Mitogen-stimulated Human Lymphocytes Research Articles

Antitubercular and immunomodulatory activities of Eugenia astringens n-hexane fraction

BackgroundIn Brazil, the Eugenia genus is traditionally used to treat several types of human diseases, including diabetes, gastric ulcers and rheumatoid arthritis. Previous studies demonstrated that medicinal properties of Eugenia astringens Cambess (Myrtaceae), popularly known as “baguaçu" in Brazil, were associated with anti-inflammatory and antimicrobial activities exhibited by the plant extracts. Anti-tubercular effects of E. astringens have not been elucidated yet. Aim of the studyTo study anti-tubercular and immunomodulatory potential of the E. astringens leaves extracts (syn. E. umbelliflora O. Berg), as well as the chemical profiles of active fractions. MethodsThe plant material was collected in the Parque Nacional da Restinga de Jurubatiba (Rio de Janeiro) and the leaves were dried and extracted with ethanol. The extracts were partitioned, to yield n-hexane (Hex), dichloromethane (DCM), ethyl acetate (EtOAc) and n-butanol (BuOH) fractions. The crude extract and fractions were evaluated for antitubercular effects in broth cultures of M. tuberculosis strains and assessed for cytotoxicity in mammalian cell cultures by the MTT method. The Hex fraction, exhibiting low cytotoxicity and stronger inhibitory effect on mycobacterial growth relative to other fractions, was selected for the further studies. Immunomodulatory effects of the Hex fraction were evaluated in the RAW 264.7 macrophages, stimulated by LPS to produce inflammatory mediators, such as TNF-α, IL-6 and nitric oxide. Additionally, the effects on mitogen-stimulated human lymphocyte functions were estimated by measuring cell proliferation and IFN-γ production. ResultsThe E. astringens Hex fraction inhibited growth of mycobacterial strains both in the bacterial culture and infected macrophages, demonstrating strong anti-tubercular potential. In addition, this fraction suppressed secretion of inflammatory cytokines and reduced production of nitric oxide through inhibition of iNOS expression in the LPS-stimulated macrophages. In the mitogen-stimulated lymphocyte assay, Hex fraction inhibited cell proliferation but did not alter IFN-γ production. GC–MS analyses showed that the Hex fraction is composed by caryophyllene-type, eudesmane-type, cadinene-type, and aromadendrene-type sesquiterpenes; and 28 compounds were identified, including spathulenol, previously described for antitubercular acitivity. It is possible that the overall biological efficiency of the Hex fraction depends on the cumulative and/or synergistic effect of these sesquiterpenes. ConclusionThe Hex fraction of E. astringens leaves showed strong antitubercular and immunomodulatory activities, demonstrating pharmacological potential of the identified compounds for the development of new anti-TB drugs.

Cytogenetically-based biodosimetry after high doses of radiation.

Dosimetry is an important tool for triage and treatment planning following any radiation exposure accident, and biological dosimetry, which estimates exposure dose using a biological parameter, is a practical means of determining the specific dose an individual receives. The cytokinesis-blocked micronucleus assay (CBMN) is an established biodosimetric tool to measure chromosomal damage in mitogen-stimulated human lymphocytes. The CBMN method is especially valuable for biodosimetry in triage situations thanks to simplicity in scoring and adaptability to high-throughput automated sample processing systems. While this technique produces dose-response data which fit very well to a linear-quadratic model for exposures to low linear energy transfer (LET) radiation and for doses up for 5 Gy, limitations to the accuracy of this method arise at larger doses. Accuracy at higher doses is limited by the number of cells reaching mitosis. Whereas it would be expected that the yield of micronuclei increases with the dose, in many experiments it has been shown to actually decrease when normalized over the total number of cells. This variation from a monotonically increasing dose response poses a limitation for retrospective dose reconstruction. In this study we modified the standard CBMN assay to increase its accuracy following exposures to higher doses of photons or a mixed neutron–photon beam. The assay is modified either through inhibitions of the G2/M and spindle checkpoints with the addition of caffeine and/or ZM447439 (an Aurora kinase inhibitor), respectively to the blood cultures at select times during the assay. Our results showed that caffeine addition improved assay performance for photon up to 10 Gy. This was achieved by extending the assay time from the typical 70 h to just 74 h. Compared to micronuclei yields without inhibitors, addition of caffeine and ZM447439 resulted in improved accuracy in the detection of micronuclei yields up to 10 Gy from photons and 4 Gy of mixed neutrons-photons. When the dose-effect curves were fitted to take into account the turnover phenomenon observed at higher doses, best fitting was achieved when the combination of both inhibitors was used. These techniques permit reliable dose reconstruction after high doses of radiation with a method that can be adapted to high-throughput automated sample processing systems.

