Search

Macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage (GM)-CSF stimulate the differentiation of rat alveolar macrophages (AM) into multinucleated giant cells (MGC) with distinct phenotypes (type 1 and type 2 MGC). In the present study, we analyzed the profile of cytokine gene expression induced respectively, by M-CSF and GM-CSF during rat AM differentiation using reverse transcription-PCR. Enhanced mRNA expression for IL-1alpha, TNF-alpha, and TGF-beta1 was observed 3 h after treatment with M-CSF (50 U/ml) or GM-CSF (50 U/ml). In contrast, IL-6 mRNA expression was increased by GM-CSF but not M-CSF. Kinetic analysis indicated that GM-CSF stimulated IL-6 expression early (1.5 h), with maximal effect observed at 24 h and up to 5 days thereafter. Increased mRNA levels for IL-6 were associated with higher IL-6 activity in the culture media of differentiating AM. IL-6 activity was stimulated 3 h after treatment with GM-CSF and increased with time (up to 5 days). Interestingly, addition of exogenous IL-6 (20-100 ng/ml) alone or in combination with GM-CSF to AM cultures increased slightly and selectively the formation of MGC with type 2 phenotype. Conversely, neutralization of endogenous IL-6 during AM differentiation into MGC inhibited significantly (up to 50%) the formation of type 2 MGC. These results suggest a role for IL-6 in the formation of type 2 MGC and provide some insights into the mechanisms of MGC formation and the processes that regulate it positively.

National audience

Induced pathogenicity in animals and humans differs considerably. This review is devoted to the relations between Brucella spp. and professional phagocytes, particularly macrophages and macrophagic cell lines in vitro. Although numerous studies have been reported, the type of ingestion by macrophages, the receptor involved, and the molecular mechanisms, are poorly understood. The ability of most Brucella species to actively inhibit their ingestion by neutrophils or macrophages has been proposed as an explanation for the poor rate of in vitro phagocytosis and in vivo alteration of the phagocytic cells. Oxidative burst plays a significant role in the antibacterial processes of phagocytic cells. The effects of whole or fractioned B. abortus on the ability of neutrophils to induce an oxidative burst in response to stimulation with opsonized zymosan particles were examined. Besides oxygen-based killing, the phagocytic cells have developed other types of defence, including hydrolytic enzymes and reactive halides. Inside the cell, the bacteria encounter new environmental conditions. Their survival is conditioned by an adaptation to this new situation. Pathogens that have acquired the ability to multiply within macrophages should synthesize products specifically interacting with the host cell defence system. Survival of intracellular pathogens is closely linked to the mechanisms of evasion from cellular defences. Brucellae stay in membrane bound vacuoles called phagosomes, but the exact nature and the maturation pathway of this compartment have not yet been understood. Macrophages play a central role in the evolution of brucellosis; this first interaction between the pathogens and the cell will determine the course of the disease. There are natural differences between brucellae species regarding macrophage response to the bacteria.

Brucella species can establish themselves and cause disease in humans, but the mechanisms by which brucellae evade the antibacterial defenses of their host remain largely unknown. We have previously reported that, unlike Escherichia coli K12, intracellular pathogens from the genus Brucella survive and multiply within U937-derived phagocytes, and live Brucella organisms failed to induce TNF-alpha release upon infection. Moreover, exogenously added TNF-alpha restricted intracellular growth of Brucella species. Herein, we demonstrate that Brucella-infected U937 cells are activated to express IL-1 beta and IL-6 at both the mRNA and protein levels, while they cannot accumulate TNF-alpha mRNA. When physically separated from macrophages, live brucellae impaired TNF-alpha production in E. coli-infected cells. Moreover, in agonist-activated macrophages, supernatants from Brucella cultures promoted an inhibition of the induction of both TNF-alpha expression and release, without affecting IL-1 beta or IL-6 induction. These phenomena, observed whatever the Brucella strain assayed, show that brucellae release some high m.w. factor(s) that specifically inhibits TNF-alpha expression in activated human macrophages. The proteic nature of the factor(s) was demonstrated by its heat and protease sensitiveness, and this could explain why U937-derived macrophages did release TNF-alpha when infected with chloramphenicol-treated brucellae. We also found that the Brucella factor(s) specifically acts on human macrophagic cells, but not on murine macrophage-like cells. Our findings provide direct evidence that a secreted Brucella virulence factor(s) inhibiting TNF-alpha expression might contribute to the evasion of Brucella organisms from human antimicrobial defenses.

