Role of IFN-gamma and alpha in IL 1 synthesis and secretion of in vitro differentiated human macrophages: a comparative study

Human blood monocytes (Mo) cultured in vitro differentiate to macrophages (Mx) and lose the capacity to secrete interleukin 1 (IL 1) in response to endotoxin (LPS). Incubation of Mo with interferon gamma or alpha (IFN-gamma or IFN-alpha) prevented this loss of IL 1 secretory potential during the first 24 h of culture. However, there were marked differences between the two interferons if culture period was extended beyond 24 h. Incubation of Mo with IFN-gamma for 48 or 72 h induced IL 1 release in response to LPS in all the donors without exception. In contrast, 48-h incubation of Mo with IFN-alpha alpha caused IL 1 secretion (in response to LPS) in only a minority of donors, while 72-h incubation resulted in very little or no IL 1 release in all the individuals tested. Moreover, only IFN-gamma had the capacity to reinduce IL 1 secretory potential in Mx which had lost the capacity to secrete IL 1 during previous culture. These and other results suggest that IFN-alpha differs from IFN-gamma in being: a less potent IL 1 inducer, ineffective in maintaining IL 1 secretory capacity of fresh Mo for more than 48-72 h, completely unable to reinduce IL 1 secretory potential in culture-derived Mx. Thus, the two species of IFN appear to have a markedly different role in IL 1 synthesis and secretion.     

Regulation of interferon production by human monocytes: requirements for priming for lipopolysaccharide-induced production.

Macrophages are uniquely responsive to bacterial lipopolysaccharide (LPS) for activation of a number of host defense functions and production of bioactive mediators. One potentially important mediator produced by LPS-stimulated macrophages is interferon (IFN-alpha/beta). In contrast to murine observations, we have observed that freshly isolated human monocytes, purified by counter-current centrifugal elutriation, do not produce interferon in response to LPS. This is not due to a lack of response to LPS, as assessed by the induction of other monokines, or to an incapacity for IFN production, since IFN was inducible by poly-I,C treatment of monocytes in the absence of any other exogenous stimulus. However, human monocytes can be primed for the production of IFN in response to LPS if they are cultured in the presence of either granulocyte-macrophage colony stimulating factor (GM-CSF) or interferon-gamma (IFN-gamma). The IFN secreted is of the alpha subtype. Monocytes primed with GM-CSF or IFN-gamma also maintained LPS responses for production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1). M-CSF did not prime monocytes for LPS-induced IFN production, although it did enhance production of TNF-alpha and promoted monocyte survival. Northern analysis indicated that the induction of IFN-alpha by LPS was regulated primarily at the mRNA level. The highly regulated production of IFN-alpha by monocytes/macrophages has important implications for autocrine action of interferons in the activation and differentiation of these cells.     

Interferon-induced indoleamine 2,3-dioxygenase activity in human mononuclear phagocytes

Interferon (IFN)-induced tryptophan degradation, catalyzed by indoleamine 2,3-dioxygenase (IDO), has been shown to mediate antimicrobial activity in epithelial cells. IDO activity has also been augmented in peripheral blood mononuclear cells (PBMC) treated with IFN or interleukin-2 (IL-2). The effector cells in this population have now been further characterized. PBMCs were isolated from normal donors, separated into monocyte and lymphocyte populations by plastic adherence, treated with IFN or IL-2, and cultivated in medium supplemented with [3H]tryptophan. Culture supernatants were collected after a 48-h incubation and fractionated by high-performance liquid chromatography; radioactivity was determined in fractions corresponding to tryptophan and its metabolites. IFN-gamma and IFN-beta induced IDO activity only in monocytes (plastic-adherent, nonspecific esterase-positive PBMCs). The induction of IDO activity by IL-2 required both monocytes and lymphocytes. Interaction was required between these populations for induction of IDO by IL-2, due to production of IFN-gamma by T lymphocytes, with subsequent IFN-gamma-mediated induction of IDO in monocytes. A number of myeloid cell lines as well as monocyte-derived macrophages were also tested for their ability to be induced to degrade tryptophan in response to IFN treatment. Monocyte-derived macrophages were found to retain their capacity to be induced by IFN-gamma and IFN-beta to degrade tryptophan after differentiation, and to possess seven times more IDO activity per cell than IFN-induced monocytes. However, the presence of lipopolysaccharide (LPS) in the culture medium was required for the maximum induction of IDO activity by IFN-beta. Furthermore, higher concentrations of LPS were sufficient to induce IDO activity in macrophages in the absence of exogenous IFN.     

