The influence of interleukin-2, feeder cells, and timing of irradiation on the radiosensitivity of human T lymphocytes assessed by the colony-forming assay

The radiosensitivity of human lymphocytes was investigated by the method of colony formation in the absence of interleukin-2 (IL2) and feeder cells, both of which enhance growth of T-cell colonies. The shape of the survival curve and the radiosensitivity was shown to depend upon the ability of lymphocytes to produce IL2: the survival curve for lymphocytes that were the most competent producers of IL2 is the closest to linearity; the lymphocytes that were poor producers show biphasic survival curves. The radiosensitivity of the lymphocytes from the first group is less than that of the latter, when the comparison is based on the first part of the biphasic survival curve. This is more easily seen when cultures are irradiated 24 h after stimulation by phytohemagglutinin (the time of the peak IL2 production) than when cultures are irradiated 2 h before stimulation. This study demonstrates that growth conditions influence the response of lymphocytes to irradiation and that optimal growth conditions result in a linear survival curve.     

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.     

The influence of cell cycle kinetics on the radiosensitivity of Down's syndrome lymphocytes

In agreement with previous work, [60Co]gamma-irradiation shortly after phytohemagglutinin (PHA) stimulation, induces higher frequencies of chromosomal aberrations in trisomy 21 lymphocytes compared to normal controls. However, equal frequencies of chromatid aberrations are induced in fully-stimulated trisomy 21 and normal lymphocytes by irradiation during G2. We have observed that trisomic lymphocytes respond more rapidly to PHA stimulation than normal lymphocytes. Furthermore, we have observed that chromosomal radiosensitivity increases as a function of time after PHA stimulation in normal lymphocytes. When normal lymphocytes are irradiated 8 h after PHA stimulation, the frequencies of chromosomal aberrations induced are comparable to those induced in trisomy 21 lymphocytes irradiated 30 min after PHA stimulation.     

X射线对人外周血淋巴细胞DNA损伤修复和凋亡的影响

目的 研究不同时间诱导X射线照射的淋巴细胞进入细胞周期DNA损伤修复与凋亡的影响.方法 X射线（0.5 Gy）作用于正常人外周血淋巴细胞,以照射后不同时间点（0、4 h）分别加入PHA并分成两组,即照射后0 h加PHA组（A组）和照射后4 h加PHA组（B组）,再分别培养0、0.5、2 h,用流式细胞术和免疫印迹法检测A组和B组γ-H2AX蛋白的表达,Annexin-V/PI法分析A、B两组的细胞凋亡率.结果 流式细胞术及免疫印迹结果均显示A组的γ-H2AX蛋白表达高于B组（P＜0.05）,且均先升高后降低.A组细胞凋亡率亦大于B组.结论 不同时间诱导被打击的淋巴细胞进入周期其可能发生DNA修复并同时伴随细胞凋亡的发生.     

Translational activity of messenger ribonucleic acid isolated from unstimulated and phytohaemagglutinin-activated lymphocytes.

Purified cytoplasmic poly(A)+ RNA isolated from unstimulated pig lymphocytes has the same ability to direct translation in a range of cell-free systems as the corresponding mRNA from 20h phytohaemagglutinin-activated lymphocytes. Additional methylation of the mRNA is not required for maximum protein synthesis in the wheat-germ cell-free system. Misleading results are obtained if the mRNA preparations used are not adequately purified, and a method suitable for routine assessment of the degree of purification achieved is described. Cell-free protein-synthesizing systems from unstimulated lymphocytes translate added lymphocyte mRNA with lower efficiency than do comparable systems from phytohaemagglutinin-activated lymphocytes, whatever the source of the mRNA used.     

