Deactivation of the respiratory burst in activated macrophages: evidence for alteration of signal transduction

Enhanced function of the respiratory burst, measured as stimulated release of superoxide anion (O2-) or hydrogen peroxide, characterizes activated macrophages. Activated macrophages undergo a decline in their capacity to release O2- (a deactivation) when placed in culture for 3 days. To better understand the molecular basis for the enhanced respiratory burst of activated macrophages, we explored the mechanisms underlying deactivation of activated mouse peritoneal macrophages. Deactivation was observed when the assay was performed in a physiologic Na+ buffer, and by day 3 of culture, release of O2- from activated macrophages stimulated with phorbol myristate acetate (PMA) was almost identical to that in resident (nonactivated) macrophages. In contrast, when the assay was performed in a buffer in which Na+ was replaced by K+, release of O2- from activated macrophages on day 3 was equal to or greater than that on day 0, suggesting that the enzyme responsible for the respiratory burst was not altered during culture. The number and affinity of PMA receptors were not changed during culture and were not affected by high external K+. Continuous assay of O2- release by coverslip-adherent macrophages in a cuvette indicated that the lag time between addition of stimulus and release of O2- was reduced, and the initial rate of O2- release was enhanced in K+ buffer. The potency of monovalent cations to support O2- release was K+ greater than Rb+ greater than choline+ greater than Cs+ = Na+ greater than Li+, suggesting that characteristics such as ionic radius or molecular size influence this effect, and the effect is not due simply to absence of Na+. Extracellular Ca2+ or Mg2+ was required for the maximal effect of high external K+, and enhancement by high K+ and divalent cations increased progressively during culture. These findings suggest that deactivation is caused primarily by changes in signal transduction from PMA receptors to the respiratory burst enzyme, rather than by changes in these receptors or the enzyme itself, and that signal transduction can differ in different macrophage populations.     

Quantitation of nitric oxide-derived nitrite from activated macrophages using microdialysis sampling

An HPLC method for detecting nitrite in microdialysis samples obtained from activated RAW 264.7 macrophages in cell culture has been developed. Nitrite was quantified using a pre-column derivatization with 2,4-dinitrophenylhydrazine (2,4-DNPH) followed by HPLC-UV analysis of the azide product. For dialysates, the detection limit of nitrite was 750 nM and the quantitation limit was 2.5 microM. The microdialysis relative recovery of nitrite in the macrophage cell culture medium was determined to be 86+/-2% (n=3) at a flow rate of 0.7 microl/min. Nitrite produced from activated macrophages was measured immediately after lipopolysaccharide (LPS) stimulation using microdialysis sampling.     

Plasmalemmal H+ extruders in mammalian alveolar macrophages

The distribution of plasmalemmal V-type H+-pumps (V-ATPase) among mammalian macrophages (mvarphi) is uncertain and, hence, the functional significance of mvarphi plasmalemmal V-ATPase is unclear. This study investigated the role of V-ATPase in the regulation of intracellular pH (pH(i)) by resident alveolar mvarphi from sheep, pigs, dogs and rabbits. The fluorescent probe 2',7'-biscarboxyethyl-5,6-carboxyfluorescein was used to monitor baseline pH(i) and the rate of pH(i) recovery (dpH(i)/dt) from intracellular acid-loads (NH(4)Cl prepulse). Baseline pH(i) was 7.1-7.2. In sheep, pig and dog studies, 10 microM bafilomycin A(1) (a selective V-ATPase inhibitor) caused a rapid fall in baseline pH(i) (0.15-0.20 units); baseline values were unaffected by 0.1 mM amiloride (a Na+ transport inhibitor). V-ATPase activity (bafilomycin-sensitive component of dpH(i)/dt) was solely responsible for pH(i) recovery from intracellular acid-loads at acid-loaded pH(i) values >6.8-6.9. Na+/H+ exchange (amiloride-sensitive component of dpH(i)/dt) was detected only at acid-loaded pH(i) values <6.8. The activity of both H+ extruders increased at lower pH(i) values, albeit the Na+/H+ exchanger was more pH-sensitive than was V-ATPase. In rabbit studies, 10 microM bafilomycin A(1) and 1 mM N-ethylmaleimide (a non-specific H+-pump inhibitor) produced similar falls in baseline mvarphi pH(i), but had significantly larger effects than did the selective V-ATPase inhibitor concanamycin A (相似文献   