Selective induction of mononuclear phagocytes to produce neopterin by interferons

Interferon-gamma (IFN-gamma) has been shown to be a potent inducer of neopterin secretion by human peripheral blood monocytes/macrophages (1). In this paper, it is shown that other known stimuli of monocytes (e.g., to secrete proteases or to migrate) such as zymosan-activated human serum, lipopolysaccharide, human C3/iC3 and zymosan coated with complement were unable to trigger monocytes/macrophages to release neopterin. Monocytes/macrophages could be stimulated solely by IFN-gamma (25 U/ml) and IFN-alpha at very high concentrations (10,000 U/ml). In the case of human peripheral blood mononuclear cells (PBMNC), basically the same pattern was observed. If however, in the buffer controls PBMNC showed some neopterin release, all stimuli triggered an increase of neopterin secretion: 10,000 U/ml IFN-alpha induced the same amount of secreted neopterin as did 25 U/ml of IFN-gamma. Both caused higher levels of neopterin secretion than ZAS, LPS and C3/iC3. Amongst the supernatants from PBMNC, only those which were obtained from cells activated with IFN-gamma or -alpha stimulated monocytes/macrophages to produce neopterin. Supernatants from lymphocytes activated with zymosan, lipopolysaccharide and interferon did not contain neopterin, nor did the latter induce monocytes/macrophages to generate and secrete neopterin. Antibodies against IFN-gamma inhibited the triggering effect of the supernatants except when generated by IFN-alpha at 10,000 U/ml. These results demonstrate that both interferons, IFN-gamma and IFN-alpha, the latter only at a 400-fold higher concentration, can trigger monocytes/macrophages directly to secrete neopterin. ZAS, LPS and C3/iC3 are weakly effective only on a mixture of lymphocytes and monocytes/macrophages, provided this cell mixture shows already a basic spontaneous neopterin release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of LPS, IFN-gamma and TNF alpha on the secretion of lysozyme by individual human mononuclear phagocytes: relationship to cell maturity.

Human mononuclear phagocytes can be activated to perform a variety of complex functions by exposure to the immunomodulators, lipopolysaccharide (LPS), interferon-gamma (IFN-gamma) and tumour necrosis factor alpha (TNF alpha). Although such activation often involves the release of various cytokines by monocytes and macrophages, little is known of the effects of such signals on their secretion of lysozyme (LZM). In this study, a reverse haemolytic plaque assay for LZM secretion is coupled with immunocytochemistry for the pan macrophage (CD68) marker, EBM/11. This enabled the direct effects of LPS, IFN-gamma and TNF alpha on the secretion of LZM by individual, immunoidentified human mononuclear phagocytes to be investigated. The overall secretion of this peptide by populations of freshly isolated or 3-day cultured monocytes was augmented by exposure for 6 hr to bacterial LPS, recombinant human IFN-gamma or recombinant human TNF alpha. Extension of the culture period for monocytes from 3 to 7 days prior to use in the assay resulted in higher levels of LZM secretion, which could be further increased by TNF alpha but not by LPS or IFN-gamma. Individual peritoneal macrophages activated by inflammation in vivo were uniform in their augmented LZM responses to TNF alpha, but a small subpopulation of human peritoneal macrophages, which may represent younger 'inflammatory' exudate macrophages, was seen to be preferentially responsive to the LZM-stimulating effects of LPS and IFN-gamma. These studies suggest that (i) secretion of LZM by human mononuclear phagocytes can be regulated by LPS and IFN-gamma, although the effects of these agents may be dependent upon the state of maturation and/or differentiation of the cells, and (ii) TNF alpha is a potent stimulant of LZM secretion by monocytes and macrophages irrespective of cell maturity.     