High homocysteine levels prevent via H2 S the CoCl2 -induced alteration of lymphocyte viability.

High homocysteine (HCy) levels are associated with lymphocyte‐mediated inflammatory responses that are sometimes in turn related to hypoxia. Because adenosine is a potent lymphocyte suppressor produced in hypoxic conditions and shares metabolic pathways with HCy, we addressed the influence of high HCy levels on the hypoxia‐induced, adenosine‐mediated, alteration of lymphocyte viability. We treated mitogen‐stimulated human lymphocytes isolated from healthy individuals and the human lymphoma T‐cell line CEM with cobalt chloride (CoCl2)to reproduce hypoxia. We found that CoCl2‐altered cell viability was dose‐dependently reversed using HCy. In turn, the HCy effect was inhibited using DL‐propargylglycine, a specific inhibitor of the hydrogen sulphide (H2S)‐synthesizing enzyme cystathionine‐γ‐lyase involved in HCy catabolism. We then addressed the intracellular metabolic pathway of adenosine and HCy, and the role of the adenosine A2A receptor (A2 AR). We observed that: (i) hypoxic conditions lowered the intracellular concentration of HCy by increasing adenosine production, which resulted in high A2 AR expression and 3′, 5′‐cyclic adenosine monophosphate production; (ii) increasing intracellular HCy concentration reversed the hypoxia‐induced adenosinergic signalling despite high adenosine concentration by promoting both S‐adenosylhomocysteine and H2S production; (iii) DL‐propargylglycine that inhibits H2S production abolished the HCy effect. Together, these data suggest that high HCy levels prevent, via H2S production and the resulting down‐regulation of A2 AR expression, the hypoxia‐induced adenosinergic alteration of lymphocyte viability. We point out the relevance of these mechanisms in the pathophysiology of cardiovascular diseases.

Apoptotic Efficacy of Etomoxir in Human Acute Myeloid Leukemia Cells. Cooperation with Arsenic Trioxide and Glycolytic Inhibitors, and Regulation by Oxidative Stress and Protein Kinase Activities

Fatty acid synthesis and oxidation are frequently exacerbated in leukemia cells, and may therefore represent a target for therapeutic intervention. In this work we analyzed the apoptotic and chemo-sensitizing action of the fatty acid oxidation inhibitor etomoxir in human acute myeloid leukemia cells. Etomoxir caused negligible lethality at concentrations up to 100 µM, but efficaciously cooperated to cause apoptosis with the anti-leukemic agent arsenic trioxide (ATO, Trisenox), and with lower efficacy with other anti-tumour drugs (etoposide, cisplatin), in HL60 cells. Etomoxir-ATO cooperation was also observed in NB4 human acute promyelocytic cells, but not in normal (non-tumour) mitogen-stimulated human peripheral blood lymphocytes. Biochemical determinations in HL60 cells indicated that etomoxir (25–200 µM) dose-dependently inhibited mitochondrial respiration while slightly stimulating glycolysis, and only caused marginal alterations in total ATP content and adenine nucleotide pool distribution. In addition, etomoxir caused oxidative stress (increase in intracellular reactive oxygen species accumulation, decrease in reduced glutathione content), as well as pro-apoptotic LKB-1/AMPK pathway activation, all of which may in part explain the chemo-sensitizing capacity of the drug. Etomoxir also cooperated with glycolytic inhibitors (2-deoxy-D-glucose, lonidamine) to induce apoptosis in HL60 cells, but not in NB4 cells. The combined etomoxir plus 2-deoxy-D-glucose treatment did not increase oxidative stress, caused moderate decrease in net ATP content, increased the AMP/ATP ratio with concomitant drop in energy charge, and caused defensive Akt and ERK kinase activation. Apoptosis generation by etomoxir plus 2-deoxy-D-glucose was further increased by co-incubation with ATO, which is apparently explained by the capacity of ATO to attenuate Akt and ERK activation. In summary, co-treatment with etomoxir may represent an interesting strategy to increase the apoptotic efficacy of ATO and (with some limitations) 2-deoxy-D-glucose which, although clinically important anti-tumour agents, exhibit low efficacy in monotherapy.