Cytoplasmic pH regulation in macrophages by an ATP-dependent and N,N'-dicyclohexylcarbodiimide-sensitive mechanism. Possible involvement of a plasma membrane proton pump

Cytoplasmic pH (pHi) regulation was studied in thioglycolate-elicited murine macrophages using fluorescent probes. Acid-loaded macrophages regained normal pHi by extrusion of H+ equivalents across the plasma membrane. A fraction of this recovery was due to Na+/H+ exchange, as evidenced by its partial Na+ dependence and amiloride sensitivity. The residual, Na+-independent pHi recovery (approximately 50% of the total) persisted in the nominal absence of HCO3- and was insensitive to disulfonic stilbenes, ruling out mediation by anion exchange. In contrast, intracellular alkalinization and H+ extrusion from the cells were inhibited by N-ethylmaleimide, by N,N'-dicyclohexylcarbodiimide or by prior depletion of intracellular ATP. These observations are consistent with the existence of a H+-pumping ATPase in the plasma membrane of macrophages. The mechanism of activation of the ATP-dependent H+ extrusion process was also investigated. In other systems, Ca2+ mobilization has been suggested to signal an exocytic insertion of H+ pumps into the plasma membrane. Acid loading of macrophages was accompanied by an elevation of the cytosolic Ca2+ concentration ([Ca2+]i), measured using indo-1. These results are consistent with a role for Ca2+ mobilization in the activation of H+ extrusion.     

Intracellular pH transients in squid giant axons caused by CO2, NH3, and metabolic inhibitors

The intracellular pH (pHi) of squid giant axons has been measured using glass pH microelectrodes. Resting pHi in artificial seawater (ASW) (pH 7.6-7.8) at 23 degrees C was 7.32 +/- 0.02 (7.28 if corrected for liquid junction potential). Exposure of the axon to 5% CO2 at constant external pH caused a sharp decrease in pHi, while the subsequent removal of the gas caused pHi to overshoot its initial value. If the exposure to CO2 was prolonged, two additional effects were noted: (a) during the exposure, the rapid initial fall in pHi was followed by a slow rise, and (b) after the exposure, the overshoot was greatly exaggerated. Application of external NH4Cl caused pHi to rise sharply; return to normal ASW caused pHi to return to a value below its initial one. If the exposure to NH4Cl was prolonged, two additional effects were noted: (a) during the exposure, the rapid initial rise in pHi was followed by a slow fall, and (b) after the exposure, the undershoot was greatly exaggerated. Exposure to several weak acid metabolic inhibitors caused a fall in pHi whose reversibility depended upon length of exposure. Inverting the electrochemical gradient for H+ with 100 mM K- ASW had no effect on pHi changes resulting from short-term exposure to azide. A mathematical model explains the pHi changes caused by NH4Cl on the basis of passive movements of both NH3 and NH4+. The simultaneous passive movements of CO2 and HCO3-cannot explain the results of the CO2 experiments; these data require the postulation of an active proton extrusion and/or sequestration mechanism.     

A spectrophotometric method for the direct detection and quantitation of nitric oxide, nitrite, and nitrate in cell culture media

