Matrix metalloproteinases in natural killer cells. Expression of MMPs, IL-2 activation and killer cell interactions with Matrigel® and model tumours

Abstract

Introduction: IL-2 activated natural killer (A-NK) cells can recognize malignant cells and exert tumouricidal activities by multiple mechanisms that require close contact with the target cells. Upon adoptive transfer, some A-NK cells accumulate in tumours by migrating from the vascular bed to a position inside the malignant tissue, forming close contacts with target cells. This process requires that naturally occurring barriers are passed, i.e. the endothelial lining with the underlying basement membrane and the adjacent extracellular matrix, ECM. Thus, tumour infiltration by A-NK cells is believed to depend in part on proteolytic matrix degradation, in analogy with malignant cell invasion during formation of metastasis. This focuses on expression, release, and possible functions of ECM degrading proteases in NK cells. Specific aims: to explore the repertoire of matrix metalloproteinases (MMPs) in NK/A-NK cells of various species; to examine whether MMPs are utilised in the process of basement membrane transmigration; to describe the morphology of A-NK cell infiltration in ECM and in model tumours as related to proteolytic mechanismsMaterials & Methods: Gelatin zymography, Western blotting and reverse transcriptase (RT-) PCR were used to detect and identify the various MMPs in NK/A-NK cell membranes and culture supernatants. Transmigration assays using Matrigel (reconstituted basement membrane ECM) covered invasion-chambers. A-NK-to-matrix and A-NK-to-tumour cell interactions were investigated by means of light and fluorescence microscopy, and electron microscopy. Model B16 melanoma tumours formed in vitro and growing intraperitoneally were used.Results: Expression and release of multiple MMPs were demonstrated in NK cells from different species. In rat MMP-2 and MMP-9 were found, in mice MMP-2, MMP-9, MMP-11, MMP-13, MT1-MMP, MT2-MMP and the specific inhibitors TIMP-1 and TIMP-2. In human NK cells MMP-2, MMP-8, MMP-9 and MT1-MMP as well as TIMP-1 were detected. A-NK cell transmigration through a basement membrane material was inhibited to about 50% (rat cells) or 90% (murine cells) by the MMP inhibitor batimastat (BB94). Human A-NK cell migrated across the membranes in a seemingly IL-2-dependent manner. Microscopy demonstrated that infiltrating A-NK cells disintegrate B16 tumour cell aggregates in vitro, which was not related to cytolysis. Further, an A-NK cell age-dependent alteration of Matrigel was detected. Short-term cultured cells caused a general digestion of the matrix material which contrasted to the effect of older A-NK cells that created large excavations around the cells with little alteration on the remaining Matrigel. A-NK cells infiltrated, but did not disintegrate, intraperitoneal tumours.Conclusions: NK/A-NK cells express and release a broad array of MMPs including their inhibitors. Further, MMPs are involved in A-NK cell basement membrane transmigration. Disintegration of tumour cell aggregates in vitro and digestion of Matrigel are interpreted as proteolytic effects. The additional matrix-dilating effect may be explained as a release of proteoglycans. The present studies confirm that intravital tumours largely resist derangement of the tumour structure in spite of A-NK cell infiltration.