Macrophage activation to kill Leishmania tropica: kinetics of macrophage response to lymphokines that induce antimicrobial activities against amastigotes

Lymphokine (LK) treatment of resident peritoneal macrophages from C3H/HeN mice induced two antimicrobial activities against Leishmania tropica: increased resistance of activated macrophages to infection with amastigotes and intracellular destruction of those parasites that entered activated cells. The onset and duration of these two antimicrobial activities were quite different. Resistance to infection was observed as early as 4 hr after the addition of LK, became maximal at 8 hr, and persisted in a subpopulation of treated cells for as long as 72 hr. In contrast, intracellular killing occurred with as little as 4 hr of LK treatment after infection, and maximal killing was observed in cultures exposed to LK 24 hr. Intracellular killing capacity of lymphokine-treated cells was progressively lost in macrophages treated longer than 12 hr before exposure to parasites. This decay in ability to destroy intracellular L. tropica was also seen in macrophages cultured longer than 12 hr before LK treatment, and may reflect loss of macrophage responsiveness to LK with increasing time in vitro. Thus, treatment of macrophages with lymphokines induced both a stable change in cell-parasite interactions, resistance to infection, and a short-lived capacity to destroy intracellular amastigotes.     

Macrophages in resistance to rickettsial infection: macrophage activation in vitro for killing of Rickettsia tsutsugamushi.

Rickettsia tsutsugamushi, strain Gilliam, replicates in cultures of resident peritoneal macrophages from BALB/c mice. Macrophage cultures treated with culture supernatants of spleen cells from rickettsial-infected mice stimulated with heat-killed rickettsiae markedly suppressed macrophage infection by rickettsiae. Rickettsiacidal activity of activated macrophages was dependent upon both lymphokine concentration and time of incubation in lymphokines. Treatment of macrophage cultures with lymphokines before exposure to viable rickettsiae resulted in an immediate decrease in percent macrophages infected and numbers of viable intracellular rickettsiae. In these cultures, enhanced intracellular killing was also apparent with further incubation (24 hr). The immediate effect of lymphokine-pretreated macrophages was dissociated from intracellular killing by infecting macrophage cultures first and adding lymphokines after infection. In these cultures, both percent macrophages infected and titers of viable intracellular rickettsiae were dramatically reduced as well.     

Macrophage activation to kill Leishmania tropica: defective intracellular killing of amastigotes by macrophages elicited with sterile inflammatory agents

Resident peritoneal macrophages and macrophages elicited by injection of C3H/HeN mice with sterile inflammatory agents were exposed to amastigotes of Leishmania tropica in vitro and treated with lymphokines. Resident macrophages developed the capacity to kill intracellular parasites; microbicidal activity of activated resident cells ranged between 60 and 80%. In contrast, inflammatory macrophages responded poorly to lymphokines for intracellular killing of amastigotes; microbicidal activity of cells elicited with chronic inflammatory agents ranged between 0 and 45%. Defective intracellular killing of L. tropica by inflammatory macrophages was independent of the agent used to elicit the cells, but was clearly associated with the number of immature macrophages in the population. That intracellular killing capacity may reflect the presence of a killing mechanism in tissue-derived cells that is not yet developed in undifferentiated macrophages is supported by studies with peripheral blood monocytes: these cells were also incapable of eliminating intracellular amastigotes in the presence of potent activating factors. These observations on inflammatory macrophage interactions with amastigotes may provide important insights into the chronic nature of leishmanial disease.     

Differential susceptibility of activated macrophage cytotoxic effector reactions to the suppressive effects of transforming growth factor-beta 1