Control by IFN-gamma and PGE2 of TNF alpha and IL-1 production by human monocytes.

There have been suggestions that the production of pro-inflammatory mediators by human monocytes in response to interferon-gamma (IFN-gamma) may be controlled by changes in prostaglandins. Therefore we investigated tumour necrosis factor alpha (TNF alpha) and interleukin-1 (IL-1) activities and prostaglandin E2 (PGE2) levels in the supernatants of highly purified human monocytes cultured for 18 hr with recombinant human IFN-gamma. IFN-gamma (100 U/ml) did not stimulate monocytes isolated by counter-current centrifugal elutriation for detectable TNF alpha or IL-1 activities, or PGE2 production. However, IFN-gamma synergistically enhanced lipopolysaccharide (LPS)-induced TNF alpha and IL-1 activities. In contrast, there was no consistent change in PGE2 levels upon addition of IFN-gamma to LPS-treated monocyte cultures. The TNF alpha and IL-1 activities induced by LPS and by LPS with IFN-gamma were reduced by PGE2, and stimulated by indomethacin. As reported previously for IL-1 activities, the regulation by cyclo-oxygenase products of TNF alpha activities reflected predominantly a control of the production of immunoreactive TNF alpha, rather than the measurement of TNF alpha bio-activity. However, the addition of indomethacin or PGE2 to monocyte cultures did not change the extent of IFN-gamma synergy with LPS for increased TNF alpha and IL-1 activities. The results of this study suggest that, despite control by cyclo-oxygenase products of TNF alpha and IL-1 production in human monocytes, IFN-gamma may enhance TNF alpha and IL-1 activities independently of this regulatory mechanism. These findings are contrary to those suggested for the regulation by prostanoids of IL-1 production by murine macrophages.     

Monocyte-derived dendritic cells release neopterin

Increased neopterin concentrations in body fluids are found in diseases associated with activated, cell-mediated immunity including infections, autoimmune diseases, and certain malignancies. Monocytes/macrophages are known to secrete large amounts of neopterin upon stimulation with interferon-gamma (IFN-gamma). Ontogenetically, the major part of dendritic cells (DC) belongs to the myeloid lineage. Therefore, we investigated whether cultured monocyte-derived DC can elaborate neopterin. Cells were treated with cytokines in the presence or absence of monocyte-conditioned medium as a maturation stimulus. DC secreted an average 3.5 nmol/l neopterin. In response to IFN-gamma, cells significantly increased their output of neopterin. In distinction to monocytes/macrophages, neopterin production in DC was highly sensitive to IFN-alpha and IFN-beta. Further, lipopolysaccharides (LPS) enhanced neopterin synthesis, whereas tumor necrosis factor alpha, interleukin (IL)-1beta, IL-2, IL-10, and IL-18 were ineffective. Simultaneously, tryptophan degradation by induction of indoleamine (2,3)-dioxygenase (IDO) was tested in stimulated cells. Our results showed that IFN-gamma as well as LPS are inducers of IDO in DC.     

