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In a previous study, we analysed the synthesis and properties of a series of imidazo[1,2-a]quinoxalines designed in our laboratory as possible imiquimod analogues. We found that these imidazo[1,2-a]quinoxalines were in fact potent inhibitors of phosphodiesterase 4 enzymes (PDE4). PDE4 inhibition normally results in an increase in intracellular cAMP which, in PBMC, induces the suppression of TNF-α mRNA transcription and thus cytokine synthesis. Such an effect is antagonistic to that of imiquimod. Furthermore, some TNF-α-induced activity, such as cell apoptosis which is dependent on the intracellular cAMP levels might also be affected. Therefore, by counteracting the properties of TNF-α and/or its production, the imidazo[1,2-a]quinoxalines could be considered as potential antiinflammatory drugs. The present study was performed to confirm or refute this hypothesis. For this, we characterized the effects of imidazo[1,2-a]quinoxalines both on TNF-α activity and synthesis in regard to their ability to act as inhibitors of PDE4 (IPDE4). We found that the imidazo[1,2-a]quinoxalines dose-dependently prevented the TNF-α-triggered death of L929 cells, with the 8-series (-NHCH3 in R4) being the most potent. Moreover, when the effect of the 8-series on TNF-α production was investigated using γ9δ2 T cells, it was observed that these compounds impaired the TCR:CD3-triggered TNF-α production. Structure-activity analysis revealed that these properties of the drugs did not coincide with their IPDE4 properties. This prompted further exploration into other signalling mechanisms possibly involved in TNF-α action and production, notably the p38 MAPK and the PI3K pathway. We demonstrate here that the imidazo[1,2-a]quinoxalines targeted these pathways in a different way: they activated the p38 MAPK pathway whilst inhibiting the PI3K pathway. Such effects on cell signalling could account for the imidazo[1,2-a]quinoxalines effects on 1) action and 2) production of TNF-α, which define these drugs as potential anti-inflammatory agents.

During the complex interaction between an infectious agent and a host organism, the pathogen can interfere with the host cell's programmed death to its own benefit. Induction or prevention of host cell apoptosis appears to be a critical step for determining the infection outcome. Members of the gram-negative bacterial genus Brucella are intracellular pathogens which preferentially invade monocytic cells and develop within these cells. We investigated the effect of Brucella suis infection on apoptosis of human monocytic phagocytes. The present study provides evidence that Brucella infection inhibited spontaneously occurring apoptosis in human monocytes. Prevention of monocyte apoptosis was not mediated by Brucella lipopolysaccharide and required bacterial survival within infected cells. Both invaded and noninvaded cells were protected, indicating that soluble mediators released during infection were involved in the phenomenon. Analysis of Brucella-infected monocytes revealed specific overexpression of the A1 gene, a member of the bcl-2 family implicated in the survival of hematopoietic cells. Brucella infection also rendered macrophage-like cells resistant to Fas ligand- or gamma interferon-induced apoptosis, suggesting that Brucella infection protected host cells from several cytotoxic processes occurring at different steps of the immune response. The present data clearly show that Brucella suis modulated the monocyte/macrophage's apoptotic response to the advantage of the pathogen, thus preventing host cell elimination. This might represent a strategy for Brucella development in infected hosts.

All trans retinoic acid and vitamin D3 derivatives are well known for their antileukemic activity, while the precise mechanism of this effect remains to be clarified. Using human leukemic U937 and THP-1 promonocytic cell lines, we analyzed the effect of all-trans retinoic acid (RA) and/or 1,25-dihydroxyvitamin D3 (VD) on the generation of nitric oxide (NO), a potent antitumoral mediator. U937 cell differentiation with VD or with both RA and VD (RA/VD) correlated with gene transcription and functional expression of inducible nitric oxide synthase (iNOS). Nitrites and L-citrulline were also detected in U937 cell supernatants as soon as 24 hours following cell incubation with VD or RA/VD, but not in cells treated with RA alone. Inhibition of iNOS activity by NG-monomethyl-L-arginine (LNMMA) significantly decreased in vitro U937 cell differentiation with VD and RA/VD as shown by the expression of cell differentiation markers (CD14 and CD68) and by the capacity of these cells to undergo a luminol-dependent chemiluminescence in response to opsonized zymosan. Similar results were obtained using the THP-1 cell line strengthening the role of NO in the VD- and RA/VD-induced growth arrest and terminal differentiation of promonocytic leukemia cells.

