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Secreted and Membrane-Associated Matrix Metalloproteinases of IL-2-Activated NK Cells and Their Inhibitors¹

Myoung H. Kim^*, Richard P. Kitson^*, Per Albertsson^,, Ulf Nannmark, Per H. Basse^§, Peter J. K. Kuppen^¶, Marianne E. Hokland^|| and Ronald H. Goldfarb^2,*

^* Department of Molecular Biology and Immunology, University of North Texas Health Science Center at Fort Worth and Institute for Cancer Research, Fort Worth, TX 76107; Departments of Anatomy and Cell Biology and Oncology, University of Göteborg, Göteborg, Sweden; ^§ University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213; ^¶ Department of Surgery, University of Leiden Medical Center, Leiden, The Netherlands; and ^|| Institute of Medical Microbiology, University of Aarhus, Denmark

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have previously documented that rat IL-2-activated NK (A-NK) cells produce matrix metalloproteinase-2 (MMP-2) and MMP-9. In this study, we describe mouse A-NK cell-derived MMPs, including MT-MMPs, and also TIMPs. RT-PCR analysis from cDNA of mouse A-NK cells revealed mRNA for MMP-2, MMP-9, MMP-11, MMP-13, MT1-MMP, MT2-MMP, TIMP-1, and TIMP-2. MMP-2 and MMP-9 expression was confirmed by gelatin zymography. Moreover, we report for the first time that MT-MMPs are expressed by NK cells, i.e., large granular lymphocytes as determined by both RT-PCR and Western blots. TIMP-1 expression was detected as a 29-kDa protein in Western blots. It is intriguing that TIMP-2 protein from A-NK cells was also detected as a 29-kDa protein, which is clearly different from the previously reported molecular mass of 21 kDa in mouse and human cells. In addition, inhibition of MMPs by BB-94, a selective inhibitor of MMP, significantly inhibited the ability of mouse A-NK cells to migrate through Matrigel, a model basement membrane. Taken together, these findings suggest that A-NK cells may therefore use multiple MMPs in various cellular functions, including degradation of various extracellular matrix molecules as they extravasate from blood vessels and accumulate within cancer metastases following their adoptive transfer.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The matrix metalloproteinases (MMPs)³ are members of a family of at least 21 Zn²⁺-dependent endopeptidases, of which 16 are soluble, secreted enzymes, while the other 5 are membrane bound (1, 2, 3, 4). The expression of most MMPs is highly regulated by several mechanisms: at mRNA level transcriptionally by cytokines, hormones, and growth factors, and at protein level by proteolytic activation of latent enzymes and inhibition of active enzymes by endogenous inhibitors (3, 5, 6, 7, 8). They play important roles in many normal biological processes, including postpartum uterine involution, wound healing, and angiogenesis as well as in pathological processes, including arthritis, emphysema, and cancer metastasis (3). The main characteristic of MMPs is the degradation of the extracellular matrix of basement membranes, thus enabling cells to invade into tissues. MMPs are secreted as proenzymes and subsequently activated by proteolytic cleavage. MMP activity is regulated by the naturally occurring inhibitors such as -macroglobulins and the tissue inhibitors of MMPs (TIMPs) (3, 7). There are four members of the TIMP family determined to date (9). Among them, TIMP-1 and TIMP-2 are most well characterized as inhibitors of all known MMPs. TIMP-1 is a glycoprotein of 28.5 kDa (9, 10). TIMP-2 is an unglycosylated protein of 21 kDa with 39% homology to TIMP-1 (11). TIMP-2 from different species, i.e., mouse, rat, and bovine, have 97%, 98%, and 91% homology to human TIMP-2, respectively (11). TIMP-3 and TIMP-4 proteins have apparent molecular masses of 24 and 23 kDa, respectively (12, 13). The balance between the production and activation of latent enzymes, and inhibition of active enzymes seems to play a critical role determining the invasive potential of many solid tumors and inflammation caused by tissue-infiltrating immune effector cells (3).