Constitutive secretion of interleukin 1 by human monocytes

The constitutive and lipopolysaccharide (LPS)-induced secretion of interleukin 1 (IL1) by cultured human monocytes and macrophages has been studied. Both freshly obtained monocytes and their culture-derived macrophages were induced by LPS to secrete similar amounts of IL1. Such induction, however, was accompanied by the secretion of dialyzed inhibitory activity. Constitutive secretion of IL1 was detected in concentrated supernatants of monocyte cultures. The factor obtained constitutively did not manifest significant inhibitory activity. A method is described for the recovery of IL1-containing supernatants in serum- and other stimulant-free medium. The biological activities of the constitutively secreted IL1 were similar to the LPS-induced activities. The constitutive secretion of IL1 was not equally distributed in the entire monocyte population. We found that a small fraction of loosely adherent monocytes secreted higher amounts of IL1 than the strongly adherent monocytes. However, the property of higher secretion of IL1 was not stable and disappeared following monocyte cultivation. Thus, constitutive activity of IL1 could be recovered either by concentrating the culture supernatants or by enriching a subset of monocytes with higher IL1 activity.     

Rapid lipopolysaccharide-induced differentiation of CD14(+) monocytes into CD83(+) dendritic cells is modulated under serum-free conditions by exogenously added IFN-gamma and endogenously produced IL-10.

We showed previously that about half of purified CD14(+) peripheral blood monocytes cultured under serum-free conditions and treated with GM-CSF and bacterial LPS rapidly (2 - 4 day) differentiate into CD83(+) dendritic cells (DC). The remaining cells retain the CD14(+)/CD83(-) monocyte/macrophage phenotype. In order to identify factors that influence whether monocytes differentiate into DC or remain on the monocyte/macrophage developmental pathway, we evaluated the effects of exogenously added IFN-gamma and endogenously produced IL-10 on the proportion and function of CD14(+) monocytes that adopt DC characteristics in response to LPS. IFN-gamma priming dramatically increased the proportion of monocytes that adopted stable DC characteristics in response to LPS, improved their T cell allosensitizing capacity, and enhanced levels of secreted IL-12 heterodimer. IFN-gamma priming also suppressed the production of IL-10, a cytokine known to have inhibitory effects on DC differentiation. When monocytes were treated with LPS plus IL-10-neutralizing antibodies, dramatically enhanced DC differentiation, IL-12 secretion, and T cell allosensitizing capacity were observed, mimicking in many respects the effects of IFN-gamma priming. IFN-gamma primed cells still displayed appreciable sensitivity to exogenously added IL-10, suggesting that attenuated IL-10 secretion is partially responsible for the enhancing effects of IFN-gamma. These studies therefore identify IFN-gamma as a DC differentiation co-factor for CD14(+) monocytes, and IL-10 as an autocrine/paracrine inhibitor of DC differentiation, linking these agents for the first time as mutually opposed regulators that govern whether CD14(+) cells differentiate into DC upon contact with LPS or remain on the monocyte/macrophage developmental pathway.     

Interleukin 1 secretion by human monocytes and macrophages

Interleukin 1 (IL-1) is generally regarded as a major regulator of T lymphocyte proliferation. Macrophages from animals and cloned tumor cell lines have been shown to produce this monokine in response to a variety of stimuli. The ability of human monocytes and macrophages to generate IL-1 is much less well characterized. We previously demonstrated that human monocytes cultured for 1-6 days transformed to macrophages but retained their capacity to support concanavalin A-driven T cell proliferation. However, cultured macrophage capacity to support antigen-driven T cell proliferation began to decline after 3 days of culture and was markedly deficient by 6 days of culture. To determine if this loss of accessory cell function was due to the inability to secrete IL-1, we measured the monokine produced by normal fresh human monocytes and macrophages cultured in vitro from monocytes. IL-1 was assayed by the mouse thymocyte proliferation method. Fresh monocytes secreted IL-1 readily in response to lipopolysaccaride and latex particles. Macrophages cultured from fresh monocytes, however, lost this ability after greater than or equal to 2 days in culture. Mixing experiments failed to demonstrate an inhibitor present in the macrophage supernatants that would suppress thymocyte proliferation. Stimulated T cells incubated with monocytes and 3-day cultured macrophages failed to prolong or promote IL-1 secretion.     

Regulation of LPS induced IL-12 production by IFN-gamma and IL-4 through intracellular glutathione status in human alveolar macrophages.

