The present study demonstrates that with time in culture blood monocytes (MO) lose their ability to express procoagulant activity (PCA) and secrete tumor necrosis factor-alpha (TNF alpha) in culture medium in response to lipopolysaccharide (LPS) stimulation. Thus, upon 10 micrograms/ml LPS stimulation for 4 hours 2-day-old MO produced lower levels of PCA and TNF alpha than fresh MO. The decrease in responsiveness was not caused by cell death, since in the case of TNF alpha it was fully reversible by interferon-gamma (IFN-gamma). Compared with cells pre-incubated in medium alone, the responsiveness of MO pre-incubated in LPS was further decreased. Thus, in MO LPS pre-incubation was followed by an LPS refractory state. It was expected that the decrease in responsiveness induced by cultivation in medium alone was mediated by LPS contamination of culture medium. However, as we were unable to prevent this decrease by neutralizing LPS contamination of the culture medium with polymyxin B, the loss in LPS-induced activities of cultured MO is likely to be mediated by culture conditions other than LPS contamination. Taken together the present data demonstrate that LPS-dependent as well as LPS-independent pathways of MO desensitization to LPS exist.

SUMMARYWe have investigated TNF-a secretory response of peripheral blood mononuclear eells (PBMC) from 13 uraemic patients undergoing regular haemodiaiysis with cuprophane membrane (CM). Sixteen healthy subjects and five uraemic patients under conservative therapy were also studied as controls. Cells ofhacmodialy.sis patients exhibited increa.sed TNF-a release//irf/rrnn the absence of activating siimuli other than culture conditions, as compared with normal and uraemic controls. In contrast to normal cells, this spontaneous secretion of TNF-i Irom dialysis PBMC could not be significantly reduced by addition of polymyxin B to culture medium, thus indicating its mdependence of trace amount of lipopoiysaccharide (LPS) present in the medium as contaminant. Furlhcrmorc. predialysis PBMC were considerably more sensitive to stimulation with 10'pg/ml of LPS under in vitro culture conditions than normal and uraemic controls. To elucidate a role of direct contact with CM in stimulation of TNF'-j release from monoeytes, PBMC were cultured on CM in vitro. Contact with CM stimulated TNF-::( secretion from PBMC above the level ofcells cultured on tis,sue culture plastic. This response persisted with time in culture in contrast to a transient LPS-induced TNF-x release. Furthermore. PBMC stimulated by contact with CM for 2 days did not lose the capacity to secrete TNF-a in response to a subsequent LPS stimulation, while a 2-day treatment ofceils with LPS was followed by LPS refractory state. Therefore, direct conuici with CM induces in PBMC a long-lasting TNF-2 response which is not down-regutated by the acquisition ofrefractorincss in a manner similar to that which occurs in the case of LPS stimulation. These in vitro findings provide a possible explanation ofthe observalion ihat predialysis PBMC exhibit elevated TNF'-a secretory capacity.