1,25-Dihydroxyvitamin D3 increases the toxicity of hydrogen peroxide in the human monocytic line U937: the role of calcium and heat shock.

1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) increases synthesis of heat shock proteins in monocytes and U937 cells and protects these cells from thermal injury. We examined whether 1,25-(OH)2D3 would also modulate the susceptibility of U937 cells to H2O2-induced oxidative stress. Cell viability was assessed by trypan blue exclusion and [3H]thymidine incorporation into DNA. Prior incubation for 24 h with 1,25-(OH)2D3 (25 pM or higher) unexpectedly increased H2O2 toxicity. Since cellular Ca2+ may be a mediator of cell injury we investigated effects of altering extracellular Ca2+ ([Ca2+]e) on 1,25-(OH)2D3-enhanced H2O2 toxicity as well as effects of 1,25-(OH)2D3 and H2O2 on cytosolic free Ca2+ concentration ([Ca2+]f). Basal [Ca2+]f in medium containing 1.5 mM Ca as determined by fura-2 fluorescence was higher in 1,25-(OH)2D3-pretreated cells than control cells (137 versus 112 nM, P less than 0.005). H2O2 induced a rapid increase in [Ca2+]f (to greater than 300 nM) in both 1,25-(OH)2D3-treated and control cells, which was prevented by a reduction in [Ca2+]e to less than basal [Ca2+]f. The 1,25(OH)2D3-induced increase in H2O2 toxicity was also prevented by preincubation with 1,25-(OH)2D3 in Ca2+-free medium or by exposing the cells to H2O2 in the presence of EGTA. Preexposure of cells to 45 degrees C for 20 min, 4 h earlier, partially prevented the toxic effects of H2O2 particularly in 1,25-(OH)2D3-treated cells, even in the presence of physiological levels of [Ca2+]e. Thus 1,25-(OH)2D3 potentiates H2O2-induced injury probably by increasing cellular Ca2+ stores. The 1,25-(OH)2D3-induced amplification of the heat shock response likely represents a mechanism for counteracting the Ca2+-associated enhanced susceptibility to oxidative injury due to 1,25-(OH)2D3.

1,25-Dihydroxyvitamin D3 maintains adherence of human monocytes and protects them from thermal injury.

Adherence to a substratum is a characteristic feature of monocyte-macrophages which may be required for several effector functions. Human peripheral blood monocytes selected by adherence were found to readhere preferentially at 1 h to fibronectin or to a biological matrix. There was then a progressive decrease in the number of adherent cells, and by 48 h only 8-20% of monocytes remained adherent. This loss of adherence occurred while monocytes remained viable by criteria such as exclusion of trypan blue or release of lactate dehydrogenase. 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) maintained the adherence of cultured monocytes to tissue culture plastic as well as to the biological matrix. This effect was concentration- and time-dependent, and suppressed by inhibitors of protein synthesis. Cellular proteins were labeled after incubation with [35S]methionine. Analysis by two-dimensional gel electrophoresis revealed increased labeling of several distinct proteins in 1,25-(OH)2D3-treated monocytes compared with control monocytes. The increased loss of adherence and decreased overall protein synthesis observed in monocytes incubated at 45 degrees C was partially prevented by preincubation of the cells with 1,25-(OH)2D3. We further evaluated the effects of thermal stress and 1,25-(OH)2D3 on protein synthesis by monocytes, and found that 1,25-(OH)2D3 increased the synthesis of heat shock proteins, protected normal protein synthesis, and increased the rate of recovery of normal protein synthesis after the thermal stress. These observations suggest that 1,25-(OH)2D3 influences monocytes by preserving the synthesis of proteins, including those critical for the maintenance of cell adherence.

1,25-Dihydroxyvitamin D3 induces collagen binding to the human monocyte line U937.