We examined the effects of TGF-beta 1 on induction of several activated macrophage antimicrobial activities against the protozoan parasite Leishmania, and on induction of tumoricidal activity against the fibrosarcoma tumor target 1023. TGF-beta by itself did not affect the viability of either the intracellular or extracellular target in concentrations up to 200 ng/ml. As little as 1 ng/ml TGF-beta, however, suppressed more than 70% of the intracellular killing activity of macrophages treated with lymphokines. In contrast, more than 100 ng/ml TGF-beta was required to suppress intracellular killing by cells activated with an equivalent amount of recombinant IFN-gamma. Addition of TGF-beta for up to 30 min after exposure to activation factors significantly reduced macrophage killing of intracellular parasites. Pretreatment of macrophages with TGF-beta was even more effective: treatment of cells with TGF-beta for 4 h before addition of activation factors abolished all macrophage intracellular killing activity. Regardless of treatment sequence, however, TGF-beta had absolutely no effect, at any concentration tested, on activated macrophage resistance to infection induced by lymphokines or by the cooperative interaction of IFN-gamma and IL-4. Effects of TGF-beta on tumoricidal activity of activated macrophages was intermediate to that of its effects on intracellular killing or resistance to infection. Lymphokine-induced tumor cytotoxicity was marginally (25%) affected by TGF-beta; 200 ng/ml was able to suppress IFN-gamma-induced tumoricidal activity by 40%. Thus, TGF-beta dramatically suppressed certain activated macrophage cytotoxic effector reactions, but was only partially or not at all effective against others, even when the same activation agent (IFN-gamma) was used. The biochemical target for TGF-beta suppressive activity in these reactions may be the pathway for nitric oxide production from L-arginine, because TGF-beta also inhibited the generation of nitric oxide by cytokine-activated macrophages.     

Macrophage activation for microbicidal activity against Leishmania major: inhibition of lymphokine activation by phosphatidylcholine-phosphatidylserine liposomes

Resident peritoneal macrophages from untreated mice develop microbicidal activity against amastigotes of the protozoan parasite Leishmania tropica (current nomenclature = Leishmania major) after in vitro exposure to LK from antigen-stimulated leukocyte culture fluids. This LK-induced macrophage microbicidal activity was completely abrogated by addition of 7:3 phosphatidylcholine: phosphatidylserine liposomes. Liposome inhibition was not due to direct toxic effects against the parasite or macrophage effector cell; factors in LK that induce macrophage microbicidal activity were not adsorbed or destroyed by liposome treatment. Other phagocytic particles, such as latex beads, had no effect on microbicidal activity. Moreover, liposome inhibition of activated macrophage effector function was relatively selective: LK-induced macrophage tumoricidal activity was not affected by liposome treatment. Liposome inhibition was dependent upon liposome dose (5 nmoles/culture) and time of addition of leishmania-infected, LK-treated macrophage cultures. Addition of liposomes through the initial 8 hr of culture completely inhibited LK-induced macrophage microbicidal activity; liposomes added after 16 hr had no effect. Similarly, microbicidal activity by macrophages activated in vivo by BCG or Corynebacterium parvum was not affected by liposome treatment. Liposome treatment also did not affect the increased resistance to infection induced in macrophages by LK. These data suggest that liposomes interfere with one or more early events in the induction of activated macrophages (macrophage-LK interaction) and not with the cytotoxic mechanism itself (parasite-macrophage interaction). These studies add to the growing body of data that implicate cell lipid in regulatory events controlling macrophage effector function.     

Macrophage activation to kill Leishmania tropica: characterization of P/J mouse macrophage defects for lymphokine-induced antimicrobial activities against Leishmania tropica amastigotes

Macrophages from P/J mice demonstrated both quantitative and qualitative defects in lymphokine (LK)-induced activated macrophage antileishmanial effector reactions: a) these cells recognized the same LK signals that generated resistance to infection in responsive C3H/HeN macrophages, but more signal was required to observe maximal activity; b) LK-induced intracellular destruction of Leishmania tropica by P/J macrophages was minimal (less than 20%), and was induced by only one of three LK signals that regulate antimicrobial activities in C3H/HeN macrophages. The defective microbicidal activity of P/J macrophages observed with LK activation in vitro could also be demonstrated in vivo. Macrophages from P/J mice exposed to the macrophage-activating agent Mycobacterium bovis strain BCG in vivo were capable of restricting the intracellular replication of L. tropica but could not eliminate intracellular parasites, even with further incubation with LK during the 72-hr culture period. The defect of P/J macrophages for intracellular destruction of L. tropica, then, occurred in the activation sequence before the triggering stage that characterizes the macrophage defect of C3H/HeJ mice. Genetic regulation of the P/J macrophage defect appears to be by a single autosomal gene, with defective microbicidal activity as a recessive trait in these animals.     

