Cytokine and chemokine regulation of proMMP-9 and TIMP-1 production by human peripheral blood lymphocytes.

The production and activation of matrix-degrading proteinases such as the matrix metalloproteinases (MMP) by lymphocytes is likely to be an important factor in facilitating lymphocyte trafficking through the endothelial barrier and the extracellular matrix. Leukocyte infiltration into inflammatory sites occurs as a response to members of the chemokine superfamily and other inflammatory mediators. In the present study, highly purified leukocyte subpopulations were cultured with or without chemokines or cytokines, and their ability to express gelatinolytic MMPs and their inhibitors, the tissue inhibitors of metalloproteinase (TIMPs), was analyzed. In the absence of exogenous stimuli, purified CD4+ T lymphocytes produced similar quantities of proMMP-9 and elevated levels of TIMP-1 compared with PBMC, while purified CD8+ and CD3+ populations exhibited less MMP-9 and TIMP-1 activity. In comparison, CD56+ (NK) cells secreted barely detectable levels of proMMP-9 and TIMP-1. The secretion of proMMP-9 by PBMC and purified CD3+, CD4+, and CD8+ lymphocytes was selectively modulated by beta chemokines and proinflammatory cytokines. ProMMP-9 secretion by CD3+ and CD4+, but not by CD8+ T cells was augmented in response to TNF-alpha and IL-1, and down-regulated by IFN-gamma, while macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES (regulated upon activation and normally T cell expressed and secreted) (beta chemokines) up-regulated the secretion of proMMP-9 by all of the lymphocyte subsets tested. These results demonstrate that a number of proinflammatory cytokines and chemokines differentially regulate proMMP-9 secretion from purified CD4+ and CD8+ lymphocytes, while for purified CD3+ T cells (consisting of CD4+ and CD8+ cells in approximately a 2:1 ratio), a predominantly CD4+ lymphocyte response profile was demonstrated.

Alterations in endothelial cell proteinase and inhibitor polarized secretion following treatment with interleukin-1, phorbol ester, and human melanoma cell conditioned medium.

Polarized secretion of matrix metalloproteinases and plasminogen activators by monkey aortic endothelial cells was studied in vitro, using transwell inserts. The endothelial cells constitutively expressed matrix metalloproteinase-2, tissue inhibitors of metalloproteinases 1 and 2, urokinase, and tissue plasminogen activator, all with basal preference. Matrix metalloproteinase-9 activity was induced by phorbol 12-myristate 13-acetate (apical), interleukin-1 alpha (basal), and by conditioned medium from DX3 human melanoma cells (basal). The DX3 melanoma conditioned medium also stimulated basal secretion of matrix metalloproteinase-2, urokinase, tissue plasminogen activator, and tissue inhibitors of metalloproteinases. The rise in proteolytic activity in the basal direction was reflected by increased capacity to degrade subendothelial basement membrane type IV collagen, shown immunohistologically, using monkey kidney tissue sections and basement membrane deposited by endothelial cells into the transwell membrane. Thus, IL-1 alpha and DX3 melanoma conditioned medium can stimulate endothelial cells in vitro to concentrate secretion of proteinases spatially onto the underlying basement membrane. We suggest that the stimulation of endothelial cell proteinase activity by tumor cells may facilitate tumor cell extravasation.

Expression and characterization of human tissue inhibitor of metalloproteinases-1 in a baculovirus-insect cell system.

The baculovirus expression system was used to overexpress human tissue inhibitor of metalloproteinases-1 (TIMP-1). Approximately 5 mg of recombinant TIMP-1 was produced per 10(9) Sf9 insect cells infected with the recombinant baculovirus. The optimum time point for the production of biologically active rTIMP-1 was 20 hours postinfection. TIMP-1 activity was demonstrated by a soluble collagenase inhibition assay and reverse zymography. The baculovirus system has the advantage over previously described methods for generating human rTIMP-1 in that it generates large amounts of a fully glycosylated and active protein.

