Mitogen-treated Lymphocytes Research Articles

Effect of Salvia mirzayanii on the immune system and induction of apoptosis in peripheral blood lymphocytes

In this study, the immunomodulatory effects of Salvia mirzayanii were investigated. Study of the effect of this plant on activated human peripheral blood lymphocytes showed stimulatory effects at lower concentrations and inhibitory effects at higher ones (p < 0.01). In flow cytometry analysis, accumulation of apoptotic cells in the sub-G1 phase of the cell cycle of the mitogen-treated lymphocytes exposed to the inhibitory doses of the extract was observed. DNA fragmentation analysis of these cells showed a typical DNA laddering. Immunisation of extract-treated mice with the antigen decreased delayed hypersensitivity skin reaction as well as the antibody titre at higher concentrations (p < 0.007). These results indicated the presence of immunomodulatory compounds in the extract of S. mirzayanii and suggest that the induction of apoptosis in lymphocytes might be the mechanism responsible for the inhibitory effect of the extract that was observed at higher concentrations.

Calycopterin, an immunoinhibitory compound from the extract of Dracocephalum kotschyi

Medicinal plants have been widely investigated for their various effects. Dracocephalum kotschyi Boiss (Labiatae) is used in Iranian traditional medicine for the treatment of rheumatoid diseases. The inhibitory effect of D. kotschyi on the lectin-induced cellular immune response has been demonstrated previously. In this study, mitogen-treated lymphocytes were exposed to the extract of D. kotschyi and analysed for the induction of apoptosis using flow cytometry and gel electrophoresis. The data obtained indicated a dose-dependent increase of cells in the sub-G1 phase of cell cycle. Study of internucleosomal DNA fragmentation showed a typical DNA laddering in agarose gels. A bioactivity-guided fractionation assay to find the active components responsible for the inhibitory effect of D. kotschyi on mitogen-induced lymphocyte proliferation led to the isolation of calycopterin from the ethyl acetate extract of D. kotschyi. Its structure was identified by spectroscopic methods including( 1)H-NMR, (13)C-NMR, MS and UV spectra. Calycopterin inhibited lymphocyte proliferation in a dose-dependent manner with an IC(50) value of 1.7 microg/mL. In conclusion, the results of this study suggest that D. kotschyi extract has the capacity to induce apoptosis in the lymphocytes and that isolated calycopterin is responsible for the inhibitory effect of D. kotschyi on lymphocyte proliferation.

Stimulatory effects of Euphorbia cheiradenia on cell mediated immunity and humoral antibody synthesis.

Studies have demonstrated that plant extracts possess various biological characteristics including immunomodulatory activity. Euphorbia cheiradenia Boiss et Hohen (Euphorbiaceae), a medicinal herb native to Iran was investigated for its immunomodulatory effects. The methanolic extract of the plant was prepared and added to mitogen-induced human peripheral blood lymphocyte cultures at different concentrations. Effect of E. cheiradenia on in vivo cell-mediated immunity was measured by delayed type hypersensitivity (DTH) reaction. The effect of the extract on humoral antibody synthesis was also measured in immunized mice treated with different extract concentrations. The stimulation index (SI) for cultures treated with 0.01 to 200 microg/ml of the extract ranged from 1.3+/-0.04 to 2.4+/-0.06, (p<0.01) showing a significant stimulatory effect of E. cheiradenia on the lymphocytes. IL-2 secreted from lymphocytes treated with the extract was significantly higher than that from the non-treated cells (p<0.001). Cell cycle analysis on mitogen-treated lymphocytes exposed to different concentrations of the extract showed an increase in the percentage of cells at G2M phase with increases in the concentration of the extract, but the results was not significant. In DTH skin test, the mean footpad thickness of all mice groups treated with 1, 50 and 100 mg/kg of the extract at 24 hours after immunization with antigen was 3.5+/-0.6 mm compared to 2.5+/-0.5 mm for the non-treated group (p=0.005). Moreover, an increase in production of specific antibody in mice immunized with different extract concentrations was also demonstrated. Results of this study showed the ability of the E. cheiradenia extract to induce proliferation of lymphocytes and enhance both cellular and humoral specific immune responses.

