Rapid activation of ornithine decarboxylase by mitogenic (but not by nonmitogenic) ligands in human T lymphocytes

The T cell mitogens, concanavalin A and the monoclonal antibody OKT3, cause a rapid activation of ornithine decarboxylase (ODC) activity in human T lymphocytes, maximal within 10 min of mitogen addition. Nonmitogenic ligands to T cell surface structures do not induce ODC. The enzyme induction is dependent upon an intact mobility of ligand-receptor complex, requires a functioning energy metabolism, but is independent of de novo protein synthesis. The early induction of pre-existing ODC molecules appears to be specifically linked to the initiation of T lymphocyte proliferation.     

MODIFIED RESPONSES TO PGE2 IN POLYAMINE BIOSYNTHESIS BY T LYMPHOCYTES OF GASTRIC- AND CONJUNCTIVA BASAL CELL-CARCINOMA PATIENTS

The response to Prostaglandin (PG) E₂ of T cells from gastric carcinoma (GC)- and conjunctiva-basal cell carcinoma (conjunctiva-BCC)-bearing patients has been studied in relation to polyamine metabolism. Polyamines are crucial cofactors in cell growth as well as differentiation and many works report that lymphocyte spermine (SP), spermidine (SPD) and putrescine (PUT) levels may be related to tumor proliferation. The present work aims to detect the basal and PGE₂ induced concentrations of these polyamines and cAMP, ornithine decarboxylase (ODC), spermine N1-acetyltransferase (SAT) activities of T lymphocytes drawn from patients suffering from GC and conjunctiva-BCC since many carcinomas are characterized by high levels of PGE₂. Data obtained from lymphocytes of neoplastic subjects were compared with those derived from PGE₂-treated control lymphocytes. Results highlight a very significant increase of all the polyamine metabolites in PGE₂-treated T cells from neoplastic patients in respect to the untreated and PGE₂-treated control lymphocytes. Therefore, it is conceivable that the PGE₂ content increase, often occurring during the epithelial tumour development, may contribute, through enhancement of polyamine metabolism, to tumor progression.     

MODIFIED RESPONSES TO PGE2 IN POLYAMINE BIOSYNTHESIS BY T LYMPHOCYTES OF GASTRIC- AND CONJUNCTIVA BASAL CELL-CARCINOMA PATIENTS

The response to Prostaglandin (PG) E₂ of T cells from gastric carcinoma (GC)- and conjunctiva-basal cell carcinoma (conjunctiva-BCC)-bearing patients has been studied in relation to polyamine metabolism. Polyamines are crucial cofactors in cell growth as well as differentiation and many works report that lymphocyte spermine (SP), spermidine (SPD) and putrescine (PUT) levels may be related to tumor proliferation. The present work aims to detect the basal and PGE₂ induced concentrations of these polyamines and cAMP, ornithine decarboxylase (ODC), spermine N1-acetyltransferase (SAT) activities of T lymphocytes drawn from patients suffering from GC and conjunctiva-BCC since many carcinomas are characterized by high levels of PGE₂. Data obtained from lymphocytes of neoplastic subjects were compared with those derived from PGE₂-treated control lymphocytes. Results highlight a very significant increase of all the polyamine metabolites in PGE₂-treated T cells from neoplastic patients in respect to the untreated and PGE₂-treated control lymphocytes. Therefore, it is conceivable that the PGE₂ content increase, often occurring during the epithelial tumour development, may contribute, through enhancement of polyamine metabolism, to tumor progression.     

Immunosuppressive effects of (2R,5R)-6-heptyne-2,5-diamine an inhibitor of polyamine synthesis: I. Effects on mitogen-induced immunoglobulin production in human cultured lymphocytes.

