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Phorbol myristate acetate (PMA)-activated neutrophils were found to destroy B lymphoblast turnout cells (Raji) as determined by the 51Cr release assay. The target cell lysis was prevented by azide, suggesting the involvement of the myeloperoxidase enzyme. Catalase and cytochrome c caused a marked impairment of the neutrophil-mediated cytolysis, whereas superoxide dismutase significantly enhanced the target cell destruction. These data indicate that hydrogen peroxide plays a key role in the target cell injury; superoxide anion appears to be devoid of direct cytotoxic activity, despite its requirement as a precursor of hydrogen peroxide. The target cell destruction required the presence of the iodide ion as oxidizable co-factor for the myeloperoxidase-hydrogen peroxide system. The chloride ion alone was uneffective. Inhibition of target cell metabolic pathways, involved in the cellular de fences against oxidative injury, by the anti-neoplastic agent 1,3-bis-(2-chloroethyl)-1-nitrosurca (BCNU) resulted in an increased neutrophil-mediated cytolysis. Under the experimental conditions employed, PMA-activated neutrophils incubated with BCNU-treated Raji cells became cytotoxic also in the presence of the chloride ion alone as myeloperoxidase co-factor. Our results suggest that Raji target cell destruction by PMA-aetivated neutrophils depends on the myeloperoxidase-hydrogen peroxide-halide system. The cytolyric event is influenced by target cells themselves, which should be regarded as an active component of the cytotoxic system, capable of interfering with the lytic mediators of the effector cells. [ABSTRACT FROM AUTHOR]

Neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) against Raji target cells and neutrophil degrantilation during the ADCC process were evaluated in the presence and in the absence of different agents able to interfere with the neutrophil release of granule components (anion channel blockers, colchicine, isoproterenol, dimethylxanthine, cAMP). When used at concentrations incapable of preventing the target cell recognition by neutrophils, the majority of these agents inhibited both the ADCC and the release of myeloperoxidase (MPO, primary granule marker) and lysozyme (LZM, primary and secondary granule marker). The inhibition of the ADCC correlated strictly with the inhibition of the MPO release. Thus, the results are consistent with the hypothesis that neutrophil primary granules play a major role in the cytolytic process. [ABSTRACT FROM AUTHOR]

The requirement for serine esterase activity in antibody-dependent cellular cytotoxicity (ADCC) in human neutrophils against Raji target cells has been investigated. The lysis was prevented when the serine esterase inhibitors TPCK and TLCK (chloromethyl-ketone derivatives of tosylamino acids) were introduced into the system. Moreover, neutrophils pretreated with TPCK or TLCK and washed were inhibited as well, via a process unaffected by the presence of adequate amounts of enzymatic substrates. This suggests that the inhibition mediated by TPCK and TLCK is independent of serine esterase blockade, therefore implying the inactivation of some other step crucial to the lysis. The addition of synthetic chymotrypsin substrates (tyrosine and phenylalanine esters) impaired the Raji cell lysis in a dose-related manner without altering the constitution of neutrophil-target conjugates. Trypsin ester substrates were ineffective. These results are in agreement with the involvement of a serine esterase activity with chymotrypsin-like specificity, which should participate in the lysis at a post-binding step. We conclude that neutrophil-mediated ADCC, as developed in our model system, needs the intervention of a serine esterase or esterases, like other systems of cell-mediated cytotoxicity. [ABSTRACT FROM AUTHOR]

The hypochlorous acid (HOCI)-dependent lysis of human red blood cells (HRBC) targets by neutrophils, activated with opsonized zymosan particles (OPZ), was increased by inhibiting HRBC catalatic activity with aminotriazole (AT; HRBCAT). The inhibition of HRBC glutathione cycle activity with carmustine (BCNU;HRBCBCNU) had no effect. In addition, the recovery of hydrogen peroxide (H2H2) and HOCI from neutrophils, activated unde conditions similar to those used for cytotoxicity assay, was reduced by the presence of HRBC and restored by replacing HRBC with HRBCAT, but not with HRBCBCNU. Linear relationships were found between the increments in the neutrophil-mediated lysis, observed by using HRBCAT instead of HRBC, and the increments in the H2O2 or HOCI recovery, detected by replacing HRBC with HRBCAT, but not with HRBCBCNU. Linear relationships were found between the increments in the neutrophil-mediated lysis, observed by using HRBCAT instead of HRBC, and the increments in H2O2 or HOCI recovery, detected by replacing HRBC with HRBCAT. Together these data, coupled with the results obtained by robing neutrophil cytolysis with chemical agents, suggest that the increased cytolytic efficiency displayed by neutrophils against HRBCAT, inhibited in their catalatic activity, is due to an enhanced availability of neutrophil-derived H2O2 with a consequent enhancement in the HOCI production (according to the following reaction:H2O2+CI-MPO/→H+ HOCI+H2O2. Thus it appears that HRBC catalase restrains the neutrophil cytolytic activity, by interfering with an early step of the pathway through which neutrophils generate cytotoxins. [ABSTRACT FROM AUTHOR]

The chemotactic peptide N-fomyl-methionyl-leucyl-phenylalanine (FMLP), per se incapable of triggering cyototoxicity, was found to significantly enhance phorbol myristate acetate (PMA)-dependent monocyte-mediated cytolysis of human red blood cell (HRBC) targets. Target cell lysis by PMA triggered monocytes was related to the release of superoxide anion and hydrogen peroxide, since cells from patients with chronic granulomatous disease and cells from normal donors, in the presence of superoxide dismutase or catalase, failed to exert significant cytotoxicity. An increased release of these mediators was found to be responsible for the FMLP-Dependent amplification of the cytolytic reaction. The results indicate that chemotactic factors are able to enhance the release of cytotoxic mediators by monocytes and raise the possibility that cellular processes during monocytes chemotaxis could modulate the subsequent cytolytic activity. [ABSTRACT FROM AUTHOR]

Human red blood cells (HRBC) were efficiently lysed when incubated with neutrophil polymorphonuclears (PMN) in the presence of phorbol- myristate-acetate (PMA), as detected by a 4-hr 51Cr release assay. The lysis was virtually absent in the presence of catalase, azide or cyanide and in the absence of chloride ions. These findings indicate the involvement of the myeloperoxidase (MPO)-hydrogen peroxide (H2O2)-chloride (Cl) system in the cytolytic process. As the MPO--H2O2--Cl system is capable of generating the powerful oxidant hypochlorous acid (HOCl), cytotoxicity assays were performed in the presence of taurine, glycine, serine and valine to scavenge this potentially lytic agent. Each of these compounds efficiently inhibited the H RBC lysis by PMA-triggered PMN, as well as the lysis caused by HOC1 in a cell-free system. Thus, the results suggest that HOC1, or an agent with similar reactivity, plays a key role in the PMA-dependent PMN-mediated cytotoxicity against HRSC targets. [ABSTRACT FROM AUTHOR]