The relationship between CYP1A1 aryl hydrocarbon hydroxylase activity and lung cancer in a Japanese population.

Because aryl hydrocarbon hydroxylase (AHH) is considered to be responsible for the activation of benzo(a)pyrene and other polyaromatic hydrocarbons in cigarette smoke to carcinogens, it is important to examine CYP1A1 (AHH) activity in the determination of susceptibility to lung cancer. We investigated AHH activity in peripheral mitogen-treated lymphocytes in 108 lung cancer patients and 95 healthy control individuals. Non-induced AHH activity was detectable in all the samples. AHH inducibility (3-methylcholanthrene-induced/non-induced AHH activity) showed a very wide interindividual variation as well as non-induced AHH activity. No significant associations were found between adjusted AHH activity and histologic type of tumor among lung cancer patients. Adjusted AHH inducibility of genotype C [geometric mean and 95% confidence interval (CI); 15.56 and 11.69-20.71] in MspI polymorphism was significantly higher than those of the other two genotypes (P = 0.0001), while no significant difference was observed between genotypes A (4.76 and 3.82-5.93) and B (5.60 and 4.57-6.86). On the other hand, non-induced AHH activity of genotype Val/Val (0.121 and 0.082-0.178 pmol/min/10(6) cells) in isoleucine-valine (Ile-Val) polymorphism was significantly higher than those of genotypes Ile/Ile (0.042 and 0.034-0.052 pmol/min/10(6) cells) and Ile/Val (0.040 and 0.030-0.053 pmol/min/10(6) cells) (P < 0.0001). Even after controlling for age, cigarettes smoked per day and season of the year, high AHH inducibility (7.0 < versus 0 < < or = 3.0: OR and 95 %CI, 12.4 and 2.88-53.4) was an independent risk factor for lung cancer. The data indicate that high AHH inducibility may strongly associate with the susceptibility to lung carcinogenesis.

Molecular Physiology, Biochemistry, and Pharmacology of Alzheimer's Amyloid Precursor Protein (APP)a

The function of APP is not yet known in detail but growing evidence exists that APP may mediate cell interactions with the cell surface or soluble glycoproteins and defense mechanisms in the CNS involving the immune system. We describe here the finding that almost all CD4+ lymphocytes and the majority of CD8+ lymphocytes were positive for A beta and the antibodies against A beta or APP did not inhibit the [3H]-thymidine uptake of mitogen-treated lymphocytes significantly. There were no differences in the A beta immunoreactivity on the cell surface of lymphocytes between Alzheimer's disease (AD) and control samples. Excessive amyloidogenic pathway of APP processing may be the final common pathway involved in the pathogenesis of AD. Thus, the identification of proteases or factors leading to aberrant proteolysis which process APP to yield a variety of potentially amyloidogenic fragments would promise pharmacological targets to develop anti-AD drugs. In attempts to define the proteases or factors which alter the balance between nonamyloidogenic and amyloidogenic processing pathways, our study indicates that thrombin or acetylcholinesterase(AChE)-associated protease may be involved in the amyloidogenic processing pathway of APP in vivo to generate amyloidogenic intermediates linked to amyloid deposition. Highly specific and dose-dependent direct modulation of APP processing by biologically available metal ions including Ca2+, Zn2+, Fe2+/Fe3+ and Al3+ suggest the disrupted metal homeostasis as factors leading to overaccumulation of APP and subsequent aberrant proteolysis utilizing excessive amyloidogenic processing pathway. There is mounting evidence that at least some of the neurotoxicity associated with AD is due to fragments from APP. Most research has focused on the toxic effect and the ion channel activity of A beta in causation of the disease. The possible role of other cleaved products of APP is less clear. We investigated the channel-forming ability of various products of APP when applied to Xenopus oocytes and their neurotoxicity in vitro. CT105 peptide was found to be exceedingly potent at 500 nM concentration in forming nonselective ion channels during application from either outside or inside the oocyte and more toxic than either of the A beta fragments, A beta 25-35, or A beta 1-40. Taken together, these results suggest the possible involvement of CT peptide in inducing the neurotoxicity characteristic of AD through the direct damage on the cell membrane. Therefore, we hypothesize that amyloidogenic CT may make nonselective ion channels or pores in the membrane and may cause neuronal death in the early stage of AD and then further metabolized to more stable and less toxic A beta which may be finally deposited in the brain where it could inflict further toxicity to neurons. Here we report successful inhibition of APP gene expression by antisense oligodeoxynucleotides at the mRNA or the protein level in in vitro and cell culture systems.

