Effect of liposomal and free bisphosphonates on the IL-1 beta, IL-6 and TNF alpha secretion from RAW 264 cells in vitro

PURPOSE: In order to evaluate the possible antiinflammatory action of bisphosphonates, the effect of the drugs on the secretion of proinflammatory cytokines (IL-1 beta, IL-6 and TNF alpha) from macrophages was studied. Liposomes or high concentration of extracellular calcium was used to enhance the intracellular delivery of bisphosphonates. METHODS: RAW 264 cells were used as macrophage model, and they were induced with lipopolysaccharide to produce the cytokines. The cytokine concentrations in the culture supernatants were measured with time-resolved fluoroimmunoassay. RESULTS: As a free drug, clodronate and pamidronate, but not etidronate, inhibited LPS-stimulated secretion of the cytokines from macrophage-like RAW 264 cells. Low concentrations of pamidronate, however, induced the IL-6 secretion, and the cytokine inhibitory action at the higher concentrations of pamidronate was attributed to cytotoxicity of the compound. The cytokine induction or toxic effects were not observed with clodronate or etidronate. When the drugs were encapsulated in negatively charged unilamellar liposomes, the inhibitory potency of both clodronate and etidronate enhanced by a factor of 10-20, while that of pamidronate was not increased. The complex formation of bisphosphonates with extracellular calcium, although enhancing the uptake of the compounds by macrophages, did not considerably increase their cytokine inhibitory potency. CONCLUSIONS: Bisphosphonates have inhibitory action on cytokine secretion by macrophages. The non-cytotoxic cytokine inhibition by liposome encapsulated clodronate could be beneficial in local inflammatory diseases, where the inflammation is sustained by the excessive amounts of inflammatory cytokines produced by activated macrophages.     

A detailed protocol for the measurement of TGF-beta1 in human blood samples

Bisphosphonates are compounds derived from pyrophosphate, a byproduct of cellular cleavage of adenosine triphosphate (ATP), and are resistant to alkaline phosphatase by virtue of replacement of oxygen by carbon. The high affinity of the P-C-P structure for hydroxyapatite accounts for deposition in bone. Modification of the two side chains of carbon alters the potency of the drugs. Of those that have either completed or are undergoing clinical trials, the order of increasing potency for inhibition of bone resorption is etidronate, clodronate, tiludronate, pamidronate, alendronate, residronate and ibandronate (potency range: 1 to 10,000). Less than 5% of bisphosphonates are absorbed and the half life is a few hours. The drugs must be given on an empty stomach because food and beverages interfere with gastrointestinal absorption. Of the absorbed fraction, as much as 60% is taken up by the skeleton and the remainder is excreted unchanged in the urine. Etidronate, tiludronate, residronate, and alendronate are given orally, clodronate intravenously, and pamidronate and ibandronate by either route. At lower concentrations, bisphosphonates inhibit osteoclatic bone resorption, whereas at higher concentrations they may inhibit mineralization and cause osteomalacia. Inhibition of mineralization diminishes with increasing potency. In postmenopausal women, etidronate and alendronate for 3 yr were shown to inhibit bone resorption, increase bone mineral density (BMD) of the lumbar spine and hip, and prevent fractures without producing osteomalacia. Bone formation also is reduced as a consequence of diminished bone resorption but reduction is less than the reduction of bone resorption. In higher doses bisphosphonates may cause upper gastrointestinal disturbances but in recommended doses they generally are well tolerated and have an excellent safety profile.     

Clodronate and liposome-encapsulated clodronate are metabolized to a toxic ATP analog, adenosine 5'-(beta, gamma-dichloromethylene) triphosphate, by mammalian cells in vitro