The consequences of specific inhibition of polyamine biosynthesis by (2R,5R)-6-heptyne-2,5-diamine (MAP) a potent inhibitor of L-ornithine decarboxylase (ODC), on immunoglobulin (Ig) production were studied in cultured human peripheral blood lymphocytes stimulated with pokeweed mitogen (PWM). MAP inhibits the usual PWM-induced increase of polyamine (putrescine, spermidine and spermine) concentrations and reduces concomitantly cell replication. In parallel with these biochemical effects, IgG and IgM production are diminished, a 95% decrease being observed at 100 microM MAP concentration. That the suppressive effects of the ODC inhibitor result from polyamine deficiency, and not from unrelated pharmacological effects, is demonstrated by the restoration of normal Ig production when 10 microM putrescine or spermidine are added to the culture medium. These findings established that the cellular immunological response can be affected by specific inhibition of polyamine biosynthesis and deserve further consideration both under in vitro and in vivo conditions.     

Renal polyamine metabolism in rats with renovascular hypertension

A standardized stenosis was induced by applying a silver clip around the left renal artery in male rats. This resulted in arterial hypertension within 10 days (as determined by increase in heart weight). Ornithine decarboxylase (ODC) activity was determined in the right (untouched) kidney, the left kidney, and the adrenal glands 1 day, 10 days, and 3 months after the operation. There was no difference in ODC activity in the right kidney of the operated animals when compared with matched controls. In the left kidney (with artery stenosis), ODC activity decreased to 40% after 1 day. A partial recovery was seen after 10 days (ODC activity 70% of normal), and after 3 months ODC activity had normalized. Removal of the clip 1 day prior to killing induced in the 3-month group a more than two-fold increase in ODC activity in the previously clipped kidney; ODC activity in the contralateral kidney was not affected. Only minor changes in ODC activity occurred in the adrenal glands following the operation. Contents of putrescine and spermidine were increased in the left (stenotic) kidney, and after clip removal, also in the right (untouched) kidney. Our observations thus indicate that alterations in renal blood flow are rapidly followed by changes in ODC activity. Contents of putrescine, spermidine and spermine seemed to a great extent to be independent of the ODC activity.     

Control of human T cell proliferation by platelet-activating factor

Platelet-activating factor, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF), is a membrane phospholipid with immunomodulatory functions. We studied the influence of PAF on mitogen-stimulated proliferation of human peripheral blood mononuclear cells (PBMC), purified T cells and T cell subsets. High concentrations of PAF suppressed the proliferation of all cell populations studied (44% mean inhibition with 5 microM PAF and 78% inhibition with 10 microM PAF). In contrast, the deacetylated metabolite of PAF, lyso-PAF, had no effect on proliferation at micromolar concentrations. Lower, and presumably physiologically relevant, concentrations of PAF (10(-14) to 10(-8) M) stimulated a small increase in the proliferation of unfractionated T cells. When T cells were fractionated into CD4+ and CD8+ subsets, a difference in sensitivity to PAF was observed. PAF stimulated a modest, yet statistically significant, increase in the proliferation of CD4+ T cells at concentrations ranging from 10(-14) to 10(-10) M, while either having no effect or inhibiting the proliferation of CD8+ cells across the entire concentration range. Addition of indomethacin to the cultures further enhanced CD4+ proliferation, possible due to the blockade of PAF-induced PGE2 production by monocytes. The PAF receptor antagonist BN 52021 did not block the PAF effects in this system, and the PAF receptor antagonist SRI 63-675 caused a dose dependent inhibition of T cell subset proliferation. These findings suggest that while high concentrations of PAF suppress T cell proliferation, low concentrations selectively stimulate proliferation of the CD4+ subset, an effect which is partially counteracted by PAF-induced monocyte PGE2 production.     

Trichomonas vaginalis polyamine metabolism is linked to host cell adherence and cytotoxicity