Interactions of cells with components of the extracellular matrix can modulate cellular functions. We measured binding of a major matrix protein to U937 cells, a human promonocytic line. Radioiodinated type I or type III human collagen was bound only to U937 cells differentiated to a more mature phenotype with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). Binding was observed at 4 degrees C and was saturable; Scatchard analysis of the binding to 1,25-(OH)2D3-pretreated U937 cells indicated a single class of high-affinity binding sites. Preincubation of U937 cells with interferon gamma did not induce collagen binding. Collagen binding did not appear to be dependent on fibronectin binding. Surface proteins of U937 cells were 125I labeled and cell membrane proteins resolved by affinity chromatography on collagen-Sepharose. Major specifically labeled bands of 180, 155, and 125 kD were identified in membrane fractions from 1,25-(OH)2D3-pretreated U937 cells only. 1,25-(OH)2D3 appears to specifically regulate collagen binding to monocyte precursors.

Heat-shock proteins in host-pathogen interactions: implications for cystic fibrosis.

The expression of heat-shock proteins by both pathogen and host cells during the phagocytosis of Staphylococcus aureus and Pseudomonas aeruginosa, two bacterial species that colonize the airways of patients with cystic fibrosis, probably contributes to pulmonary inflammation in cystic fibrosis. Here, we discuss the likely signals for heat-shock-protein induction within host and bacterial cells.

Cultured human alveolar macrophages from smokers with lung cancer: resolution of factors that stimulate fibroblast proliferation, production of collagenase, or prostaglandin E2.

Marked connective tissue remodelling involves both destruction and repair in inflammatory lung diseases. Throughout the remodelling event, it was reasoned that alveolar macrophages may release substances similar to those produced by blood monocyte-macrophages that affect fibroblast functions, ie, the interleukin 1 family of monokines (or cytokines). We have examined human alveolar macrophage cultures obtained after bronchoalveolar lavage of freshly excised lungs from heavy smokers with bronchial carcinoma. Crude culture media contained fibroblast proliferative activity and collagenase- and PGE2- production-stimulating activity. The main peak of these biological activities was located around approximately 18 kilodaltons (kD) on gel filtration chromatography. Resolution of this peak by high performance liquid chromatography showed the presence of three distinct peaks, with quantitative and qualitative differences in biological activities. This suggests the presence of heterogeneous factors.

1,25-dihydroxyvitamin D3 induces responsiveness to the chemotactic peptide f-Met-Leu-Phe in the human monocytic line U937: dissociation between calcium and oxidative metabolic responses.

In the human premonocytic line U937, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) induces a functional NADPH oxidase, that is responsive to both phorbol esters and opsonized zymosan. The chemotactic peptide f-Met-Leu-Phe (fMLP) did not, however, induce superoxide generation by these cells. This was not due to the absence of receptors for fMLP. Although there was no significant binding of [3H]-fMLP to undifferentiated U937 cells, preincubation with 1,25-(OH)2D3 induced expression of specific and saturable binding sites. Moreover, fMLP induced a rapid and reversible rise in cytosolic free Ca2+ concentration ([Ca2+]i) in 1,25-(OH)2D3-treated U937 cells, but not in control or 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3)-treated cells. This [Ca2+]i response was dependent on concentrations of both fMLP and 1,25-(OH)2D3 and was observed at physiologic concentrations of the hormone (approximately 25 pM). The rise in [Ca2+]i induced by fMLP in 1,25-(OH)2D3-treated U937 cells was blocked by pertussis toxin and presumably mediated by inositol (1,4,5)-trisphosphate generation. These results indicate that in U937 cells differentiated with 1,25-(OH)2D3, inositol phosphate-mediated [Ca2+]i responses to fMLP are uncoupled from NADPH oxidase activation.

Cellular basis of ECD brain retention.