Clodronate, alendronate, and other bisphosphonates are widely used in the treatment of bone diseases characterized by excessive osteoclastic bone resorption. The exact mechanisms of action of bisphosphonates have not been identified but may involve a toxic effect on mature osteoclasts due to the induction of apoptosis. Clodronate encapsulated in liposomes is also toxic to macrophages in vivo and may therefore be of use in the treatment of inflammatory diseases. It is generally believed that bisphosphonates are not metabolized. However, we have found that mammalian cells in vitro (murine J774 macrophage-like cells and human MG63 osteosarcoma cells) can metabolize clodronate (dichloromethylenebisphosphonate) to a nonhydrolyzable adenosine triphosphate (ATP) analog, adenosine 5'-(beta, gamma-dichloromethylene) triphosphate, which could be detected in cell extracts by using fast protein liquid chromatography. J774 cells could also metabolize liposome-encapsulated clodronate to the same ATP analog. Liposome-encapsulated adenosine 5'-(beta, gamma-dichloromethylene) triphosphate was more potent than liposome-encapsulated clodronate at reducing the viability of cultures of J774 cells and caused both necrotic and apoptotic cell death. Neither alendronate nor liposome-encapsulated alendronate were metabolized. These results demonstrate that the toxic effect of clodronate on J774 macrophages, and probably on osteoclasts, is due to the metabolism of clodronate to a nonhydrolyzable ATP analog. Alendronate appears to act by a different mechanism.     

Expansions of T-cell subsets expressing particular T-cell receptor variable regions in chronic beryllium disease

Bisphosphonates are currently the most important class of antiresorptive drugs used for the treatment of metabolic bone diseases. Although the molecular targets of bisphosphonates have not been identified, these compounds inhibit bone resorption by mechanisms that can lead to osteoclast apoptosis. Bisphosphonates also induce apoptosis in mouse J774 macrophages in vitro, probably by the same mechanisms that lead to osteoclast apoptosis. We have found that, in J774 macrophages, nitrogen-containing bisphosphonates (such as alendronate, ibandronate, and risedronate) inhibit post-translational modification (prenylation) of proteins, including the GTP-binding protein Ras, with farnesyl or geranylgeranyl isoprenoid groups. Clodronate did not inhibit protein prenylation. Mevastatin, an inhibitor of 3-hydroxy-3-methylglutatyl (HMG)-CoA reductase and hence the biosynthetic pathway required for the production of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, also caused apoptosis in J774 macrophages and murine osteoclasts in vitro. Furthermore, alendronate-induced apoptosis, like mevastatin-induced apoptosis, could be suppressed in J774 cells by the addition of farnesyl pyrophosphate or geranylgeranyl pyrophosphate, while the effect of alendronate on osteoclast number and bone resorption in murine calvariae in vitro could be overcome by the addition of mevalonic acid. These observations suggest that nitrogen-containing bisphosphonate drugs cause apoptosis following inhibition of post-translational prenylation of proteins such as Ras. It is likely that these potent antiresorptive bisphosphonates also inhibit bone resorption by preventing protein prenylation in osteoclasts and that enzymes of the mevalonate pathway or prenyl protein transferases are the molecular targets of the nitrogen-containing bisphosphonates. Furthermore, the data support the view that clodronate acts by a different mechanism.     

Differential regulation of cytokine production by nitric oxide

Nitric oxide (NO) has recently been identified as a potent and pleiotropic intracellular mediator produced by and acting on many cells of the body. Although considerable attention has been devoted to the regulation of NO by inflammatory cytokines, and also to the role of NO as an important effector molecule in immune function, there is very little information on the role of this mediator in modulating T-cell-dependent cytokine production. In this study we show that physiological levels of NO (either produced by activated macrophages or by the addition of exogenous NO donors) can selectively down-regulate interleukin-3 (IL-3) production by spleen cells from contact-sensitized mice, while leaving IL-2 activity unaffected. Thus NO may have an important role as an immunomodulatory as well as effector molecule in the immune system.     

