Distinct modulation by interferon-gamma (IFN-γ) of CD23 expression on B and T lymphocytes of atopic subjects

The low-affinity IgE receptor (FcεRII/CD23) plays a role in IgE production. Cytokines participating in IgE synthesis also modulate CD23 expression on lymphocytes, but whether this modulation is different in atopic subjects remains unclear. We studied CD23 expression on B and T lymphocytes in 10 asthmatic patients with Dermatophagoides pteronyssinus hypersensitivity and 10 healthy non-atopic subjects. Studies were performed by flow cytometry, in phytohaemagglutinin (PHA) or IL-4-stimulated mononuclear cell cultures, alone or in the presence of IFN-γ. Soluble CD23 (sCD23) released in the culture supernatants was measured by enzyme-linked immunoassay. Both PHA and IL-4 induced the expression of CD23 on lymphocytes of atopic and non-atopic subjects. Whereas PHA increased both the percentage and mean fluorescence intensity of CD23⁺ B and T cells, IL-4 alone did not increase the percentage of CD23⁺ T cells. The effects of IFN-γ were different in both groups, since it was able to reduce the percentage of PHA-stimulated CD23⁺ T cells only in non-atopic individuals. In non-atopic subjects more than atopic, levels of sCD23 were increased in the supernatants of PHA and IL-4 cultures. These results show that the modulation of CD23 expression is different on B and T cells, and that IFN-γ acts differently in atopic and non-atopic individuals.     

Sensitivity of cultured lymphocytes from patients with nevoid basal cell carcinoma syndrome to ultraviolet light and phytohemagglutinin stimulation

DNA repair and replication after in vitro UV irradiation were determined in cultured peripheral blood lymphocytes from 6 patients with nevoid basal cell carcinoma syndrome (NBCCS) and from a group of control donors. DNA repair synthesis (UDS) was measured in unstimulated lymphocytes by incubation with 3H-TdR in the presence of hydroxyurea for 3 and 6 h after UV irradiation (6-48 J/m2). DNA replication was measured in PHA-stimulated lymphocytes, UV-irradiated or mock-irradiated, by incubation with 3H-TdR for 24 h. The effect of the mitogen was followed during 5 days after stimulation by determining the incorporation of 3H-TdR, the increase of cell number, and the mitotic index. NBCCS and control lymphocytes showed equal sensitivity to UV light in terms of UDS and reduced response to PHA. On the contrary, the mitotic index and the number of cells in stimulated cultures were significantly lower in the affected subjects. These data suggest an altered progression along the cell cycle, which could be characteristic of stimulated NBCCS lymphocytes.     

The stimulation of human B and T lymphocytes by various lectins

The response of human peripheral blood lymphocytes to different lectins was tested in vitro by monitoring DNA synthesis, blast transformation, and mitotic activity. One group of lectins - RCA, VGA, HPA, PNA, and UEA - showed no stimulating effects at all. WGA and VVA induced DNA synthesis and blast transformation but failed in stimulating mitosis. The mitogens PHA, ConA, LCA, and PWM showed peaks of mitotic activity at 50-60 hours for PHA, 70 hours for ConA, 80 hours for LCA, and between days 4 and 5 for PWM. The stimulation of different subpopulations of lymphocytes was investigated by immunological methods for the detection of B- and T-cell-specific surface structures during the whole incubation period. PHA proved to be a predominantly T cell stimulating agent, whereas ConA seemed to activate a higher proportion of B cells than yet known. PWM and the so-called T cell mitogen LCA turned up to stimulate a large number of B cells, but lead also to a T cell activation. The analysis of SCE events in stimulation experiments with these two lectins showed the early proliferation of a cell population with low SCE frequencies and the late propagation of a cell population with higher SCE rates. It could be assumed that the first population is represented by B- and the second by T-cells.     

