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PubMed1980

Production of interferon and other lymphokines during murine tumour growth. I. Lymphokines in cell--free fluid of rat Zajdela ascites hepatoma.

Cell-free ascites of rat Zajdela hepatoma was assayed for the presence of lymphokine-like factors. Macrophage migration inhibitory and microbial growth inhibitory cytokines were detected with peak activities at day 4-8 and 10-11 after tumour inoculation, respectively. Occasionally, skin reactive activity was found, whereas only borderline titres of an interferon-like substance were demonstrated. Preliminary studies indicated that both migration inhibitory and microbial growth inhibitory factors are proteins resembling the corresponding lymphocyte-derived lymphokines. The cellular site of formation of these factors remains to be determined.

Animals↗
PubMed1983

Cyclosporin A inhibits lymphokine production but not the responses of macrophages to lymphokines.

Cyclosporin A (Cs A) exerted a dose-related inhibitory effect on antigen (ovalbumin, OVA) and phytohaemagglutinin (PHA)-induced transformation of guinea-pig lymph node cells (LNC). Whereas 0.05 micrograms/ml was sufficient to depress these responses markedly, it required 100-fold this concentration of Cs A to inhibit the production of lymphocyte activating factor (LAF) by lipopolysaccharide (LPS) stimulated peritoneal macrophages. Addition of Cs A together with insoluble concanavalin A (iCon A) to LNC cultures resulted in suppressed lymphokine production, as assessed by measurement of migration inhibition factor (MIF), the generation of macrophage procoagulant activity (MPCA) and the release of lymphocyte-derived-macrophage chemotactic factor (LDCF). Cs A also inhibited MIF and procoagulant production by sensitized peritoneal exudate cells in response to antigen, at the same concentrations which blocked lymphocyte transformation. In contrast, Cs A had no direct effect on the migration of peritoneal cells from capillary tubes, or on the responses of macrophages to preformed MIF, the lymphokine inducing MPCA or LDCF. Overnight incubation of macrophages with Cs A did, however, result in mild inhibition of their basal level of procoagulant activity.

Animals↗
PubMed1984

Ia-restricted interaction of normal lymphoid cells and SJL lymphoma (reticulum cell sarcoma) leading to lymphokine production. III. Relative roles of reticulum cell sarcoma and normal lymphoid cells in lymphokine production.

Production of interleukin 2 (IL-2) in mixed lymphocyte cultures of SJL/J lymph node (LN) and gamma-irradiated, syngeneic lymphoma cells of transplantable reticulum cell sarcoma (gamma-RCS) was abolished by pactamycin pretreatment of gamma-RCS. LN cells needed for this interaction were Lyt-2 T-cells, not adherent to Sephadex G-10. The effect of separation of T-cells and gamma-RCS after the first 24 hours of culture suggested that both components contributed to the IL-2 production. Exogenous IL-2, but not interleukin 1 (IL-1), restored the ability of pactamycin-treated gamma-RCS to induce syngeneic T-cell proliferation. The inability of T-cells from "nonresponder" F1 hybrids of SJL to proliferate to gamma-RCS was not corrected by addition of IL-1 or IL-2. Interferon (IFN) production also required the presence of untreated gamma-RCS and LN cells, but it was dependent on a Sephadex G-10 and plastic adherent cell in LN. IFN-free supernatant of LN cells plus gamma-RCS induced IFN production in fresh normal lymphoid cells, suggesting a possible indirect induction of this lymphokine. In addition, unirradiated RCS cells (la+ cells) produced immune IFN over a period of 3 weeks in vitro, after which the cells lost their viability. Prolonged IL-2 production was not observed in these cultures. The possible biological importance of the lymphokine production during the exaggerated syngeneic mixed lymphocyte reactions induced by RCS cells is discussed.

Animals↗
PubMed1989

IL-4 regulation of murine lymphokine-activated killer activity in vitro. Effects on the IL-2-induced expansion, cytotoxicity, and phenotype of lymphokine-activated killer effectors.

