Human Blood-derived Macrophages Research Articles

Tumor necrosis factor α stimulates mycobactericidal/ mycobacteriostatic activity in human macrophages by a protein kinase C-independent pathway

Tumor necrosis factor (TNF) is a 17-kDa protein produced by endotoxin-stimulated macrophages. We have demonstrated that recombinant human TNF activates human macrophages to kill intracellular bacteria of the Mycobacterium avium complex (MAC) in a dose-related manner. TNF also primed macrophages to produce superoxide anion (O − 2) following treatment with phorbol esther PMA (0. l μg/ml). To investigate the intracellular pathway involved in the TNF-mediated activation of mycobacteriostatic/mycobactericidal activity in macrophages, we used two different protein kinase C (PKC) inhibitors: H7 (10 −5–10 −7 M) and staurosporine (10 −7–10 −9 M). Mellitin (1 and 100 m M) was used as a calmodulin inhibitor. Human peripheral blood-derived macrophages cultured for 7 days were treated with H7, mellitin, or staurosporine for 1 hr prior to incubation with TNF (10 3 U/ml). Twenty-four hours after treatment with TNF the O − 2 release was measured spectrophotometrically following exposure to PMA. Macrophages were infected with MAC and the viable intracellular bacilli were quantitated following 4 days of treatment with TNF. All PKC inhibitors suppressed O − 2 production after incubation with PMA. However, treatment with either PKC or calmodulin inhibitors did not influence the intracellular killing of M. avium by TNF-stimulated macrophages. Exposure of the macrophages to cGMP inhibitor but not to cAMP inhibitor significantly impaired the response to the stimulation with TNF. In contrast, incubation of macrophages with protein kinase A (PKA) had no effect on TNF-mediated mycobacteriostatic/mycobactericidal activity. These results suggest that the TNF-mediated mycobactericidal activity in cultured macrophages probably occurs by a PKC-independent mechanism.

Blood-derived macrophages produce IL-1, but not TNF-α, after infection with HIV-1 isolates from patients at different stages of disease

Interleukin 1 (IL-1) and tumor necrosis factor α (TNF-α) are not constitutively produced by human mononuclear phagocytes. In the present study we have investigated the production of these cytokines in human blood-derived macrophages (BDM) after infection with 16 primary HIV-1 blood isolates obtained from individuals at different stages of disease. In addition, we monitored the replicative capacity of these primary isolates in blood-derived macrophages over a 3-month period. Production of IL-1α was detected in two cultures, IL-β was positive in two other cultures, and both IL-1α and IL-β were present in three additional macrophage cultures. IL-1α production was also detected in BDMs infected with the laboratory strain HIV-1 HIB. In contrast, TNF-α was not found in any of the culture supernatants tested. All primary HIV-1 isolates used in these experiments were able to infect BDM productively irrespective of the clinical stage of the patients at the time of virus isolation. The production of IL-1 was mostly found in chronically infected cultures displaying low levels of HIV-1 replication. These results indicate that macrophages tropism is a general feature of all HIV-1 isolates. Furthermore, release of IL-1 by mononuclear phagocytes upon HIV-1 infection may contribute to the pathogenesis of HIV-1 related diseases.

Interleukin-3 induces antimicrobial activity against Leishmania amazonensis and Trypanosoma cruzi and tumoricidal activity in human peripheral blood-derived macrophages.

The ability of interleukin-3 (IL-3) to induce antimicrobial and tumoricidal activity was evaluated. Macrophages infected with two intracellular protozoa, Leishmania amazonensis or Trypanosoma cruzi, were treated with cytokines. IL-3 induced a dose-dependent enhancement of microbistasis against leishmanias, and the activity of IL-3 (100 ng/ml) was comparable to that of gamma interferon (IFN-gamma) (1,000 U/ml). In addition, IL-3 in combination with either granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage CSF (M-CSF) or with IFN-gamma reduced infection and lowered the required dose. IL-3 similarly activated macrophages to inhibit intracellular replication of T. cruzi. Furthermore, IL-3 induced antibody-independent tumoricidal activity against melanoma cells that was dose dependent and comparable to that of lipopolysaccharide and GM-CSF. The mechanisms by which IL-3 induced antimicrobial activity may involve at least the augmentation of oxidative capacity. IL-3, at concentrations of 0.5 ng/ml or greater, led to a significantly increased oxidative burst which paralleled the inhibition of protozoan replication. The enhancement of oxidative capacity by IL-3 (5 ng/ml or higher) was comparable to that of IFN-gamma. The induction of tumoricidal activity was associated with the production of tumor necrosis factor alpha (TNF-alpha), which in this system may feed back to enhance the macrophage inhibition of leishmanias, as demonstrated by neutralization of IL-3 activation by anti-TNF-alpha antibody. Thus, peripheral blood macrophages remain responsive to IL-3, as demonstrated by enhanced antimicrobial and tumoricidal activity. IL-3 may have potential clinical applications because of these properties and its effect on myelopoiesis.

