Brief Definitive Report |
Address correspondence to Joseph A. Trapani, Cancer Immunology Program, Research Division, Peter MacCallum Cancer Centre, Locked Bag 1, A'Beckett St., 8006, Australia. Phone: 61-3-9656-3726; Fax: 61-3-9656-1411; email: joe.trapani{at}petermac.org
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Abstract |
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Key Words: HLH cytotoxic granule NK cell CTL immunodeficiency
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Introduction |
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A syndrome of perforin deficiency was recently described in humans, in that 30% of immunodeficient children presenting with the autosomal recessive disorder hemophagocytic lymphohistiocytosis (HLH) carry mutations in both their perforin alleles (1113). The CLs of these children are unable to impart a lethal hit to target cells and thus fail to clear APCs, resulting in uncontrolled activation and accumulation of macrophages and overproduction of proinflammatory cytokines. Clinically, this is manifested as fever, liver and spleen enlargement, and hemophagocytosis in the spleen, liver, and bone marrow (14). The CLs of these patients show a marked reduction of perforin content, which is thought to indicate failed expression, inaccurate trafficking and/or instability of mutated perforin. Overall, the clinical and pathological findings in HLH are strikingly reminiscent of perforin-deficient mice infected with lymphocytic choriomeningitis virus, as these animals also show expansion of T- and antigen-presenting cell numbers and are unable to down-regulate the immune response (10).
Perforin is released from the secretory granules of CLs with granzymes, a family of proapoptotic serine proteases, and is thought to mediate their trafficking to the target cell cytosol where the proteolytic activity of granzymes imposes apoptotic cell death (15). Purified perforin applied alone at high concentration can also induce osmotic lysis, which may also occur under some physiologically relevant conditions (15). However, at the molecular and cellular levels very little is known of how perforin achieves its functions. This sizeable gap in our knowledge exists largely because it has not been possible to develop robust prokaryotic or eukaryotic models for expressing perforin or its putative domains. Some years ago, it was shown that rat basophil leukemia (RBL) cells transfected to express perforin acquired the capacity to lyse erythrocytes (16), whereas cells that coexpressed perforin and granzyme B were able to kill some nucleated target cells (17). However, these RBL transfectants failed to express perforin in sufficient quantity, or expression was too unstable to enable ongoing studies. A further study reported expression and partial purification of perforin expressed in baculovirus-infected insect cells (18); however, the yield was low and the recovered perforin was denatured and had poor activity.
In the current study, we revisited perforin expression in RBL cells and developed robust expression systems to investigate, for the first time, the molecular and cellular basis for perforin dysfunction associated with two missense mutations reported in an HLH patient. Our results indicate the feasibility of using RBL-based and other perforin expression modalities to study both the basis of the HLH immune deficiency and, more broadly, structurefunction relationships for perforin.
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Materials and Methods |
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Transient Transfection of RBL Cells.
Mature human and mouse perforin each have 534 amino acids. However, the leader sequence of human perforin is one amino acid longer than that of the mouse. This results in a difference in conventional amino acid numbering such that amino acids at positions 225 and 429 mutated in HLH Patient #5 (11) correspond to residues 224 and 428 in the mouse protein, as noted throughout Results and Discussion. Importantly, arginine 225 is a nonconserved residue with threonine being present in mouse perforin. To demonstrate the equivalence of arginine and threonine at this position, we generated the T224R variant and, subsequently, the T224W mutant, which corresponds to R225W in Patient #5 (11). The mutations were introduced using the Transformer (Stratagene) site-directed mutagenesis system according to the manufacturer's instructions. The resultant and the WT cDNA were cloned into the pIRES2-EGFP expression vector (CLONTECH Laboratories, Inc.). Fc receptorexpressing RBL cells were grown to near confluence in 175-cm2 flasks, harvested, washed twice, and resuspended at 108 cells/ml in serum-free DMEM. 200 µL of the cell suspension was mixed with 20 µg pIRES2-EGFP containing the WT or mutated perforin cDNA or vector DNA alone, incubated at room temperature for 10 min, and electroporated in 4-mm electroporation cuvettes and Bio-Rad Laboratories pulser at 500 µF and 0.25 V. After 10 min at RT, the cells were transferred into complete DMEM. Cells were harvested 1820 h later, and GFP-expressing cells were sorted by flow cytometry (FACStar; Beckton Dickinson).
