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.     

IFN-gamma-activated human alveolar macrophages inhibit the intracellular multiplication of Legionella pneumophila

Human alveolar macrophages activated by human rIFN-gamma inhibit the intracellular multiplication of Legionella pneumophila, an intracellular bacterial pathogen and the agent of Legionnaires' disease. Activation of alveolar macrophages with IFN-gamma is dose dependent; significant inhibition of L. pneumophila multiplication (mean 1.60 +/- 0.20 logs) is achieved consistently with concentrations of IFN-gamma of greater than or equal to 2 x 10(-2) micrograms/ml (220 U/ml). Activation of alveolar macrophages is also time dependent. In macrophages treated continuously after explantation, macrophages infected at 48 to 96 h after explantation are more inhibitory than macrophages infected at 24 h after explantation. In macrophages not treated continuously after explantation but treated for various lengths of time before infection, the longer their exposure to IFN-gamma before infection, the greater the inhibition of L. pneumophila multiplication (96 greater than 72 greater than 48 greater than 24 h). IFN-gamma-activated alveolar macrophages exhibit morphologic signs of activation, including increased size, spreading, and aggregation. This paper demonstrates that a human resident macrophage can be activated with IFN-gamma such that it exhibits enhanced antimicrobial activity against a relevant pathogen.     

Tumor necrosis factor, alone or in combination with IL-2, but not IFN-gamma, is associated with macrophage killing of Mycobacterium avium complex

Organisms belonging to the Mycobacterium avium complex (MAC) are the most common bacterial pathogens in patients with AIDS but factors associated with the activation of cellular defense mechanisms against this atypical mycobacterium have not been defined. Peritoneal macrophages harvested from a chronic MAC infection in C57 black mice are able to kill approximately 86% of intracellular MAC in contrast to 0 to 20% killing by unstimulated human and mouse macrophages in vitro. The availability of human rTNF-alpha, rIFN-gamma, and rIL-2 permitted evaluation of the role of each of these lymphokines/monokines, alone or in combination, in activating macrophages in vitro to kill MAC. Human monocyte-derived macrophages were cultured in vitro, stimulated with rIL-2, rIFN-gamma, or rTNF, and then infected with MAC (serovars 1 and 8). Mouse peritoneal macrophages were harvested, cultured in vitro, and stimulated with rIFN-gamma. rTNF (10(4) U/ml) was associated with a modest increase of intracellular killing of MAC (58 +/- 5%) even when utilized 24 or 48 h after macrophage infection or when administered for 5 consecutive days after infection (78.1 +/- 4%). Both human and murine IFN-gamma were associated with increased intracellular growth of MAC (32 +/- 4% for murine and 38 +/- 3% for human macrophages). However, intracellular killing (53 +/- 6% compared with control) was observed after 6 days of treatment with IFN-gamma. This latter effect was fully blocked by anti-TNF antibody, whereas rIL-2 alone did not augment the intracellular killing of MAC by human macrophages. rTNF plus either rIFN-gamma or rIL-2 triggered significant increases in superoxide anion production, but subsequent MAC killing was no greater than with rTNF alone. Treatment of macrophages with 10 U/ml of rTNF followed by rIL-2 (200 U/ml) was associated with 68% of intracellular killing. TNF seems to be an important monokine, promoting activation of mycobactericidal mechanisms in human macrophages.     

In vitro and in vivo activation of human mononuclear phagocytes by interferon-gamma. Studies with normal and AIDS monocytes