A method for the spectrophotometric determination of nitric oxide, nitrite, and nitrate in tissue culture media is presented. The method is based on the nitric oxide-mediated nitrosative modification of sulfanilic acid that reacts with N-(1-naphthyl)ethylenediamine dihydrochloride forming an orange-colored product absorbing at 496 nm. Nitric oxide levels were determined in culture media from this absorbance measurement using chemiluminescence standardization. Extinction coefficients of 5400 and 6600 M(-1) cm(-1) were determined for the nitric oxide product in assay solutions containing 0.1 or 100 mM KPO4 buffer (pH 7.4), respectively, with a limit of detection of 1 microM. Acidification of these reactions (pH 2.4) generated a pink-colored product absorbing at 540 nm allowing for quantitation of total nitric oxide/nitrite levels using extinction coefficients of 38,000 and 36,900 M(-1) cm(-1), for the assay solutions described. The limit of detection of this assay was approximately 300 nM. Using the 100 mM KPO4 buffer system, nitrate levels were determined following reduction to nitrite using a copper-coated cadmium reagent with an extinction coefficient of 29,500 M(-1) cm(-1) and a detection limit of 0.5 microM. The utility of these assays was demonstrated in the standardization of nitric oxide-saturated cell culture media, and the release of nitric oxide by the NONOate compound DEA/NO.     

Immunoregulatory role of in vitro differentiated macrophages on human natural killer (NK)-cell activity

Immunoregulatory effects of human macrophages on natural killer (NK) activity were studied. Monocytes were isolated by adherence to plastic, after leukapheresis of normal blood donors, and cultured for 1 to 14 days. In vitro-differentiated (5-7 days) human macrophages consistently and significantly (P less than 0.01) augmented NK activity of fresh autologous or allogeneic PBMNC. During culture, these macrophages also developed increased antitumor cytostatic activity. The optimal time for both the expression of cytostatic activity and up-regulation of NK activity was 5-7 days in culture. In contrast, 12- to 14-day macrophages significantly suppressed NK activity and had less cytostatic activity. Macrophages in culture demonstrated shifts in Leu-M3+HLA-DR+ phenotype from the mean of 60% +/- 11 (SD) in fresh monocytes to 90% +/- 5 between Days 5 and 7 in culture and then down to 10% +/- 5 in 14-day cultures. The activity of NK (CD56+CD3-) cells, purified by Percoll gradient centrifugation and flow cytometry, was up-regulated directly by in vitro-differentiated macrophages at low macrophage to NK cell ratios, and this up-regulation was not dependent on T lymphocytes or other accessory cells. The modulation of NK activity by differentiated macrophages was not MHC-restricted and depended on the viability and cellular integrity of macrophages. Sonicated macrophages could no longer up-regulate NK activity. This study shows that antitumor effects mediated by human in vitro differentiated LeuM3+HLA-DR+ macrophages may simultaneously involve more than one mechanism, namely direct cytostasis of tumor cells and activation of NK cells.     

Micellar electrokinetic capillary chromatography determination of +S and -R arotinolol in serum using UV detection and solid phase extraction.

A method for the simultaneous determination of +S and -R arotinolol in serum by micellar electrokinetic capillary chromatography is described. Stereoselective resolution of the arotinolol enantiomers was achieved using 5 mM sodium taurocholate in 10 mM sodium dihydrogen phosphate buffer of pH 2.5. A 72-cm uncoated fused-silica capillary at a constant voltage of 15 kV was used for the analysis. The analytes of interest were extracted from serum using solid phase extraction. An octadecyl cartridge gave good recoveries in excess of 87% for both +S and -R arotinolol without any interference. The calibration curves were linear over the range of 50-500 ng ml(-1) with +S propranolol as the internal standard and the coefficient of determination was greater than 0.999 (n = 3). The limit of quantitation was 50 ng ml(-1) for each enantiomer and the detection limit using 1 ml serum and a UV detection set et 220 nm was 25 ng ml(-1) (S/N = 2). Precision and accuracy of the method were in the range 0.8-2.7% and 1.2-6.4%, respectively, for +S arotinolol and 1.1-3.9% and 2.2-6.5%, respectively, for -R arotinolol.     