Macrophage activation for tumor cytotoxicity: induction of tumoricidal macrophages by supernatants of PPD-stimulated Bacillus Calmette-Guérin-immune spleen cell cultures.

Resident peritoneal macrophages from normal mice were activated for tumor cytotoxicity in vitro by co-cultivation with BCG1-immune spleen cells and PPD and by incubation with supernatants of PPD-stimulated BCG-immune spleen cell cultures (lymphokine supernatants). Lymphokine activation of macrophages occurred in unfractionated PC suspensions as well as in macrophage monolayers depleted of nonadherent PC. Tumor cytotoxicity by lymphokine-activated macrophages was evident by 3 to 4 hr of culture in active supernatants, reached maximal levels by 8 to 12 hr. and was absent by 20 hr. Continued incubation in lymphokines or even re-exposure after washing did not maintain macrophage cytotoxicity. The capacity of normal resident macrophages to be activated by lymphokines in vitro progressively decreased and was absent by 20 hr in culture. This decrease did not necessarily reflect cell death; macrophage viability as estimated by exclusion of trypan blue or by phagocytic responses did not change over the 20-hr culture period. The short lived nature of both macrophage tumoricidal capacity and capacity of precursor cells to be activated by lymphokines may function as negative feedback mechanisms in immune reactions.     

Antileishmanial activities of macrophages from C3H/HeN and C3H/HeJ mice treated with Mycobacterium bovis strain BCG

C3H/HeN and C3H/HeJ mice were infected ip with viable BCG, a macrophage-activating agent, and their peritoneal exudate macrophages exposed to Leishmania tropica amastigotes. Macrophages from BCG-infected C3H/HeN mice had both leishmanicidal activities described for lymphokine activation of C3H/HeN macrophages in vitro: increased resistance to L. tropica infection, followed by intracellular killing of the parasite. Macrophages from BCG-infected C3H/HeN mice were also activated to kill tumor cells in vitro. In contrast, macrophages from BCG-treated C3H/HeJ mice were not resistant to L. tropica infection, did not kill intracellular amastigotes over 72 hr in culture, and were not cytotoxic to tumor cells.     

Colony-stimulating factors and regulation of macrophage tumoricidal and microbicidal activities

Conditioned medium from antigen- or mitogen-stimulated spleen cells, lymphokines, contained factors that induced formation of granulocyte and macrophage colonies in cultures of bone marrow cells (CSF). Lymphokines also contained factors that induced macrophage non-specific tumoricidal activity against fibrosarcoma 1023, antibody-dependent tumoricidal activity against lymphoma 18-8, and antimicrobial activities against amastigotes of the protozoan parasite, Leishmania tropica. The factors that regulated macrophage effector functions, however, were different from those that induced colony formation, and could be distinguished from CSF by Sephadex gel chromatography or heat sensitivity. To further analyze a role for CSF in induction of macrophage effector activities, conditioned medium from several nonlymphoid cell sources (L-929, WEHI-3, and endotoxin-treated lung cells) were assayed for CSF activities and capacity to induce tumoricidal and microbicidal activities. Conditioned medium that contained either macrophages CSF (CSF-1) or the factor that induced formation of both macrophage and granulocyte colonies failed to activate macrophages for effector activities against fibrosarcoma 1023, lymphoma 18-8, and L. tropica amastigotes (either resistance to infection or intracellular destruction). These data suggest that CSF has no direct role in activation of macrophages for tumoricidal and microbicidal activities against these targets.     

Suppression of macrophage antimicrobial activity by a tumor cell product

Medium conditioned by tumor cells (TCM) and certain nonmalignant cells contains a trypsin-sensitive factor that suppresses macrophage oxidative metabolism. Because the killing of intracellular pathogens such as Toxoplasma gondii and Leishmania donovani by macrophages is largely oxygen-dependent, we tested the effect of TCM on the antiprotozoal activity of mouse peritoneal macrophages. After 24 hr of cultivation with TCM, in vivo and in vitro activated macrophages could no longer kill toxoplasmas or inhibit their replication. In vivo administration of TCM resulted in similar impairment. The leishmanicidal activity of resident and activated macrophages, when measured 6 hr after infection, was markedly suppressed by in vitro exposure to TCM. The addition of exogenous H2O2 in the form of glucose-glucose oxidase reconstituted the capacity of TCM-exposed macrophages to kill L. donovani promastigotes as quickly as control cells. Thus, TCM appears to deactivate macrophages by the functional criteria of suppressed antitoxoplasmal and antileishmanial activity, as well as by the biochemical criterion of suppressed oxidative metabolism.     