MMPs in immune cells serve numerous specialized immunologic functions in addition to extracellular matrix degradation (5). T lymphocytes have been shown to produce MMP-9 constitutively, whereas MMP-2 expression is induced by IL-2 and VCAM-1-dependent adhesion to endothelial cells (14, 15). These MMPs contribute to the ability of T cells to migrate through model subendothelial basement membranes (14, 16). Neutrophils have been shown to store MMP-8 and MMP-9 intracellularly in specific granules and to secrete these enzymes upon stimulation (17, 18, 19). Macrophages express MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, and MT1-MMP as well as MMP-12 (20, 21, 22, 23, 24). These MMPs mediate secretion of Fas ligand and TNF- by cleavage of their membrane-bound forms, and generation of angiostatin from plasminogen by proteolytic cleavage (25, 26, 27, 28).

Cells of the immune system, including IL-2-activated NK (A-NK) cells as well as macrophages and cytolytic T cells, have generated much interest for their immunotherapeutic potential for established cancer (29, 30, 31, 32, 33, 34). Previous studies have shown that fluorescently labeled A-NK cells, following their adoptive transfer, can accumulate within established pulmonary and hepatic tumor metastases (35, 36). To reach the tumor cells, the circulating A-NK cells must adhere to endothelial cells and penetrate through the subendothelial extracellular matrix and actively migrate into the perivascular tissue space. Once in contact with target cells, A-NK cells exert their cytotoxic effects by secreting various proteolytic enzymes and cytolytic proteins, including granzymes and perforin (37). These observations have led to the hypothesis that A-NK cells might produce matrix-degrading enzymes. Indeed, rat A-NK cells have been shown to produce MMP-2 and MMP-9, and RNK-16 cells, a rat NK tumor cell line, to produce MMP-3 and MMP-13 (38, 39).

In this study, we examine an array of soluble and MT-MMPs and TIMPs produced by mouse A-NK cells and their potential role in the NK cell migration through a model basement membrane.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals

Nude mice were from Harlan Sprague-Dawley (Indianapolis, IN). The animals were housed in a specific pathogen-free animal facility.

Reagents and chemicals

Tissue culture medium and FBS were purchased from Life Technologies (Grand Island, NY). rIL-2 was a generous gift of Chiron (Emeryville, CA). All reagents were of the highest available commercial purity. Abs to MT1-MMP (clone 114-1F2) were obtained from Oncogene Research Products (Cambridge, MA). Abs to TIMP-1 (polyclonal Ab), TIMP-2 (clone 67-4H11), and MT2-MMP (clone 67-4H111) were obtained from Chemicon International (Temecular, CA).

Preparation of mouse A-NK cells

A-NK cells were prepared essentially as described previously (40). Spleens were harvested from nude mice, and splenocytes were incubated in nylon wool column preequilibrated with warm complete medium (CM; RPMI 1640 with 10% FBS, 55 µM 2-ME, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, 2 mM glutamine, 0.1 mM MEM nonessential amino acids, and 1 mM sodium pyruvate). Nonadherent cells were washed off by 2 column volumes of CM, counted, and cultured in CM containing 6000 IU/ml IL-2. On day 2, nonadherent cells were removed, and the flasks were gently washed with prewarmed (37°C) CM to remove cells not firmly attached to the plastic surface. New CM containing 6000 IU/ml IL-2 was added, and the adherent cells were cultured for additional days, as indicated in figure legends.

Concentration of conditioned medium

After 7 days in culture, A-NK cells from nude mice were placed in Opti-MEM (Life Technologies) supplemented with 6000 IU/ml human rIL-2 and 100 ng/ml of PMA (Sigma, St. Louis, MO) for an additional 24 h. HT-1080 and B16F1 mouse melanoma cells were grown to 80% confluence and stimulated with 100 ng/ml PMA for 24 h before collection of supernatants. PMA was added to enhance the production of MMPs (41, 42). Culture supernatants were collected, centrifuged to remove debris, and concentrated in Amicon (Beverly, MA) Centriprep concentrators up to 60-fold. Aliquots were frozen at -80°C. HT-1080 and B16F1 supernatants were prepared in the same manner and stored at -80°C.