UNLABELLED: Clinical observations have shown discrepancies between ECD and HMPAO regional cerebral perfusion, particularly in brain tumors and during stroke recovery. We investigated the nature of the process(es) involved in ECD accumulation in vitro at the cellular level. METHODS: Time course incorporation of ECD was studied in a fast-growing human premonocytic line, U937, in a human astrocytic-derived cell line, U373, and a human hybridized endothelial cell line, EaHy926. Cells were further used in experiments aiming to correlate esterase activity and ECD retention. RESULTS: Significant differences in ECD retention between these cell lines were observed: %UECD (cpm cells/cpm standard of injected) plateaued within 2 hr in all cases but %UECD was significantly higher in U937 cells (25.1 +/- 3.9% at 120 min) than in the other cell lines (6.1 +/- 0.7% and 8.2 +/- 2.0% at 120 min for U373 and EaHy926, respectively). Contrary to what we expected, total cellular esterase activity (EATOT) was inversely correlated to %UECD.EATOT was 5-fold lower in U937 cells than in U373 and 20-fold lower than in EaHy926. Thus, we compared the membranar to the cytosolic esterase activity of U937 and analyzed the influence of temperature and diisopropylfluorophosphate (DFP, an inhibitor of cytosolic esterase activity) on both ECD retention and enzymatic activities. When cells were exposed to DFP, %UECD was reduced by 80%; while when cells were maintained at 4 degrees C, %UECD continuously increased, corresponding to a passive diffusion since both cytosolic and membranar esterase activities were inhibited. CONCLUSION: For optimal uptake of ECD, the membranar fraction of the esterase activity has to be low, while, in contrast, the cytosolic fraction of the esterase activity plays an important role in ECD cell retention. ECD-SPECT is likely able to reflect regional cerebral blood flow in normal and pathological states accurately, but in the event of unusual observations, the membranar esterase activity should be considered to explain reduced ECD retention.

Oxido-reductive state: the major determinant for cellular retention of technetium-99m-HMPAO.

UNLABELLED: Several clinical observations have suggested that HMPAO cerebral uptake might be related not only to regional cerebral perfusion but also to the nature of the lesion. Our aim was to investigate at the cellular level the nature of the process(es) involved in HMPAO accumulation in vitro. METHODS: Time-course incorporation of HM-PAO was studied in a fast-growing human premonocytic line, U937, in a human astrocytic-derived cell line, U373 and a human hybridized endothelial cell line, EaHy926. Minimal differences of HMPAO retention between these cell lines were observed and plateau of %U(HMPAO) (cpm cells/cpm standard of injected) were achieved within 2 hr. Because HMPAO cell retention was related to the intracellular content in glutathione, experiments studying effects of redox were conducted by preexposing U937 cells to D, L dithiothreitol or 2-Mercaptoethanol. RESULTS: Overnight incubation with NAC or BSO did not significantly modified the kinetic of 99mTc-HMPAO incorporation while overnight incubation with NAC resulted in a 2-fold increase in intracellular glutathione content and overnight incubation with BSO nearly abolished the intracellular glutathione content. At the opposite, presence of these reducing agents in the medium during the experiments completely abolished 99mTc-HMPAO retention. CONCLUSION: Our data thus provide in vitro evidence to support that overall intracellular retention of HMPAO is more dependent upon the redox activity of the tissue than the intracellular glutathione content. SPECT-HMPAO may accurately reflect regional cerebral blood flow in a normal state but possibly not in all pathological situations in which cell metabolism disturbances are characterized by alterations in the redox status.

Evaluation of [iodine-125]N,N,N'-trimethyl-N'-[2-hydroxy-3-methyl-5-iodobenzyl]-1,3- propanediamine lung uptake using an isolated-perfused lung model.

Lung uptake of N,N,N'-trimethyl-N'-[2-Hydroxy-3-methyl-5-iodobenzyl]-1,3- propanediamine (HIPDM) has been reported, but the mechanism of this process has not yet been established. Thus, single-pass [125I]HIPDM accumulation was studied in rat lungs perfused with a Krebs-Ringer bicarbonate buffer containing 4.5% bovine albumin. Iodine-125 HIPDM lung accumulation was monitored by the percent of extraction per gram of lung tissue. Iodine-125 HIPDM lung uptake did not appear to occur by simple diffusion. As the time of perfusion was increased from 2 to 15 min, the rate of uptake of 2 microM [125I]HIPDM decreased by 40%. During a 2-min perfusion, 98.6% +/- 6.7 (n = 8) extraction was observed with 2 microM [125I]HIPDM, but only 38% +/- 2.0 (n = 3) was extracted when the [125I]HIPDM concentration was 1 mM. The addition of 1 mM chlorpromazine, propranolol or imipramine also decreased [125I]HIPDM lung uptake to 43.0% +/- 1.5, 51.4% +/- 2.2, and 49.8% +/- 0.8, respectively, (each n = 4 - 6, p less than 0.001). Cold (4 degrees C) had little effect on pulmonary accumulation (77.7% +/- 7.4, n = 5, p less than 0.01), and the addition of ouabain or the use of sodium-free medium had no effect. Thus, pulmonary [125I]HIPDM accumulation does not appear to occur by sodium-dependent active transport. Rather, its uptake appears to be similar to the uptake of other basic amines, such as propranolol and imipramine, which are known to bind by physico-chemical interactions to pulmonary endothelial cell membranes and reflect pulmonary vascular surface area.