Relative proportions of mitotic T and B cells in PHA-stimulated lymphocyte cultures

Peripheral blood lymphocytes from six healthy adults were stimulated with phytohemagglutinin (PHA) and cultured for 3 days. The relative proportions of mitotic lymphocyte subsets were determined by a method that allows both cytogenetic and immunologic characterization of a mitotic cell. The results directly indicated that not only T cells but also B cells undergo mitosis in PHA-stimulated lymphocyte cultures. The frequency of the mitotic B cells varied between 6% and 20%.     

Binding and functional effects of thyroid stimulating hormone on human immune cells

The expression and functional relevance of thyroid stimulating hormone (TSH) receptors on human immune cells were studied. Flow cytometric analysis was used to study the binding of biotinylated TSH to human peripheral blood mononuclear cells (PBMC) and various purified lymphoid populations. Our results indicate that the hormone binds well to monocytes and natural killer (NK) cells and marginally to purified tonsillar T and B lymphocytes. There was a significant increase in the binding of TSH to purified B cells that were activatedin vitro withStaphylococcus aureaus Cowan. In contrast, the binding of TSH to T cells was unaltered when they were stimulated with phytohemagglutinin (PHA). While TSH increases DNA synthesis and intracellular cAMP levels of FRTL-5 rat thyroid cells, it did not have such stimulatory effects on lymphocytes. However, there was a moderate increase in Ig production by activated B lymphocytes when they were cultured in the presence of the hormone. A possible function for TSH as a link between the immune system and the thyroid is discussed.     

Induction of 6-thioguanine-resistant lymphocytes in Fischer 344 rats following in vivo exposure to N-ethyl-N-nitrosourea and cyclophosphamide

We have developed a limiting dilution clonal assay for determining the frequency of 6-thioguanine-resistant (TGr) lymphocytes produced in rats by in vivo exposure to genotoxic agents. Spleen lymphocytes were isolated from female Fischer 344 rats and were cultured with 1 microgram/ml of phytohemagglutinin (PHA) for 40 hr. Northern blot analysis revealed that this procedure resulted in increased hprt and beta-actin mRNA synthesis. Conditions for optimum cloning were established by culturing four PHA-primed lymphocytes/well in 96-well round-bottom microtiter plates containing a medium supplemented with interleukin-2. These cultures also contained autologous and/or TK6 feeder cells inactivated with different doses of irradiation. Lymphocyte cloning efficiencies (CEs) were highest in plates containing both irradiated TK6 cells (5 x 10(3) cells/well; 90 Gy) and irradiated autologous feeder cells (5 x 10(4) cells/well; 50 Gy). CE did not depend on the number of primed lymphocytes/well when four or fewer target cells/well were cloned. To measure the effects of chemical mutagens on the frequency of TGr lymphocytes, rats were given a single i.p. injection of 0-150 mg/kg of N-ethyl-N-nitrosourea (ENU), a direct-acting alkylating agent, or 0-50 mg/kg of cyclophosphamide (CP), an indirect acting alkylating agent. Lymphocytes were isolated, primed, and cloned at 4 weeks after CP treatment and at 1, 2, 4 and 6 weeks after ENU treatment. CE in these cultures ranged from 12% to 27%. Cultures were also established for measuring CE in the presence of 6-thioguanine (TG) and these contained 5 x 10(3) irradiated TK6 cells and 5 x 10(4) primed rat lymphocytes/well. The frequency of TGr lymphocytes was calculated by correcting the CE in the presence of TG with the CE measured in its absence. ENU exposure produced a higher frequency of TGr lymphocytes than CP, but both chemicals produced a dose-dependent increase in TGr cells. In addition, the frequency of ENU-induced TGr lymphocytes increased with time after treatment. The TGr cells are presumed to be hprt mutants, but further analysis at the DNA level is required to establish this. The dose-dependent responses obtained with both ENU and CP treatments suggest that rat lymphocytes are sensitive to direct- and indirect-acting alkylating agents administered in vivo and that the rat lymphocyte assay is a useful complement to the in vivo/in vitro mouse assay for determining the mutagenicity of environmental toxicants.     

