Monocyte Priming Research Articles

Monocyte trafficking to the brain with stress and inflammation: a novel axis of immune-to-brain communication that influences mood and behavior.

HIGHLIGHTS Psychological stress activates neuroendocrine pathways that alter immune responses.Stress-induced alterations in microglia phenotype and monocyte priming leads to aberrant peripheral and central inflammation.Elevated pro-inflammatory cytokine levels caused by microglia activation and recruitment of monocytes to the brain contribute to development and persistent anxiety-like behavior.Mechanisms that mediate interactions between microglia, endothelial cells, and macrophages and how these contribute to changes in behavior are discussed.Sensitization of microglia and re-distribution of primed monocytes are implicated in re-establishment of anxiety-like behavior.Psychological stress causes physiological, immunological, and behavioral alterations in humans and rodents that can be maladaptive and negatively affect quality of life. Several lines of evidence indicate that psychological stress disrupts key functional interactions between the immune system and brain that ultimately affects mood and behavior. For example, activation of microglia, the resident innate immune cells of the brain, has been implicated as a key regulator of mood and behavior in the context of prolonged exposure to psychological stress. Emerging evidence implicates a novel neuroimmune circuit involving microglia activation and sympathetic outflow to the peripheral immune system that further reinforces stress-related behaviors by facilitating the recruitment of inflammatory monocytes to the brain. Evidence from various rodent models, including repeated social defeat (RSD), revealed that trafficking of monocytes to the brain promoted the establishment of anxiety-like behaviors following prolonged stress exposure. In addition, new evidence implicates monocyte trafficking from the spleen to the brain as key regulator of recurring anxiety following exposure to prolonged stress. The purpose of this review is to discuss mechanisms that cause stress-induced monocyte re-distribution in the brain and how dynamic interactions between microglia, endothelial cells, and brain macrophages lead to maladaptive behavioral responses.

Identification of structural features of ursolic acid required to protect monocytes against metabolic stress‐induced dysfunction (249.5)

We showed that ursolic acid (UA), a pentacyclic triterpenoid with anti‐inflammatory properties, reduces atherosclerotic lesion size and improves kidney function in diabetic mice. We proposed that the atheroprotective effects of UA are due to its ability to protect monocytes against metabolic stress‐induced dysfunction or “priming”, i.e. the Nox4‐mediated hyper‐responsiveness to chemokine‐induced cell adhesion and migration. The aim of this study was to test eight of UA’s naturally occurring structural analogs and identify the structural features of UA required to inhibit monocyte priming. We determined in metabolically primed THP‐1 monocytes the doses (0.03 ‐ 3 μM) at which UA and its analogues blocked metabolic stress‐induced Nox4 induction and accelerated chemotaxis in response to MCP‐1. Only the UA analogs uvaol and corosolic acid protected monocytes against metabolic priming. We found that altering the position of one methyl group in position 19 of UA abolishes its protective effects. Furthermore, the carboxyl group in position 17 appears to be critical but not sufficient for UA to prevent monocyte priming by metabolic stress. In summary, our data suggest a highly selective molecular mechanism for the protective effects of UA on monocyte priming and dysfunction and UA’s anti‐atherogenic properties. Identifying the functional groups responsible for UA’s beneficial effects will allow us to develop more potent, low‐cost dietary supplements for the prevention of atherosclerosis and other monocyte‐mediated diabetic complications.Grant Funding Source: Supported by grants to RA from the NIH (R01‐AT006885)

Group VIA Phospholipase A 2 Mediates Enhanced Macrophage Migration in Diabetes Mellitus by Increasing Expression of Nicotinamide Adenine Dinucleotide Phosphate Oxidase 4