The effect of silica treatment on accessory cell-dependent rat T lymphocyte proliferation

Previous work has shown that purified rat macrophages lack both accessory activity for T lymphocyte responses to mitogens and stimulatory activity in a mixed leukocyte reaction, in marked contrast to the potent activity of dendritic cells. This study was designed to re-evaluate macrophages as accessory cells by treating various cell preparations with either silica or Lleucine methyl ester, which have been reported to be toxic to macrophages, then determining the effect of the treated cells on responses to the mitogens, sodium periodate or concanavalin A. These studies indicated that treatment with L-leucine methyl ester failed to kill rat macrophages or dendritic cells, whereas silica was specifically toxic for rat macrophages. The studies therefore focused on silica. Co-culturing mitogen-treated lymph node cells with silica over a wide range of concentrations had no effect on responses. The same results were obtained if mitogen-treated lymphocytes were enriched with lymph node macrophages and dendritic cells and then co-cultured with silica. Preparations containing both macrophages and dendritic cells were incubated with silica for 24 h to ensure the death of virtually all macrophages; upon the addition of mitogen-treated lymphocytes, the macrophage-depleted accessory cells induced vigorous proliferative responses. Peritoneal exudate cells showed variable, but low accessory activity that increased after incubation with silica. Elimination of more than 90 % of the macrophages from peritoneal exudate cells, as determined by staining for non-specific esterase, failed to eliminate this accessory activity. Taken together, these findings confirm and extend the conclusion that rat macrophages lack or have exceedingly low accessory activity.

Blastogenic response of peripheral blood lymphocytes from multiparous pregnant ewes.

The immunocompetence of pregnant multiparous ewes was investigated with respect to the blastogenic response of peripheral blood lymphocytes of (PBL) to three mitogens: PHA, Con A, and PWM. The profile of PBL responses shows 1) progressive suppression at 36 and 66 days of gestation, 2) enhanced response at 97 days of gestation, which approaches the mean values observed at the premating period, and 3) a redecline of the response at 137 days of gestation to depressed values lower than observed at 36 and 66 days of gestation. The results suggest that mitogen-treated lymphocytes were depressed and that the immunodepressive factor(s), which can influence lymphocytes at the systemic level, may be involved in the maintenance of the ovine fetal semiallograft.

Biochemical changes taking place in mitogen-treated lymphocytes

This chapter focuses on biochemical changes taking place in mitogen-treated lymphocytes. Lymphocytes are metabolically activated by a variety of agents acting at their surfaces, including antigens, antibodies to membrane components, lectins, heavy metal ions, ionophores, proteolytic enzymes, and microorganisms or their products. These stimulatory agents produce a complex series of biochemical alterations leading after a period of many hours to physical alterations and in many instances cell division. Other changes include the expression or release of new surface and secreted proteins as required for cell regulation, function, and the mitogenic process itself. The most notable of the polyclonal activators are the mitogenic lectins. Because of their commercial availability and marked stimulatory activity lectins—such as phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed (PW)—have gained wide popularity for metabolic studies.PHA and Con A are the mitogens used in the majority of the studies described in this chapter. Among the earliest changes seen in stimulated cells are increases in transport of important nutriments and ions.

Highly purified human T-lymphocytes do not respond to mitogens--including calcium ionophore and phorbol ester.