To determine the potential immunotherapeutic role of interferon-gamma (IFN-gamma) as a mononuclear phagocyte-activating agent, we examined the effector cell function of peripheral blood monocytes from healthy donors and acquired immunodeficiency syndrome (AIDS) patients after either in vitro and/or in vivo treatment with recombinant (r) IFN-gamma. When assayed immediately after a 24-hr in vitro pulse with 300 U/ml, normal and AIDS monocytes behaved similarly with little augmentation of their intrinsically high levels of H2O2 release and activity against Toxoplasma gondii; in contrast, activity toward the more resistant intracellular pathogen, Leishmania donovani, was appreciably enhanced by rIFN-gamma. In addition, upon testing 4 to 6 days after in vitro pulsing, both normal and AIDS monocytes showed clear evidence of persistent activation in all three assays. The capacity of IFN-gamma to similarly activate monocytes in vivo was confirmed in all ten treated AIDS patients by examining cells before and after 24-hr infusions of 0.03 and 0.5 mg of rIFN-gamma/square meter (M2) of body surface area. For postinfusion monocytes tested after 1 day in culture, H2O2 release and antitoxoplasma activity were essentially unchanged, but antileishmanial effects were augmented. After 5 to 7 days in culture, monocytes from treated patients showed 3.2- to 5.9-fold increases in H2O2-releasing capacity and increases of 49 to 68% and 35 to 61% in intracellular activity against T. gondii and L. donovani, respectively. These results indicate that the human monocyte can be induced by rIFN-gamma to express signs of both immediate and persistent activation and suggest that, as a direct activator of mononuclear phagocytes, rIFN-gamma may also have potential as an immunotherapeutic agent for patients with intracellular infections.     

In vivo and in vitro activation of alveolar macrophages by recombinant interferon-gamma

In vivo administration of recombinant interferon-gamma (rIFN-gamma) was previously shown to result in activation of the microbicidal activities of peritoneal macrophages (PM phi). Because macrophages at different anatomical sites vary in their functional capacities, we considered it of interest to determine whether administration of murine rIFN-gamma, either in vitro or in vivo, can enhance the microbicidal activity of resident alveolar macrophages (AM phi) and to compare the effects of rIFN-gamma on AM phi and PM phi. After incubation in vitro with rIFN-gamma, the antimicrobial activities of both murine AM phi and PM phi were enhanced, as assessed by their ability to inhibit replication of the intracellular parasite, Toxoplasma gondii. This effect was dose dependent for AM phi over a range of 0.1 to 1 U/ml and for PM phi over a range of 0.5 to 1000 U/ml. In this assay, the minimum dosage required for in vitro activation of AM phi was one-half that required for activation of PM phi, suggesting a greater sensitivity of AM phi to the in vitro activity of rIFN-gamma. Macrophages from both anatomical sites were also activated when rIFN-gamma was administered in vivo. This effect was dose dependent over a range of 10(3) to 10(5) U/mouse. Freshly harvested AM phi and PM phi from mice injected 24 hr earlier with 10(4) U rIFN-gamma by either the i.v. or i.p. routes markedly inhibited intracellular multiplication of Toxoplasma. In contrast, AM phi and PM phi from control mice permitted fourfold to ninefold increases in numbers of intracellular Toxoplasma. The anti-toxoplasma activity of AM phi and PM phi gradually diminished over a period of 3 days when assayed at successive 24 hr periods after a single i.v. injection of rIFN-gamma. At 3 days after injection, a substantial loss of anti-toxoplasma activity was observed with PM phi as compared with controls; residual anti-toxoplasma activity was still demonstrable in AM phi at 3 days. These results demonstrate that in vitro as well as in vivo treatment with rIFN-gamma confers on AM phi an enhanced antimicrobial activity. These findings provide a rationale for evaluating rIFN-gamma in the treatment of pulmonary infections, especially those due to opportunistic pathogens against which AM phi play a major role in host defense.     

Lymphokine stimulation of human macrophage C2 production is partially due to interferon-gamma

Monocyte complement stimulator (MCS), a product of T lymphocytes, is defined by its ability to stimulate the synthesis and secretion of the second complement component (C2) by monocytes. Most macrophage-activating factor (MAF) activity present in lymphokine-rich culture supernatants has recently been found to be due to interferon-gamma (IFN-gamma). We therefore hypothesized that IFN-gamma may have MCS activity as well. We tested recombinant, E. coli-derived, human IFN-gamma (rIFN-gamma) for its effects on C2 production by adherent peripheral blood monocytes and U937 cells, a human monocytic cell line. Recombinant IFN-gamma in concentrations ranging from 0.1 to 300 U/ml (0.003 to 8.8 ng/ml) stimulates C2 production by both cell populations. Exposure of responding cells for at least 24 hr is required for maximal stimulation. To determine the contribution of IFN-gamma toward total MCS activity in crude lymphokine-rich supernatants, we employed a solid-phase immunoabsorption technique with the use of a monoclonal anti-IFN-gamma antibody. This technique removed all IFN-gamma detectable by a sensitive ELISA, but MCS activity was decreased by only 40 to 50%. Additionally, MCS activity of these supernatants did not correlate with IFN-gamma content as determined by ELISA. By using another method to eliminate IFN-gamma activity, acid dialysis destroyed all rIFN-gamma activity, as measured by stimulation of U937 C2 synthesis, but eliminated only 30 to 67% of MCS activity from crude lymphokine preparations. Thus IFN-gamma stimulates C2 production by monocytes and U937 cells and apparently accounts for some, but not all, MCS activity present in lymphokine-rich supernatants. Other lymphokines are present in such supernatants that also possess this activity.     

