Secretion Of Reactive Oxygen Intermediates Research Articles

Repeatedly administered antidepressant drugs modulate humoral and cellular immune response in mice through action on macrophages.

Depression is associated with an altered immune response, which could be normalized by antidepressant drugs. However, little is known about the influence of antidepressants on the peripheral immune response and function of macrophages in individuals not suffering from depression. Our studies were aimed at determining the influence of antidepressant drugs on the humoral and cellular immune response in mice. Mice were treated intraperitoneally with imipramine, fluoxetine, venlafaxine, or moclobemide and contact immunized with trinitrophenyl hapten followed by elicitation and measurement of contact sensitivity by ear swelling response. Peritoneal macrophages from drug-treated mice were either pulsed with sheep erythrocytes or conjugated with trinitrophenyl and transferred into naive recipients to induce humoral or contact sensitivity response, respectively. Secretion of reactive oxygen intermediates, nitric oxide, and cytokines by macrophages from drug-treated mice was assessed, respectively, in chemiluminometry, Griess-based colorimetry and enzyme-linked immunosorbent assay, and the expression of macrophage surface markers was analyzed cytometrically. Treatment of mice with fluoxetine, venlafaxine, and moclobemide results in suppression of humoral and cell-mediated immunity with a reduction of the release of macrophage proinflammatory mediators and the expression of antigen-presentation markers. In contrast, treatment with imipramine enhanced the humoral immune response and macrophage secretory activity but slightly suppressed active contact sensitivity. Our studies demonstrated that systemically delivered antidepressant drugs modulate the peripheral humoral and cell-mediated immune responses, mostly through their action on macrophages. Imipramine was rather proinflammatory, whereas other tested drugs expressed immunosuppressive potential. Current observations may be applied to new therapeutic strategies dedicated to various disorders associated with excessive inflammation.

Ability of Curcuminoid from Curcuma domestica Val. in Reducing the Secretion of Reactive Oxygen Intermediates by Synovial Fluid Monocytes in Patients with Osteoarthritis

Increasing the secretion of reactive oxygen intermediates (ROI) by monocytes in the synovial fluid is anindicator to determine the severity of joint inflammation. Previous studies have shown that curcumin inhibitthe osteoarthritis progression with its ability to inhibite the activity of the nitric oxide synthase (NOS) enzymefrom macrophages. In this prospective randomized open end blinded evaluations = PROBE study, 80 patientswith knee osteoarthritis were eligable. The subject were devided in to two group: group who received 3 x 30mg of curcuminoid from Curcuma domestica Val. extract (curcuminoid group) and group who received 3 x 25mg of diclofenac sodium (diclofenac group) as comparison. The treatment was for 4 weeks time. The secretionof ROI by sinovial fluid monocytes was calculated by scoring the amount of formazan formation after neutralred staining in nitrobleu tetrazolium reduction assay. The result of this study showed that the secretion of ROIby synovial fluid monocytes was significantly decreased in both groups (p <0.001) respectively. There wasno significant difference in decreasing of ROI secretion of synovial fluid monocytes between both treatmentgroups (p = 0.92).

Proteinase-activated receptors induce nonoxidative, antimicrobial peptides and increased antimicrobial activity in human mononuclear phagocytes