The efficacy of all-trans retinoic acid (RA) in the treatment of acute promyelocytic leukemia results from the ability of RA to differentiate these peculiar leukemic cells. The efficacy of differentiation therapy could be improved and extended to other forms of leukemia by associating retinoids with other differentiating agents. Here we have compared the effects of different combinations of retinoids with 1 alpha,25-dihydroxyvitamin D3 (VD3) analogs on myelomonocytic cell lines HL-60, U937 and THP-1. All-trans RA, its natural isomer 9-cis RA and the arotinoid TTNPB, which differ by their respective specificities for the RA receptor families (retinoic acid receptor and retinoid X receptor), were found to cooperate with VD3 in inhibiting cell growth of the leukemic cell lines. Although the three cell lines displayed different susceptibilities to retinoids, each molecule was able to cooperate with VD3 in inducing U937 cell differentiation. Because the effects of VD3 on calcium metabolism limit its therapeutic use, we studied the effects of two synthetic analogs, MC903 and KH1060. Both agents cooperate with RA, acting more efficiently than the natural molecule in inhibiting cell growth and inducing some parameters of U937 cell differentiation. These results extend our previous data demonstrating that RA and VD3 exert synergistic effects on the differentiation of the myelomonocytic cell line U937. They demonstrate that combinations of agents able to inhibit leukemia cell growth with limited side effects may be found among a wide array of retinoids and vitamin D3 analogs.

We have previously described a synergism between the two physiological hormones, retinoic acid (RA) and 1 alpha,25-dihydroxyvitamin D3 (VD) in the induction of U937 cell differentiation towards a more mature state. Herein, we investigated the regulation of cytokine production during RA and/or VD treatment of U937 cells. Cell differentiation was followed by measurement of their capacity to give oxidative responses, and interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha) and IL-6 gene and protein expression were determined in RA/VD-treated cells, activated or not with lipopolysaccharide (LPS). The undifferentiated and RA-treated U937 cells were unable to produce monokines even when they were stimulated by LPS. VD induced the monokine mRNA expression in U937 cells but failed to induce protein release. However, unlike RA, it primed the cells to secrete monokines upon endotoxin stimulation. A large enhancement of the production of the monokines both at mRNA and protein levels was observed in the U937 cells exposed to the combination of RA + VD. Nevertheless, protein release required a further step of activation of the RA + VD-primed cells. The co-inducer effect of RA and VD was not observed in HL-60 or THP-1 cells and seems to be restricted to U937 cells. These results on cytokine expression support our previous finding that a combination of RA and VD brings the U937 cells to a high stage of myeloid differentiation with major characteristics of monocytes/macrophages.

Pretreatment of human lymphocytes for 2 days in 2 × 10−6 M ouabain caused irreversible loss of their subsequent capacity to stimulate in the mixed lymphocyte reaction (MLR). Pretreatment for the same period with 10−7 M ouabain resulted in an enhanced incorporation of thymidine into DNA of the responding cells in the MLR; this effect was also on the stimulating cells, as previously reported by Christen et al. (Cell. Immunol. 19, 137–142 (1975)). Pretreatment of stimulating lymphocytes with 10−7 M ouabain caused a persistent but reversible inhibition of the synthesis of RNA and protein in the MLR; peak incorporation of labelled uridine or alanine reached the same level as that of the control cultures, but 24 h later. Exactly the same persistent but reversible inhibition was found in the case of DNA synthesis of cells pretreated with 10−7 M ouabain and then stimulated by antigens (streptolysin-O and varidase) or by mitogens (phytohemagglutinin and concanavalin A); the same level of incorporation of labelled thymidine occurred but 24–48 h later than in the case of the controls. Pretreatment with the cardiotonic steroid under these conditions also resulted in a pronounced inhibition of the basal, unstimulated levels of RNA and protein synthesis in the case of both control lymphocytes and those which had been treated with mitomycin C. The effects of ouabain pretreatment on basal RNA and protein synthesis were identical for both 2 × 10−6 M and for 10−7 M; the effect of pretreatment of stimulating cells with these two concentrations was completely opposite: irreversible inhibition of the proliferative response of allogeneic responding cells at the former concentration and delayed activation at the latter.

Phytohaemagglutinin (PHA), phorbol myristate acetate (PMA) and PHA + PMA stimulation of T-enriched peripheral blood lymphocytes (PBL) and the Jurkat malignant T-cell line leads to oxidative-product formation, as evaluated by flow cytofluorometric studies, an increase in K+ flux across the membrane, cGMP production and a depolarization of the cell membrane. Irradiation (20 Gy), which enhances IL-2 synthesis by activated T-enriched PBL and Jurkat cells, also increases oxidative product formation, K+ flux, cGMP production, and induces cell membrane depolarization. Conversely, irradiation does not produce a rise in intracellular free Ca2+, as measured in PHA-stimulated Jurkat cells. PMA is also without effect on intracellular free Ca2+, added before or after PHA stimulation. Thus, except for the rise in intracellular free Ca2+, irradiation and stimulation exert similar effects on some of the events observed in IL-2-producing Jurkat cells, but these effects are not additive. Stimulation and irradiation effects are shown to be additive or synergistic only for cGMP production. It is proposed that irradiation may increase IL-2 synthesis by participating in an additional signal related to the oxidative metabolism of arachidonic acid (AA).