Long-term regulation of Na,K-ATPase pump during T-cell proliferation

The aim of the study was to elucidate the mechanism responsible for the proliferation-related regulation of Na,K-ATPase pump. Our data demonstrate that in mitogen-stimulated human blood lymphocytes, enhanced ouabain-sensitive Rb(K) fluxes in the middle/late stage of G(0)/G(1)/S transit are associated with the increased number of Na,K-ATPase pumps expressed at the cell surface (as determined by the [(3)H]ouabain binding). Analysis of total RNA (reverse transcription-polymerase chain reaction) and protein (Western blotting) showed a threefold increase in the level of Na,K-ATPase alpha1-subunit and beta1-subunit mRNAs and significant increase in the Na,K-ATPase alpha1-subunit protein during the first day of mitogen-induced proliferation. The elevated K transport as well as the increased expression of Na,K-ATPase is closely associated with the IL-2-dependent stage of T-cell response. The pharmacological inhibition of IL-2-induced MEK/ERK or JAK/STAT cascades suppressed the IL-2-induced proliferation and reduced the functional and protein expressions of Na,K-ATPase. It is concluded that during the lymphocyte transition from resting stage to proliferation, (1) long-term activation of Na,K-ATPase pump is due to the enhanced expression of Na,K-ATPase protein and mRNA, and (2) the cytokine signaling via the IL-2 receptor is necessary for the cell cycle-associated upregulation of Na,K-ATPase.

Cytometric detection of chromatin relaxation, an early reporter of DNA damage response

One of the early events of the DNA damage response (DDR), particularly if the damage involves induction of DNA double-strand breaks, is remodeling of chromatin structure characterized by its relaxation (decondensation). The relaxation increases accessibility of the damaged DNA sites to the repair machinery. We present here a simple cytometric approach to detect chromatin relaxation based on the analysis of the proclivity of DNA in situ to undergo denaturation after treatment with acid. DNA denaturation is probed by the metachromatic fluorochrome acridine orange (AO) which differentially stains single-stranded (denatured) DNA by fluorescing red and the double-stranded DNA by emitting green fluorescence. DNA damage was induced in both human leukemic TK6 cells and mitogen-stimulated human peripheral blood lymphocytes by exposure to UV light or by treatment with H2O2. Chromatin relaxation was revealed by diminished susceptibility of DNA to denaturation, likely reflecting decreased DNA torsional stress, seen as soon 10 min after subjecting cells to UV or H2O2. While cells in all phases of the cell cycle showed a comparable extent of chromatin relaxation upon UV or H2O2 exposure, H2AX was phosphorylated on Ser139 predominantly in S-phase cells. The data are consistent with the notion that chromatin relaxation is global, affects all cells with damaged DNA, and is a prerequisite to the subsequent steps of DDR that can be selective to cells in a particular phase of the cell cycle. The method offers a rapid and simple means of detecting genotoxic insult on cells.

A combination of the micronucleus assay and a FISH technique for evaluation of the genotoxicity of 1,2,4-benzenetriol