Trichomonas vaginalis secretes putrescine that is readily detected in vaginal secretions. We wanted to examine the effect of decreased putrescine synthesis by inhibition of ornithine decarboxylase (ODC) on T. vaginalis. One reason is because inhibition of Tritrichomonas foetus ODC results in growth arrest, destruction of hydrogenosomes, and decreased amounts of hydrogenosomal enzymes. Treatment of T. vaginalis T016 with >/=20 mM 1,4-diamino-2-butanone (DAB) to inhibit ODC resulted in growth arrest, which was reversed by addition of exogenous putrescine. No similar reversal of growth arrest was achieved with the polyamines spermine or spermidine or with iron. Electron microscopic examination of control versus DAB-treated trichomonads did not reveal any adverse effects on the number and integrity of hydrogenosomes. Further, the adhesins AP65, AP51, and AP33 mediating binding to immortalized vaginal epithelial cells (VECs) share identity to enzymes of the hydrogenosome organelle, and there was no difference in amounts of adhesins between control versus DAB-treated T. vaginalis parasites. Likewise, similar patterns and extent of fluorescence were evident for the prominent AP65 adhesin. Surprisingly, DAB treatment increased by 4- to 20-fold above untreated trichomonads handled identically the level of adherence mediated by adhesins. Interestingly, the enhanced attachment to VECs was reversed by exogenous putrescine added to DAB-treated trichomonads. Equally noteworthy was that DAB-treated T. vaginalis with enhanced adherence did not possess the previously reported ability to kill host cells in a contact-dependent fashion mediated by cysteine proteinases, and total cysteine proteinase activity patterns were identical between control and DAB-treated trichomonads. Overall, these data suggest that polyamine metabolism and secreted putrescine are linked to host cell adherence and cytotoxicity.     

PGE2 和 NF-κB信号途径在骨再生中的相互作用

目的　本研究旨在利用MC3T3-E1 成骨细胞株与大鼠骨折模型来研究骨折愈合过程中PGE2和NF-κB信号途径的相互作用。 方法　利用PGE2与NF-κB 抑制剂 BAY 11-7082处理MC3T3-E1成骨细胞和大鼠骨折模型，采用碱性磷酸酶活性测定、Western blot 分析、EMSA分析以及ELISA测定等研究手段，研究PGE2、NF-κB、BMP-7、 Id2以及SOD2在骨再生中的作用。 结果　利用10 μmol/l PGE2处理MC3T3-E1细胞10 min, 30 min 和2h后，能够显著地促进成骨细胞增殖与分化，当细胞用5 μmol/L BAY 11-7082处理后，PGE2诱导作用受到抑制，表明 PGE2 增加ALP的表达是通过NF-κB 途径来介导。局部注射NF-κB 抑制剂后 PGE2 的生物合成明显减少；此外，抑制骨折部位ALP的活性，在骨折的损伤修复过程中，NF-κB、BMP-7以及SOD2 的表达均明显上升，而Id2的表达明显下降；加入NF-κB活性抑制剂后，BMP-7与Id2的表达恢复到正常水平，而SOD2的表达没有改变。　结论 　PGE2能够同时通过抑制Id2细胞因子和激活NF-κB信号途径诱导成骨细胞分化。     

The inhibition of T-lymphocyte proliferation by fatty acids is via an eicosanoid-independent mechanism.

Eicosanoids, in particular prostaglandin E2 (PGE2), are potent inhibitors of a number of immune responses, including lymphocyte proliferation. We have previously shown that fatty acids, especially polyunsaturated fatty acids (PUFA), inhibit mitogen-stimulated proliferation of lymphocytes. One mechanism by which fatty acids could exert their inhibitory effect is via modulation of eicosanoid synthesis. This possibility was investigated in the present study. PGE2 concentrations in the medium taken from lymphocytes cultured in the presence of a range of different fatty acids did not correlate with the inhibitory effects of the fatty acids upon lymphocyte proliferation. Although PGE2 at concentrations above 10 nM caused inhibition of lymphocyte proliferation, PGE2 at the concentration measured in lymphocyte culture medium (0.3-4 nM) was not inhibitory. PGE3 did not inhibit lymphocyte proliferation, except at high concentrations (greater than 250 nM). The maximal inhibition of proliferation caused by PGE2 or PGE3 was less than the inhibition caused by each of the fatty acids except myristic or palmitic acids. Inclusion of inhibitors of phospholipase A2, cyclo-oxygenase or lipoxygenase in the culture medium did not prevent the fatty acids from exerting their inhibitory effect on lymphocyte proliferation. The eicosanoids present in lymph node cell cultures originate from macrophages rather than lymphocytes. Depletion of macrophages from the cell preparation by adherence did not prevent fatty acids from inhibiting proliferation. Proliferation of thoracic duct lymphocytes, which are devoid of macrophages, is inhibited by fatty acids to a similar extent as proliferation of lymph node lymphocytes. These observations provide convincing evidence that the inhibition of lymphocyte proliferation by fatty acids is independent of the production of eicosanoids. Therefore, other mechanisms must be investigated if the effect of fatty acids upon lymphocyte proliferation is to be understood at a biochemical level.     