Interleukin-12 (IL-12) is secreted from monocytes and macrophages; it exerts pleiotropic effects on T cells and natural killer (NK) cells, and stimulates interferon-gamma (IFN-gamma) secretion. Glutathione tripeptide regulates the intracellular redox status and other aspects of cell physiology. We examined whether IFN-gamma and IL-4 affect the balance between intracellular reduced glutathione (GSH) and oxidized (GSSG) glutathione, as this may affect IL-12 production in human alveolar macrophages (AM). We used both AM from healthy non-smokers obtained by bronchoalveolar lavage and the monocytic THP-1 cell line in this study. Incubation of AM for 2 h with the GSH precursor N-acetylcysteine (NAC) increased the intracellular GSH/GSSG ratio, and enhanced lipopolysaccharide (LPS)-induced IL-12 secretion by AM. In THP-1 cells, NAC increased the GSH/GSSG ratio and the expression of LPS-induced IL-12 mRNA, whereas L-buthionine-[S,R]-sulphoximine (BSO) decreased these. NAC and BSO offset their own effects on the intracellular GSH/GSSG ratio and the expression of LPS-induced IL-12 mRNA. Furthermore, exposure of AM to the helper T cell type 1 (Th1) cytokine IFN-gamma or the helper T cell type 2 (Th2) cytokine IL-4 for 72 h increased and decreased the GSH/GSSG ratio, respectively. Lipopolysaccharide (LPS)-induced secretion of IL-12 in AM was enhanced by IFN-gamma but inhibited by IL-4. These results suggest that IFN-gamma and IL-4 oppositely affect the GSH/GSSG balance, which may regulate IL-12 secretion from AM in response to LPS.     

Cytokine secretion patterns of NK cells and macrophages in early human pregnancy decidua and blood: implications for suppressor macrophages in decidua

PROBLEM: Local immune modulation has been shown to be of considerable importance for the maintenance of successful pregnancy. We have previously reported the secretion of interferon-gamma (IFN-gamma), interleukin-4 (IL-4) and IL-10 in human decidua from early normal pregnancy. The aim of this study was to investigate the cellular source of cytokine secretion in the decidua, and compare this to secretion patterns in peripheral blood. METHOD OF STUDY: Decidual tissue and peripheral blood was collected from 20 women undergoing surgical abortion during first trimester pregnancy. Monocytes/macrophages and NK cells were enriched by immunomagnetic cell separation and cytokine secretion was detected by enzyme-linked immunosorbent spot-forming cell assay. RESULTS: Decidual and peripheral monocytes/macrophages and NK cells spontaneously secrete IFN-gamma, IL-4 and IL-10. The number of IL-10 secreting cells was significantly higher in decidual macrophages compared with decidual non-monocytic cells as well as compared with blood monocytes/macrophages. These differences were not seen for IFN-gamma or IL-4. CONCLUSIONS: Our results indicate that decidual macrophages subserve important suppressive functions in the pregnant uterus.     

FcR+ and FcR- monocytes differentially secrete monokines during pokeweed mitogen-induced T-cell-monocyte interactions.

Monocyte subpopulations which differ in the expression of Fc receptor for human IgG (FcRI) differentially regulate the T-cell-dependent, pokeweed mitogen (PWM)-induced, polyclonal B-cell response. We, thus, studied the cytokine production in human peripheral blood monocyte and T-lymphocyte cultures activated with this lectin. Monocytes or their FcR+ and FcR- subpopulations stimulated with PWM were cultured with or without T lymphocytes or their CD4+ and CD8+ subsets. Both monocyte subpopulations cultured alone produced similar amounts of tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), but FcR- monocytes showed significantly enhanced ability to secrete interleukin-1 (IL-1). T cells, especially CD4+, added to monocyte cultures enhanced IL-1 production. This enhancement was presumably due to interferon-gamma (IFN-gamma) release by T lymphocytes, since this lymphokine enhanced IL-1 secretion when added to PWM-stimulated cultures of monocytes. Addition of monocytes, in particular the FcR+ subpopulation, greatly enhanced production of IFN-gamma by T lymphocytes. Although both T-cell subsets produced IFN-gamma, the CD4+ cells were more efficient. These results indicate that in PWM-stimulated cultures subpopulations of monocytes differ in secretion of cytokines, which might explain their differential effect on T-cell-dependent immune responses in vitro.     