As thrombin and SFLLRNPNDKYEPF (SFLLRN-14), a synthetic ligand, mainly of the proteinase-activated receptor-1 (PAR-1), induce in monocytes the synthesis and secretion of chemokines, the PAR pathway can be viewed as a mononuclear phagocyte-activating principle. Classically, antimicrobial activity of mononuclear phagocytes is the measure for activation. Here, we investigated whether thrombin or SFLLRN-14 increases the antimicrobial activity of human monocytes and compared these effects to those of IFN-gamma. Furthermore, we measured the effects of these agents on the secretion of reactive oxygen intermediates and the antimicrobial activity of acid peptide extracts from monocytes. Human monocytes were exposed to maximally active concentrations of thrombin, SFLLRN-14, and IFN-gamma. Human monocytes treated with thrombin or SFLLRN-14 and then challenged with Salmonella enterica serovar typhimurium, including its attenuated mutant phoP, or Listeria monocytogenes killed, within 3 h, significantly more bacteria than control cells, an effect comparable with or surpassing the effect of IFN-gamma. This finding establishes the proteinase-PAR pathway as a potent, alternate activation pathway of mononuclear phagocytes. Thrombin and SFLLRN-14 had no significant effects on the amount of H(2)O(2) secreted by monocytes. This was in contrast to IFN-gamma, which as expected, increased the secretion of H(2)O(2) by approximately fourfold. Thrombin and SFLLRN-14, but not IFN-gamma, however, significantly increased the antimicrobial activity of acid peptide extracts of monocytes in a radial diffusion assay. Taken together, these findings suggest that IFN-gamma and thrombin differentially regulate oxidative and nonoxidative killing systems of human monocytes.

Deactivation of the respiratory burst of human and murine macrophages by lipopolysaccharide is a receptor mediated effect independent from autocrine mechanisms

Endotoxin blunts secretion of reactive oxygen intermediates by macrophages. Because transforming growth factor β (TGFβ) or interleukin 10 (IL-10) were shown to deactivate macrophages, and are secreted by macrophages in response to LPS, we sought for autocrine mechanisms of macrophage deactivation by endotoxin. TGFβ did not deactivate the respiratory burst of human blood-derived and resident or thioglycollate-induced murine peritoneal macrophages. According to previous reports, suppression of H202 secretion by TGFβ was restricted to periodate-elicited murine peritoneal macrophages.In contrast to TGFβ, IL-10 deactivated systems producing reactive oxygen intermediates in human blood-derived human macrophages, but neither anti-TGFβ1 nor anti-IL-10 antibodies restored LPS-mediated deactivation of macrophages.Supernatants from LPS-treated human blood-derived macrophages could not confer deactivation to homologous macrophages.Deactivation by LPS required the presence of serum proteins and appeared to be mediated by the CD14 antigen-related LPS receptor, because anti-CD14 antibody and IL-4, which downregulates CD14, antagonized the LPS effect.Taken together, these observations indicate that the suppressive effect of LPS on the respiratory burst of macrophages is lipid A receptor-dependent, and results in a direct deactivation response of the cell without mediation by autocrine mechanisms.

Role of Fcγ receptors in cancer and infectious disease

Receptors for the Fc domain of immunoglobulin G (Fc gamma R) provide an interface between specific humoral immunity and the cellular branch of the immune system through their interaction with antibody. Cross-linking Fc gamma R on myeloid cells triggers such diverse functions as clearance of immune complexes, phagocytosis of opsonized pathogens, secretion of reactive oxygen intermediates, and antibody-dependent cellular cytotoxicity. The Fc gamma R play a major role in the removal of antibody-coated infectious agents and are the exclusive trigger molecules for tumor cell killing by human myeloid cells. Studies of Fc gamma R function have been aided by the use of Fc gamma R specific monoclonal antibodies, self-directed target cells, and bispecific antibodies that link target cells or pathogens to specific host cell molecules, including Fc gamma R. These reagents have contributed significantly to our understanding of the role of the different classes of Fc gamma R in mediating protection from various infectious agents and in mediating tumor cell killing. Taken together, these approaches have provided insight into the utility of manipulating Fc gamma R function in the therapy of cancer and infectious disease.

