By
From the Departments of Medicine, Surgery, Anatomy, and Cell Biology and the Transplantation Immunology and Immunogenetics Laboratories, State University of New York Health Science Center at Brooklyn, Brooklyn, New York 11203
A receptor-ligand interaction exclusive to natural killer (NK) cell-mediated recognition and
triggering of tumor cell destruction has not yet been identified. In contrast, molecules that are
involved in cellular adhesion and regulation of NK cytolysis have been well studied. In this report, a novel tumor surface protein is identified that exhibits characteristics of a recognition
structure for naive NK cells. A tagged ligand-cell adsorption technique revealed a 38.5-kD
plasma membrane protein (p38.5) from a prototypical NK-susceptible cell line (K562) that preferentially bound to NK cells (CD3CD5
CD16+) relative to T lymphocytes (CD3+CD5+
CD16
). The molecule was purified to apparent homogeneity for further characterization. An
amino acid sequence of an 11-mer internal peptide of p38.5 did not exhibit homology to
known proteins. Affinity-purified antibody generated against this peptide (anti-p38.5) reacted
with a single protein of 38.5 kD on Western blots of whole cell extracts of K562. Flow cytometry and immunoprecipitation studies of surface-labeled tumor cells demonstrated expression of
p38.5 on NK-susceptible tumor cell lines (K562, MOLT-4, Jurkat), whereas p38.5 was not detected on NK-resistant tumor cell lines (A549, Raji, MDA-MB-231). Significantly, p38.5 loss
variants derived from wild-type Jurkat and Molt-4 cell lines exhibited decreased susceptibility
to NK cell-mediated lysis demonstrating a strong association between cell surface expression of
p38.5 and cytotoxicity. Purified p38.5 retained preferential binding to NK cells and inhibited
NK activity in a dose-dependent manner, thereby providing direct evidence of a role in the
lytic process. Binding studies identified a 70-kD membrane protein from NK cells as a possible
receptor for the p38.5 tumor ligand. Consistent with cellular adsorption studies, the 70-kD,
p38.5 binding protein was not detected on T lymphocytes. Based on studies demonstrating selective binding of p38.5 to NK cells, lack of expression on NK-resistant tumor cell lines and ability of the purified molecule to block cytolysis, we conclude that p38.5 may serve as a recognition/triggering ligand for naive human NK cells.
Naive NK cells (CD3 A possible feature of NK cell tumor-specific recognition
structures is that the molecules may be exclusively expressed on NK cells and their susceptible target cells, respectively. We examined this possibility by using a tagged
ligand-cell adsorption technique (13) to reveal surface molecules of human tumor cells that preferentially bind to NK
cells. Results from these studies identified a 38.5-kD tumor
membrane protein (p38.5) that bound to NK cells and not
at all to T lymphocytes. Functional studies suggest that this
interaction is necessary for naive NK cell-mediated cytotoxicity.
Chemicals, Antibodies, and Cell Lines.
N-hydroxy succinamide
ester of biotin (biotin-NHS) was purchased from Calbiochem-
Novabiochem Corp. (La Jolla, CA). Streptavidin alkaline phosphatase and all cell culture media and reagents were from GIBCO BRL
(Gaithersburg, MD). Electrophoresis reagents and chemicals were
purchased from Bio-Rad Laboratories (Melville, NY). All other
chemicals used were from Sigma Chemical Co. (St. Louis, MO).
, CD16+, TCR
), unlike CTL
(CD3+, CD16
, TCR+) provide cell-mediated lytic
activity against virus-infected cells and certain tumors without requirement for activation (1). Such unactivated lymphocytes, also referred to as resting or naive NK cells, are
capable of destroying a relatively limited spectrum of tumor
cells (1, 4). Upon activation with lymphokines such as IL-2,
NK cells acquire broad anti-tumor lytic activity (lymphokineactivated killer cells, i.e., LAK cells). The mechanism(s) by
which naive NK cells recognize their target cells is not
completely understood. Interaction of cellular adhesion
molecules and recognition of specific target structure(s) have
been proposed as critical initial events in the cell-mediated
lytic process (1, 5). For example, it is well known that the
initiation of target cell lysis by CTL is due to interaction of
the MHC class I molecules (plus bound peptide) with the
TCR (6, 7). Analogous molecular structures that initiate
the lytic process between NK cells and tumor cells have not
been defined. Although MHC molecules may serve a regulatory function for NK cells (8), it is clear that their
presence on the surface of tumor cells is not required for cytolysis, because NK-susceptible cell lines do not express
MHC gene products (11, 12).
