytosis (12). NK cell cytolytic function is substantially reduced in these patients (13). However, its role in NK cell-mediated rejection of incompatible bone marrow grafts is somewhat controversial. Previous observations have proposed that perforin is not necessary to mediate rejection of allogeneic BMC grafts (14). However, using donor BMC from a more susceptible mouse strain (TAP-1 KO), we have shown that NK cells from perforin knockout (PKO) mice have a decreased ability to reject these class I-deficient BMC grafts (15). Specifically, 129:B6 PKO mice are unable to reject TAP-1 KO BMC grafts, whereas B6 PKO are able to reject. Thus, it is possible that perforin may play a major role in acute bone marrow rejection by NK cells.

Within the cytoplasm of NK cells, granules containing perforin and granzymes can be exocytosed toward their target cell for the induction of cell death (16, 17). Perforin is stored within these specialized lytic, secretory granules (18). Initially, perforin is synthesized in the endoplasmic reticulum as a larger precursor protein (70 kDa) that is inactive (19). After passage through the endoplasmic reticulum and Golgi, this precursor form of perforin is proteolytically cleaved to the mature, active form (60 kDa) within the secretory lysosomes. This bioactive form of perforin is then stored in the acidic environment of the lytic granules. Thus, the low pH in the secretory lysosomes is essential to maintaining functionally active perforin.

The importance of acidic lytic granules in NK cell function was also demonstrated in studies using chloroquine (CHQ). CHQ is a primary amine that prevents the acidification of lysosomes by raising the lysosomal pH (20). It also interferes with NK cell cytotoxicity, presumably by inhibiting lysosomal enzyme function (21, 22, 23). The exact inhibitory mechanism of NK cell cytotoxicity by CHQ is unknown. However, because CHQ neutralizes the lysosomal pH in the lytic granules of NK cells, we hypothesize that CHQ prevents the processing of inactive perforin to its active form and weakens the rejection of incompatible donor BMC by NK cells from CHQ-treated mice.

Using CHQ to inhibit NK cell cytolytic function, we demonstrate the inhibitory effects of CHQ on NK cells in the clearance of tumor cells from the lungs and in acute NK cell-mediated rejection of bone marrow grafts in mice. In addition, we show that CHQ treatment inhibits the processing of perforin to its active form, thereby, reducing NK cell cytotoxicity. Although the addition of CHQ may interfere with perforin processing, other important cytotoxic mechanisms, such as Fas/FasL interactions, appear to remain functional. Therefore, similar to our previous results with PKO mice, treatment with CHQ emphasizes the significance of perforin in the acute rejection of bone marrow grafts by NK cells. Of particular interest was the observation that C57BL/6 mice are more resistant to CHQ than are 129 mice, mirroring similar findings in PKO mice.


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

CHQ was purchased from Sigma (St. Louis, MO). Anti-Fas ligand mAb (clone MFL3) was purchased from PharMingen (San Diego, CA). Anti-asialo-GM1 antiserum was purchased from Wako Bioproducts (Richmond, VA). Purified rabbit anti-rat perforin mAb was purchased from Torrey Pines Biolabs (San Diego, CA). Human rIL-2 was obtained from Chiron (Emeryville, CA).

Cell culture

LAK cells were cultured in 500 U/ml of human rIL-2 as previously described (24). YAC-1 and Jurkat tumor cell lines were maintained in vitro in RPMI 1640 medium supplemented with 10% FCS, 1% L-glutamine, 1% nonessential amino acids, 1% sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin.

Mice

As previously described in detail (15), all mice were bred and maintained at the University of Texas Southwestern Medical Center. All mice were housed in a conventional colony except for certain 129:B6 PKO mice housed in a specific-pathogen-free (spf) facility. TAP-1 KO mice were obtained from The Jackson Laboratory (Bar Harbor, ME). 129:B6 PKO mice and TAP-1 KO mice have a mixed background of 129 and B6 genes.

Bone marrow transplantation

This assay has been previously described (15, 25, 26). Most groups contained five mice. Briefly, recipient mice were lethally irradiated (800 cGy) and injected i.v. with 2.5 x 106 TAP-1 KO donor BMC. Five days after cell transfer, growth of donor-derived BMC was assessed by splenic incorporation of [125I]UdR (Amersham Life Science, Arlington Heights, IL), which is a specific DNA precursor and a thymidine analogue. The results are expressed as the geometric mean (95% confidence level) percentage uptake of [125I]UdR. A high % [125I]UdR uptake represents growth of the BMC graft and a low % [125I]UdR uptake denotes rejection. Using log10 values, parametric and nonparametric statistical analyses were performed to determine significance of differences between geometric mean values. The Student t test was used to compare two groups, and the Newman-Keules Multiple Comparison test was used to determine significantly different groups at p = 0.05 level for experiments that consist of multiple recipient groups of a given donor. Values significantly (p < 0.05) different from another group by nonparametric and parametric analysis are indicated in the figure legends.

Lung clearance assay

Details of the lung clearance assay have been described previously (27). Each group contained five mice. Briefly, YAC-1 target cells were labeled with [125I]UdR (Amersham Life Science), then 5 x 105 cells were injected intraveneously into recipient mice. Two to three hours later, the lungs were excised from the mice, and the amount of 125I remaining in the lungs was measured. Results are expressed as the geometric mean (95% confidence limits) of the percentage of injected radioactivity remaining in the lungs. Values in Fig. 3GoA are expressed as a percent of [125I]UdR retention, which is inversely related to NK cell lytic activity. High percent retention denotes low or no NK cell lytic activity, whereas a low percent retention indicates high NK cell lytic activity. Values in Fig. 3GoB are expressed as a percent of [125I]UdR retention determined by the kinetic study in Fig. 3GoA. The same statistical analyses were performed as described in the bone marrow transplantation assays above.



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  		FIGURE 3. CHQ reduces the clearance of YAC-1 tumor cells from the lungs. A, Where indicated, 1 day before infusion, B6+/+ mice were injected i.p. with 15 µl of anti-asialo-GM1 to deplete NK cells. Mice were infused with 5 x 105 [125I]UdR-labeled YAC-1 cells. Lung clearance capability was assessed at the indicated time points after injection by determining % [125I]UdR retention. Results are expressed as geometric means. B, Where indicated, mice were injected i.p. with 1.5 mg of CHQ per day for 3 days before infusion. One day before infusion, B6+/+ mice were injected i.p. with 15 µl of anti-asialo-GM1 to deplete NK cells. Mice were infused with 5 x 105 [125I]UdR-labeled YAC-1 cells. Lung clearance capability was assessed 3 h after injection by determining % [125I]UdR retention. Results are expressed as geometric means. Geometric mean values in the 129 + CHQ group is significantly different (p < 0.05) from each of the other groups. Geometric mean values in the B6 + anti-asialo-GM1 group are significantly different (p < 0.05) from each of the other groups, except the 129:B6 PKO group.

 
Cytotoxicity assay

As previously described (27), target cells were radiolabeled with 100–150 µCi of sodium chromate (51Cr) (Amersham Life Science) for 1.5 h at 37°C. At various E:T ratios, effectors and radiolabeled targets were added to each well in triplicates. Preincubation of effectors with 50 or 100 µg/ml CHQ was performed at 37°C for 60 min. Following chloroquine treatment and before addition to targets, 10 µg/ml anti-FasL mAb was added to effectors. After a 4-h incubation, 100 µl of supernatant were removed and the 51Cr released was counted in a liquid scintillation counter. Specific lysis was expressed as the mean ± SEM and calculated as follows: percent specific lysis = 51Cr cpm, (ER - SR)/(MR - SR) x 100, where ER is the experimental 51Cr released in the presence of effectors, SR is the spontaneous 51Cr released in the presence of medium only, and MR is the maximum 51Cr release in the presence of 0.5% Triton X-100.

Western blot analysis

Cells were washed once in PBS, resuspended at 108 cells/ml in lysis buffer (50 mM TrisCl, pH 8; 150 mM NaCl; 1 mM MgCl2; 2% Nonidet P-40) and incubated on ice for 30 min. Lysates were spun for 15 min at 14,000 rpm at 4°C, and aliquots of 10 µl were resolved by SDS-PAGE on gels of 10% acrylamide. Proteins were transferred to nitrocellulose and stained with Ponceau dye. Filters were blocked for 30 min in 5% nonfat dried milk in PBS, 0.1% Tween 20, then incubated with primary Ab (1:1000) diluted in PBS, 0.1% Tween 20 for 1 h. Filters were washed three times in PBS, 0.1% Tween 20, then incubated with anti-rabbit IgG HRP-labeled secondary Ab (Amersham Life Sciences) for 1 h. After washing, blots were developed with the Super Signal chemiluminescence kit from Pierce (Rockford, IL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CHQ prevents the rejection of class I-deficient BMC by 129 wild-type mice