Objective— We previously demonstrated that nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) mediates increased monocyte priming and chemotaxis under conditions of diabetic metabolic stress, and emerging data indicate that group VIA phospholipase A 2 (iPLA 2 β) also participates in regulating monocyte chemotaxis. Here, we examined relationships between iPLA 2 β expression and Nox4 action in mouse peritoneal macrophages subjected to diabetic metabolic stress. Approach and Results— Increased iPLA 2 β expression and activity were observed in macrophages from low-density lipoprotein receptor knockout mice that were fed a high-fat diet, and this was associated with time-dependent increases in atherosclerotic lesion size and macrophage content. Incubating macrophages with 30 mmol/L D-glucose, 100 μg/mL low-density lipoprotein, or both (D-glucose+low-density lipoprotein) induced a robust increase in iPLA 2 β expression and activity and in cell migration in response to monocyte chemoattractant protein-1. The increases in iPLA 2 β activity and cell migration were prevented by a bromoenol lactone iPLA 2 β suicide inhibitor or an iPLA 2 β antisense oligonucleotide. Incubating macrophages under conditions that mimic diabetic metabolic stress ex vivo resulted in increased Nox4 expression and activity and hydrogen peroxide generation compared with controls. Bromoenol lactone prevented those effects without affecting Nox2 expression. Nox4 inhibition eliminated diabetic metabolic stress–induced acceleration of macrophage migration. Lysophosphatidic acid restored Nox4 expression, hydrogen peroxide generation, and migration to bromoenol lactone–treated cells, and a lysophosphatidic acid receptor antagonist abrogated iPLA 2 β-mediated increases in Nox4 expression. Conclusions— Taken together, these observations identify iPLA 2 β and lysophosphatidic acid derived from its action as critical in regulating macrophage Nox4 activity and migration in the diabetic state in vivo and under similar conditions ex vivo.

Regulation of Monocyte Adhesion and Migration by Nox4

We showed that metabolic disorders promote thiol oxidative stress in monocytes, priming monocytes for accelerated chemokine-induced recruitment, and accumulation at sites of vascular injury and the progression of atherosclerosis. The aim of this study was to identify both the source of reactive oxygen species (ROS) responsible for thiol oxidation in primed and dysfunctional monocytes and the molecular mechanisms through which ROS accelerate the migration and recruitment of monocyte-derived macrophages. We found that Nox4, a recently identified NADPH oxidase in monocytes and macrophages, localized to focal adhesions and the actin cytoskeleton, and associated with phospho-FAK, paxillin, and actin, implicating Nox4 in the regulation of monocyte adhesion and migration. We also identified Nox4 as a new, metabolic stress-inducible source of ROS that controls actin S-glutathionylation and turnover in monocytes and macrophages, providing a novel mechanistic link between Nox4-derived H2O2 and monocyte adhesion and migration. Actin associated with Nox4 was S-glutathionylated, and Nox4 association with actin was enhanced in metabolically-stressed monocytes. Metabolic stress induced Nox4 and accelerated monocyte adhesion and chemotaxis in a Nox4-dependent mechanism. In conclusion, our data suggest that monocytic Nox4 is a central regulator of actin dynamics, and induction of Nox4 is the rate-limiting step in metabolic stress-induced monocyte priming and dysfunction associated with accelerated atherosclerosis and the progression of atherosclerotic plaques.

Abstract 466: Redox Regulation of 14-3-3zeta Controls Monocyte Migration

Cofilin regulates actin turnover and is inactivated by phosphorylation at Ser-3 by LIM kinase (LIMK) and reactivated by the cofilin-phosphatase Slingshot-1L (SSH1L). We recently reported that protein-S-glutathionylation induced by metabolic disorders enhances monocyte recruitment into vascular lesions. Because monocyte priming and accelerated migration involves increases actin turnover, we tested the hypothesis that these regulators of actin assembly/disassembly are redox-regulated. Metabolic priming was induced in human THP-1 monocytes by exposing the cells to diabetic conditions, i.e. human native LDL plus high glucose concentrations (LDL+HG). Phosphorylation of cofilin in response to MCP-1 was completely blocked in metabolically primed monocytes, suggesting that hyperactivation of cofilin may contribute to the enhanced chemotactic activity of primed monocytes. Yet, neither LIMK1 nor SSH1L levels were affected by metabolic stress. However, metabolic priming resulted in a 3.8-fold increase the S-glutathionylation of 14-3-3zeta, a SSH1L binding protein which maintains the cofilin phosphatase in an inactive state. S-glutathionylation of 14-3-3zeta resulted in its caspase-dependent degradation and a 35% reduction in 14-3-3zeta protein levels. Overexpression of glutaredoxin 1 in monocytes inhibited both LDL+HG-induced S-glutathionylation and degradation of 14-3-3zeta, and normalized cofilin phosphorylation in response to MCP-1. The C25S mutant of 14-3-3zeta was resistant to S-glutathionylation and degradation induced by LDL+HG, and restored MCP-1-induced cofilin phosphorylation in metabolically primed monocytes. 14-3-3zeta S-glutathionylation did not affect binding of 14-3-3zeta to SSH1L, suggesting that loss of 14-3-3zeta increases the pool of free SSH1L phosphatase, thus preventing the phosphorylation and deactivation of cofilin in response to chemokine activation. In conclusion, we identified a novel redox-sensitive mechanism for the regulation of monocyte migration that links (thiol) oxidative stress induced by metabolic disorders to monocyte dysfunction and the conversion of blood monocytes into a hypermigratory, proinflammatory and proatherogenic phenotype.