Functional analysis of cloned macrophage hybridomas. VII. Modulation of suppressor T cell-inducing activity

We have previously characterized a macrophage hybridoma clone, termed clone 59, which induced immunity but consistently failed to induce Ts responses. Macrophage 59 cells were cultured with supernatants from several activated T cell clones to determine if lymphokines could modulate the activity of this macrophage hybridoma to generate effector Ts. Culture supernatants from Th1 clones and from one atypical IL-4 and IFN-gamma-producing T cell clone successfully modulated clone 59 cells to induce effector Ts cells. In contrast, supernatants from activated Th2 cells failed to generate Ts-inducing activity in macrophage 59 cells. Culture with recombinant derived IFN-gamma was sufficient to cause modulation of Ts-inducing activity in macrophage 59 cells. The data imply that the differential functional activities ascribed to various macrophage hybridoma clones reflect macrophage heterogeneity instead of independent macrophage lineages. The suppression induced by clone 59 macrophages was genetically restricted to the putative I-J region. The ability of IFN-gamma containing supernatants to endow macrophage 59 with the capacity to induce effector suppressor cells was specifically abrogated by addition of an anti-IJk-idiotype antibody, which also reacts with IJ-interaction molecules, indicating that the mechanism of modulation most likely involves expression of IJ-interaction molecule determinants on antigen presenting cells.     

Activation of mouse peritoneal macrophages in vitro and in vivo by interferon-gamma

To determine the role of IFN-gamma in the activation of resident mouse peritoneal macrophages, crude macrophage-activating lymphokines were incubated with a monoclonal anti-murine IFN-gamma antibody. This treatment abolished the capacity of mitogen-induced lymphokines to enhance either H2O2 release or activity against the intracellular protozoa Toxoplasma gondii and Leishmania donovani. All macrophage-activating factor detected by these assays was also removed by passing the lymphokines over a Sepharose column to which the monoclonal anti-IFN-gamma antibody had been coupled. Therefore, pure murine rIFN-gamma was tested both in vitro and in vivo as a single activating agent. After 48 hr of pretreatment in vitro with 0.01 to 1 antiviral U/ml, macrophage H2O2-releasing capacity was enhanced an average of 6.4-fold; half-maximal stimulation was induced by 0.03 U/ml. Resident macrophages infected with T. gondii half-maximally inhibited parasite replication after 24 hr of preincubation with 0.14 U/ml of rIFN-gamma, and near complete inhibition was achieved by pretreatment with 100 U/ml. Half-maximal leishmanicidal activity was induced by 0.08 U/ml of rIFN-gamma, and 67 to 75% of intracellular L. donovani amastigotes were killed after macrophages were preincubated with 10 to 100 U/ml. Eighteen hours after parenteral injection of rIFN-gamma, peritoneal macrophages displayed a dose-dependent enhancement of H2O2-releasing capacity and antiprotozoal activity. Half-maximal enhancement required 85 to 250 U or rIFN-gamma given i.p. Peritoneal macrophages were also activated by rIFN-gamma injected i.v. and intramuscularly. These results suggest that, in the mouse model, IFN-gamma is likely to be a primary factor within mitogen-induced lymphokines responsible for activating macrophage oxidative metabolism and antiprotozoal activity, and indicate that rIFN-gamma is a potent activator of these effector functions both in vitro and in vivo. These findings provide a rationale for evaluating rIFN-gamma in the treatment of systemic intracellular infections, and indicate that murine models are appropriate for such studies.     

ATP-mediated killing of intracellular mycobacteria by macrophages is a P2X(7)-dependent process inducing bacterial death by phagosome-lysosome fusion.