Effects of the bisphosphonate tiludronate on bone resorption, calcium balance, and bone mineral density

Bone resorption inhibitors, such as bisphosphonates, are potentially useful in treatments aimed at increasing bone mass. Among bisphosphonates, tiludronate has proven efficacious in preventing bone loss in postmenopausal women. However, it is not clearly established whether bisphosphonates are more potent when given intermittently or continuously. We investigated the effects of tiludronate on (1) retinoid-stimulated bone resorption in thyroparathyroidectomized rats, (2) calcium balance in intact rats, and (3) bone mineral density (BMD) as measured by dual-energy x-ray absorptiometry at the levels of the lumbar spine, tail, and tibia in 6-month-old rats made osteoporotic by ovariectomy (OVX), in which an intermittent cyclic schedule of treatment was compared to continuous administration. Tiludronate induced a dose-dependent decrease in retinoid-stimulated bone resorption. It increased the intestinal absorption and body retention of calcium. In OVX rats it caused a time- and dose-dependent increase in BMD at the level of the three investigated sites, the effects being maintained for at least 8 weeks after the end of therapy. Continuous and intermittent cyclic regimens appeared to induce similar increases in BMD. These results indicate that tiludronate is efficacious in decreasing bone resorption and increasing calcium balance and bone mineral density in rats.     

Macrophage inflammatory protein-1 alpha production in lipopolysaccharide-stimulated human adherent blood mononuclear cells is inhibited by the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine

The release of chemokines such as macrophage-inflammatory protein-1 alpha (MIP-1 alpha) from activated macrophages is a crucial step in cell recruitment necessary for establishing local inflammatory responses. To ascertain the importance of the L-arginine/nitric oxide (NO) pathway in LPS-induced MIP-1 alpha release, we stimulated human adherent PBMC with LPS in the presence of the NO synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA). L-NMMA decreased LPS-induced MIP-1 alpha protein release (45.5% inhibition) and steady state levels of mRNA (48% inhibition) in adherent PBMC. The concentration of L-NMMA for inhibition of MIP-1 alpha release was dependent on the concentration of L-arginine in the cell culture medium, emphasizing the L-arginine-related action of the drug. Most of the MIP-1 alpha release was attributed to the activity of IL-1 and TNF, since coincubation of LPS-stimulated PBMC with IL-1R antagonist and TNF-binding protein abrogated LPS-induced MIP-1 alpha release (by 76.8%). Analysis of cytokine secretion revealed that, in addition to MIP-1 alpha, L-NMMA inhibited the release of mature IL-1 beta (by 70%) and TNF-alpha (by 53%). In contrast, release of macrophage chemoattractant protein-1 was unaffected; IL-10 was augmented (123.4%) by L-NMMA. In the presence of exogenous NO provided by NO donors, LPS-induced MIP-1 alpha release was enhanced. We concluded that endogenous NO acts as a mediator of inflammation. Since IL-10 is a potent anti-inflammatory cytokine, these data also suggest that L-NMMA acts as an anti-inflammatory agent by specifically altering the balance of pro- and anti-inflammatory cytokines released from LPS-stimulated human PBMC.     

Interleukin-13 is a more potent inhibitor of the expression of inducible nitric oxide synthase in smooth muscle cells than in macrophages: a comparison with interleukin-4 and interleukin-10

This study compares the effects of interleukin (IL)-13, a cytokine with anti-inflammatory properties, with those of IL-4 or IL-10 on the expression of inducible nitric oxide synthase (iNOS) protein and activity in 1) a murine macrophage cell line (J774.2) activated with lipopolysaccharide (LPS) and 2) rat aortic smooth muscle cells (RASM) activated with LPS plus interferon-gamma. Pretreatment of macrophages with IL-4 or IL-13 caused a similar, concentration-dependent inhibition of the formation of nitrite and the expression of iNOS protein elicited by LPS. In contrast, IL-13 was a much more potent inhibitor of the formation of nitrite and the expression of iNOS protein in activated RASM than IL-4. IL-10 caused only a small, but significant, inhibition of the nitrite formation induced by LPS in macrophages and RASM. Pretreatment of J774.2 macrophages, but not of RASM, with the phosphatidylinositol-3-kinase inhibitor, wortmannin (10-100 nM), attenuated the inhibition by either IL-13 or IL-4 of the LPS-induced increase in nitrite in a dose-related fashion. Thus, IL-13 is more potent than IL-4 in preventing the expression of iNOS protein and activity in activated RASM, whereas IL-13 and IL-4 are equipotent in inhibiting the expression of iNOS protein and activity in J774.2 macrophages.     