Tumor necrosis factor-alpha triggers antitoxoplasmal activity of IFN-gamma primed macrophages

IFN-gamma is an important mediator of cellular resistance against microbial pathogens and tumor cells due in part to its potent capacity to activate macrophages for enhanced cytotoxicity. The present study demonstrates that TNF-alpha regulates the expression of enhanced antimicrobial activity by triggering IFN-gamma primed macrophages to kill or inhibit intracellular Toxoplasma gondii. Resident mouse macrophages stimulated with rIFN-gamma at levels up to 2500 U/ml failed to display enhanced antitoxoplasmal activity when cultured in vitro under low endotoxin conditions (less than 10 pg/ml), but were triggered by addition of small amounts of LPS (0.1 ng/ml). A similar requirement for LPS as a second signal necessary to trigger antitoxoplasmal activity was observed when IFN-gamma was administered to mice in vivo. The essential nature of this triggering step allowed us to explore the role of cytokines that act as endogenous regulators of macrophage activation. rTNF-alpha, although unable to confer antitoxoplasmal activity when used alone to treat macrophages, was capable of triggering IFN-gamma-primed macrophages cultured under low endotoxin conditions. The ability of TNF-alpha to trigger IFN-gamma-primed macrophages was blocked by rabbit anti-TNF-alpha polyclonal antisera but was not affected by polymyxin B indicating that TNF-alpha triggering was not due to contamination with LPS. Collectively, these findings demonstrate that TNF-alpha performs an important regulatory role in the expression of enhanced anti-microbial activity by IFN-gamma-primed macrophages.     

Inability of recombinant interferon-gamma to activate the antibacterial activity of mouse peritoneal macrophages against Listeria monocytogenes and Salmonella typhimurium

The effect of recombinant murine interferon-gamma (rIFN-gamma) as single stimulus for the activation of antibacterial activity of macrophages was investigated on the basis of the rate of intracellular killing of Listeria monocytogenes and Salmonella typhimurium by normal and rIFN gamma-activated peritoneal macrophages of CBA and C57BL/10 mice, which differ in natural resistance to infection by these bacteria. Eighteen hours after i.p. injection of 10 to 1 X 10(4) U rIFN-gamma, resident and exudate peritoneal macrophages which had phagocytosed L. monocytogenes or S. typhimurium in vivo, killed both species in vitro just as efficiently as did resident macrophages of normal mice. Similar results were obtained after 18 hr of in vitro incubation of resident or exudate peritoneal macrophages with 0.1 to 1 X 10(4) U/ml rIFN-gamma. Consistent with the in vitro findings, two i.v. injections of 5 X 10(4) U rIFN-gamma did not affect the rate of in vivo proliferation of L. monocytogenes or S. typhimurium in the spleens of mice during the first 2 days after i.v. injection of the bacteria. Compared with the effect on the controls, two i.p. injections of 5 X 10(2) to 5 X 10(4) U rIFN-gamma did not decrease the numbers of viable S. typhimurium in either the peritoneal cell suspension or the spleen 24 hr after i.p. injection of the bacteria. Checking the state of activation of rIFN-gamma-activated macrophages on the basis of two commonly used criteria for macrophage activation showed that rIFN-gamma-activated macrophages inhibited the intracellular replication of Toxoplasma gondii and displayed enhanced O2 consumption and H2O2 release after stimulation with phorbol myristate acetate compared with macrophages from normal CBA and C57BL/10 mice. The present findings show that as single activating stimulus, rIFN-gamma is not capable of activating the antibacterial effector functions of peritoneal macrophages against facultative intracellular pathogens such as L. monocytogenes and S. typhimurium.     