HIV infection of monocytes inhibits the T-lymphocyte proliferative response to recall antigens, via production of eicosanoids.

Human monocytes infected in vitro with human immunodeficiency virus (HIV) soon after adherence to plastic substrate demonstrated a significantly decreased ability to restimulate autologous immune T-lymphocyte proliferation after exposure to soluble (tetanus toxoid) and particulate [herpes simplex virus (HSV)] antigen. Incubation with the cyclo-oxygenase inhibitor, indomethacin (2-5 microM), prevented inhibition of antigen-stimulated lymphocyte proliferation. The inhibitory activity was identified in ultrafiltrates containing the low molecular weight fraction (less than 3000 MW) of supernatants from HIV-infected monocyte cultures. This activity was significantly and markedly reduced in similar ultrafiltrates prepared from indomethacin-treated cultures. Increased concentrations of prostaglandin E2 (PGE2) were detected in ultrafiltrates from HIV-infected monocyte cultures compared with uninfected cultures and cultures preincubated with indomethacin. Ultrafiltrates were inhibitory when added during the presentation of antigen to T lymphocytes but not when removed from monocyte cultures prior to the addition of lymphocytes. In addition, ultrafiltrates inhibited antigen-stimulated lymphocyte proliferation and PHA-induced lymphocyte proliferation to the same extent. These data indicate that cyclo-oxygenase products of arachidonic acid, including PGE2, are produced in excess by HIV-infected monocytes and that PGE2 and perhaps other cyclo-oxygenase products are implicated in the inhibition of antigen-stimulated lymphocyte proliferation via a direct effect on T lymphocytes.     

Stimulation of cell division in mouse fibroblast line 3T3 by an extract derived from Triticum vulgare

The ability of an extract derived from Triticum vulgare, the common wheat plant, to stimulate cellular proliferation of mouse 3T3 fibroblast cells was investigated. Cellular response to Triticum extract (TE) was most evident in sparse cultures made quiescent by growing cells on low concentrations (0.6%) of calf serum. The growth-promoting activity in the extract was lost after dialysis but was resistant to heat treatment and digestion with trypsin or chymotrypsin, suggesting a low-molecular-weight non-protein substance(s). Growth-curve experiments showed that TE was capable of supporting continuous cell division. Cellular proliferation showed a dose-dependent response in the range of 2%-10% TE, and addition of 10% TE to cell culture medium caused a level of cell-growth stimulation approximately 72% that of 20 ng/ml fibroblast growth factor (FGF). Measurement of ornithine decarboxylase (ODC) activity of 3T3 cells after addition of 10% TE showed a significant rise in the specific activity of the enzyme.     

A new immunomodulator, LF 1695--I. In vitro effects on the T lymphocyte lineage in man

A new immunomodulator, LF 1695, was analyzed in vitro for its capacity to induce T cell markers (HTLA and OKT3, 4 and 8 antigens), to enhance the mitogen-induced lymphocyte proliferation, and to increase the Con A-induced suppressor activity. The inductive activity was compared with that of thymic hormone (thymosin fraction V). LF 1695 was capable of significantly augmenting the percentages of HTLA+ cells (10-15%) and of OKT 3+,T4+, or T8+ cells (13-28%) in human bone marrow prothymocytes. Maximum induction was observed at the concentration of 0.5 microgram/ml. In addition, LF 1695 significantly augmented the proliferation of human peripheral blood lymphocytes stimulated by mitogens (Concanavalin A, phytohemagglutinin, pokeweed mitogen and phorbol myristate acetate). LF 1695 also increased the Con A-induced suppressor activity of human lymphocytes. These data indicate that LF 1695 is active both on T cell precursors and on more mature T cells.     

