Effect of arabinosyl cytosine on the level of DNA polymerase and thymidine kinase activity in PHA-stimulated human tonsillar lymphocytes

Summary The effect of arabinosyl cytosine (ara-C) was studied on the uptake, phosphorylation and incorporation of ³H-thymidine in human tonsillar lymphocyte cultures is described along with its effect on the level of DNA polymerase and thymidine kinase activities induced by phytohaemagglutinin (PHA). Freshly isolated tonsillar lymphocytes are stimulated cells with a remarkably high activity of DNA polymerase a and thymidine kinase. During in vitro culture, these stimulated cells are transformed to the resting state with low DNA polymerase and thymidine kinase activity. However, a new DNA synthesising cycle can be induced by PHA with maximum at 48 h.10^–6 M ara-C inhibited the incorporation of ³H-thymidine by 90–95%. This inhibition may be reversed by rinsing the cells. The inhibition of the transport of ³H-thymidine seems to be only a consequence of the inhibitory effect of ara-C on the DNA polymerisation reaction, because at 10 °C, where DNA synthesis was arrested, ara-C does not influence the uptake and the phosphorylation of ³H-thymidine.Ara-C (10^–6 M) abolished also the PHA induced elevation of DNA polymerase a and thymidine kinase activities without influencing protein synthesis of the cell. This supports a coordinated regulation mechanism between DNA synthesis and the synthesis of enzymes involved in DNA replication.     

Enhanced uptake of calcium by transforming lymphocytes

Phytohemagglutinin caused a rapid increase in calcium accumulation by lymphocytes. The enhanced uptake was observed within 1 hr of initiation of transformation in both human lymphocyte and mouse spleen cell cultures. Increased uptake was also found in mixed lymphocyte cultures although not until late in the response. The rate of calcium uptake increased with time after stimulation and depended upon the PHA concentration. The lowtemperature coefficient (Q₁₀) for calcium permeability in unstimulated cells was indicative of a passive diffusion process, but the Q₁₀ was slightly greater for PHA-stimulated cells. Various chemical agents which alter membrane properties and/or cellular metabolism inhibited uptake to a greater extent in stimulated cultures than in control cultures. Ouabain did not affect the calcium permeability of controls or stimulated cells within 1 hr after PHA addition, but it partially inhibited calcium uptake 12 hr after PHA treatment. Cyclic AMP, dibutyryl cyclic AMP, and theophylline also altered calcium transport providing evidence for an effect of cyclic AMP on an early event in the transformation process.     

Studies on the regulation of lymphocyte reactivity by normal and activated macrophages.

To determine the potential role of macrophages as regulators of the immune response, the effect of mouse peritoneal macrophages on transforming mouse spleen lymphocytes was investigated. Mitogen and antigen stimulated lymphocyte transformation, as measured by DNA synthesis, was enhanced by all concentrations of normal macrophages tested, but only by low concentrations of activated macrophages. High concentrations of activated macrophages markedly inhibited lymphocyte transformation. This inhibition occurred whether lymphocyte DNA synthesis was measured by incorporation of [³H]TdR or of ³²P. Activated macrophages cultured with lymphocytes within 4 hr of being removed from the peritoneal cavity inhibited lymphocyte transformation. When activated macrophages were cultured alone for 24 or more hours before addition of lymphocytes, enhancement of transformation was noted. Once lymphocytes were exposed to activated macrophages, they could not be induced to undergo transformation in the presence of Con A. Whereas heat-killed activated macrophages, which appeared intact morphologically, lost their capacity to inhibit lymphocyte transformation, macrophages treated with mitomycin C to inhibit DNA synthesis retained this capacity. Syngeneic and allogeneic macrophages had similar inhibitory ability. Supernatants from cultures of many cell types (including normal or activated macrophages, lymphocytes, lymphocytes plus macrophages, and L cells) inhibited [³H]TdR incorporation by both mitogen stimulated lymphocytes and tumor cells. These studies demonstrate the capacity of macrophages to regulate lymphocyte transformation in vitro and suggest a role for these cells as regulators of cell-mediated immunity in vivo.     