Tetrahydrobiopterin biosynthetic activities in human macrophages, fibroblasts, THP-1, and T 24 cells. GTP-cyclohydrolase I is stimulated by interferon-gamma, and 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase are constitutively present.

Interferon-gamma induces tetrahydrobiopterin biosynthesis in human cells and cell lines. Macrophages are peculiar in the formation of large amounts of neopterin derivatives as compared to tetrahydrobiopterin (Werner, E. R., Werner-Felmayer, G., Fuchs, D., Hausen, A., Reibnegger, G., and Wachter, H. (1989) Biochem J. 262, 861-866). Here we compare the impact of interferon-gamma treatment on activities of GTP-cyclohydrolase I (EC 3.5.4.16), 6-pyruvoyl tetrahydropterin synthase, and sepiapterin reductase (EC 1.1.1.153) in human peripheral blood-derived macrophages, normal dermal fibroblasts, THP-1 myelomonocytic cells, and the T 24 bladder transitional-cell carcinoma line. Upon interferon-gamma treatment, GTP-cyclohydrolase I activity is increased 7- to 40-fold, whereas 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase activities, which are constitutively present in all four investigated cells, remain unchanged. In fibroblasts and T 24 cells GTP cyclohydrolase I activity is the rate-limiting step of tetrahydrobiopterin biosynthesis. In macrophages and in THP-1 cells, however, the induced GTP cyclohydrolase I activity is higher than the 6-pyruvoyl tetrahydropterin synthase activity, leading to the accumulation of neopterin and neopterin phosphates.

Endotoxin suppresses the generation of O2- and H2O2 by "resting" and lymphokine-activated human blood-derived macrophages.

In evaluation of macrophage-activating principles other than lymphokines, we systematically investigated the effects of endotoxin on the formation of reactive oxygen intermediates measured by chemiluminescence. Surprisingly, endotoxin exposure of human blood monocytes cultured in vitro for 36 h lessened in a dose-dependent manner the amount of O2- and H2O2 secreted in response to phagocytosis of opsonized particles or to PMA, a soluble stimulant. Blunting of the respiratory burst by endotoxin was independent from the state of macrophage activation. Endotoxin thus impaired formation of reactive oxygen metabolites before, during, or after activation of macrophages by IFN-gamma. The median effective concentration (EC50) was 1.95 ng/ml LPS in resting macrophages and 7.22 ng/ml in IFN-gamma-activated macrophages with as little as 0.1 ng/ml reproducibly giving detectable inhibition. Lipid A, but not "detoxified" monophosphoryl lipid A gave an inhibition comparable to that of complete LPS. The inhibitory effect of endotoxin was attenuated by dexamethasone, but not by inhibitors of arachidonic acid metabolism. Because endotoxin induces and dexamethasone inhibits production of some monokines, it is tempting to speculate that endotoxin is part of an autoregulatory system of mononuclear phagocytes for the control of excessive production of potentially harmful oxidants. The two monokines identified to be secreted in response to LPS and to be inhibited by dexamethasone, IL-1 and TNF, had, however, no comparable effect on chemiluminescence.

Inhibition by normal human serum of Mycobacterium avium multiplication in cultured human macrophages

Normal human serum used as a protein supplement in RPMI 1640 medium inhibited growth in blood-derived human macrophages (MP) of virulent Mycobacterium avium serovars 4 and 8, derived from patients with acquired immunodeficiency syndrome, but not virulent Mycobacterium tuberculosis. A defined serum substitute (SS) promoted the intramacrophage growth of M. avium but not of M. tuberculosis. The effects of serum or SS were measured by counting viable bacteria in lysates of the MP at 0, 4, and 7 days after their infection by the bacteria. Neither serum nor SS inhibited or enhanced M. avium growth in the absence of MP. The results suggest that a nutrient essential for intracellular replication of M. avium is made by MP from a pronutrient present in both SS and serum and that something in serum inhibits MP conversion of the pronutrient to nutrient. This inhibition may be an important mechanism of native resistance against M. avium in normal people.