The in vitro incubation of B6 splenocytes with purified, mouse rIL-4 for 4 to 5 days was sufficient to generate lymphokine-activated killer (LAK) activity. In addition, rIL-4 augmented LAK cytotoxic activity when combined with rIL-2, as measured in a 4 h 51Cr-release assay against fresh, syngeneic MCA-sarcoma (MCA-102 and MCA-105) cells. Interestingly, this augmentation was not observed against the cultured YAC-1 target. LAK generation and augmentation of cytotoxicity by rIL-4 was species-specific, because human rIL-4 (up to 20,000 U/ml) failed to elicit these effects in the mouse splenocyte cultures. When 5-day B6 LAK cells (splenocytes incubated in rIL-2 at 1000 U/ml for 5 days) were split and recultured in the combination of rIL-2 plus rIL-4 for 4 additional days at least a twofold greater expansion in cell number resulted compared to similar cells cultured in either rIL-2 or rIL-4 alone. Moreover, LAK cells expanded in rIL-2 plus rIL-4 exhibited substantial increases in in vitro cytolytic activity (on a per cell basis) against MCA-102 and MCA-105 sarcoma cells, but not against YAC-1 targets. FACS analysis or negative selection using Lyt-2 or NK-1.1 mAb plus C revealed no differences in effector phenotype(s) of LAK cells expanded in rIL-2 alone compared to rIL-2 plus rIL-4 to account for the differences observed in both expansion and cytolytic activity by rIL-4. The majority of cells was Thy-1+, Lyt-2+, T3+, and ASGM-1+. However, a marked increase in the granule-associated serine esterase, BLT-E, was found only in LAK cells expanded in the combination of both lymphokines. Collectively, these studies show that rIL-4 has potent regulatory activities on splenic LAK generation, expansion, and cytotoxic function in the mouse.

Adjuvants, Immunologic↗
PubMed1989

Culture supernatants of lymphokine-activated killer (LAK) cells contain a high-molecular-weight cytotoxic lymphokine.

Supernatants of human lymphokine-activated killer (LAK) cells grown in vitro were tested for cytotoxic activity against several mouse and human neoplastic cell lines. All LAK preparations tested (14/14) exhibited cytotoxic activity (40-90% killing of the target cells). Sephacryl S-300 Gel filtration experiments indicated that the biological activity of the LAK supernatant is associated with molecular moieties ranging from 800 kDa or more, to less than 10 kDa. The finding of strong cytotoxic activity in LAK supernatants against several tumor lines points to the possibility of employing soluble products of these cells, rather than the living cells themselves, for therapeutic purposes.

Animals↗
PubMed1990

A high molecular weight cytotoxic lymphokine in supernatants of lymphokine-activated killer cells cross-reacts with transforming growth factor beta.

Supernatants of lymphokine-activated killer (LAK) cells were highly cytotoxic for melanoma A375 cells. A high-molecular-weight fraction was isolated from such supernatants by gel filtration on an S-300 Sephacryl column (Fraction 1; Fr1). The cytotoxic activity in Fr1 was heat- and acid-resistant and was completely abolished by a rabbit antibody against TGF-beta. We conclude that Fr1 contains TGF-beta or a cross-reactive molecule, associated with a high-molecular-weight carrier.

Animals↗
PubMed1983

Immunoregulation by lymphokines: immune interferon and lymphotoxin induction of lymphokine activity in human peripheral blood leukocyte cultures.

Supernatants of mitogen-induced human PBL cultures contain several lymphokine activities. Analysis of kinetic studies has suggested a correlation in the appearance in culture supernatants of lymphotoxin (LT) and immune interferon (Im-IF). We investigated this interaction between LT and Im-IF in the serum-free human PBL culture system. Partially purified LT was derived from a normal cell line, RPMI 1788. This LT, which had no contaminating IF activity, induced production of significant levels of IF activity as early as 15 hours after initiation of culture. This induced IF activity was identified as Im-IF by criteria of pH 2, SDS and heat sensitivity, as well as neutralization by AB to Im-IF. LT activity, in excess of the units added to the culture, was also measured in the LT-induced cultures. In another group of experiments, partially purified human Im-IF, produced by expression of a cloned gene in monkey COS-7 cells, was added to human PBL cultures. This Im-IF, at concentrations of 160 and 320 units/ml, caused induction of LT activity. The induced LT activity was neutralized by AB raised to 1788-derived LT. Im-IF treated cultures also contained IF activity in excess of the titers added to the cultures. These results would suggest an interaction between LT and Im-IF at the level of production by lymphoid cells.

Adjuvants, Immunologic↗
PubMed2004

Lymphokine activated killer cells from umbilical cord blood show higher antitumor effect against anaplastic astrocytoma cell line (U87) and medulloblastoma cell line (TE671) than lymphokine activated killer cells from peripheral blood.

