Effect of zinc chloride on hamster lymphoid cells: mitogenicity and differential enhancement of lipopolysaccharide stimulation of lymphocytes.

ZnCl2 over a very narrow concentration range was found to be mitogenic for hamster lymph node cells but not for thymocytes or splenocytes. Maximal leucine, [3H]uridine, and [3H]thymidine incorporation. Addition of 10 micron ZnCl2 was found to greatly enhance the stimulation observed with the B-lymphocyte mitogen lipopolysaccharide but not with dextran sulfate or the T-lymphocyte mitogen lipopolysaccharide but not with dextran sulfate or the T-lymphocyte mitogen concanavalin A. Although not mitogenic for splenocytes, 10 to 25 micron ZnCl2 slightly enhanced lipopolysaccharide stimulation but not concanavalin A or dextran sulfate stimulation. The effect of ZnCl2 on lipopolysaccharide stimulation was also confirmed with outbred Hartley guinea pig splenocytes and lymph node cells. Zinc chloride (50 micron) was mitogenic for both of these tissues; the response to lipopolysaccharide was enhanced by addition of 50 micron ZnCl2, but the concanavalin A response was unaffected. The possibility that the zinc effect is mediated by proteolytic mechanisms is discussed.     

Regulation of mitogen- and antigen-stimulated lymphocyte blastogenesis by prostaglandins.

Exogenously added prostaglandin E1 or E2 inhibited the blastogenic response of Mycobacterium bovis-sensitized bovine peripheral blood lymphocytes stimulated with concanavalin A, phytohemagglutinin, or M. bovis purified protein derivative as measured by [3H]thymidine uptake. The kinetics of the response showed that prostaglandins must be added to lymphocyte cultures within hours after mitogen or antigen addition to achieve maximum suppression of [3H]thymidine uptake. Addition of prostaglandins 24 h after the addition of mitogens or antigens resulted in considerably less suppression, supporting a hypothesis that prostaglandins initiate an early series of events which ultimately control lymphocyte blastogenesis rather than directly inhibit deoxyribonucleic acid synthesis.     

Characterization of the spontaneous DNA-synthesizing and/or IgG-secreting cells in peripheral blood from patients with systemic lupus erythematosus

Characterization of the cells responsible for spontaneous DNA synthesis and/or IgG secretion in systemic lupus erythematosus (SLE) was undertaken by fractionation of the peripheral blood mononuclear cells (PBM). Each fraction was analyzed for its capacity to incorporate [3H]thymidine [( 3H]TdR) and secrete IgG without mitogen. The non-E rosette-forming cell (non-ERRC) fraction, which consisted of the surface immunoglobulin-positive [sIg(+)] cells and null cells, revealed a markedly increased spontaneous DNA synthesis (620.0 +/- 586.9 cpm) during the first hour of culture and an elevated spontaneous IgG secretion (8639 +/- 2630 ng/ml) during 9 days of culture. Of particular interest was the finding that both increased responses were conducted by the null cells; the null cell population had approximately a fourfold relative increase of [3H]TdR incorporation and a 60-fold relative increase of IgG secretion compared with the sIg(+) cell population. These results suggest that SLE patients have a small population of preactivated B-cell lineage cells, which lack sIg or have a very low density of sIg.     

Effect of the ratio of follicle-stimulating hormone to luteinizing hormone on rat granulosa cell proliferation and oestradiol-17 beta secretion.