Redox regulation of MAPK phosphatase 1 controls monocyte migration and macrophage recruitment.

Monocytic adhesion and chemotaxis are regulated by MAPK pathways, which in turn are controlled by redox-sensitive MAPK phosphatases (MKPs). We recently reported that metabolic disorders prime monocytes for enhanced recruitment into vascular lesions by increasing monocytes' responsiveness to chemoattractants. However, the molecular details of this proatherogenic mechanism were not known. Here we show that monocyte priming results in the S-glutathionylation and subsequent inactivation and degradation of MKP-1. Chronic exposure of human THP-1 monocytes to diabetic conditions resulted in the loss of MKP-1 protein levels, the hyperactivation of ERK and p38 in response to monocyte chemoattractant protein-1 (MCP-1), and increased monocyte adhesion and chemotaxis. Knockdown of MKP-1 mimicked the priming effects of metabolic stress, whereas MKP-1 overexpression blunted both MAPK activation and monocyte adhesion and migration induced by MCP-1. Metabolic stress promoted the S-glutathionylation of MKP-1, targeting MKP-1 for proteasomal degradation. Preventing MKP-1 S-glutathionylation in metabolically stressed monocytes by overexpressing glutaredoxin 1 protected MKP-1 from degradation and normalized monocyte adhesion and chemotaxis in response to MCP-1. Blood monocytes isolated from diabetic mice showed a 55% reduction in MKP-1 activity compared with nondiabetic mice. Hematopoietic MKP-1 deficiency in atherosclerosis-prone mice mimicked monocyte priming and dysfunction associated with metabolic disorders, increased monocyte chemotaxis in vivo, and accelerated atherosclerotic lesion formation. In conclusion, we identified MKP-1 as a central redox-sensitive regulator of monocyte adhesion and migration and showed that the loss of MKP-1 activity is a critical step in monocyte priming and the metabolic stress-induced conversion of blood monocytes into a proatherogenic phenotype.

NADPH Oxidase 4 Mediates Monocyte Priming and Accelerated Chemotaxis Induced by Metabolic Stress

Metabolic disorders increase monocyte chemoattractant protein-1 (MCP-1)-induced monocyte chemotaxis in mice. The goal of this study was to determine the molecular mechanisms responsible for the enhanced responsiveness of monocytes to chemoattractants induced by metabolic stress. Chronic exposure of monocytes to diabetic conditions induced by human LDL plus high D-glucose concentrations (LDL+HG) promoted NADPH Oxidase 4 (Nox4) expression, increased intracellular H(2)O(2) formation, stimulated protein S-glutathionylation, and increased chemotaxis in response to MCP-1, platelet-derived growth factor B, and RANTES. Both H(2)O(2) added exogenously and overexpression of Nox4 mimicked LDL+HG-induced monocyte priming, whereas Nox4 knockdown protected monocytes against metabolic stress-induced priming and accelerated chemotaxis. Exposure of monocytes to LDL+HG promoted the S-glutathionylation of actin, decreased the F-actin/G-actin ratio, and increased actin remodeling in response to MCP-1. Preventing LDL+HG-induced protein S-glutathionylation by overexpressing glutaredoxin 1 prevented monocyte priming and normalized monocyte chemotaxis in response to MCP-1. Induction of hypercholesterolemia and hyperglycemia in C57BL/6 mice promoted Nox4 expression and protein S-glutathionylation in macrophages, and increased macrophage recruitment into MCP-1-loaded Matrigel plugs implanted subcutaneous in these mice. By increasing actin-S-glutathionylation and remodeling, metabolic stress primes monocytes for chemoattractant-induced transmigration and recruitment to sites of vascular injury. This Nox4-dependent process provides a novel mechanism through which metabolic disorders promote atherogenesis.

