A comparative study of [3H]thymidine and [125I]iodo-2'-deoxyuridine uptake as indicators of in vivo cell proliferation induced by graft-versus-host reaction in the mouse

Since [3H]thymidine ([3H]TdR) and [125I]iododeoxyuridine ([125I]UdR) appear to label very different cell populations in different tissues, we carried out the present investigation to determine the value and limitations of the use of [125I]UdR as a DNA precursor in a study of grafted cell proliferation during the course of a graft-versus-host reaction in mice. Results showed that the iodine radioisotope was as efficient as [3H]thymidine when endogenous thymidylate synthetase was inhibited by fluorodeoxyuridine. The use of 125I as an in vivo labelling compound constitutes a simple technique for evaluating DNA synthesis, provided the unfixed labelled iodine is eliminated by in vitro ethanol washing of tissues. The optimal conditions for elution are defined but may vary from one tissue to another. [125I]UdR and [3H]TdR give similar pictures of the very intricate phenomena accompanying a chronic graft-versus-host reaction (GVHR) induced in mice by minor histoincompatible antigens.     

The differentiation of T lymphocytes. V. Evidence for intrathymic death of most thymocytes.

The DNA of CBA mouse thymocytes was simultaneously labelled with the efficiently reutilized precursor [3H]TdR and the poorly reutilized precursor [125I]UdR, and the subsequent rate of loss of the two thymidine analogues was used to compute the extent of local thymidine reutilization. This should provide a minimum estimate of thymocyte death within the thymus. In agreement with published data, reutilization of thymidine in the "steady state' adult mouse thymus was about 61%. Adrenalectomy was used to show that this turnover was not due to steroid mediated stress effects; nor could it be attributed to direct effects of [125I]UdR itself. Most of the initial uptake and the subsequent turnover was within the major "high theta' thymus subpopulation. A similar study with the growing thymus of 7-day-old mice, when the demand for T cells in the periphery should be high, also revealed a high level (63%) of thymidine reutilization. The results support the view that a minimum of 60% of newly formed thymocytes die within the tyhmus, and that this is a constant and normal aspect of thymus function. This would be compatible with some form of negative selection for the appropriate developing T cells.     

Cell cycle-specific effects of glucocorticoids on phytohaemagglutinin-stimulated lymphocytes.

Effects of steroid hormones and colchicine on the response of pig lymphocytes to phytohaemagglutinin (PHA) were assessed by measurement of [6-3H]thymidine incorporation. At steroid concentrations of 1 microM and below, only glucocorticoids and progesterone inhibited PHA-stimulated [6-3H]thymidine incorporation but at 100 microM inhibition was also produced by oestrogens, androgens and physiologically inactive steroids. Measurement of [6-3H]thymidine incorporation 18-24 hr, 6-12 hr or 0-6 hr after the delayed addition of the synthetic glucocorticoid, dexamethasone, to PHA-stimulated lymphocytes revealed a succession of alternating phases of sensitivity and insensitivity to the effects of the steroid which suggested that it was acting, perhaps indirectly, in a cell cycle stage-specific manner to arrest the progression of activated lymphocytes from G1 to S. Similar effects were observed with colchicine, but 100 microM 11-epicortisol inhibited [6-3H]thymidine incorporation in a non-cycle-specific manner. Glucocorticoid receptor levels in pig lymphocytes were increased 2-5-fold within 24 hr of PHA stimulation.     

Conditions for measuring DNA synthesis in PHA stimulated human lymphocytes in 20 μl hanging drops with various cell concentrations and periods of culture

We have studied conditions for measuring the uptake of [³H]thymidine ([³H]Tdr) by human lymphocytes in inverted microcultures, varying cell concentrations and periods in culture. Analysis of variance of the log values for [³H]Tdr uptake may be used to separate effects of variables and their interactions. A pulse time of 2 h, a total thymidine concentration of about 1 μg/ml and a specific activity of [³H]Tdr of 2 Ci/mmole were optimal. Variables such as cell concentration, period of culture, type of serum and dose of PHA were shown to interact, suggesting that these variables should be examined together especially when comparing different samples of lymphocytes. Conditions for doing this simply in small volumes are now available for culturing, thymidine pulsing, harvesting and analysis of data.     