The heat shock response in human phagocytes.

During the last 10 years the intriguing field of the heat-shock response and stress proteins has switched from a particular case to a phenomenon of general interest. Discovered in Drosophila, these proteins were observed in every living organism with a surprisingly high sequence homology. In addition, these proteins are not only inducible by stress or pathophysiological situations but are also expressed in unstressed cells. These are arguments for crucial roles of heat-shock proteins. Here we discuss some aspects of the heat shock/stress response that we observed in phagocytic cells after phagocytosis with regard to their physiologic functions such as oxygen-free radical generation, antigen processing and presentation.

1,25-Dihydroxyvitamin D3 increases the toxicity of hydrogen peroxide: the role of calcium and heat shock.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] increases synthesis of heat shock proteins in monocytes and U937 cells and protects these cells from thermal injury. We therefore examined whether 1,25-(OH)2D3 would also modulate the susceptibility to H2O2-induced oxidative stress. Prior incubation for 24 h with 1,25-(OH)2D3 (25 pM or higher) produced unexpected increased H2O2 toxicity. Since cellular Ca2+ may be a mediator of cell injury, we investigated the effects of altering extracellular Ca2+ ([Ca2+]e) on 1,25-(OH)2D3-enhanced H2O2 toxicity, as well as the effects of 1,25-(OH)2D3 and H2O2 on cytosolic-free Ca2+ concentration ([Ca2+]f). Basal [Ca2+]f in medium containing 1.5 mM Ca2+ as determined by fura-2 fluorescence was higher in 1,25-(OH)2D3-pretreated cells than control cells (137 versus 112 nM, p less than 0.005). H2O2 induced a rapid increase in [Ca2+]f (to greater than 300 nM) in both 1,25-(OH)2D3-treated and control cells, which was prevented by a reduction in [Ca2+]e to less than basal [Ca2+]f. The 1,25-(OH)2D3-induced increase in H2O2 toxicity was also prevented by preincubation with 1,25-(OH)2D3 in Ca2(+)-free medium or by exposing the cells to H2O2 in the presence of EGTA. Preexposure of cells to 45 degrees C for 20 min, 4 h earlier, partially prevented the toxic effects of H2O2 particularly in 1,25-(OH)2D3-treated cells, even in the presence of physiological levels of [Ca2+]e. Thus, 1,25-(OH)2D3 potentiates H2O2-induced injury probably by increasing cellular Ca2+ stores. The protective effects of heat shock are probably exerted at a site distal to the toxic effects of Ca2+. The 1,25-(OH)2D3-induced amplification of the heat shock response likely represents a mechanism for counteracting the Ca2(+)-associated enhanced susceptibility of oxidative injury due to 1,25-(OH)2D3.

Therapy by taking away: the case of iron.

The recent finding of the beneficial effects of iron deprivation in the outcome of muscle necrosis in an animal model of genetic myopathy served as the basis of this commentary. Here, "taking away" iron by controlled dietary deprivation is proposed as a reasonable, feasible, cheap, and efficient clinical approach to many diverse diseases, all of which have a free radical component. Indeed, iron potentiates the generation of the highly reactive and toxic hydroxyl radical, and, thus, of oxidative damage. Iron deprivation may represent the first really efficient antioxidant, preventing oxidative stress in all subcellular compartments, tissues, and organs. Iron/iron deprivation also modulates programmed cell death (apoptosis), which should be the subject of further studies to better define the mechanisms mediating these complex effects. Finally, related to its antioxidant effects, iron deprivation may find applications in the anti-aging field, whether programmed or premature aging, and whether in cosmetics or in gerontology.