Differential cytotoxicity of activated lymphocytes on allogeneic and xenogeneic target cells. II. Activation by phytohemagglutinin

In the preceding paper it has been shown that human or mouse lymphocytes stimulated by a variety of agents, damaged allogeneic target cells while damage of xenogeneic target cells was weak or absent. In this study, the species specificity of the cytotoxicity of PHA activated lymphocytes has been studied in greater detail. Effector cells were purified lymphocytes either from human peripheral blood, or from spleen or lymph nodes of inbred mice. Target cells were ⁵¹Cr-labeled human Chang liver cells or mouse L cells.PHA stimulated human or mouse lymphocytes were significantly more cytotoxic to allogeneic than to xenogeneic target cells. At low PHA doses at which damage of allogeneic target cells was significant, damage of xenogeneic target cells was very weak or absent. At higher PHA doses, damage of xenogeneic target cells became also significant but always remained at a lower level than that of allogeneic target cells.Prestimulation of human lymphocytes with PHA for 3 days increased their cytotoxic efficiency. Furthermore, damage of human Chang cells by human lymphocytes had a dose-response relationship similar to that valid for stimulation of DNA synthesis. However, damage of mouse L cells by human lymphocytes increased at PHA-doses at which stimulation of DNA-synthesis declined. For mouse lymphocytes, these doseresponse relationships were less clear-cut, probably due to differences in origin and survival of the effector cells. This confirms previous observations that cytotoxicity and DNA-synthesis are different but probably interdependent expressions of lymphocyte activation.     

Stimulation of chicken lymphocytes by T- and B-cell mitogens.

Cultures of chicken spleen, peripheral blood, thymus, and bursal lymphocytes were tested for mitogenic stimulation by phytohaemagglutinin (PHA), concanavalin A (ConA), pokeweed mitogen (PWM), bacterial lipopolysaccharide (LPS), trypsin, and insulin. Spleen and blood leukocytes were stimulated by both the lectins and LPS, and also to some degree by trypsin and insulin as judged by increased incorporation of [³H]thymidine into acid-insoluble material. This was observed in cultures incubated in serum-free medium as well as in the presence of foetal bovine serum or autologous plasma. Thymus cells were reproducibly stimulated by high concentrations of PHA. No significant responses were obtained in bursal cell cultures with any of the compounds tested. Removal of cotton wool-adherent cells from the spleen cell suspensions resulted in a subpopulation of cells which were stimulated by PHA but showed little response to ConA, PWM, or LPS. This procedure did not remove surface immunoglobulin-bearing cells from the original suspension. Both these enriched spleen lymphocytes and the unfractionated spleen, blood and thymus leukocyte cultures were effectively stimulated by a partially purified PHA but with a highly purified PHA preparation only at very high concentrations. These and other results suggest that the mitogenic components in crude PHA preparations are different for chicken and human or mouse cells.     

Mastocytoma-medicated suppression of mixed lymphocyte culture and mitogen responsiveness.

Murine spleen cells, stimulated in vitro by allogeneic spleen, display a strong proliferative response with the subsequent development of cytotoxic cells. This proliferation and sensitization can be abrogated by the addition of mitomycin-treated or X-irradiated murine DBA/2 mastocytoma cells (P-815). The substance required for this depression of lymphocyte responsiveness is present in the cell-free supernatant fluids of P-815 cultures. The suppression appears to be due to interference with cell proliferation in the mixed lymphocyte culture, because the P-815 also prevents spleen cells from proliferating in response to the mitogens concanavalin A (Con A), lipopolysaccharide (LPS), and phytohemagglutinin (PHA). The significance of these findings is discussed.     