Increase of bovine alveolar macrophage superoxide anion and hydrogen peroxide release by dusts of different origin

The release of reactive oxygen intermediates (ROI) from bovine alveolar macrophages (BAM) after stimulation with heavy metal-containing dusts was investigated. BAM were obtained by postmortem lavages of bovine lungs. The dusts were collected from waste incineration, sewage sludge incineration, an electric power station, and from two different factories. Three quartz dusts were used as heavy metal-free controls. The dusts were fractionated by sieving and sedimentation and analyzed by electron microscopy, atomic absorption spectrometry (AAS), and atomic emission spectrometry with inductively coupled plasma (AES-ICP). Incubation of BAM with the dusts (12.5-1000 micrograms/ml medium) led to concentration-dependent increases in ROI release. The secretion of ROI was already seen after 15 min and lasted throughout the experiment up to 90 min, with the exception of a waste incinerator ash, which contained the highest contents of some heavy metals and where the release of ROI ceased after 60 min. We suggest that this dust exhibits simultaneously stimulating and inhibiting effects. The ratio of the secreted O2- and H2O2 varied, depending on the dust being investigated. The release of hydrogen peroxide correlated best, in descending order, with the content of iron, manganese, chromium, vanadium, and arsenic in the dusts.

Lipopolysaccharide primes human alveolar macrophages for enhanced release of superoxide anion and leukotriene B4: self-limitations of the priming response with protein synthesis.

Human alveolar macrophages (AM) can produce potent reactive oxygen intermediates (ROI) and arachidonic acid metabolites (eicosanoids), which have important roles in host defense and the pathogenesis of some diseases of the lung. Bacterial lipopolysaccharide (LPS) is believed to cause profound lung injury and can prime mouse peritoneal macrophages for the enhanced secretion of ROI and eicosanoids. Therefore, we investigated the effect of LPS pretreatment on the ability of AM to release superoxide anions (O2-) and leukotriene B4 (LTB4). LPS can prime AM for the enhanced secretion of O2- and LTB4, regardless of whether they are derived from nonsmokers or smokers. Moreover, judging from the time-response characteristics, this priming for LTB4 release could be inhibited in the later stages of pretreatment, when the O2(-)-releasing capacity was enhanced. The priming inhibition was prevented, at least in part, by cycloheximide, but not by SOD and/or catalase. In addition, cycloheximide also inhibited the priming for O2- release. Hence, protein synthesis might be necessary for the priming for O2- release and for inhibiting the priming for LTB4 release. This phenomenon of self-limiting the priming response with LPS seems to be very important when we consider the high oxygen tension in the lungs and the many bacterial substances inspired into alveoli.

Surface contact modulation of inflammatory macrophage antibody dependent cytotoxicity and prostanoid release.

Adherence to extracellular matrix proteins modulates the functional and secretory activities of mononuclear phagocytes, although the mechanisms regulating these adherence-dependent changes are poorly understood. In this study, the ability of rat inflammatory peritoneal macrophages (PM) to adhere to an endothelial cell-derived extracellular matrix or a denatured collagen/fibronectin-coated surface and perform antibody dependent cell cytotoxicity (ADCC) and secrete reactive oxygen intermediates was compared with PM adherent to tissue culture plastic. Prostaglandin E2 (PGE2) and thromboxane B2 (TxB2), two major cyclooxygenase products released by inflammatory macrophages, were also measured by PM adherent to the protein coated surfaces. Rat exudate PM were equally adherent to tissue culture plastic or wells coated with either endothelial cell derived matrix or denatured collagen (gelatin)/fibronectin. PM adherent to a denatured collagen/fibronectin-coated wells demonstrated significantly less cytolytic activity (15 +/- 2% lysis) when compared with either tissue culture plastic adherent PM (43 +/- 7% lysis) or PM adherent to extracellular matrix (59 +/- 11% lysis). PM adherent to extracellular matrix released twofold more TxB2 than plastic adherent PM, while PM adherent to denatured collagen/fibronectin released 40% more PGE2 than cells adherent to tissue culture plastic or 80% more PGE2 than PM adherent to the extracellular matrix. PM adherent to denatured collagen/fibronectin release less superoxide anion (27 +/- .9 nmoles/10(6) PM) than PM adherent to either tissue culture plastic (43 +/- 1 nmoles/10(6) PM) or the extracellular matrix (60 +/- 0.5 nmoles/10(6) PM). Furthermore, incubation of plastic adherent PM with exogenous PGE2 reduced superoxide production in a dose-dependent manner. These results demonstrate that the inhibition of ADCC and secretion of reactive oxygen intermediates by PM adherent to a denatured collagen/fibronectin surface correlated with an increased release of the immunosuppressive prostanoid PGE2. Furthermore, the addition of exogenous PGE2 to plastic adherent PM reproduced the depression in ADCC and superoxide anion production observed by PM adherent to a denatured collagen/fibronectin surface. These studies suggest that the increased production and release of PGE2 by inflammatory macrophages adherent to a denatured collagen surface may act to suppress cytotoxic mechanisms and thereby constitutes part of an autocrine feedback mechanism regulating macrophage function during wound injury.