Preparation of whole cell lysates from mouse A-NK cells

Day 7 nude mouse A-NK cells were stimulated with 100 ng/ml PMA and 6000 IU/ml IL-2 for 24 h before harvest. Then cells were washed once with PBS, and lysed in PBS containing 1% Triton X-100. Cell lysates were cleared by centrifugation at 4500 x g for 15 min. Resulting lysates were frozen at -80°C.

Gelatin zymography

SDS-PAGE gelatin zymography was performed as previously described with some modification (38). Briefly, after electrophoresis, the gel was washed at room temperature for 3 h in washing buffer (50 mM Tris-HCl, pH 7.5, 5 mM CaCl₂, 1 µM ZnCl₂, 2.5% Triton X-100) and then incubated for 20 h at 37°C in the same buffer containing only 1% Triton X-100. The gel was stained with a solution of 0.25% Coomassie brilliant blue R-250 and destained in 7% acetic acid and 10% methanol.

Western blot analysis

HT-1080, B16F1, and mouse A-NK media supernatants (for TIMP analysis) or mouse A-NK cell lysates (for MT-MMP analysis) were separated on 10% SDS polyacrylamide gels under reducing conditions and then transferred to a nitrocellulose membrane using a Mini trans-blot electrophoretic transfer cell (Bio-Rad, Hercules, CA). The membranes were blocked for 30 min at room temperature in T-PBS, pH 7.5 (PBS with 0.2% Tween-20) with 10% nonfat dry milk and 1% goat serum. After washing, the blot was incubated with primary Ab as indicated in figure legends for 1 h at room temperature. The blots were washed five times in T-PBS and incubated with peroxidase-coupled goat anti-mouse IgG (H+L) (for mAb detection; Pierce Chemical, Rockford, IL) or goat anti-rabbit IgG (for polyclonal Ab detection; Sigma), according to manufacturer’s instruction for 1 h at room temperature. After extensive washing, the bands were detected using SuperSignal CL-HRP Substrate System (Pierce Chemical). The resulting chemiluminescence was recorded on ECL Hyper film (Amersham Pharmacia Biotech, Piscataway, NJ).

Immunodepletion of TIMP-1

Mouse A-NK supernatant (1 ml) was incubated with 2 µg of polyclonal TIMP-1 Ab for 1 h on a rotating shaker at 4°C. Protein A beads (30 µl of settled bead volume; Sigma) were washed extensively with PBS, added to the Ab supernatant, and incubated for 30 min on a rotating shaker at 4°C to capture TIMP-1-Ab complexes. The beads were removed by centrifugation, and the resulting supernatants were concentrated in Microcon 10 microconcentrators (Amicon). Control A-NK supernatants (1 ml) were concentrated in the same manner. Concentrated supernatants were analyzed by Western blot, as described above and in figure legends.

RT-PCR

Total RNA was isolated from mouse A-NK cells using RNeasy columns (Qiagen, Chatsworth, CA). cDNA synthesis was performed using the RT-PCR kit from Stratagene (La Jolla, CA). For each cDNA synthesis, total RNA from 10⁶ cells were reverse transcribed using random hexamer or oligo(dT)₁₆ primer in a volume of 50 µl each, according to the protocol supplied by Stratagene. The two reactions were combined after heat inactivation of reverse transcriptase, and 2 µl of the cDNA was used for each PCR amplification in a buffer stated in figure legends. PCR primers are described in Table I.

The assay was performed as previously described (38). Briefly, day 6 A-NK cells prepared from nude mice were harvested, washed with RPMI 1640, and resuspended in Opti-MEM containing 6000 IU/ml of IL-2. A total of 500,000 cells in 0.2 ml were loaded into the top well, and 1 ml of Opti-MEM containing 6000 IU/ml of IL-2 was added to the bottom chamber. For chemotaxis, RANTES (10 ng/ml), MIP-1 (1 ng/ml), or IP-10 (1 ng/ml) was added to the bottom chamber. For inhibition studies, 10 µM BB-94 or 100 µM benzamidine was added to the top well. Determination of cell numbers invaded through Matrigel was done essentially as described previously (38) by labeling invaded cells with 1 µM calcein AM. All determinations were performed in triplicate.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
RT-PCR analysis of cDNAs from A-NK cells