Trypanosoma cruzi: Responses by cells from infected mice to alloantigens

In unidirectional mixed lymphocyte cultures containing (as responders, stimulators, or regulators) spleen cells from mice infected with Trypanosoma cruzi, alloantigen responses were less than in cultures containing normal spleen cells only. Depletion of plastic adherent cells from infected spleen cells (stimulators or regulators) reversed their inhibitory effect on normal spleen cells (responders); removal of adherent responder cells and/or B lymphocytes did not alter the low alloantigen responses of normal spleen cells (stimulated by infected spleen cells) or infected spleen cells (stimulated by normal spleen cells). Infected spleen cells were effective in regulating mixed lymphocyte cultures only when added at the initiation of the culture. Serum from infected mice suppressed mixed lymphocyte cultures containing responder spleen cells syngeneic to the serum donor if added up to 24 hr after initiation of cultures, whereas the “suppressor serum” had to be present at the initiation of cultures when responder cells were allogeneic to the serum donor. Cultures of infected spleen cells (whole or macrophage enriched) produced a factor which was suppressive when added to mixed lymphocyte cultures containing syngeneic responder cells at initiation. It is proposed that the serum suppressor substance regulates cell-mediated immune responses directly by suppressing the response-potential of cells and indirectly by triggering the release of a factor from adherent splenic cells which induces a hyporesponsive state in T lymphocytes.     

Serum factors in lymphocyte proliferation. I. The requirement for serum in mixed lymphocyte reactions

An evaluation was made of the serum requirement for mixed lymphocyte reactions. Allogeneic mixtures, totalling 3.0 × 10⁶ mouse lymphoid cells, showed DNA synthesis when serum was removed after the first few hours of cultivation. Cultures initiated in the absence of serum failed to incorporate ³H-thymidine, except when high cell concentrations (6.0 × 10⁶ cells/ml) were used. Partial stimulation was supported by as little as 0.5% serum and full stimulation occurred with a concentration of 2% serum. The results indicate that serum facilitates an early point in recognition or blastogenesis in mixed lymphocyte reactions.     

Mixed lymphocyte reactions: absence of stimulation by irradiated allogeneic cells

¹³⁷Cs-irradiated mouse spleen cells, in contrast to mitomycin-blocked cells, do not stimulate a mixed lymphocyte reaction (MLR) when cocultured with normal allogeneic lymphocytes. Attempts to find an irradiation dose which blocks the DNA-synthetic capability of alloantigenic cells, but which does not also render them unstimulatory in mixed cultures, have not been successful. Low-level irradiation (100–500 rad) does not completely block mitogen (PHA) reactivity, hence the target cells may be capable of participating in a two-way MLR. High-level irradiation (> 1000 rad), however, thoroughly blocks PHA-stimulated DNA synthesis and eliminates the capacity of these cells to stimulate an MLR. Potentiation of MLR by irradiated cells syngeneic with the reacting cell population was not possible in these experiments. Cell death and lysis in irradiated suspensions occurred during the 72-hr culture period. It is believed that a combination of cell lysis and irradiation-blocked metabolic events normally necessary for the MLR sufficies to explain the poor or nonexistent MLR's obtained in these experiments.     

Stimulation of uptake of tritiated thymidine into mouse marrow cells in culture by a factor from L-cell conditioned medium

Uptake of ³H-thymidine into suspension cultures of mouse marrow cells was stimulated by the addition of serum-free conditioned medium harvested from cultures of mouse L-cells. Characterization of the “conditioning factor activity” (CFA) by gel filtration, ion exchange chromatography, velocity sedimentation, polyacrylamide gel electrophoresis and susceptibility to trypsin digestion indicated that the CFA detected by stimulation of ³H-thymidine uptake is the same as the CFA detected previously by its ability to stimulate colony formation by marrow cells in vitro. The ³H-thymidine uptake assay was used to investigate the kinetics of disappearance of CFA as a function of time in the presence of mouse marrow cells. The CFA recoverable from the cultures decreased rapidly during the first day, and approached background levels by the fifth day. There was no evidence of inhibitory substances in the depleted media. Even if the cultures continued to receive fresh conditioned medium at daily intervals, ³H-thymidine uptake decreased sharply after the fifth day, indicating that the marrow cells had lost their capacity to respond to CFA.     