Lymphocytes. Human peripheral blood lymphocytes (HPBL)1 were isolated as described previously (13). In brief, blood obtained from healthy volunteers was diluted 1:1 with HBSS, layered on Ficoll-Hypaque (Pharmacia, Piscataway, NJ) and centrifuged at 200 g for 20 min. The mononuclear leukocyte fraction (Ficoll-Hypaque interface) was passed through nylon wool columns to obtain HPBL depleted of B cells and monocytes.
Isolation of Naive NK Cells and T Lymphocytes.
Naive human
NK cells are defined as freshly isolated CD3, CD5
, CD16+
that exhibit limited target cell specificity, i.e., lytic activity against
K562, but not A549 tumor cells. Activated NK cells, i.e., those
that are stimulated with lymphokines such as IL-2 (LAK), are able
to lyse NK-resistant tumor cells such as A549 (1, 4). Only those
preparations of HPBL that lacked LAK cell activity were used
for the isolation of naive NK cells and T lymphocytes. Isolation
of naive NK cells and T lymphocytes was performed as follows.
Freshly isolated HPBL were suspended in PBS containing 2% fetal bovine serum (FBS) at 20 × 106 cells/ml and incubated (4°C for
30 min) with anti-CD5, 20 µl/1 × 106 cells (Becton-Dickinson,
San Jose, CA). Anti-CD5 reactive lymphocytes were captured by
incubating the mixture with sheep anti-mouse IgG-coated magnetic beads, 30 µl/1 × 106 cells (Dynal Inc., Lake Success, NY).
The magnetic beads containing adherent CD5+ cells were washed,
suspended in RPMI supplemented with 15% FBS, and incubated
overnight at 37°C in 5% CO2. Subsequently, CD5-enriched lymphocytes (T lymphocytes) were separated from the magnetic
beads by vortexing 2 min and placing the mixture in a magnetic
field. The supernatant containing CD5
HPBL was incubated
with anti-CD3, 20 µl/106 cells (Becton-Dickinson, San Jose, CA)
at 4°C for 30 min. Anti-CD3-reactive lymphocytes were separated using magnetic beads as described above. The supernatant
was centrifuged to obtain cells that were depleted of CD3+ and
CD5+ lymphocytes (i.e., CD 16+ enriched naive NK cells). The
average phenotype of the T lymphocyte enriched fraction was
<3% CD16+, 50% CD4+, and 93% CD3+. These cells did not
exhibit NK or LAK cytolytic activity. The average phenotype of
the negatively selected naive NK cell enriched fraction was 85%
CD 16+, <3% CD3+. These cells exhibited increased NK cell
lytic activity (60% cytotoxicity at an effector to target E/T ratios
of 100:1) compared with unfractionated HPBL (32% cytotoxicity).
Binding of Tumor Plasma Membrane Proteins to Lymphocytes. Plasma membrane proteins of viable tumor cells were labeled with biotin as described previously (13). In brief, cells ( >99% viable by trypan blue dye exclusion) were washed three times with a solution containing 10 mM Hepes, 145 mM NaCl, 4 mM KCl, 11 mM glucose, pH 8.0 (buffer A) and then incubated in the same buffer with biotin-NHS (1 mM biotin-NHS, 10 × 106 cells/ml) for 1 h at 4°C. Cells were then washed three times with 20 vol of buffer A to remove unreacted biotin. The biotinylated cells were suspended in 250 mM sucrose with 10 mM Hepes (pH 8.0) and disrupted by N2 cavitation (1,000 psi) in a Parr's chamber. The resulting suspension was centrifuged at 200 g for 5 min to remove undisrupted cells and the supernatant was centrifuged at 30,000 g for 20 min to obtain a crude membrane fraction. Membranes were washed once with 10 mM sodium borate, 10 mM benzamidine, 1 mM EDTA, 1 mM iodoacetamide, 1 mM PMSF, pH 8.0 (buffer B), and incubated in the same solution with 1% Triton X-100 at 4°C overnight. Solubilized membrane proteins were obtained as the 30,000 g supernatant of the detergent treated membranes and dialyzed extensively against buffer B containing 0.05% Triton X-100, as described previously (13).
