Search

Thrombomodulin (TM) is an essential cofactor for the physiologic activation of the anticoagulant protein C by thrombin. We have observed that the expression of TM mRNA in response to retinoic acid was markedly increased in human U937 monoblast-like cells, and human MEG01 megakaryocyte-like cells, but not in human umbilical vein cells, murine hemangioma cells, human K562 erythroblast-like cells, and murine HSD fibroblast-like cells. TM activity in U937 cells and MEG01 cells was not detectable in untreated cells, but developed rapidly after treatment with retinoic acid. In endothelial cells there was minimal change in TM activity in response to retinoic acid treatment. We have isolated clones for the genes for murine and human TM and have identified potential retinoic acid response elements in the 5'-flanking region of the human gene. In U937 cells the increase in mRNA levels was associated with increased transcription, and transient transfection studies with reporter plasmids demonstrate functional retinoic acid response elements present in the 5'-flanking region of the gene. Deletion of, and mutations introduced into, the potential retinoic acid response element confirm the functional response in transient transfections.

A monoclonal antibody, designated 3.2.3, which recognizes a novel Mr 60,000 disulfide-linked lytic triggering structure present on rat large granular lymphocytes and natural killer (NK) cells was recently described (W. H. Chambers et al., J. Exp. Med., 169: 1373-1389, 1989). The present study describes the use of 3.2.3 to identify the in situ tissue distribution of large granular lymphocytes/NK cells in different organs from normal and tumor-bearing F344 rats. Frozen tissue sections were prepared and stained with monoclonal antibody 3.2.3 using an avidinbiotin immunoperoxidase technique. 3.2.3+ NK cells were easily identified using this technique, and quantitative analysis of various tissues of normal rats demonstrated that (a) in the spleen, most NK cells were located, sometimes as aggregates, in the red pulp (12.4% of total nucleated cells in that organ compartment) with relatively few noted in the white pulp (0.2-2.3%); (b) in the liver, 3.2.3+ cells were rare, sparsely distributed, and located primarily in the sinusoids (1.2%); (c) in the lungs, 3.2.3+ cells were located in the interstitium (3.7%); (d) in the thymus, 3.2.3+ cells were found primarily in the medulla (1.8%) adjacent to the cortex but not in the cortex itself (0.2%); (e) in the lymph node, most 3.2.3+ cells were contained in the paracortex (6.9%); and finally (f) in the small bowel, 3.2.3+ cells were present in the lamina propria (8.6%) and as aggregates in the interfollicular zone of Peyer's patches (0.7%). To study the distribution of 3.2.3+ NK cells in developing tumor metastases, we induced liver metastases by intrasplenic injection of MADB106 mammary adenocarcinoma cells and prepared frozen tissue sections of the liver 10-14 days later. We found that the frequency of 3.2.3+ cells in the developing liver metastases was 3-6 times higher than in the surrounding normal liver tissue. Moreover, the frequency of 3.2.3+ NK cells was equivalent to the frequency of tumor-infiltrating CD5+ T-cells identified in the same tumor lesions. This suggests specific infiltration of 3.2.3+ NK cells in early developing metastatic lesions. These results indicate that monoclonal antibody 3.2.3 will be valuable in analyzing the involvement of NK cells in various pathological states.