Cytokine stimulation of T lymphocytes regulates their capacity to induce monocyte production of tumor necrosis factor-alpha, but not interleukin-10: possible relevance to pathophysiology of rheumatoid arthritis

Previous studies in the laboratory have shown that the pro-inflammatory cytokine tumor necrosis factor (TNF)-alpha plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA). The mechanisms involved in regulating monocyte/macrophage cytokine production are not yet fully understood, but are thought to involve both soluble factors and cell/cell contact with other cell types. We and others have previously demonstrated that T cells activated through the T cell receptor/CD3 complex induce monocyte TNF-alpha production by contact-mediated signals. In this report, we investigated further whether T cells activated by cytokines in the absence of T cell receptor stimulation also regulate monocyte cytokine production. T cells were activated in an antigen-independent manner using the cytokines interleukin (IL)-15 or IL-2 alone, or in combination with IL-6 and TNF-alpha. Subsequently, T cells were fixed and incubated with monocytes. Fixed, cytokine-stimulated T cells induced monocytes to secrete TNF-alpha in a dose-dependent manner, but did not induce secretion of IL-10, a potent endogenous down-regulator of TNF-alpha and other pro-inflammatory cytokines. Stimulation of monocyte TNF-alpha was markedly inhibited when T cells were physically separated from monocytes within the tissue culture well, confirming that T cell contact is necessary. T cell acquisition of monocyte-activating capacity was shown to be dependent on the period of cytokine stimulation, with T cells activated for 8 days more effective than T cells activated for shorter periods. Addition of interferon-gamma or granulocyte/macrophage colony-stimulating factor to the T cell/monocyte cultures enhanced T cell induction of monocyte TNF-alpha by threefold and ninefold, respectively. The results from this model of cognate interaction suggest that cytokine-stimulated T cells, interacting with macrophages in the rheumatoid synovial membrane, may contribute to the continuous excessive production of TNF-alpha observed in the RA joint, and to the imbalance of pro-inflammatory cytokines over anti-inflammatory cytokines.     

Increased in vitro induced CD4+ and CD8+ T cell IFN-gamma and CD4+ T cell IL-10 production in stable relapsing multiple sclerosis

Multiple sclerosis (MS) is presumed to be a T-cell mediated chronic inflammatory disease of the central nervous system. Investigators previously demonstrated increased IFN-gamma (pro-inflammatory) and IL-10 (counterregulatory anti-inflammatory) in MS. The balance of pro-inflammatory and counterregulatory anti-inflammatory cytokines may be important in the stabilization of disease activity. Purified CD4+ and CD8+ T cells from patients with clinically definite, stable relapsing MS (RRMS) were stimulated by anti-CD3 mAb or Con A for 48 hours and cytokine supernatants analysed for production of IL-2, IL-6, IFN-gamma, TNF-alpha (potential pro-inflammatory) and IL-4, IL-10, and TGF-beta (potential counterregulatory anti-inflammatory). Con A activated CD4+ and CD8+ T cell proinflammatory cytokine IL-2 secretion, CD4+ T cell IL-6 secretion, CD4+ and CD8+ T cell TNF-alpha secretion and CD8+ T cell IFN-gamma secretion was decreased significantly in RRMS subjects compared to controls. CD3 activated CD4+ and CD8+ T cell IL-6 secretion and CD4+ T cell TNF-alpha secretion was significantly decreased in MS subjects compared to controls. In contrast, there was increased CD3-induced IFN-gamma in both CD4+ and CD8+ T cells and counterregulatory anti-inflammatory CD3-induced IL-10 secretion in CD4+ T cells in RRMS compared to controls. These data suggest that an equilibrium of a pro-inflammatory (IFN-gamma) and a counterregulatory anti-inflammatory (IL-10) cytokine may define stable clinically definite early RRMS.     