Monocyte factors modulate in vitro T-lymphocyte mitogenesis in protein malnutrition.

This study investigated changes in T-lymphocyte mitogenesis and immunoregulatory cytokines during protein malnutrition. In vitro T cell response to concanavalin A was compared among protein deprived (PD), energy restricted pair fed control (PF), and ad libitum control (C) rabbits. Cell cultures were supplemented with crude monocyte supernatants (CMS) from PD, PF or C animals at either 1% or 8% final concentration in culture. Prostaglandin E2 (PGE2) concentration of unstimulated or stimulated lymphocyte culture supernatants and CMS was determined. Lymphocyte cultures from PD, PF and C animals had enhanced 3H-thymidine incorporation when supplemented with C and/or PF derived CMS. Addition of 8% CMS from PD rabbits inhibited proliferation below levels observed in mitogen-only stimulated groups in all cultures. At the 1% concentration, inhibition was seen in PD and C derived cells cultures and modest enhancement was seen in PF cultures. PGE2 concentration in supernatants from stimulated and unstimulated lymphocyte cultures from PD rabbits were higher than in C and PF cell cultures. These results suggest (a) that under appropriate culture conditions lymphocytes from PD donors are capable of enhanced proliferation and (b) that depressed T cell mitogenesis observed in protein malnutrition may reflect alterations in immunoregulatory signals. The role of interleukin 1 (IL-1) and PGE2 in the modulation of this response is discussed.     

The combined effect of prostaglandin E2 and interleukin 1 on interleukin 2 production and lymphocyte proliferation

In this study the combined effect of prostaglandin E2 (PGE2) and interleukin-1 (IL-1) on the proliferation of concanavalin A (Con A) stimulated mouse spleen lymphocytes was studied. IL-1 in concentrations ranging from 0.5 to 5.0 U/ml consistently and significantly enhanced Con A activity. However, to be effective, IL-1 needed to be added at the time of initiation of the culture. If added 24 h later, IL-1 failed to enhance the proliferative response. PGE2 at concentrations ranging from 0.1 to 5.0 ng/ml effectively inhibited or antagonized this enhancing effect of IL-1, with the majority of this IL-1 augmentation abrogated by 5.0 ng/ml PGE2. Unlike IL-1, PGE2 was as effective if added 24 h after initiation of the culture as if added simultaneously with IL-1. PGE2 was also found to markedly suppress the enhanced production of IL-2 resulting from the addition of IL-1 to Con A stimulated lymphocytes, however, the amount of IL-2 produced in the cultures containing both IL-1 and PGE2 was always greater than that produced in the cultures which contained only PGE2. This finding indicates that IL-1 could partially reverse or antagonize the suppressive effect of PGE2 on IL-2 production. In addition, PGE2 at concentrations of 1.0 and 5.0 ng/ml was also found to inhibit the proliferation of IL-2 stimulated cultured T lymphocytes, but by only about 15-20%. The addition of IL-1 to these cultured T cells neither altered the response of the culture T cells to IL-2 nor altered the sensitivity of these cells to PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha-difluoromethylornithine resistance in Leishmania donovani is associated with increased ornithine decarboxylase activity