The present study examined the effects of the ratio of follicle-stimulating hormone (FSH) to luteinizing hormone (LH) on granulosa cell proliferation and oestradiol-17 beta secretion. For these studies, ovarian segments from either immature rats or those primed with pregnant mares serum gonadotrophin (PMSG) were incubated for 5 h with [3H]thymidine and FSH (0-100 mIU/ml) with or without equivalent doses of LH. After incubation, granulosa cells were isolated and their mitotic activity estimated by determining the amount of [3H]thymidine incorporated into the DNA. The amount of oestradiol secreted into the media was measured by radioimmunoassay. Compared to granulosa cells from immature ovaries, granulosa cells from PMSG-primed ovaries required significantly less FSH to stimulate incorporation of [3H]thymidine, had a 9-fold higher basal level of oestradiol production and increased oestradiol secretion in response to gonadotrophins. At pharmacological serum levels (10-20 mIU of total gonadotrophin), FSH:LH ratios of less than or equal to 2 increased oestradiol secretion from PMSG-primed ovaries but did not increase the rate of [3H]thymidine incorporation. Conversely, FSH:LH ratios of greater than or equal to 3 stimulated [3H]thymidine incorporation without altering oestradiol secretion. These data demonstrate that granulosa cells of immature follicles not secreting oestradiol are relatively unresponsive to gonadotrophins at any dose tested. Once the capacity for oestradiol secretion develops, then both the dose and ratio of FSH and LH play major roles in determining whether the follicle will grow or secrete oestradiol.     

Modulation in vitro of human natural cytotoxicity, lymphocyte proliferative response to mitogens and cytokine production by essential fatty acids.

Essential fatty acids (EFA) have been shown in animal studies to have a differential effect on various aspects of immune reactivity. However, there have been few studies in humans. Therefore, we elected to investigate the effects of a variety of EFA [gamma-linolenic acid (GLA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] in vitro on human blood lymphocyte reactivity, cytokine secretion and natural cytotoxicity. The proliferative response to polyclonal mitogens (phytohaemagglutinin, pokeweed mitogen, concanavalin A), as measured by [3H]thymidine incorporation into newly synthesized lymphocytes, was inhibited (P < 0.05) by all EFAs tested, in a dose-dependent manner (3-15 micrograms/ml). The greatest inhibition of proliferation was caused by EPA and DHA. Similarly, EPA, DHA and GLA significantly reduced cytotoxic activity [expressed as lytic units, using 51 chromium-release assays natural killer (NK) (K562 cells) and lymphokine-activated (LAK) (Daudi cells) cells] (P < 0.05) in a concentration-dependent manner (5-50 micrograms/ml), without affecting cell viability. EPA and DHA exhibited greater suppression than GLA. Furthermore, the inhibition of cell proliferation and suppression of natural cytotoxicity was associated with marked decrease in cytokine [interleukin-1 (IL-1), IL-2, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma)] production in vitro. Our findings demonstrate that EFAs (GLA, EPA, DHA) have the potential to inhibit significantly various aspects of human lymphocyte cell-mediated and humoral immune reactivities.     

The differential effect of cyclic AMP on lymphocyte stimulation by T- or B-cell mitogens.

Dibutyryl-cyclic adenosine 3'5'-monophosphate (cAMP) added to mouse spleen cell cultures under serum free conditions inhibited the stimulation of [3H]thymidine incorporation induced by T-cell mitogens much more than the one induced by B-cell mitogens. The inhibition was most impressive with phytohaemagglutinin, followed by concanavalin A, pokeweed mitogen and the specific antigen Keyhole limpet haemocyanin (KLH). The response to bacterial lipopolysaccharide (LPS) was clearly less susceptible to suppression and the effect on the stimulation induced by trypsin and suramin, both B-cell mitogens, was marginal. Similar results were obtained by addition of isoproterenol or therophyllin to the cultures.     

Initiation of the blastogenic response of lymphocytes by hyperoptimal concentrations of concanavalin A.

The blastogenic response of human lymphocytes in vitro to hyperoptimal concentrations of concanavalin A (Con A) has been studied by means of volume spectroscopy (measuring cellular and nuclear volume), flow cytofluorometry (measuring cellular DNA content) and incorporation of [3H]thymidine ([3H]dThd). The optimal Con A dose with respect to [3H]dThd incorporation was about 30 micrograms/ml. In cultures given hyperoptimal doses, e.g. 100 micrograms/ml, [3H]dThd incorporation was strongly inhibited, whereas the number of cells entering S-phase and significantly increasing their cellular and nuclear volume was considerably larger than with 30 micrograms/ml. With 200 micrograms/ml Con A, which induced negligible [3H]dThd incorporation, the percentage of responding cells was even larger. Hence, doses of Con A, which were hyperoptimal with regard to [3H]dThd incorporation, induced blastogenic response, including DNA synthesis, in a larger percentage of the cells than did the optimal dose. However, in cultures with hyperoptimal Con A doses, the progression of the cell cycle stagnated mainly during S- and G2-phase and few cells completed mitosis. Thus, the blocking effect of hyperoptimal doses was not confined to any particular point of the cell cycle. The reduced [3H]dTd incorporation, seen with hyperoptimal doses, is attributed partly to a failure of this assay under such conditions.     