Increased chromosomal instability in lymphocytes from elderly humans

Lymphocytes from young and old donors were incubated with PHA for 96 h and exposed to [3H]Tdr during the last 24 h of culture. Comparable amounts of [3H]Tdr were incorporated into chromosomes of old and young lymphocytes as measured by autoradiography of metaphase chromosomes. However, chromosomal damage and cell-cycle arrest were far greater in lymphocytes from old as compared to young humans. The frequency of chromosome breaks, fragments, exchange figures and dicentric chromosomes induced by [3H]Tdr was greater in cultures from old than in cultures from young humans. Lymphocytes from old donors exposed to 20 microM BrdU during the last 24 h of culture showed significantly more sister-chromatid exchanges than did lymphocytes from young donors. These data suggest that chromosomes in lymphocytes from old donors express more damage after exposure to [3H]Tdr or BrdU than do chromosomes in lymphocytes from young donors.     

Identification of IL-8 as a locomotor attractant for activated human lymphocytes in mononuclear cell cultures with anti-CD3 or purified protein derivative of Mycobacterium tuberculosis.

On culture of human blood mononuclear cells for 24 to 48 h with anti-CD3 (aCD3) or purified protein derivative of Mycobacterium tuberculosis, chemoattractants are released into the medium which induce polarization and locomotion of activated (G1) lymphocytes but not resting lymphocytes. Here we show that, during a period of up to 72 h of culture, IL-8 is released in nanomolar quantities into the supernatant and that the lymphocyte chemoattractant activity of these supernatants is inhibited by incubation with anti-IL-8. Examination of the cultured mononuclear cells by immunofluorescence suggests that many monocytes, but almost no lymphocytes in aCD3 cultures contain IL-8 in cytoplasmic organelles, yet few monocytes direct from blood stained for IL-8. IL-8 is an attractant for only a small proportion (ca 10%) of lymphocytes direct from blood. The proportion of responding cells is increased after culture for 24 to 48 h in aCD3 or purified protein derivative of Mycobacterium tuberculosis, and these are a phenotypically distinct subpopulation consisting of large lymphocytes enriched for CD45RO. These cells respond to their own culture supernatants and to IL-8 in polarization assays and by invasion of collagen gels into which the attractants are incorporated. They also show orientation to a source of IL-8 in a chemotactic gradient. These responses are consistent with in vivo observations that the lymphocytes which migrate selectively into inflammatory sites are activated. The fact that many lymphocytes do not respond to IL-8 may reflect the diversity of migratory pathways shown by lymphocytes in vivo, the locomotion of small, recirculating, lymphocytes being regulated by other, unknown, locomotor stimuli.     

Differentiated cells from BALB/c mice differ in their radiosensitivity

Various isolated cells of an inbred mouse strain (BALB/c) differed widely in their sensitivity to gamma irradiation: fibroblasts are five times more resistant than peripheral lymphocytes. Among lymphocytes, T cells are more resistant than B cells. Cell lines derived from the primary cells conserved their radiosensitivity. Cytofluorometric measurements show that the differential reaction of a cell to gamma irradiation can be detected already 2–3 h after the irradiation event. Radiation-sensitive cells are delayed for a longer time in S phase and G2 phase of the cell cycle than radiation-resistant cells. No difference in the capacity of the cells to perform single-strand break repair, double-strand break repair or unscheduled DNA synthesis could yet be detected.     

High-potentiality preliminary selection criteria and transformation time-dependent factors analysis for establishing Epstein-Barr virus transformed human lymphoblastoid cell lines

Infection of freshly isolated and cryopreserved lymphocytes with Epstein-Barr virus (EBV) leads to the establishment of human B lymphoblastoid cell lines (LCLs). Techniques for optimal infection of the lymphocytes are vital for the establishment of a human biobank. The present study found that more than half (58-86%) of such established LCLs had transport times of less than 48 h, cell densities exceeding 10(6) cells/ml and cell viabilities greater than 90%. After EBV infection, 3306 freshly isolated lymphocytes required 30.0 +/- 0.1 days to become LCLs. Conversely, 1210 cryopreserved lymphocytes required 36.2 +/- 0.4 days. Cell density and viability of the culture affected transformation time in freshly isolated lymphocytes. On the other hand, blood transport time, cryopreservation time and initial cell viability were major factors in cryopreserved specimens. These results contribute to general information concerning the establishment of a human biobank for EBV infected cells.     

