Two categories of lymphocyte unresponsiveness to phytohemagglutinin

Peripheral lymphocytes from healthy subjects, sarcoidosis and influenza patients were studied in vitro by measurement of the tritiated thymidine uptake of unstimulated and phytohemagglutinin. (PHA) stimulated cells. When the mitogen induced metabolic response is defined as the ratio between thymidine uptake by stimulated and unstimulated cells (stimulation index), PHA responsiveness was significantly decreased in both diseases and varied inversely with the level of isotope incorporated by unstimulated cells (p = 0.0002). The uptake of isotope by unstimulated cells from influenza patients was significantly increased (p = 0.0001). Isotope incorporation by mitogen stimulated cells from the same patients did not differ significantly from controls (p = 0.0925). In contrast, the impaired PHA responsiveness of lymphocytes from sarcoidosis patients was associated with levels of isotope incorporation in unstimulated cell cultures similar to those observed in healthy controls (p = 0.6444). These observations suggest that two different mechanisms may be responsible for low lymphocyte PHA stimulation indices associated with disease states. Methods are presented for minimizing variation of replicate observations and identification of both categories of lymphocyte unresponsiveness.     

Cytokine production by human colonic intraepithelial lymphocytes in controls and ulcerative colitis

Using an ELISA technique, concentrations of gamma-interferon and interleukin-2 were assayed in the supernatants of colonic intraepithelial lymphocytes cultured with or without phytohaemagglutinin (PHA). IntraepitheHal lymphocytes produced low concentrations of gamma-interferon and interleukin-2 when stimulated with PHA, but significantly more than when unstimulated (p < 0.05). There was no difference in production of these cytokines by IEL from control or inflammatory bowel disease.     

5-氮胞苷对贵州小型猪淋巴细胞DNA损伤及修复的影响

目的　研究贵州小型猪淋巴细胞对化学物或药物引起的DNA损伤及修复影响的反应。方法　用单细胞凝胶电泳技术检测比较 5 氮胞苷对PHA刺激和未刺激淋巴细胞的DNA损伤及其修复过程。结果　 5 氮胞苷引起未刺激淋巴细胞明显的DNA泳动 (彗星尾 ) ,经修复孵育 2h后 ,DNA泳动与孵育前比较无显著差异 ,而 5 氮胞苷引起的刺激细胞DNA泳动经 2h修复孵育后与孵育前比较显著减少。结论　 5 氮胞苷引起贵州小型猪未刺激淋巴细胞DNA损伤经 2h孵育未能修复 ,而刺激细胞的DNA损伤明显修复。     

Induction of premature chromosome condensation by a phosphatase inhibitor and a protein kinase in unstimulated human peripheral blood lymphocytes: a simple and rapid technique to study chromosome aberrations using specific whole-chromosome DNA hybridization probes for biological dosimetry

We developed a simple and rapid method to study chromosome aberrations involving specific chromosomes using unstimulated human peripheral blood lymphocytes (HPBL). Premature chromosome condensation (PCC) was induced by incubating unstimulated HPBL in the presence of okadaic acid (OA, a phosphatase inhibitor), adenosine triphosphate (ATP), and p34(cdc2)/cyclin B kinase [an essential component of mitosis-promoting factor (MPF)], which eliminated the need for fusion with mitotic cells. OA concentration and duration of incubation for PCC induction was optimized using mitogen-stimulated HPBL; a final concentration of 0.75 microM incubated for 3 h was optimum, resulting in approximately 20% PCC yield. In unstimulated HPBL, PCC was induced by the addition of p34(cdc2)/cyclin B kinase at concentrations as low as 5 units/ml to a cell culture medium containing OA. Increases in the concentration of p34(cdc2)/cyclin B kinase from 5 to 50 units/ml resulted in a concentration-dependent increase in PCC yield (30% to 42%). We demonstrate that this technique of inducing PCC in unstimulated HPBL is suitable for studying radiation-induced aberrations involving a specific chromosome (chromosome 1) after 24 h repair using a whole-chromosome in situ hybridization probe and chromosome painting. Cells with aberrant chromosome number 1 are characterized with more than two chromosome spots. The frequency of cells with aberrant chromosome 1 increased with 60Co gamma-radiation doses in the region 0-7.5 Gy. The observed dose-effect relationship for the percentage of cells with aberrant chromosome 1 (Y) was explained by using both a linear [Y=(2.77+/-0.230)D+0.90+/-0.431, r(2)=0.966] and a nonlinear power [Y=(5.70+/-0.46)D((0.61+/-0.05)), r(2)=0.9901) model. This technique can be applied to biological dosimetry of radiation exposures involving uniform whole-body low linear energy transfer (LET) exposures.     

