Inhibition of pinocytosis in rat embryo fibroblasts treated with monensin.

Rat embryo fibroblasts cultured in the presence of monensin exhibited an inhibited uptake of horseradish peroxidase. The inhibition was detected after 3 h, after which time the cells became increasingly vacuolated; the concentration of monensin required to inhibit pinocytosis (0.4 microM for half-maximum inhibition at 18 h) was similar to that found by others to inhibit secretion. Both the exchange of 5'-nucleotidase between the membranes of cytoplasmic organelles and the cell surface and the internalization of anti-5'-nucleotidase bound to the cell surface were inhibited by approximately 90% in monensin-treated cells. The effects of monensin were reversible: cells cultured first with monensin, and then in fresh medium, exhibited control levels of horseradish peroxidase uptake, exchange of 5'-nucleotidase, and internalization of anti-5'-nucleotidase bound to the cell surface. After monensin treatment, the median density of both galactosyl transferase and 5'-nucleotidase increased from 1.128 to 1.148, and the median density of both N-acetyl-beta-glucosaminidase and horseradish peroxidase taken up by endocytosis decreased from 1.194 to 1.160. The results indicate that monensin is a reversible inhibitor of pinocytosis and, presumably, therefore, of membrane recycling. They suggest that the inhibition of membrane recycling occurs at a step other than the fusion of pinocytic vesicles with lysosomes and is perhaps a consequence of an effect of the ionophore on the Golgi complex.

Evidence for involvement of B lymphocytes in the surveillance of lung metastasis in the rat.

These studies examined the composition of lymphocytes within the lung after the introduction of tumor cells that metastasize to the lung in rats. i.v. delivery of MADB106 tumor cells into syngeneic Fischer 344 rats caused dose- and time-dependent development of lung tumors, with surface metastases evident 7 days after injection and markedly increased 11 days after injection. The total number of lymphocytes recovered from the lung was increased 11 days after injection but not 7 days after injection. When lymphocytes from the lung, spleen, and blood were subjected to fluorescence-activated cell sorting analysis, the most conspicuous change was an increase in the percentage of CD45RA+ cells (i.e., B lymphocytes in the rat) in the lung, with no changes seen in the percentage of natural killer (NKR-P1+), CD4+, or CD8+ cells in the lung. Analysis of the time course showed that B lymphocytes increased in the lung soon after i.v. tumor injection, with an initial peak seen 6 h after injection. Rapid influx of B lymphocytes into lung after i.v. tumor cell injection was also observed in another syngeneic tumor model, i.e., after injection of CC531 cells into WAG rats. To determine whether the influx of B lymphocytes into the lung might participate in tumor surveillance, a high dose of antibody (100 microg) to rat B lymphocytes was given to immunoneutralize these cells; this produced an increase in lung tumors in both models. Finally, Fischer 344 rats were given a s.c. injection of MADB106 tumor cells that made them resistant to lung tumors when given a later i.v. injection of these tumor cells. These animals were found to have an elevated level of B lymphocytes residing in the lung associated with the resistance to lung tumor. These findings suggest that early responses of B lymphocytes are important in protection against tumor development in two rat models of cancer.

Immunocytochemical localization of multicatalytic protease complex (proteasome) during generation of murine IL-2-activated natural killer (A-NK) cells.

Murine IL-2-activated, adherent natural killer (A-NK) cells produce proteolytic activities (including a chymase and a tryptase) which appear to be components of the proteasome/multicatalytic proteinase complex and appear to represent the mouse homologues of the rat A-NK cell A-NKP 2 and A-NKP 1 protease components. The chymase is readily inhibited by Z-Gly-Gly-Phe chloromethylketone (Z-GGF) and to a lesser extent by N-tosyl-L-phenylalanyl-chloromethylketone (TPCK). In addition, this activity is inhibited by 3,4-dichloroisocoumarin (DCI), a suicide inhibitor for both chymotryptic and tryptic proteolytic enzymes. Protease inhibitors reduced A-NK cell-mediated cytotoxicity against P815 target cells, most prominently with inhibitors of chymotryptic and tryptic enzymes, including TPCK, DCI and Z-GGF. A polyclonal rabbit antibody raised against rat liver proteasome immunofluorescently labeled the cytoplasm of 4-day-cultured murine A-NK cells, multiple granules in 5 to 6-day cultures and large intracytoplasmic pools in cells cultured longer. Ultrastructurally this shift in labeling over time corresponded to an immunogold redistribution to non-membrane delineated mucoid masses. A minor nuclear labeling was noted at all time points, whereas the cisternae of the endoplasmic reticulum or Golgi region were negative. It is concluded that murine A-NK cells synthesize and accumulate proteasome in large intracytoplasmic pools, non-delineated by membranes which can occupy up to 80% of the A-NK cellular volume. The potential function of the proteasome produced by A-NK cells including cell-mediated cytotoxicity against tumor target cells remains to be elucidated.