HIV-1 does not alter in vitro and in vivo IL-10 production by human monocytes and macrophages

The present study analyses the ability of HIV-1 to modulate IL-10 production in cells of monocyte-macrophage lineage cultured in the presence of macrophage colony-stimulating factor (M-CSF). Both monocytes and macrophages spontaneously produced low amount of IL-10. Lipopolysaccharide (LPS) induced a strong IL-10 response in fresh monocytes and in M-CSF-treated macrophages. In contrast, macrophages cultured in the absence of M-CSF exhibited a marked decrease in their susceptibility to LPS stimulation. M-CSF increased the IL-10 response of macrophages to LPS by enhancing both the expression of membrane-bound CD14, the protein that serves as LPS receptor, and the sensibility of CD14-expressing cells to LPS stimulation. Neither spontaneous nor LPS-induced expression of IL-10 was modulated in monocytes and macrophages by infection with eight monocytotropic strains, as demonstrated by ELISA and cytofluorimetric analysis. In contrast, all the HIV-1 strains primed macrophages for an increased IL-6 response to LPS stimulation. To determine whether IL-10 production was associated with in vivo infection, monocytes from AIDS individuals were analysed for IL-10 production. We found that neither spontaneous nor LPS-induced IL-10 production were different between healthy controls and HIV-infected patients. Taken together, these data strongly suggest that HIV-1 infection of monocytes-macrophages does not play a significant role in the regulation of IL-10 in infected patients. This study also emphasizes the role of M-CSF activation in the regulation of the cytokine response in macrophages.     

Modulation of anti-Candida activity of human alveolar macrophages by interferon-gamma or interleukin-1-alpha

The fungicidal and bactericidal activities of human alveolar macrophages (AM) and peripheral blood monocytes (PBM) from 18 healthy volunteers were evaluated. The results showed that AM were able to phagocytize and kill Candida albicans, Pseudomonas aeruginosa, and Staphylococcus aureus. However, killing of the bacteria was already complete in 2 h, whereas killing of Candida required 4 to 6 h despite an early phagocytosis of yeast cells. The fungicidal activity of freshly collected AM and PBM was also tested after effector cell exposure to interferon-gamma (IFN-gamma), interleukin-1-alpha (IL-1 alpha), endotoxin lipopolysaccharide (LPS), or interleukin 2 (IL-2). It was found that treatment with IFN-gamma, IL-1 alpha, or LPS significantly augmented macrophage and PBM candidacidal activity, whereas the addition of IL-2 was ineffective. We also evaluated killing of C. albicans by AM cultured in vitro for different times. While phagocytosis was apparently unaffected, the candidacidal activity progressively decreased over the in vitro culture period, an effect that was largely reversed by cell exposure to IFN-gamma, IL-1 alpha, or LPS. In an experimental model in which mice infected with an agerminative C. albicans strain (PCA-2) resisted lethal microbial challenge, freshly harvested AM showed increased cytotoxic activity to Aspergillus fumigatus in vitro as well as enhanced IL-1 production. In conclusion, present data confirm the crucial role of AM in the surveillance of bacterial and fungal infections and indicate that treatment of these cells with IFN-gamma or IL-1 alpha is able to enhance their antimicrobial capability.     

Regulation of interleukin 1 production in human monocytes. I. Effects of gamma-interferon and cycloheximide.

