Study Of The Inhibition By Polymorphonuclear Leukocytes Of TNF-αrelease From Human Mononuclear Cells And Its Mechanism

M-a is a proinflammatory cytokine produced by mononuclear phagocytes,which plays a pivotal role in enhancing the host defense against infection. However,the poteniial toxic effect of TWh-a, when excessively produced by the host uponstimulation by pathogens, is an imPortaflt factor in the pathogenesis of septic shock.Previous investigation found that human po1ymorPhonuclear leukocy'tes (PMNs)suppressed the TNF-a release from phagocytic cell U937. The present study wasundertaken to further investigate the effect of human Pnss on the production ofM-ca by the human peripheral blood mononuc1ear cells (PBMCs) and to elucidateits tentatve mechanism.Human PMNs and PBMCs were isolated from the venous b1ood of hea1thydonors by dextran sedimentation aJnd density gradient centrifugation. After they werecocultured at the ratio of 2fl in the presence of 1ipopo1ysaccharide (LPS), theconcefltrdtion of TM-a in the supernatam was measured by enzyme-linkedimmunosorbent assay. The binding rate of monocytes with the fluoresceinisothiocyanate-labeled LPS (FITC-LPS) and the mean surface fluorescence intensityof monocy''tes were anaIyzed by flow cytometry. The results showed that fl .PBMCs secreted wr-a functionally on stimu1ation by LPS whi1e no TN'F-afrom PMNs was detected in the same condition. PMNs were caPable of inhibiting theTNF-a release in PBMCs (P<0.05). PMNs suppressed the TNF-a re1ease fromPBMCs by 45% on average when Pams and PBMCs cocu1tllred at the ratio of 2f l.This inhibitory effect was further confirmed to be specific, as erythfocytes did notdemonstrate similar inhibitory effect (P>0.05).2.ParafOrmaldehyde-fiXed Pams still demonstrated the same inhibition (P<0.05), which proved that the inhibition was dePendellt on cell-to-cell contact andsuggested that effector mo1ecu1es responsib1e fOr this effect existed on the cell surfaceof PMNs.3. Serine proteinase inhibitor eglin C significafitly suppressed the TN'F-aproduction by PBMCs (P<0.05). Eglin C did not restore the inhibitory effect of PMNs3on the wi-a release by PBMCs, but the inhibitory rate of Pwns was higher in theabsence of eglinC than th&t in the presence of eglin C (P<0.05).4. FITC-LPS and flow cytometry were used to determine if PMNs blocked thebinding of LPS to monocytes. ln the presence of PMNs, the binding rate of monocyteswith LPS and the mean surface fluorescence intensity of monocytes were not affectedcomPared with PBMCs alone (P>0.05). As incubation time increased, the binding ofLPS to monocytes also increased (P<0.05). Thus Puns did not block the binding ofLPS with PBMCs to exert the inhibitory effect.In summary the results showed that Prms suppressed the TNF-a re1ease byPBMCs via ce1l~to-cell interaction. In a cell-contact dependot manner, PMNs mayinterfere with the signal transduction pathWay through which LPS activates PBMCs,thus attenuating the response of PBMCs to LPS and downregUlating the TN'F-arelease.