We have shown that perforin plays a fundamental role in NK cell-mediated rejection of incompatible bone marrow grafts (15). Depending upon environmental conditions or genetic background, certain strains of PKO mice are unable to reject class I-deficient or allogeneic donor BMC. For instance, B6 mice are strong rejectors of incompatible BMC grafts, whereas 129 mice are poor rejectors. Deleting perforin in the 129 strain further weakens their rejection capacity. B6 PKO mice, in contrast, retain the ability to effectively reject incompatible BMC. In an attempt to mimic the lytic defects of these PKO mice, we chose to administer CHQ to 129+/+ and B6+/+ mice i.p. before performing the BMC graft. CHQ was considered an ideal candidate reagent because previous studies have indicated that CHQ was an inhibitor of CTL and NK cell cytotoxicity in vitro (16, 17, 18). We also proposed the application of CHQ because of its chemical characteristics as a nontoxic, weak base, which could interfere with the cleavage of perforin to its mature, functional form (19). Given this rationale, we administered CHQ i.p. to lethally irradiated B6+/+ and 129+/+ mice to inhibit NK cell cytotoxicity. The chosen CHQ dosages were equivalent to those dosages given to human subjects for anti-malarial therapies (28). We then challenged these CHQ-treated mice with TAP-1 KO donor BMC, expressing low or no levels of MHC class I on the cell surface. As shown in Fig. 1Go, B6+/+ and 129+/+ are able to effectively reject TAP-1 KO BMC. However, CHQ-treated 129+/+ mice accepted the BMC graft as well as syngeneic recipients. This is significantly different from those CHQ-treated B6+/+ mice that can mediate rejection. These results suggest that CHQ can act as an inhibitor of acute NK cell-mediated rejection of class I-deficient BMC by 129+/+ mice, but not by B6+/+ mice. In addition, these findings with CHQ administration are similar to those results observed when PKO mice are challenged with class I-deficient BMC grafts (15).



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  		FIGURE 1. CHQ prevents the rejection of class I-deficient BMC by 129+/+ mice. Lethally irradiated hosts were injected i.v. with 2.5 x 106 Tap-1 KO donor BMC. 129+/+ and B6+/+ mice were injected i.p. with 1 mg CHQ per day for 3 days before bone marrow transplantation. Host spleens were harvested 5 days after injection of donor BMC to assess splenic hemopoietic cell repopulation. Growth of transplants is represented as % [125I]UdR uptake. Geometric mean values in the 129 + CHQ group are significantly greater (p < 0.05) than 129+/+, B6 + CHQ, and B6+/+ groups, and are similar to values in Tap-1 KO syngeneic controls (p > 0.05).

 
We have previously shown that 129:B6 PKO mice housed in a spf environment are able to reject TAP-1 KO BMC by a perforin-independent mechanism; although those 129:B6 PKO mice housed in a conventional (conv) environment are unable to reject TAP-1 KO BMC (15). To determine whether CHQ interferes with perforin-independent mechanisms of bone marrow graft rejection, we administered CHQ to 129:B6 PKO mice (spf) before challenging with TAP-1 KO BMC. Fig. 2Go demonstrates the inability of CHQ to prevent rejection of TAP-1 KO BMC by these PKO mice. Therefore, CHQ does not appear to have pleiotropic effects on acute bone marrow graft rejection by 129:B6 PKO mice (spf). These results provide further evidence that the effects of CHQ appear to mimic the lytic defects observed in PKO mice.



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  		FIGURE 2. CHQ does not interfere with perforin-independent mechanisms of bone marrow graft rejection. Lethally irradiated hosts were injected intraveneously with 2.5 x 106 Tap-1 KO donor BMC. 129:B6 PKO mice (spf) were injected i.p. with 1 mg CHQ per day for 3 days before bone marrow transplantation. Host spleens were harvested 5 days after injection of donor BMC to assess splenic hemopoietic cell repopulation. Growth of transplants is represented as % [125I]UdR uptake. Geometric mean values in the 129:B6 PKO (conv) group are significantly greater (p < 0.05) than B6+/+, 129:B6 PKO (spf), and 129:B6 PKO (spf) + CHQ groups, and are similar to values in Tap-1 KO syngeneic controls (p > 0.05).