OBJECTS: The aims of this study were to assess the cytotoxic capability of lymphokine-activated killer (LAK) cells from umbilical cord blood (UCB), to compare them with those of peripheral blood (PB)-derived cells against anaplastic astrocytoma cell line (U87) and medulloblastoma cell line (TE671), and to identify which mechanism and genes were involved in cytotoxicity. METHODS: The effector cells were generated by interleukin-2 from UCB and PB. The antitumor property of effector cells against the target cells (U87, TE671) were estimated using a visual survival cell assay. The mixed target and effector (UCB) cells were analyzed for whether DNA fragmentation was present or not. Reverse transcription polymerase chain reaction analysis was then performed to estimate the statement of the perforin and FasL genes in activated and inactivated cells from UCB. RESULTS: The higher in vitro antitumor properties of the LAK cells from UCB were observed in comparison to the LAK cells from PB against the U87 and the TE671 ( p<0.05). Apoptosis may be one of the lysis mechanisms of target cells by the LAK cells from UCB. The contributing genes could be FasL and perforin. CONCLUSIONS: This study suggests that UCB may be used as a source of LAK cells in adults and children suffering from anaplastic astrocytoma or medulloblastoma.

Adult↗
PubMed1989

Lymphokine-activated killer cells in rats: generation of natural killer cells and lymphokine-activated killer cells from bone marrow progenitor cells.

The coculture of rat bone marrow cells with recombinant interleukin-2 induced the generation of cells mediating natural killer (NK) activity and subsequent lymphokine-activated killer (LAK) activity depending upon the dose of IL-2 and time of culture. NK activity was detected as early as 4 to 5 days after the addition of IL-2 and could be evoked with as little as 5 to 50 U/ml. The induced NK cells had large granular lymphocyte (LGL) morphology and expressed 0X8 and asialo GM1 surface markers but did not express 0X19 or W3/25 markers. LAK activity was detected only after 5 days of culture, and required above 100 U/ml IL-2. Cells mediating LAK activity also expressed 0X8 and asialo GM1 but not 0X19. The generation of detectable NK and subsequent LAK activity was due to induction of early progenitor cells and not contaminating mature LGL/NK cells within the bone marrow population since of removal of such mature NK cells with L-leucine methyl ester (L-LME) did not affect the subsequent generation of either activity. Moreover, the removal of actively dividing cells as well as mature NK cells from the bone marrow by treatment with 5-fluorouracil (5-FU) in vivo enriched the remaining bone marrow population for both NK and LAK progenitor cells. The phenotype of the L-LME- and 5-FU-resistant NK and LAK progenitor cells within populations of bone marrow was determined by antibody plus complement depletion analysis. Although treatment of normal bone marrow with anti-asialo GM1 + C reduced the induction of NK and LAK activity in 5-day cultures, treatment of 5-FU marrow with anti-asialo GM1 + C did not affect either activity. Treatment with a pan-T cell antibody + C did not affect the development of NK or LAK activity under any conditions. Thus, the 5-FU-resistant NK/LAK progenitors were asialo GM1 negative but became asialo GM1+ after induction by IL-2. Finally, evidence that bone marrow-derived LAK cells were generated directly from the IL-2-induced NK cells was obtained by treating the IL-2-induced LGL/NK cells with L-LME.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1977

Lymphokines of T and B cells. I. Demonstration that T and B cells of tumor antigensensitised mice produce different lymphokines changing surface charge of target cells.

Lymphocytes from MC sarcoma-bearing mice were exposed to a soluble tumor antigen. The lymphokine-containing supernatant thus obtained was fractionated on Sephadex G-75. Fractions were tested for their activity to reduce the surface charge of indicator macrophages by cell electrophoresis technique. When unseparated spleen or lymph node cells were used the charge-reducing activity of the supernatant was found in three regions according to molecular weights of about 100,000, 60,000 - 35,000 and 13,000 Daltons. The appearance of activity was found to be time-dependent. As indicated by tests with separated lymphocytes both B and T cells appear to release charge-reducing activity.

Animals↗
PubMed1991

Lymphokine-activated killer traffic assay and our preliminary clinical results of regional arterial infusion of lymphokine-activated killer cells for renal cell carcinoma.