Biotin-labeled solubilized tumor plasma membrane proteins were reacted with freshly isolated naive NK cells and T lymphocytes at a ratio of 2:1 (on the basis of cell numbers) in RPMI1640 supplemented with 15% FBS at 4°C for 2 h. The reacted cells were washed twice with media and three times with buffer A. Finally, cells were solubilized in Laemmli sample buffer and the solubilized proteins were subjected to SDS-PAGE and Western blotting. Biotinylated tumor membrane proteins that bound to lymphocytes were identified with a streptavidin biotin detection system as described previously (13).Purification of Tumor Membrane Proteins. Membrane proteins were purified as described previously (13). Approximately 1011 cells of erythroleukemia cell line (K562) were washed three times with PBS and used to prepare plasma membrane proteins as described above. Solubilized membrane proteins were extensively dialyzed against buffer B and subjected to preparative SDS-PAGE. A vertical portion of the gel was sliced and stained with Coomassie blue to locate desired proteins. Protein bands of interest were cut from the gel, eluted, and further purified by SDS-PAGE. A portion of the purified protein was extensively dialyzed against 10 mM Tris-HCl, pH 8.0 and used for cytotoxicity inhibition studies or dialyzed against buffer B and labeled with biotin as described above for study of its binding properties to various lymphocyte subsets. A portion of the purified protein was also isolated on nitrocellulose paper after SDS-PAGE using the method described by Aebersold et al. (14) for the purpose of internal amino acid sequence analysis of the protein subsequent to in situ protease digestion (Harvard Microchemistry Laboratory, Cambridge, MA).
Immunoprecipitation of Surface-labeled Proteins. Immunoprecipitation of plasma membrane proteins was performed by initially incubating anti-p38.5 with anti-rabbit IgG coupled to CNBr- Sepharose 4B at 4°C for 90 min in PBS containing 1% BSA (13). The reacted beads were then washed three times with buffer and incubated at 4°C for 90 min with surface-biotinylated tumor plasma membrane proteins. Beads were then washed three times with PBS containing 1% BSA and two times with PBS alone, suspended in Laemmli SDS-PAGE sample buffer, and boiled for 10 min. The supernatant was subjected to SDS-PAGE and proteins transblotted to Immobilon-P membrane (Millipore Corp., Bedford, MA). Anti-p38.5 immunoprecipitated biotinylated tumor membrane proteins were identified on Western blots by reaction with streptavidin-biotin detection system.
Fluorescence-activated Flow Cytometry.
Approximately, 10 6 cells
were washed three times with PBS containing 0.1% BSA (PBS-
BSA) and incubated with appropriately diluted anti-p38.5 at 4°C
for 30 min. Cells were then washed three times with PBS-BSA
and reacted with FITC-F(ab)2 goat anti-rabbit IgG for 30 min at
4°C. Finally, cells were washed three times with PBS-BSA, resuspended to a concentration of 106/ml, and analyzed on a FACSort® Flow Cytometer (Becton-Dickinson, San Jose , CA).
Lymphocyte-mediated Cytotoxicity.
Cytolysis of tumor cells by
lymphocytes was measured as described previously (13, 15). In
brief, 100 µl of 51Cr-labeled target cells (2 × 104 cells/ml) was
mixed with 100 µl of lymphocytes (2 × 106 cells/ml) to yield serial twofold dilutions of E/T cell ratios of 100:1 to 6:1. Cells were
incubated at 37°C for 3 h in 5% CO2. Incubation of target cells
without the effector cells and in media alone served as control for
spontaneous release of 51Cr, and wells without effector cells but
with 1% SDS provided the maximum amount of radioactivity
present. Cytolysis was calculated as percent cytotoxicity = 100 × ([CPMexp CPMspont.] / [CPMmax
CPMspont.]).
A tagged ligand-cell adsorption technique (13) was used to identify membrane proteins from a
susceptible tumor cell line that preferentially bind to NK
cells. Solubilized membrane proteins from surface biotinlabeled K562 cells were reacted with immunomagnetic bead-
enriched freshly isolated naive NK cells or T lymphocytes. Approximately 14 common bands were detected on Western blots of K562 plasma membrane proteins that bound to
both NK cells and T lymphocytes (Fig. 1). However, a single band of 38.5 kD (p38.5) was detected from NK cell adsorbed preparations, but was not observed in T lymphocyte
adsorbed preparations. This initial result suggested the possibility that p38.5 interacted with a receptor on the surface of naive NK cells that was not expressed on T lymphocytes. Accordingly, p38.5 was purified by preparative SDSPAGE to apparent homogeneity for additional studies. The
purified protein resolved as a single band of 38.5 kD molecular mass upon SDS-PAGE (Fig 2, lane A). p38.5 was
labeled with biotin for use in studying its binding property
to lymphocyte subsets (in a similar manner as that described for proteins from the crude membrane preparation). Biotinylated p38.5 bound extensively to the NK cell-enriched
fraction (Fig. 2, lane D) and not at all to the enriched T lymphocytes (Fig. 2, lane C). In this experiment, unfractionated cells bound a slight amount of p38.5 (Fig. 2, lane B),
which is consistent with the low percentage of NK cells in
peripheral blood lymphocyte specimens. These data demonstrate that SDS-PAGE-purified p38.5 retained its preferential binding property for naive NK cells.
