Prostaglandin E2 regulates macrophage colony stimulating factor secretion by human bone marrow stromal cells.

Bone marrow stromal cells regulate marrow haematopoiesis by secreting growth factors such as macrophage colony stimulating factor (M-CSF) that regulates the proliferation, differentiation and several functions of cells of the mononuclear-phagocytic lineage. By using a specific ELISA we found that their constitutive secretion of M-CSF is enhanced by tumour necrosis factor-alpha (TNF-alpha). The lipid mediator prostaglandin E2 (PGE2) markedly reduces in a time- and dose-dependent manner the constitutive and TNF-alpha-induced M-CSF synthesis by bone marrow stromal cells. In contrast, other lipid mediators such as 12-HETE, 15-HETE, leukotriene B4, leukotriene C4 and lipoxin A4 have no effect. EP2/EP4 selective agonists (11-deoxy PGE1 and 1-OH PGE1) and EP2 agonist (19-OH PGE2) inhibit M-CSF synthesis by bone marrow stromal cells while an EP1/EP3 agonist (sulprostone) has no effect. Stimulation with PGE2 induces an increase of intracellular cAMP levels in bone marrow stromal cells. cAMP elevating agents (forskolin and cholera toxin) mimic the PGE2-induced inhibition of M-CSF production. In conclusion, PGE2 is a potent regulator of M-CSF production by human bone marrow stromal cells, its effects being mediated via cAMP and PGE receptor EP2/EP4 subtypes.     

Role of tyrosyl phosphorylation in neutrophil priming by tumor necrosis factor-alpha and granulocyte colony stimulating factor.

The ability of human tumor necrosis factor-alpha (TNF-alpha) and human granulocyte colony stimulating factor (G-CSF) to induce phosphorylation of protein tyrosyl residues in human peripheral neutrophils (PMN) was investigated by Western blot analysis with antiphosphotyrosine antibody. Both TNF-alpha and G-CSF increased the tyrosyl phosphorylation of various proteins, such as species of 54-, 63-, 72-, 83-, 98-, 108-, and 115-kDa proteins. The ligand-stimulated tyrosyl phosphorylation of the 115-kDa protein was time- and concentration-dependent. When the 115-kDa protein was phosphorylated, it was recovered from membrane fractions. The phosphorylation of the 115-kDa protein was inhibited by genistein and alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide (ST 638), inhibitors of tyrosine kinase (TK), and was enhanced by 1-(5-isoquinoline-sulfonyl) methyl-piperazine dihydrochloride (H-7) and staurosporine, inhibitors of Ca(2+)- and phospholipid-dependent protein kinase (PKC). Similar inhibition by the TK inhibitors and stimulation by the PKC inhibitors were also observed with formylmethionyl-leucyl-phenylalanine (FMLP)-induced superoxide (O2.-) generation by TNF-alpha- or G-CSF-primed PMN. Phosphorylation of the 115-kDa protein occurred in parallel with the ligand-dependent generation of O2.-. These and other observations suggested that substrate proteins for tyrosine kinase, such as the 115-kDa protein, might play critical roles in the mechanism for priming of neutrophils. This is the first report describing that tyrosyl phosphorylation is involved in the priming of neutrophils by G-CSF and TNF-alpha.     

Proteomic analysis of human eosinophil activation mediated by mast cells,granulocyte macrophage colony stimulating factor and tumor necrosis factor alpha