Azaspiracids modulate intracellular pH levels in human lymphocytes

The azaspiracids (AZAs) are a group of marine toxins implicated in several intoxications whose mechanism of action is unknown. These phycotoxins include the five compounds shown in : AZA-1 (1), AZA-2 (2), AZA-3 (3), AZA-4 (4), and AZA-5 (5). The aim of this work was to study the effects of the five naturally occurring azaspiracids (AZA-1 to -5, Fig. 1) and four synthetic analogues (6-9, Fig. 2) on intracellular pH, and the influence of Ca2+ upon this effect. The AZAs (1-5) were found to modulate cytosolic Ca2+ levels in human lymphocytes, while some of them, but not all, had effects on the intracellular pH. AZA-1 (1) and AZA-2 (2) did not modify intracellular pH in a Ca2+-containing or a Ca2+-free medium. AZA-3 (3) increased intracellular pH by 0.16 units in the presence of extracellular Ca2+, an effect that was blocked when a 1 mM solution of Ni2+ was added. In a Ca2+-free medium, the increase in pH induced by AZA-3 (3) was reduced to 0.08 pH units. AZA-4 (4) inhibited the basal pH increase even in the presence of a 1 mM solution of Ni2+. In a Ca2+-free medium, the inhibition caused by AZA-4 (4) was small, but when Ca2+ was added back to the medium, the pH basal increase was again significantly inhibited. The alkalinization was also inhibited when AZA-4 (4) was added simultaneously, 10 min before or 10 min after thapsigargin (Tg), and also when the Ca2+-influx induced by Tg was inhibited by Ni2+. AZA-5 (5), on the other hand, did not modulate the intracellular pH profile in either a Ca2+-containing or a Ca2+-free medium. Finally, we investigated four synthetic analogues (6-9, Fig. 2) whose structures were based on the four originally proposed structures of azaspiracid-1, with an opened E-ring. Compound 6 induced a small cytosolic Ca2+ increase, but did not modify intracellular pH in saline solution. In a Ca2+-free medium, compound 6 blocked the pH fall when Ca2+ was added back to the medium. Compound 7 also did not modify intracellular pH in saline solutions, however it significantly blocked basal pH increases in a Ca2+-free medium. Compound 8 did not alter intracellular pH, however compound 9 induced a small acidification when Ca2+ was present in the extracellular medium. These results point to a structure-activity relationship in AZAs pH effect that affects the modulation and the coupling of intracellular pH and Ca2+.     

Role of CC chemokine receptor 2 in bone marrow cells in the recruitment of macrophages into obese adipose tissue

The MCP-1 (monocyte chemoattractant protein-1)/CCR2 (CC motif chemokine receptor-2) pathway may play a role in macrophage infiltration into obese adipose tissue. Here we investigated the role of CCR2 in the recruitment of bone marrow-derived macrophages into obese adipose tissue in vitro and in vivo. Using the TAXIScan device, which can measure quantitatively the directionality and velocity of cell migration at time lapse intervals in vitro, we demonstrated that bone marrow cells (BMCs) from wild type mice migrate directly toward MCP-1 or culture medium conditioned by adipose tissue explants of genetically obese ob/ob mice, which are efficiently suppressed by pharmacological blockade of CCR2 signaling. The number of F4/80-positive macrophages was reduced in the adipose tissue from high fat diet-fed obese KKAy or ob/ob mice treated with a CCR2 antagonist propagermanium relative to vehicle-treated groups. We also found that the number of macrophages is reduced in the adipose tissue from ob/ob mice reconstituted with CCR2(-/-) BMCs (ob/ob + CCR2(-/-) BMCs) relative to those with CCR2+/+ BMCs (ob/ob + CCR2+/+ BMCs). Expression of mRNAs for CD11c and TLR4 (Toll-like receptor 4) markers of proinflammatory M1 macrophages was also decreased in the adipose tissue from ob/ob + CCR2(-/-) BMCs relative to ob/ob + CCR2+/+ BMCs, whereas mannose receptor and CD163, markers of anti-inflammatory M2 macrophages, were unchanged. This study provides in vivo and in vitro evidence that CCR2 in bone marrow cells plays an important role in the recruitment of macrophages into obese adipose tissue.     