Lymphocyte reactivity in pregnant women and newborn infants.

The mitotic response to phytohaemagglutinin (PHA) was determined in lymphocytes of mothers and their newborn infants obtained at delivery and seven days later by measuring the rate of 125 I-idoxuridine uptake into DNA in lymphocytes cultured in their own plasma and after washing and resuspension in fetal bovine serum. There was no difference in the unstimulated counts of maternal lymphocytes taken at delivery, whether unwashed or washed, compared with those from nonpregnant controls. With PHA stimulation the mitotic response of the maternal lymphocytes cultured in their own plasma was reduced compared with that of the control lymphocytes but washed maternal cells showed a similar response to the controls. These findings suggest that the reduced lymphocyte mitotic response to PHA in pregnancy is due to a plasma inhibitory factor This inhibition was not evident in maternal blood taken seven days after delivery. DNA synthesis in unstimulated cultures from newborn infants at birth and seven days after birth was greater than that in adult control cultures. With PHA stimulation the mitotic response of cord-blood lymphocytes cultured in their own plasma paralleled that of control lymphocytes but washed newborn cells showed a greater response. Thus plasma suppression similar to that observed in the mother seems also to affect infants at birth. This inhibition was not demonstrable in blood taken from infants of 7 days.     

Direct analysis of the clastogenic effect of formaldehyde in unstimulated human lymphocytes by means of the premature chromosome condensation technique

The cytogenetic effect of formaldehyde (FA) on unstimulated human lymphocytes was studied by means of conventional chromosome analysis and the premature chromosome condensation (PCC) technique. In first post-treatment metaphases no significantly increased yields of chromosomal changes could be observed. The analysis of PCCs, however, showed high yields of chromosome fragments. Bleomycin (BLM) used as positive control was also highly clastogenic in PCCs and resulted in significantly increased yields of chromosome-type aberrations. As recently argued, a premitotic selection against heavily damaged cells could be an explanation for the discrepancy between the chromosome findings in metaphase and PCC analysis after FA treatment. In addition, a differential effectiveness may exist in unstimulated lymphocytes to convert multiple fragmentation into chromatid- or chromosome-type aberrations through S-phase-dependent or S-phase-independent mechanisms.     

The chronic lymphatic leukemia lymphocyte: Correlation of functional,metabolic, and surface immunoglobulin characteristics

This study determined the correlation between the functional capacity of chronic lymphatic leukemia lymphocytes as determined by their response to nonspecific mitogens with their glucose metabolism and surface immunoglobulin characteristics. A majority of patients (12) were found to have lymphocytes with impaired transformation to both PHA and pokeweed mitogens. These cells also had impaired glucose metabolism in unstimulated cultures and failed to have the striking increase in glucose metabolism in response to mitogens which is characteristic of normal lymphocytes. Most of these lymphocytes had IgM surface immunoglobulins. However, we were not able to demonstrate surface immunoglobulins on the lymphocytes of one patient in this group. Two patients were found to have lymphocytes with normal lymphoblastic transformation to PHA and impaired transformation to pokeweed suggesting cells of T origin. The glucose metabolism of these lymphocytes were less impaired in unstimulated cultures than those of the other patients and had a striking increment in glucose metabolism in response to PHA similar to normal lymphocytes. Unexpectedly, these lymphocytes were found to have IgG on their surface suggesting cells of B origin. These results indicate that there may be two groups of CLL patients with clinically similar disease in whom the functional and metabolic characteristics of the lymphocytes are distinct and that the surface immunoglobulin characteristic of lymphocytes may not always predict their functional characteristic.     

Tyrosine phosphorylation of a 66,000 Mr soluble protein in lectin-activated human peripheral blood T lymphocytes

Protein phosphorylation was studied in human T lymphocytes stimulated with the mitogenic lectins phytohemagglutinin (PHA) and concanavalin A (Con A). The T lymphocytes were prepared from the venous blood of normal volunteers, their intracellular ATP pools were labeled with [32P]orthophosphate, and protein phosphorylation was assayed in the soluble fraction by two-dimensional gel electrophoresis and autoradiography. When lymphocytes stimulated with PHA or Con A were compared to unstimulated control cells, there was a general increase in protein phosphorylation and the specific phosphorylation of a soluble protein with Mr = 64.9 to 69 KD and pI = 5.6 to 5.8. Phosphorylation of this protein, designated TPP-66, was observed as early as 2 min after the addition of lectin with a gradual increase in the level of phosphorylation over the next 120 min. In the majority of experiments, there was no phosphorylation seen in the unstimulated lymphocytes; however, in some experiments, there was appreciable phosphorylation, which was seen beginning 60 min after the labeling period. When the TPP-66 spot from stimulated lymphocytes was excised from gels, was eluted, and was subjected to limited base hydrolysis followed by single-dimension high voltage electrophoresis, the major phosphorylated residue migrated with phosphotyrosine. In some experiments, there was phosphorylation of serine residues in both the stimulated and control cells; tyrosine phosphorylation was never seen in the unstimulated cell population. These data suggest that, like other stimuli for cell growth, the induction of lymphocyte growth by lectins is associated with the activation of a tyrosine-specific kinase. Thus, tyrosine phosphorylation may play a key role in the transmission of the signal for lymphocyte growth from the exterior to the interior of the cell.     