The cytokinesis-block micronucleus (CBMN) assay has emerged as one of the preferred methods for assessing chromosome damage. Micronuclei (MN) are small, extranuclear bodies that are formed in mitosis from acentric chromosomal fragments or chromosomes that are not included in each daughter nucleus. Thus, MN contain either chromosomal fragments or whole chromosomes. The CBMN assay, together with a fluorescence in situ hybridization (FISH) technique using specific centromeric probes for chromosomes 7 and 8, were employed in mitogen-stimulated human lymphocytes pretreated with the benzene metabolite, 1,2,4-benzenetriol (BT). Treatment of human lymphocytes resulted in the induction of MN in a dose-dependent manner. The frequency of MN in control lymphocytes was 4.5 per 1000 binucleated (BN) cells and this increased to 9.5, 14, 28 and 40 per 1000 BN cells at 10, 25, 50 and 100 μM BT, respectively. The frequency of aneuploidy 7 and 8 in BN cells also increased at each concentration. Aneuploidy 8 was more frequent than aneuploidy 7, suggesting that chromosome 8 is more sensitive to aneuploidy induction by BT. The frequency of MN containing centromere positive signals for chromosomes 7 and 8 increased with the concentration of BT. The frequency of MN with centromere positive signals was higher for chromosome 8 than for chromosome 7, also suggesting a greater sensitivity of chromosome 8 to this agent. These results suggest that combined application of the CBMN assay with a FISH technique, using chromosome-specific centromeric probes, would allow the detection of aneuploidy in human lymphocytes and identify the mechanistic origin of MN induced by a clastogen or aneugen.

Differential effects of the antioxidant α-lipoic acid on the proliferation of mitogen-stimulated peripheral blood lymphocytes and leukaemic T cells

The effects of the antioxidant α-lipoic acid (LA) on the proliferation of mitogen-stimulated human peripheral blood lymphocytes (HPBL) were investigated in comparison to its effects on the proliferation of two leukaemic T cell lines, Jurkat and CCRF-CEM. At low mM concentrations, LA inhibited in a dose-dependent manner DNA synthesis of HPBL stimulated with either phorbol myristate acetate (PMA) in combination with ionomycin (IoM), or phytohaemagglutinin (PHA). At similar concentrations, LA inhibited the proliferation of Jurkat and CCRF-CEM cells. However, LA was preferentially cytotoxic to the leukaemic cell lines. The selective toxicity of LA to Jurkat cells was shown by electron microscopy (EM) to be due to the induction of apoptosis. Furthermore, LA had different effects on the secretion of interleukin-2 (IL-2) and steady-state levels of IL-2 mRNA in mitogen-stimulated HPBL depending on the mitogens used. LA dramatically increased the induction of IL-2 mRNA and IL-2 protein secretion in PMA/IoM-stimulated HPBL, whereas it inhibited these in HPBL stimulated with PHA. The differential effects of LA on normal and leukaemic T lymphocytes may indicate a new route towards development of therapeutic agents.

Cleistanthin A, a diphyllin glycoside fromCleistanthus collinus, is cytotoxic to PHA-stimulated (proliferating) human lymphocytes

An ideal anticancer drug would be one that preferentially kills tumor cells with the least toxicity to normal cells. Cleistanthin A, a diphyllin glycoside of the tropical plant Cleistanthus collinus, was found to possess cytotoxic and tumor regressing properties. To find out whether this compound acts selectively on proliferating cells it was tested against quiescent and proliferating human lymphocytes. Mitogen-stimulated and unstimulated human lymphocytes were treated with cleistanthin A. A cytotoxicity assay using MTT was used to assess the viability of the cells. Percentage viability of the unstimulated and treated cells were normalized to that of the untreated and unstimulated cells and percentage viability of stimulated and treated cells were normalized to that of stimulated and untreated cells. Quiescent lymphocytes were refractory to the action of cleistanthin A. Only proliferating cells were killed. Cell death was proportional to the percentage of cells in the proliferating stage and was also dose-dependent. Quiescent lymphocytes pretreated with cleistanthin A had the ability to proliferate upon subsequent stimulation with PHA. These results indicate that cleistanthin A does not affect the viability of quiescent cells. Also, it did not affect the proliferating potential of quiescent cells. However, this compound drastically affected proliferating cells by reducing their viability to 10-20%. Our results therefore indicate that the antiproliferative property of cleistanthin A could be used in regimens for treating tumors with extensive proliferative potencies.