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.     

Activation and proliferation of normal resting human T lymphocytes in serum-free culture: role of IL-4 and IL-6

Purified human T lymphocytes, completely depleted of accessory cells [i.e. monocytes, large granular lymphocytes (LGL) and B lymphocytes], have been grown in serum-free culture in presence of a mitogenic lectin (phytohaemagglutinin, PHA) and different recombinant cytokines. Only IL-2 and IL-4 induced a marked stimulation of [3H] thymidine ([3H]TdR) uptake, cell proliferation and expression of activation markers [transferrin receptor (TrfR), IL-2R]. The other cytokines (IL-1 alpha, IL-1 beta, IFN-gamma, GM-CSF, TNF-alpha) had no significant effect, except for a moderate, but significant, stimulation of [3H]TdR uptake induced by IL-3. Simultaneous addition of IL-4 and anti-IL-2 neutralizing monoclonal antibodies (mAb) did not modify the effects induced by IL-4 alone. Furthermore, IL-2 was not detected in the supernatant of T cells grown in the presence of PHA and IL-4. Thus, our results indicate that IL-4 acts on T lymphocytes independently of IL-2. We also observed that IL-6 moderately activates DNA synthesis in PHA-stimulated T lymphocytes, but markedly potentiates the proliferative effect of suboptimal amounts of IL-2. In conclusion, the present study suggests that B-cell growth factors, in addition to IL-2, control the proliferation of normal circulating T lymphocytes.     

Distribution of lung-associated lymphocytes from the caudal mediastinal lymph node: effect of antigen.

Lymphocytes from the efferent lymph of the caudal mediastinal lymph node (CMLN) were labelled in vitro with 125I-iododeoxyuridine [125I]UdR and Na2(51)CrO4. The labelled cells were re-infused i.v. and their distribution in organs/tissues was determined 20-24 hr later. As indicated by tissue 125I-activity, pulmonary lymphoblasts had a marked tendency to relocate in the lung, regional pulmonary lymph nodes and spleen. Localization of efferent CMLN lymphoblasts was greater in antigenically stimulated segments compared to unstimulated segments of the lung. Dual antigen experiments indicated that the increased localization was not specific for the antigen which stimulated production of lymphoblasts used for in vitro labelling and reinfusion. Intranodal labelling of blasts by the direct injection of [125I]UdR supported the results obtained from in vitro labelling. In these studies, comparisons were made with the localization of lymphocytes obtained from thoracic duct lymph.     

Hydrophobic labelling of membrane-embedded proteins with lipophilic reagents. Incorporation of [125I]INA and [125I]TID into B lymphocyte membrane immunoglobulins

Hydrophobic labelling is frequently used in the study of membrane-inserted domains of intrinsic proteins. However, the published procedures, fail to incorporate sufficient radioactivity into membrane immunoglobulins of B lymphocytes to permit investigation of their subunit structures and associations with other proteins. In order to increase the specific radioactivity of [125I]iodonaphthylazide ([125I]INA), an improved method for the synthesis of the reagent was developed. In addition, the optimal conditions for labelling B lymphocytes with [125I]INA and the commercially available reagent 3-(trifluoromethyl)-3-(3'-[125I]iodophenyl)diazirine ([125I]TID) were elaborated. Under these optimized conditions, Ig molecules labelled with [125I]INA and [125I]TID were isolated and analysed in detail by SDS-PAGE. The usefulness of the two reagents for the investigation of lipid-embedded domains of membrane proteins is discussed.     