Nitric oxide prevents IL-1beta and IFN-gamma-inducing factor (IL-18) release from macrophages by inhibiting caspase-1 (IL-1beta-converting enzyme)

Procytokine processing by caspase-1 is required for the maturation and release of IL-1beta and IFN-gamma-inducing factor (IGIF) (or IL-18) from activated macrophages (Mphi). Nitric oxide (NO) has emerged as a potent inhibitor of cysteine proteases. Here, we tested the hypothesis that NO regulates cytokine release by inhibiting IL-1beta-converting enzyme (ICE) or caspase-1 activity. Activated RAW264.7 cells released four to five times more IL-1beta, but not TNF-alpha, in the presence of the NO synthase inhibitor N(G)-monomethyl-L-arginine. Stimulated peritoneal Mphi from wild-type mice (inducible NO synthase (iNOS)+/+) also released more IL-1beta if exposed to N(G)-monomethyl-L-arginine, whereas Mphi from iNOS knockout mice (iNOS-/-) did not. Inhibition of NO synthesis in stimulated RAW264.7 cells also resulted in a threefold increase in intracellular caspase-1 activity. The NO donor S-nitroso-N-acetyl-DL-penicillamine inhibited caspase-1 activity in cells as well as the activity of purified recombinant caspase-1 and also prevented the cleavage of pro-IL-1beta and pro-IGIF by recombinant caspase-1. The inhibition of caspase-1 by NO was reversible by the addition of DTT, which is consistent with S-nitrosylation as the mechanism of caspase-1 inhibition. An in vivo role for the regulation of caspase-1 by NO was established in iNOS knockout animals, which exhibited significantly higher plasma levels of IL-1beta and IFN-gamma than their wild-type counterparts at 10 h following LPS injection. Taken together, these data indicate that NO suppresses IL-1beta and IGIF processing by inhibiting caspase-1 activity, providing evidence for a unique role for induced NO in regulating IL-1beta and IGIF release.     

Current use of bisphosphonates in oncology. International Bone and Cancer Study Group

PURPOSE: The purpose of this article is to review the recent data on bisphosphonate use in oncology and to provide some guidelines on the indications for their use in cancer patients. DESIGN: The group consensus reached by experts on the rationale for the use of bisphosphonates in cancer patients and their current indications for the treatment of tumor-induced hypercalcemia and metastatic bone pain in advanced disease and for the prevention of the complications of multiple myeloma and of metastatic bone disease are reviewed. RESULTS: Bisphosphonates are potent inhibitors of tumor-induced osteoclast-mediated bone resorption. They now constitute the standard treatment for cancer hypercalcemia, for which we recommend a dose of 1,500 mg of clodronate or 90 mg of pamidronate; the latter compound is more potent and has a longer lasting effect. Intravenous bisphosphonates exert clinically relevant analgesic effects in patients with metastatic bone pain. Regular pamidronate infusions can also achieve a partial objective response by conventional International Union Against Cancer criteria and enhance the objective response rate to chemotherapy. In breast cancer, the prolonged administration of oral clodronate 1,600 mg daily reduces the frequency of morbid skeletal events by more than one fourth, whereas monthly pamidronate infusions of 90 mg for only 1 year in addition to chemotherapy reduce by more than one third the frequency of all skeletal-related events. The use of bisphosphonates to prevent bone metastases remains experimental. Last, bisphosphonates in addition to chemotherapy are superior to chemotherapy alone in patients with stages II and III multiple myeloma and can reduce the skeletal morbidity rate by approximately one half. CONCLUSION: Bisphosphonate use is a major therapeutic advance in the management of the skeletal morbidity caused by metastatic breast cancer or multiple myeloma, although many questions remain unanswered, notably regarding the optimal selection of patients and the duration of treatment.     