We have analysed Con A-stimulated mouse lymphocytes for the presence of 2'-5'-linked oligoadenylates using a radioimmunoassay based on a monoclonal antibody raised against adenylyl (2'-5') adenosine (A2'pA). Time-course and Con A dose dependence were performed.We found that Con A induced, in a dose-dependant manner, the accumulation of immunoreactive material, together with the incorporation of 3H-thymidine in DNA. We showed that the immunoreactive material was constituted for the essential, by a novel 2'-5' oligoadenylate. It was isolated and characterized as adenylyl 2'-5'adenylic acid (2' and 3'P) according to the combination of criteria such as immunoreactivity, enzyme susceptibility, chromatographic behaviour and comparison with A2'pA3'(32P)p, A2'pA3'p acid A2'pA2'p that we have chemically synthetized. This is the first example of significant variations of a 2'-5' oligoadenylate level in circumstances other than the antiviral mechanism of interferon.

Vascular endothelium produces and/or interferes with various cytokines. Previous studies have demonstrated interactions of these inflammatory and immunological mediators with oxygen-derived free radicals. The present work examines the relationship between hypoxia/reoxygenation (H/R) and cytokine production by cultured endothelial cells. Human umbilical vein endothelial cell (HUVEC) monolayers were incubated for 24 h in normoxia or submitted to 5 h hypoxia/19 h reoxygenation. Then, interleukin-1 (IL-1) α and β, and interleukin-6 (IL-6), were measured in culture supernatants by specific enzyme immunoassays and bioassays, respectively.

Vascular endothelium is known to closely interact with leukocytes and immunocompetent cells. We report here that cultured bovine aortic endothelial cells (BAEC) synthesize both interleukin 1 (IL-1) and interleukin 6 (IL-6) like activities in response to bacterial lipopolysaccharide stimulation. Our results agree with previous data obtained from human venous endothelia and support the concept that IL-1 and IL-6 synthesis are properties common to endothelial cells from different vascular beds. The IL-1 activity was measured by murine thymocyte proliferation assay and by an indirect bioassay using NOB1 cells, which evidenced higher IL-1 amounts than the former. This discrepancy appeared to be partly due to the simultaneous production of one or more inhibitor(s) of the thymocyte proliferation by BAEC. The IL-6 assay was performed with the murine hydridoma cell line B9. In other respects, the cyclooxygenase inhibitor indomethacin enhanced the IL-1 like production, but was ineffective on IL-6 like production. The present study provides additional evidence that endothelial cells from large arteries may also participate in inflammatory and immunological processes.

Lymphocytes possess membrane adenosine receptors through which this nucleoside or analogs activate adenylate cyclase. Several adenosine analogs - 5'-N-ethylcarboxamide adenosine (NECA), N6-(L-2-phenyl-isopropyl) adenosine (PIA), 2-chloro adenosine (Cl-Ado) - were tested on the adenylate cyclase of mouse thymocyte homogenates; the order of potencies: NECA > Cl-Ado > PIA is consistent with the order which has been assumed to be characteristic of Ra type adenosine receptors. The stimulation of thymocyte adenylate cyclase by NECA was biphasic, while the responses to Cl-Ado and PIA were not, which suggests the existence of Ra sub-types of adenosine receptors. Moreover, these adenosine receptors were found to be present in several lymphocyte subclasses: mature and immature mouse thymocytes, B and T mouse splenocytes, human peripheral blood lymphocytes, and also in lymphoblastoid cell lines. No crucial differences were observed between the adenylate cyclase stimulations of the various populations by NECA.