Effects of concanavalin A, phytohemagglutinin, pokeweed mitogen, and lipopolysaccharide on the replication and immunoglobulin synthesis by canine peripheral blood lymphocytes in vitro

Canine peripheral blood lymphocytes (cPBLs) were used to investigate the mitogenic effects of Con A (concanavalin A), LPS (lipopolysaccharide), PHA (phytohemagglutinin), and PWM (pokeweed mitogen) in vitro by measuring tritium-labeled thymidine [( 3H]thymidine) incorporation and immunoglobulin (Ig) secretion. An ELISA specific for canine IgG and IgM showed that cPBLs secreted significantly more IgG than IgM in response to mitogen concentrations from 30,000 to 0.03 ng/10(5) cells. The optimal stimulating dose of mitogen for lymphocyte response measured by IgG secretion was over a much narrower range of concentration than was the [3H]thymidine incorporation measured response. At a concanavalin A dose where there was increased [3H]thymidine incorporation with a decrease in IgG secretion, it appeared that an active suppression of the IgG response was induced.     

Inhibition of human lymphocyte stimulation by steroid hormones: cytokinetic mechanisms.

The steroid hormones estradiol, progesterone and testosterone, in addition to cortisol, inhibited stimulation of human peripheral blood lymphocytes by phytohaemagglutinin (PHA) and Con A. This effect upon lymphocyte transformation was assayed by three methods: quantitation of [3H]thymidine incorporation into acid precipitable material, microscopic assessment of blastic transformation and determination of the labelling index. Addition of steroid hormones at the initiation of culture resulted in a marked inhibition in all three parameters, which was observed with lower concentrations of cortisol than the other hormones. The inhibition was not attributable to cell death and could be partially reversed by removing hormones from the incubation medium after culture for 48-72 hr. Late addition of steroid hormones, 52 hr after addition of mitogen and 18 hr prior to pulse-labelling with [3H]thymidine, also resulted in reduced [3H]thymidine incorporation, accompanied by a nearly 50% reduction in the labelling indices and only a minimal decrease in the per cent transformed cells. Inhibition of lympohcyte stimulation by steroid hormones operates by the following cytokinetic mechanisms: (1) suppressed recruitment of cells from G3 to G1 phase of the cell cycle, as indicated by the diminished per cent blasts; (2) inhibition of progression from G1 phase into S phase, as evidenced by the reduced ratio [labelling index/blasts]; and, in the case of estradiol and progesterone, (3) reduced rate of DNA replication or altered intracellular [3H]thymidine specific activity as shown by the decreased ([3H]thymidine incorporation/labelling index) ratio. Late addition of steroid hormones to stimulated cultures reduced the per cent of cells in S phase, but did not revert previously transformed cycling lymphocytes to the G3 state.     

IL-2 responsiveness of lectin-induced lymphoblasts: soluble IL-2 receptor release and differential in vitro effects of immunosuppressants.

We examined the mode of action of different immunosuppressants on the responsiveness of phytohemagglutinin (PHA)-induced lymphoblasts further stimulated by recombinant interleukin-2 (rIL-2). The stimulation of PHA blasts with rIL-2 resulted in an enhancement of tritiated thymidine ([3H]TdR) incorporation and of soluble interleukin-2 receptor (sIL-2R) release. Cyclosporin A (CsA) and prednisolone inhibited in different ways the responsiveness of PHA pre-stimulated blood mononuclear cells (PBMC) to rIL-2, as measured by [3H]TdR incorporation. The addition of CsA resulted in considerable enhancement of the release of sIL-2R, whereas the addition of prednisolone was associated with a similar enhancement only when the higher concentrations of rIL-2 were employed. EGTA, a calcium (Ca2+) chelator, and verapamil, a Ca2+ channel blocker, inhibited [3H]TdR incorporation in a concentration-dependent manner. EGTA inhibited sIL-2R release in the same manner when used alone, and reversed the CsA- and prednisolone-induced enhancement of sIL-2R release by rIL-2 induced lymphoblasts, when used in combination with CsA or prednisolone. Verapamil had a similar but less striking effect. The effects of CsA and prednisolone were also studied in PHA-induced blasts originating from purified CD4+ or CD8+ lymphocytes. Stimulation of these blasts with rIL-2 resulted in higher [3H]TdR incorporation by CD8+ blasts than by CD4+ blasts: however, no sIL-2R release was detected in supernatants of either CD4+ or of CD8+ blasts. Both CsA and prednisolone inhibited the rIL-2-induced enhancement of [3H]TdR incorporation by both T-cell subsets.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of lymphocytes isolated from the gastric mucosa of the mouse.