一株高效菲降解菌的筛选及降解条件研究

从南京某石化厂排污口附近采集土样,以菲为碳源的选择性培养基分离筛选到一株菲高效降解菌F10a,根据形态和生理生化特性初步鉴定为芽孢杆菌属,并对其降解菲的特性及各种影响因素进行了研究.结果表明,F10a在50 mg·L^-1的条件下,28 ℃振荡培养27 h,菲的降解率达到98.12%;静置培养8 h,菲的降解率达到98.7%.pH值分别为、6、8时,F10a对菲具有良好的降解效能;pH值为10时F10a不生长.Zn²⁺与Pb²⁺的存在不影响F10a的降解效能,Cu²⁺可以延缓菲的降解,Cr²⁺对F10a有毒性.F10a在菲浓度为200 mg·L^-1时,28 ℃振荡培养8 h,降解率为99.6%.菲的降解程度与细菌数量的增长呈正相关关系.     

Effects of nutrient and non-nutrient stimuli on cytosolic pH in cultured insulinoma (HIT-T15) cells

Intracellular pH (pHi) was measured in the insulin-secreting HIT-T15 cell line using the pH-sensitive fluorescent dye, 2',7'-bis(carboxyethyl)-5'(6')-carboxyfluorescein (BCECF). It was observed that the addition of a weak acid (e.g., acetate or propionate) caused a rapid decrease in pHi, followed by a slower recovery to the resting pH value. Conversely the addition of N4Cl caused an increase in pHi followed by recovery. The addition of amiloride caused a fall in pHi; however, in this case no recovery to basal pH levels was observed. Subsequent addition of a weak acid caused a further fall in pHi with no recovery. The addition of glucose caused a transient acidification followed by alkalinization. When glucose was added to cells which had been pretreated with amiloride, the initial acidification was not followed by recovery or alkalinization. Addition of glyceraldehyde, alpha-ketoisocaproate, lactate or pyruvate to HIT cells also resulted in intracellular acidification followed by recovery. Similarly, depolarisation of HIT cells by treatment with high K+ or with Ba2+ was associated with a pronounced fall in pHi, followed by a gradual recovery. Insulin secretion from HIT cells was stimulated by glucose, glyceraldehyde, alpha-ketoisocaproate, lactate, pyruvate and KCl, whilst amiloride and weak acids exerted only modest effects in the absence of glucose, but amiloride in particular markedly potentiated glucose-induced insulin release. Thus, HIT cells appear to have an amiloride-sensitive mechanism for the extrusion of protons, probably Na+-H+ exchange. Whilst intracellular acidification appears to potentiate secretory responses to nutrient stimuli, it seems unlikely that the activation of HIT cells by these nutrients occurs as a result of intracellular acidification. The mechanisms by which various nutrient and non-nutrient stimuli might exert distinct effects on pHi are discussed.     

Low extracellular pH stimulates the production of IL-1β by human monocytes

The development of acidic environments is a hallmark of inflammatory processes of different etiology. We have previously shown that transient exposure to acidic conditions, similar to those encountered in vivo, induces the activation of neutrophils and the phenotypic maturation of dendritic cells. We here report that extracellular acidosis (pH 6.5) selectively stimulates the production and the secretion of IL-1β by human monocytes without affecting the production of TNF-α, IL-6 and the expression of CD40, CD80, CD86, and HLA-DR. Stimulation of IL-1β production by pH 6.5-treated monocytes was shown to be dependent on caspase-1 activity, and it was also observed using peripheral blood mononuclear cells instead of isolated monocytes. Contrasting with the results in monocytes, we found that pH 6.5 did not stimulate any production of IL-1β by macrophages. Changes in intracellular pH seem to be involved in the stimulation of IL-1β production. In fact, monocytes cultured at pH 6.5 undergo a fall in the values of intracellular pH while the inhibitor of the Na+/H+ exchanger, 5-(N-ethyl-N-isopropyl)amiloride induced both, a decrease in the values of intracellular pH and the stimulation of IL-1β production. Real time quantitative PCR assays indicated that monocytes cultured either at pH 6.5 or in the presence of 5-(N-ethyl-N-isopropyl)amiloride expressed higher levels of pro-IL-1β mRNA suggesting that low values of intracellular pH enhance the production of IL-1β, at least in part, by stimulating the synthesis of its precursor.     