Perforin, granzyme B, and FasL expression by peripheral blood T lymphocytes in emphysema

Background

It is generally accepted that emphysematous lungs are characterized by an increase in the numbers of neutrophils, macrophages, and CD8⁺ T lymphocytes, the lasts having increased cytotoxic activity. Because systemic inflammation is also a component of emphysema, we hypothesize that peripheral CD8⁺ T lymphocytes of emphysematous smokers who show evidence of systemic inflammation will have higher expression of cytotoxic molecules.

Methods

We assessed parameters of systemic inflammation in normal individuals (smokers or non-smokers) and in emphysematous subjects with an active smoking history by measuring serum interleukine-6, C-reactive protein, and tumor necrosis factor. Expression of perforin, granzyme B, and FasL protein by CD8⁺ T lymphocytes, CD4⁺ T lymphocytes, and natural killer cells were assessed by flow cytometry while perforin, granzyme B, and FasL mRNA expression were measured on purified systemic CD8⁺ T lymphocytes by real-time PCR.

Results

Emphysematous smokers had higher levels of serum interleukine-6 than normal subjects. Even with the presence of systemic inflammation in emphysematous smokers, the percentage of peripheral CD8⁺ T lymphocytes, CD4⁺ T lymphocytes, and NK cells expressing perforin and granzyme B protein was not different between the three groups.

Conclusion

Despite evidence of systemic inflammation, peripheral T lymphocytes of emphysematous smokers did not show higher levels of cytotoxic markers, suggesting that increase of activated T lymphocytes in the emphysematous lung may be due to either activation in the lung or specific peripheral recruitment.     

Molecular distribution of cytoplasmic zinc in phytohemagglutinin-stimulated and unstimulated human lymphocytes

Human peripheral blood lymphocytes were incubated with [⁶⁵Zn] zinc transferrin and with and without phytohemagglutinin for 1, 2, 4, 10, 24, 48, 72, and 96h. Gel filtration of cytoplasmic fractions obtained from these lymphocytes was then performed to determine the molecular distribution of incorporated zinc as a function of time in culture. The data obtained indicated that: (1) transferrin-bound zinc incorporated by human lymphocytes is associated with a variety of soluble molecules whose molecular weights range from less than 5,000 to greater than 70,000 daltons; (2) there is a time-dependent change in the distribution of cytoplasmic zinc for both phytohemagglutinin-stimulated and unstimulated lymphocytes; and (3) for all times studied, there is a difference in the elution profiles obtained for phytohemagglutinin-stimulated and unstimulated lymphocytes. Furthermore, lymphocytes from a donor with untreated hairy cell leukemia exhibited a totally different pattern of cytoplasmic zinc distribution than did lymphocytes from apparently healthy donors.     

Effect of treatment with azathioprine on the responses of rat lymphocytes to phytohaemagglutinin.

The proliferative responses of rat peripheral blood lymphocytes (PBL) and spleen cells to phytohaemagglutinin (PHA) were studied after single or multiple (daily for 4 days) injections of azathioprine (AZ). Lymphopenia developed within 4 h of a single dose (78 mg/kg) of AZ and persisted for at least 72 h. There was no lymphopenia 24 h after the last of 4 daily injections. In vitro, PBL were more sensitive than spleen cells to the inhibitory effect of AZ. Likewise, the responses of PBL were relatively more depressed than those of spleen cells after single or multiple injections of AZ. The degree of depression was less than was expected from the effect of AZ in vitro. Multiple small doses were more depressive than multiple large doses. Serum from treated rats, used at 20% concentration, was more depressive than normal. Thus, rat lymphocytes are quite sensitive to AZ in vitro, but appear to be relatively resistant in vivo, this resistance resembling the resistance of the primary antibody response to AZ treatment.     