The role of serine hydroxymethyltransferase in cell proliferation: DNA synthesis from serine following mitogenic stimulation of lymphocytes

It is shown that the time-course of incorporation of radioactivity from [3-¹⁴C]serine into nucleic acids parallels DNA synthesis following mitogenic stimulation of human peripheral blood lymphocytes by phytohaemagglutinin (PHA). The activity of serine hydroxymethyltransferase was elevated about four-fold in PHA-stimulated lymphocytes compared to that in unstimulated control ceils. It is suggested that lymphocytes, in common with other proliferating cell systems:, may synthesize serine de novo for utilization in pathways of nucleotide biosynthesis following mitogenic stim--ulation.     

Delayed repair of DNA single-strand breaks does not increase cytogenetic damage

DNA damage and cytogenetic effects of ionizing radiation were investigated in Chinese hamster ovary (CHO) cells and unstimulated human peripheral blood lymphocytes. DNA damage and repair were analysed by alkaline elution under conditions that predominantly measured DNA single-strand breaks (ssb). X-radiation (2.5 Gy) induced ssb in both CHO cells and unstimulated lymphocytes, and the breaks were repaired within 30 and 90 min, respectively. This rapid repair was delayed by the poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide (3AB). The cytogenetic effects of the 3AB-induced delay in DNA repair were examined by analysing sister chromatid exchange (SCE) frequency in CHO cells and fragmentation of prematurely condensed chromosomes (PCC) in unstimulated human lymphocytes after 2.5 Gy of X-rays. Although 3AB delayed the rejoining of DNA ssb, this delay did not result in increased cytogenetic damage manifested as either SCE or fragmentation of PCC. These results indicate that the rapidly rejoining DNA ssb are not important in the production of chromosome damage.     

Characteristics of calcium accumulation by lymphocytes and alterations in the process induced by phytohemagglutinin

Calcium uptake by normal human lymphocytes was found to be a saturable process which was competitively inhibited by manganese indicating the existence of a carrier-mediated mechanism for calcium uptake. Exchange diffusion was not observed, Phytohemagglutinin (PHA) significantly stimulated calcium uptake within minutes after treatment. The increased uptake was attributed to a decreased K_m for the proposed membrane carrier rather than to an increased V_max. Also PHA did not stimulate a normally unused exchange diffusion process, nor did it affect calcium efflux. Uptake by both unstimulated and PHA-treated lymphocytes was not influenced by magnesium or by cycloheximide or actinomycin D.     

Mammalian cell fusion. 3. A HeLa cell inducer of premature chromosome condensation active in cells from a variety of animal species

The phenomenon of premature chromosome condensation (PCC) is induced in unstimulated horse lymphocytes, bovine spermatozoa, Chinese hamster ovary cells, embryonic chick fibroblasts and erythrocytes, Xenopus kidney and mosquito cells by fusing each of these cell types with HeLa cells blocked in mitosis. Thus it becomes possible to visualize chromosomes even from non-multiplying cells of heterologous species, such as, chick erythrocytes and bovine spermatozoa.     

Modulation of SCE induction and cell proliferation by 2-mercaptoethanol in phytohemagglutinin-stimulated rat lymphocytes

