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.     

Continuous activation of primitive hematopoietic cells in long-term human marrow cultures containing irradiated tumor cells.

Human hematopoietic cells can be maintained in vitro for many weeks in the absence of exogenously provided hematopoietic growth factors if an adequate stromal cell containing adherent layer is present. We have now extended the use of this type of long-term culture (LTC) system to create a model of perturbed hematopoiesis in which human tumor cells that constitutively produce a variety of factors are co-cultured together with normal human marrow cells. In the present study, we used the human bladder carcinoma cell line (5637) because these cells were known to produce not only a variety of factors active directly on hematopoietic cells but also factors that can stimulate hematopoietic growth factor production by human marrow stromal cells. Analysis of mRNA extracted from the adherent layer and measurement of growth factor bioactivity in the medium of established LTC of human marrow containing irradiated 5637 cells, showed increased levels of interleukin-1 and -6, as well as granulocyte and granulocyte-macrophage colony-stimulating factor production by comparison to control cultures. As in normal cultures, high proliferative potential clonogenic hematopoietic cells were found almost exclusively in the adherent layer of these co-cultures, but these primitive cells were maintained in a state of continuous turnover, in contrast to control cultures where the same cell types showed the expected oscillation between a quiescent and a proliferating state following each weekly change of the medium. A similar perturbation of primitive progenitor cycling was achieved by adding medium conditioned by 5637 cells twice a week to otherwise normal LTC. The presence of irradiated 5637 cells in the LTC or the addition of 5637 conditioned medium also resulted in modest (2- to 3-fold) but sustained increases in the total hematopoietic progenitor population, as well as in the final output of terminally differentiated granulocytes and macrophages. These findings indicate that primitive hematopoietic cells in LTC can be kept in a state of continuous activation for many weeks by appropriate endogenous or exogenous hematopoietic growth factor provision and that this does not necessarily lead either to their rapid exhaustion or to a large amplification in output of mature progeny.     

Human long-term bone marrow cultures in aplastic anemia

Long-term bone marrow cultures (LTMC) were initiated with marrow from five normal subjects and eight patients with aplastic anemia (AA). Near confluent to confluent adherent layers developed in all cultures from normal subjects and AA patients. When present, the 'cobblestone' areas in LTMC from AA subjects were smaller than those observed in the LTMC from normal subjects. The decline in total and viable cell numbers in the LTMC was similar for both normal subjects and AA patients. Granulocyte-macrophage colony-forming units (CFU-gm) were present in nonadherent cells (NAC) from normal LTMC for a mean of 5.2 weeks. CFU-gm were present in the NAC of only two of the eight AA cultures for one week. The absent or small 'cobblestone' areas and the absence of CFU-gm production in AA-LTMC suggest a decrease in the reproductive potential of adherent hematopoietic stem cells, which may be the result of either an abnormal hematopoietic stem cell or an abnormal stromal microenvironment or both.     

Effect of prostaglandin E2 on PMA-induced macrophage differentiation

Major trauma such as severe bums and extensive surgery could result in accelerated macrophage differentiation and hyperactivation causing an excessive release of proinflammatory cytokines and prostaglandin E2 (PGE2) with consequent severe impairment of immunologic reactivity. HL-60 cells stimulated with phorbol 12-myristate 13-acetate (PMA) have been used as a model to asses the PGE2 role in the macrophage differentiation observed after major trauma. Cell adhesion, matrix metalloproteinase-9 (MMP-9) and tumor necrosis factor-alpha (TNF-alpha) production were measured after 24 h of PMA treatment in the presence of PGE2 (1 nM - 1 microM). PGE2 increased both the PMA-induced cell adhesion and MMP-9 production via EP2/EP4 receptors while it had no effect on the induced TNF-alpha release. The cAMP/PKA pathway, usually linked to EP2/EP4 activation, was not involved in the phenomenon, suggesting that an alternative signalling pathway could be linked to a PKC-activated enzyme. In fact PGE2 activity was partially inhibited by Wortmannin, a phosphoinositide-3 kinase (PI-3K) inhibitor indicating that PGE2 act as a co-factor able to increase macrophage differentiation in vitro via a PI-3K dependent pathway that could be also involved in the immunosuppression observed in the aftermath of trauma.     