Differential sensitivities of human blood monocytes and alveolar macrophages to the inhibition of prostaglandin endoperoxide synthase-2 by interleukin-4

Interleukin-4 (IL-4) is a potent immunomodulatory cytokine synthesized and released by Th2 lymphocytes, mast cells and basophils. It has important effects on monocyte/macrophage cell lines, regulating the secretion of several cytokines, and the production of eicosanoids. In human monocytes and macrophages, IL-4 increases the expression of 15-lipoxygenase and 15-HETE production, but suppresses the inducible isoform of the prostaglandin H synthase (PGHS-2) enzyme and prostanoid synthesis. Prostanoids, in particular prostaglandin E2 (PGE2) have important functions in modulating inflammatory and fibrotic processes. We compared the effect of IL-4 on the expression of PGHS-2 in human alveolar macrophages (AM) and blood monocytes (BM) activated with physiologically distinct stimuli, lipopolysaccharide (LPS) or IL-1 in vitro. The induction of PGHS-2 mRNA and protein, and prostanoid synthesis by all stimuli was inhibited by exogenous IL-4 in both cell types. However, monocytes were more susceptible to this effect of IL-4 than alveolar macrophages.     

Differential regulation of IL-4 and IL-13 secretion by human basophils: their relationship to histamine release in mixed leukocyte cultures

Human basophils are an important source of IL-4, a cytokine that is central to the pathogenesis of allergic inflammation. Recent reports have indicated that these cells also generate IL-13, which shares a number of biologic properties with IL-4. We found basophils to be the major source of IL-13 produced in mixed leukocyte cultures following 20-h activation with a variety of stimuli. While the magnitude of IL-4 protein generated correlated with the percent histamine secreted (r = 0.8; p = 0.007), there was no relationship between the levels of IL-13 detected and the amount of either IL-4 or histamine in cultures activated with IL-3/anti-IgE. The induction of IL-13 secretion also occurred in response to IL-3 alone, without concomitant secretion of either IL-4 or histamine. Although previously shown to inhibit IL-4 secretion, the phorbol ester PMA was a potent stimulus for IL-13 generation from basophils, and this secretion was sensitive to the protein kinase C inhibitor, bisindolylmaleimide. In contrast, bisindolylmaleimide did not prevent cytokine secretion induced by either anti-IgE or IL-3. The immunosuppressant, FK506, while strikingly inhibiting the accumulation of IL-4 mRNA and the secretion of protein in response to IL-3/anti-IgE, had no effect on the generation of IL-13 in these cultures; the resistance was attributed to the IL-3-dependent signaling. Similarly, FK506 had no effect on the secretion of IL-13 in basophil cultures stimulated with PMA. This study suggests that multiple intracellular mechanisms control the generation of IL-13 in basophils, some of which are distinct from those regulating IL-4.     

Macrophage-derived nitric oxide regulates T cell activation via reversible disruption of the Jak3/STAT5 signaling pathway

Nitric oxide (NO) has been invoked as an important pathogenic factor in a wide range of immunologically mediated diseases. The present study demonstrates that macrophage-derived NO may conversely function to fine tune T cell-mediated inflammation via reversible dephosphorylation of intracellular signaling molecules, which are involved in the control of T cell proliferation. Thus, T cells activated in the presence of alveolar macrophages are unable to proliferate despite expression of IL-2R and secretion of IL-2. This process is reproduced by the NO generator S-nitroso-N-acetylpenicillamine and is inhibitable by the NO synthase inhibitor N(G)-methyl-L-arginine. Analysis of T cell lysates by immunoprecipitation with specific Abs and subsequent immunoblotting indicated marked reduction of tyrosine phosphorylation of Jak3 and STAT5 mediated by NO. Further studies indicated that NO-mediated T cell suppression was reversible by the guanylate cyclase inhibitors methylene blue and LY-83583 and was reproduced by a cell-permeable analogue of cyclic GMP, implicating guanylate cyclase activation as a key step in the inhibition of T cell activation by NO.     

Evidence for the involvement of interleukin 10 in the differential deactivation of murine peritoneal macrophages by prostaglandin E2