A method for the isolation of lymphocytes and epithelial cells from the murine gastric mucosa has been developed. Gastric lymphocytes were stimulated by T-cell mitogens in vitro. When mice were sensitized systemically with human gammaglobulin (HGG) and given the antigen orally 4 days before cell isolation, both the number of mucosal lymphocytes and their capacity to incorporate [3H]thymidine ([3H]TdR) was enhanced. Gastric epithelial cells added to cultures of syngeneic spleen cells enhanced both phytohaemagglutinin (PHA) responsiveness and mixed lymphocyte reactions (MLR) in low numbers, but significantly suppressed these responses at high concentrations. These findings suggest that gastric lymphocytes, like those of the gut, are involved in the immune response against antigens taken in by mouth, and that there may be in vivo interactions between gastric mucosa lymphocytes and epithelial cells.     

A new ultra-microculture system. I. Stimulation of human T lymphocytes by phytohemagglutinin (PHA)

We have developed an ultra-microtechnique for culturing lymphocytes in glass capillary tubes at a final culture volume of 1 microliter or 2 microliter. The advantage of the method is that a substantially lower number of cells and minute amounts of culture medium are required. The cultures are premixed in microtubes, sucked into glass capillary tubes and incubated for an appropriate culture period. For determination of [3H]thymidine ([3H]TdR) incorporation, the cells are transferred into the wells of microtiter plates. Some special accessories have been developed which allow routine use of this system for large numbers of cultures. Optimal culture conditions for stimulation of human T lymphocytes by PHA are described.     

Distribution of [125I]omega-conotoxin GVIA and [3H]isradipine binding sites in the central nervous system of rats of different ages

Potential age-related changes in L- and N-type voltage-sensitive calcium channels (L- and N-VSCCs) were assessed by the in vitro binding of [3H]isradipine ([3H]ISR, 150 pM) and [125]omega-conotoxin GVIA ([125I]omega-CT, 4 pM) to membranes prepared from discrete central nervous system regions of 0.5-, 2-, and 18-month-old rats. The rank orders of [3H]ISR and [125I]omega-CT binding, although differing, indicated that the highest binding was in neocortex, corpus striatum, and hippocampus; radioligand binding was generally not affected by the variable of age. These results suggest that the nonidentical [3H]ISR and [125I]omega-CT binding sites are concentrated in those regions characterized by high densities of synaptic connections, and that these sites, as presumed components of L- and N-VSCCs, are relatively stable during the aging process.     

Suppression of T-lymphocyte blastogenesis by ovine trophoblast protein-1 and human interferon-alpha may be independent of interleukin-2 production

Cells derived from the trophoblast tissue of a day 15 sheep conceptus released substances that inhibit incorporation of [3H]thymidine into phytohemagglutinin (PHA)-stimulated ovine lymphocytes. This effect was partially reversed by addition of antiserum to ovine trophoblast protein-1 (oTP-1), a major secretory product of day 13-21 sheep conceptuses and a protein structurally and functionally related to alpha-interferons (IFN-alpha). Human IFN-alpha, unlike dexamethasone, inhibits phytohemagglutinin-induced lymphocyte blastogenesis without reducing interleukin-2 (IL-2) production by the cultures, and conditioned medium containing IL-2 does not promote [3H]thymidine incorporation into ovine lymphocytes when oTP-1 is present. Thus, oTP-1, by virtue of being an IFN, may have a local immunomodulatory role by selectively inhibiting the proliferative responses of certain maternal immune cells to IL-2.     

Recombinant human gamma interferon enhances in vitro activation of lymphocytes isolated from patients with acquired immunodeficiency syndrome.