Metabolism of polyadenylated mRNA in growing human lymphocytes.

The kinetics of degradation of newly synthesized, cytoplasmic polyadenylated RNA have been examined in normal human lymphocytes stimulated to grow with phytohemagglutinin. A single class of poly(A)-bearing RNA was identified with a half-life of approximately 50 h. In the presence of actinomycin D, the half-life was 5 to 6 h, and virtually no decay of pulse-labeled material was detectable after 6 h of chase incubation with cordycepin. These findings contrast sharply with data obtained from other growing human cells used as controls: polyadenylated mRNA in MOLT-4 cells, a cultured line of T lymphocytes, had a half-life of 2 h in the presence of actinomycin D. The stability of poly(A)-containing RNA in stimulated lymphocytes from normal donors is therefore not simply a manifestation of cell proliferation. In normal resting lymphocytes, Berger and Copper [(1975) Proc. Natl. Acad. Sci. U.S. 72, 3873--3877] reported the existence of 2 classes of polyadenylated mRNA with half-lives of under an hour and greater than 20 h, respectively. Since short-lived poly(A)-bearing mRNA is absent from mitogen-stimulated lymphocytes, the data suggest that stabilization of previously labile poly(A)-bearing RNA is one of many carefully regulated processes accompanying growth induction in normal lymphoid cells.     

Exposure to asbestos: correlation between blood levels of mesothelin and frequency of micronuclei in peripheral blood lymphocytes

Inhalation of asbestos, a mineral extensively used in a variety of applications, is strongly associated with malignant mesothelioma (MM), a fatal cancer of the pleura. Soluble mesothelin-related peptides (SMRP) are a promising biomarker suggested for the screening of MM in healthy asbestos-exposed subjects. In the present study a comparison of micronucleus (Mn) frequencies in peripheral blood lymphocytes (PBL) between 44 asbestos-exposed and 22 control individuals has been performed, and the correlation with serum SMRP has been examined. SMRP levels were found to be significantly higher in subjects exposed to asbestos and in their various subgroups than in controls. Concerning micronucleated lymphocytes, a statistically significant difference from controls was seen in the percentages of both micronucleated mononucleated lymphocytes (MnMNL) and micronucleated binucleated lymphocytes (MnBNL), but the difference was markedly higher for the percentage of micronucleated polynucleated lymphocytes (MnPNL). With respect to the correlation between the frequency of the three types of micronucleated lymphocytes and serum-SMRP values of asbestos-exposed subjects, it was statistically significant for MnMNL, but not for MnBNL and MnPNL.     

Low molecular weight nuclear RNA in human lymphocytes : A comparison of PHA-treated and control cells

Seven species of low molecular weight nuclear (LMN) RNA were identified in human peripheral lymphocytes. These were designated as A, B, C, D, D′, E and F. The same 7 species of LMN RNA were obtained from resting lymphocytes and from lymphocytes exposed to PHA for 16 and 60 h, respectively. Thus, no detectable qualitative changes were seen in the spectrum of LMN RNAs during PHA-induced lymphocyte transformation. However, the amount of species A, D-D′, E and F per nucleus in fully transformed cells was greater than in untreated lymphocytes. This increase had not yet occurred after 16 h treatment with PHA. ³H-Uridine was incorporated into all species of LMN RNA of resting and PHA-treated lymphocytes. Furthermore, all species of LMN RNA except C (5S RNA) were methylated in both resting and transformed cells. The ³H and ¹⁴C specific activities of LMN RNAs following an 8 h exposure to ³H-uridine and ¹⁴C-methyl methionine were higher in PHA-treated cells than in untreated lymphocytes. For several species of LMN RNA (A, D-D′, E, F) the highest ³H and ¹⁴C spec. act. were observed after 16 h exposure to PHA. The possibility that quantitative alterations in the synthesis and methylation of LMN RNAs may occur during lymphocyte transformation is discussed.     