Glucosamine and chitin accumulation in cell walls of the yeast Rhodotorula glutinis CBS 3044. Influence of culture conditions

During culture of Rhodotorula glutinis the fall in pH from 4.5 to 2 stimulates the synthesis of chitin; alternatively keeping the pH at 4.5 causes the chitin content to fall largely.However there is no proportional value between the chitin content fall and that of total hexosamines.     

Inorganic carbon limitation and mixotrophic growth in Chlamydomonas from an acidic mining lake

Plankton communities in acidic mining lakes (pH 2.5-3.3) are species-poor because they face extreme environmental conditions, e.g. 150mg l(-1) Fe2+ +Fe3+. We investigated the growth characteristics of the dominant pigmented species, the flagellate Chlamydomonas acidophila, in semi-continuous culture experiments under in situ conditions. The following hypotheses were tested: (1) Low inorganic carbon (IC) concentrations in the epilimnion (e.g. 0.3 mg l(-1)) arising from the low pH limit phototrophic growth (H-1); (2) the additional use of dissolved organic carbon (mixotrophy) leads to higher growth rates under IC-limitation (H-2), and (3) phagotrophy is not relevant (H-3). H-1 was supported as the culture experiments, in situ PAR and IC concentrations indicated that IC potentially limited phototrophic growth in the mixed surface layers. H-2 was also supported: mixotrophic growth always exceeded pure phototrophic growth even when photosynthesis was saturated. Dark growth in filtered lake water illuminated prior to inoculation provided evidence that Chlamydomonas was able to use the natural DOC. The alga did not grow on bacteria, thus confirming H-3. Chlamydomonas exhibited a remarkable resistance to starvation in the dark. The compensation light intensity (ca. 20 micromol photons m(-2) s(-1)) and the maximum phototrophic growth (1.50 d(-1)) fell within the range of algae from non-acidic waters. Overall, Chlamydomonas, a typical r-strategist in circum-neutral systems, showed characteristics of a K-strategist in the stable, acidic lake environment in achieving moderate growth rates and minimizing metabolic losses.     

Medium components and culture conditions affect the thermotolerance of aerial conidia of fungal biocontrol agent Beauveria bassiana

AIMS: To produce more thermotolerable conidia of Beauveria bassiana, a well-known fungal biocontrol agent, by optimizing the medium components and culture conditions. METHODS AND RESULTS: The conidia produced on media including 0.5-6% glucose, sucrose or starch as carbon source and 50-300-microg ml(-1) Cu2+, Zn2+, Mn2+ or Fe3+ as additive to Sabouraud dextrose medium at 15-30 degrees C, pH 4-8 or KCl-adjusted water availabilities were exposed to 30-min wet heat stress at 48 degrees C. The medium components for conidial production with greatly enhanced thermotolerance included 4% glucose as optimum or 1% starch as alternative for the carbon source and < or =50-microg ml(-1) Mn2+ for the metal additive. The culture conditions were optimized as 25 degrees C and pH 5-6. Conidial thermotolerance decreased remarkably when sucrose and Fe3+ or Cu2+ were used in the cultures, but altered slightly when 50-200-microg ml(-1) Zn2+ were included. CONCLUSIONS: The tolerance of B. bassiana conidia to the thermal stress was significantly affected by the medium composition and culture conditions under which the conidia were produced. SIGNIFICANCE AND IMPACT OF THE STUDY: Proper treatment of small grains as mass production substrates for more glucose release and supplement of glucose or 50-microg ml(-1) Mn2+ are possible means to enhancing conidial thermotolerance and field persistence for improved insect control.     

The selective release of phospholipase A2 by resident mouse peritoneal macrophages.