Suppression of lymphocyte proliferation by parainfluenza virus type 3-infected bovine alveolar macrophages.

Lymphocytes stimulated with concanavalin A (Con A) or antigen in the presence of bovine parainfluenza virus type 3 (PIV-3) infected bovine alveolar macrophages (BAM) or monocytes, had depressed [3H]thymidine incorporation. This failure of lymphocytes to incorporate radiolabel required live virus, was time dependent and was most pronounced when BAM were infected for 48 hr prior to the addition of lymphocytes. The rate of infection of alveolar macrophages and the release of infectious virus into culture supernatants paralleled suppression of lymphocyte mitogenesis by PIV-3. However, the peak titre of exogenous, live or inactivated virus was not suppressive when added to lymphocyte macrophage cultures just prior to Con A stimulation. Neither the loss of viable alveolar macrophages nor a shift in antigen or mitogen dose response in virally infected cultures could account for the deficit in [3H]thymidine incorporation by lymphocytes. Despite the presence of lymphocyte-associated virus antigen detected by direct immunofluorescence, no increase in PIV-3 titre above baseline was seen from infected lymphocytes, irrespective of mitogen stimulation. Likewise, lymphocytes did not contribute to the extracellular virus pool in lymphocyte-macrophage cultures as the increases in viral titre above basal levels in supernatants were equal to levels released by macrophages alone. The expression of viral antigen on lymphocytes stimulated in the presence of PIV-3-infected BAM suggests a non-productive or abortive infection of lymphocytes mediated through contact with infected macrophages.     

Enhancement of lymphocyte blastogenic and delayed hypersensitivity skin responses by indomethacin.

Incubation of Mycobacterium bovis-sensitized bovine peripheral blood lymphocytes with concanavalin A or M. bovis purified protein derivative and indomethacin caused a consistent, statistically significant increase in [3H] thymidine uptake as compared to cultures without indomethacin. The kinetics of the response showed that indomethacin must be added to lymphocyte cultures within hours after mitogen or antigen addition for enhancement of [3H]thymidine uptake to occur. Lymphocyte blastogenic responses to purified protein derivative were enhanced in both normal and M. bovis-sensitized lymphocyte cultures. However, enhancement of sensitized lymphocyte responses was significantly (P less than 0.01) greater than that in normal animals. Additionally, indomethacin treatment of M. bovis-sensitized guinea pigs singnificantly augmented delayed skin reactions to tuberculin. Delayed hypersensitive skin reactions were only enhanced when indomethacin was administered simultaneously with tuberculin.     

Determination of human T cell activity in response to allogeneic cells and mitogens. An immunochemical assay for gamma-interferon is more sensitive and specific than a proliferation assay

T lymphocytes proliferate and secrete lymphokines in response to allogeneic cells, mitogens and other stimuli. Cell proliferation as measured by [3H]thymidine ([3H]Tdr) incorporation into DNA has been routinely used to determine T cell responses in research and clinical laboratories. We have compared the sensitivity of an immunoradiometric assay (IRMA) for human gamma-interferon (IFN-gamma) (Chang et al., 1984), with that of the conventional [3H]Tdr incorporation assay in the measurement of T cell responses to antigens and mitogens in culture. Peripheral blood mononuclear cells (PBMs) were incubated in the presence and absence of phytohemagglutinin (PHA) or mononuclear cells from another individual for various periods of time. The culture fluids were collected for determining IFN-gamma and the cells were assayed for [3H]Tdr incorporation. Results of measurements were expressed in terms of stimulation indices. Both IFN-gamma secretion and thymidine incorporation were measurable in mixed lymphocyte cultures after incubation for 3 days, and in PHA stimulated culture after 24 h of incubation. The stimulation indices reflecting increased gamma-interferon were found to be more pronounced and more consistent than those of [3H]Tdr incorporation.     