Distribution of actin content in human B and T lymphocytes by DNAse 1 inhibition test

The DNAse inhibition assay, which allows for the titration of two forms of soluble actin in cell extracts, has been applied to normal human lymphocytes from peripheral blood. Actin-like activities associated with the membrane-rich fraction of extracts from thymus dependent (T) lymphocytes are significantly higher than those from bone marrow derived (B) lymphocytes, on a per cell basis, provided care is paid to the elimination of monocytic and polymorphonuclear cells which produce significantly higher DNAse 1 inhibition. The apparent discrepancy with a recent report that normal T and B lymphocytes do not differ in their total actin content is discussed with respect to the functional uniqueness of membrane-associated DNAse 1 inhibitions and the importance of using purified lymphocyte populations.     

Differentiation of human lymphocytes by pokeweed mitogen in vitro: Studies in normal and in immunodeficient subjects

Summary Immunoglobulin (Ig) synthesis and secretion by peripheral blood lymphocytes activated by pokeweed mitogen (PWM), and by phytohemagglutinin (PHA) was measured in normal individuals and in subjects with primary immunodeficiency. Unstimulated cultures demonstrated stable, low Ig synthesis of all major Ig classes; PWM showed a peak of Ig synthesis at day 5; PHA cultures demonstrated a late peak of Ig production at day 9. Three cases of common variable immunodeficiency showed different patterns of data when the percentage of B cells in blood and Ig production in vitro were used as parameters. Two persons with immunodeficiency and thymoma showed decreased Ig production in vitro, and had suppressor cells capable of blocking Ig production by normal lymphocytes in co-cultivation experiments.

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Abbreviations used PWM pokeweed mitogen - PHA phytohemagglutinin - Ig immunoglobulin - IDD immunodeficiency disease - GARIg goat-anti-rabbit immunoglobulin serum - PBS phosphate buffered saline Supported by: United States Public Health Service Grants AI 09239, AM 20122, AM 11796     

Stimulation of chronic lymphatic leukaemia cells by pokeweed mitogen after treatment with neuraminidase-galactose oxidase.

CLL lymphocytes gave a low response upon stimulation with PHA or PWM in 3-day cultures. However, after treatment with neuraminidase-galactose oxidase (NGO), in the presence of PWM, CLL lymphocytes transformed into blasts and incorporated 3H-thymidine in 3-day cultures. This response of CLL lymphocytes was similar to that given by normal lymphocytes to PWM in 3-day cultures. The best stimulation of CLL lymphocytes was achieved when conditioned medium (CM) from normal T lymphocytes was present in PWM cultures. Purified B lymphocytes from CLL (T lymphocytes and monocytes removed) did not respond to PHA or PWM. However, after NGO treatment these cells were stimulated by PWM, but only in the presence of CM. PHA failed to stimulate NGO-treated CLL lymphocytes or purified B lymphocytes. This study shows that CLL lymphocytes, which usually fail to respond to mitogens, can be stimulated by PWM to proliferate after treatment with neuraminidase-galactose oxidase (NGO). This technique of B cell stimulation has been found useful in cytogenetic studies of B cell proliferative disorders.     

Cytogenetic and chemical detection of human exposure to polyhalogenated aromatic hydrocarbons