Structural Characteristics of p38.5.
Initial attempts to obtain a partial amino acid sequence of the purified p38.5 were unsuccessful because the NH2 terminus of the molecule was blocked. Consequently, p38.5 was transferred onto nitrocellulose paper and subjected to in situ protease digestion (14). Amino acid sequence analysis of resultant peptides was performed at the Harvard Microchemistry Laboratory (Cambridge, MA). An 11-mer amino acid sequence of an internal peptide was obtained with a high degree of confidence (Table 1). Comparison of this sequence with known sequences of proteins in Gen Bank did not reveal significant homology. Treatment of p38.5 with sulfhydryl reducing agents (2-mercaptoethanol and dithiothreitol) or deglycosylating enzymes (O-glycosidase, N-acetylneuraminidase II and peptide-N-glycosidase F, deglycosylation kit; Glyko, Inc., Novato, CA) did not alter the molecular mass of the molecule, suggesting that the isolated protein is a monomer and nonglycosylated (Das, B., unpublished data).
|
Because p38.5 (derived from K562 cells) exhibited selective binding to naive NK cells, it was of interest to examine the expression of this molecule on the plasma membrane of NK-resistant cell lines as well as other NK-sensitive tumor cell lines. Affinity-purified antibody raised against the internal 11-mer synthetic peptide of p38.5 (anti-p38.5) was used for this purpose. Anti-p38.5 was reacted with three NK-sensitive and three NK-resistant tumor cell lines and analyzed separately by flow cytometry. As shown in Fig. 3 A, anti-p38.5 reacted with the surface of NK-sensitive tumor cell lines, K562 (erythroleukemia), Jurkat (T cell lymphoma), and Molt-4 (T cell leukemia). In contrast, anti-p38.5 did not bind to the three NK-resistant tumor cell lines A549 (lung adenocarcinoma), Raji (Burkitt lymphoma), and MDA-MB231 (breast carcinoma). Plasma membrane expression of p38.5 was also studied by anti-p38.5 immunoprecipitation of surface-labeled (biotin) membrane proteins of the above tumor cells. Western blot analysis of the immunoprecipitates showed the presence of a single 38.5-kD biotinylated protein from NK-sensitive cell lines, whereas biotinylated proteins were not observed when the NK-resistant cell lines were employed (Fig. 3 B). These results demonstrate an association between the expression of p38.5 on the surface of tumor cell lines and their susceptibility to NK cell-mediated lysis.
Cell Surface Expression of p38.5 on NK-resistant Variants of Jurkat and Molt-4 Cell Lines.
Though the expression of
p38.5 is associated with susceptibility of well-established
tumor cell lines to NK cell-mediated cytolysis, one must
interpret such data with caution as the cell lines are derived
from different tissues and are likely to have been transformed by different mechanisms. This issue could however be more readily addressed by examining the association of
NK susceptibility and p38.5 expression in variants derived
from wild-type tumor cells. After long-term culture of Jurkat and Molt-4 cell lines, we observed a substantial reduction in their susceptibility to NK cell-mediated cytotoxicity. These variant cell lines were then analyzed for surface
expression of p38.5 by anti-p38.5 immunoprecipitation. As
shown in Fig. 4, variant cell lines exhibited decreased susceptibility to lysis by NK cells and expressed significantly
lower amounts of p38.5 on their surfaces as compared with
the wild-type parental cell lines.
Effect of Purified p38.5 on NK Cell-mediated Cytolysis.
The preferential binding of p38.5 to NK cells suggested
that the molecule may be involved as a ligand in the cytolytic process mediated by naive NK cells. To examine
this possibility directly, a purified preparation of p38.5 was
incubated with human lymphocytes and then tested in a
standard cytotoxicity assay against 51Cr-labeled K562 target
cells. Incubation of lymphocytes with the purified p38.5
preparation inhibited naive NK activity in a concentrationdependent manner (Fig. 5). A K562 membrane protein of
40 kD, purified by the same procedure as p38.5, did not affect NK activity. These data provide direct evidence that
interaction of tumor surface protein p38.5 with its NK cell
receptor is necessary for cytotoxicity, because binding of
soluble ligand to the NK cells before contact with K562
cells inhibited their lysis.