We assessed mast cell influence on eosinophils, the prominent cells in late and chronic allergic reactions, by comparing the proteomic pattern of eosinophils incubated with mast cells, tumor necrosis factor alpha (TNF-alpha) or granulocyte macrophage colony stimulating factor (GM-CSF). Eosinophils were incubated with the human mast cell line HMC-1 cellular sonicate and their survival and GM-CSF production were evaluated. For proteomic studies, eosinophils were cultured with HMC-1 sonicate, TNF-alpha or GM-CSF in the presence of [(35)S]methionine, solubilized and submitted to isolelectric focusing separation and sodium dodecyl sulfate polyacrylamide gel electrophoresis in the ISODALT system, followed by radiofluorography and computer image analysis. HMC-1-incubated eosinophils displayed increased survival partly mediated by mast cell-associated TNF-alpha, and produced GM-CSF. Metabolically labeled eosinophils incubated with either HMC-1, TNF-alpha or GM-CSF released eosinophil peroxidase. Comparison of two-dimensional gel spots from the eosinophils revealed that each of the three activating signals yielded a distinctly different proteomic pattern of labeled polypeptides. GM-CSF provided the strongest signal and the highest rate of protein synthesis (1,018 spots) followed by TNF-alpha (747 spots) and HMC-1 sonicate (611 spots). A portion of spots differed both in terms of quality and quantity. Although each stimulus induced similar functional effects, the resulting biosynthetic programs of the eosinophils greatly differed. The presented proteomic analysis is the first step in the exploration of molecular mechanisms involved in eosinophil activation.     

Regulation of prostaglandin secretion from epithelial and stromal cells of the bovine endometrium by interleukin-1 beta, interleukin-2, granulocyte-macrophage colony stimulating factor and tumor necrosis factor-alpha.

Bovine endometrium was obtained on day 16 of pregnancy (estrus = 0) and separated into epithelial and stromal cell populations. When confluent, the two cell populations were treated for 24 h with cytokines at 1, 10 and 100 ng/ml. Prostaglandin (PG) E2 was the major prostaglandin produced by both cell types. For control cultures, more PGE2 was secreted into medium by stromal cells than by epithelial cells, whereas secretion of PGF was similar for epithelial and stromal cells. Interleukin-1 beta had no effect on prostaglandin production by stromal cell cultures but increased epithelial production of PGE2 and, to a lesser extent, PGF. Conversely, granulocyte-macrophage colony stimulating factor had no effect on epithelial cells but reduced secretion of PGE2 and PGF from stromal cells. There were no effects of interleukin-2 or tumor necrosis factor-alpha on prostaglandin secretion. Results indicate that certain cytokines can regulate endometrial prostaglandin secretion in a cell type-restricted manner.     

Potentiation of lymphokine-induced macrophage activation by tumor necrosis factor-alpha

In this study, we examined the possible role of TNF-alpha and lymphotoxin (TNF-beta) as cofactors of macrophage activation. The results demonstrate that both TNF were capable of enhancing the cytostatic and cytolytic activity of murine peritoneal macrophages against Eb lymphoma cells. The potentiation of tumor cytotoxicity became apparent when macrophages from DBA/2 mice were suboptimally activated by either a T cell clone-derived macrophage-activating factor or by IFN-gamma plus LPS. Neither TNF-alpha nor TNF-beta could induce tumor cytotoxicity in IFN-gamma-primed macrophages, indicating that TNF cannot replace LPS as a triggering signal of activation. In LPS-resistant C3H/HeJ macrophages, which were unresponsive to IFN-gamma plus LPS, a supplementation with TNF fully restored activation to tumor cytotoxicity. Furthermore, TNF-alpha potentiated a variety of other functions in low-level activated macrophages such as a lactate production and release of cytotoxic factors. At the same time, TNF-alpha produced a further down-regulation of pinocytosis, tumor cell binding and RNA synthesis observed in activated macrophages. These data demonstrate new activities for both TNF-alpha and TNF-beta as helper factors that facilitate macrophage activation. In particular, the macrophage product TNF-alpha may serve as an autocrine signal to potentiate those macrophage functions that were insufficiently activated by lymphokines.     

