Cytokine release pathway in mononuclear cells stimulated in vitro by dialysis membranes.
BACKGROUND
Among the possible variables associated with cytokine activation in hemodialysis, filter membrane has been reported to be a major factor and monocytes adherent to the membrane have been suggested as a possible source for cytokine synthesis.


METHODS
In order to exclude variables other than the direct cell-to-membrane interaction, suspensions of peripheral blood monocytes isolated from donors' blood were incubated for 180 min at 37 degrees C in Petri dishes coated with cuprophan (Cu) or polyacrylonitrile (AN69S) or polysulfone (PS). Total RNA was purified, reverse transcribed in cDNA and amplified by polymerase chain reaction (PCR) primed with specific oligomers for determining IL-1beta and TNF-alpha gene expression. For Western blot analysis, cell homogenates and supernatants were electrophoresed and transferred to a polyvinylidene difluoride membrane and the membrane was then incubated with polyclonal antibodies specific for the detection of IL-1beta and TNF-alpha.


RESULTS
Semi-quantitation of targets for PCR (using the technique of limiting dilutions and referring to actin and glyceraldehyde-3-phosphate dehydrogenase as noninducible molecule) revealed a comparable increase at 180 min (p < 0.05) in IL-1beta and TNF-alpha mRNA in monocytes incubated with polyacrylonitrile, PS and Cu membranes. In 2 of 10 experiments pro-IL-1beta was detected in monocytes interacting with Cu, PS and AN69S membrane. However, the extent of extracellular release of mature protein was greater for Cu.


CONCLUSIONS
Some dissociation between transcriptional and translational events was detected in experiments using donors' blood in vitro. More specifically, synthetic membranes (both polyacrylonitrile and PS) were found to be as active as Cu in inducing IL-1beta and TNF-alpha mRNA expression, but less effective in promoting the extracellular release of proinflammatory cytokine products.
Menegatti
Elisa
E
Cattedre e Scuola di Specializzazione di Patologia Clinica e Dipartimento di Medicina e Oncologia Sperimentale, Università di Torino, Torino, Italia.
Rossi
Daniela
D
Chiara
Marica
M
Alpa
Mirella
M
Sena
Luigi M
LM
Roccatello
Dario
D
D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D007375:Interleukin-1 / Q000096:biosynthesis* / Q000235:genetics; D007688:Kidneys, Artificial; D008567:Membranes, Artificial; D009000:Monocytes / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics
D007375:Interleukin-1; D008567:Membranes, Artificial; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha