Adenosine inhibits the release of arachidonic acid in activated human peripheral mononuclear cells. A proposed model for physiologic and pathologic regulation in systemic lupus erythematosus.

In the current work, the pathways are presented and reviewed showing how adenosine acts on the production and release of arachidonic acid (AA) in activated human monocytes by the involvement of various phospholipase A2 (PLA2) and protein kinase C (PKC) enzymes in physiological (normal) conditions and in a pathologic state in systemic lupus erythematosus (SLE). Two molecules of activated monocytes mainly determine the actual amounts of AA released: (1) interleukin-1 beta (IL-1 beta) increasing and (2) adenosine (Ado) suppressing this process. The AA production of monocytes mainly depends on two (IV and VI) types of PLA2 enzymes. PKC alpha phosphorylates the cytosolic, Ca2+-dependent and steroid-sensitive PLA2 (type IV), whereas PKC delta phosphorylates the Ca2+-independent PLA2 (type VI). By the suppression of IL-1 beta production in the activated human monocytes, adenosine can decrease the release of AA causing a diminished phosphorylation of both PKC isoenzymes. In SLE monocytes, the disease-specific decreased release of AA that we found earlier could be related to the decreased expression of PKC delta. These pathways are summarized in a proposed model.

Lysis of endogenously infected CD4+ T cell blasts by rIL-2 activated autologous natural killer cells from HIV-infected viremic individuals.

Understanding the cellular mechanisms that ensure an appropriate innate immune response against viral pathogens is an important challenge of biomedical research. In vitro studies have shown that natural killer (NK) cells purified from healthy donors can kill heterologous cell lines or autologous CD4+ T cell blasts exogenously infected with several strains of HIV-1. However, it is not known whether the deleterious effects of high HIV-1 viremia interferes with the NK cell-mediated cytolysis of autologous, endogenously HIV-1-infected CD4+ T cells. Here, we stimulate primary CD4+ T cells, purified ex vivo from HIV-1-infected viremic patients, with PHA and rIL2 (with or without rIL-7). This experimental procedure allows for the significant expansion and isolation of endogenously infected CD4+ T cell blasts detected by intracellular staining of p24 HIV-1 core antigen. We show that, subsequent to the selective down-modulation of MHC class-I (MHC-I) molecules, HIV-1-infected p24(pos) blasts become partially susceptible to lysis by rIL-2-activated NK cells, while uninfected p24(neg) blasts are spared from killing. This NK cell-mediated killing occurs mainly through the NKG2D activation pathway. However, the degree of NK cell cytolytic activity against autologous, endogenously HIV-1-infected CD4+ T cell blasts that down-modulate HLA-A and -B alleles and against heterologous MHC-I(neg) cell lines is particularly low. This phenomenon is associated with the defective surface expression and engagement of natural cytotoxicity receptors (NCRs) and with the high frequency of the anergic CD56(neg)/CD16(pos) subsets of highly dysfunctional NK cells from HIV-1-infected viremic patients. Collectively, our data demonstrate that the chronic viral replication of HIV-1 in infected individuals results in several phenotypic and functional aberrancies that interfere with the NK cell-mediated killing of autologous p24(pos) blasts derived from primary T cells.

Apoptotic cell death in activated monocytes following incorporation of clodronate-liposomes.

The present study was performed to elucidate whether sterically stabilized liposomes laden with clodronate, which lead to depletion of macrophages (Mphis) and amelioration of experimental autoimmune arthritis in vivo, selectively affect cells of the mphi lineage in vitro. The rates of incorporation of drug-free, fluorescent liposomes and the rates of cell death following exposure to clodronate-liposomes were assessed in human peripheral blood monocytes, as well as in polymorphonuclear leukocytes (PMNs), T cells, endothelial cells, and fibroblasts, both at rest and following activation. Gel electrophoresis of nuclear extracts and ultrastructural analyses were performed to identify the modality of cell death. Monocytes, particularly upon activation, were more efficient in incorporating sterically stabilized liposomes than all other cells except PMNs. Twenty percent of resting monocytes and up to 65% of activated monocytes died within 24 h of exposure to clodronate-liposomes, whereas the other cell types, including PMNs, remained unaffected. Activated monocytes exposed to clodronate-liposomes, but not resting or activated monocytes exposed to drug-free liposomes, showed clear signs of apoptotic cell death. In most of the assays, sterically stabilized liposomes were more efficient than conventional phosphatidylcholine-liposomes. Sterically stabilized clodronate-liposomes preferentially affect cells of the mphi lineage, particularly if activated. Selective elimination of activated Mphis by apoptosis may explain both therapeutic efficacy and safety of clodronate-liposomes in experimental models of autoimmunity.

Effect of cytokines on the in vitro fungicidal activity of monocytes from paracoccidioidomycosis patients.

Peripheral blood monocytes obtained from paracoccidioidomycosis patients and healthy individuals were preactivated with recombinant gamma interferon (IFN-gamma) in different concentrations (250, 500 and 1000 U/ml) and evaluated for fungicidal activity against Paracoccidiodes brasiliensis strain 18 (Pb 18, high-virulence strain) and strain 265 (Pb 265, low-virulence strain) by plating of cocultures and counting of colony-forming units, after 10 d. Monocytes from healthy individuals failed to present fungicidal activity against P. brasiliensis even after IFN-gamma activation at the three concentrations. However, patient monocytes activated with IFN-gamma (1000 U/ml) showed a significant fungicidal activity when compared to that obtained with non-activated or activated cells with other IFN-gamma concentrations (250 and 500 U/ml). Moreover, patient monocytes presented higher fungicidal activity than the control, even before the activation process. These results may be explained by the activation state of patients' cells as a function of the in vivo contact with the fungus, which was confirmed by their higher capacity to release H(2)O(2) in vitro. Unlike the results obtained with Pb 18, patient and control cells presented a significant fungicidal activity against Pb 265, after priming with IFN- gamma. These results are explained by the higher levels of TNF-alpha in supernatants of cultures challenged with Pb 265. Moreover, higher levels of the cytokine were obtained in patient cell supernatants. Taken together, our results suggest that for effective killing of P. brasiliensis by monocytes, an initial activation signal induced by IFN-gamma is necessary to stimulate the cells to produce TNF-alpha. This cytokine may be involved, through an autocrine pathway, in the final phase activation process. The effectiveness of this process seems to depend on the virulence of the fungal strain and the activation state of the challenged cells.

Augmented 18F-FDG uptake in activated monocytes occurs during the priming process and involves tyrosine kinases and protein kinase C.

UNLABELLED: Activated monocytes with a high (18)F-FDG accumulation can affect the results of clinical PET studies. To better understand the mechanisms regulating monocytic (18)F-FDG uptake, we investigated the effect of priming and respiratory-burst generation and further evaluated the role of potential protein kinase pathways. METHODS: Purified human monocytes were primed with interferon-gamma (IFN-gamma), and respiratory burst was generated by stimulation of primed cells with phorbol-12-myristate-13-acetate (PMA). Oxygen-intermediate generation was assessed by luminescence measurements after the addition of lucigenin. (18)F-FDG uptake after 30 min of incubation was measured for unprimed control cells, primed cells, and PMA-stimulated cells. The role of protein kinases was investigated using respective inhibitors. RESULTS: PMA stimulation of primed monocytes dramatically increased oxygen-intermediate generation, leading to a 42.2 +/- 1.1 fold higher level of cumulative luminescence compared with unprimed control cells, whereas IFN-gamma priming alone resulted in low luminescence levels (13.9% +/- 4.6% of PMA-stimulated cells). In contrast, priming alone was sufficient to augment monocytic (18)F-FDG uptake to 273.3% +/- 16.7% of control levels (P < 0.001), and it was not further increased by PMA stimulation. The tyrosine kinase inhibitor, genistein, and the specific protein kinase C inhibitor, staurosporine, completely abolished the priming-induced enhancement of (18)F-FDG uptake and lowered uptake to control levels. Under the same conditions, wortmannin, a phosphatidylinositol 3 kinase (PI3 kinase)-specific inhibitor, and cycloheximide, a protein synthesis inhibitor, were associated with only minor reductions in the enhanced-uptake effect of priming. CONCLUSION: IFN-gamma priming alone, without stimulation of respiratory-burst activity, is sufficient to induce maximal augmentation of (18)F-FDG uptake in monocytes. Furthermore, this metabolic effect appears to involve tyrosine kinases and the protein kinase C pathway but is independent of the PI3 kinase pathway.