Resident mouse peritoneal macrophages have three phospholipase activities: a phospholipase A2 active at pH 4.5, a Ca2+-dependent phospholipase A2 active at pH 8.5 and a phosphatidylinositol-specific phospholipase C activity. When macrophages are exposed to zymosan in culture, the cellular activity of pH-4.5 phospholipase A2 is diminished in a manner dependent on zymosan concentration and time of exposure, whereas the cellular activities of pH-8.5 phospholipase A2 and phospholipase C remain unchanged. The depletion of pH-4.5 phospholipase A2 activity from the cell is paralleled by a quantitative recovery of this activity in the culture medium in a manner similar to the cellular depletion and extracellular recovery of two lysosomal enzymes. This release is specifically elicited by an inflammatory substance such as zymosan, since macrophages incubated with 6 micrometer latex spheres retain pH-4.5 phospholipase A2 activity and lysosomal enzyme activities intracellularly.     

Secretion of L-glutamate from osteoclasts through transcytosis

Osteoclasts are involved in the catabolism of the bone matrix and eliminate the resulting degradation products through transcytosis, but the molecular mechanism and regulation of transcytosis remain poorly understood. Upon differentiation, osteoclasts express vesicular glutamate transporter 1 (VGLUT1), which is essential for vesicular storage and subsequent exocytosis of glutamate in neurons. VGLUT1 is localized in transcytotic vesicles and accumulates L-glutamate. Osteoclasts secrete L-glutamate and the bone degradation products upon stimulation with KCl or ATP in a Ca2+-dependent manner. KCl- and ATP-dependent secretion of L-glutamate was absent in osteoclasts prepared from VGLUT1-/- knockout mice. Osteoclasts express mGluR8, a class III metabotropic glutamate receptor. Its stimulation by a specific agonist inhibits secretion of L-glutamate and bone degradation products, whereas its suppression by a specific antagonist stimulates bone resorption. Finally, it was found that VGLUT1-/- mice develop osteoporosis. Thus, in bone-resorbing osteoclasts, L-glutamate and bone degradation products are secreted through transcytosis and the released L-glutamate is involved in autoregulation of transcytosis. Glutamate signaling may play an important role in the bone homeostasis.     

Induction of interleukin 1 beta expression from human peripheral blood monocyte-derived macrophages by 9-hydroxyoctadecadienoic acid.

Oxidatively modified low density lipoproteins (LDL) have recently been proposed to play a role in atherogenesis by promoting foam cell formation and endothelial cell toxicity. The purpose of the present study was to determine whether modified LDL could also induce macrophage release of interleukin 1 beta (IL-1 beta), a cytokine which enhances vascular smooth muscle cell proliferation, another feature of the atherosclerotic process. LDL were oxidatively modified by incubation with either Cu2+ (Cu(2+)-LDL) or human peripheral blood monocyte-derived macrophages (M-LDL). Incubation of these modified LDL with macrophages (6 x 10(6) cells/culture) resulted in a dose-dependent induction of IL-1 beta release. At 300 micrograms protein/ml, Cu(2+)-LDL and M-LDL induced 422 and 333 pg of IL-1 beta/culture, respectively. Saponified Cu(2+)-LDL and M-LDL were shown to contain 9- and 13-hydroxyoctadecadienoic acid (HODE), lipid oxidation products of linoleate. When tested for activity in macrophage culture (3 x 10(6) cells/culture), it was found that 9-HODE and 13-HODE (final concentration 33 microM) induced the release of 122 and 43 pg of IL-1 beta/culture, respectively, whereas untreated cells released only 4 pg of IL-1 beta/culture. Incubation of macrophages with cholesteryl-9-HODE also induced IL-1 beta release; however, the degree of induction of IL-1 beta release by 9-HODE or its cholesteryl ester relative to modified LDL suggests that other components in oxidized LDL may also contribute to IL-1 beta induction. 9-HODE was rapidly taken up by macrophages, and the kinetics were similar to IL-1 beta release. A 1.5- to 6-fold increase in the level of IL-1 beta mRNA was detected as little as 3-h post-9-HODE treatment. The induction of IL-1 beta release from human monocyte-derived macrophages by 9-HODE and cholesteryl-9-HODE suggests a role for modified LDL, and its associated linoleate oxidation products, in vascular smooth muscle cell proliferation.