Expression of neutrophil collagenase (MMP-8) in Jurkat T leukemia cells and its role in invasion.

BACKGROUND: Previous studies have shown that MMP-8, the neutrophil collagenase, was expressed in neutrophils, chondrocytes and rheumatoid synovial fibroblasts. MATERIALS AND METHODS: We used semi-quantitative RT-PCR analysis, Western blotting, and immunofluorescence assays to determine the expression of MMP-8 in Jurkat T cells. RESULTS: We have determined the expression of MMP-8 from Jurkat cells and the down-regulation of its expression by genistein, a principal soy isoflavone. Genistein inhibited the invasion of Jurkat cells through a model basement membrane by about 75%, similar to the inhibition by BB-94, a synthetic MMP inhibitor. Genistein also down-regulated the expression of MMP-13, but slightly up-regulated the expression of TIMP-1 and TIMP-2. CONCLUSIONS: Our findings documented for the first time the expression of the neutrophil collagenase by a T-cell line. We also determined the inhibition of Jurkat cell invasion by genistein, which was in part mediated through the regulation of the expression of MMPs and TIMPs.

uPA and uPAR contribute to NK cell invasion through the extracellular matrix.

BACKGROUND: The urokinase plasminogen activator (uPA) system has been implicated in cellular invasiveness of tumor cells and immune cells. Herein we provide evidence for the production by natural killer (NK) cells of both uPA and its receptor (uPAR). MATERIALS AND METHODS: Western blot analysis, RTPCR, casein/plasminogen zymography, and fluorescence microscopy were employed to detect uPA and uPAR on NK cells. NK cell invasiveness was examined using Matrigel invasion assays. RESULTS: NK cell uPA appeared at its characteristic molecular weights, is enzymatically active in casein/plasminogen zymography, and is recognized by monoclonal antibodies. uPAR was detected by RTPCR and fluorescence microscopy. Matrigel invasion assays demonstrated an active role of uPA in NK cell invasion. CONCLUSION: The uPA system contributes to extracellular matrix (ECM) degradation by NK cells, which may be essential for NK cell accumulation into metastases, and may be prerequisite for their killing of tumor cells following NK cell adoptive transfer.

Augmentation of IL-2 activated natural killer cell adoptive immunotherapy with cyclophosphamide.

UNLABELLED: We have previously documented that adoptively transferred, IL-2 activated natural killer (A-NK) cells can accumulate within established pulmonary metastases. Since we have observed that increases in the accumulation of A-NK cells do not always lead to increases in therapeutic efficacy, we examined the ability of cyclophosphamide to enhance the therapeutic efficacy of A-NK cells. Animals with established B16 melanoma or Lewis lung carcinoma pulmonary metastases were treated with A-NK cell adoptive immunotherapy, either alone or following treatment with chemotherapeutic doses of cyclophosphamide. Adoptive immunotherapy studies with A-NK cells yielded at most a 30% reduction in the number of pulmonary metastases; however, cyclophosphamide (300 mg/kg) consistently reduced the size of metastatic colonies. In contrast, the combination therapy of A-NK cells plus cyclophosphamide was more effective than adoptive immunotherapy alone. In addition, polyethylene glycol IL-2 is superior to IL-2 in these studies. CONCLUSIONS: Our studies suggest that chemoimmunotherapy with A-NK cells plus cyclophosphamide may be more effective than adoptive immunotherapy alone since it results in the reduction in both the size and number of pulmonary metastases.

Enhanced anti-metastatic efficacy of IL-2 activated NK (A-NK) cells with novel benzothiazoles.