The Lavage Fluid from a Patient with Alveolar Proteinosis Inhibits theIn VitroChemiluminescence Response and Arachidonic Acid Metabolism of Normal Guinea Pig Alveolar Macrophages

Pulmonary alveolar proteinosis is characterized by the accumulation of a lipoproteinaceous material within the alveoli of the lung. It is well established that patients with pulmonary alveolar proteinosis have a high incidence of complicating pulmonary infections possibly resulting from defects of antibacterial functions of alveolar macrophages. Moreover, for unclear reasons, an inflammatory response in the airways is frequently absent. In order to investigate the role of the lipoproteinaceous material in these two patterns, we tested the in vitro effects of a lavage fluid from a human pulmonary alveolar proteinosis on the secretion of reactive oxygen intermediates and arachidonic acid metabolites by normal guinea pig alveolar macrophages. After incubation with the lipoproteinaceous material, the luminol-enhanced chemiluminescence of zymosan-triggered alveolar macrophages was reduced in a dose-dependent fashion. The lipoproteinaceous material similarly reduced the chemiluminescence response in a cell-free xanthine-xanthine oxidase system generating superoxide anions. This latter observation suggests that the lipoproteinaceous material acts as a scavenger for superoxide anions produced by alveolar macrophages. Its purified protein or phospholipid fractions also resulted in a general inhibition of the secretion of arachidonic acid metabolites by alveolar macrophages challenged in vitro with zymosan. Our results suggest that the alveolar filling material of pulmonary alveolar proteinosis may inhibit the action of antibacterial and/or proinflammatory agents produced by alveolar macrophages. We speculate that such effects of the lipoproteinaceous material may play a role in vivo in the high incidence of pulmonary infections and in the absence of discernible interstitial or intraalveolar inflammation seen in pulmonary alveolar proteinosis.

Effect of anticancer drugs on macrophage-mediated antibody-dependent cytotoxicity and secretion of reactive oxygen intermediates

The in vitro effects of methotrexate, vincristine, dexamethasone and adriamycin to modulate the antibody dependent cellular cytotoxicity (ADCC) reaction and secretion of superoxide (O 2 −) and hydrogen peroxide (H 2O 2) by phorbol myristate acetate (PMA) stimulated rat peritoneal macrophages were studied. Macrophage-mediated ADCC, as measured by the lysis of 51Cr-labeled IgG-coated sheep red blood cells in an 18 h assay, was significantly enhanced in the presence of 100 ng/ml of PMA. Treatment of macrophage monolayers for 24 h with vincristine (10 −5–10 −7 M), dexamethasone (10 −5–10 −9 M) and adriamycin (10 −6M) inhibited ADCC by PMA stimulated macrophages. Vincristine, dexamethasone and adriamycin also inhibited the secretion of O 2 − and H 2O 2 by PMA stimulated macrophages at similar concentrations that reduced the ADCC reaction. Methotrexate (10 −3–10 −7 M) did not inhibit either ADCC or O 2 − and H 2O 2 secretion by PMA stimulated macrophages. These results suggest that select anticancer drugs can inhibit macrophage mediated cytotoxicity of antibody coated target cells by suppressing the release of reactive oxygen intermediates by activated macrophages.