Previously, we have detected MMP-2 and MMP-9 in rat IL-2-activated NK cells using zymography, RT-PCR, and Western blot. Because most of our in vivo tumor model and immunotherapy studies are done in mouse systems, it was of interest to see whether mouse A-NK cells also produced MMPs. To prepare a pure population of mouse A-NK cells, we used splenocytes from nude mice passed through nylon wool column to eliminate B cells and macrophages. Flow-cytometric analysis of day 8 cultured A-NK cells showed that greater than 99% of A-NK cells are negative for CD3, CD19, and macrophage marker staining (data not shown). Total RNA was prepared from day 8 A-NK cells and subjected to RT-PCR analysis using primers for known mouse MMPs and TIMPs (Table I). Results in Fig. 1 showed that mouse A-NK cells expressed two gelatinases, MMP-2 and MMP-9, similar to rat A-NK cells as well as MMP-11 (a stromelysin) and MMP-13 (an interstitial collagenase). In addition, we were able to detect the expression of two MT-MMPs, MT1- and MT2-MMP, and two TIMPs, TIMP-1 and TIMP-2.

View larger version (59K): [in this window] [in a new window]   FIGURE 1. RT-PCR analysis of MMPs and TIMPs of mouse A-NK cells. Total RNA was isolated from day 8 A-NK cells cultured from nude mouse splenocytes, as described in Materials and Methods. Primers used for PCR were described in Table I. PCR for MMP-2 and MMP-11, and MT1- and MT2-MMP done in buffer containing 60 mM Tris-HCl (pH 9), 15 mM (NH4)2SO4, 2 mM MgCl2. PCR for MMP-9, and MMP-13, and TIMP-1 and TIMP-2 were done in 20 mM Tris-HCl (pH 8.8), 2 mM MgSO4, 10 mM KCl, 10 mM (NH4)2SO4, 0.1% Triton X-100, and 0.1 mg/ml BSA.

To determine the enzymatic activities of MMPs identified in RT-PCR, we performed SDS-PAGE gelatin zymography. Supernatants isolated from mouse A-NK cells grown in serum-free medium were concentrated, as described in Materials and Methods, and analyzed by SDS-PAGE gelatin zymography in conjunction with HT-1080 supernatant concentrated in the same way (Fig. 2). The results indicated that there are three major gelatin-cleaving activities present in mouse A-NK cells, two of which correspond to the 72-kDa MMP-2 and the 92-kDa MMP-9 of HT-1080 cells. The third gelatinolytic band showed at higher molecular mass than MMP-9 in HT-1080 sample. It has been shown that a latent form of mouse MMP-9 has an apparent molecular mass at about 105 kDa that is larger than human MMP-9 (43). Our Western blot using mAb against human MMP-9 (clone 1-11c) also reacted with the protein in A-NK supernatant at the similar molecular mass, thus suggesting this higher molecular mass might be a latent form of MMP-9 (data not shown). A weak but detectable gelatinolytic band was also noticed at about 30 kDa. Incubation with BB-94 (also called Batimastat) (44), a broad inhibitor of MMPs, resulted in the complete ablation of enzymatic activity in all bands, while incubation with 3,4-dichloroisocoumarin, a general inhibitor of serine proteases that is known not to react with MMPs, had no effect on the pattern of bands observed in the zymograms of samples from either HT1080 or A-NK cells. These results confirmed the identity of these bands as MMPs.

View larger version (79K): [in this window] [in a new window]   FIGURE 2. Gelatin zymographic analysis of MMP-2 and MMP-9 from mouse A-NK cell supernatants. Day 7 mouse A-NK cells and HT-1080 cells were incubated in Opti-MEM containing 6000 IU/ml IL-2 (for A-NK cells only) and 100 ng/ml PMA for 24 h. Media supernatants were concentrated, as described in Materials and Methods. MMP-9 and MMP-2 indicate the 92- and 72-kDa gelatinolytic bands. The enzymatic activity marked with * may indicate the latent form of mouse MMP-9 (also see Results).