The objective of these studies was to investigate mechanisms of regulation of interleukin 1 (IL-1) production by human monocytes. IL-1 production was measured by augmentation of phytohemagglutinin-induced proliferation of murine thymocytes. Adherent human monocytes incubated in medium for one day exhibited a marked decrease in lipopolysaccharide (LPS)-induced IL-1 production over a second day. Cells pre-incubated in 100 U/ml gamma interferon (gamma-IFN) for 24 h displayed a partial to complete maintenance of LPS-induced IL-1 production. Studies with inhibitors of cyclo-oxygenase or lipoxygenase indicated that prostaglandins or leukotrienes were not responsible for the alterations in IL-1 production observed with cultured cells or for the effects of gamma-IFN. Monocytes were pre-incubated in cycloheximide for 24 h and the drug was washed out. These cells exhibited an enhancement in IL-1 production over a second 24 h culture in the presence of 2 ng/ml LPS. Furthermore, the partial maintenance of LPS-induced IL-1 production seen after cells were pre-incubated in gamma-IFN was markedly increased by the inclusion of 0.25 microgram/ml cycloheximide during the 24 h pre-incubation. These results indicate that IL-1 production may be inhibited by newly-synthesized proteins during maturation in vitro or differentiation of monocytes into macrophages. Pre-incubation in gamma-IFN and cycloheximide leads to separate but synergistic effects on the maintenance of LPS-induced IL-1 production in cultured monocytes.     

Differences in cytokine secretion by intestinal mononuclear cells, peripheral blood monocytes and alveolar macrophages from HIV-infected patients.

Mononuclear cells of the lamina propria (LpMNC), isolated from endoscopically taken biopsies of the large bowel from AIDS patients, were analysed for their ability to secrete tumour necrosis factor-alpha (TNF-alpha), IL-1 beta and IL-6. Stimulation of LpMNC from normal controls with pokeweed mitogen (PWM) led to a time- and dose-dependent enhancement of TNF-alpha, IL-1 beta and IL-6 secretion. In contrast, PWM stimulation of LpMNC from AIDS patients resulted in only a small increase in TNF-alpha release. Constitutive secretion of IL-1 beta and IL-6 in these patients was already increased to the concentration range of stimulated cells from normal controls and could not be further increased, probably due to maximal in vivo stimulation. Secretion of TNF-alpha, IL-1 beta and IL-6 by peripheral blood monocytes (PBM) and alveolar macrophages from AIDS patients was elevated with or without stimulation compared with normal controls. Obviously, the regulation of TNF-alpha secretion is dependent on the microenvironment. Since it is known that interferon-gamma (IFN-gamma) may induce the production of TNF-alpha, the secretion of this cytokine was examined. Release of IFN-gamma was constitutively and under stimulation lowered in LpMNC from AIDS patients compared with normal controls. Addition of IFN-gamma to LpMNC did not result in enhanced TNF-alpha secretion. Our data indicate a defective function of intestinal mononuclear cells in AIDS patients as shown by the diminished TNF-alpha secretion.     

Human monocytes tumoricidal activity: the role of interferon-gamma and bacterial lipopolysaccharide in its stimulation, preservation and decay

The effect of interferon-gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS) on the cytotoxic activity of cultured monocytes was studied in a 6-h51Cr release assay with Actinomycin-D-treated tumor cells as targets. In this system, the lysis of target cells is mediated by a soluble factor (CF) which is similar or identical to human tumor necrosis factor (TNF). The spontaneous cytotoxic activity of freshly isolated monocytes declined after their maturation to macrophages during in vitro culturing. The decrease in the ability of cultured monocytes to lyse the targets is explained by a decrease in their ability to produce the soluble cytolytic factor. Both LPS and IFN-gamma modulated the effect of culturing. LPS exhibited a dual effect. Within 2-3 h after its addition, LPS enhanced the cytotoxic activity of monocytes by increasing the synthesis of CF. However, upon a longer incubation, the decay of the activity was more pronounced in the presence of LPS. IFN-gamma did not augment the cytotoxic activity of monocytes above the basal level, yet it prevented the loss of activity which accompanies the process of monocyte maturation to macrophages.     