In vivo distribution of radiolabeled lymphokine-activated killer (LAK) cells that were administered either through the caudal vein or the abdominal aorta of Lewis rats was studied. The LAK traffic study showed remarkable increase in radioactivity in several organs peripheral to the arterial injection site. We began treating the patients with metastatic renal cancer by means of regional arterial administration of LAK cells. This treatment was supported by the results of the traffic assay. Our regional LAK therapy, which involves a weekly leukapheresis in combination with daily systemic low dose interleukin-2 administration, has been in practice for over 2.5 years. Seven out of 12 metastatic lesions in the 7 patients treated showed regression. Two responding cases have been alive for over 2 years. Responding lesions were bone, muscle, para-aortic lymph node, and/or retroperitoneum. The regional arterial administration of LAK cells is local therapy. However, this mode of therapy could be useful for those patients with extrapulmonary nonresectable metastasis and at least one injectable feeding artery. This study shows our laboratory data concerning the localization of LAK cells transferred via arterial route and our clinical cases treated with regional arterial administration of LAK cells.

Animals↗
PubMed1992

Mechanism of interferon gamma-induced protection of human gliosarcoma cells from lymphokine-activated killer lysis: division of lymphokine-activated killer cells into natural killer- and T-like cells.

The mechanism by which interferon gamma (IFN-gamma) decreases the susceptibility of the established cultured gliosarcoma line Gl-1 to lymphokine-activated killer (LAK) lysis was analyzed. The results of monolayer depletion and lectin-dependent cellular cytotoxicity assays by LAK cells revealed that the resistance to LAK lysis of IFN-gamma-treated Gl-1 cells is manifested at the stage of LAK cell target recognition alone. We have also divided LAK cells into populations of phenotypically natural killer (NK)- and T-like cells with monoclonal antibodies and complement, respectively. We have used these cells to examine the mechanism of IFN-gamma-induced protection of Gl-1 cells from LAK lysis in cold target inhibition, monolayer depletion, and direct binding assays. The results revealed that NK-like cells do not recognize IFN-gamma-treated Gl-1 cells as efficiently as they do untreated targets, whereas T-like cells show the opposite tendency. In conclusion, we have demonstrated that the IFN-gamma induced protection of tumor cells from LAK lysis is predominantly regulated by the target recognition of NK-like cells. On the other hand, IFN-gamma-treated tumor cells may bind to T-like cells but fail to trigger them to initiate further stages for lysis as effectively as NK-like cells.

Cell Division↗
PubMed1976

Antibodies to guinea pig lymphokines. IV. Suppression of the mixed leukocyte culture reaction by anti-lymphokine globulin.

An antiserum, (anti-lymphokine globulin, ALyG) directed against highly purified products of activated lymphocytes, inhibits the proliferation of responding cells in guinea pig mixed leukocyte cultures ((MLC). This serum recognizes three newly synthesized lymphocyte products (one of them being migration inhibition factor (MIF) which are involved in the mediation of delayed hypersensitivity reactions in vivo. Since ALyG does not appear to contain cytotoxic antibodies against guinea pig lymphocyte antigens and its inhibitory activity cannot be removed by absorption with lymphoid cells it seems likely that the inhibition of MLC reactivity is not mediated by the lysis of stimulator or responder cells and its target is not the lymphocyte per se but possibly some factor elaborated during MLC response. By contrast, antisera with specificity for histocompatibility (H) antigens can inhibit the MLC when the appropriate H antigens are present on the responding and/or stimulating cell population. However, this inhibitory of the antisera can be effectively absorbed with lymphoid cells bearing the appropriate H antigens. The addition of ALyG even 48 hr after the initiation of culture results in a marked inhibition of MLC reactivity. This finding is consistent with the elaboration of a mitogenic factor or signal during the first 48 hr of culture and the delivery of this signal to the responding cell population. Thus, ALyG does not appear to interfere with the synthesis of this factor but must be present after its release in order to block responder cell proliferation. Furthermore, the stimulatory effect of this MLC mitogenic factor, but not of PPD-induced mitogenic factor on "third party" cells can be completely inhibited when the cells are cultured in the presence of ALyG. These findings suggest that the MLC-mitogenic factor in this system is identical to or cross-reactive with one or both of the newly synthesized molecules recognized by the ALyG.

Animals↗
PubMed1992

Regulation of lymphokine-activated killer cell induction by human recombinant IL-1 receptor antagonist. Obligate paracrine pathway of IL-1 during lymphokine-activated killer cell induction.