Peripheral lymphocytes from Taiwanese women (n = 35) exposed to polychlorinated aromatic hydrocarbons and from matched controls (n = 24) were assessed for the levels of sister chromatid exchanges (SCEs) after a 72-hour incubation of whole blood in the presence or absence of alpha-naphthoflavone (ANF) and for chromosome aberrations after 48 hours of incubation. Serum levels of polychlorinated biphenyl (PCB) congeners were measured for all individuals, and serum levels of several polychlorinated dibenzofurans (PCDFs) were measured for 12 exposed individuals by gas chromatography-mass spectometry. Blood concentrations of total PCBs in the exposed population averaged approximately 15 ppb, whereas mean PCDF values were 14 ppt. Major PCB congeners detected were 2,2' 4,4', 5,5'-hexa CB and 2,2'3,4,4',5-hexa CB. PCDFs detected were primarily 1,2,3,4,7,8,-hexachlorodibenzofuran (10.8 ppt) and 2,3,4,7,8-pentachlorodibenzofuran (2.7 ppt). Average SCE frequencies were 7.61 for controls and 7.30 for exposed individuals when assays were conducted in the absence of ANF, whereas respective values were 8.85 and 10.75 in the presence of ANF. Differences in the level of ANF-induced SCEs between the two populations were highly significant (P less than .001). Moreover, the ANF-induced SCEs were highly correlated with the serum concentrations of total PCBs and of several PCB congeners (P less than .001). Increases in ANF-induced SCEs appeared to be linear up to a PCB concentration of approximately 30 ppb. Chromosome aberration frequencies were similar in control and exposed populations. These studies demonstrate that in vivo exposure to PCBs and PCDFs result in an enhanced sensitivity of lymphocytes to the SCE-causing actions of ANF.     

Measurements of the equivalent whole-body dose during radiation therapy by cytogenetic methods.

Estimates of equivalent whole-body dose following partial body exposure can be performed using different biophysical models. Calculations should be compared with biodosimetry data, but measurements are complicated by mitotic selection induced in target cells after localized irradiation. In this paper we measured chromosomal aberrations in peripheral blood lymphocytes during radiotherapy, and estimated the equivalent whole-body dose absorbed, by using the novel technique of interphase chromosome painting. Premature chromosome condensation was induced in stimulated lymphocytes by incubation in calyculin A, and slides were hybridized in situ with whole-chromosome DNA probes specific for human chromosomes 2 and 4. Reciprocal exchanges were used to estimate the equivalent whole-body dose, based on individual pre-treatment in vitro calibration curves. Equivalent whole-body dose increased as a function of the number of fractions, and reached a plateau at high fraction numbers. Chromosomal aberration yields were dependent on field size, tumour position and concurrent chemotherapy. Results suggest that interphase chromosome painting is a simple technique able to give a reliable estimate of the equivalent whole-body dose absorbed during therapeutic partial-body irradiation.     

Heterogeneity of concanavalin A-induced suppressor T cells in man defined with monoclonal antibodies.

Human peripheral blood T lymphocytes were enriched for OKT4+ or OKT8+ subpopulations using complement mediated lysis with OKT8 or OKT4 monoclonal antibodies. These subpopulations and unfractionated T cells were separately stimulated with concanavalin A (Con A) for a period of 48 hr and were then examined for their suppressive influence on proliferative response of autologous T cells to phytohaemagglutinin (PHA) or allogeneic non-T cells. Con A-activated unfractionated T cells, OKT4+ and OKT8+ T cell subsets markedly suppressed both these responses. Both OKT4+ and OKT8+ T cell subsets when enriched following Con A-activation of unfractionated T cells also caused significant suppression of proliferative responses of autologous T cells to PHA and allogeneic non-T cells in mixed lymphocyte cultures. The suppressive influence of Con A-activated T subsets was abolished by irradiation (2,000 rad) of activated cells. These studies indicate that Con A-induced suppressor T cells are heterogeneous. Precursors of Con A-induced suppressor T cells appear to reside in both OKT4+ and OKT8+ T cell populations.     

Effect of normal mouse serum on mouse lymphocyte transformation in vitro

Mouse spleen cells were cultured in the presence or absence of one of the following mitogens: phytohemagglutinin (PHA), concanavalin A, allogeneic spleen cells or bacterial lipopolysaccharide. The addition of normal mouse serum (NMS) to the cultures usually depressed mitogen-induced lymphocyte transformation as measured by DNA, RNA or protein synthesis, whether calculated as gross or net synthesis. The degree of depression was increased as the concentration of NMS serum increased. DNA and RNA synthesis in unstimulated cultures were also usually depressed by NMS. NMS added 24 hours or more after the start of cultures with PHA or allogeneic lymphocytes was less depressive than serum present from the start and in some cases, actually increased DNA synthesis. The presence of NMS in cultures containing supra-optimal doses of concanavalin A could also increase DNA synthesis. Cells incubated with NMS for 24 hours survived as well as cells incubated without NMS, but subsequently responded less well to PHA. Cells passed through a column of glass wool had a lower baseline DNA synthesis, which was not inhibited by NMS; they responded less well to mitogens and this response was further depressed by NMS. It is suggested that NMS contains a factor which damps the proliferation of mouse T and B cells, without exerting an overt cytotoxic effect, and that it acts directly on lymphocytes.     