To confirm the presence of MT-MMPs and TIMPs, we performed Western blot analyses. For the Western blots of MT-MMPs, whole cell lysates were prepared from mouse A-NK cells, as described in Materials and Methods. Mouse mAb raised against human MT1-MMP recognized mouse proteins at an apparent molecular mass of 70 kDa (Fig. 3). Smaller bands at about 43 and 40 kDa may represent the proteolytically processed forms of MT1-MMP, as noted previously (45, 46). Mouse mAb to mouse MT2-MMP specifically recognized mouse MT2-MMP as a 73-kDa band (Fig. 3). Molecular masses of MT-MMPs in mouse A-NK samples are in good agreement with those reported previously (43, 47, 48).

View larger version (45K): [in this window] [in a new window]   FIGURE 3. Western blot analysis of MT1-MMP and MT2-MMP in mouse A-NK cells. Blots of SDS-PAGE gels of whole cell lysates were blocked and incubated with mouse mAb to human MT1-MMP (2 µg/ml, A), or to mouse MT2-MMP (1 µg/ml, B). Closed arrows indicate MT1-MMP. Bands indicated by * are thought to be proteolytically processed forms of MT1-MMP. An open arrow indicates MT2-MMP.

TIMP-1 protein in mouse A-NK cell culture supernatant was identified in Western blots using polyclonal Ab against human TIMP-1 (Fig. 4A). This Ab recognized human TIMP-1 in HT-1080 culture supernatants at 29 kDa. TIMP-1 in mouse A-NK supernatants also appeared as a major protein band of 29 kDa and as a minor protein band of about 36 kDa. It has been shown that the molecular mass of TIMP-1 protein can range from 30 to 34 kDa, depending on the degree of glycosylation (49). Mouse mAb against human TIMP-2 specifically recognized mouse TIMP-2 in B16F1, a mouse melanoma cell line, and human TIMP-2 in HT-1080 sample as a 24-kDa protein (Fig. 4B). This TIMP-2 Ab has been characterized previously to cross-react with TIMP-2 species from mouse, rat, guinea pig, and rabbit, but not to recognize TIMP-1 (11). However, this TIMP-2 mAb specifically recognized TIMP-2 in mouse A-NK supernatant as a 29-kDa protein. Because this molecular mass is very similar to TIMP-1 protein, we performed Western blot analysis after immunodepletion of TIMP-1 protein in A-NK cell supernatants to exclude any possibility that the 29-kDa band recognized by TIMP-2 mAb is TIMP-1 protein. TIMP-1 was depleted from mouse A-NK supernatant, as described in Materials and Methods, using rabbit polyclonal TIMP-1 Ab against C-terminal domain of human TIMP-1. TIMP-1 depletion was confirmed by Western blot (Fig. 5A). However, TIMP-2 mAb was still able to detect the 29-kDa band in TIMP-1-depleted supernatant, confirming that this 29-kDa band is not TIMP-1 and indeed TIMP-2 protein (Fig. 5B).

View larger version (39K): [in this window] [in a new window]   FIGURE 4. Western blot analysis of TIMP-1 and TIPM-2. Media supernatants from mouse A-NK, HT-1080, and B16F1 cells (for TIMP-2 only) were prepared as in Fig. 2, except HT-1080 supernatants used for TIMP-2 Western blot are not PMA stimulated. Blots were blocked and incubated with rabbit polyclonal Ab to human TIMP-1 (1 µg/ml, A) or to mouse mAb to human TIMP-2 (1 µg/ml, B). Molecular mass markers are as indicated Std.

View larger version (51K): [in this window] [in a new window]   FIGURE 5. Immunodepletion of TIMP-1. TIMP-1 protein was depleted from mouse A-NK supernatants, as described in Materials and Methods. Blot was done as in Fig. 4. A, Blot with polyclonal TIMP-1 Ab; B, blot with TIMP-2 mAb. Lanes: Control, no immunodepletion; -TIMP-1, immunodepletion of TIMP-1; Std, molecular mass markers. *, Indicates TIMP-1 protein, and indicates TIMP-2 protein.