The regulation of macrophage protein turnover by a colony stimulating factor (CSF-1)

CSF-1 is a hemopoietic growth factor that specifically regulates the survival, proliferation, and differentiation of mononuclear phagocytic cells. A homogeneous population of mononuclear phagocytes, bone marrow derived macrophages (BMM), were used to study the regulation of protein turnover by CSF-1. Removal of CSF-1 (approximately 0.4 nM) from exponentially growing BMM cultured in 15% fetal calf serum containing medium decreases the rate of DNA synthesis by more than 100-fold. Addition of CSF-1 to these cells causes them to resume DNA synthesis within 12 h. More immediate effects of CSF-1 were observed on BMM protein metabolism. BMM cultured for 24 h in the absence of CSF-1 reduce their protein synthetic rate by 50-60%. The protein synthetic rate commences to decrease at 2-3 h after CSF-1 removal. Readdition of CSF-1 to BMM previously incubated in its absence causes a return to the protein synthetic rate of exponentially growing cells within 2 h. In the presence of CSF-1, BMM synthesize protein at a rate of approximately 8.7%/h and degrade it at a rate of approximately 0.9%/h. Removal of CSF-1 results in a decrease in the protein synthetic rate to approximately 3.4%/h and an increase in the rate of protein degradation to approximately 3.4%/h. The rate of protein synthesis by BMM increases linearly with CSF-1 concentration over the range of concentrations stimulating both survival and proliferation, while the rate of protein degradation decreases exponentially over the range of concentrations stimulating survival without proliferation. Therefore, it appears that the stimulation of the rate of protein synthesis and inhibition of the rate of protein degradation are two distinct effects of CSF-1, both part of the pleiotropic response to this growth factor. The inhibition of the rate of protein degradation by CSF-1 may be most significant for its survival inducing effect.     

Role of first stimulating agents in the production of tumor necrosis factor

Summary The conditions and kinetics of tumor necrosis factor (TNF) production were examined. For TNF production, dual stimulation is necessary. Priming agents such as BCG, Corynebacterium parvum, and zymosan, which can stimulate the reticuloendothelial system (RES), are good substances for TNF production with the aid of lipopolysaccharide. Wide differences are observed in TNF producibility among different priming agents. The producibility of TNF depends on the degree of stimulation of the RES by the priming agents. Those priming agents, e.g., Propionibacterium acnes and Corynebacterium anaerobium, that are able to induce substantial RES hyperplasia are also able to induce high levels of TNF activity. Following administration of large doses of BCG or zymosan, mice were found to produce TNF activity. However, PPD, OK 432, PSK, and Choreito were unable to induce TNF activity.     

Role of tumor necrosis factor in monocyte/macrophage tumor cytotoxicity in vitro

The ability of activated monocytes/macrophages to exert cytotoxic effects in vitro which are preferentially manifest on target cells displaying a transformed phenotype has elicited intense efforts aimed at a molecular characterization of the underlying mechanism. This multistep reaction is typified by an apparently stringent requirement for conjugation between the effector and target to facilitate cytotoxicity, which has therefore long caused bias against the role of soluble effector molecules in mediating target cell damage. However, several laboratories have recently demonstrated a compelling role for at least one such mediator, tumor necrosis factor (TNF), in cell-mediated cytotoxicity exerted against certain target cells; these studies indicated that specific anti-TNF antibodies could block direct monocyte/macrophage-mediated cytotoxicity of TNF-sensitive targets. More recently we have shown that some targets which are completely resistant to soluble or fluid-phase TNF are effectively lysed by a TNF-dependent mechanism upon coculture with activated macrophages under conditions in which conjugation is facilitated. Furthermore, macrophage-mediated cytolysis of both TNF-sensitive and TNF-resistant targets occurs independently of the action of secreted TNF via this mechanism. The purpose of this review is to consider the implications of distinct modes of effector cell delivery of TNF to the target for molecular characterization of the target injury phase of macrophage-mediated tumor cytotoxicity.     