Mycobacterium tuberculosis survives within host macrophages by actively inhibiting phagosome fusion with lysosomes. Treatment of infected macrophages with ATP induces both cell apoptosis and rapid killing of intracellular mycobacteria. The following studies were undertaken to characterize the effector pathway(s) involved. Macrophages were obtained from p47(phox) and inducible NO synthase gene-disrupted mice (which are unable to produce reactive oxygen and nitrogen radicals, respectively) and P2X(7) gene-disrupted mice. RAW murine macrophages transfected with either the natural resistance-associated macrophage protein gene 1 (Nramp1)-resistant or Nramp1-susceptible gene were also used. The cells were infected with bacille Calmette-Guérin (BCG), and intracellular mycobacterial trafficking was analyzed using confocal and electron microscopy. P2X(7) receptor activation was essential for effective ATP-induced mycobacterial killing, as its bactericidal activity was radically diminished in P2X(7)(-/-) macrophages. ATP-mediated killing of BCG within p47(phox-/-), inducible NO synthase(-/-), and Nramp(s) cells was unaffected, demonstrating that none of these mechanisms have a role in the ATP/P2X(7) effector pathway. Following ATP stimulation, BCG-containing phagosomes rapidly coalesce and fuse with lysosomes. Blocking of macrophage phospholipase D activity with butan-1-ol blocked BCG killing, but not macrophage death. ATP stimulates phagosome-lysosome fusion with concomitant mycobacterial death via P2X(7) receptor activation. Macrophage death and mycobacterial killing induced by the ATP/P2X(7) signaling pathway can be uncoupled, and diverge proximal to phospholipase D activation.     

Kinetics and requirements for activation of macrophages for fungicidal activity: effect of protein synthesis inhibitors and immunosuppressants on activation and fungicidal mechanism.

Peritoneal-and pulmonary macrophages can be activated in vitro with lymphokines (LK) or IFN-gamma, without exogenous lipopolysaccharide, for fungicidal activity against several pathogenic fungi. However, neither the biochemical nor metabolic events of the activation process or of the effector phase have been defined. In the present work we sought to elucidate these events with time-course studies using inhibitors of protein synthesis as well as immunosuppressive agents. We found that protein synthesis inhibitors abrogated the activation process, because cycloheximide (CHX) (1-2 micrograms/ml) prevented activation of macrophages for fungicidal activity against Candida albicans, Blastomyces dermatitidis, and Paracoccidioides brasiliensis. Blocking of the activation process by CHX was not due to macrophage cytotoxicity, and CHX did not impair the ability of nonactivated macrophages to kill Candida parapsilosis. In kinetic studies we showed that activation of macrophages was induced in 4 hr of LK treatment and that CHX had no effect if added after this time. In contrast to CHX, therapeutic concentrations of hydrocortisone (HC), such as less than or equal to 5 micrograms/ml, or cyclosporin A (CsA), 5 micrograms/ml, did not significantly inhibit LK activation of macrophages for killing of fungi. In the effector phase, the fungicidal capacity of activated macrophages in short-term (less than or equal to 4 hr) killing assays could not be abrogated by CHX (5 micrograms/ml), HC (100 micrograms/ml), or CsA (10 micrograms/ml). These results demonstrate that the activation but not the effector mechanism of macrophages for fungicidal activity is blocked by inhibition of protein synthesis. In contrast, therapeutic concentrations of HC or CsA may not interfere with activation of macrophages or their killing mechanisms, thus providing a rationale for antifungal immunotherapy in certain clinical situations (e.g., infection in the immunosuppressed patient).     