We have previously shown that A-NK cells when locoregionally administered accumulate within established cancer metastases and establish direct contact with both tumor cells and microvascular endothelial cells. Nevertheless, the accumulation of adoptively transferred A-NK cells into established cancer metastases is not sufficient for therapeutic efficacy in the B16 melanoma model. We have therefore attempted to enhance the anti-metastatic therapeutic efficacy of adoptively transferred A-NK cells with standard anticancer chemotherapeutic agents. We have found that chemoimmunotherapy with A-NK cells plus cyclophosphamide to be more effective than A-NK cell adoptive immunotherapy alone. We have now built on these findings, by examining the ability of novel biologic response modifiers (low molecular weight benzothiazole compounds) to augment adoptive immunotherapy with A-NK cells. Two compounds KB-R4107 (4-methoxy-2-(4-t-butylphenyl)benzothiazole) and KB-R4250 (4-methoxy-2-(4-trifluoromethylphenyl)benzothiazole) enhanced reduction of B16 melanoma pulmonary metastases mediated by A-NK cell adoptive immunotherapy. Both compounds were administered for 5 days prior to administration of A-NK cells at 100 mg/kg p.o. All experimental groups initially contained at least 7 animals and were examined for tumor burden on day 10. With B16 melanoma cells administered on day 0 and A-NK cells administered on Day 4, KB-R4107 and KB-R4250 yielded on average a 64% and 52% reduction in metastatic burden, respectively compared to an average 17% reduction using A-NK cells alone. In contrast these compounds did not diminish metastatic burden when administered alone. KB-R4107 and KB-R4250 are therefore low molecular weight, heterocyclic, biological response modifiers which can augment the anti-metastatic therapeutic effect of adoptively transferred A-NK cells.

Cytolytic activities of IL-2 activated NK cells from MMTV/v-Ha-ras transgenic oncomice during tumor progression.

IL-2 activated natural killer (A-NK) cells have the capacity to infiltrate metastatic tumors and lyse tumor cells. Nevertheless, adoptive immunotherapy with lymphokine-activated killer cells has been only modestly effective in the clinic and has not routinely provided long-term survival in patients with established cancer metastases. This may indicate the need for more carefully investigating the role of effector cells of the immune response, including A-NK cells, in models of tumor progression. Herein we describe the use of the MMTV/v-Ha-ras transgenic mouse model as a system for exploring the role of NK cells during tumor progression. We have examined the lytic capacity of A-NK cells generated from tumor-free and tumor-bearing transgenic oncomice against standard A-NK cell targets (YAC-1 and P815) in addition to tumor cells isolated from these animals. A-NK cells generated from mice without obvious tumor burden show higher lytic activity than A-NK cells generated from mice with evident tumors, i.e., those at a more advanced stage of tumor progression. Only long term (8-day) cultures of late passage A-NK cells generated from tumor-bearing mice showed significant increases in lytic activity over those generated from tumor-free mice. These results suggest that experimental protocols using transgenic oncomice at various stages of tumor growth may constitute a novel model for testing the role of A-NK cells for their capacity to interfere with cancer progression.

Flavone acetic acid enhances accumulation of IL-2 activated NK cells within established metastases.

Flavone acetic acid, an agent which has been implicated in both tumor vasculature collapse and NK cell activations, has been tested recently as a potential anti-cancer chemotherapeutic agent. We have tested this agent in combination with adoptive immunotherapy using IL-2 activated natural killer (A-NK) cells in a metastatic B16 melanoma model in C57BL/6 mice. By using rhodamine-labeled A-NK cells we have been able to quantitate both the number of A-NK cells that localize within each tumor section and the percentage of the tumor area occupied by A-NK cells. This has been accomplished using an image analysis system. Flavone acetic acid (200 mg/kg, i.p.) given one day prior to the injection of A-NK cells increased the area of the tumor occupied by A-NK cells and the area of individual A-NK cells approximately 2-fold; however, it did not appear to increase the number of A-NK cells per tumor cross-section. Nevertheless, this increase did not lead to any significant change in the therapeutic efficacy of A-NK cell adoptive immunotherapy. Our studies therefore suggest that mere enhancement of A-NK cell recruitment into tumor metastases does not necessarily translate into enhanced metastatic therapeutic efficacy. Moreover, this method may be a useful tool for pre-screening of compounds which enhance the accumulation of adoptively transferred cells into tumor metastases prior to in vivo screening for therapeutic efficacy.

Proteasome inhibitor and lymphocyte function: partial inhibition of cell-mediated cytotoxicity and implication that the lymphocyte proteasome may contain multiple chymotryptic domains.