Glucocorticoid-induced impairment of macrophage antimicrobial activity: mechanisms and dependence on the state of activation.

Experimental observations indicate that tissue macrophages deployed in great numbers at critical anatomic sites such as the liver, spleen, and lung are major targets for glucocorticoids compromising natural resistance of the host. Therapeutic concentrations of glucocorticoids appear to prevent destruction of microorganisms ingested by macrophages without interfering with phagocytosis, phagolysosomal fusion, and/or secretion of reactive oxygen intermediates. These findings indicate that at the cellular level the glucocorticoid target should be sought for in the nonoxidative armature of the phagocyte and that nonoxidative killing systems of resident tissue macrophages play an important role in natural resistance to opportunistic pathogens. Glucocorticoids do not prevent lymphokine-induced activation of oxidative killing systems. Thus, lymphokines such as interferon-gamma can restore the microbicidal activity of macrophages functionally impaired by glucocorticoids. Counterbalance of the suppressive effect of glucocorticoids by lymphokines might only be possible, however, for pathogens susceptible to oxidative killing and not for microorganisms that are more resistant to reactive oxygen intermediates such as Aspergillus spores and Nocardia, opportunists that appear to be particularly associated with hypercortisolism.

Effects of systemic treatment of a hibernator and a non-hibernator with bacterial lipopolysaccharide (LPS)

1. 1. The injection of Salmonella enteritidis lipopolysaccharide (10 μg/kg) increased body temperature in guinea pigs, with a peak of + 1.8 ± 0.2° C above baseline. Euthermic golden hamsters did not develop a thermal response (+ 0.6 ± 0.6°C). On the contrary, golden hamsters injected with high doses of endotoxin (500 μg/kg) produced a significant fever. On the basis of these findings the effects of lipopolysaccharide (LPS) injected systematically on the secretion of reactive oxygen intermediates by phagocytes—as detected by chemiluminescence (CL)—were studied. 2. 2. The phagocytes in blood of guinea pigs but not of golden hamsters were suppressed in their capacity to generate toxic oxygen species after LPS treatment in vivo. 3. 3. The results of these studies suggest bactericidal activity of blood phaghocytic cells in the hibernator golden hamster is less profoundly influenced by bacterial lipopolysaccharides than in the non-hibernator guinea pig.

Release of arachidonate and reduction of oxygen. Independent metabolic bursts of the mouse peritoneal macrophage.

Diverse particulate and soluble stimuli trigger two metabolic bursts in mouse peritoneal macrophages important in the inflammatory and/or cytotoxic actions of the cells: release, oxygenation, and further metabolism of arachidonic acid from endogenous phospholipids and reduction of molecular oxygen to reactive intermediates. We tested the hypothesis that the release of arachidonic acid or formation of its metabolites are obligatory intermediate steps in triggering the NADPH oxidase that reduces O2 to O-2. With phorbol diesters as stimuli, the following inhibitors of phospholipase A2 and lipoxygenase suppressed release of H2O2 at nontoxic concentrations (microM range): p-bromophenacyl bromide, quinacrine, eicosatetraenoic acid, nordihydroguaiaretic acid, and phenidone. Indomethacin and acetylsalicylic acid were ineffective. However, the suppressive effect of the first five agents on H2O2 release could be attributed to their suppression of macrophage glucose uptake at the same concentrations, a previously unrecognized effect of these compounds. Further, concanavalin A, wheat germ agglutinin, and thrombin each stimulated abundant arachidonate release without H2O2 release. Finally, noncytolytic concentrations of cycloheximide and/or emetine suppressed arachidonate release without affecting H2O2 secretion triggered either by phorbol esters or zymosan. Release and metabolism of arachidonic acid and secretion of reactive oxygen intermediates appear to be two frequently coincident but mutually independent metabolic pathways in the mouse peritoneal macrophage.