Effects of membrane fatty acids on thermal and oxidative injury in the human premonocytic line U937.

Heat shock (HS) proteins (HSP) function as molecular chaperones and protect cells from thermal and oxidative injury. The signals leading to HSP synthesis, i.e. the "cellular thermometer(s)," are still a matter of debate. In the human premonocytic line U937, we investigated the effects of specific modification of membrane fatty acid (FA) composition by incubation with various saturated and unsaturated fatty acids (UFA) on the HS response and on hydrogen peroxide (H2O2)-induced cell death. FA readily incorporated into U937 cell membranes. UFA did not modulate the HS response but potentiated H2O2-mediated damage, while pre-exposure to HS protected the UFA-treated cells from this increased H2O2 toxicity.

Effects of iron deprivation on the pathology and stress protein expression in murine X-linked muscular dystrophy.

Duchenne muscular dystrophy (DMD) is caused by dystrophin deficiency, which results in muscle necrosis and the upregulation of heat shock/stress proteins (HSP). We hypothesized that reactive oxygen species, and in particular hydroxyl radicals (.OH), participate in muscle necrosis and HSP expression. It was assumed that iron deprivation decreases .OH generation, restraining the disease process and reducing the oxidant-induced expression of HSP. The role of iron-catalyzed free radical reactions in the pathology of dystrophin-deficient muscle was evaluated in the murine model for Duchenne muscular dystrophy (mdx), by examining the effects of dietary deficiency and supplementation of iron on serum creatine kinase (CK), muscle morphology, lipid peroxidation and HSP levels in mice maintained on diets deficient in or supplemented with iron for 6 weeks. Iron-deprived mdx mice showed a significant decrease in the number of macrophage-invaded necrotic fibers and the expression of the 70-kDa heat shock protein (Hsp70). This suggests that the iron-dependent generation of .OH relates to muscle necrosis in the mdx mouse and modulates the expression of Hsp70 in vivo. In contrast, iron deprivation had no influence on other HSP or on lipid peroxidation in mdx mice, while maintenance on either diet caused a significant decrease in serum creatine kinase activity. The potential therapeutic effects of iron deprivation in mdx should be considered.

Protection from cellular oxidative injury and calcium intrusion by N-(2-mercaptoethyl)-1,3-propanediamine, WR 1065.

The radioprotective agent WR 2721 has been shown to prevent deterioration of renal function in a rodent model of chronic renal failure, and it has been suggested that WR 2721 may exert these protective effects by scavenging free radicals. To test this hypothesis, we investigated the effects of WR 2721 and its dephosphorylated, active metabolite, WR 1065, in an in vitro model of oxidative injury. By using the classical assay for superoxide production (reduction of ferricytochrome c), we first demonstrated that WR 1065 may act as a reducing agent. To establish the biological relevance of this reducing ability, U937 cells (a human premonocytic line) were incubated with or without WR 1065 or 2721, then exposed to increasing concentrations of H2O2. Cell viability was measured by Trypan blue exclusion and [3H]-thymidine incorporation into DNA, and cytosolic free calcium determined by fura-2 fluorescence. WR 1065 protected U937 cells from H2O2-induced cell death in a dose-dependent manner, and more efficiently than WR 2721. WR 1065 also prevented the rise in cytosolic free calcium induced in these cells by H2O2. These in vitro molecular and cellular events may account, at least in part, for the in vivo protective effects of WR 2721.

Transcriptional regulators of oxidative stress-inducible genes in prokaryotes and eukaryotes.

It appears that redox regulation is an important mechanism for the control of transcription factor activation. The role of oxidation-reduction is probably determined in part by the structure of the transcription factors. For example, the presence of cysteine residues within the DNA binding sites may sensitize a transcription factor to ROS. The ROS-mediated regulation of transcription factors is specific, some ROS are more efficient than other ROS in activating defined regulators. While the protective antioxidant responses induced by ROS in prokaryotes and eukaryotes are rather conserved (for example, SOD, HSP...), the regulators for these genes do not appear to be conserved. Further studies designed to fully characterize these regulators and understand the subtle mechanisms involved in redox gene regulation are ongoing, and should provide the theoretical basis for clinical approaches using antioxidant therapies in human diseases in which oxidative stress is implicated.