 
CHQ reduces the clearance of YAC-1 tumor cells from the lungs

Another in vivo method to assay for NK cell cytotoxicity is based upon their ability to rapidly clear tumor cell targets from the lungs following i.v. injection (29). We have noticed that different tumor targets have variable retention times in the lungs of mice. Fig. 3GoA is a time course lung clearance assay of YAC-1 to detect the optimum retention time. Based upon results from Fig. 3GoA, we chose to observe lung clearance of YAC-1 tumor cells at 3 h. NK cells in control B6+/+ mice possess normal lytic activity; thus, they are able to optimally clear labeled YAC-1 cells, a murine NK sensitive tumor target, from the lungs 3 h after injection (Fig. 3GoA). However, treating B6+/+ mice with anti-asialo-GM1 serves to deplete their NK cells, preventing the NK cell-mediated clearance of tumor cells from the lungs of these mice (as previously reported (27); and Fig. 3GoA). Similar to B6+/+ mice, 129+/+ mice also possess normal NK cell lytic activity (Fig. 3GoB). Resembling those results observed after challenging with class I-deficient BMC (15), the lytic activity of NK cells from B6 PKO and 129:B6 PKO mice are different (Fig. 3GoB). B6 PKO mice rapidly clear labeled YAC-1 tumor cells, whereas 129:B6 PKO mice cannot. In fact, the clearance capability of 129:B6 PKO mice is similar to those of NK cell-depleted B6+/+ mice. As observed in this lung clearance assay, the NK cells of B6+/+ mice treated with CHQ appear to retain normal lytic activity. However, CHQ-treated 129+/+ mice were defective in their clearance of YAC-1 cells. The retention levels of labeled targets in these mice appear to be more than the levels retained in CHQ-treated B6+/+ mice, but slightly less than the levels retained in 129:B6 PKO mice. Using the lung clearance assay, the NK cell cytotoxicity observed of CHQ-treated 129+/+ and B6+/+ mice seems to simulate those results observed in the rejection of class I-deficient BMC grafts. Therefore, the effects of CHQ on NK cell function as determined by bone marrow transplantion and lung clearance assays mimic the lytic defects observed in PKO mice.

CHQ inhibits NK cell cytotoxicity without interfering with Fas/FasL interactions

Because Fas/FasL interactions have been shown to participate in NK cell-mediated cytotoxicity (7, 8), we wanted to test whether CHQ inhibited this cytolytic mechanism, using in vitro chromium release assays. Two types of tumor targets were used: 1) YAC-1 cells, which are perforin-sensitive, yet FasL-insensitive targets and 2) Jurkat cells, a human T cell leukemia that is sensitive to both perforin- and Fas/FasL-mediated killing (30). In a 4-h chromium release assay, IL-2 activated B6+/+ LAKs lysed YAC-1 tumor cells, which express low levels of Fas (Fig. 4GoA). The addition of anti-FasL mAb did not significantly reduce this NK cell lytic activity, suggesting that YAC-1 tumor targets are predominately killed by the perforin pathway. Consistent with previously described data, B6 PKO LAKs in the presence or absence of anti-FasL mAb, do not lyse YAC-1 tumor targets (5). In a dose-dependent manner, CHQ treatment of 50 or 100 µg/ml diminished cytolytic activity by B6+/+ LAKs. Moreover, anti-FasL mAb had no additional inhibitory effect when added to CHQ-treated B6+/+ LAKs. These results were consistent at both the low and high doses of CHQ, implying that CHQ may be interfering with perforin-mediated cytolytic activity.



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  		FIGURE 4. CHQ inhibits NK cell cytotoxicity without interfering with Fas/FasL interactions. In a 51Cr release assay, B6+/+ or B6 PKO IL-2-activated LAK effectors were preincubated for 1 hour with indicated amounts of CHQ. A total of 10 µg/ml anti-FasL mAb was added to effectors before addition of targets, where indicated. Untreated or CHQ-treated LAK effectors were then incubated for 4 h with labeled YAC-1 targets (A) at 100:1 E:T ratio or labeled Jurkat targets (B) at 200:1 E:T ratio.

 
To determine whether CHQ affects other killing mechanisms, killing assays using the FasL-sensitive Jurkat targets were performed (Fig. 4GoB). B6+/+ and B6 PKO LAKs are able to lyse Jurkat targets, although killing by the latter is somewhat reduced. Moreover, in the presence of anti-FasL mAb, reduced lysis of Jurkat tumor cells was observed, especially in B6 PKO LAKs where lysis was inhibited completely. Lysis of Jurkat targets was decreased slightly by CHQ-treated effectors. However, the addition of anti-FasL mAb lowered NK cell lytic activity even further. CHQ is unable to inhibit the major cytotoxic mechanism of Jurkat target cell lysis which is the engagement of FasL by Fas receptor. When NK cells are treated with a high concentration of CHQ, residual lysis of Jurkat targets can be attributed to Fas/FasL interactions. Consequently, CHQ must not function to paralyze all of the critical cytolytic pathways, such as those governed by Fas/FasL interactions.