Secretion of toxic oxygen products by macrophages: regulatory cytokines and their effects on the oxidase.

We are attempting to identify cytokines that regulate macrophage secretion of reactive oxygen intermediates (ROI) and to analyse the biochemical basis of their effects. In both humans and mice, interferon-gamma (IFN-gamma) appears to be the chief factor secreted by clonally unselected lymphocytes that enhances macrophage oxidative metabolism and antiprotozoal activity. In vivo administration of recombinant IFN-gamma enhances the ROI secretory capacity of monocytes in humans, and the secretion of ROI and killing of protozoa by peritoneal macrophages in mice. A protein secreted by murine tumours and certain non-malignant cells exerts opposing effects. This macrophage deactivation factor (MDF) both blocks the induction of activation by IFN-gamma and reverses pre-existent activation. MDF action is non-toxic and selective, suppressing the secretion of ROI, killing of intracellular protozoa, and expression of Ia antigen, without inhibiting secretion of several other products, or synthesis of protein, ingestion of particles or adherence to culture vessels. The suppressive effect of MDF is reversed over several days after its removal. This reversal is hastened by IFN-gamma. Profound suppression of oxidative metabolism accompanies the differentiation of murine monocytes into Kupffer cells. The capacity of Kupffer cells to secrete ROI and kill intracellular protozoa remains deficient even after exposure to IFN-gamma. Thus, four states of macrophage activation can provisionally be discerned: the transition of mouse peritoneal macrophages from the non-activated to the activated state is accompanied by a ninefold increase in affinity of the superoxide-producing enzyme for NADPH, without a marked increase in cellular Vmax or content of cytochrome b559. The MDF-induced transition of mouse peritoneal macrophages from the activated to the deactivated state is accompanied by both an increase in Km and a decrease in apparent V max of the oxidase. There are no changes in the phorbol myristate acetate receptor number or affinity, glucose transport, NADPH levels, cytochrome b559 content, catalase (EC 1.11.1.6) GSH, GSH peroxidase (EC 1.11.1.9), GSH reductase (EC 1.6.4.2) or myeloperoxidase, consistent with the suppressed ROI secretory capacity and antiprotozoal activity of these cells. The Kupffer cell, whose non-responsiveness to IFN-gamma may mark it as inactivated, appears to lack detectable NADPH oxidase activity, despite the probable presence of cytochrome b559, and in this regard differs from both non-activated and deactivated macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)

Administration of recombinant interferon gamma to cancer patients enhances monocyte secretion of hydrogen peroxide.

Recombinant interferon gamma (rIFN-gamma) activates macrophage antimicrobial and antitumor functions and related metabolic processes, including secretion of reactive oxygen intermediates in mice and in cultured mouse and human macrophages. To look for similar actions in man, we monitored the H2O2 secretory capacity of monocytes from cancer patients receiving intravenous rIFN-gamma at 0.1, 0.5, or 1.0 mg/m2 of body area over 6 hr daily or over 1 hr on alternate days. Monocytes taken just before the first infusion served as controls and were comparable to normal donor monocytes in secretion of H2O2. Monocytes from 11 of the 13 subjects (85%) studied through 20 treatment cycles responded to rIFN-gamma with elevation in H2O2 secretion in greater than or equal to 67% of the tests conducted greater than 1 hr after the start of treatment. Five of the five subjects tested had monocytes with diminished H2O2 secretory capacity when tested immediately after a 1-hr infusion of rIRN-gamma, at which time the amount of adherent mononuclear cell protein recovered from the blood averaged only 24% of the control. At all other times tested (from 6 hr to 5 days after infusion) combined results for all subjects showed enhancement of H2O2 releasing capacity. Statistically significant mean increases ranged from 1.4- to 2.8-fold above the control and included the sets in which monocytes collected 24 hr following a single infusion were assayed the same day or the next. By the criterion of enhanced H2O2 secretory capacity, the ability of rIFN-gamma to activate mononuclear phagocytes is manifest upon its administration to patients with advanced malignancy.

