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.

Selective IgA deficiency in children and adults with systemic lupus erythematosus.

The objective of this study was to determine the frequency and clinical characteristics of selective IgA deficiency (SIgAD) in children and adults with systemic lupus erythematosus (SLE), and evaluate potential differences in presentation and course of the SLE. IgA deficiency was defined as a serum IgA concentration < or =0.01 mg/mL determined on two sera by radial diffusion. SLE was classified by the 1982 criteria of the American College of Rheumatology. Seventy-seven children with SLE followed prospectively for > or =20 years and 152 adults surveyed during a one-year period were assayed for serum IgA levels. Disease characteristics were compared among the deficient patients and the IgA-normal patients. Twelve patients with SIgAD were identified: 1) Juvenile(J)-SLE: four children with juvenile onset (< or =18 years) and four others encountered as adults; and 2) Adult(A)-SLE: four patients with adult onset. No significant differences were found in clinical presentation or course except for a possible increase in recurrent infections and the observation that there were only two African-Americans. Five patients had received blood transfusions with no reactions; three of these patients had serum anti-IgA antibodies. One pediatric patient developed low levels of IgA (

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.

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.

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.

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.

2B4 stimulation of YT cells induces natural killer cell cytolytic function and invasiveness.

2B4 is a surface molecule found on all human natural killer (NK) cells, a subset of CD8+ T cells, monocytes and basophils. It was originally identified on mouse NK cells and the subset of T cells that mediate non-major histocompatibility complex (MHC)-restricted killing. Recently,9 we have cloned the human homologue of 2B4 (h2B4) and found h2B4 to also mediate non-MHC-restricted cytotoxicity. In this study, we examine h2B4 in regulating various functions of NK cells using a human NK cell line YT, with monoclonal antibody (mAb) C1.7, an antibody that specifically recognizes h2B4. Ligation of surface 2B4 with mAb C1.7 increases YT's ability to destroy tumour cells. In the presence of mAb C1.7, the production of interferon-gamma (IFN-gamma) by YT cells is greatly enhanced. Engagement of surface 2B4 by mAb C1.7 downregulates the expression of h2B4 at the cell surface as well as the expression of h2B4 mRNA. Also, signalling through h2B4 causes the increased expression of matrix metalloproteinase-2, a member of the matrix degrading proteinase family. Thus, in addition to modulating cytolytic function and cytokine production of NK cells, activation through surface 2B4 may play a role in upregulating the machinery for degradation of extracellular matrices to promote invasion of the tumour by NK cells.

Secreted and membrane-associated matrix metalloproteinases of IL-2-activated NK cells and their inhibitors.

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.

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.

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.

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.

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.

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.

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.

Matrix metalloproteinases produced by rat IL-2-activated NK cells.

We have previously documented that adoptively transferred IL-2-activated NK (A-NK) cells can accumulate within cancer metastases. Electron microscopic studies of pulmonary metastases have revealed that adoptively transferred A-NK cells that accumulate within metastases bind to endothelial cells and are able to traverse basement membranes. We have now extended these morphologic studies. We report that rat A-NK cells produce two matrix metalloproteinases: MMP-2 and MMP-9, as determined by SDS-PAGE gelatin zymography. These activities are inhibited following incubation with BB-94 (batimastat), a specific inhibitor of matrix metalloproteinases but not with 3,4-dichloroisocoumarin, an inhibitor of neutral serine proteases. The identity of MMP-2 was confirmed by Western blots using a polyclonal Ab against human MMP-2, whereas reverse transcriptase-PCR analysis of mRNA extracts of A-NK cells has confirmed the presence of MMP-9. In addition, we report for the first time that A-NK cells can migrate through a model basement membrane-like extracellular matrix. Moreover, the ability of A-NK cells to migrate through this model basement membrane was partially inhibited by BB-94; however, BB-94 has no effect on A-NK cell-mediated cytotoxicity, suggesting that matrix metalloproteinases do not contribute to cytolytic function of A-NK cells. In sum, our studies show that A-NK cells employ BB-94-inhibitable matrix metalloproteinases to degrade extracellular matrices. This suggests that matrix metalloproteinases may play a role in the accumulation of A-NK cells within cancer metastases.

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.