CHQ prevents the processing of perforin to its active form

Because CHQ has been shown to effectively diminish NK cell cytotoxicity, we wanted to determine the biochemical mechanism of this inhibition. One possibility is that CHQ prevents the processing of perforin to its active form by neutralizing the lysosomal pH in the lytic granules of NK cells. Other lysosomotropic agents, such as NH4Cl, have provided evidence for this inhibition of perforin processing (19). Furthermore, Uellner et al. (19) have emphasized the significance of acidity in the lytic granules to provide an environment for the cleavage of perforin to its mature, functional form. To investigate whether CHQ treatment affects perforin processing, we incubated B6+/+ LAKs in the absence (lane 1) or presence of 100 µg/ml CHQ for 1 (lane 2) or 5 (lane 3) h (Fig. 5Go). Immunoblots of untreated B6+/+ LAKs cell lysates revealed that the majority of perforin is in the 60-kDa bioactive, processed form. However, CHQ treatment resulted in the accumulation of the 70-kDa inactive form of perforin, indicating a decrease in the processing of newly synthesized perforin (Fig. 5Go, compare lane 1 to lanes 2 and 3). Similar results were also seen using CHQ-treated 129+/+ LAKs (data not shown). It is not surprising that a residual amount of the 60-kDa perforin remains after 5 h of CHQ treatment. Presumably, perforin made before CHQ treatment is stored in the cells and has a moderate turnover time. Interestingly, CHQ decreased perforin processing more effectively than NH4Cl (lane 4), which also has been shown to increase the pH of endosomal compartments substantially (19). These results indicate that perforin processing to its active form is inhibited by CHQ treatment. In addition, this biochemical mechanism explains why CHQ treatment simulates perforin genetic defects as described in the assays above.



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  		FIGURE 5. CHQ prevents the processing of perforin to its active form. B6+/+ or B6 PKO IL-2-activated LAKs were incubated in the absence or presence of inhibitors at various time points. Cell lysates were resolved by SDS-PAGE under nonreducing conditions and immunoblotted with anti-perforin. The 70-kDa band corresponds to inactive (nonproteolytically cleaved) perforin and the 60-kDa band corresponds to active (proteolytically cleaved) perforin. B6+/+ LAKs were used in lane 1, untreated; lane 2, 1 h of 100 µg/ml CHQ treatment; lane 3, 5 h of 100 µg/ml CHQ treatment; lane 4, 5 h of 10 mM NH4Cl treatment. B6 PKO LAKs were used in lane 5, 5 h of 100 µg/ml CHQ treatment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In recent years, several mechanisms of lymphocyte cell-mediated cytotoxicity have been studied extensively. Particular emphasis has been placed on perforin as the key component in NK cell and CTL-mediated cytolysis. Perforin has been shown to aid in the elimination of virally infected and tumorigenic cells (6). Our previous studies have demonstrated perforin-mediated killing by NK cells to be a fundamental mechanism involved in the acute rejection of incompatible bone marrow grafts (15). When housed in a conventional animal colony, 129:B6 PKO mice are incapable of rejecting MHC class I-deficient donor BMC, whereas B6 PKO mice are able to reject. These findings support the concept that 129+/+ and B6+/+ mice differ genetically in their ability to mediate perforin-independent NK cell functions. Thus, the inability of perforin-deficient NK cells to acutely reject incompatible BMC provides further insight into the significance of perforin as a primary effector molecule mediating NK cell cytotoxicity.