Co-trimoxazole effect on human alveolar macrophages of AIDS patients

Compelling evidence suggests that co-trimoxazole prophylaxis reduces mortality in HIV-infected patients, although it is unclear whether these effects are directly related to antimicrobial activities. We evaluated in vitro phagocytosis and killing of Staphylococcus aureus in alveolar macrophages (AM) obtained from AIDS patients who smoke, treated (n=19) or not treated (n=13) with co-trimoxazole, as compared to non-HIV-infected healthy smokers (n=15). Phagocytosis and killing of Staphylococcus aureus by AM obtained from non-co-trimoxazole treated AIDS patients were significantly lower compared to non-HIV-infected healthy smokers. In contrast, AIDS patients treated with co-trimoxazole prophylaxis showed phagocytosis and killing levels similar to those of healthy controls. These results might help to clarify the observed positive effect of co-trimoxazole on survival in HIV-infected patients.     

Effect of gamma-interferon on phagosome-lysosome fusion in Salmonella typhimurium-infected murine macrophages

Effect of recombinant gamma-interferon (rIFN-gamma) on phagosome-lysosome fusion in Salmonella typhimurium-infected murine macrophages was examined. rIFN-gamma enhanced phagosome-lysosome fusion in macrophages infected with S. typhimurium in a dose-dependent manner, and over a range of 10(2) to 10(3) U/ml of rIFN-gamma exhibited maximum phagosome-lysosome fusion, although phagocytosis was slightly decreased. The enhancement of phagosome-lysosome fusion occurred greater than 3 h post-treatment with rIFN-gamma. Furthermore, the macrophage activation for phagosome-lysosome fusion was found to persist for 4 days even when rIFN-gamma had been removed. These results demonstrate that IFN-gamma may serve as a mediator for the activation of phagosome-lysosome fusion in murine macrophages.     

Recombinant granulocyte-macrophage colony-stimulating factor down-regulates expression of IL-2 receptor on human mononuclear phagocytes by induction of prostaglandin E

Recent studies have shown that normal human alveolar macrophages and blood monocytes, as well as HL-60 and U937 monocyte cell lines, newly express IL-2R after stimulation with rIFN-gamma or LPS. In addition, macrophages transiently express IL-2R in vivo during immunologically mediated diseases such as pulmonary sarcoidosis and allograft rejection. We therefore investigated in vitro factors that modulate macrophage expression of IL-2R. IL-2R were induced on normal alveolar macrophages, blood monocytes, and HL-60 cells using rIFN-gamma (24 to 48 h at 240 U/ml), and cells were cultured for an additional 12 to 24 h with rIL-2 (100 U/ml), recombinant granulocyte-macrophage CSF (rGM-CSF, 1000 U/ml), rGM-CSF plus indomethacin (2 X 10(-6) M), PGE2 (0.1 to 10 ng/ml), 1 X 10(-6) M levels of caffeine, theophylline, and dibutyryl cyclic AMP, or medium alone. IL-2R expression was quantitated by cell ELISA (HL-60 cells) or determined by immunoperoxidase staining (alveolar macrophages, blood monocytes, and HL-60 cells), using anti-Tac and other CD25 mAb. PGE production was assayed by RIA. We found greater than 95% of alveolar macrophages, monocytes, and HL-60 cells expressed IL-2R after rIFN-gamma treatment and remained IL-2R+ in the presence of IL-2R or medium alone. By comparison, greater than 95% of cells induced to express IL-2R became IL-2R- after addition of rGM-CSF, and the culture supernatants from GM-CSF-treated cells contained increased levels of PGE. This inhibition of macrophage IL-2R expression by rGM-CSF was blocked by indomethacin, and IL-2R+ macrophages became IL-2R- after addition of PGE2 alone. These findings indicate GM-CSF down-regulates IL-2R expression by human macrophages via induction of PGE synthesis. Moreover, a similar down-regulation of IL-2R expression was seen after stimulation with caffeine, theophylline, or dibutyryl cyclic AMP. Hence, GM-CSF, PGE, and other pharmacologic agents that act to increase intracellular levels of cAMP may play a modulatory role, antagonistic to that of IFN-gamma on cellular expression of IL-2R by human inflammatory macrophages in vivo.     