The promastigote form of Leishmania donovani is sensitive to growth inhibition by DL-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC), the first enzyme of the polyamine biosynthetic pathway, with an EC50 value of approximately 30 microM. Exposure of a wild type (DI700) cell population to gradually increasing concentrations of DFMO resulted in the selection of a strain of Leishmania, DFMO-10, which was capable of proliferating in 10 mM DFMO. DFMO-10 cells possessed an EC50 value for DFMO greater than 4 mM, and were cross-resistant to alpha-methylornithine, alpha-monofluoromethyl-3,4-dehydroornithine methyl ester, and delta-methyl-acetylenic putrescine, three other inhibitors of ODC activity. DI700 and DFMO-10 cells accumulated and/or transported [3H]DFMO and a spectrum of basic, neutral, and acidic amino acids at comparative rates. However, the DFMO-resistant Leishmania, if suspended in culture medium in the absence of DFMO for several days, expressed up to 15-fold greater levels of ODC activity than did wild-type cells. The overexpressed ODC in mutant cells appeared kinetically normal, since the ODC activities from DI700 and DFMO-10 cells possessed similar apparent Km values for ornithine and were equally sensitive to inactivation by DFMO. Incubation of extracts of DFMO-10 cells, but not of wild-type parental cells, with [3H]DFMO for 1 h resulted in the labeling of a polypeptide, presumably ODC, which migrated with a molecular weight of 76,000 +/- 4000 on SDS-gel electrophoretograms. As a consequence of the elevated ODC activities, the levels of putrescine in mutant cells released from DFMO exposure were also elevated by about 15-fold over those of wild-type cells, although spermidine levels in DI700 and DFMO-10 cells were similar. In the absence of prolonged selective pressure, the resistance to DFMO, the ODC activity, and the putrescine levels of DFMO-10 cells all returned to those of wild type cells, indicating that the mutant phenotype of DFMO-selected L. donovani was unstable.     

Role of Ca2+ in prostaglandin E2-induced T-lymphocyte proliferative suppression in sepsis.

Prostaglandin E2 (PGE2) has been known to modulate immune responses by inhibiting T-cell activation following hemorrhagic and traumatic injury. Recently, we documented a sepsis-related depression in concanavalin A (ConA)-induced T-cell proliferation and intracellular Ca2+ (Ca2+i) mobilization. The present study evaluated the potential role of PGE2 in the sepsis-related attenuation in Ca2+ signaling and proliferation in T cells. Sepsis was induced in rats by implanting into their abdomen fecal pellets containing Escherichia coli (150 CFU) and Bacteroides fragilis (10(4) CFU). A group of rats implanted with septic pellets were treated with indomethacin at three consecutive time points. Levels of PGE2 in blood were measured with a radioimmunoassay kit. ConA-induced [Ca2+]i mobilization in T cells obtained from indomethacin-treated and untreated rats was measured with Fura-2 and microfluorometry. We observed a 10-fold increase in PGE2 levels in the circulation of septic rats compared with levels in rats implanted with bacterium-free sterilized pellets. The proliferative response and Ca2+i mobilization were significantly depressed in T cells obtained from septic rats 48 h after implantations compared with those in rats implanted with sterile pellets. However, treatment of rats with the cyclooxygenase inhibitor indomethacin prevented the sepsis-related depression in ConA-induced T-cell Ca2+i mobilization as well as proliferation. Further, incubation of T cells from nonimplanted control rats with PGE2 resulted in a substantial depression in both T-cell proliferation and Ca2+i mobilization. The restoration of T-cell proliferation and Ca2+ signaling after indomethacin treatment of septic rats and the depression in the mitogen responsiveness in T cells previously exposed to PGE2 suggest that the PGE2 does play a significant role in the modulation of T-cell responses in septic rats and that such PGE2-induced suppression in T-cell activation is likely due to an attenuation in Ca2+ signaling.     

糖皮质激素对前列腺素E2介导的主动脉平滑肌细胞cAMP生成的影响

目的观察地塞米松对前列腺素Ｅ２介导的离体培养兔主动脉平滑肌细胞的ｃＡＭＰ生成的影响。方法用蛋白竞争结合法测定细胞ｃＡＭＰ含量,并确定腺苷酸环化酶的活性。结果从１０ｐｍｏｌ／Ｌ到１０ｎｍｏｌ／Ｌ地塞米松对培养细胞的ｃＡＭＰ生成有明显抑制作用并呈现剂量依赖性,作用时间愈长,该抑制作用愈强。经地塞米松预处理８小时的培养细胞,其前列腺素Ｅ２介导的腺苷酸环化酶活性显著下降。结论糖皮质激素在腺苷酸环化酶水平抑制前列腺素Ｅ２介导的培养平滑肌细胞ｃＡＭＰ生成。另外还观察到地塞米松拮抗前列腺素Ｅ２的抑制培养细胞增生效应,但未发现地塞米松对培养平滑肌细胞增生的直接促进作用