Employing both in vitro and in vivo assays for NK cell cytotoxicity, we have shown that the administration of CHQ models the perforin lytic defect. NK cell cytotoxicity was suppressed significantly after the addition of CHQ. Attributed to its properties as a weak base and a lysosomotropic agent, CHQ prevented the acidification of the secretory, lytic granules. This biochemical characteristic of CHQ caused the accumulation of the unprocessed, inactive form of perforin in NK cells. By inhibiting the processing of this major cytotoxic molecule, NK cell cytotoxicity was severely diminished. Even after treatment with high concentrations of CHQ, B6+/+ LAKs were somewhat able to lyse perforin-sensitive YAC-1 tumor cells (Fig. 4GoA). This lack of complete inhibition of NK cell cytolysis can be best explained by the residual, processed perforin detected after incubation of the NK cells with CHQ for 5 h (Fig. 5Go). This observation also explains why CHQ treatment of 129+/+ mice does not completely suppress their ability to clear YAC-1 tumor cells from the lungs (Fig. 3GoB). CHQ given for a period of 3 days may not permit the complete inhibition of perforin processing in all NK cells of 129+/+ mice. Consequently, a small population of activated NK cells treated with CHQ may release remaining bioactive perforin, resulting in the diminished, yet incomplete clearance of some tumor targets. Longer CHQ treatment would hypothetically reduce or completely prevent perforin-mediated NK cell lysis. Thus, the ability of CHQ to cause the accumulation of unprocessed perforin in the secretory granules of NK cells provides an excellent mechanism for controlling the release of functionally active proteins involved in regulating the cytolytic immune response.

Previous studies have detected the colocalization of bioactive FasL and perforin within the lytic granules of the human NK cell line YT (31). These results suggest that newly synthesized, intact FasL is secreted together with perforin from the secretory, lytic granules after NK cells recognize their targets. Upon mitogenic stimulation, bioactive FasL and TRAIL were also released from microvesicles within Jurkat and normal human T cell blasts as nonproteolyzed proteins (32). Because CHQ is a lysosomotropic amine, we addressed the prospect that CHQ may modify or affect the function of perforin-independent molecules stored within the lytic granules. In vitro killing assays using Jurkat tumor cells confirmed that CHQ suppressed only the processing of perforin, allowing perforin-independent mechanisms, such as Fas/FasL, to remain functionally active. In addition, we have shown that 129:B6 PKO mice maintained in the spf colony and B6+/+ mice were effectively able to reject class I-deficient BMC grafts following CHQ treatment (Figs. 2Go and 1Go, respectively). These data further emphasize the inability of CHQ to interfere with perforin-independent cytotoxic mechanisms.

As depicted in Fig. 3GoB, B6 PKO mice are similar to B6+/+ mice in that both are able to optimally clear labeled YAC-1 cells from the lungs after 3 h. However, in an in vitro killing assay, B6 PKO LAKs were completely inhibited in their ability to lyse YAC-1 tumor cells. This discrepancy suggests that there are other perforin-independent elements within the host which can facilitate lysis of tumor targets by NK cells. These perforin-independent mechanisms must not be employed in our in vitro system.

Other immunomodulatory effects of CHQ have been described previously. By increasing the pH within the intracellular vacuoles of APCs, low doses of CHQ have also been shown to decrease the processing of MHC class II Ags (33). As a result of altering antigenic protein degradation and preventing the assembly of peptides with MHC class II proteins, CHQ diminishes the stimulation of CD4+ T cells (34). These findings implicate CHQ as a model reagent for antirheumatic therapy because it down-regulates the immune response against autoantigenic peptides. CHQ has also been used to inhibit the development of graft-vs-host disease by preventing T cell responses to minor and MHC Ags (35). Aside from its antirheumatic and anti-graft-vs-host disease qualities, CHQ can act as an antimalarial agent. After accumulating within intracellular acid vesicles, CHQ diminishes heme polymerase activity, which is a crucial metabolic step specific to the life cycle of Plasmodium flaciparum trophozites (36). Presenting another clinically relevant application of CHQ, we have shown that CHQ can be administered before bone marrow transplantation as a specific inhibitor of NK cell-mediated rejection. By mimicking perforin genetic defects, long-term administration of CHQ to patients before injection of donor BMC may provide a relatively nontoxic, immunosuppressive therapy for the prevention of BMC rejection.


   Acknowledgments
 
We thank Jennifer Klem for the critical reading of this manuscript and Silvio and Maria Peña for the maintenance of the animal facilities.


   Footnotes
 
1 This work was supported by the National Institutes of Health Grants CA36922, CA70134, and AI38938. Back

2 Address correspondence and reprint requests to Mesha Austin Taylor, Department of Pathology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9072. Back

3 Abbreviations used in this paper: BMC, bone marrow cell; PKO, perforin knockout; FasL, Fas ligand; CHQ, chloroquine; spf, specific pathogen free; LAK, lymphokine-activated killer. Back

Received for publication May 5, 2000. Accepted for publication August 10, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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