Leishmania major amastigotes initiate the L-arginine-dependent killing mechanism in IFN-gamma-stimulated macrophages by induction of tumor necrosis factor-alpha

Macrophages exposed to IFN-gamma and infected with amastigotes of Leishmania major develop the capacity to eliminate the intracellular pathogen. This antimicrobial activity of activated macrophages correlates with the initiation of nitrogen oxidation of L-arginine, yet other reports suggest that two signals are required for induction of this biochemical pathway for effector activity. In the present studies, macrophages treated with up to 100 U/ml IFN-gamma, or 100 ng LPS, or 10(7) amastigotes produced minimal quantities (less than 9 microM) of NO2- and failed to develop cytotoxic effector activities. In contrast, the combination of IFN-gamma and either LPS (greater than 0.1 ng) or amastigotes (10(6) induced high concentrations (much greater than 30 microM) of NO2- and macrophage cytotoxicity against intra- and extracellular targets. The induction of nitrogen oxidation by amastigotes could be dissociated from LPS-induced events by 1) performing the assays in the presence of polymyxin B (which blocked LPS effects, but not amastigote effects), 2) determining the threshold of IFN-gamma required to prime cells for subsequent trigger (1 U/ml for LPS trigger effects; 10-fold higher for amastigotes), and 3) determining the heat sensitivity of the two trigger agents (amastigote effects abolished at 100 degrees C; LPS effects unaffected at this temperature). Further, culture fluids from amastigote-infected macrophages did not contain detectable LPS (less than 6 pg/ml). Possible parasite and cell-associated factors that could contribute to the induction of nitrogen oxidation and cytotoxic activity of IFN-gamma treated macrophages were examined: only certain intact microorganisms, LPS from a variety of bacteria, and the cytokine TNF alpha were effective. Both NO2- production and intracellular killing were abolished by the addition of anti-TNF-alpha mAb in the assay. TNF-alpha was produced by amastigote-infected macrophages and IFN-gamma dramatically enhanced secretion of this cytokine; IFN-gamma alone had no effect. Endogenous TNF-alpha produced during infection of macrophages with L. major acted in an autocrine fashion to trigger the production of L-arginine-derived toxic nitrogen intermediates that killed the intracellular parasites.     

IFN-gamma mediates increased glucocorticoid receptor expression in murine macrophages.

Exposure of the murine macrophage cell line, RAW 264.7, to murine rIFN-gamma resulted in a significant increase in the number of glucocorticoid receptors (GcR). A doubling in the number of GcR was observed as early as 24 h after rIFN-gamma treatment, and receptor number was maximal by 36 h after rIFN-gamma treatment and represented approximately a fourfold increase. Scatchard analysis indicated that a twofold increase in GcR affinity was concomitant with the rIFN-gamma-induced increase in GcR number in RAW 264.7 cells. Increased GcR numbers were induced after exposure of RAW 264.7 cells to as little as 0.1 U/ml rIFN-gamma, and optimal expression was observed at 5 U/ml. Treatment of peritoneal exudate macrophages from C3H/OuJ mice and the LPS hyporesponsive mouse strain, C3H/HeJ, with rIFN-gamma induced an approximately twofold increase in the GcR with no concomitant change in receptor affinity. These results suggest that IFN-gamma may be essential not only for macrophage activation, but also for increasing macrophage sensitivity to feedback inhibition by glucocorticoids by increasing the number and/or affinity of available GcR.     

Vaccination of brushtail possums, Trichosurus vulpecula, with Bacille Calmette-Guerin induces T lymphocytes that reduce Mycobacterium bovis replication in alveolar macrophages via a contact-dependent/nitric oxide-independent mechanism