Synthesis and inhibitory effects of pinosylvin derivatives on prostaglandin E2 production in lipopolysaccharide-induced mouse macrophage cells

A series of natural stilbenoids, pinosylvin and its derivatives, were synthesized and evaluated for the inhibitory activity of prostaglandin E(2) production in lipopolysaccharide-induced RAW 264.7 cells. Potential inhibitors, including 3,5-dimethoxy-trans-stilbene and 3-hydroxy-5-benzyloxy-trans-stilbene, have been newly identified, and thus providing chemical leads for the further development of anti-inflammatory or cancer chemopreventive agents.     

2-Carba cyclic phosphatidic acid inhibits lipopolysaccharide-induced prostaglandin E2 production in a human macrophage cell line

Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator that contains a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. Using mouse models for multiple sclerosis (cuprizone-induced demyelination and experimental autoimmune encephalomyelitis) and traumatic brain injury, we revealed that cPA and its metabolically stabilized cPA derivative, 2-carba-cPA (2ccPA), have potential to protect against neuroinflammation. In this study, we investigated whether 2ccPA has anti-inflammatory effect on peripheral immune function or not using inflammation-induced macrophages-like cell line, THP-1 monocytes differentiated by phorbol 12-myristate 13-acetate (PMA). Lipopolysaccharide (LPS)-stimulated THP-1 cells were found to have higher expression of the mRNAs of several inflammation-related cytokines and of the enzyme cyclooxygenase-2 (Cox-2); however, when THP-1 cells were stimulated by LPS in the presence of 2ccPA, the increase in the expression of pro-inflammatory cytokine and Cox-2 mRNA was attenuated. 2ccPA treatment also decreased the amount of prostaglandin E2 (PGE2) produced by LPS-stimulated THP-1 cells and decreased expression of the mRNA of prostaglandin E receptor 2 (EP2, PTGER2), a PGE2 receptor that mediates inflammation. These results indicate that 2ccPA has anti-inflammatory properties.     

Major role of adipocyte prostaglandin E2 in lipolysis-induced macrophage recruitment

Obesity induces accumulation of adipose tissue macrophages (ATMs), which contribute to both local and systemic inflammation and modulate insulin sensitivity. Adipocyte lipolysis during fasting and weight loss also leads to ATM accumulation, but without proinflammatory activation suggesting distinct mechanisms of ATM recruitment. We examined the possibility that specific lipid mediators with anti-inflammatory properties are released from adipocytes undergoing lipolysis to induce macrophage migration. In the present study, we showed that conditioned medium (CM) from adipocytes treated with forskolin to stimulate lipolysis can induce migration of RAW 264.7 macrophages. In addition to FFAs, lipolytic stimulation increased release of prostaglandin E₂ (PGE₂) and prostaglandin D₂ (PGD₂), reflecting cytosolic phospholipase A₂ α activation and enhanced cyclooxygenase (COX) 2 expression. Reconstituted medium with the anti-inflammatory PGE₂ potently induced macrophage migration while different FFAs and PGD₂ had modest effects. The ability of CM to induce macrophage migration was abolished by treating adipocytes with the COX2 inhibitor sc236 or by treating macrophages with the prostaglandin E receptor 4 antagonist AH23848. In fasted mice, macrophage accumulation in adipose tissue coincided with increases of PGE₂ levels and COX1 expression. Collectively, our data show that adipocyte-originated PGE₂ with inflammation suppressive properties plays a significant role in mediating ATM accumulation during lipolysis.     

Residual radiation injury exhibited in long-term bone marrow cultures

Residual radiation injury was demonstrated in long-term primary cultures of mouse bone marrow. Control cultures underwent three phases of hematopoietic activity as distinguished by initial establishment, steady high (plateau) production of granulocytes, and gradual decline. Irradiation with 50, 300, or 550 rads, given at the end of the initial phase, did not prevent any culture flasks from entering the plateau phase. However, actual production levels and the time they were maintained varied inversely with the radiation dose so that the accumulated postradiation cell production corresponded to an exponential dose-response relationship at any time after treatment. The accumulated cell productions were found to be similar in all groups when expressed by the number of stem cell doublings necessary to produce them. The findings cannot be explained by reproductive cell death and are consistent with the notion of a limited division capacity in hematopoietic stem cells.     

Production of colony stimulating factor in long-term bone marrow cultures

Previous studies have shown no detectable colony-stimulating factor (CSF) in media harvested from long-term bone marrow cultures. In the present experiments supernatants from long-term cultures established in three laboratories were assayed for CSF by colony assay and by radioimmunoassay (RIA). Most samples were devoid of biologic activity but all contained CSF as judged by RIA. Biologic activity was found in the majority of samples after diafiltration to remove low molecular weight inhibitors or 5-fold concentration by ultrafiltration. Samples that remained inactive in the colony assay were subjected to gel filtration on Sephadex G-150 to remove potential high molecular weight inhibitors. Biologic activity remained lower than that by RIA in two of three samples tested. Thus, most long-term cultures appear to contain biologically active CSF but this activity is masked by various types of inhibitors. In addition some media appear to contain material that is only detected by RIA.     