The effects of purified mitogenic proteins (Pa-1 and Pa-2) from pokeweed on human T and B lymphocytes in vitro.

Purified proteins (Pa-1 and Pa-2) from pokeweed have been compared with commercial pokeweed mitogen (PWM-G) and other mitogens in their ability to stimulate human lymphocytes. With cultures of T and B cells separated from tonsil lymphocytes, thymidine uptake, blast transformation and immunoglobulin (Ig) synthesis have been measured. IgM and IgG was measured in supernates of stimulated cultures by radioimmunoassay. Pa-1, Pa-2 and PWM-G were found to be potent mitogens for unseparated tonsil lymphocytes or nylon column purified T cells. Pa-2 was found to be active at lower concentrations than Pa-1, and PWM-G was less potent than the purified mitogens. These three mitogens all stimulated unseparated lymphocytes to secrete large quantities of Ig (20-100 mug/ml) during 7 days in culture. With increasing amounts of mitogens severe decreases in immunoglobulin synthesis were observed at day 6 even with doses which were still optimal for stimulation of thymidine uptake at days 3 and 6. With purified B cells (less than 2% T cells) Pa-1 was the best mitogen for thymidine incorporation. However, the secretory response was very variable. In some experiments B cells did not secrete Ig in response to mitogens; in others Pa-1 was clearly more effective at stimulating secretion than Pa-2 or PWM-G and in some experiments B cells were stimulated by all three. In one experiment Pa-1 stimulated prolymphocytic leukaemia cells to blast transformation and the secretion of IgM. It is concluded that Pa-1, Pa-2 and PWM-G are much better activators of Ig synthesis in human cultures than either PHA or LPS and that Pa-1 is the most reliable B-cell stimulant of the three.     

Production and Secretion of Immunoglobulins by in Vitro-Activated Human B Lymphocytes

Activation induced by pokeweed mitogen in cultures of mononuclear cells from human blood was followed sequentially by simultaneous quantitation of live cells, thymidine incorporation, cells displaying cytoplasmic IgM, IgG, IgA or IgD, cells secreting IgM, IgG or IgA and cumulated IgM secretion. Maximal cellular activity was found after 7 days of culture, with means of 16000 IgM-, 20700 IgG- and 9900 IgA-secreting cells per 10⁶ originally cultured cells. The cumulated IgM secretion after 21 days of culture averaged 10400 ng per 10⁶ originally cultured cells. A close correlation was found between the number of IgM-secreting cells and the cumulated IgM secretion.     

The demonstration in vitro of the mitogenic effects of trypanosomal antigen on the spleen cells of normal, athymic and cyclophosphamide-treated mice

When the spleen cells of normal NIH mice were cultured with pokeweed mitogen (PWM), staphylococcal filtrate (SF) and trypanosomal antigen (TAg), and tritiated thymidine ([³H]Tdr) incorporation was used as a measure of mitogenic activity, the TAg (at a level of 25 μg/ml of spleen cell suspensions containing 2–3 × 10⁶ cells/ ml) was found to be a better mitogen than SF. PWM, however, was more effective than either of the two. [³H]Tdr incorporation by the spleen cells of Nu/nu (`athymic') mice was greater than that by the spleen cells of normal NIH mice when equal numbers of both cells were cultured with TAg. Pretreatment of NIH mice with cyclophosphamide suppressed [³H]Tdr incorporation by their cells when TAg was added to the cultures. The TAg used was derived from T. brucei TREU 226 obtained from Edinburgh University.     

Blastogenic responses of lymphocytes from mice immunized by sublethal infection with yeast cells of Histoplasma capsulatum.