We used a Matrigel invasion assay to determine the role of MMPs in the ability of mouse A-NK cells to invade through a model basement membrane. Day 6 nude mouse A-NK cells were placed in a Matrigel invasion chamber in the presence of BB-94 or benzamidine, an inhibitor of neutral serine proteases. As shown in Fig. 6, BB-94 at 10 µM inhibited about 90% of the invasion of mouse A-NK cells through Matrigel in a 24-h period; however, no significant inhibition of mouse A-NK invasion was observed by treatment with benzamidine. BB-94 has been shown to not affect the viability or cytolytic activity of A-NK cells (38). Various chemokines such as MIP-1, RANTES, and IP-10 at the concentration tested had no significant effect on migration of mouse A-NK cells. These results suggest that MMPs produced by A-NK cells are essential for the ability of these cells to degrade and migrate through the basement membrane.

View larger version (19K): [in this window] [in a new window]   FIGURE 6. Inhibition of mouse A-NK cell migration across a model basement membrane by BB-94, an inhibitor of MMPs. A-NK cells (500,000) in 0.2 ml were placed in the top well of Matrigel invasion chambers with either no addition, 10 µM BB-94, or 100 µM benzamidine. In addition, RANTES (10 ng/ml), MIP-1 (1 ng/ml), or IP-10 (1 ng/ml) was added to the bottom chamber of some wells. Results are expressed as cell numbers invaded through Matrigel chambers. Each bar represents the average of triplicate determinations (±SD).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we have expanded on our previous findings and have determined the presence of mRNAs for MMP-2, MMP-9, MMP-11, MMP-13, MT1-MMP, MT2-MMP, TIMP-1, and TIMP-2 in mouse A-NK cells by RT-PCR analysis. The activities of MMP-2 and MMP-9 in mouse A-NK cell culture supernatant were shown by gelatin zymography. A weak but detectable gelatinolytic band was present at 30 kDa. Among the MMPs determined by RT-PCR, mouse MMP-11, which has lost its propeptide and the majority of its C-terminal domain, has a molecular mass at 28 kDa. This form has demonstrated proteolytic activity against casein, fibronectin, laminin, and gelatin (50, 51). This suggests that the gelatinolytic band at the low molecular mass might be a truncated form of MMP-11.

Western blot analysis confirmed the presence of MT1-MMP, MT2-MMP, TIMP-1, and TIMP-2 in mouse A-NK cells. It is intriguing to notice that the apparent molecular mass of TIMP-2 from mouse A-NK cells is 29 kDa, which is different from those of human HT-1080 cells, or mouse B16F1 melanoma cells (see below). It has been shown that TIMP-2 cDNAs cloned from cultured colon 26 mouse carcinoma cells and from human heart tissue have 92% homology at the cDNA level and 97% homology at the protein level; these are also known to be nonglycosylated proteins (52). Moreover, TIMP-2 proteins purified from human and mouse serum showed similar molecular mass at 24 kDa in Western blot by the TIMP-2 mAb (11). Using the same mAb, we demonstrated that TIMP-2 proteins from HT-1080 and B16F1 mouse melanoma cells showed the same molecular mass of 24 kDa in Western blots. Thus, these results suggest that the difference in molecular mass may be A-NK cell specific. The cause for this discrepancy is under active investigation in our laboratory. The RT-PCR results showed that the PCR-amplified TIMP-2 band is the same size as that predicted from mouse fibroblast cDNA clone. However, it cannot be excluded that there might be additional sequences at the N or C terminus of the protein, or that there are some point mutations that might introduce glycosylation sites. The best known role of TIMPs is in inhibiting matrix degradation by MMPs. It is also noteworthy that at least TIMP-1 and TIMP-2 have other reported functions, such as erythroid-potentiating activity and growth-promoting activities on various cultured cell lines (53, 54, 55, 56). TIMP-2, but not TIMP-1, inhibits basic fibroblast factor-induced human microvascular endothelial cell proliferation in culture, which is unrelated to its metalloproteinase-inhibitory activity (57). It has been shown that separate domains of TIMP-1 are responsible for the MMP-inhibitory and erythroid-potentiating activities (58). Thus, it will be interesting to determine whether this 29-kDa TIMP-2 has any specific modifications or extra sequences, and its MMP inhibitory as well as other functions.