Intracellular Salmonella dublin induces substantial secretion of the 40-kilodalton subunit of interleukin-12 (IL-12) but minimal secretion of IL-12 as a 70-kilodalton protein in murine macrophages.

The induction by intracellular pathogens of interleukin-12 (IL-12) secretion is of particular importance since this cytokine has been shown to be necessary for optimal cell-mediated immune responses. Several recent investigations have suggested that cultured macrophages are a significant source of IL-12 following intracellular infection with pathogens such as Salmonella spp. In an effort to critically evaluate the magnitude of the IL-12 response in cultured macrophages following interaction with Salmonella dublin, enzyme-linked immunosorbent assays specific for the 40- and 70-kDa subunits of IL-12 (IL-12p40 and IL-12p70) and a sensitive bioassay for IL-12p70 were used. Using BALB/c macrophages, S. dublin at various challenge doses was a potent inducer of IL-12p40 secretion (>6,000 pg/10(7) macrophages). However when secretion of IL-12p70 was evaluated, S. dublin did not induce comparable IL-12p70 production (<80 pg/10(7) macrophages) at any time, despite varying the challenge dose of Salmonella. The limited ability of BALB/c (Ity(s)) macrophages to secrete IL-12p70 in response to Salmonella was not a strain-specific phenomenon since similar results were demonstrated for macrophages isolated from CBA/J (Ity(r)) and C3H/HeJ (lipopolysaccharide [LPS]-hyporesponsive) mice. While intracellular infection with Salmonella was not a potent stimulus for IL-12p70 secretion in these mouse strains, macrophages from these mice responded significantly to a stimulus of gamma interferon plus LPS. Taken together these results demonstrate a limited capacity for intracellular Salmonella to stimulate murine macrophage secretion of IL-12p70, despite being a significant stimulus for IL-12p40 secretion. Furthermore, our results suggest that Salmonella-induced IL-12p40 secretion by macrophages is not solely an LPS-mediated event.     

Role of endogenous IFN-gamma in macrophage programming induced by IL-12 and IL-18.

Besides the established role of interleukin-12 (IL-12) and IL-18 on interferon-gamma (IFN-gamma) production by natural killer (NK), T, and B cells, the effects of these cytokines on macrophages are largely unknown. Here, we investigated the role of IL-12/IL-18 on nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production by CD11b(+) adherent peritoneal cells, focusing on the involvement of endogenously produced IFN-gamma. C57BL/6 cells released substantial amounts of NO when stimulated with IFN-gamma or lipopolysaccharide (LPS), but failed to respond to IL-12 or IL-18 or both. However, IL-12/IL-18 pretreatment was able to program these cells to release 6-8-fold more NO and TNF-alpha in response to LPS or Trypanosoma cruzi stimulation, with NO levels directly correlating with macrophage resistance to intracellular parasite growth. Analysis of IL-12/IL-18-primed cells from mice deficient in IFN-gamma, IFNGR, and IFN regulatory factor-1 (IRF-1) revealed that these molecules were essential for LPS-induced NO release, but TNF-alpha production was IFN-gamma independent. Conversely, the myeloid differentiation factor 88 (MyD88)-dependent pathway was indispensable for IL-12/IL-18-programmed LPS-induced TNF-alpha production, but not for NO release. Contaminant T and NK cells largely modulated the IL-12/IL-18 programming of LPS-induced NO response through IFN-gamma secretion. Nevertheless, a small population of IFN-gamma(+) cells with a macrophage phenotype was also identified, particularly in the peritoneum of chronically T. cruzi-infected mice, reinforcing the notion that macrophages can be an alternative source of IFN-gamma. Taken together, our data contribute to elucidate the molecular basis of the IL-12/IL-18 autocrine pathway of macrophage activation, showing that endogenous IFN-gamma plays an important role in programming the NO response, whereas the TNF-alpha response occurs through an IFN-gamma-independent pathway.