We report the effect of CBS-211A, a synthetic retinoid analog, designed for topical eye administration, on the growth and differentiation of myelomonocytic cells. This compound was assayed alone or in combination with 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), since we previously evidenced a synergism of retinoic acid (RA) and 1α,25(OH)2D3 in the induction of U937 cell differentiation. Unlike RA, CBS-211A neither affected the growth of myelomonocytic cells nor differentiated them. Nevertheless, when it was associated with 1α,25(OH)2D3, CBS-211A strongly potentiated the 1α,25(OH)2D3-induced inhibition of U937 cell proliferation and caused a dramatic increase in their differentiation toward monocytes/macrophages. The co-inducing effect of CBS-211A was restricted to U937 cells.

We examined the expression and activity of inducible nitric oxide synthase (iNOS) in both gamma interferon (IFN-gamma)-treated and untreated murine macrophages infected with the gram-negative bacterium Brucella suis. The bacteria were opsonized with a mouse serum containing specific antibrucella antibodies (ops-Brucella) or with a control nonimmune serum (c-Brucella). The involvement of the produced NO in the killing of intracellular B. suis was evaluated. B. suis survived and replicated within J774A.1 cells. Opsonization with specific antibodies increased the number of phagocytized bacteria but lowered their intramacrophage development. IFN-gamma enhanced the antibrucella activity of phagocytes, with this effect being greater in ops-Brucella infection. Expression of iNOS, interleukin-6, and tumor necrosis factor alpha (TNF-alpha) mRNAs was induced in both c-Brucella- and ops-Brucella-infected cells and was strongly potentiated by IFN-gamma. In contrast to that of cytokine mRNAs, iNOS mRNA expression was independent of opsonization. Similar levels of iNOS mRNAs were expressed in IFN-gamma-treated cells infected with c-Brucella or ops-Brucella; however, expression of iNOS protein and production of NO were detected only in IFN-gamma-treated cells infected with ops-Brucella. These discrepancies between iNOS mRNA and protein levels were not due to differences in TNF-alpha production. The iNOS inhibitor N omega-nitro-L-arginine methyl ester increased B. suis multiplication specifically in IFN-gamma-treated cells infected with ops-Brucella, demonstrating a microbicidal effect of the NO produced. This observation was in agreement with in vitro experiments showing that B. suis was sensitive to NO killing. Together our data indicate that in B. suis-infected murine macrophages, the posttranscriptional regulation of iNOS necessitates an additive signal triggered by macrophage Fcgamma receptors. They also support the possibility that in mice, NO favors the elimination of Brucella, providing that IFN-gamma and antibrucella antibodies are present, i.e., following expression of acquired immunity.

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.

Tumor necrosis factor alpha (TNF-alpha) plays a central role in activation of first-line defenses of a host against foreign organisms. To determine whether Brucella infection modulated TNF-alpha production, we measured the biological activity of this cytokine in supernatants of U937 cell-derived macrophages and of fresh human monocytes infected with Brucella spp. Neither the smooth nor rough Brucella strains used induced any measurable TNF-alpha excretion upon infection. On the contrary, as reported before for other gram-negative bacteria, phagocytosis of nonpathogenic Escherichia coli was followed by a rapid and transient induction of TNF-alpha release, suggesting an involvement of this cytokine in some autocrine process. As expected, the Brucella strains tested survived and/or multiplied within U937-derived macrophages, whereas E. coli was rapidly eliminated after phagocytosis. Immunoglobulin G opsonization of E. coli strains enhanced their intracellular killing and strongly potentiated TNF-alpha secretion. Immunoglobulin G opsonization of Brucella strains, in contrast, did not lead to TNF-alpha production, although their rate of intracellular multiplication was reduced. Killed brucellae, however, promoted a significant excretion of TNF-alpha from U937-derived macrophages into cell culture supernatants. We finally demonstrated that pretreatment of U937-derived macrophages with exogenous TNF-alpha significantly inhibited intracellular multiplication of Brucella spp. These results and experiments performed on fresh human monocytes or with isolated lipopolysaccharide (LPS) showed that (i) differences in TNF-alpha production observed during macrophage infection by Brucella spp. and E. coli were not due to differences in LPS structure but resulted from active inhibition of TNF-alpha production by a specific process linked to Brucella spp. and (ii) the capacity of Brucella spp. to use pathways avoiding TNF-alpha production during infection may be considered a major attribute of virulence.