The proliferative, and other, in vitro responses of human lymphocytes are dependent upon interactions with accessory cells. Studies of the nature of these required interactions are dependent upon the ability to isolate functionally pure populations of lymphocytes and accessory cells. We describe here a four-step separation procedure using plastic adherence, passage through columns of nylon wool and Sephadex G-10, and treatment with L-leucine methyl ester which produced T-lymphocyte populations which were functionally free of accessory cells, and accessory cell populations which were highly enriched for macrophages. T-lymphocytes were 91-98% Leu-4-positive, contained fewer than 0.05% Mo1- or Mo2-positive cells and fewer than 0.1% nonspecific esterase-positive cells, showed relatively little change in the T4/T8 ratio, and gave no proliferative response to a panel of mitogens. The accessory cell populations were 72-98% nonspecific esterase-positive, 91-99% OKM-1-positive and only 0-4% Leu-4-positive, and could fully restore the proliferative responses of the mitogen-treated lymphocytes. Using these purified cell populations, we have found that, contrary to a number of published observations, both the calcium ionophore A23187 and the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate were dependent on the presence of accessory cells to produce a mitogenic response by human lymphocytes.

Early mitogen-induced metabolic events essential to proliferation of human T lymphocytes: dependence of specific events on the influence of adherent accessory cells.

Adherent accessory cells (AC) are required for the proliferative response of T lymphocytes to antigens and various mitogens. A current model of AC-T cell cooperation is that commitment to growth of mitogen activated T lymphocytes occurs via sequential action of IL 1 and IL 2. Initial mitogen action on T lymphocytes in the presence of AC is followed by a sequence of metabolic changes which culminate in DNA replication and mitosis. Many of these early events are critical to DNA replication. We studied several of these mitogen-induced events in experiments designed to define the specific influence of AC on T cell metabolism before initiation of DNA replication. By using human peripheral T lymphocytes depleted of AC to the extent that the proliferative response is essentially ablated, we found that the sequence of early events is divided into two phases: an early activated state in which certain events are stimulated directly by mitogen and independently of AC, and an AC-dependent state in which other events occur in mitogen-treated lymphocytes only in the presence of the numbers of AC necessary to support the proliferative response. We partially support the proliferative response. We partially characterized the nature of the metabolic activation that pulse neuraminidase-galactose oxidase treatment induces in lymphocytes in the presence and functional absence of AC. Stimulated uptake of [3H] uridine and [3H]-leucine into cellular precursor pools and incorporation into macromolecules apparently requires the presence of AC, but stimulated influx of both [3H]3-O-methyl glucose and [3H]alpha-amino isobutyric acid are independent of the presence of AC. These data suggest that stimulated influx of glucose and a certain class of essential amino acids are events of the early activated state, whereas increased RNA and protein synthesis are events of the AC-dependent state. All of these events are critical to the T cell's commitment of DNA replication and mitosis. The early activated state is consistent with AC-T cell cooperation via IL 2. It is possible that IL 2 mediates passage of IL 2 receptor-bearing T cells from the early activated state to the AC-dependent state, which then leads directly to DNA replication and mitosis.

Inhibitory effect of leukemic plasma on periodate-induced lymphocyte transformation

Plasma from nine out of 18 patients with untreated acute lymphoblastic leukemia (ALL) depressed the transformation of normal blood lymphocytes induced by sodium periodate (Na10 4) as judged by reduction of blast cell formation and [ 3H]thymidine and [ 3H]uridine incorporation into DNA and RNA respectively. The depressed mitogen responsiveness of lymphocytes cultured in the presence of leukemic plasma was due to the presence of inhibitory factor(s) present in the plasma rather than the absence of components present in normal plasma. The inhibitory effect of leukemic plasma on periodate-induced cell stimulation indicated that the leukemic plasma inhibitory factor(s) exert their action very rapidly and directly on the cell. Transfer of the inhibitory factor(s) from the leukemic plasma to the cultured cells was supported by the finding that the depressive action of leukemic plasma on lectin and non-lectin mitogen-induced transformation of lymphocytes was reduced if the leukemic plasma was preincubated with either resting or mitogen-treated lymphocytes prior to testing with lymphocytes not previously exposed to leukemic plasma or mitogen.

Rat dendritic cells function as accessory cells and control the production of a soluble factor required for mitogenic responses of T lymphocytes.