The permissiveness of alveolar macrophages from brushtail possums for the replication of Mycobacterium bovis was examined. Mycobacterium bovis replication was indirectly measured by assessing bacterial metabolism via the incorporation of [3-H]-uracil by bacilli released from lysed macrophages previously infected with mycobacteria. Alveolar macrophages allowed substantial replication of virulent M. bovis, in contrast to Bacille Calmette-Guerin (BCG) Pasteur, which replicated poorly. The addition of crude lymphokines enhanced the metabolic activity of phagocytosed M. bovis in possum macrophages. Possum lymphokines enhanced the ability of possum macrophages to generate reactive oxygen intermediates, measured by the reduction of nitroblue tetrazolium, which is indicative of an activation process. Similarly, the addition of recombinant possum TNF-alpha enhanced the permissiveness of alveolar macrophages for M. bovis. In contrast to mouse peritoneal macrophages, possum alveolar macrophages did not release significant levels of nitric oxide (NO) after stimulation with M. bovis and/or lymphokines. However, the uptake of virulent M. bovis by possum macrophages was associated with an enhanced ability of cells to release TNF-alpha, whereas very low levels of TNF-alpha were released after infection with BCG. The addition of a selective inhibitor of inducible NO synthase had no impact on the replication of M. bovis or BCG in possum macrophages in the presence or absence of lymphokines. Co-culturing infected possum alveolar macrophages with autologous blood mononuclear cells from BCG-vaccinated possums led to a significant decrease in the metabolic activity of intracellular M. bovis. This effect was contact dependent and NO independent and was mediated by a population of CD3+ cells. In addition, adding scavengers of reactive oxygen intermediates did not abrogate this phenomenon.     

Tumor necrosis factor-independent protective effect of recombinant IFN-gamma against acute toxoplasmosis in T cell-deficient mice

rIFN-gamma conferred remarkable resistance against acute infection with Toxoplasma gondii in T cell-deficient (athymic nude) mice. Mice that received an i.p. injection of rIFN-gamma every other day beginning 24 h before infection for a total of eight doses survived significantly longer than untreated control mice although all of the treated mice died after the lymphokine was discontinued. Mice that received 14 doses of rIFN-gamma survived significantly longer than those that received eight doses of the lymphokine although mice started dying soon after the final (14th) injection of rIFN-gamma and eventually all of the treated mice died. Histologic study revealed that the IFN-gamma treatment prevented proliferation of the organisms in all organs examined, including brain, lung, heart, liver, and spleen. The treatment was effective even when started 1 day after infection. Peritoneal macrophages obtained from mice injected with rIFN-gamma were activated and effectively killed tachyzoites of T. gondii in vitro. TNF activity could not be detected in sera of the infected mice during treatment with rIFN-gamma. Administration of anti-TNF antibody did not affect the protective effect of rIFN-gamma against T. gondii infection. These facts indicate that rIFN-gamma can confer resistance to acute infection with T. gondii without collaboration of lymphokines derived from T cells and TNF. This suggests that rIFN-gamma may be effective for therapy of toxoplasmosis in immunosuppressed patients who have impaired activity of T cell function, especially those with AIDS.     

Activation of alveolar macrophages after lower respiratory tract infection.

Alveolar macrophage function has been studied in relation to bacterial infection of the lower respiratory tract. First, LRT macrophages were examined after exposure of rabbits to Listeria monocytogenes aerosols. Macrophages obtained from the LRT of animals 10 to 48 days after infection were activated, as evidenced by greater adherence to culture dishes and increased ability to ingest and kill both the original infecting organism and unrelated organisms, when compared to normal alveolar macrophages. Next, the in vitro effects on normal alveolar macrophages of incubation supernatants of control and antigen-stimulated lymphocytes (LRT and lymph node) from animals infected with L. monocytogenes or Streptococcus pneumoniae were evaluated. As manifested by increased adherence and phagocytosis, and an enhanced nonspecific bactericidal activity, alveolar macrophages were activated by the antigen-stimulated supernatants. These stimulated lymphocyte supernatants contain lymphokines (MIF), but the exact nature of the alveolar macrophage activating factor(s) remains to be determined. These observations, together with recent evidence that alveolar macrophages respond to lymphokines (MIF), suggest that the effector mechanism for cell-mediated immunity in the LRT is intact.     

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.     

Interleukin 2 enhances the natural killer cell activity of acquired immunodeficiency syndrome patients through a gamma-interferon-independent mechanism