Proteome of monocyte priming by lipopolysaccharide, including changes in interleukin-1beta and leukocyte elastase inhibitor

BackgroundMonocytes can be primed in vitro by lipopolysaccharide (LPS) for release of cytokines, for enhanced killing of cancer cells, and for enhanced release of microbicidal oxygen radicals like superoxide and peroxide. We investigated the proteins involved in regulating priming, using 2D gel proteomics.ResultsMonocytes from 4 normal donors were cultured for 16 h in chemically defined medium in Teflon bags ± LPS and ± 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), a serine protease inhibitor. LPS-primed monocytes released inflammatory cytokines, and produced increased amounts of superoxide. AEBSF blocked priming for enhanced superoxide, but did not affect cytokine release, showing that AEBSF was not toxic. After staining large-format 2D gels with Sypro ruby, we compared the monocyte proteome under the four conditions for each donor. We found 30 protein spots that differed significantly in response to LPS or AEBSF, and these proteins were identified by ion trap mass spectrometry.ConclusionWe identified 19 separate proteins that changed in response to LPS or AEBSF, including ATP synthase, coagulation factor XIII, ferritin, coronin, HN ribonuclear proteins, integrin alpha IIb, pyruvate kinase, ras suppressor protein, superoxide dismutase, transketolase, tropomyosin, vimentin, and others. Interestingly, in response to LPS, precursor proteins for interleukin-1β appeared; and in response to AEBSF, there was an increase in elastase inhibitor. The increase in elastase inhibitor provides support for our hypothesis that priming requires an endogenous serine protease.

Oxidized LDL specifically promotes the initiation of monocyte invasion at cell‐cell junctions by upregulating PECAM‐1

Oxidized LDL (oxLDL) has been reported to activate vascular endothelial cells and promote monocyte diapedesis. But how and on which steps oxLDL affects the monocyte dynamics has been poorly addressed. Using human umbilical vein endothelial cells and freshly-isolated human monocytes, we developed an in vitro proatherogenic model with oxLDL embedded in very thin (30–50 μm) subendothelial collagen gels. This model resembled the subendothelial accumulation of oxLDL in atherosclerotic plaques in vivo, and enabled to track 3D-kinetics of individual living monocytes by confocal laser scanning microscopy. We divided the monocyte dynamics into three steps (1. adhesion, 2. diapedesis at cell-cell junctions, 3. migration under endothelium), and found that oxLDL specifically and significantly increased the number of adherent monocytes that started diapedesis at junctions (step 1–2), but had no effect thereafter (step 2–3). Migration speed of each monocyte was not affected by oxLDL throughout the whole process (step 1–3). To explore the role of oxLDL, we examined the expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) by western blotting, and showed that both hydrophobic (membrane-bound) and hydrophilic (cytosolic) PECAM-1 significantly increased in the presence of oxLDL. Upregulation of PECAM-1 by oxLDL may promote monocyte priming at junctions to start diapedesis. Source of research support: The Ministry of Education, Culture, Sports, Science and Technology of Japan (K.H. and K.T.).

Failure of monocyte priming in clinical protein malnutrition: [formula omitted], C.W. Ramsden, P.J. Guillou and J.V. Reynolds. Academic Surgical Unit, St. Jame's University Hospital, Leeds LS9 7TF U.K.

Failure of monocyte priming in clinical protein malnutrition: [formula omitted], C.W. Ramsden, P.J. Guillou and J.V. Reynolds. Academic Surgical Unit, St. Jame's University Hospital, Leeds LS9 7TF U.K.