Induction of nitrite/nitrate synthesis in murine macrophages by BCG infection, lymphokines, or interferon-gamma

Macrophage synthesis of nitrite and nitrate after activation by BCG infection or by treatment in vitro with both T cell-derived (lymphokines (LK) or recombinant murine interferon-gamma (IFN-gamma] and bacterial (lipopolysaccharide (LPS) and heat-killed bacillus Calmette-Guerin (hk BCG] agents was studied by using macrophages from C3H/He and C3H/HeJ mice. Spleen and peritoneal macrophages isolated from BCG-infected donors that were producing nitrate continued to synthesize nitrite and nitrate in culture. LPS treatment in vitro (25 or 50 micrograms/ml) additionally increased this nitrite/nitrate synthesis. Thioglycolate-elicited macrophages from non-infected C3H/HeJ mice treated with LK also produced nitrite/nitrate, and concurrent LPS (0.1 to 50 micrograms/ml) treatment resulted in enhanced synthesis. Recombinant IFN-gamma also stimulated nitrite/nitrate synthesis by C3H/He and CeH/HeJ macrophages as did LPS (C3H/He only) and hk BCG. When given concurrently with either LPS or hk BCG, IFN-gamma enhanced C3H/He and C3H/HeJ macrophage nitrite/nitrate synthesis over that produced by macrophages treated with either LPS or hk BCG alone. Macrophages activated in vitro exhibited a 4 to 12 hr lag time before engaging in nitrite/nitrate synthesis, which then proceeded for 36 to 42 hr at linear rates. Daily medium renewal did not alter the synthesis kinetics but increased the total amount of nitrite/nitrate produced. Nitrate and nitrite were stable under the conditions of culture and when added did not influence additional macrophage synthesis. Taken together, these results indicate that T cell lymphokines and IFN-gamma are powerful modulators of macrophage nitrite/nitrate synthesis during BCG infection and in vitro, and nitrite/nitrate synthesis appears to be common property of both primed and fully activated macrophage populations.     

Resistance to macrophage-mediated killing as a factor influencing the pathogenesis of chronic cutaneous leishmaniasis

Cutaneous leishmaniasis can be either a spontaneously healing or chronic disease, depending upon the strain of parasite and the immunological status of the host. We have investigated parasite factors responsible for the variable pathogenesis observed in leishmanial infections by testing the sensitivity of several leishmanial strains to intracellular killing in lymphokine (LK) activated mouse macrophages. Significant microbicidal activity against Leishmania tropica, a strain which heals in C57BL/6 (B6) mice, was found. In contrast, a strain (Maria) which has previously been shown to induce chronic nonhealing cutaneous lesions in B6 mice was resistant to killing in activated macrophages. This resistance to killing was observed in macrophages activated by LK obtained from either Bacille Calmette-Guérin-, L. tropica, or the Maria strain infected mice. The inability of LK activated macrophages to kill the Maria strain was shown not to be due to parasite induced inhibition of killing mechanisms, since Maria strain infected, LK treated macrophages exhibited tumoricidial activity similar to uninfected macrophages. Furthermore, LK activated macrophages simultaneously infected with the Maria strain and another intracellular pathogen, Toxoplasma gondii, killed Toxoplasma, but not the Maria strain. Temperature was also found to significantly influence the multiplication and killing of Leishmania parasites. As would be expected from their cutaneous nature, L. tropica and Maria strain parasites multiplied better at 35 degrees C than at 37 degrees C. Also consistent with the failure of cutaneous strains to visceralize in immunocompetent mice was the observation that the killing of leishmanial parasites was enhanced at the higher temperature. Thus, the temperature dependent growth capacity and sensitivity to killing of a given leishmanial strain in macrophages may be important factors influencing the pathogenesis of cutaneous leishmaniasis.     

Activation of tissue macrophages from AIDS patients: in vitro response of AIDS alveolar macrophages to lymphokines and interferon-gamma

To test the hypothesis that tissue macrophages from AIDS patients have no intrinsic defects in either antimicrobial activity or in the capacity to respond to T cell-derived activating stimuli, alveolar macrophages from 11 patients were treated with crude lymphokines produced by healthy donors. After 72 hr of pretreatment with 10% mitogen- or antigen-induced crude lymphokines (which contained 300 U/ml of interferon-gamma [IFN-gamma]), AIDS alveolar macrophages generated twofold to threefold more H2O2 and readily inhibited the replication of the intracellular pathogens Toxoplasma gondii and Chlamydia psittaci. These responses were indistinguishable from those displayed by activated alveolar cells from 12 non-AIDS patients and three healthy volunteers. As judged by the abrogating effects of a neutralizing anti-human IFN-gamma monoclonal antibody, lymphokine-induced alveolar macrophage activation appeared to be largely IFN-gamma-dependent; thus, macrophages were also stimulated with recombinant (r)IFN-gamma alone. Seventy-two hours of treatment with 300 U/ml of rIFN-gamma resulted in both enhanced oxidative and antimicrobial activity comparable to that achieved by crude lymphokines, and the responsiveness of AIDS alveolar macrophages to rIFN-gamma was identical to control cells. These in vitro results suggest that tissue mononuclear phagocytes from AIDS patients a) are free of apparent defects in intracellular antimicrobial activity, b) are fully responsive to activating T cell products, and c) support the use of IFN-gamma as a potential macrophage-activating immunotherapeutic agent in AIDS-related opportunistic infections.     