Decreased responses to antigens and lectins are a characteristic feature of peripheral blood lymphocytes isolated from patients with the acquired immunodeficiency syndrome (AIDS). The in vitro addition of recombinant gamma interferon (IFN-gamma) to cultures of peripheral blood lymphocytes obtained from patients with AIDS resulted in an augmented proliferative response [( 3H]thymidine uptake) to phytohemagglutinin (PHA) and an enrichment in CD4+ and CD8+ T lymphocytes. In AIDS cultures stimulated with PHA, IFN-gamma increased the release of T-cell growth factors and enhanced the expression of interleukin-2 receptors on activated lymphocytes. Responsiveness to PHA was augmented, albeit to a lesser extent, by IFN-gamma in cultures derived from normal donors. Proliferation to microbial antigens including herpes simplex virus, cytomegalo virus, and Candida albicans was increased by IFN-gamma in cultures established from a group of AIDS patients with Kaposi's sarcoma who had no history of opportunistic infection.     

Structure and biological functions of human IgD. XII. Anti-IgD enhancement of PHA responsiveness in patients with chronic lymphocytic leukaemia.

Specifically purifed anti-human delta stimulated the in vitro incorporation of [3H]thymidine by human peripheral lymphocytes from patients with chronic lymphocytic leukaemia (CLL). The peak response varied between individuals; those with 5--52% IgD-bearing lymphocytes exhibited maximum stimulation at 3 days, whereas a patient with only 1% IgD-bearing cells showed optimal activation at 6 days. In agreement with others, our data indicated that, in most instances, lymphocytes from patients with CLL respond poorly to PHA. One of the most important findings in this study is the enhancement of PHA responsiveness by anti-delta. Lymphocytes that exhibited reduced responsiveness to PHA alone, when pre-treated with anti-delta, showed transformation greater than the sum of the anti-delta plus PHA responses.     

Mitogen-activated Xenopus laevis lymphocytes produce a T-cell growth factor.

Mitogen-free and serum-free supernatants (SNs) from cultures of phytohaemagglutinin (PHA)-stimulated Xenopus splenocytes, co-stimulated thymocytes, induced proliferation of splenic and thymic lymphoblast and supported growth of alloreactive T-cell lines. These SNs had no effect on 'resting' splenocytes, as measured by uptake of tritiated thymidine ([3H]TdR). Growth-promoting activity was also detected in SNs of cultures containing alloreactive T-cell lines and either PHA or irradiated stimulator cells that expressed the original priming alloantigens. Thus, T lymphocytes appear to be involved in producing, as well as responding to, a Xenopus T-cell growth factor (TCGF). TCGF activity could be absorbed from these active SNs with PHA-activated splenic blasts. No functional cross-reactivity among different mammalian interleukin-2 (IL-2) and Xenopus TCGF preparations was detected.     

Specific in vitro elimination of histocompatibility antigen reactive cells (HARC)

Human lymphocytes proliferating in response to allogeneic cells in mixed lymphocytes cultures (MLC), can be selectively eliminated by incubating the culteres initially with [³H] thymidine of high specific activity. After such treatment the remaining cells are vitually incapable of responding towards the initial stimulating cell donor. The response towards other allogeneic cells was unaltered, except for the non-specific effects of radiation damage. The addition of 0.25 μl/ml of PHA to the cultures during the period of [³H] thymidine incorporation, appeared to enhance the elimination of the proliferating cells.     

Lymphocyte transformation and production of a human mononuclear leucocyte chemotactic factor in patients with Hodgkin''s disease

Human peripheral lymphocytes stimulated in vitro with phytohaemagglutinin (PHA) produce a factor with chemotactic activity for homologous monocytes. The production of this factor and lymphocyte transformation was investigated in patients with Hodgkin's disease. It has been found that the PHA response as measured by incorporation of [³H]thymidine was markedly depressed in patients when compared to the response of normal lymphocytes. In contrast, the production of the chemotactic factor was not significantly depressed in patients with Hodgkin's disease.