Crohn's disease monocytes are primed for accentuated release of toxic oxygen metabolites

Background: Inflammatory bowel disease occurs in regions of the intestine characterized by a bowel content high in bacteria. Intestinal bacteria synthesize cell wall products such as lipopolysaccharide; when normal monocytes or macrophages come in contact with these products, they can be primed to release a number of inflammatory mediators. Mediators such as toxic oxygen metabolites released as part of the respiratory burst may contribute to inflammatory tissue damage. The aim of this study was to determine if monocytes from patients with Crohn's disease are primed by lipopolysaccharide for a greater respiratory burst. Methods: The generation of superoxide anion was measured by superoxide dismutase inhibitable reduction of ferricytochrome c. Results: Freshly isolated monocytes from active untreated Crohn's disease patients (n = 8) showed enhanced stimulated release of superoxide anion when compared with normal monocytes (n = 15; 3.80 ± 0.12 vs. 1.02 ± 0.06 nmol/5 min; P < 0.001). We tested the hypothesis that the monocyte priming factor in Crohn's disease serum may be lipopolysaccharide by showing that Crohn's disease serum lost its ability to prime normal monocytes after lipopolysaccharide was removed (0.25 ± 0.25 nmol/5 min, P < 0.001). Conclusions: These studies indicate that bacterial cell wall products may be important proinflammatory molecules involved in the initiation and/or perpetuation of Crohn's disease.

Immunostimulatory, but not antiendotoxin, activity of lipid X is due to small amounts of contaminating N,O-acylated disaccharide-1-phosphate: in vitro and in vivo reevaluation of the biological activity of synthetic lipid X.

Lipid X, a precursor in the biosynthesis of lipid A, has been claimed to possess most of the immunostimulatory activity but none of the toxicity of endotoxin. However, recent work shows that synthetic lipid X can be contaminated with small amounts of N,O-acylated disaccharide-1-phosphate (H. Aschauer, A. Grob, J. Hildebrandt, E. Schuetze, and P. Steutz, J. Biol. Chem. 265:9159-9164, 1990). Because the impurities themselves exhibit immunostimulatory properties, it was necessary to establish whether chemically pure synthetic lipid X exhibits any of the endotoxinlike biological activities previously attributed to the native compound extracted from the Escherichia coli MN7 mutant. In the present study, two batches of synthetic lipid X were used: batch A contained the contaminating disaccharide, and batch B was pure lipid X. Batch A, previously believed to be pure on the basis of spectroscopic and microanalysis data, induced murine peritoneal macrophages to secrete tumor necrosis factor, interleukin-1, and prostaglandin E2 at a minimal dose of 10 micrograms/ml in vitro. Batch B, further purified by Sephadex LH 20 chromatography, was found virtually inactive in these in vitro assays. Furthermore, batch A was pyrogenic in rabbits at a dose of 0.05 mg/kg, whereas batch B was not pyrogenic at doses of up to greater than or equal to 2 mg/kg. However, both batches were equally tolerated by galactosamine-loaded mice at doses of up to 100 mg/kg. Surprisingly, while only batch A protected neutropenic mice against lethal infection with Pseudomonas aeruginosa (50% effective dose, 12.4 mg/kg), both batches were equally protective against infection with herpes simplex virus type 1 in mice and guinea pigs, even when lipid X was administered therapeutically. Interestingly, both batches of lipid X blocked endotoxin-induced expression of monocyte procoagulant activity and priming of human neutrophils for superoxide anion release. Similarly, both batches protected galactosamine-sensitized mice from otherwise lethal endotoxemia when administered prophylactically or simultaneously with the lipopolysaccharide challenge. Thus, our findings suggest that chemically pure lipid X (batch B) is devoid of the immunostimulatory properties of lipid A or endotoxin. Rather, it behaves as a competitive inhibitor of lipopolysaccharide. We conclude, therefore, that previous studies which attributed immunostimulatory activities to any batch of synthetic lipid X should be interpreted with caution.