Differentiation of lymphocytes in mouse bone marrow. II. Kinetics of maturation and renewal of antiglobulin-binding cells studied by double labeling

DNA labeling by ³H-thymidine in vitro and antiglobulin-¹³¹I binding in vitro were used to determine the development and turnover of immunoglobulin-bearing lymphocytes in mouse bone marrow.Bone marrow cells from CBA mice previously injected repeatedly with ³H-thymidine for 1–84 hr were exposed to ¹³¹I-labeled rabbit-antimouse globulin for 30 min at 0 °C, and examined radioautographically. The antiglobulin-binding cells in bone marrow were predominantly (97–98%) nondividing small lymphocytes. Some plasmacytoid and monocytoid cells, but not the proliferating large lymphoid cells, also bound antiglobulin. The ³H-thymidine labeling index of the small lymphocyte population showed a rapid exponential increase (50% in 32 hr). The first small lymphocytes to show ³H-thymidine labeling were those lacking antiglobulin-binding capacity, reaching approximately 90% ³H-thymidine labeling after 2 days. Small lymphocytes which bound antiglobulin-¹³¹I at a concentration of 1.0 μg/ml became labeled with ³H-thymidine only after a lag of approximately 1.5 days. More avid antiglobulinbinding cells were delayed a further 12 hr in ³H-thymidine labeling. During in vitro culture the proportion of antiglobulin-binding small lymphocytes increased progressively in bone marrow but decreased in spleen cell suspensions.The results demonstrate a continuous, rapid renewal of immunoglobulin-bearing small lymphocytes in adult mouse bone marrow. Surface immunoglobulin molecules are not detectable when marrow small lymphocytes are first formed, but they appear and increase progressively in density as the cells mature.     

Partial purification of uterine secretory protein capable of suppressing lymphocyte reactivity

Porcine uterine flushings obtained on day 15 of the estrous cycle were fractionated using gel filtration, and preparative poly-acrylamide gel electrophoresis was employed to separate one molecular weight fraction into two groups of small uterine-specific proteins designated pAP and pLAP. The two groups were assayed for immuno-suppressive ability using phytohemagglutinin (PHA) stimulated porcine lymphocyte cultures and incorporated ³H-thymidine. It was found that the pAP preparation which was composed of two proteins inhibited lymphocyte reactivity to PHA (p<.001) while the pLAP preparation failed to exhibit a similar activity at levels as high as 1000 μg/ml. The immunosuppressive effect was determined to be independent of cytotoxicity, PHA inactivation by binding and other non-specific phenomena. The results of this study indicate that the immunosuppressive activity of porcine uterine flushings is caused, at least in part, by one or both of these proteins present in the pAP preparation.     

A differential effect of IgM and IgG antibodies on the blastogenic response of lymphocytes to rubella virus

Peripheral blood lymphocytes of rabbits immunized with live rubella vaccine respond to rubella virus antigens in tissue culture with increased DNA synthesis as measured by incorporation of ³H-thymidine. This reaction can be inhibited by rubella antibody. A dose dependent effect was observed when antibodies in whole serum were mixed with virus prior to addition to lymphocyte cultures. When antisera were fractionated and their individual immunoglobulins tested, a paradoxical effect was obtained. Immune IgG although it was highly effective in neutralizing the virus was incapable of inhibiting the lymphocyte response and at times caused an increased response. In contrast, immune IgM which was less efficient in neutralizing virus caused significant suppression of the blastogenic reaction. By themselves these results might have signified that IgG and IgM antibodies have different specificities or different binding properties with respect to viral surface antigens. However, immune complexes consisting of virus and IgM reduced response of both rubella immune and normal rabbit lymphocytes to PHA. This nonspecific inhibitory action required a specific step of antigen and IgM antibody interaction and normal IgM-virus mixtures or mixtures of anti-rubella IgM and poliovirus or influenza virus did not suppress lymphocyte response to PHA. Anti-rubella IgG complexed with rubella virus did not suppress the PHA response. The IgG function was apparently limited to neutralization of the infectivity of rubella virus whereas the major role of IgM was manifested through its suppressive effect on lymphocyte reactions.     

Folate requirement for blast cell transformation in mixed leukocyte cultures.

