Induction of tissue factor (TF) expression on monocytes and endothelial cells is central to the development of septic coagulopathy. Serum concentrations of endotoxin in septic patients who develop disseminated intravascular coagulation (DIC) do not, however, reach the levels that would directly stimulate TF expression on either monocytes or endothelium. We show, using an in vitro coculture system, that the interaction of monocytes with endothelium induces the expression of significant levels of TF. Unstimulated cocultures of monocytes (2 x 10(4)/well) and endothelial cells (2 x 10(4)/well) produced 35.3 +/- 8.5 mU of PCA/well, representing a 5-fold increase over the combined PCA of each cell type cultured alone (7.1 +/- 1.5 mU, n = 6, P < 0.001). Significant enhancement was also found in the presence of low concentrations of LPS. Induction of TF protein was confirmed by Western blotting. Fixation of monocytes with paraformaldehyde completely abolished TF induction in cocultures, whereas fixation of endothelium had no effect, suggesting that TF induction occurred in monocytes rather than endothelial cells. Induction of TF in cocultures could be further augmented by preincubating the endothelial cells with IFN-gamma. When endothelium was prestimulated with 500 U/ml IFN-gamma there was 142 +/- 11% increase over unstimulated cocultures (n = 5, P < 0.01). TF induction was inhibited by 32 +/- 6% in the presence of anti-ICAM-1 mAb (n = 5, P < 0.01). Our results suggest that monocyte interactions with vascular endothelium, regulated by inflammatory cytokines, and mediated by adhesive ligand binding, leads to the induction of functional monocyte TF protein, which may be responsible for the initiation of DIC in sepsis.

There is now good evidence that tumour necrosis factor [TNF] stimulates DNA synthesis of B-chronic lymphocytic leukaemia (B-CLL) cells. The malignant clone produces TNF, and addition of exogenous TNF up-regulates the TNF mRNA in B-CLL cells. Interleukin-6 (rIL-6) may also be important in this growth loop. We studied the interaction of TNF and IL-6 in the regulation of DNA synthesis (3H-TdR uptake), cytokine release and cell survival in CLL cells in vitro. Addition of TNF (100 U/ml over 5 days) enhanced DNA synthesis from 718 +/- 284 (mean cpm +/- SE) to 2730 +/- 545 compared to cells cultured in medium alone (n = 16, p < 0.01). TNF-alpha induced DNA synthesis was inhibited in all cases studied by the addition of anti-TNF monoclonal antibody (5 micrograms/ml) to cell cultures. Spontaneous IL-6 protein release was enhanced in the presence of TNF (100 U/ml and 250 U/ml) by CLL cells at 48 hours of culture 143.6% and 172% (p < 0.05, n = 6). At 120 hours of culture, the increase was 323% and 412.5% (4 of 7 cases) of the control respectively. IL-6 (100 U/ml or greater) increased spontaneous DNA synthesis (3H-TdR uptake) but, in the presence of high concentrations of TNF-alpha, inhibited TNF induced DNA synthesis in a dose dependent manner. Cell survival was reduced in the presence of anti-IL-6 mAb, while IL-6 was able to protect CLL cells from spontaneous apoptosis. These results suggest that IL-6 in an autocrine manner may inhibit DNA synthesis but prolongs survival in CLL cells. Increased serum IL-6 levels were detected in 27 of 50 cases of CLL, the mean level being significantly higher in Rai Stage III and IV cases compared to Rai Stage O-II cases.

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