Modelling of peripheral lymphocyte migration: system identification approach

This is the first application of the prediction error method (PEM) of system identification to modelling lymphocyte migration through peripheral lymphoid tissue. The PEM was applied to the emergence of labelled lymphocytes from the efferent lymphatic of a lymph node following their intravenous administration. Advantages of PEM included the capacity to calculate the response to a unit impulse stimulus, unavailable to direct observation, and to allow for the return to the node of labelled cells that had already recirculated once. Calculation of the system delay (time between introduction of cells into the blood and their first appearance in lymph) indicated 4.67 +/- 1.05 h for the total lymphocyte population. The peak in efferent lymph occurred at 11.91 +/- 4.68 h, much earlier than previous reports, which were affected by cells that had already recirculated. While 75% of labelled cells had emerged in efferent lymph by 20.77 +/- 5.62 h, 86.38 +/- 29.44 h was required for 100% emergence. The considerable heterogeneity in migratory behaviour is likely to reflect frequency and duration of binding of lymphocytes by dendritic cells in paracortical cord corridors. It is proposed that differences in the speed with which lymphocytes pass along corridors depend on their functional status, in particular whether they are na?ve or memory cells.     

Aberrant O-linked oligosaccharide biosynthesis in lymphocytes and platelets from patients with the Wiskott-Aldrich syndrome

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive immunodeficiency affecting B lymphocytes, T lymphocytes, and platelets. Previous studies on lymphocytes from WAS patients have revealed that leu-kosialin (CD43), a cell-surface glycoprotein bearing approximately 90 O-linked oligosaccharide chains, shows an aberrant electrophoretic mobility. To determine whether this finding reflects a different pattern of O-linked glycosylation in WAS cells, we have compared healthy individuals and WAS patients with respect to glycosyltransferase activities in T lymphocytes, platelets, and Epstein-Barr virus (EBV)-immortalized B cell lines. Stimulation of peripheral T cells from normal individuals in vitro with anti-CD3 antibodies and interleukin-2 was associated with a 3-fold increase in UDP-GlcNAc:Gal beta 3GalNAc-R (GlcNAc to GalNAc) beta 6-N-acetylglucosaminyltransferase (core 2 GlcNAc-T) from 0.8 to 2.2 nmol/mg/h. In contrast, peripheral T lymphocytes from WAS patients showed an inversion of this phenotype with high core 2 GlcNAc-T activity in unstimulated cells (2.3 nmol/mg/h) and a 2-3-fold decrease in activity following stimulation. Core 2 GlcNAc-T activity was also three times higher in platelets from WAS patients than in normal platelets. Glycosyltransferase activities were measured in immortalized B cell lines established from WAS and normal subjects by infection with EBV. Core 2 GlcNAc-T was less than 0.4 nmol/mg/h in WAS EBV-B cell lines compared to 2.4 nmol/mg/h in EBV-B cell lines from healthy individuals, In contrast, CMP-SA:SA alpha 2-3Gal beta 1-3GalNAc-R (where SA represents sialyl (sialic acid to GalNAc) alpha 6-sialyltransferase II activity was 2.0 nmol/mg/h in the WAS EBV-B cell and less than .01 nmol/mg/h in EBV-B cell lines derived from normal subjects. Eleven other glycosyltransferase activities were measured and found to be similar in EBV-B cell lines from WAS and normal individuals. Polylactosamine sequences were much reduced in the O-linked oligosaccharides of CD43 from WAS EBV-B cells consistent with decreased core 2 GlcNAc-T activity and expression of core 1 oligosaccharides in the cells. In conclusion, B cells, T cells, and platelets in WAS patients show abnormal expression of two developmentally regulated glycosyltransferases, consistent with the idea that the WAS immunodeficiency is due to a failure of normal lymphocyte maturation.     