Characterization of NK Cell Receptor of p38.5.
To delineate further the preferential interaction of p38.5 with NK
cells relative to T lymphocytes, we conducted investigations to identify an interactive molecular species on effector
cells. In direct binding assays, biotin-labeled purified p38.5
was reacted with either NK cell or T lymphocyte membrane proteins that had been previously immobilized on
Western blots. Labeled p38.5 was detected as a single band
at 70 kD on blots of NK cell membrane proteins, whereas
reactive bands were not detected on blots of T lymphocyte
membrane proteins (Fig. 6). This result suggests that a protein of 70 kD from naive NK cells binds cell surface p38.5
from NK cell-susceptible tumors and that the 70-kD protein is either not present or is present in substantially lower
amounts in T lymphocytes.
To establish the immunologic identity of the ligand bound
to the NK cell receptor, anti-p38.5 serum was used. A Western blot of peripheral blood lymphocyte membrane proteins was initially incubated with a solubilized crude extract
of K562 membrane proteins, washed, and then reacted
with anti-p38.5. The antiserum reacted with a band at 70 kD rather than at 38.5 kD (Fig. 7, compare lanes C and D
to lane A). Western blots of lymphocyte membrane proteins that were not preincubated with the tumor proteins
did not react with anti-p38.5 (Fig. 7, lane B). These results
suggest that the 38.5-kD surface molecule from K562 cells
that binds to the 70-kD naive NK cell protein contains the
11-mer peptide to which antibody was raised. We conclude that the 11-mer epitope of p38.5 is probably not directly involved in the receptor-ligand interaction. (Presumably, if the p38.5-70-kD interaction included the 11-mer
epitope of p38.5, antibody to the latter determinant could
not bind to the complex).
The following experiment was performed to confirm
that a 70-kD NK cell protein that specifically binds p38.5 is
localized on the plasma membrane. Solubilized membrane
proteins from surface-biotinylated naive NK cells were applied to a p38.5-Sepharose 4B column. After extensive washing with high salt buffer, specifically bound protein was extracted in denaturing buffer and subjected to SDS-PAGE
and Western blot analysis. A single discreet biotinylated band
of 70 kD from NK cells was detected on the blots suggesting that this p38.5 binding protein is located on the exterior of the plasma membrane (Fig. 8, lane B). One possibility is that the 70-kD, p38.5 binding protein is the inducible
form of heat shock protein (Hsp 70). We performed flow
cytometry studies using antibody to Hsp 70 (Stressgen Biotech. Corp., Victoria, BC Canada; Clone C92F3A-5) to determine surface expression of this moleule on T and NK
cells. Such experiments indicated that neither NK nor T cells
expressed Hsp70 on their plasma membranes in contrast with
K562 cells that served as a positive control (Norin, A.J., unpublished data).
Previous attempts during the past two decades to identify recognition structures exclusive to NK cell-tumor interaction have been unsuccessful (reviewed in reference 8), though important components on both NK cells and on tumor cells that contribute to cellular adhesion and regulation of the cytolytic process have been revealed (5, 8). However, these receptor-ligand interactions do not appear to be unique to NK cells, because they also occur between T lymphocytes and respective target cells (10). The limited number of NK cells available for biochemical studies (<5% in peripheral blood) has undoubtedly contributed to the difficulty in analysis of NK cell-specific receptor-ligand interactions. Many investigators have addressed this issue by the expansion of freshly isolated NK cells in vitro using a variety of culture systems employing growth factors and/or feeder cells (11, 12, 16). Such techniques have provided sufficient numbers of cells for immunologic and biochemical studies but at the expense of altering the phenotype of the freshly isolated NK cells from a naive state to an activated one, as demonstrated by induction of the Lag 3 protein (17). It is possible that activation results in decreased expression of naive NK cell recognition structure(s), thereby preventing their detection. In the present study, several strategies were used to overcome the difficulties of heterogeneity and the low frequency of NK cell in HPBL preparations. Only freshly isolated NK cells were used. Cells from individuals that demonstrated activity against NK-resistant LAK-sensitive tumor cell lines were not used, because the activated NK cells in these fresh preparations might mask the detection of naive NK cell-specific ligands. Finally, a tagged ligand-cell adsorption technique (13) was used with enriched NK cell preparations to enhance the likelihood of detection of tumor proteins that selectively bound to the naive killer cells.