Cryopreservation induces macrophage colony stimulating factor from human periodontal ligament cells in vitro

Cryopreservation is used to protect vital periodontal ligaments during the transplantation of teeth. We investigated which gene products implicated in root resorption are upregulated in human periodontal ligament cells by cryopreservation, and whether cryopreservation affects the expression of macrophage-colony stimulating factor (M-CSF) in human periodontal ligament cells. We used customized microarrays to compare gene expression in human periodontal ligament cells cultured from teeth immediately after extraction and from cryopreserved teeth. Based on the result of these assays, we examined M-CSF expression in periodontal ligament cells from the immediately extracted tooth and cryopreserved teeth by real-time PCR, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence. We also investigated whether human bone marrow cells differentiate into tartrate-resistant acid phosphatase (TRAP) positive osteoclasts when stimulated with RANKL (Receptor Activator for Nuclear Factor κ B Ligand) together with any secreted M-CSF present in the supernatants of the periodontal ligament cells cultured from the various groups of teeth. M-CSF was twofold higher in the periodontal ligament cells from the rapid freezing teeth than in those from the immediately extracted group (p < 0.05). Cryopreservation increased M-CSF expression in the periodontal ligament cells when analyzed by real time PCR, ELISA, Western blotting, and immunofluorescence (p < 0.05). TRAP positive osteoclasts were formed in response to RANKL and the secreted M-CSF present in the supernatants of all the experimental groups except negative control. These results demonstrate that cryopreservation promotes the production of M-CSF, which plays an important role in root resorption by periodontal ligament cells.     

Regulation of macrophage tumor necrosis factor production by prostaglandin E2

We have studied the role of prostaglandin E2 on the modulation of tumor necrosis factor by immunologically elicited and lipopolysaccharide treated murine macrophages. Indomethacin, a potent inhibitor of prostaglandin E2 production, caused a dose dependent augmentation of lipopolysaccharide induced tumor necrosis factor production (2-3 fold at 10(-7) molar). Tumor necrosis factor was released into the extracellular environment and no activity was found to be associated with membrane or cytosolic fractions. Prostaglandin E2 added to the lipopolysaccharide treated cultures suppressed tumor necrosis factor in a dose dependent manner. In these studies, 10(-7) molar PGE2 reduced tumor necrosis factor production to basal levels. These data suggest that PGE2 may be a potent autoregulatory factor that dramatically influences tumor necrosis factor production.     

Induction of colony stimulating factor in vivo by recombinant interleukin 1 alpha and recombinant tumor necrosis factor alpha 1

In response to a potent inflammatory challenge, such as Gram-negative endotoxin, a number of cytokines are induced that, in turn, mediate many of the pathophysiologic alterations associated with endotoxicity. In this study, we have observed two endotoxin-associated monokines, recombinant interleukin-1 alpha (rIL 1 alpha) and recombinant tumor necrosis factor alpha (rTNF alpha), to induce colony stimulating factor (CSF) in vivo. The CSF activities produced in response to rIL 1 alpha or rTNF alpha gave rise to a mixture of granulocyte-macrophage colonies and were induced in a dose- and time-dependent fashion, peaking within 3 hr of cytokine injection (preceding peak CSF induction by endotoxin by several hours). Combined injection of suboptimal concentrations of rIL 1 alpha and rTNF alpha were additive, and simultaneous injection of optimal concentrations of each failed to increase CSF levels over that observed with either cytokine alone. Unlike endotoxin, neither cytokine induced interferon in vivo. These findings extend our understanding of the cytokine cascade that is operative in an inflammatory response and may account for many of the observed hematopoietic alterations that accompany inflammation.     