IL-2-stimulated human lymphocytes, referred to as lymphokine-activated killer (LAK) cells, can develop a broad range of lytic activity against fresh tumor cells and cultured tumor cell lines. IL-1, a pleiotropic cytokine shown to synergize with IL-2 on LAK induction, is endogenously synthesized and secreted by LAK cells. Immunoblot analysis demonstrated that IL-2-stimulated PBL produced the 31- to 34-kDa pro-molecules of IL-1 within 24 h and maintained their expression for at least 96 h. The role of secreted IL-1 has been examined using rIL-1R antagonist (IL-1ra). The addition of IL-1ra to LAK activation culture resulted in dose-dependent inhibited lytic activity, which was more apparent in LAK cells cultured with higher doses of IL-2. However, IL-1ra had no effect on proliferative responses elicited in LAK cells by IL-2. Moreover, when IL-1 binding was blocked by IL-1ra, the expression of the IL-2R p55 subunit was reduced compared with control LAK cells. The effect of IL-1 binding blockade on expression of other cytokine mRNA was further examined by polymerase chain reaction analysis, and, specifically, inhibition of both TNF-alpha and TNF-beta mRNA expression by IL-1ra was observed in PBL stimulated with IL-2. The reduced biologic activity of TNF in culture supernatants correlated well with the inhibition of mRNA expression. These findings suggest that autocrine/paracrine IL-1 is involved in the initial generation of LAK activity and, in particular, that TNF expression could be induced via an IL-1 autocrine pathway.

Base Sequence↗
PubMed1985

Selective regulation of chemotactic lymphokine production. I. Selective potentiation of eosinophil chemotactic lymphokine production in alum hydroxy gel- and Bordetella pertussis vaccine-treated guinea pigs.

Delayed tissue eosinophilia in DNP-ovalbumin-induced allergic inflammatory skin lesions of guinea pigs was markedly enhanced by previous treatment with alum hydroxy gel (Alum) or Bordetella pertussis vaccine. This enhancement seemed due to increased production of a lymphocyte-derived eosinophil chemotactic factor (ECF) at the skin site. Treatment of animals with Alum potentiated antigen-induced in vitro ECF production by lymphoid cells from spleen and mesenteric lymph node of sensitized animals. The co-culture supernatants of lymphoid cells from Alum-treated animals also potentiated concanavalin A (Con A)-induced in vitro ECF production. The potentiating effect of Alum on ECF production seemed to be ascribed to the release of soluble factors from macrophages of the Alum-treated animals. The macrophage-derived soluble factor ECF-potentiating factor (ECF-PF) selectively potentiated ECF production but not macrophage chemotactic lymphokine production by Con A-stimulated lymphoid cells from normal animals. ECF-PF activity was associated with two separate m.w. fractions: one was 50,000 to 70,000 and the other was 10,000 to 20,000. The present study provides one of the explanations for enhanced ECF production by adjuvants, such as Alum and Bordetella pertussis vaccine.

Adjuvants, Immunologic↗
PubMed1986

The anti-tumor efficacy of lymphokine-activated killer cells and recombinant interleukin 2 in vivo: direct correlation between reduction of established metastases and cytolytic activity of lymphokine-activated killer cells.

Our previous studies demonstrated that the incubation of human peripheral blood lymphocytes or murine splenocytes in recombinant interleukin 2 (RIL 2) resulted in the generation of lymphokine-activated killer (LAK) cells capable of lysing a broad spectrum of fresh tumors in short-term chromium-release assays. Moreover, injections of LAK cells plus RIL 2 were highly effective in eliminating established 3 day metastases in the lung and liver (1-3). We have examined several parameters to define whether or not the cytolytic activity of LAK cells as measured in vitro correlated directly with the in vivo anti-tumor efficacy of adoptively transferred LAK cells. LAK cells plus RIL 2 could mediate marked reductions of established pulmonary metastases in mice rendered T cell deficient by adult thymectomy and lethal, total body irradiation followed by reconstitution with T cell-depleted bone marrow and spleen cells. Thus there was no requirement for additional T lymphocytes of host origin for successful therapy with adoptively transferred LAK cells plus RIL 2. Fresh splenocytes depleted of T cells by anti-Thy-1.2 monoclonal antibody plus complement generated LAK cells that were as highly lytic to fresh tumor in vitro and were as effective in reducing established pulmonary metastases as those generated from untreated or complement-treated splenocytes. Thus the precursor to LAK cells with anti-tumor activity in vitro and in vivo did not express the Thy-1 antigenic marker. In contrast, treatment of LAK effector cells (those generated from a 3-day incubation of fresh, normal splenocytes in RIL 2) with anti-Thy-1.2 antibody plus complement reduced or abolished their in vitro cytolytic activity. However, when combined with the systemic administration of RIL 2, these T cell-depleted, non-lytic LAK cells remained as effective in reducing the number of established pulmonary metastases upon adoptive transfer as untreated or complement-treated lytic LAK cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

The mechanism of action of lymphokines. VIII. Lymphokine-enhanced spontaneous hydrogen peroxide production by macrophages.