Stress proteins in inflammation.

Inflammation provides those searching in the field with a number of "models" allowing them to study, in vivo, in humans and in animals, the regulation and the functions of HSP, which are being considered as a new and promising marker for the severity and the prognosis of inflammatory diseases. HSP are differentially regulated according to the type of inflammation, whether acute or chronic, whether self-limiting (inflammatory cell elimination by apoptosis) or self-perpetuating (inflammatory cell death by necrosis). We propose that mitochondria are a key organelle in determining the outcome of inflammation, because they are both the cellular "switchboard" for apoptosis and a selective target for the protective effects of HSP against the cytotoxic effects of TNF alpha and ROS. On the other hand, HSP exert multiple protective effects in inflammation, including self/non-self discrimination, enhancement of immune responses, immune protection, thermotolerance and protection against the cytotoxicity of inflammatory mediators. The latter protective effects against the deleterious effects of the mediators of inflammation, including ROS and cytokines, open new avenues for the development of original anti-inflammatory therapies, such as non-toxic inducers of a complete HS response. It may well be that the "beneficial effects of fever" already described by Hippocrates actually relate to increased HSP expression during fever, and to their protective effects....

Stress symposium in Johannesburg: plants versus humans.

On 18 February 1998, a 'Stress symposium' was held at the Rand Afrikaans University (RAU) in Johannesburg, South Africa. The meeting brought together people from both the plant and the human oxidative stress field, which was exemplified by a talk entitled 'Heat shock proteins in host-pathogen interactions: plants versus humans'. There were moments when it appeared as if the main difference between plants and humans was, as sung by Julos Beaucarne, that 'the human plant is the only one to be able to water itself...'

Induction of stress proteins by tobacco smoke in human monocytes: modulation by antioxidants.

Tobacco smoke (TS) induced in human monocytes the synthesis of both the classical heat shock proteins (HSP) (Hsp70, Hsp90, Hsp110) and the oxidation-specific stress protein (SP) heme oxygenase (HO). To determine the role of reactive oxygen species in SP induction by TS, we incubated the monocytes with various antioxidants before exposure to TS. Quercetin and N-acetylcysteine (NAC) both prevented the induction of HO by TS but not, or less so, than that of the classical HSP, while the nitric oxide synthase inhibitor L-nitroarginine had no effect. Thus, at least two mechanisms appear involved in SP induction by TS; (i) the induction of HO (oxidation-dependent), which was prevented by quercetin and NAC; and (ii) the induction of Hsp70, which was, at least in part, oxidation-independent. SP induction might represent an adequate biosensor for TS and other radical-mediated environmental exposures.

Flow cytometry is a rapid and reliable method for evaluating heat shock protein 70 expression in human monocytes.

The increasing interest in stress/heat shock proteins (Hsps) as markers of exposure to environmental stress or disease requires an easily applicable method for Hsp determination in peripheral blood cells. Of these cells, monocytes preferentially express Hsps upon stress. An appropriate fixation/permeabilization procedure was developed, combined with immunofluorescence staining and flow cytometry for the detection of the inducible, cytosolic, 72 kDa Hsp (Hsp70) in human monocytes. Higher relative fluorescence intensity was observed in cells exposed to heat shock (HS), reflecting a higher expression of Hsp70 in these cells as compared with cells kept at 37 degrees C. The heat-inducible increased Hsp70 expression was temperature- and time-dependent. Expression of Hsp70 was not uniform within the monocyte population, indicating the presence of subpopulations expressing variable levels of Hsp70 in response to HS. Simultaneous measurements of intracellular Hsp70 and membrane CD14 expression revealed that the higher Hsp70 inducibility coincided with the higher CD14 expression. Comparisons performed with biometabolic labelling, Western blotting, immunofluorescence and immunoperoxidase microscopic analysis, showed a high concordance between these different methods; however, cytometry was more sensitive for Hsp70 detection than Western blotting. Flow cytometric detection of intracellular Hsp70 is a rapid, easy and quantitative method, particularly suited for the determination of protein levels in individual cells from an heterogeneous population such as peripheral mononuclear blood cells, and applicable to cohort studies.