Interleukin 1 and interleukin 1 inhibitor production by human macrophages exposed to influenza virus or respiratory syncytial virus. Respiratory syncytial virus is a potent inducer of inhibitor activity.

Respiratory viral infections are commonly associated with altered immune responses, such as proliferative responses to mitogens and antigens. To examine potential mechanisms, we examined production of IL-1 and IL-1 inhibitors by purified human peripheral blood-derived macrophages exposed to influenza virus or respiratory syncytial virus (RSV). IL-1 and IL-1 inhibitor activities in supernatant fluids from macrophages exposed to the viruses 24 h previously were measured using the standard mouse thymocyte comitogen assay. Crude fluids from macrophages exposed to influenza virus contained substantial IL-1 activity, whereas crude fluids from macrophages exposed to RSV contained marked IL-1 inhibitor activity. Assays with gel filtration-separated fractions revealed that both influenza virus and RSV induced production of both IL-1 and IL-1 inhibitors. Neither IL-2 nor IL-2 inhibitor activities were detected. Thus, effects of human macrophage-derived factors on thymocyte proliferation, or potentially on human lymphocyte proliferation, may reflect the total or net activity of multiple composite factors, the balance of which varies according to the challenge. The data raise the possibility that marked production of IL-1 inhibitor activity in response to RSV plays a role in the clinical recurrence of RSV infection despite the absence of clear evidence for antigenic shift or drift of the virus.

Therapeutic concentrations of glucocorticoids suppress the antimicrobial activity of human macrophages without impairing their responsiveness to gamma interferon.

By exposing human blood-derived macrophages and alveolar macrophages in vitro to dexamethasone, we showed in these studies that glucocorticoids markedly suppress the antimicrobial activity of macrophages but not macrophage activation by lymphokines. As little as 2.5 X 10(-8) mol/liter of dexamethasone prevented macrophages from inhibiting germination of Aspergillus spores or from eliminating ingested bacteria such as Listeria, Nocardia, or Salmonella. Damage to macrophage function was inhibited by progesterone and appeared to be receptor-mediated. In accordance with in vivo observations, dexamethasone required 24-36 h to suppress antimicrobial activity. While glucocorticoids interfered with base-line activity of macrophages, dexamethasone concentrations comparable to drug levels in patients had no effect on macrophage activation. Proliferating lymphocytes and gamma-interferon thus increased the antimicrobial activity of phagocytes exposed to glucocorticoids over that of control cells. Macrophage activation and correction of the dexamethasone effect by gamma-interferon, however, was dependent on the pathogen. The lymphokine enhanced the antimicrobial activity of dexamethasone-treated macrophages against Listeria and Salmonella but not against Aspergillus or Nocardia. Dexamethasone-induced damage to the antimicrobial activity of human macrophages in vitro parallels observations that glucocorticoids render laboratory animals susceptible to listeriosis and aspergillosis by damaging resident macrophages. Suppression of macrophage antimicrobial activity should thus be considered when treating patients with glucocorticoids; its prevention by gamma-interferon might be beneficial for some but not all pathogens.

Aryl hydrocarbon hydroxylase induction in mouse peritoneal macrophages and blood-derived human macrophages.

SummaryMacrophages of human and mouse origins were shown to contain inducible aryl hydrocarbon hydroxylase activity. Mononuclear phagocytic cells derived from circulating blood monocytes of healthy human donors and peritoneal macrophages from untreated C57 B1/6 mice exhibited low levels of enzyme activity. For mice, aryl hydrocarbon hydroxylase specific activity of freshly explanted macrophages was 1/70th the enzyme activity found in liver. Aryl hydrocarbon hydroxylase activity for both human and mouse macrophages maintained in vitro in the presence of benzanthracene exceeded by 2-3 fold the enzyme specific activity of cells cultivated in the absence of inducer.