Essay on the nucleoli survey by the α- and β-satellite DNA probes of the acrocentric chromosomes in mitogen-stimulated human lymphocytes

The two constitutive heterochromatin (α- and β-satellite DNA) probes of human acrocentric chromosomes were essayed separately to label the nucleoli in the phytohemagglutinin (PHA)-stimulated human lymphocytes. Fluorescent in situ hybridisation (FISH) results have shown that: a) whole (100 %) signal–nucleoli overlapping was obtained with both heterochromatin probes in maximally activated nuclei (MANs); b) partial overlapping was observed in non-activated or slightly activated nuclei; c) random signal–nucleolus overlapping (background level) was found to be ∼6 % by the NOR-irrelevant euchromatic probe (D5S23); d) Yq–nucleolus association in the MANs was found to be ∼97 % without the subtraction of the background level. We concluded that: a) acrocentric α- or β-satellite DNA probes may be used as nucleolar markers only in the MANs and not in slightly activated or non-activated nuclei; b) the distances between rDNA loci and α-/β-satellite DNA on human acrocentrics are short enough to permit their observation on the same nucleolus.

Lymphocyte suppression by glucocorticoids with cyclosporine, tacrolimus, pentoxifylline, and mycophenolic acid.

Methylprednisolone has been found to be significantly more suppressive than prednisolone (the pharmacologically active metabolite of prednisone) of mitogen-stimulated human lymphocyte proliferation. In this study, peripheral blood mononuclear cells (PBMC) from end stage renal disease patients were cultured with phytohemagglutinin (PHA) alone and with methylprednisolone and prednisolone individually, as well as each glucocorticoid (10(-7) mol/L) in combination with 300 ng/ml cyclosporine, 10 ng/ml tacrolimus, 25 microg/ml pentoxifylline, and 10(-7) mol/L mycophenolic acid. Under each experimental condition, the mean +/- SD % inhibition of PHA-stimulated 3H-thymidine incorporation was significantly greater with methylprednisolone than with prednisolone: methylprednisolone 55 +/- 17 versus prednisolone 28 +/- 14, p < 0.001; methylprednisolone + cyclosporine 76 +/- 18 versus prednisolone + cyclosporine 52 +/- 18, p < 0.001; methylprednisolone + tacrolimus 74 +/- 18 versus prednisolone + tacrolimus 50 +/- 20, p = 0.001; methylprednisolone + mycophenolic acid 69 +/- 14 versus prednisolone + mycophenolic acid 46 +/- 15, p < 0.001. These results confirm and extend previous observations and suggest that methylprednisolone might be more effective than prednisone in treatment protocols used to suppress allograft rejection.

Estrogenic xenobiotics affect the intracellular activation signal in mitogen-induced human peripheral blood lymphocytes : immunotoxicological impact

The present study was an attempt to elucidate the effect of estrogenic xenobiotics on the proliferation of mitogen-stimulated human peripheral blood lymphocyte (PBL). Our findings follow : (a) the proliferation of PBL in response to phytohemagglutinin (PHA) was mediated by protein kinase C activity, but estrogenic xenobiotics had a strong inhibitory effect on protein kinase C activity of PHA-stimulated PBL ; (b) cytoplasmic extracts from PHA-stimulated PBL greatly activated DNA replication, but estrogenic xenobiotics had a strong inhibitory effect on these activities. The results suggest that the cytoplasmic signal-generating system in mitogen-treated PBL is inhibited by estrogenic xenobiotics, and that the defect occurs at all stages in the sequence of events leading to DNA synthesis and cell proliferation.

Sex hormones affect the intracellular activation signal in mitogen-stimulated human blood lymphocytes

The present study was an attempt to elucidate the effect of sex steroids (estradiol-17β and 5α-dihydrotestosterone) on the mitogen-stimulated human peripheral blood lymphocytes (PBL) proliferation. Our findings were as follows: (a) the proliferation of PBL in response to phytohemagglutinin (PHA) was mediated by protein kinase C (PKC) activity, but a strong inhibitory effect of sex steroids on the PKC activity of PHA-stimulated PBL was observed; (b) cytoplasmic extracts of PHA-stimulated PBL showed high activity as an activator of DNA replication (ADR) as well as increased levels of the mitosis stimulator p34cdc2 kinase, and the steroid hormones studied all significantly reduced these activities; and (c) these effects of sex steroids are similar to those found with hydrocortisone in parallel experiments. The results suggest that the cytoplasmic signal-generating system stimulating DNA synthesis and mitosis is inhibited in PHA-stimulated PBLs by several steroid hormones. The inhibition affects different factors stimulating DNA replication and cell proliferation.