Effects of 17 beta-oestradiol on function of lymphocytes from normal donors and patients with common variable immunodeficiency (CVID).

Effects of oestradiol (E2) have been studied on the in vitro T cell-dependent differentiation of B cells from peripheral blood and spleen using normal donors and patients with the antibody deficiency disease CVID. We also studied whether it modifies T cell DNA synthesis. The effect of E2 was examined on cultures of B cells with T cells for IL-2-driven immunoglobulin secretion or of T cells for phytohaemagglutinin (PHA)-driven DNA synthesis. Interestingly, in control experiments without E2, the normal sex difference in immunoglobulin production is reversed in CVID. The data show that for normal individuals there is no major difference between male and female donors in the in vitro actions of E2 on blood B and T lymphocytes. With normal blood B cells, E2 failed to affect IgM production, but it did inhibit IgG. In normal splenic cells, E2 increased both IgM and IgG secretion in a similar way to the tonsillar cell data previously reported. E2 on normal blood T cell DNA synthesis was stimulatory. With blood cells from CVID patients an interesting contrast was seen. As with normal B cells, E2 had no effect on IgM secretion by those CVID blood B cells able to secrete IgM. However, a difference between patients and normals was that E2 did not inhibit the IL-2-driven IgG production by those CVID B cells able to secrete IgG. For T cell function, the stimulatory effect of E2 on CVID T cell DNA was as in normal T cells. However, E2 failed to restore CVID B and T cell function to normal levels. These data suggest that there may be subtle defects in the pathway of action of E2 in CVID lymphocytes.     

Radiosensitivity of isolated subsets of human lymphocytes (E+, OKT4+, OKT8+): protective role of monocytes and monokines.

The sensitivity of human peripheral blood T lymphoid populations to 60Co ionizing radiation was investigated. Dose-response values were determined for populations that are commonly identified by their ability to form spontaneous rosettes with sheep red blood cells (E+ cells), helper T lymphocytes (OKT4+ cells) and suppressor T lymphocytes (OKT8+ cells). OKT4+ and OKT8+ T cell subsets were negatively selected by complement (C)-mediated cytolysis using the C fixing OKT4 and OKT8 monoclonal antibodies (MoAb). The irradiation-induced damage was assessed by the lymphoblast transformation test, using the polyclonal T cell mitogen, phytohaemagglutinin (PHA) and the OKT3 MoAb. (The OKT3 antibodies are mitogenic for T cells only in the presence of monocytes). No significant differences were evident between dose-response values of E+, OKT4+ and OKT8+ lymphoid subpopulations when using PHA as a mitogen. On the other hand, when OKT3 was used to trigger resting irradiated peripheral blood T lymphocytes, e.g. E+ cells, OKT3 stimulated T cells proved to be markedly radioresistant as compared to PHA stimulated cell cultures. This was found to result from the fact that purified T cell cultures were co-cultured with non-irradiated monocytes when OKT3 was employed as a motogen. Similarly co-culturing of irradiated E+, OKT4+ and OKT8+ cells with non-irradiated autologus monocytes partially corrected the irradiation damage, regardless of the mitogen employed. More important, however, was the observation that macrophage derived supernatants containing (interleukin-1) IL-1 could confer a high degree of radioprotection on irradiated E+ cells. It is concluded that monocytes and monocyte products partially protect against irradiation damage.     