Apoptotic neutrophils release macrophage migration inhibitory factor upon stimulation with tumor necrosis factor-alpha

Macrophage migration inhibitory factor (MIF) is an important cytokine involved in the regulation of innate immunity and present at increased levels during inflammatory responses. Here we demonstrate that mature blood and tissue neutrophils constitutively express MIF as a cytosolic protein not associated with azurophil granules. Functionally active MIF, but not proteases stored in azurophil granules, was released from apoptotic neutrophils following short term tumor necrosis factor (TNF)-alpha stimulation in a caspase-dependent manner and prior to any detectable phagocytosis by monocyte-derived macrophages. Moreover, TNF-alpha-mediated MIF release was blocked by glyburide and propenicide, both inhibitors of ATP-binding cassette-type transporters, suggesting that this transporter system is activated during neutrophil apoptosis. Taken together, apoptotic mature neutrophils release MIF upon short term TNF-alpha stimulation. Therefore, apoptosis may not always occur without the induction of pro-inflammatory mechanisms.     

Role of SODD in regulation of tumor necrosis factor responses

Signaling from tumor necrosis factor receptor type 1 (TNFR1) can elicit potent inflammatory and cytotoxic responses that need to be properly regulated. It was suggested that the silencer of death domains (SODD) protein constitutively associates intracellularly with TNFR1 and inhibits the recruitment of cytoplasmic signaling proteins to TNFR1 to prevent spontaneous aggregation of the cytoplasmic death domains of TNFR1 molecules that are juxtaposed in the absence of ligand stimulation. In this study, we demonstrate that mice lacking SODD produce larger amounts of cytokines in response to in vivo TNF challenge. SODD-deficient macrophages and embryonic fibroblasts also show altered responses to TNF. TNF-induced activation of NF-kappaB is accelerated in SODD-deficient cells, but TNF-induced c-Jun N-terminal kinase activity is slightly repressed. Interestingly, the apoptotic arm of TNF signaling is not hyperresponsive in the SODD-deficient cells. Together, these results suggest that SODD is critical for the regulation of TNF signaling.     

NF-kappaB-dependent regulation of tumor necrosis factor-alpha gene expression by CpG-oligodeoxynucleotides

Immunostimulatory activities of synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODNs) have gained attention as potentially useful immunotherapeutics. However, CpG-ODNs induce harmful and lethal shock effects because they greatly enhance the sequence-dependent induction of tumor necrosis factor-alpha (TNF-alpha). We have shown that phosphorothioate-modified oligodeoxynucleotides (PS-ODNs) of the CpG-ODN 1826 stimulate TNF-alpha gene expression, TNF-alpha promoter activity, IkappaB degradation, and NF-kappaB activation at higher levels compared with its phosphodiester ODN (PO-ODN). In contrast to the effects of CpG-ODN 1826, PS-ODN of the CpG-ODN 2006 showed lower stimulatory activities than its PO-ODN. Using transient transfection, it was found that myeloid differentiation protein (MyD88) and tumor necrosis factor receptor-associated factor 6 are commonly required for activation of the TNF-alpha promoter by various CpG-ODNs with different potencies. These results strongly suggest a possibility to optimally activate the innate immune responses by modulating the potency of CpG-ODNs via sequence rearrangement and phosphorothioate backbone modification.     

Interferon-tau blocks the stimulatory effect of tumor necrosis factor-alpha on prostaglandin F2alpha synthesis by bovine endometrial stromal cells