Among other effects, prostaglandins (PG) of the E series are known to inhibit several acute and chronic inflammatory conditions in vivo and proinflammatory cytokine production by activated macrophages in culture. The research presented here demonstrates that the inhibitory effect of PGE2 on tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) production by lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages involves IL-10. In a dose-dependent manner, PGE2 inhibits LPS-induced release of TNF-alpha and IL-6, but not of lactate or nitric oxide. The decrease in the level of these cytokines is inversely proportional to the increase in immunoreactive IL-10. This differential inhibitory effect of PGE2 is mimicked by agents that elevate intracellular levels of cAMP, but not cGMP. Neutralizing anti IL-10 antibody but not neutralizing antibodies against other macrophage secretory products (IL-6, leukemia inhibitory factor, and transforming growth factor beta [TGF-beta]), significantly reverse the potent inhibitory effect of PGE2. In vivo, the administration of PGE2 before LPS challenge significantly reduces circulating TNF-alpha and IL-6 levels. Anti-IL-10 antibody substantially enhanced the LPS-induced TNF-alpha and IL-6 levels in mice that received either LPS alone or LPS plus PGE2. These results suggest that the anti-inflammatory effect of PGE2 on mononuclear phagocytes is mediated in part by an autocrine feedback mechanism involving IL-10.     

Inhibition of growth of Dictyostelium discoideum amoebae by bisphosphonate drugs is dependent on cellular uptake

PURPOSE: The aim of the study was to determine whether bisphosphonates are internalised by Dictyostelium amoebae and whether cellular uptake is required for their growth-inhibitory effects. Bisphosphonates inhibit growth of amoebae of the slime mould Dictyostelium discoideum, by mechanisms that appear to be similar to those that cause inhibition of osteoclastic bone resorption. METHODS: Cell-free extracts prepared from amoebae that had been incubated with bisphosphonates were analysed by 31P-n.m.r, spectroscopy or ion-exchange f.p.l.c., to identify the presence of bisphosphonates or bisphosphonate metabolites respectively. The growth-inhibitory effect of bisphosphonates towards Dictyostelium amoebae was also examined under conditions in which pinocytosis was inhibited. RESULTS: All of the bisphosphonates studied were internalised by Dictyostelium amoebae, probably by fluid-phase pinocytosis, and could be detected in cell-free extracts. Amoebae that were prevented from internalising bisphosphonates by pinocytosis were markedly resistant to the growth-inhibitory effects of these compounds. In addition, bisphosphonates encapsulated within liposomes were more potent growth inhibitors of Dictyostelium owing to enhanced intracellular delivery of bisphosphonates. CONCLUSIONS: All bisphosphonates inhibit Dictyostelium growth by intracellular mechanisms following internalisation of bisphosphonates by fluid-phase pinocytosis. It is therefore likely that bisphosphonates also affect osteoclasts by interacting with intracellular, rather than extracellular, processes.     

Reprogramming of lipopolysaccharide-primed macrophages is controlled by a counterbalanced production of IL-10 and IL-12

We studied the potential role of a cytokine regulatory mechanism(s) in LPS-dependent reprogramming and modulation of TNF-alpha and nitric oxide (NO) responses in mouse peritoneal macrophages. Reciprocal regulation of TNF-alpha and NO production by LPS-primed and LPS-stimulated macrophages was found to be dependent on the presence of soluble secretory products released by the cells during the initial LPS priming interaction. Pretreatment of naive macrophages with different mouse recombinant cytokines such as rIL-10, rIL-12, and rIFN-gamma dose dependently and differentially regulated subsequent LPS-induced production of TNF-alpha, IL-6, and NO by cytokine-primed cells. Analysis of IL-12 and IL-10 levels present in culture supernatants of LPS-primed and LPS-stimulated macrophages revealed a high degree of correlation between the profiles of TNF-alpha and IL-12 as well as NO and IL-10. Furthermore, LPS priming of macrophages in the presence of anti-IL-12-neutralizing mAb attenuated TNF-alpha responses while at the same time up-regulated NO production. In contrast, neutralization of endogenous IL-10 with anti-IL-10 mAb resulted in considerable TNF-alpha response at LPS priming doses under conditions that would otherwise strongly inhibit TNF-alpha production. We also found that the initial LPS priming of naive macrophages differentially and dose dependently regulates expression of mRNAs for IL-10, IL-12, and IFN-gamma in LPS-primed macrophages. Collectively, our data provide experimental support for the hypothesis that a cytokine regulatory network, most probably autocrine, tightly controls the reciprocal modulation of TNF-alpha and NO responses in LPS-primed macrophages.