Generation of Recombinant Retroviruses and Stable Expression of Perforin in RBL Cells.
The missense perforin mutation, G428E, corresponding to the human G429E (identified in another perforin allele in Patient #5; [reference 11]), was generated using the Quick-Change sitedirected mutagenesis system (Stratagene) according to the manufacturer's instructions. The cDNAs encoding mouse WT and G428E perforin were subcloned into the retroviral expression vector MSCV, which contains an internal ribosome entry site for GFP expression (19). For retroviral transduction of RBL cells, viral supernatant was generated by cotransfecting the MSCV plasmids with an amphotropic packaging plasmid into 293T cells by calcium phosphate precipitation. After 48 h, the viral supernatant was harvested and added to RBL cells every 12 h for 3 d. The population of cells with the greatest GFP expression (up to 5% of total cells) was subsequently purified by flow cytometry and analyzed for perforin expression.
Assessing the Cytotoxicity of Transfected RBL Cells.
The cytotoxic capacity of RBL cells was analyzed using Jurkat T cell targets in a 4-h 51Cr release assay as described previously (16). Briefly, the surface of 51Cr-labeled Jurkat cells was derivatized with a 1 mM solution of trinitrobenzosulfonic acid in PBS, (pH 7.4) for 15 min at 37°C and washed with unsupplemented DMEM three times. The transfected RBL cells were harvested and incubated with antitrinitrophenol IgE mAb (2 µg/ml) at 37°C for 15 min and washed with unsupplemented DMEM three times. RBL and Jurkat cells were coincubated at various effector to target (E:T) ratios at 37°C in 200 µl serum-free DMEM supplemented with 1% BSA for 4 h in 96-well plates. The supernatant was then harvested and the released 51Cr measured in a gamma counter. The total 51Cr content of Jurkat cells was estimated using 5% Triton X-100lysed cells. The percentage-specific chromium release was calculated as 100x ([experimental release spontaneous release]/[total release spontaneous release]) and is shown as mean ± SD.
Isolation of Lysosomal Granules from RBL Cells.
Perforin was isolated from 109 stably expressing RBL cells by nitrogen cavitation and Percoll density fractionation as described (20). To distinguish granule-enriched fractions from other subcellular fractions, the activity of the RBL granule marker enzyme, ß-hexosaminidase, was measured as follows. 50 µL of each fraction was mixed with 30 µl 8 mM p-nitrophenyl N-acetyl-ß-D-glucosaminide (Sigma-Aldrich) and 10 µl 0.5 M sodium acetate, pH 5.0, at RT for 30 min. The reaction was stopped by adding 150 µl 50 mM NaOH, and the absorbance was measured at 405 nm.
Expression of Recombinant Perforin and Membrane-binding Assay.
Perforin cDNA was cloned into the pFastBac vector and overexpressed in Sf-21 cells using a Back-to-Back kit (Invitrogen) and perforin was purified, all according to the manufacturer's instructions. Small amounts of recombinant WT and the G428E perforin mutant protein were obtained. To study the calcium-dependent membrane binding of perforin, 2 x 107 sheep RBCs were resuspended in 200 µl 20 mM Hepes-150 mM NaCl buffer (pH 7.4) supplemented with 1 mM CaCl2. An aliquot of the purified perforin was added to the cell suspension for 5 min on ice. The cells were pelleted at 16,000 g for 10 s, the supernatant promptly removed, and the cells lysed in ice cold water. The lysate was centrifuged for 20 min at 16,000 g at 4°C. The pellet was washed once, dissolved in SDS-PAGE loading buffer, and analyzed by Western blotting.
Immunoperoxidase Staining.
Approximately 1,000 RBL cells were seeded in each well of an 8-well chamber slide 1 d before staining and cultured overnight. In some experiments, cells were induced to undergo degranulation by transient incubation with TNP-labeled tumor target cells. The RBL cells were fixed for 10 min at RT in 3.7% paraformaldehyde, washed three times in PBS, permeabilized in 0.1% Triton X-100, 0.5% BSA for 5 min, and then washed as before. The cells were treated with periodic acid (0.5%) for 10 min, and endogenous peroxidase activity was quenched with 0.3% H2O2 for 15 min. Blocking buffer (1% BSA/1% skim milk powder in PBS) was added for 30 min before the rat antiperforin mAb P1-8 (21). Bound Ig was detected with biotinylated donkey antirat IgG (Jackson ImmunoResearch Laboratories), streptavidin-HRP (Dako) for 10 min, and the chromogen diaminobenzidine (Dako). Finally, cells were counterstained with eosin and viewed by light microscopy.