Tumor invasion, proteolysis, and angiogenesis.

In this review, some of the current literature on the regulation of proteolysis and angiogenesis during tumor invasion is discussed. Due to the critical location of brain tumors, an understanding of tumor cell interactions with the local environment is particularly relevant. Tissue breakdown during tumor invasion is associated with proteolytic activity, mediated by tumor cells, and surrounding host cells. This review covers two classes of proteinases and inhibitors that have commonly been associated with tumor invasion i.e., plasminogen activator (PA)/plasmin and matrix metalloproteinases (MMP) with special emphasis on the MMP inhibitors, TIMP-1 and TIMP-2. At different steps of the metastatic process, tumor cells interact with endothelial cells. Tumor cells also stimulate the formation of new vessels through the expression of specific angiogenic molecules. At least eight angiogenic molecules have been purified, sequenced and cloned, four of which are discussed here. Regulation of angiogenic activity has been the focus of intense studies recently, and a wide range of synthetic and natural angiogenesis inhibitors have been discovered. Targeting of angiogenic molecules and tumor vasculature may prove useful in future cancer therapeutic strategies.

Identification of the 72-kDa (MMP-2) and 92-kDa (MMP-9) gelatinase/type IV collagenase in preparations of laminin and Matrigel.

EDTA inhibitable type IV collagenolytic activity copurified with laminin preparations from the Engelbreth-Holm-Swarm (EHS) tumor. Several gelatinolytic and type IV collagenolytic matrix metalloproteinase (MMP) species were visualized in EHS laminin from three different sources by gelatin and type IV collagen substrate gel electrophoresis. Incubation with 4-aminophenylmercuric acetate and trypsin suggested that laminin contained both active and latent MMPs. EHS-derived reconstituted basement membrane, Matrigel, was found to possess an MMP profile identical to that of laminin. The presence of 72-kDa (MMP-2) and 92-kDa (MMP-9) gelatinases/type IV collagenases was demonstrated in laminin and Matrigel preparations by Western blot analysis. A rough quantitation of MMP-2 and MMP-9 in 30 micrograms of laminin and 100 micrograms of Matrigel was between 0.3 and 0.6 ng. The presence of these contaminants must be considered in experiments addressing the effects of EHS laminin or Matrigel on cell behavior and, in particular, stimulation of cellular proteolytic activity.

Non-random abnormalities of chromosomes 3, 6, and 8 associated with posterior uveal melanoma.

We present ten cases of posterior uveal melanoma which were karyotyped after short-term culture. One tumour had a normal chromosome complement. The remaining nine tumours were cytogenetically abnormal, with chromosomes 3, 6, 8, 11, and 13 most frequently involved. Abnormalities of chromosome 13 were seen in two cases, chromosome 11 in three cases, and chromosomes 3, 6, and 8 in five cases. Four tumours, all derived from the ciliary body, demonstrated monosomy 3 and i(8q), confirming the involvement of these aberrations with a subgroup of uveal melanomas arising from the ciliary body.

Gelatinolytic metalloproteinase secretion patterns in ocular melanoma.

Fifteen posterior uveal melanoma cell lines were analyzed qualitatively for gelatinolytic and caseinolytic proteinase activity after one to five in vitro passages. All 15 cell lines secreted a gelatinolytic metalloproteinase, with an apparent molecular weight of 72 kD, into protein-free culture media; nine of these secreted an additional gelatinolytic metalloproteinase with an apparent molecular weight of 92 kD. Neither species had the ability to degrade casein. This approach may provide insight into the mechanisms of tumor metastasis in uveal melanoma.

Cytogenetic findings in six posterior uveal melanomas: involvement of chromosomes 3, 6, and 8.

Six posterior uveal melanomas were karyotyped after short-term culture. One had a normal chromosome complement; the remaining five had limited chromosome changes. Involvement of chromosomes 1 and 6 was noted in two and four cases, respectively, and three ciliary body tumours demonstrated both monosomy 3 and i(8q).