Oil-elicited guinea-pig peritoneal macrophages (MPs) cultured for 2-3 days in medium containing supernatant of concanavalin A-activated lymphocytes (lymphokine, LK) generated large amounts of hydrogen peroxide (H2O2), as detected by the horseradish peroxidase (HRP)-dependent oxidation of phenol red, in the absence of further stimulation. H2O2 production increased with the duration of exposure to LK and was evident at high dilutions of supernatant (1/64). Parallel cultures of MPs in medium or a supernatant of non-activated lymphocytes also increased their H2O2 production during culture but levels at all time intervals were significantly lower than those measured in LK treated cultures. The marked increase in H2O2 production was associated with only a moderate increment in superoxide (O-2) liberation and this was not specific for LK treated cells. Detection of LK-dependent H2O2 production was dependent on ongoing pinocytosis during the assay. This and other arguments suggest that the HRP-phenol red assay, as applied here, detects H2O2 generation occurring at the level of intracellular vesicles and it is concluded that LK elicits H2O2 production that is limited to the intracellular compartment. H2O2 is, apparently, derived by non-enzymatic dismutation of O-2 taking place within the cell; LK treatment of MPs also resulted in a significant reduction in catalase activity.

Animals↗
PubMed1986

The mechanism of action of lymphokines. IX. The enzymatic basis of hydrogen peroxide production by lymphokine-activated macrophages.

The purpose of this study was to elucidate the biochemical basis of the enhanced hydrogen peroxide (H2O2) production by guinea pig peritoneal macrophages (MP) cultured in lymphokine (LK)-containing medium. The markedly augmented H2O2 generation by these cells, demonstrable by the horseradish peroxidase (HRP)-catalyzed oxidation of phenol red, is distinguished by its lack of dependence on a second stimulus. We demonstrate that H2O2 production is truly spontaneous and is not caused by a stimulant present among the H2O2 assay reagents. The principal candidate for such a role was HRP type II (a mixture of five isoenzymes) that was reported to be capable of eliciting an oxidative burst in MP. Four distinct HRP isoenzymes that were found incapable of provoking an oxidative response were nevertheless adequate for demonstrating H2O2 production by LK-activated MP. Blocking the MP receptor for mannose by the addition of mannan to the assay system resulted in enhanced detection of H2O2 by low concentrations of HRP type II and by three out of four HRP isoenzymes. Treatment of MP with LK-containing medium for 72 hr did not result in a significant change in the activity of cellular superoxide dismutase (SOD) compared with MP cultured for the same length of time in control medium. By using the specific inhibitor of copper, zinc-containing SOD, sodium diethyldithiocarbamate (DDC), and the universal SOD inhibitor, sodium nitroprusside, we found that the predominant enzyme in guinea pig peritoneal MP is probably manganese-containing SOD. Incubation of LK-activated MP with nitroprusside resulted in almost total inhibition of H2O2 production and a simultaneous switch to superoxide (O2-) liberation. Similar exposure to DDC had no effect. These data indicate that H2O2 produced by LK-activated MP is derived exclusively by enzymatic dismutation of O2- mediated by a manganese-containing SOD. The increase in spontaneous H2O2 production induced by LK is therefore secondary to augmented O2- production that occurs at a cellular location where O2- is accessible to SOD. The enzymatic basis of the enhanced oxygen radical production was investigated by determining the kinetic parameters of the O2- -forming NADPH oxidase of resting LK-treated MP in a cellfree system in which O-2 production was induced by sodium dodecyl sulfate. The Km for NADPH and the Vmax of the enzyme of LK-treated MP were not different from those of the enzyme of MP incubated in control medium. We conclude that LK treatment of MP does not modulate the NADPH oxidase itself but, most likely, a process related to activation of the enzyme.

Animals↗