Monocyte activation for enhanced tumour necrosis factor-alpha and interleukin 6 production during chronic renal allograft rejection.

To evaluate the contribution of immune mechanisms in the initiation and progression of chronic renal allograft rejection we investigated monocyte-derived cytokine synthesis in vitro in 16 patients with histologically proven chronic rejection; 22 transplant patients with stable function served as controls. Basal tumour necrosis factor-alpha (TNF-alpha) production, measured by L 929 bioassay, was low and not significantly different in both groups. By triggering TNF-alpha formation in vitro by lipopolysaccharide (LPS), high concentrations of TNF-alpha (155.1 +/- 243.0 ng/ml) were measured in monocyte cultures from chronic rejection patients which greatly exceeded the TNF-alpha levels of 11.5 +/- 14.5 ng/ml in the control group. Measurement of interleukin 6 (IL-6) levels by enzyme immunoassay gave similar results, with significantly higher IL-6 concentrations in LPS-triggered monocyte cultures from chronic rejection compared with stable function patients. Additional stimulation of LPS-treated monocytes with interferon-gamma (IFN-gamma) as a priming agent enhanced TNF-alpha formation in stable function patients and, in contrast, slightly reduced monokine formation in chronic rejection patients, which suggests that in this group a high activation level of monocytes has already been reached in vivo by T cell factors such as IFN-gamma. Treatment of monocyte cultures in vitro with prednisolone reduced TNF-alpha formation differently in most but not all cultures from chronic rejection and stable function patients; thus this in vitro test system might be helpful in predicting the benefit of an intensified immunosuppressive regimen for chronic rejection patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumoricidal activity of interferon-r activated peripheral monocytes in colorectal cancer patients.

Peripheral blood monocytes obtained from 8 colorectal cancer patients and 6 normal controls were incubated in vitro with interferon-r (IFN-r) in the presence of bacterial lipopolysaccharide (LPS). The cytotoxic properties of the monocyte were determined subsequent to the interaction with radiolabeled autologous, allogeneic, as well as cultured colorectal cancer cells. Monocytes from normal controls and all colorectal cancer patients were activated in vitro to become tumoricidal; monocytes lysed tumorigenic cells but not nontumorigenic cells. Activators of protein kinase C (e.g. phorbol esters, PMA) and Ca2+ ionophores (A23187) when added alone did not effect the activation state of the monocyte. Whereas, PMA and A23187 cooperatively reproduced the ability of IFN-r to prime monocytes for tumoricidal activity. In the presence of PMA, A23187, and EGTA, the addition of excessive Ca2+ was sufficient for priming, whereas the addition of excessive Mg2+ was much less efficient. Priming by IFN-r, however, was not blocked by EGTA. An efflux of Ca2+ from preloaded monocytes was significantly increased by A23187 and by IFN-r. Quin-2/AM, an intracellular chelator of Ca2+, blocked priming by IFN-r. The results suggest that priming of monocytes for tumoricidal function by IFN-r may be involved in the activation of protein kinase C and mobilization of intracellular Ca2+.

Near eradication of clinically relevant concentrations of human tumor cells by interferon-activated monocytes in vitro.

We have previously reported that low concentrations of interferon (IFN)-activated monocytes exert near-eradicative cytocidal activity against low concentrations of several human tumor cells in vitro. In the present study, we examined 7 human tumor cell lines and 3 diploid lines in the presence or absence of 10 ng/mL IFNα2a and monocytes. The results confirmed strong cytocidal activity against 4 of 7 tumor lines but none against 3 diploid lines. To model larger in vivo tumors, we increased the target cell concentration and determined the concentration of IFNα2a and monocytes, required for cell death. We found that increasing the tumor cell concentration from 10- to 100-fold (10(5) cells/well) required an increase in the concentration of IFNs by over 100-fold and monocytes by 10-fold. High concentrations of monocytes could sometimes kill tumor or diploid cells in the absence of IFN. We may conclude that killing of high concentrations of tumor or diploid cells required high concentrations of monocytes that could sometimes kill in the absence of IFN. Thus, high concentrations of tumor cells required high concentrations of IFN and monocytes to cause near eradication of tumor cells. These findings may have clinical implications.