Mixed leukocyte cultures from normal donors were set up in standard tissue culture media. Blast cell transformation was measured 6 days later by ³H-thymidine uptake and assessed qualitatively with stained smears. Media RPMI 1629 and RPMI 1640 allowed much more intense blastogenesis than Medium 199, the difference being due to the low folic acid content of Medium 199 (0.01 mg/liter) compared with RPMI 1629 (10 mg/liter) and RPMI 1640 (1 mg/liter). Further experiments indicated that folic acid increased the multiplication rate of blast cells but did not promote the induction or initial transformation phases of the mixed leukocyte reaction. The effect of folic acid on the PHA response was entirely different: ³H-thymidine uptake in 3 day PHA cultures was decreased, and there was no apparent effect on the number or percentage of blast cells seen on the smears. The reason for this difference is at present unknown, but some possibilities are discussed.     

Further studies on the thymocyte stimulating factor.

The thymocyte-stimulating activity produced by spleen cells cultured in the presence of PHA (cell. Immunol.17, 495, 1975) was further investigated. It was found that this activity is caused by a factor having the chemical properties of a protein with a molecular weight of 30,000–32,000 that, at high concentration, probably dimerizes to a molecule of molecular weight about 55,000 as measured by Sephadex gel filtration. This factor is produced also in mixed lymphocyte cultures of CBA and C57 spleen cells. The observation that the factor produced in the presence of PHA and the factor produced in the mixed lymphocyte cultures have the same molecular weight and the same rate of denaturation at two different temperatures suggests that they are the same, or very similar, substance. The fact that spleen cells of nu/nu mice do not produce thymocyte-stimulating factor suggests that thymus-dependent cells are involved in the production of this factor. This assumption is consistent with the observation that spleen cells of mice less than two weeks old (which show a very small response to PHA) do not produce significant amounts of thymocyte-stimulating factor. On the other hand, results of experiments with hydrocortisone-injected mice suggest that the target cell for thymocyte-stimulating factor is the immature, cortisone-sensitive thymocyte. The properties of thymocyte-stimulating factor are discussed and compared to those of factors with similar actions reported in the literature.     

Enhanced inhibition of splenic lymphocyte proliferation by 2,3,7,8-tetrachlorodibenzo-p-dioxin in C57B1/10 (Ah+Ah+) mice compared to DBA/2 (AhAh) mice

In concanavalin A-treated cultures TCDD (10(-8)M) inhibited proliferation of mouse spleen lymphocytes in cells of C57B1/10/mice by 65% and in cells of DBA/2 mice--by 42%. In phytohaemagglutinin-treated cultures of spleen lymphocytes of both strains TCDD produced no significant decrease in incorporation of 3H-thymidine into DNA. These observations indicate that effects of TCDD on lymphocyte proliferation are mediated by pleiotropic activity of Ah gene.     

Partial purification of uterine secretory protein capable of suppressing lymphocyte reactivity invitro

Porcine uterine flushings obtained on day 15 of the estrous cycle were fractionated using gel filtration, and preparative poly-acrylamide gel electrophoresis was employed to separate one molecular weight fraction into two groups of small uterine-specific proteins designated pAP and pLAP. The two groups were assayed for immuno-suppressive ability using phytohemagglutinin (PHA) stimulated porcine lymphocyte cultures and incorporated ³H-thymidine. It was found that the pAP preparation which was composed of two proteins inhibited lymphocyte reactivity to PHA (p<.001) while the pLAP preparation failed to exhibit a similar activity at levels as high as 1000 μg/ml. The immunosuppressive effect was determined to be independent of cytotoxicity, PHA inactivation by binding and other non-specific phenomena. The results of this study indicate that the immunosuppressive activity of porcine uterine flushings is caused, at least in part, by one or both of these proteins present in the pAP preparation.     

Regulation of T-lymphocyte mitogenesis by the leukocyte product 15-hydroxy-eicosatetraenoic acid (15-HETE)