Transformation of T lymphocytes, induced by treatment with periodate or with neuraminidase plus galactose oxidase, requires the participation of accessory cells. Procedures were developed for the fractionation of rat lymph node cells, by which most of the lymphocytes can be recovered as a major population of cells that do not respond to mitogenic stimulation unless accessory cells from a separated minor population are added. Further purification led to a 1000-fold overall increase in accessory activity per cell, with a 50-70% yield. The purest preparations were virtually free of macrophages and contained more than 90% typical dendritic cells. Maximum responses occurred at a ratio of only one dendritic cell per 200 periodate-treated lymphocytes. This evidence thus indicates strongly that in rats, dendritic cells--not macrophages--function as accessory cells. Further, the number of dendritic cells in a preparation governed the magnitude of the mitogenic response and was limiting in the case of unfractionated lymph node cells. In addition, when oxidized with periodate or with neuraminidase plus galactose oxidase, the dendritic cell served as a very potent indirect stimulator of untreated responder lymphocytes. Both functions of the dendritic cell appeared to lack species specificity, since mouse dendritic cells were very active when tested with rat responder lymphocytes. A soluble factor (accessory cell-replacing factor), produced by cultures of lymph node or spleen cells subjected to oxidative mitogenesis, enabled otherwise unresponsive mitogen-treated lymphocytes to respond. Dendritic cells were required for the production of this factor but may not be solely responsible for its production.

Regulation of Sodium and Potassium Transport in Phytohemagglutinin-Stimulated Human Blood Lymphocytes

Phytohemagglutinin (PHA) or concanavalin A treatment of lymphocytes causes an increase in membrane permeability so that the leak rates of Na and K increase 1.5- to 2-fold. Active Na and K transport increase proportionately in response to the increased membrane permeability. We have examined the role of lymphocyte Na concentration in sustaining the increased Na and K transport observed after PHA treatment. Cell Na concentration increases from 14.8 to 20.5 mmol/liter cell water in PHA-treated lymphocytes (P < 0.001). Four lines of evidence suggest that the 5-6 mmol/liter cell water increase in lymphocyte Na accounts for the increase in active Na and K transport in mitogen-treated lymphocytes. First, PHA does not increase directly the maximal Na, K-ATPase activity of isolated lymphocyte membrane vesicles. Second, when the Na concentration is increased by 6 mmol/liter cell water in unstimulated lymphocytes, Na and K transport increase nearly twofold. Third, the cell Na concentration (15 mmol/liter cell water) is near the K(m) for Na activation of the Na, K-ATPase in lymphocyte membranes. The ATPase activity thus, is capable of increasing as the cell Na rises above normal. Fourth, if lymphocytes are incubated in a medium containing a low Na concentration, K transport does not maintain the internal K concentration and the fall in cell K is accentuated in PHA-treated lymphocytes. These studies indicate that the adaptive acceleration of Na and K transport in mitogen-treated lymphocytes is mediated by a small increase in cell Na.

Stimulation and inhibition of human T-lymphocyte colony cell proliferation by hemopoietic cell factors

Human lymphocytes, isolated from peripheral blood and stimulated with phytohemagglutinin M (PHA) prior to being seeded on a two-layer medium of soft agar which contained the mitogen, developed into colonies 3–4 days after seeding in the culture system. The cloning potential of PHA-treated lymphocytes is significantly enhanced by adding, to the soft agar culture, culture fluid (CF) obtained from mitogen-treated lymphocytes or a feeder layer (FL) prepared either from lymphocytes isolated from peripheral blood or from T-cell enriched populations. PHA seems to stimulate the release of lymphocyte colony enhancing factor (LCEF) from the T-sensitized lymphocytes. The addition of CF or FL to the culture medium appears to increase the amount of LCEF, resulting in enhancement of the number and size of lymphocyte colonies. When CF derived from spleen cells or from the peripheral blood adherent-cell population was added to the lower layer of the soft agar culture, the growth and development of lymphocyte colonies was inhibited. This suggests that monocyte-macrophages release a lymphocyte colony inhibiting factor (LCIF) into the CF. The extent of inhibition or stimulation of colony formation is a function of the number and type of cells used to prepare the CF or FL and the concentration of CF in the culture medium. The presence of FL or CF derived from spleen non-adherent cells, white blood cells, bone marrow cells, or a B-cell enriched population had no effect on colonies growing in the culture. This may possibly be due to the paucity of T lymphocytes and monocyte-macrophages present in these materials. A control system in which LCIF, produced by monocyte-macrophages, and LCEF, produced by T lymphocytes, participate in the regulation of lymphocyte production is postulated.