Activation of human monocytes by interleukin 2: Role of T lymphocytes

The effect of interleukin 2 (IL2) on the capability of human monocytes to secrete reactive oxygen species triggered via Fc-gamma receptor (Fc-gamma R) function had been investigated by measurement of chemiluminescence (CL). IL 2 did not activate highly purified (hp) monocytes to respond to Fc-gamma R mediated phagocytic stimulation with an enhanced respiratory burst activity unless low numbers of T cells had been co-cultured with hp monocytes. Supernatants from IL 2 treated PBMC contained interferon-gamma (IFN-gamma) and monocyte activating factor (MAF) activity. The secretion of both cytokine activities was strongly enhanced by cooperative function of monocytes. The correlation of IL 2 induced secretion of IFN-gamma and MAF activity was striking, however, monoclonal antibody (mAb) anti-human IFN-gamma failed to abrogate IL 2 stimulated and lymphocyte dependent monocyte activation. Although IL 2 had no direct monocyte activating effect, pretreatment of hp monocytes with IL 2 led to monocyte priming: subsequent co-culture with autologous control T cells enhanced the monocyte Fc-gamma R mediated CL response. The priming of monocytes by IL 2 was dependent on the interaction of IL 2 with the monocytic IL 2 receptor as shown by inhibition experiments with anti IL 2 R monoclonal antibody. Thus the IL 2 driven monocyte/T-cell interaction leads to an increased Fc-gamma R mediated monocytic respiratory burst activity and to the secretion of a soluble MAF activity, but there were no detectable amounts of IFN-gamma.

IFN-gamma and LPS overcome glucocorticoid inhibition of priming for superoxide release in human monocytes. Evidence that secretion of IL-1 and tumor necrosis factor-alpha is not essential for monocyte priming.

Abstract We examined the interaction between IFN-gamma, LPS, and glucocorticoids on release of oxygen radicals by human monocytes cultured in vitro. After 48 h culture, monocytes released low amounts of superoxide anion (O2-) when stimulated by PMA or FMLP. Monocytes incubated with either IFN-gamma or LPS became "primed" and released greater amounts of O2- in response to stimuli. Monocytes incubated with hydrocortisone, methylprednisolone, dexamethasone, or prednisolone alone showed decreased release of O2-. Prednisone and progesterone, which are not active glucocorticoids, had no effect. When glucocorticoids were co-incubated with IFN-gamma or LPS, the effect of hydrocortisone and other active steroids was blocked, and the monocytes released high O2-. However, when monocytes were preincubated with hydrocortisone for 24 h before addition of IFN-gamma or LPS, priming for enhanced O2- production by LPS was partially inhibited whereas there was no effect on IFN-gamma priming. We suggest that IFN-gamma and LPS can block the anti-inflammatory effects of glucocorticoids, contributing to increased inflammation at tissue sites; however, the mechanism of this effect may differ for the two macrophage activators. To investigate the mechanisms of priming by IFN-gamma and LPS, we examined the effects of these agents and of hydrocortisone on secretion of IL-1 and TNF-alpha. Both IL-1 and TNF-alpha primed monocytes for enhanced release of O2- in response to PMA. LPS caused monocytes to secrete both IL-1 beta and TNF-alpha. LPS-induced secretion of TNF-alpha and IL-1 beta was completely blocked by hydrocortisone, but the priming effect of LPS on O2- release was only partly blocked. IFN-gamma did not cause monocytes to secrete IL-1 beta or TNF-alpha, under our culture conditions (mononuclear cells cultured in Teflon in endotoxin-free modified Earle's salt solution without serum). Therefore, priming by LPS and IFN-gamma, and the inhibition of priming by glucocorticoids involve mechanisms that extend beyond regulation of secretion of IL-1 and TNF-alpha.

The Effect of Interleukin 2 on Fc-γ Receptor Function of Human Monocytes Requires Specific Intercellular Interaction