Rapid killing of actinomycin D-treated tumor cells by human monocytes. II. Cytotoxicity is independent of secretion of reactive oxygen intermediates and is suppressed by protease inhibitors.

Pretreatment with Actinomycin D (ActD, 1 microgram/ml for 3 hr) rendered WEHI 164 tumor cells susceptible to killing by human monocytes in a 6-hr 51Cr release assay. The present study was designed to elucidate the role of reactive oxygen intermediates (ROI) and of proteolytic enzymes in this reactivity. ActD-treated WEHI 164 cells did not trigger any measurable release of O-2 or H2O2 from monocytes. Monocytes exposed to phorbol-12-myristate-13-acetate, which enhanced release of ROI, did not show augmented killing of ActD-treated tumor cells. Scavengers of oxygen metabolites (catalase, superoxide dismutase, gluthatione, and mannitol), which inhibited ROI-mediated PMA-induced monocyte cytotoxicity against erythrocytes, did not affect monocyte killing of ActD-treated WEHI 164 cells. Enzymatically generated ROI with xanthine/xanthine-oxidase glucose/glucose-oxidase did not show preferential killing of ActD-treated WEHI 164 cells. Two patients with chronic granulomatous disease had normal levels of monocyte cytotoxicity against ActD-treated tumor cells. To determine the possible role of proteolytic enzymes in mediating this reactivity, we studied various antiproteases. Organophosphorous agents (DFP and PMSF), chloromethyl-ketone derivatives of tosylamino acids (TLCK and TPCK), Actinomyces products (pepstatin and chymostatin), and the synthetic protease substrate TAME inhibited monocyte-mediated cytotoxicity against ActD-treated WEHI 164 cells. The macromolecular protease inhibitors alpha-1 antitrypsin, bovine pancreatic trypsin inhibitor (BPTI), soybean trypsin inhibitor, and the synthetic protease substrate ATEE had little effect on monocyte cytotoxicity. When monocytes were preincubated with drugs for 1 hr and washed, TLCK, TPCK, and PMSF inhibited cytolysis, whereas the less effective chymostatin and TAME and the inactive BPTI had no effect under these conditions. Inhibition by preincubation with TLCK, PMSF, and TPCK was completely reversed after 6 hr of culture. Supernatants of monocyte cultures had lytic activity against ActD-treated WEHI 164 but not against untreated cells. Antiproteases inhibited the lytic activity of monocyte supernatants. These results strongly suggest that ROI do not play a critical role in monocyte-mediated rapid killing of drug-treated tumor cells, and that proteolytic enzymes are involved in this reactivity.

Murine Kupffer cells. Mononuclear phagocytes deficient in the generation of reactive oxygen intermediates.

Murine Kupffer cells (KC) were isolated by a high yield collagenase perfusion technique. The morphology, surface markers, and secretory products were typical of macrophages in other tissues. However, KC released negligible levels of H2O2 and O-2, in contrast to peritoneal macrophages. KC oxygen consumption was not increased by agents triggering a respiratory burst in peritoneal cells. Moreover, KC capacity to secrete reactive oxygen intermediates (ROI), in contrast to Ia antigen expression, was not enhanced by exposure to lymphokines or recombinant gamma interferon. The selective defect in KC oxidative response was paralleled by impaired in vitro killing of Toxoplasma gondii trophozoites and Leishmania donovani promastigotes and amastigotes. Deficient secretion of ROI by KC might protect hepatocytes and erythrocytes from injury during endocytosis by KC, but might render the liver more susceptible to parasitization by organisms that are primarily killed through oxygen-dependent mechanisms.