Energy levels in resting and mitogen-stimulated human lymphocytes during treatment with FK506 or cyclosporin A in vitro

By employing microcalorimetry to assess overall metabolic activity in combination with other assays for specific metabolic events, we have investigated the influence of cyclosporin A and FK506 on the metabolic status of resting and mitogen-stimulated human peripheral lymphocytes. Both cyclosporin A and FK506 significantly reduced heat output from resting lymphocytes. This reduction could not be correlated with effects on DNA synthesis, lactate production, ATP levels or mitochondrial uptake of Rhodamine 123. These two drugs also potently reduced the increase in heat output seen during mitogen stimulation of lymphocytes. Both cyclosporin A and FK506 also prevented the increase in DNA synthesis, lactate production and ATP levels seen in response to mitogen stimulation. The increase in mitochondrial uptake of Rhodamine 123 during blastoid transformation was significantly reduced only by cyclosporin A. We ascribe the major part of the effects of these compounds to inhibition of the glycolytic pathway in both resting and mitogen-stimulated lymphocytes. These results indicate that the immunosuppressants cyclosporin A and FK506 exert other effects on lymphocytes than their well-established inhibitory action on calcineurin.© 1997 Elsevier Science B.V. All rights reserved.

Effect of melatonin and corticosteroid on in vitro cellular immune function in humans.

It is well accepted that the immune system shows circadian rhythmicity and that circadian disruption can significantly alter indices of immune function. Recently, a functional link between the endocrine and immune systems has been proposed to explain circadian rhythms in immune activity. Of particular interest is the finding that hormones such as melatonin and corticosteroid are able to exert modulating effects on lymphocyte proliferation. Previous research examining the effects of melatonin in vitro, however, has produced equivocal results. The aim of this study, therefore, was to examine the effects of melatonin and corticosteroid, both separately and together, on mitogen-stimulated human lymphocyte proliferation. Purified human lymphocytes were stimulated with concanavalin A (Con A, 4 micrograms/mL). Melatonin and/or corticosteroid were added to the culture medium during incubation. All cultures were done in quadruplicate. As expected, corticosteroid (25-1,000 ng/mL) significantly reduced proliferation by between 30 and 60% in a dose-related manner (P < 0.0001). Melatonin alone (10-1,000 fmol/mL) did not significantly affect lymphocyte proliferation. However, when lymphocytes were cultured in the presence of melatonin and corticosteroid, a significant decrease in proliferative function of 50-85% was observed (P < 0.0001). Hence, the effect of melatonin and corticosteroid combined was significantly greater than that observed with corticosteroid alone (P < 0.0001). Therefore, it appears that the in vitro effect of corticosteroid on immune function may be modulated by melatonin in physiological to pharmacological concentrations.

Trypanosoma cruzi Downregulates the Production of Interleukin-2, Interferon-g, Interleukin-4 and Interleukin-5 by Activated Human Lymphocytes

The intracellular levels of interleukin (IL)-2, IL-4, IL-5 and interferon-gamma (IFN-gamma) produced by mitogen-stimulated normal human blood lymphocytes were found to be markedly reduced in cultures containing purified Trypanosoma cruzi trypomastigotes. This was evidenced by marked decreases in the proportion of lymphocytes whose cytoplasm was stained with fluorochrome-conjugated monoclonal antibodies specific for the selected cytokines and a somewhat small but systematically demonstrable reduction in the mean fluorescence intensity of the stained lymphocytes relative to values obtained with parasite-free cultures. The percentage of activated CD3+ cells containing both IL-2, IL-4, i.e., THO-like cells, was also much smaller in the cultures containing trypomastigotes, indicating that T. cruzi can concomitantly inhibit the production of more than 1 cytokine by individual activated T lymphocytes. These results evidence the ability of T. cruzi to down-regulate the production of several important cytokines and raise the possibility of a direct role for this parasite in altering cytokine production during infection.

