Tumour necrosis factor-alpha (TNF alpha) is up-regulated by cross-linking of major histocompatibility complex (MHC) class II [human leucocyte antigen (HLA)-DR] antigens on monocytes. This is done by a bacterial superantigen or anti-HLA-DR monoclonal antibody (MAb). We have previously shown that HLA-DR cross-linking results in inhibition of haematopoiesis and apoptosis. TNF alpha acts as a negative regulator of haematopoiesis. Here we investigated whether HLA-DR-mediated inhibition of haematopoiesis involved TNF alpha and TNF alpha-dependent secondary signals. Anti-HLA-DR MAb H81.9 up-regulated TNF alpha, as well as transforming growth factor beta, interleukin (IL)-1beta and IL-6 in human marrow cells at the ribonucleic acid (RNA) and protein level. The effect on TNF alpha was investigated further. Up-regulation was blocked by herbimycin A, consistent with a tyrosine kinase-dependent mechanism. Up-regulation was also blunted by the soluble TNF-receptor fusion protein TNFR:Fc, suggesting an autocrine amplification loop. Following TNF alpha up-regulation, there was increased expression of Fas (CD95) and Fas-ligand (Fas-L). Up-regulation of Fas and Fas-L was blocked by TNFR:Fc. Furthermore, MAb H81.9-induced apoptosis was prevented by anti-TNF alpha MAb and by the soluble Fas receptor, Fas-Ig, providing further evidence that the TNF effect was mediated via Fas. At the transcriptional level, cross-linking of HLA-DR by MAb H81.9 affects nuclear localization of NFkappaB, which is involved in the transcription of TNF alpha. NFkappaB activity is modified by changes in cellular redox potential, and we have shown that H81.9 affects redox potential as determined by the generation of nitric oxide. These data show that HLA-DR-initiated signals are able to trigger a cascade of negative regulators of haematopoiesis. This model provides an opportunity to dissect signalling pathways that may be involved in the development of spontaneous marrow failure, and to devise interventions aimed at protecting haematopoiesis.