Western Blotting.
Cell lysates from stable or transiently transfected RBL cells or granule extracts were resolved on 10% SDS-PAGE (Tris-Glycine) gels, transferred to PVDF membranes, and assayed for perforin content using rat antiperforin mAb PI-8 (provided by Dr. Hideo Yagita, Juntendo Univeristy, Tokyo, Japan) (21) and antirat HRP-conjugated Ig. The signal was detected using chemiluminescence (Amersham Biosciences).
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Results and Discussion |
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We initially devised a rapid method for transient transfection of RBL cells, using the vector pIRES2-EGFP. 1 d after electroporation, fluorescent cells were sorted and immediately used in a 51Cr release assay with Jurkat target cells to which they were conjugated. The efficiency of electroporation was as high as 40%, and up to 106 GFP-expressing cells were obtained per electroporation. Although G429 is conserved in human, mouse, and rat perforin, R225 is not invariant and corresponds to T224 in mouse perforin. To confirm the functional equivalence of arginine and threonine at this position, we generated RBL cells expressing T224R mouse perforin and found they were as efficient in the 51Cr release assay as WT perforin-transfected cells. However, expressing perforin with tryptophan at the same position (T224W) resulted in complete loss of cytolytic function (Fig. 1). As expected, the WT protein had an apparent molecular mass of 67 kD; however, the introduction of tryptophan resulted in the appearance of truncated (
45 kD) perforin (Fig. 1), suggesting the mutation facilitated proteolytic cleavage/ processing of perforin. Furthermore, immunohistochemistry analysis of transfected cells indicated mislocalisation of T224W, possibly due to a loss of putative signaling motif(s). Whereas WT perforin produced a punctate appearance consistent with packaging in secretory granules, T224W perforin produced diffuse staining throughout the RBL cell cytoplasm (Fig. 2 A).
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Since G428E perforin was expressed at equivalent levels to WT perforin and correctly targeted to, and released from, granules (Fig. 2 and Fig. 3, B and C), the mutation was likely to affect a postsynaptic function of perforin. To test this possibility, we generated and purified recombinant WT and G428E perforin using a baculovirus expression system and tested their ability to bind to sheep RBC membranes in a calcium-dependent manner. Whereas WT perforin displayed strong calcium-dependent plasma membrane binding with essentially all the added perforin bound, the binding of G428E perforin was markedly reduced (Fig. 4). Consistent with this observation, the cytolytic activity of the recombinant G428E mutant was 5% of that of WT perforin (unpublished data). Although RBL cells have been used as a read-out of perforin function for many years, a perceived weakness of the model is that perforin exerts its cytolytic effects in the absence of granzyme B. Exposure of target cells to recombinant G428E-perforin with granzyme B did not rescue the perforin phenotype (unpublished data). Therefore, our findings strongly suggested that the diminished activity of G428E-perforin was due to diminished target cell membrane binding, rather than the absence of granzymes. Based on modeling studies and biochemical analysis of CTL clones, Uellner and colleagues found that perforin's COOH terminus strongly resembles that of calcium-dependent membrane-binding C2 family of protein domains, some of which are involved in vesicular trafficking at neuronal synapses (23). Importantly, G428 is located in the C2 domain of perforin, immediately adjacent to one of five highly conserved aspartate residues believed to be essential for calcium binding (23). We postulate that the G428E substitution may interfere with calcium binding to the C2 domain, leading to decreased affinity of perforin for lipids in the target cell membrane (23). The apparent quantitative differences in G428E-perforininduced cytolysis in the RBL-based and recombinant models probably reflects differences in the kinetics of perforintarget cell membrane interaction in the context of a cell conjugate (RBL model) and in solution (purified recombinant perforin).
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Acknowledgments |
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This work was supported by program grants from the National Health and Medical Research Council (NHMRC), Australia (to M.J. Smyth and J.A. Trapani) and the Juvenile Diabetes Research Foundation (to J.A.T). M.J. Smyth, P.K. Darcy, and J.A. Trapani also receive fellowships from NHMRC.
The authors have no conflicting financial interests.
Submitted: 20 April 2004
Accepted: 15 July 2004
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References |
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