The multicatalytic proteinase complex or proteasome possesses at least 4 distinct proteolytic activities. We have previously reported that the chymotrypsin-like activity of the rat natural killer cell proteasome may play a role in natural killer (NK) cell-mediated cytotoxicity or IL-2 activated NK (A-NK) cell-mediated cytotoxicity. Using a series of novel, Cephalon, Inc, synthetic proteasome inhibitors (CEP-1508, CEP-1612 and CEP-3117) which have been reported to be specific for the chymotrypsin-like activity of the proteasome, we have further investigated the possible role of the proteasome, with emphasis on the chymotryptic activity components, in cell-mediated cytotoxicity. We now report that these compounds can inhibit the rat NK proteasome in a dose dependent manner. Nevertheless, there is only a 50% inhibition of A-NK cell-mediated cytotoxicity. These results confirm and extend our previous results that the proteasome contributes, at least in part, to cell-mediated cytotoxicity. However, as anticipated, since multiple molecular pathways contribute to cell-mediated cytotoxicity, the proteasome contributes only partially to NK cell-mediated cytolytic reactivity. The exact role of the proteasome in NK cell-mediated killing, and whether single or multiple chymotryptic domains function directly or indirectly, remains to be fully determined.

Cooperation of urokinase plasminogen activator and matrix metalloproteinases in NK cell invasion.

We have previously investigated the role of the urokinase plasminogen activator (uPA) system in NK cell invasion. We have also studied NK cell-derived matrix metalloproteinases (MMPs) in extracellular matrix (ECM) degradation. We now report that both enzyme systems cooperate in NK cell invasion. Zymographic analyses detected uPA in RNK-16 cell conditioned media (CM) with the same molecular weights as the uPA we have previously shown to be associated with the rat NK cell urokinase plasminogen activator receptor. The combination of aprotinin, an inhibitor of plasmin, and Batimastat (BB94), an inhibitor of MMPs, in Matrigel invasion assays showed a more potent inhibitory effect on NK cell invasion than either inhibitor alone. Finally, a down regulation of uPA mRNA was noted following RNK-16 stimulation with collagen IV, fibronectin, and laminin.

Expression of matrix metalloproteinases and their inhibitors by rat NK cells: inhibition of their expression by genistein.

In this study, we describe rat NK cell-derived MMPs including membrane-type MMPs (MT-MMPs) and tissue inhibitors of MMP (TIMPs). RT-PCR analysis from cDNA of rat A-NK cells revealed mRNA for MMP-2, MMP-9, MMP-7, MMP-10, MMP-11, MMP-13, MT1-MMP, MT2-MMP, TIMP-1, and TIMP-2. The RNK-16 cells expressed mRNA for MMP-7, MMP-10, MMP-11, MT1-MMP, MT2-MMP, TIMP-1, and TIMP-2, in addition to MMP-3 and MMP-13. Western blot analysis confirmed proteins for MT1-MMP and MT2-MMP in RNK-16 cells. TIMP-1 in rat A-NK cells was present at molecular mass of 34-kDa protein which may represent a highly glycosylated form. Genistein, a natural isoflavone found in soybeans, inhibited proliferation of RNK-16 cells in dosage dependent manner. In addition, it down-regulated the expression of MMP-13, MT1-MMP, TIMP-1 and TIMP-2. Moreover, genistein greatly impaired the ability of RNK-16 cells to invade through a model basement membrane. This effect might be mediated by the observed down-regulation of MMP-13 and MT1-MMP.

Neuroendocrine modulation of tumor metastases. I. Effect of adrenalectomy on B16 melanoma metastases.

There is increasing evidence to suggest a link between the neuroendocrine system and the immune system. Since it is well known that the immune response influences the establishment, progression or elimination of malignancy, we have examined the effects of adrenalectomy on B16 melanoma pulmonary tumor metastases and immune function in C57BL/6 mice to investigate the role of adrenal corticosteroids. Adrenalectomized mice were injected with 10(5) B16 melanoma cells on day 0. On day 9 the mice were sacrificed and the number of lung colonies counted. Adrenalectomized animals had a greater than 3 to 4-fold increase in the number of metastases as compared with sham operated animals. Steroid replacement therapy using dexamethasone delivered at 1 microgram/hr did not lead to any reduction in tumor metastases in adrenalectomized animals. These studies indicate that normal levels of adrenal steroids may influence the ability of tumor cells to colonize target organs and/or the ability of the immune system to mount an effective response.

Zymographic analysis of extracellular, released and cell-associated proteases of rat interleukin 2-activated natural killer (A-NK) cells.