In vitro killing of schistosomula of Schistosoma mansoni by BCG and C. parvum-activated macrophages.

Resistance to Schistosoma mansoni infection in the mouse has been induced either specifically by a primary infection with this parasite or nonspecifically by a variety of immunostimulants such as BCG. In the present study we developed an in vitro system to examine the effector mechanism of nonspecifically induced resistance. Activated macrophage monolayers obtained from BCG- or Corynebacterium parvum treated mice killed a respective mean 32 +/- 6% and 48 +/- 5% of schistosomula after 24 hr incubation. The killing of the parasites was verified by their inability to mature to adult worms upon injection into normal mice. The activated macrophage-mediated killing was related to cell:parasite ratio, and was partially lost if the macrophage monolayers were kept in cultures for 24 hr before incubation with the organism. Supernatants of macrophages cultured in the presence of schistosomula killed a mean of 51 +/- 3% of the organisms whereas those from cells cultured alone resulted in a mean killing of 25 +/- 3%. Furthermore, toxic supernatants could be generated equally well on incubation with S. mansoni schistosomula or Trichinella spiralis larvae. Our data show that activated macrophage monolayers through soluble mediators destroy a significant proportion of the multicellular parasite S. mansoni schistosomula in vitro.     

Suppression of lymphokine production: II. Macrophage-dependent inhibition of production of macrophage activating factor

The ability of different populations of macrophages to affect the production of macrophage activating factor (MAF) by stimulated T lymphocytes was investigated. We found that activated macrophages, infiltrating MSV-induced regressing tumors or macrophages recovered from the peritoneum of mice injected with Corynebacterium parvum, were able to actively suppress the production of MAF. MAF production by antigen-stimulated MSV-immune or -alloimmune spleen cells and by normal spleen cells stimulated by Con A was susceptible to macrophage-dependent suppression to a similar extent. In contrast, resident macrophages or those elicited by light mineral oil or proteose-peptone did not affect MAF production. While suppressor macrophages added at the time of the lymphocyte stimulation inhibited MAF production, the same cells added 4–6 hr after stimulation were ineffective. Therefore, it seems that the macrophages suppressed the early events of lymphocyte activation leading to MAF production. Suppressor macrophages, by inhibiting MAF production, may limit the expansion of the cytotoxic activity. This regulation of macrophage functions, mediated by the effects of suppressor macrophages on T lymphocytes, could be responsible for an insufficient antitumor cytotoxic response by macrophages.     

Echinococcus multilocularis in the mouse: The in vitro protoscolicidal activity of peritoneal macrophages

Baron R. W. and Tanner C. E. 1977. Echinococcus multilocularis in the mouse: the in vitro protoscolicidal activity of peritoneal macrophages. International Journal for Parasitology7: 489–495. The larvae of Echinococcus multilocularis are susceptible to the protoscolicidal activity of infected A/J mouse peritoneal cells. It is shown that the effector cell in this response is an activated macrophage. Preincubation of protoscolices in ‘immune’ serum increases their susceptibility to the protoscolicidal activity of infected mouse peritoneal cells. Macrophages activated nonspecifically by BCG or Taenia crassiceps infections also exhibit protoscolicidal activity in vitro. The identity of the effector cell was confirmed by scanning electron microscopy. It is shown that ‘immune’ macrophages adhere to and form close cellular contacts with the protoscolex surface. It is concluded that resistance to hydatid infections is mediated by activated macrophages.