Synthesis of lipoxygenase metabolites of [¹⁴C]arachidonic acid by mouse spleen lymphocyte cultures was inhibited by the leukocyte product 15-hydroxy-eicosatetraenoic acid (15-HETE) in a dose-dependent manner. In parallel experiments, the influence of 15-HETE on mitogenesis in spleen lymphocyte cultures was examined. 15-HETE at concentrations similar to those which inhibited cellular lipoxygenases progressively inhibited mitogenesis induced by the T-cell mitogen PHA but had no significant effect on the mitogenic response to the B-cell mitogen LPS. The inhibitory response was maximal when 15-HETE was added within 8 hr of exposure to PHA. Several analogs of 15-HETE having progressively fewer double bonds were tested in the same systems. 15-OH,20:3 had approximately the same potency as 15-HETE in inhibiting both mitogenesis and formation of metabolites from [¹⁴C]arachidonic acid. 15-OH, 20:2 and 15-OH,20:0 were much less active in either assay. Mitogenesis, induced in spleen cell cultures by the tumor promoter phorbol myristate acetate, was also blocked by 15-HETE. These experiments indicate that lipoxygenase metabolites of arachidonic acid may play an important role in T-lymphocyte blastogenesis and suggest that 15-HETE, via its ability to selectively inhibit cellular lipoxygenases, may function as an endogenous regulator of T-lymphocyte responses.     

Dipetalonema viteae: in vitro blastogenesis of hamster spleen and lymph node cells to phytohemagglutinin and filarial antigens

Hamsters of the randomly bred LAKZ and inbred LSH strains were infected with Dipetalonema viteae, and the in vitro responses of lymph node and spleen lymphocytes to male and female worm antigens and phytohemagglutinin (PHA) were measured by a [³H]-thymidine-uptake assay at various times after infection. The PHA response remained unchanged at the level of controls in infected LAKZ hamsters while LSH hamsters showed a depressed response to the mitogen during late infection. Stimulation of lymph node cells by filarial antigens was maximal in both strains of hamsters at Week 4 postinfection, almost reaching values obtained in PHA stimulated cultures. A similar high lymphocyte transformation reaction was measured after the injection of dead third stage larvae. During transient microfilaremia, when antibody titers reached a maximal level, the lymphocyte reactivity to filarial antigens decreased drastically and only occasionally was demonstrated in hamsters 20 and 30 weeks after infection. No correlation between lymphocyte reactivity and parasitological findings (worm load or intensity and duration of microfilaremia) could be demonstrated. The cellular unresponsiveness to filarial antigens was further analyzed in chronically infected LAKZ hamsters. No suppressor cells could be found in lymphocyte suspensions of nonresponding hamsters. A challenge infection did not restore lymphocyte reactivity. Serum of chronically infected hamsters caused marked inhibition when added to filarial antigen-sensitive lymphocytes. Lymphocytes from hamsters with a mixed D. viteae and Schistosoma mansoni infection responded as well to soluble schistosomal egg antigens at Week 30 of a D. viteae infection as lymphocytes from hamsters infected with S. mansoni alone. The humoral immune response to schistosomal antigens, however, was significantly lower in animals with a mixed infection.     

Responsiveness of Thymus Cells to Syngeneic and Allogeneic Lymphoid Cells

THE thymus is necessary for the normal development of cell-mediated immunity in mice as shown by the immunological defects after neonatal thymectomy¹. Thymus cells themselves can be stimulated by allogeneic lymphoid cells in mixed leucocyte reaction (MLR)² and become killer cells or cytotoxic lymphocytes after stimulation with allogeneic spleen cells in vitro (H. Wagner and M. Feldmann, unpublished work) and in vivo^3,4. This suggests that the thymus as well as peripheral lymphoid tissues contain T cells which can be stimulated by foreign histocompatibility antigen to divide and differentiate into the cytotoxic lymphocytes which mediate cellular immunity. There have been suggestions that thymus cells might be stimulated to divide by “self” antigen, as well as foreign cells: incorporation of ³H-thymidine above background levels has been found in cultures with syngeneic spleen and thymus cells of adult rats⁵, although the experiments do not determine whether thymus or spleen cells have been stimulated. In contrast to these experiments, Howe et al. reported that only thymus cells of neonatal CBA mice reacted to allogeneic and syngeneic spleen cells of adult animals in “one way” MLR cultures^6,7. Whether the reaction of neonatal thymus cells to syngeneic adult spleen cells is recognition of “self” antigens is uncertain, since spleens of adult mice could carry antigens which do not occur in neonatal animals and are therefore “unknown” for neonatal thymus cells. We demonstrate here that neonatal thymus cells do not react to 4-day-old CBA spleen cells, but adult thymus cells do react against both allogeneic and syngeneic adult spleen cells.