Tumor necrosis factor-alpha (TNFalpha) has been shown to be a potent stimulator of prostaglandin (PG) F2alpha synthesis in bovine endometrial stromal cells. The aims of the present study were to determine the effect of interferon-tau (IFNtau) on TNFalpha-stimulated PGF2alpha synthesis and the intracellular mechanisms of TNFalpha and IFNtau action in the stromal cells. When cultured bovine stromal cells were exposed to TNFalpha (0.006-0.6 nM) for 24 h, the production of PGF2alpha and cyclooxygenase (COX)-2 gene expression were stimulated by TNFalpha (0.06-0.6 nM, P < 0.05). Moreover, a specific COX-2 inhibitor (NS-398; 5 nM) blocked the stimulatory effect of TNFalpha on PGF2alpha production (P < 0.05). Although IFNtau (0.03-30 ng/ml) did not stimulate basal PGF2alpha production in the stromal cells, it suppressed TNFalpha action in PGF2alpha production dose dependently (P < 0.05). Moreover, the stimulatory effect of TNFalpha (0.6 nM) on COX-2 gene expression was completely blocked by IFNtau (30 ng/ml; P < 0.05), although the gene expression of COX-2 was not influenced by IFNtau. The overall results indicate that the stimulatory effect of TNFalpha on PGF2alpha production is mediated by the up-regulation of COX-2 gene expression and suggest that one of the mechanisms of the inhibitory effect of IFNtau on luteolysis is the inhibition of TNFalpha action in PGF2alpha production in the stromal cells by the down-regulation of COX-2 gene expression stimulated by TNFalpha.     

Influence of purified recombinant human macrophage colony stimulating factor in mice

The endotoxin-resistant strain of mouse, C3H/Hej, was assessed for hematological responsiveness to multiple injections of high dosages of purified recombinant human macrophage colony stimulating factor (rhu-M-CSF). Mice were administered the rhu M-CSF i.p. at dosages of 40 micrograms per injection, 2 or 3 times per day for 4 days. This resulted in significant increases in circulating leukocytes compared to control mice given sterile pyrogen-free saline. Assessment of the marrow and spleen of these mice on the 5th day noted a significant reduction in the numbers of marrow hematopoietic progenitor cells, with no change in their cycling rates. In contrast, splenic granulocyte-macrophage and erythroid progenitor cell numbers were markedly increased and the cycling rates of these progenitors plus those of multipotential progenitors were significantly enhanced. Marrow and splenic early myeloid cells (blasts, promyelocytes, and myelocytes) and macrophages were increased, while marrow and splenic PMN were decreased. The results suggest that multiple injections of high dosages of rhu-M-CSF to previously untreated mice for a short period of time has a modest enhancing effect on blood leukocyte levels. This is associated with a shift of hematopoietic cell activity from the marrow to the spleen.     

Tissue sources of bone marrow colony stimulating factor

Possible tissue sources in C57BL mice of the serum factor stimulating colony formation in vitro by mouse bone marrow cells have been investigated. A reproducible technique employing batch chromatography on calcium phosphate gel was developed for the extraction and assay of material with colony stimulating activity from mouse tissues. Sixteen hematopoietic and non-hematopoietic tissues from C57BL mice were found to vary widely in their content of extractable activity. Characterisation of the colony stimulating factors (CSF's) from these tissues by assay of stepped concentrations of eluate showed that CSF's from most tissues were similar in chromatographic behavior, but all differed significantly from those of serum in being both more disperse and more firmly bound to calcium phosphate gel. Male submaxillary salivary gland gave the richest yield of CSF. CSF from this source displayed a greater dispersity on and affinity to calcium phosphate, a lower electrophoretic mobility and a smaller average sedimentation coefficient than that from any other source investigated. Colony morphology appeared to be identical for all tissue sources investigated.     

Studies on colony formation in vitro by mouse bone marrow cells. II. Action of colony stimulating factor

An analysis was made of some of the processes involved in the stimulation by colony stimulating factor (CSF) of cluster and colony formation by mouse bone marrow cells in agar cultures in vitro. Colony formation was shown to be related to the concentration and not the total amount of CSF. The concentration of CSF determined the rate of new cluster initiation in cultures and the rate of growth of individual clusters. Colony growth depleted the medium of CSF suggesting that colony cells may utilise CSF during proliferation. Bone marrow cells incubated in agar in the absence of CSF rapidly died or lost their capacity to proliferate and form clusters or colonies. CSF appears (a) to be necessary for survival of cluster-and colony-forming cells or for survival of their proliferative potential, (b) to shorten the lag period before individual cells commence proliferation and (c) to increase the growth rate of individual clusters and colonies.