We examined the effect of interleukin 2 (IL 2) on the capacity of human monocytes tosecrete reactive oxygen species triggered via Fc-γ receptor (FcγR) function as measured by chemiluminescence (CL). IL 2 did not activate highly purified (hp) monocytes to respond to FcγR-mediated phagocytic stimulation with an enhanced respiratory burst activity, unless low numbers of T lymphocytes had been cocultured with hp monocytes. The degree of monocyte activation in peripheral blood mononuclear cells (PBMC) by IL 2 was dependent on T lymphocytes to monocytes ratios as well as on IL 2 concentrations. Optimal FcγR function was detected with IL 2 concentrations of 60 to 600 U/ml and one to two T lymphocytes per monocyte, whereas higher cellular ratios were suppressive. Supernatants from IL 2-treated PBMC contained interferon-γ), (IFN-γ) and monocyte activating factor (MAF) activity (= enhanced monocytic FcγR mediated CL). The secretion of both cytokine activities was strongly enhanced by a cooperative function of monocytes. The correlation of IL 2-induced secretion of IFN-γ and MAF activity was striking; however, monoclonal anti-IFN-γ antibodies failed to abrogate IL 2-stimulated and lymphocyte-dependent monocyte activation. Although IL 2 had no direct monocyte activating effect, pretreatment of hp monocytes withIL 2 led to a monocyte priming: subsequent coculture with autologous control T lymphocytes enhanced the monocyte FcγR-mediated CL response. The priming of monocytes by IL 2 was dependent on the interaction of IL 2 with the monocytic IL 2 receptor, as shown by inhibition experiments with anti-IL 2 receptor monoclonal antibodies. Thus, we observed an IL 2-driven monocyte/T lymphocyte interaction leading to an increased FcγR-mediated monocytic respiratory burst activity and to the secretion of a soluble MAF activity, but there were no detectable amounts of IFN-γ.

Inhibition of macrophage priming by sulfatide from Mycobacterium tuberculosis.

Sulfatide from the outer surface of Mycobacterium tuberculosis blocked priming in cultured human monocytes. Monocytes were primed in vitro with either lipopolysaccharide (LPS) or interferon-gamma. Primed monocytes released increased amounts of superoxide anion (O2-) when stimulated with formyl-methionyl-leucyl-phenylalanine or with phorbol myristate acetate. Primed monocytes also showed increased phagocytosis of sheep erythrocytes and increased release of interleukin 1. When primed monocytes were treated with 10 micrograms/ml of sulfatide, these enhanced functions, characteristic of primed monocytes, returned to levels found in unprimed monocytes. (With respect to these functions and others, monocytes or macrophages primed in vitro by exposure to LPS or interferon-gamma resemble macrophages activated in vivo by infection. In vivo, activated macrophages provide non-specific resistance to infection). Inhibition of priming by sulfatide could be detected within 10 min, but maximum effect of sulfatide required 3 to 5 hr. Sulfatide had no effect on O2- release, if it was added after the cells had been stimulated by PMA, suggesting that sulfatide did not inhibit enzymes involved in formation of O2-, but rather that sulfatide inhibited priming. Increasing the amounts of LPS or interferon-gamma did not counteract the effects of sulfatide. Sulfatide did cause monocytes to release some prostaglandin E2 (less than 1 nM), but the amount was not sufficient to inhibit monocyte functions. The effect of sulfatide was not blocked by indomethacin. Other sulfated compounds and other products of mycobacteria did not produce the sulfatide effect. We conclude that M. tuberculosis has on its outer surface a chemical that directly interferes with monocyte priming. In vivo, M. tuberculosis might use sulfatide to block macrophage activation and thereby resist being killed by macrophages.

Enhancement of production of superoxide anion by human monocytes exposed to products of HT 29 human colonic adenocarcinoma cell line.

The generation of superoxide anion (O2-) by human blood monocytes in response to stimulation by either phorbol myristate acetate (PMA) or opsonized Zymosan was greatly enhanced (range: 100-200% according to donor) by prior exposure of the peripheral blood mononuclear cells (PBM) to human colonic adenocarcinoma cells (HT 29 line) or their conditioned culture medium (DMEM-HT 29). This priming effect was observed after 5 hr and persisted for up to 15 hr of contact between PBM and endotoxin-free DMEM-HT 29. Beyond this time, primed monocytes gradually lost this ability. However, they maintained a higher capacity (about 100%) to produce O2- when compared to controls. DMEM-HT 29-induced monocyte priming requires that the tumor-active substance(s) act(s) on 2 target cells: first, on non adherent mononuclear cells (NA-PBM) to induce cytokine production and, second, on the monocyte itself. Priming activity was also found in conditioned medium from FR3T3 embryonic fibroblasts but not in conditioned medium from HT 29 repolarized cells (by culture in glucose-free medium) or from non-tumorous human colonic mucosa explants.