Expression of 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible cytochrome P4501A1 in human splenic lymphocyte cultures

The induction of cytochrome P4501A1 (P4501A1) and P4501A1-specific 7-ethoxyresorufin O -deethylase (EROD) activity by 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) was investigated in human splenic lymphocytes cultures. EROD activity was induced by TCDD in mitogen (phytohemagglutinin and pokeweed mitogen) stimulated blast cells but not in the resting cells. TCDD markedly induced EROD activity in a dose- and time-dependent manner. The expression of P4501A1 mRNA was increased by TCDD in mitogen-stimulated cells as detected by Northern blot analysis. These findings support the conclusion that TCDD induced the expression of P4501A1 gene, resulting in increased EROD activity in mitogen-stimulated human splenic lymphocytes cultures.

Expression of 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible cytochrome P4501A1 in human splenic lymphocyte cultures

The induction of cytochrome P4501A1 (P4501A1) and P4501A1-specific 7-ethoxyresorufin O-deethylase (EROD) activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated in human splenic lymphocytes cultures. EROD activity was induced by TCDD in mitogen (phytohemagglutinin and pokeweed mitogen) stimulated blast cells but not in the resting cells. TCDD markedly induced EROD activity in a dose- and time-dependent manner. The expression of P4501A1 mRNA was increased by TCDD in mitogen-stimulated cells as detected by Northern blot analysis. These findings support the conclusion that TCDD induced the expression of P4501A1 gene, resulting in increased EROD activity in mitogen-stimulated human splenic lymphocytes cultures.

Mitogenic stimulation of human lymphocytes mediated by a cell surface elastase

A ca. 45-kDa protein which was recently identified and purified to homogeneity from a solid tumor cell line as a T-cell mitogen was found to have significant sequence similarities with human monocyte/neutrophil elastase inhibitor (EI)[1]. Since EI is a known substrate for elastase, a determination of whether a cell surface expressed elastase-like molecule might be the binding protein for this 45-kDa factor and mediate mitogenic signal transduction was undertaken. First, the surface of tumor infiltrating lymphocytes, TIL 660, the indicator cell line used for the purification of this mitogen, was shown to stain positively with an anti-elastase antibody using flow cytofluorometry for quantitation. Then, after observing an inverse correlation between cell surface staining and the proliferative status of the TILs, behavior which might be expected of a growth factor receptor upon activation, mitogenic signal transduction was attempted through the elastase-like molecules of the lymphocytes' plasma membrane with the anti-elastase antibody in the role of mitogen. A greater than 4-fold mitogenic stimulation was observed when this antibody was covalently linked to latex beads; in contrast, addition of the soluble form of the same antibody did not result in any increase in [ 3H]thymidine incorporation into the cells' DNA. Hence, these data support induced clustering of an elastase-like molecule on the lymphocyte surface as a mediator of mitogenesis and suggest that the binding protein for mitogenic signal transduction induced by the 45-kDa protein, a member of the serine protease inhibitor (serpin) superfamily of proteins, is a molecule with structure similar to a serine protease.

In vitro effects of prostaglandin E1 and indomethacin on mitomycin C-induced sister-chromatid exchanges in mitogen-stimulated human lymphocytes

In this study, the individual and combined effects of prostaglandin E1 (PGE1) and Indomethacin on mitomycin C (MMC)-induced SCEs in human lymphocytes was investigated in vitro. All MMC-treated cultures showed a great increase of SCEs (approximately two-fold), indicating its ability to induce mutations. SCE data showed that MMC-induced SCEs were reduced significantly in the presence of PGE1 in pooled analysis of six experiments (60.55% reduction of SCEs at 10 −6 M, 34.13% reduction of SCEs at 10 −9 M). In contrast the presence of indomethacin in the medium during MMC treatment of cells failed to show a significant reduction of SCEs in pooled analysis (21.17%). However, individual analyses revealed only two of six donors with a significant SCE response. Thus, the findings suggest that PGE1 can modify the DNA damaging effect of carcinogens and thereby may prevent the initiation of the carcinogenic process.