Patients with the acquired immunodeficiency syndrome (AIDS) exhibit a variety of disorders of cellular immunity, including a deficient ability to generate cytotoxic T cells and depressed levels of natural killer (NK) cell activity. Interleukin 2 (IL 2) in vitro can markedly augment these depressed immune functions. Because IL 2 can induce the release of interferon-gamma (IFN-gamma) from normal peripheral blood lymphocytes (PBL), and because IFN-gamma may play a role in the regulation of NK cell activity, this study was performed to determine if the IL 2 enhancement of the NK cell activity of patients with AIDS was an IFN-gamma-dependent effect. PBL from eight healthy heterosexual donors and from nine patients with AIDS were studied for their ability to release IFN-gamma in response to IL 2 at a concentration of 100 U/ml. After 60 hr of culture, the PBL of all eight healthy donors produced IFN-gamma with a mean titer of 113 U/ml (range 40 to 320 U/ml). In contrast, the PBL from only two of nine patients with AIDS released measurable amounts of IFN-gamma (40 U/ml each) in response to IL 2 with a mean titer of 13.5 U/ml for all nine. Although the PBL from patients with AIDS were deficient in their capacity to produce IFN-gamma in response to 100 U/ml of IL 2, significant enhancement of NK cell activity could be obtained after only 1 hr of PBL treatment with 10 U/ml of IL 2, with an optimal NK enhancing effect occurring at doses of 50 to 100 U/ml of IL 2. The use of an anti-IFN-gamma monoclonal antibody resulted in complete neutralization of the IFN released from the normal PBL cultured with IL 2, but failed to inhibit the IL 2 enhancement of NK cell activity. Exogenous IFN-gamma exhibited different kinetics of enhancement of NK cell activity when compared to IL 2, requiring substantially more than 1 hr of pretreatment of PBL. These results indicate that the PBL from patients with AIDS usually do not release IFN-gamma when cultured with IL 2, and that IL 2 enhancement of the depressed NK cell activity of these patients may be an IFN-gamma-independent event. These results may have important implications for the therapy of AIDS.     

Spontaneous cytotoxicity and tumor necrosis factor production by peripheral blood monocytes from AIDS patients

Peripheral blood monocytes (PBM) from AIDS patients have exhibited defects in some but not all of the immune functions yet tested. This study has examined the capacity of AIDS PBM to lyse tumor target cells as well as their ability to secrete TNF. Untreated PBM from AIDS patients were significantly cytotoxic to U937 target cells and responded to IFN-gamma pretreatment with augmented cytotoxicity. Both the spontaneous and IFN-gamma-stimulated cytotoxic activity was significantly (p less than 0.01) higher than that observed with normal PBM. The cytotoxic activity depended on the E:T ratio used and was higher in AIDS PBM at all ratios tested (10:1 to 40:1). Because TNF has been implicated in macrophage cell-mediated cytotoxicity, we examined whether the elevated cytotoxic activity of AIDS PBM was associated with an increase in TNF production. Supernatants from PBM cultured overnight with or without IFN-gamma were tested in a bioassay measuring cytotoxicity against U937 target cells as well as in an RIA specific for TNF. Supernatants derived from either unstimulated or IFN-gamma-treated AIDS PBM exhibited significantly higher levels of cytotoxicity than supernatants from normal macrophages. Both normal and AIDS PBM produced higher levels of cytotoxic factors in response to IFN-gamma. As determined by the RIA, AIDS PBM spontaneously released high levels of TNF whereas little TNF was produced by normal PBM. Treatment with IFN-gamma augmented the level of TNF production in both AIDS and normal PBM. These results demonstrate that PBM from AIDS patients have undergone in vivo activation as manifested by both cytotoxicity against tumor target cells and production of TNF. Target cell lysis by both AIDS PBM and their supernatants was inhibited by monoclonal anti-rTNF, suggesting that the increase in PBM cell-mediated cytotoxicity was caused by an increase in TNF production. The significance of these findings in the pathogenesis of the disease is discussed.     

Defective production of interferon-gamma and tumour necrosis factor-alpha by AIDS mononuclear cells after in vitro exposure to Rhodococcus equi

The production of interferon-gamma and tumour necrosis factor-alpha was evaluated in the peripheral blood mononuclear cells (PBMCs) from healthy donors and AIDS patients after Rhodococcus equi infection in vitro. PBMCs from healthy donors secreted elevated levels of IFN-gamma and TNF-alpha when challenged in vitro with killed R. equi, whereas the release of both cytokines was impaired in supernatant cultures from AIDS patients. We conclude that the failure of IFN-gamma generation in AIDS patients in response to R. equi is not antigen-specific but it may reflect the global impairment of T-cell function. In such patients, however, the infection with R. equi, a facultative intracellular pathogen which survives and replicates within macrophages, may be responsible for the impairment in the TNF-alpha release, possibly enhancing the HIV-induced macrophage dysftmction.