2-Mercaptoethanol (2-ME) is used as a medium supplement to enhance the proliferation of lymphocytes culturedin vitro. In this study, we have examined the effects of 2-ME on cell growth and on SCE induction in cultures of unstimulated and phytohemagglutinin (PHA)-stimulated Fischer 344 rat lymphocytes. There were virtually no metaphases detected in cells cultured without PHA. In PHA-stimulated cultures, 2-ME decreased SCE-frequency but it enhanced SCE frequency in the presence of S to 12.5 µM bromodeoxyuridine (BRd U). Both mitotic and replication indices were increased in the PHA/2-ME system. The levels of incorporated exogenous thymidine, in the presence of 2-ME, were relatively low in unstimulated cells, suggesting that 2-ME is not mitogenic for T-cells. However, 2-ME enhanced PHA-induced response of T-cells as evidenced by increased levels of thymidine incorporation into cellular DNA. The growth promoting effects and the decrease in SCE frequency caused by 2-ME upon PHA stimulation indicate that 2-ME may alter the nature of interaction between PHA and cellular activating properties or the replicative processes.Abbreviations BRdU bromodeoxyuridine - FBS fetal bovine serum - SCE sister-chromatid exchanges - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - IL-2 interleukin-2 - 2-ME 2-mercaptoethanol - PBS phosphate buffered saline - PHA phytohemagglutinin - MI mitotic index - RI replication index - NADH nicotinamide adenine dinucleotide (reduced form)     

Quiescent human lymphocytes do not contain DNA strand breaks detectable by alkaline elution

On the basis of qualitative assays, quiescent lymphocytes have previously been reported to have numerous DNA strand breaks, which are thought to be repaired after mitogenic stimulation by a process associated with poly(ADP-ribosyl)ation. Using alkaline elution, a very sensitive assay for quantifying DNA single-strand breakage, we found no evidence for a high frequency of DNA strand breaks in unstimulated human peripheral blood lymphocytes. No differences in elution profiles were observed between unstimulated lymphocytes and lymphocytes 4 or 48 h after addition of the mitogen phytohemagglutinin (PHA). Furthermore, addition of 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) synthetase, or aphidicolin, an inhibitor of DNA polymerase alpha, did not increase the amount of DNA eluting from the filter after PHA stimulation. In contrast to reported studies of mouse splenic lymphocytes, we found that human lymphocytes were able to replicate and divide in the presence of the ADP-ribosylation inhibitor. Human lymphocytes were also capable of proliferating in nicotinamide-free medium, with or without 3AB, indicating that ADP-ribosylation is not a requirement for lymphocyte differentiation. We therefore consider it unlikely that peripheral human lymphocytes contain significant numbers of strand breaks that play any role in their stimulation or differentiation in response to PHA.     

Effect of increased PHA synthase activity on polyhydroxyalkanoates biosynthesis in Synechocystis sp. PCC6803

Polyhydroxyalkanoate (PHA) synthase activity in Synechocystis sp. PCC6803 was increased two-fold by introducing the PHA biosynthetic genes of Ralstonia eutropha. The resulting recombinant Synechocystis sp. PCC6803 strain was subjected to conditions that favor PHA accumulation and the effects of various carbon sources were studied. In addition, the fine structure of both wild-type and recombinant Synechocystis sp. PCC6803 was examined using freeze-fracture electron microscopy technique. The PHA granules in the recombinant Synechocystis sp. PCC6803 were localised near the thylakoid membranes. Maximum amount of PHA accumulation was obtained in the presence of acetate, where the number of granules in the recombinant cells ranged from 4 to 6 and their sizes were in the range of 70-240 nm. In comparison to wild-type Synechocystis sp. PCC6803, recombinant cells with increased PHA synthase activity showed only a marginal increase in PHA content suggesting that PHA synthase is not the rate limiting enzyme of PHA biosynthesis in Synechocystis sp. PCC6803.     

Purine nucleotide metabolism in phytohemagglutinin-induced human T lymphocytes

The comprehensive studies of purine nucleotide metabolism were done in nonstimulated and phytohemagglutinin (PHA)-stimulated human peripheral blood T lymphocytes. Nonstimulated lymphocytes synthesize nucleotides in two alternative pathways: via biosynthesis de novo and salvage pathways. Although synthesis of triphosphonucleosides in unstimulated lymphocytes was the predominant pathway, interconversion of monophosphonucleosides was also active. Exposure of cells to PHA affects differently various pathways of nucleotide metabolism. The most marked changes observed were rapid activation of purine salvage within minutes after exposure to PHA, and significant increase of 5-phosphoribosyl-1-pyrophosphate levels. In addition, significant increases were found in de novo purine biosynthesis, nucleotide interconversions, and RNA and DNA synthesis, whereas catabolism of nucleotides remained unchanged. These results indicate that PHA activation of T lymphocytes causes a rapid synthesis of nucleotides which may be required immediately for increases in energy metabolism and later as the precursors of nucleic acid synthesis.     