HIF-1 alpha expression is associated with an atheromatous inflammatory plaque phenotype and upregulated in activated macrophages.

OBJECTIVE: Angiogenesis and inflammation are important features in atherosclerotic plaque destabilization. The transcription factor hypoxia-inducible factor-1 alpha (HIF-1 alpha) is a key regulator of angiogenesis and is also involved in inflammatory reactions. We studied HIF-1 alpha expression in different atherosclerotic plaque phenotypes. METHODS AND RESULTS: HIF-1 alpha expression was observed in 18/37 (49%) carotid and in 9/15 (60%) femoral endarterectomy specimens. Expression of HIF-1 alpha was associated with the presence of a large extracellular lipid core (P=0.03) and macrophages (P=0.02). HIF-1 alpha co-localized with vascular endothelial growth factor (VEGF), an important downstream target of HIF-1 alpha. In addition, a strong association was observed between expression levels of HIF-1 alpha and VEGF (P=0.001). The average number of plaque microvessels was higher in plaques with no or minor HIF-1 alpha staining than in plaques with moderate or heavy HIF-1 alpha staining (P=0.03). In human macrophages, lipopolysaccharide activation induced HIF-1 alpha expression. In embryonic fibroblasts derived from wild-type mice, lipopolysaccharide activation induced an increase in HIF-1 alpha mRNA, whereas in Toll-like receptor 4 defective embryonic fibroblasts no effect was observed after lipopolysaccharide stimulation. CONCLUSIONS: In atherosclerotic plaque, the transcription factor HIF-1 alpha is associated with an atheromatous inflammatory plaque phenotype and with VEGF expression. HIF-1 alpha expression is upregulated in activated macrophages under normoxic conditions.

TNF-alpha activates human monocytes for Paracoccidioides brasiliensis killing by an H2O2-dependent mechanism.

Human monocytes activated by recombinant tumor necrosis factor alpha (TNF-alpha) exhibited significant fungicidal activity on the yeast cells of a highly virulent strain of Paracoccidioides brasiliensis. This process was significantly inhibited in the presence of catalase (CAT - a scavenger of H2O2), but not in the presence of superoxide-dismutase (SOD - a scavenger of superoxide anion) or NG-monomethyl-L-arginine (NG-MMLA - a nitric oxide inhibitor). Furthermore, there was a direct association between the intracellular killing of the fungus and the production of H2O2 by activated cells. These results strongly suggest a role for H2O2 in the killing of highly virulent strains of P. brasiliensis by TNF-alpha-activated human monocytes.

Increased progesterone concentrations are necessary to suppress interleukin-2-activated human mononuclear cell cytotoxicity.

Fetal trophoblast is generally resistant to lysis by cytotoxic cells. Trophoblast progesterone and estrogens may act at the choriodecidual interface, where they are present in high concentrations to provide a local, paracrine immunosuppressive effect on cellular cytotoxicity. However, interleukin activation of these cytotoxic lymphocytes enhances their ability to lyse trophoblast. Recent evidence suggests that immunoactivation occurs in certain aberrant pregnancy conditions, including preeclampsia. Preeclamptic placentas produce more progesterone in vitro than do normal placentas. To study the potential association between progesterone production and immunoactivation, we evaluated the immunomodulatory effect of progesterone on cellular cytotoxicity. Comparisons were made with the use of both normal and interleukin-2-stimulated peripheral blood mononuclear cells as effector cells in a cytotoxicity assay. Progesterone suppressed cytotoxicity in a dose-dependent manner. Interleukin-2 augmented cellular cytotoxicity, and higher concentrations of progesterone were required to attenuate this response. An additive suppression of cytotoxicity was also observed when estrone, estradiol, estriol, and progesterone were combined. We speculate that the higher placental production of progesterone seen in preeclampsia may be a trophoblast compensatory response to immunoactivated maternal effector cells.