The biochemical properties of heretofore uncharacterized extracellular proteases of rat interleukin-2-activated natural killer (A-NK) cells were investigated. Following p-aminobenzamidine- and D-phenylalanine-agarose affinity chromatography both trypsin- and chymotrypsin-like protease activities were found. Zymography revealed "trypsin-like" activities, with apparent molecular weights of approximately 42 kDa to 20.5 kDa, and "chymotrypsin-like" activities of approximately 27 kDa, to 22 kDa. These proteases might contribute to diverse NK cell functions, and the characteristics of these proteolytic activities are compared with those of granzymes of lytic granules and the proteasome of A-NK cells.

Interleukin-2 (IL-2) activated natural killer (A-NK) cells: binding to microvascular endothelial cells and BRM enhancement of cytolytic activity.

A syngeneic in vitro system was developed for investigation of the binding of purified murine interleukin-2 (IL-2) activated natural killer (A-NK) cells to murine microvascular endothelial cells. This system is potentially useful as a model to investigate biochemical and molecular events underlying the binding of A-NK cells to microvascular endothelial cells within metastases in vivo that we have previously noted using electron microscopy. A-NK/endothelial cell binding in the syngeneic system was compared with the binding of allogeneic A-NK cells to the endothelial cells. Among test agents used to pretreat the A-NK cells for modulation of binding, lipopolysaccharide (LPS) most consistently enhanced binding. Additionally, test agents were also used in concurrent assays of A-NK cytolytic activity versus YAC-1 and P815 tumor cell targets. Polyinosinic: polycytidylic acid (poly IC), and gamma interferon (gamma IFN) were among the test agents which enhanced A-NK cell cytolytic activity.

Matrix metalloproteinases of human NK cells.

We have previously reported that MMP-2 and MMP-9 are present in rat A-NK cells, and have recently documented that additional MMPs are present in rodent A-NK cells. To our knowledge only proMMP-9 has previously been reported for human NK and A-NK cells. Herein, we report for the first time the presence of MMP-2 and MT1-MMP in human NK cells. The importance of these enzymes for the migration of A-NK cells into tumor metastases is of great potential relevance. MMPs may be rate limiting in A-NK cells, following their adoptive transfer, to traverse basement membrane and accumulate within established cancer metastases, a likely pre-requisite to their cytolytic function. Human NK cells express and produce MMP-2, MMP-9, MT1-MMP and the inhibitor TIMP-1. Moreover, human A-NK cells degrade the extracellular matrix equivalent (Matrigel) in a seemingly IL-2 dependent manner. It is therefore likely that A-NK cell MMPs play crucial roles in contributing to A-NK cell localisation and positioning the cells in vivo to allow for triggering their cytolytic potential.

A novel drug delivery system using IL-2 activated NK cells and Zyn-linked doxorubicin.

Adoptively transferred IL-2 activated NK (A-NK) cells selectively accumulate within tumor metastases which recommends them as vehicles for locoregional drug delivery. Zyn-Linkers are membrane-binding lipophilic dyes which can be coupled by a variety of conjugation chemistries to therapeutic agents. We have previously demonstrated that A-NK cells labeled with PKH26 are able to accumulate within established B16 melanoma pulmonary metastases by 16 h at a concentration of over 600 cells/mm2 of tumor tissue (Basse et al. J. Exp. Med. 174: 479 1991). Zyn-205 is a prodrug in which doxorubicin is attached to a similar Zyn-Linker through an acid-sensitive bond. We have optimized the ex vivo labeling conditions and found that a 10 min incubation with 25 microM Zyn-205 results in the uptake of over 10(8) drug molecules per cell with no effect on either cell viability or cytolytic activity up to 24 h after labeling. Given these parameters, the amount of drug which may be carried to and concentrated in metastatic lesions represents a local concentration of approximately 15 microM. In addition, A-NK cells carrying Zyn-Linked doxorubicin at an equivalent dose of 25 micrograms/kg was therapeutically comparable to a systemic dose of 8 mg/kg (320x more) in the 3LL model of experimental metastasis. These data indicate that A-NK cells bearing Zyn-Linked chemotherapeutic agents represent a unique and feasible method to target chemotherapeutic agents to cancer metastases and that therapeutic doses can be attained without unwanted systemic exposure.

Differential locomotion of long- and short-term IL-2-activated murine natural killer cells in a model matrix environment.