Mast cell products stimulate collagenase and prostaglandin E production by cultures of adherent rheumatoid synovial cells

Mast cells were purified from histologically-confirmed dog mastocytomas and extracted for whole mast cell products (MCP). When added to cultures of human adherent rheumatoid synovial cells MCP induced a 50-400 fold increase in prostaglandin E synthesis and a 10-50 fold stimulation of collagenase production. The mast cell stimulatory factor has not been identified and was not due to histamine, heparin or prostaglandin E. These results indicate a novel way in which mast cells might interact with synovial cells to promote the production of inflammatory mediators and proteolytic enzymes which might contribute to connective tissue degradation.     

Condensed E. coli cultures for highly efficient production of proteins containing unnatural amino acids

Current biosynthetic methods for producing proteins containing site-specifically incorporated unnatural amino acids are inefficient because the majority of the amino acid goes unused. Here we present a universal approach to improve the efficiency of such processes using condensed Escherichia coli cultures.     

Pre-irradiation of tissue culture flasks leads to diminished stem and progenitor cell production in long-term bone marrow cultures

Abstract. Empty plastic tissue culture flasks were exposed to X-irradiation doses of 0.3–10.0 Gy, prior to the establishment of long-term bone marrow cultures. During the course of a 10 week culture period, all irradiated plastic flasks exhibited a dramatic decrease in the number of both haemopoietic stem cells and myeloid progenitor cells, in the non-adherent layer, when compared with controls. This decrease was not due to a decrease in the number of non-adherent cells produced. Histological examination of non-adherent cells showed an increase in mature granulocytic cells with few blast cells. Morphologically, the adherent layers of irradiated flasks demonstrated a delay in appearance or absence of fat cell production. X-irradiation of glass tissue culture flasks had no deleterious effect.     

Renewal of hematopoietic stem cell clones in long-term bone marrow cultures

In long-term cultures of murine bone marrow, clonal succession of hemopoietic cells was observed as measured by karyologic analysis. There were high oscillations in self-renewal of CFUs in the cultures. A close correlation between the CFUmix karyotype and mitotic non-adherent cells in culture (but not between these cell types and CFUs) was revealed.     

Microglia-specific expression of microsomal prostaglandin E2 synthase-1 contributes to lipopolysaccharide-induced prostaglandin E2 production

Microsomal prostaglandin E2 synthase (mPGES)-1 is an inducible protein recently shown to be an important enzyme in inflammatory prostaglandin E2 (PGE2) production in some peripheral inflammatory lesions. However, in inflammatory sites in the brain, the induction of mPGES-1 is poorly understood. In this study, we demonstrated the expression of mPGES-1 in the brain parenchyma in a lipopolysaccharide (LPS)-induced inflammation model. A local injection of LPS into the rat substantia nigra led to the induction of mPGES-1 in activated microglia. In neuron-glial mixed cultures, mPGES-1 was co-induced with cyclooxygenase-2 (COX-2) specifically in microglia, but not in astrocytes, oligodendrocytes or neurons. In microglia-enriched cultures, the induction of mPGES-1, the activity of PGES and the production of PGE2 were preceded by the induction of mPGES-1 mRNA and almost completely inhibited by the synthetic glucocorticoid dexamethasone. The induction of mPGES-1 and production of PGE2 were also either attenuated or absent in microglia treated with mPGES-1 antisense oligonucleotide or microglia from mPGES-1 knockout (KO) mice, respectively, suggesting the necessity of mPGES-1 for microglial PGE2 production. These results suggest that the activation of microglia contributes to PGE2 production through the concerted de novo synthesis of mPGES-1 and COX-2 at sites of inflammation of the brain parenchyma.     

Regulation of agonist-induced prostaglandin E1 versus prostaglandin E2 production. A mass analysis.