The prostaglandin analogs, misoprostol and SC-46275, potently inhibit cytokine release from activated human monocytes.

Inflammatory mediator release is one of the body's responses to tissue injury and inflammation. These mediators, such as interleukin-1 beta (I1-1 beta), tumor necrosis factor (TNF-alpha), and products of arachidonic acid metabolism, are themselves proinflammatory. Purified human monocytes stimulated in vitro with E. coli-derived lipopolysaccharide (LPS) will release these key cytokines along with various other eicosanoid mediators. Monocytes incubated with LPS and the prostaglandin E-1 analog, misoprostol, released significantly lower levels of cytokines compared to monocytes incubated with LPS alone. Eicosanoid release was also affected by misoprostol. SC-46275, a more potent mucosal protective PGE1 analog, also altered the release of cytokines and eicosanoids from human monocytes. However SC-46275 inhibited I1-1 beta release with an IC50 value of 9 microM compared to 75 microM for misoprostol. SC-46275 and misoprostol both inhibited TNF-alpha release. These data suggest there is a potential immunomodulatory role for prostaglandin analogs in the therapeutic treatment of inflammatory diseases such as ulcerative colitis, Crohn's disease, and autoimmune inflammatory diseases of the central nervous system.

[Changes in the profile of cytotoxic mediator monocytes in patients with cancer and precancerous conditions of the stomach]. / Izmenenie profilia tsitotoksicheskikh mediatorov monotsitov u bol'nykh rakom i predrakovymi sostoianiiami zheludka.

Peripheral blood monocytes from healthy subjects, patients with gastric precancer disease (chronic gastric ulcer, stomach polyps and chronic atrophic gastritis) and different stages of gastric cancer were used. Spontaneous and lipopolysaccharide (LPS)-stimulated TNF-like factors production by monocytes was significantly higher in the precancer gastric disease patients than in the healthy subjects. At the same time the spontaneous capacity of monocytes to produce NTF-like factors was 2.5 lower in the gastric cancer patients compared to the healthy subjects. Moreover, in 5/13 of the gastric cancer patients in TNF-like factors production by the LPS-stimulated and non-stimulated monocytes was 1 unit/ml less. Spontaneous and reactive CL indexes were higher in the cancer patients monocytes than in the healthy subjects. The obtained results suggest that reactive oxygen species production can be an alternative mechanism by which a cytotoxic action of monocytes is regulated.

Stress-induced enhancement of NF-kappaB DNA-binding in the peripheral blood leukocyte pool: effects of lymphocyte redistribution.

To identify signaling pathways by which the sympathetic nervous system (SNS) might alter gene expression in the immune system, we assayed activation of the inflammatory transcription factor NF-kappaB in peripheral blood mononuclear cells (PBMC) from 13 healthy young adults at rest and following 5 min of intense exercise. SNS activation was verified by changes in cardiovascular parameters and mobilization of NK cells into circulating blood. Electrophoretic mobility shift assays (EMSA) of nuclear protein extracts confirmed previous findings that SNS activation increased NF-kappaB DNA-binding activity in bulk PBMC. However, analyses of isolated leukocyte subsets failed to indicate any increase on a per-cell basis in NK cells (the major carriers of NF-kappaB activity in circulating PBMC), in the residual CD56- leukocyte pool, or in CD14+ monocytes. Regression analyses indicated a strong correlation between increasing NK cell prevalence and changes in NF-kappaB DNA-binding activity in bulk PBMC, and suggested that no change in EMSA activity would be observed in the absence of NK cell mobilization. Such results imply that SNS-induced mobilization of NK cells can rapidly (< 10 min) alter NF-kappaB DNA-binding activity in the circulating PBMC pool without generating any true change in NF-kappaB activity on a per-cell basis. Implications for future efforts to analyze stress effects on leukocyte gene expression are considered.