Tumour infiltration by activated natural killer (A-NK) cells is a pre-requisite for tumour eradication by adoptive NK cell transfer. Extravasated A-NK cells do not always succeed in reaching the crucial target cell conjugation. Therefore, we wished to study A-NK cell locomotion and interactions with melanoma cells in a matrix environment (Matrigel) by electron, confocal and fluorescence microscopy. Two distinct patterns of A-NK cell-mediated matrix disintegration were revealed during incubation of tumour cells and A-NK cells in Matrigel: (1) A-NK cells pre-cultured for 5 days altered the homogeneous texture of the Matrigel, an initial microporous appearance became a loose filamentous meshwork by 24 h. Matrix degrading protease inhibitors could not fully prevent this, but could delay the process; and (2) A-NK cells pre-cultured for 6 days or more, instead formed large excavations in the Matrigel leaving the remaining matrix less affected compared to the effects by the younger A-NK cells. By histochemical staining with Cupromeronic Blue, the excavations were shown to contain proteoglycan material. Protease inhibitors had no discernable effect on the development of the excavations. The conspicuous capacity of A-NK cells to disintegrate extracellular matrix and the formation of large excavations seems only partially to depend on matrix-degrading proteases. Formation of extracellular proteoglycan material is suggested to facilitate A-NK cell locomotion within a matrix environment.

2-Keto-4-hydroxyglutarate aldolase: purification and characterization of the homogeneous enzyme from bovine kidney.

2-Keto-4-hydroxyglutarate aldolase, which catalyzes the reversible cleavage of 2-keto-4-hydroxyglutarate, yielding pyruvate plus glyoxylate, has been purified from extracts of bovine kidney to apparent homogeneity as judged by polyacrylamide gel electrophoresis, gel filtration chromatography, sucrose density gradient centrifugation, and meniscus depletion sedimentation equilibrium experiments. The enzyme from this source has a native and a subunit mass of 144 and 36 kDa, respectively; the pH-activity optimum is 8.8. Rather than being stimulated, aldolase activity is inhibited to varying degrees by added divalent metal ions, whereas a number of metal ion-chelating agents have no effect. An absolute requirement for added thiol compounds could not be shown, but 2-mercaptoethanol enhances activity 2-fold, and added Hg2+ as well as p-mercuribenzoate or dithiodipyridine markedly inhibit catalysis. Incubation of the enzyme with either pyruvate or glyoxylate in the presence of NaBH4 causes extensive loss of aldolase activity concomitant with stable binding of approximately 1.0-1.5 mol of 14C-labeled substrate/mol of enzyme. The circular dichroism spectrum for native aldolase is characteristic of an alpha-helix; incubation of the enzyme with glyoxylate has no effect on this spectrum, but it is considerably altered by pyruvate. Bovine kidney aldolase shows no stereospecificity in catalyzing the aldol cleavage of the two optical isomers of 2-keto-4-hydroxyglutarate, and although it also catalyzes the beta-decarboxylation of oxalacetate, its decarboxylase/aldolase activity ratio is lower than that seen with the pure enzyme from either bovine liver or Escherichia coli.

Non-granular proteolytic enzymes of rat interleukin-2-activated natural killer cells. III. Enhancement of A-NKP 1, 2, and 3 proteolytic activities (cleaving after Arg, Phe and Pro) in response to interleukin-2.

Rat IL-2-activated natural killer (A-NK) cells contain a number of cytosolic proteases (A-NKP 1-3), two of which (A-NKP 2 and A-NKP 3) have been implicated in cytolytic function. The 3.2.3 monoclonal antibody directed against the NKRP-1 signal transduction molecule, a selective marker of rat NK cells, was used to select a highly purified population of rat NK cells. When the 3.2.3-positive cells were cultured in the presence of IL-2, a time-dependent increase in the specific activity of A-NKP 1, A-NKP 2 and A-NKP 3 was observed which paralleled the observed increase in lytic activity against both NK-sensitive (YAC-1) and NK-resistant (P815) targets. A transient rise was observed in the specific activity of N alpha-benzyloxycarbonyl-L-lysine thiobenzyl esterase (BLT esterase, i.e. granzyme A). These results lend further support to the hypothesis that A-NKP 2, a component of the rat A-NK cell multicatalytic proteinase complex/proteasome, and A-NKP 3 contribute to the cytolytic function of A-NK cells. Moreover, these results further suggest that proteolytic enzymes that contribute to cell-mediated cytotoxicity are not restricted to the granzymes or localized only in the lytic granules of effector cells.