Prostaglandin E1 (PGE1) and prostaglandin E2 (PGE2), derived by enzymatic oxidation of cellular dihomogammalinolenic acid (DHLA) and arachidonic acid (AA), respectively, have diverse and, at times, distinct biological actions. It has been suggested that PGE1 specifically inhibits a variety of inflammatory processes, and, in light of the potential therapeutic benefit of PGE1 and its fatty acid precursor in inflammatory disorders, there is growing interest in the biochemical mechanisms which determine the balance between PGE1 and PGE2 synthesis. Metabolic studies in this area have been hampered by the difficulties in measuring the extremely small masses of these prostaglandins which are generated in cell culture systems. We studied the regulation of PGE1 versus PGE2 synthesis using an essential fatty acid-deficient, PGE-producing, mouse fibrosarcoma cell line, EFD-1. Because EFD-1 cells contain no endogenous AA or DHLA, we were able to replete the cells with AA and DHLA of known specific activities; thus, the mass of both cellular AA and DHLA, and synthesized PGE1 and PGE2, could be accurately determined. The major finding of this study is that production of PGE2 was highly favored over production of PGE1 due to preferential incorporation of AA versus DHLA into, and release from, the total cellular phospholipid pool. Further, we correlated the selective release of AA versus DHLA from total cellular phospholipids with the selective incorporation of AA versus DHLA into specific phospholipid pools. In addition, we showed that conversion of DHLA to AA by delta 5 desaturase was enhanced by increasing the cellular mass of n-6 fatty acids and by increasing the cell proliferative activity. Together, these results indicate that the relative abundance of PGE2 versus PGE1 in vivo is not merely a function of the relative abundance of AA versus DHLA in tissues, but also relates to markedly different cellular metabolism of these two fatty acids.     

Specific production of prostaglandin E by human amnion in vitro

Prostaglandin production by intra-uterine human tissues has been investigated using a method of tissue superfusion. Tissues were obtained at elective Caesarean section and after spontaneous vaginal delivery. It was found that all the tissues studied (amnion, chorion, decidua and placenta) produced more prostaglandin E (PGE) and 13,14-dihydro-15-keto-prostaglandin F (PGFM — the major circulating metabolite of prostaglandin F) than prostaglandin F (PGF). Amnion produced significantly more PGE (but not PGF or PGFM) than any other tissue. Prostaglandin production by each tissue was similar whether it was taken at elective Caesarean section or after spontaneous vaginal delivery.     

Regulation of bone marrow cell survival in short-term cultures: a new macrophage function

The involvement of macrophages (M phi) in the regulation of bone marrow (BM) cell survival in short-term cultures was studied. We developed a system to measure the survival of fresh BM cells in vitro, by evaluating 111indium (111In) release from prelabeled BM cells. 111In release was proportional to cell death and inversely related to the number of trypan blue excluding cells. Upon 24 hr of culture in conventional medium, more than 50% of BM cells died. In order to investigate whether BM cell death could be reduced by coculture with other cell types, 111In-labeled BM cells were incubated for 24 hr with peritoneal M phi, thymocytes (THY), or polymorphonuclear cells (PMN) and then assayed for their survival. We found that coculture of BM cells with M phi dramatically increased BM survival, whereas THY or PMN consistently failed to enhance BM survival. The ability to promote BM cell survival, here designated nurse activity, represented a novel function of M phi and was further characterized. The stage of activation of M phi did not influence their nurse activity, since M phi elicited in vivo by proteose-peptone, thioglycollate, or Bacillus Calmette-Guérin, as well as resident M phi unstimulated or activated in vitro with lipopolysaccharide, equally sustained survival of BM cells. BM-derived M phi (adherent cells from BM cultures maintained in 20% L-cell-conditioned medium for 14 days) were equally effective in exerting nurse activity. Moreover, nurse activity was also exerted across the histocompatibility barriers. Supernatants from M phi cultures or killed M phi were ineffective. We propose that the nurse effect of M phi on BM is a primitive function that may play an important role in the development of the hemopoietic system.     

Enhancement of vitamin E production in sunflower cell cultures

The most biologically active component of vitamin E, -tocopherol, is synthesized in its most effective stereoisomeric form only by photosynthetic organisms. Using sunflower cell cultures, a suitable in vitro production system of natural -tocopherol was established. The most efficient medium was found to be MS basal medium with naphthaleneacetic acid and 6-benzylaminopurine with the addition of casaminoacids and myo-inositol. Culture feeding experiments using biosynthetic precursors showed that -tocopherol production improved by 30% when homogentisic acid was used. Interestingly, time-course experiments with sunflower suspension cultures showed a possible increase of 78% in -tocopherol production when using cultures of longer subculture intervals. Compared to the starting plant tissue, an overall 100% increase of -tocopherol was reached by these sunflower cell cultures.