Blastogenic responses of spleen cells to histoplasmin and ribosomal antigens and to the mitogens concanavalin A. phytohemagglutinin, and lipopolysaccharide were studied in normal and immunized mice (10(5) live yeast cells of Histoplasma capsulatum given by the subcutaneous route). Cells (10(6) per well) were cultured with and without antigens and mitogens in microtiter plates with RPMI 1640-5% heat-inactivated normal mouse serum for 72 h at 37 degrees C. Cells were harvested after a 16- to 18-h pulse with 1 microCi of [3H]thymidine (6.7 Ci/mol), and thymidine incorporation was measured by scintillation counting. The initial blastogenic response to concanavalin A (54 X 10(3) cpm) was suppressed (P less than 0.001) from 4 to 14 days post-immunization and returned to control levels on day 21. The response to phytohemagglutinin was suppressed up to 21 days. Lipopolysaccharide responses, however, were affected to a lesser degree. Blastogenic responses to histoplasmin and H. capsulatum ribosomes were similar on day 0 in normal and immune lymphocytes, but by day 4 cells from immunized mice were more responsive (P less than 0.01). The maximum response to H. capsulatum antigens was detected on day 42 and was 9- to 16-fold higher than in controls. These results demonstrate in vitro responses of primed lymphocytes on exposure to H. capsulatum antigens and suppressed responses to mitogens during early stages of the immune response.     

Antigen-Induced Proliferative Response to Murine Thymus Cells in Vitro

Thymus cells from 5- to 6-week-old normal (unimmunized) BALB/c mice showed an increased incorporation of [(3)H]thymidine in the presence of 2,4-dinitrophenyl-bovine serum albumin, fluorescein-bovine serum albumin, and bovine serum albumin (BSA) in tissue culture. The concentrations of antigen (BSA and haptenated proteins) required for stimulation were approximately 25- to 50-fold higher than those of the nonspecific mitogen, concanavalin A. In contrast to the stimulation by concanavalin A, which was maximal at 24 to 72 h, the stimulation by antigen was most marked earlier in the culture period (6 to 24 h). The BSA response was diminished to a statistically significant degree (especially at low BSA concentrations) in thymocytes from animals injected 72 h previously with BSA, indicating that the stimulation is immunologically specific.     

Inositol stimulates DNA and protein synthesis, and expansion by rabbit blastocysts in vitro.

The effect of different concentrations (0, 0.6, 3, 15, 75 and 375 microM) of myo-inositol on the development of rabbit morulae to expanded blastocysts was investigated in terms of blastocyst expansion and synthesis of DNA and protein, as measured by incorporation of [3H]thymidine and [14C]amino acids into acid-precipitable material. A concentration of 15 microM inositol caused a 2.8-fold increase in blastocyst expansion (P less than 0.01), a 9.9-fold increase in thymidine incorporation into DNA (P less than 0.01) and a 3.6-fold increase in amino acid incorporation into protein (P less than 0.01). There were no significant differences in the range from 15 to 375 microM inositol.     

Reversal of age-associated decline in immune responsiveness by dietary glutathione supplementation in mice

The potential of dietary glutathione to alter immune response in aging mice was studied. Four (young), 17 (mature) and 24 (old) month old C57BL/6Nia male mice were fed semi-purified, nutritionally adequate diets containing 0 (control) to 1.0% of reduced glutathione (GSH) for 4 weeks. Concanavalin A (Con A) stimulated proliferation of splenocytes was assessed by [3H]thymidine incorporation. Delayed-type hypersensitivity (DTH) to dinitrofluorobenzene (DNFB) was measured by a radioisotopic method. Spleen GSH and splenocyte thiol (-SH) levels were determined by HPLC and N-ethyl[14C]maleimide binding, respectively. In the control fed group, mature and old mice showed 67% and 72% reductions (P less than 0.05) in Con A stimulated [3H]thymidine incorporation compared to young mice. Dietary GSH supplementation partially, but significantly (P less than 0.05) reversed this age-associated decline in mature and old mice. DTH assays revealed that the in vivo T-cell-mediated immune function is depressed with age and that dietary GSH supplementation reverses this depression. Spleens from control-fed mature and old mice contained 12 and 19% less GSH, respectively, than young mice (P less than 0.05). This decline was also reversed (P less than 0.05) by dietary GSH supplementation. Splenocyte -SH content after incubation with Con A and responsiveness to this mitogen were positively correlated in old mice and were greater (P less than 0.05) in GSH supplemented animals. Thus, dietary GSH supplementation improves the splenic status of this tripeptide and enhances T-cell mediated immune responses in aging mice.     