Using the above approaches and additional biochemical and immunologic techniques, we demonstrate a novel 38.5- kD protein on the plasma membrane of certain tumor cell lines that preferentially reacts with a surface component of naive human NK cells. The interaction appears to be unique to NK cells, because T lymphocytes did not bind p38.5. However, binding studies have not been conducted with B cells, monocytes, or polymorphonuclear leukocytes. In further experiments, a 70-kD protein on the plasma membrane of NK cells was identified as a p38.5 binding molecule. Consistent with p38.5 cellular binding studies, a 70-kD receptor was not detected on T lymphocytes. Addition of purified p38.5 to fresh lymphocyte preparations before incubation with K562 target cells substantially decreased NK cell lytic activity. Preferential interaction of p38.5 with NK cells and its blocking activity in functional assays are properties consistent with a role in an early recognition event in NK cell-mediated tumor cytolysis.
Additional evidence of a role for p38.5 as a target ligand in naive NK cell-mediated cytotoxicity is provided by data demonstrating an association of the expression of this molecule and susceptibility to cytolysis of different tumor cell lines. Flow cytometry and immunoprecipitation studies (of surface labeled cells) revealed that p38.5 is expressed on NK susceptible targets such as K562, MOLT-4, and Jurkat, whereas this molecule was not detected on the plasma membrane of NK-resistant LAK-sensitive targets such as Raji, A549, and MDA-MB-231, suggesting that p38.5 is not involved in LAK-mediated cytotoxicity. The functional role of p38.5 in NK cell-mediated cytolysis was also demonstrated in studies of p38.5 loss variants. After long-term culture of wild-type, NK-sensitive Jurkat and Molt-4 cell lines, variants were isolated that exhibited decreased levels of p38.5 and reduced susceptibility to lysis by NK cells. This property was not due to a phenotypic alteration in the cells as a result of culture conditions, because resistant clones were obtained at limiting dilution (Norin, A.J., unpublished data). These studies clearly establish a strong association between the expression of p38.5 on the tumor plasma membrane and susceptibility to NK cell-mediated cytolysis.
Recent studies suggest that cytolytic activity is affected
by recognition of HLA class I polymorphisms on target cells
by NK cell receptors of the C-lectin family (CD94) (18) or
members of the immunoglobulin multi gene family (p50/
p58) (19). These receptors, which we note are detected
on both T cells as well as NK cells, may downregulate or
upregulate cytolytic activity (10, 20, 25) depending on the
subtype of receptor and/or the presence of cytosol associated clonotypic signaling molecules (18, 21). Inhibitory
receptors apparently interfere with proximal signaling events
such as Ca2+ flux, phospholipase C2 activity, and inhibition of specific tyrosine kinases activities (lck and ZAP-70),
whereas the activity of other tyrosine kinases may remain
unchanged (18, 26, 27). Experimental results in the current
study are likely not influenced by the above families of
molecules, because K562 cells do not express HLA class I
or class II molecules (11, 12).
Current concepts regarding the mechanism of lymphocytemediated cytolysis have focused on three alternative pathways: (a) Fas-FasL-induced apoptosis of tumor cells (28), (b) extracellular ATP-mediated osmotic lysis (13, 34), and (c) granule exocytosis of effector molecules such as perforin and granzymes (29, 37). Clearly, NK cell-induced cytolysis is not mediated by Fas-FasL interaction, because K562 tumor cells do not express Fas and naive NK cells do not express FasL (41; Das, B., unpublished data). Previous reports by us and others have suggested that NK cells use the ATP-osmotic lysis pathway (13, 35) and the granule exocytosis pathway (28, 33). Further studies are likely to reveal which lytic pathway is mediated by the interaction of tumor surface ligand p38.5 with NK cell plasma membrane proteins.
Address correspondence to Allen J. Norin, SUNY Health Science Center at Brooklyn, 450 Clarkson Avenue, Box 1197, Brooklyn, NY 11203.
Received for publication 31 October 1996 and in revised form 6 February 1997.
This work was supported, in part, by a research grant from the US Public Health Service (CA-47548).We thank Dr. W. Solomon (State University New York Health Science Center, Brooklyn, NY) for his suggestions and critical reading of the manuscript. We are grateful to Dr. S. Kamholz (SUNY Health Science Center, Brooklyn, NY) for his continuous encouragement and support. The expert word processing by S. Lamy is appreciated.
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