Oxalyl thiolester concentrations decreased in lectin and phorbol ester-stimulated lymphocytes

Oxalyl thiolesters (OTEs) are a newly discovered class of mammalian metabolites that are believed to function in controlling animal metabolism and possibly serve as intracellular mediators for some hormones. Previous correlations had suggested that the concentrations of OTEs might be decreased when cells are stimulated to proliferate, and in our research that was found to be the case. Thus, when bovine lymph node lymphocytes are stimulated either with concanavalin A (Con A) or with a combination of phytohemagglutinin (PHA) and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), the concentration of OTEs in the lymphocytes decreases within 3 h by a factor of approximately two. With either PHA or TPA alone, the decrease in OTE concentration is considerably smaller. With Con A as stimulant, the OTE levels decrease within 1 h and remain low for at least 24 h. It was also noted that the concentration of OTEs in unstimulated isolated lymphocytes is significantly lower in lymphocytes obtained from 2-year-old animals than in lymphocytes obtained from older animals. The results of the current investigation, when considered in conjunction with other recent results, suggest that OTEs may be natural cell proliferation inhibitors.     

Comparative study of intracellular glutathione content in rat lymphocyte cultures treated with 2-mercaptoethanol and interleukin-2

The level of intracellular glutathione (GSH) in mitogen-stimulated mouse lymphocytes is increased in the presence of 2-mercaptoethanol (2-ME), an enhancer of lymphocyte activation and proliferation. Since proliferation of lymphocytes in response to mitogens involves direct activation by a mitogen followed by continued proliferation in response to interleukin-2 (IL-2), we have investigated the effect of 2-ME and exogenous IL-2 on the GSH content and cell proliferation of rat lymphocytes stimulated with phytohemagglutinin (PHA). PHA stimulation increased both GSH content and the magnitude of the proliferative response, as measured by thymidine incorporation into cellular DNA. However, incubation of stimulated lymphocytes with 2-ME or IL-2 for 72 hr produced a significant further elevation of GSH levels and thymidine incorporation. 2-ME also increased the GSH content in unstimulated cultures, but it had little effect on thymidine incorporation. IL-2 increased GSH content and decreased thymidine incorporation in unstimulated lymphocytes. Exposure of cells to DL-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of GSH biosynthesis, significantly depleted GSH and lowered the proliferative response, suggesting a crucial role of de novo GSH synthesis for lymphocyte activation. The data suggest that both 2-ME and IL-2 promote lymphocyte proliferation, although the mechanisms by which intracellular GSH levels are increased by the agents are apparently different.Copies of articles are available through ISI Document Delivery Services c/o The Genuine Article, 3501 Market Street, Philadelphia, PA 19104.     

Culture conditions affecting induction and release of lymphocyte produced proliferation inhibitory factor (PIF)

Studies on the factors affecting the production of a proliferation inhibitory factor (PIF) by human lymphocytes are presented. Maximal PIF production occurred with mitogen stimulation of blood lymphocytes cultured at 1 × 10⁶/ml. Optimal cultures contained 10% fetal calf serum, but PIF could be produced in the absence of serum, and after only a 6-hr pulse exposure to PHA. PIF production was found to correlate with lymphocyte activation in response to the mitogen PHA but was not related to lymphocyte proliferation (DNA synthesis). Inhibitory activity could be detected as early as 3 hr after mitogen addition, long before DNA synthesis occurs. The mitogens Con A and PWM initiated different intensities of DNA synthesis in these cultures, but similar quantities of PIF. Antigenic stimulation of sensitive human peripheral lymphocyte populations resulted in the release of PIF. Cells from donors that gave a strong positive skin test to tuberculin (PPD) responded in tissue culture to PPD by producing PIF, while the cells from skin test negative donors did not. A small quantity of PIF was also evident in the supernatants from cultures with no known stimulus (“unstimulated”), this was found to result from activation of the lymphocytes by nonlymphoid elements and by fetal calf serum. An investigation of the PIF-producing capabilities of other lymphoid tissues showed that lymph node cells produced this humoral factor, whereas thymus cells did not. Thymus cell supernatants, in fact, were found to contain an extremely labile cytotoxin which degraded rapidly upon storage.     

Formation of multilayer rosettes in phytohemagglutinin-stimulated lymphocytes: correlation with chromatin condensation conformation in premature chromosome condensation

We found that the formation of multilayer rosettes by transformed human blood lymphocytes after phytohemagglutinin (PHA) stimulation is correlated with conformational changes of the chromatin as seen by premature chromosome condensation (PCC). The frequency distribution of grades of PCC and multilayer rosette formation suggests that changes in chromatin are a prerequisite for rosette formation. Rosette formation was most pronounced for 24-h and 48-h cultures. Chromatin decondensation and rosette formation showed identical patterns. The possibility that multilayer rosette formation is directly dependent on conformational changes of chromatin is discussed.