Comparative responses to phytohaemagglutinin of appendiceal, thymic and splenic lymphocytes of rabbits

Lymphocytes from the appendix, thymus and spleen of young rabbits were cultured in the presence of varying concentrations of phytohaemagglutinin and their proliferative response during a 72-hour culture period measured with [³H]thymidine uptake. At the time of culture initiation, appendiceal lymphocyte populations showed the highest spontaneous rate of DNA synthesis followed by thymic and splenic lymphocytes in that order. In the rabbit appendix the presence of a small nondividing population of phytohaemagglutinin-responsive small lymphocytes was demonstrated. Additional evidence suggests that low concentrations of PHA accelerated or initiated DNA synthesis during the first 24-hour culture period in a proportion of appendiceal lymphocytes which were in the mitotic cycle at the time of culture initiation. In the thymus, as in the appendix, only a small fraction of the lymphocyte population responded to phytohaemagglutinin during the 72-hour culture period. Splenic lymphocytes showed the greatest response to PHA with a peak of DNA synthesis between 48 and 60 hours. The results indicate that varying concentrations of PHA should be employed to detect optimum responses by lymphocyte populations from various lymphoid organs and that different lymphoid organs have different proportions of G₀ vs. G₁ and PHA-responsive vs. non-PHA-responsive cells.     

Measurement of cell mediated cytotoxicity by post-labeling surviving target cells

The 51Cr release assay (CRA) is the commonly accepted technique for measurement of cell mediated cytotoxicity. This assay shows some disadvantages when mononucleated cells of human peripheral blood (MNC) are used as effector and target cells. The uptake of 51Cr by PHA stimulated lymphocytes is low compared to the spontaneous release. In an attempt to develop a cytotoxicity assay suitable for human lymphocytes we used 14C-TdR to label target cells surviving after contact with effector cells. Cytotoxic lymphocytes were generated by incubation of MNC with irradiated allogeneic MNC for 6 days. On day 6 the effector cells are irradiated and co-cultured with PHA stimulated target cells. Twenty-four hours later 14C-TdR is added. After an additional 24 h the cultures are harvested and 14C-TdR taken up by target cells is measured. It is shown that the effector cells are still cytotoxic after irradiation. These cells do not take up 14C-TdR. Cell-free supernatants do not influence the uptake of 14C-TdR by target cells. The results obtained with this assay correlate very well with those obtained by the CRA, if the spontaneous release does not exceed 30%.     

A Simple Method for Specific Depletion of Lymphocytes from Bovine Peripheral Blood Mononuclear Cells

The treatment of bovine peripheral blood mononuclear cells with rabbit anti-bovine immunoglobulin, goat anti-rabbit immunoglobulin (GAR) and complement resulted in the specific lysis of all surface immunoglobulin (SIg) bearing B lymphocytes. No SIg lymphocytes were detected after 12 hours of culture following lytic treatment, whereas cells treated with antibody without complement readily stained for SIg. The percentage of cells forming E-rosettes or binding peanut agglutinin (PNA) increased following lysis. The percentage of latex-ingesting monocytes also increased after lysis even though many of these cells had cytophilic Ig. B lymphocyte-depleted (T cell-enriched) populations cultured with phytohemagglutinin (PHA) and concanavalin A (Con A) were never less reactive than unseparated cells. No differences in background mitosis was observed for these 2 cell preparations. These results suggest that bovine SIg^? cells do not require B lymphocytes to respond to PHA and Con A and that lysis of B lymphocytes does not alter the responsiveness of the recovered SIg^? cells.     

Chromosome changes in human lymphocytes after separate and combined treatment with divalent salts of lead,cadmium, and zinc

Unstimulated human lymphocytes in whole blood were treated for three hours with lead, cadmium, and zinc acetate separately and in combinations of two or three metal salts with different concentrations of between 10^?3 and 10^?8 moles, respectively. Untreated cultures and sodium acetate-treated samples served as controls. Chromosome analysis from 48 cultures revealed higher incidences of chromatid-type aberrations and gaps only for cultures exclusively treated with cadmium. The results are discussed under the aspect of heavy metal metabolism in human lymphocytes.