MMPs have also been reported in other cell types of the immune system. T cells, macrophages, and neutrophils all produce MMPs that mediate functional roles in immunity and inflammation (5). Although it has been reported that MT1-MMP was expressed from macrophages and human T cell lines (59, 60), this study is the first report documenting the presence of MT-MMPs in NK cells, i.e., large granular lymphocytes.

The extracellular matrix-degrading function of MT1-MMP is well established in tumor cell invasion and metastasis (61, 62). Acting as a plasma membrane-associated activator and receptor of MMP-2 and digesting extracellular matrix components by itself, MT1-MMP has the potential to localize matrix degradation to the vicinity of the tumor cell surface (63, 64, 65). While we have also examined the expression of other MMPs, including MMP-11 (a stromelysin), MMP-13 (an interstitial collagenase), and MT2-MMP, their exact role and scope in NK cell function are obscure and remain to be determined. Most of these MMPs have the capacity to degrade extracellular matrix and basement membrane proteins, while some of them have also been shown to mediate the proteolytic processing of other immune system regulatory molecules; in an in vitro assay, MMP-1, MMP-3, and MMP-7 were able to cleave a GST-TNF- fusion protein to 17-kDa protein that contains the same amino terminus as the mature form of TNF- (26). MMP-2 and MMP-9 also mediate this cleavage, but with less efficiency. MMP-3, MMP-7, MMP-9, and MMP-12 have been shown to cleave plasminogen to angiostatin in an in vitro system (27, 28). The release of Fas ligand from human CD4⁺ T cells or mouse T lymphoma cell lines stably transfected with human Fas ligand cDNA was inhibited by MMP-inhibitors, BB-94 and KB8112, but not by inhibitors of other proteases. This suggests that MMPs play a role in Fas ligand release (25). These observations merit the further investigation of A-NK cell MMPs for potential roles in regulation of angiogenesis and apoptosis. This may be of critical importance in the regulation of the accumulation of A-NK cells within tumor metastases, because such accumulation has been documented to be correlated in metastatic tumors with high numbers of microvessels (66).

Thus, expression of numerous MMPs and TIMPs from A-NK cells may contribute to important and multiple functions of A-NK cells, including extracellular matrix degradation, infiltration into tumor metastases, and perhaps secretion of cytokines, and potential modulation of cytolytic, apoptotic, and angiostatic effector pathways of NK cell function.

	Acknowledgments

 
We thank Chiron for providing us with the IL-2 used in these studies, and British Biotech Pharmaceuticals (Oxford, UK) for supplying us with BB-94. We also thank Yaming Xue for his expert technical assistance.

	Footnotes

 
¹ This work was supported in part by grants from the American Cancer Society (RPG-5-042-03-IM) and Bank One to R.H.G., Swedish Medical Council (K97-12RM-12142) and the Agnes and Gustav Backlund Foundation to P.A., the King Gustav V Jubilee Clinic Research Foundation to U.N. and P.A., and the Assar Gabrielsson Research Foundation to U.N. This study has been presented in preliminary form at the American Society for Biochemistry and Molecular Biology Meeting 1999, in San Francisco, CA (67 ).

² Address correspondence and reprint requests to Dr. Ronald H. Goldfarb, Department of Molecular Biology and Immunology, University of North Texas Health Science Center at Fort Worth and Institute for Cancer Research, 3500 Camp Bowie Blvd., Fort Worth, TX 76107-2699.

³ Abbreviations used in this paper: MMP, matrix metalloproteinase; A-NK, IL-2-activated NK; CM, complete medium; IP-10, IFN--inducible protein 10; MIP, macrophage-inflammatory protein; MT-MMP, membrane-type MMP; TIMP, tissue inhibitor of MMP.

Received for publication November 17, 1999. Accepted for publication March 17, 2000.

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