Effects of mitogens for mouse B lymphocytes on chicken lymphoid cells.

Lipopolysaccharide from E. coli (LPS) and purified protein derivative from tuberculin (PPD) increased the [3H]thymidine incorporation of chicken spleen cells in culture. No such stimulation was observed with dextran sulphate. Thymus and bursa lymphocytes were not stimulated by any of these compounds. Spleen cells from chickens chemically bursectomized with cyclophosphamide treatment showed decreased responses to LPS and PPD, but responded normally to the T-cell mitogen concanavalin A. None of the tested mitogens induced polyclonal antibody synthesis or directly enhanced the primary antibody response to sheep erythrocytes (SRBC) in spleen cell cultures. LPS-coated SRBC, however, enhanced the in vitro antibody response to SRBC. The results suggest a moderate proliferative response of chicken lymphocytes to LPS and PPD, possibly involving B cells, but not further effects comparable to those on mouse B lymphocytes.     

Inhibition of in vitro human lymphocyte response by the pneumococcal toxin pneumolysin

The effects of pneumolysin, a sulfhydryl-activated cytolytic toxin produced by Streptococcus pneumoniae, on the in vitro human lymphocyte response was examined. The toxin, at concentrations of one to five hemolytic units per ml, caused marked inhibition of the response of lymphocytes to concanavalin A, phytohemagglutinin, pokeweed mitogen, and protein A. The response was assessed by measuring both [3H]thymidine incorporation and the ability of lymphocytes to produce immunoglobulins and lymphokine activity. The effects of pneumolysin were irreversible, could be prevented by pretreatment of the toxin with cholesterol, and were not related to a direct cytotoxic effect on the lymphocytes. Pneumolysin appeared to act at the initiation phase of the immune response and had no effect on lymphocytes committed to DNA synthesis or to the synthesis and secretion of immunoglobulins. Furthermore, pneumolysin-mediated inhibition of the lymphocyte response was not due to the inhibition of binding of mitogens to leukocytes and is likely to be related to effects on membrane-mediated signals essential for lymphocyte triggering. This may be one means by which pneumolysin plays a role in the pathogenesis of pneumococcal infections.     

Assessment of Peripheral Blood Mononuclear Cell Proliferation by [2-3H]Adenine Uptake in the Woodchuck Model

The levels of incorporated exogenous [³H]thymidine of peripheral blood mononuclear cells (PBMC) of the woodchuck were low after stimulation with mitogens concanavalin A (ConA), phytohemagglutinin (PHA), and pokeweed mitogen (PWM) when compared with other cell systems. The use of EDTA as an anticoagulant for blood sampling and AIM-V medium for culturing of PBMC improved the [³H]thymidine uptake of PBMC. A pronounced uptake is observed after use of [³H]adenine instead of [³H]thymidine for PBMC proliferation measurement. One likely explanation for the difference in [³H]adenine versus [³H]thymidine uptake is that the alternative pathway for thymidine monophosphate synthesis is important: the conversion of uridine to uridine monophosphate and, thereafter, to thymidine monophosphate. The optimal conditions for mitogen-induced proliferation of PBMC of the woodchuck were 2 μg/ml ConA and PHA at day 4 and 0.14 μg of PWM/ml at day 5. No consistent differences of [³H]adenine uptake were observed between PBMC from four woodchuck hepatitis virus-infected woodchucks and five uninfected animals.