IL-4 induces loss of B lymphocyte Fc gamma R II ligand binding capacity

Murine B lymphocytes cultured for 24 h with rIL-4 lost (mean reduction of 88%, range 81 to 96%) the capacity to bind Ag-IgG antibody complexes to B lymphocytes as assessed by flow microfluorometry. This effect was specific in that it was not seen with IL-1, IL-2, or IFN-gamma; IL-4 did not have a similar effect on other B lymphocyte membrane molecules; and the effect was completely prevented by anti-IL-4 (mAb 11B11). More than 60% inhibition of the binding of complexes was seen with as little as 1 U/ml of IL-4 although maximal inhibition was seen with greater than or equal to 30 U/ml. IL-4-induced inhibition of the binding of complexes was time dependent (the effect was first seen after 8 h and was not maximal until 24 h), temperature dependent (it did not occur at 4 degrees C), and reversible (B lymphocytes that had lost the ability to bind complexes due to IL-4 regained this capacity when re-cultured for 24 h in the absence of IL-4). The effect could be partially prevented by IFN-gamma. The inability to bind complexes appeared to be mainly due to an alteration of Fc gamma R (Fc Receptors) II rather than down-regulation of receptor expression because IL-4 induced only a moderate reduction in the binding of two Fc gamma R II specific mAb (20% for 2.4G2 and 32% for K9.361). The IL-4-induced loss of binding of complexes to B lymphocyte Fc gamma R II appears to be a novel form of receptor regulation (function rather than expression), and likely plays a role in the up-regulation of B lymphocytes by IL-4 by preventing Fc gamma R II-mediated inhibition of B lymphocyte responses.     

Basic types and vital forms of human peripheral and PHA-stimulated lymphocytes studied in vitro

Natural diversity in peripheral and PHA-stimulated lymphocytes seen in the same donors was studied using digitized streak photo of living cells in observational camera. Cells were monitored for 5-8 h at the superior limit of optical resolution by means of phase-contrast microscopy. Intact lymphocytes were observed in autological blood plasma, and PHA-stimulated lymphocytes were examined in self-conditioned centrifuged growth medium. The majority of intact cells were small- and middle-sized floating lymphocytes with microvilli, and middle-sized caudate lymphocytes capable of stick-slip motion. The lesser part consisted of "spread-eagle" or movable forms of both large granular lymphocytes and middle-sized lymphocytes of several types: narrow-plasm lymphocytes with lamellipodia, wide-plasm lymphocytes without cytoplasmic processes, lymphocytes with single pseudopodia, and lymphocytes with single lobopodia of complex shape. On the contrary, the minor fraction of PHA-stimulated lymphocytes of 3 day old cultures contained floating cells with microvilli or floating cells with microvilli and two pseudopodia, whereas the majority of these lymphocytes were spread-eagle or movable forms of cells of different type. These substrate-adhesive PHA-stimulated lymphocytes had well defined apical and basal cell surfaces, but upon mechanical stress are easily pinched off to become ball-shaped. At least 6 different cell types were distinguished among substrate-adhesive PHA-stimulated lymphocytes, with more than half of these being heavily vacuolated spheroid lymphocytes prone to forming cell clusters. The rest PHA-stimulated lymphocytes were represented by signet-ring lymphocytes with dark or light cytoplasm, narrow-plasm lymphocytes with large prolonged nuclei and lamellipodia, lymphocytes with single lobopodia, and lymphocytes with single spiral structures in the cytoplasm. The spiral structure is 10-11 microns in length and 0.5-0.7 micron in width, being presumably a mitochondrion or a group of butt-joined mitochondria. Since some of the caudate middle-sized lymphocytes also contain this structure, these may be regarded as putative precursors of respective type of PHA-stimulated lymphocytes. Under the conditions of observation, interphase nuclei of all live PHA-stimulated lymphocytes were seen to contain numerous globular or fiber structures of condensed chromatin made of 0.3-0.8 micron beads. These beads are doubtless interphase chromomeres.