Inhibition of lymphocyte proliferative responses by ribavirin.

When added to cultures of human peripheral blood lymphocytes, ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) inhibited antigen- and mitogen-induced proliferative responses as determined by [3H]thymidine incorporation. Dose-dependent suppressive effects were obtained when concentrations of 5 to 60 microgram of ribavirin per ml were added at culture initiation or up to 96 h thereafter. Ribavirin inhibited [3H]uridine and [3H]leucine incorporation by concanavalin A-activated and normal lymphocytes although not as severely as deoxyribonucleic acid synthesis. The capacity of ribavirin to interfere with lymphoproliferative responses was entirely reversed by guanosine and, to a lesser extent, by adenosine and xanthosine. These studies demonstrate that ribavirin is a reversible inhibitor of lymphocyte nucleic acid synthesis and suggest that the drug may be immunosuppressive when administered in vivo.     

In vitro sensitivity of immature human mast cells to chemotherapeutic agents

Cells from the human immature mast cell line, HMC-1, were tested for their sensitivity to 11 chemotherapeutic agents by changes in viability and incorporation of tritiated thymidine ([3H]-thymidine) after 72 h of in vitro drug exposure. Doxorubicin hydrochloride and cytosine arabinoside were the most active agents against HMC-1 cells at the concentrations tested. Doxorubicin hydrochloride inhibited the incorporation of [3H]-thymidine and decreased the viability of HMC-1 cells by greater than 90% at a concentration of 0.06 microgram/ml. Cytosine arabinoside inhibited the incorporation of [3H]-thymidine and decreased the viability by greater than 95% at a concentration of 0.1 microgram/ml. A clone of HMC-1 cells, A4, with an enhanced percentage (70-80%) of metachromatically staining cells was equally sensitive to these two agents; however, A4 cells were more sensitive to vinblastine sulfate and less sensitive to methylprednisolone than was the parent cell line. Both HMC-1 cells and A4 cells showed approximately equal sensitivity to etoposide and to mitomycin. These results show that human mast cells are susceptible in vitro to a number of commonly used chemotherapeutic drugs.     

Detection of mycoplasma contamination through modulation (stimulation or inhibition) of thymidine incorporation by unstimulated mouse spleen cells.

In the present report we describe a rapid and sensitive assay for mycoplasma detection in cell cultures. The assay is based on the ability of contaminated culture supernatants to modulate [3H]TdR incorporation by unstimulated mouse splenocytes. Several mycoplasma species (Mycoplasma orale, culturable and non-culturable strains of Mycoplasma hyorhinis) inhibited [3H]TdR incorporation and permitted the detection of some contaminated cell cultures that would otherwise have escaped detection in assays measuring [3H]TdR incorporation by mitogen-stimulated splenocytes. On the other hand, several other mycoplasma species (Mycoplasma arginini, Mycoplasma hominis) strongly enhanced [3H]TdR incorporation by unstimulated splenocytes. This enhancement was optimally detectable on day 2 after initiation of the cultures. The sensitivity of the assay was determined for a mycoplasma species (culturable M. hyorhinis) that inhibited as well as for one (M. arginini) that enhanced [3H]TdR incorporation. In both cases, the sensitivity was such that 1-3 x 10(2) mycoplasma colony-forming units (CFU) could be detected.     

Tuftsin-enhanced thymidine incorporation by murine splenic monocytes

Tuftsin (Thr-Lys-Pro-Arg), a natural immunomodulating peptide originally found to stimulate phagocytosis by polymorphonuclear leukocytes (PMN), is now known to bind to both PMN and monocyte-macrophages, affecting many of their functions. Administration of tuftsin induces leukocytosis in vivo. We have recently observed that while tuftsin remains in the cytoplasm upon binding and internalization in human PMNs, it translocates into the human monocyte nucleus, suggesting that tuftsin may directly affect growth of monocytes. We have therefore examined the effect of tuftsin on [3H]thymidine incorporation in fractions of murine splenocytes to identify a cell population responding to tuftsin. Tuftsin showed the greatest effect in [3H]thymidine incorporation of splenocytes over controls at optimum conditions of 2% fetal bovine serum and 1 microgram/ml of tuftsin. Splenocyte fractionation by Lymphocyte Separation Medium indicated that tuftsin primarily affects the mononuclear cell fraction; further fractionation revealed that tuftsin affects mostly the monocytes that adhered to plastic. We subsequently further purified the splenic monocytes by repeated plastic adhesion and Percoll gradient separation, to show that tuftsin increases [3H]thymidine incorporation of these highly purified monocytes.     

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.     

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.     

Increased unscheduled DNA synthesis in aged human diploid fibroblasts

Using Hayflick's model, ultraviolet (UV) induced unscheduled DNA synthesis was compared in cells at a low, middle and high population doubling level (PDL). Concomitant DNA replication was prevented by arresting all cultures in the G₁ phase by lowering the serum concentration. After UV-irradiation cells at a high PDL incorporated 1.5–2 times more [³H] thymidine into DNA than cells at a low and middle PDL. These findings seem to indicate that the repair capacity of cells at a high PDL is more than those of cells at low and middle PDL. Alternatively, the higher incorporation might be explained by a difference in the pool sizes of precursors of cells at different PDL. These possibilities were examined by adding fluorodeoxyuridine to the system to reduce de novo synthesis of deoxythymidine triphosphate (dTTP), and measuring the pool size of dTTP and the specific activity of [³H] dTTP in cells at different PDL. The results indicated that the increased incorporation in fact reflects increased unscheduled DNA synthesis in cells at a high PDL.     

Mitogenic activity of staphylococcal peptidoglycan.

Staphylococcus aureus peptidoglycan displayed a marked dose-dependent mitogenic activity for mouse splenocytes and human peripheral blood lymphocytes in vitro, as measured by increased [3H]thymidine incorporation. Similarly it was mitogenic for athymic nude mouse spleen cells, whereas no blastogenic effect was observed in T cell-enriched and B cell-depleted mouse lymphocyte cultures. These data demonstrate that peptidoglycan-responding cells in mouse spleen cell cultures are B lymphocytes.     

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.     

A comparison of DNA repair synthesis in primary hepatocytes from young and old rats

DNA repair synthesis has been compared in primary hepatocyte cultures obtained from 3-month-old and 16-20-month-old rats. Several morphological and metabolic characteristics were determined to assure cultures of comparable quality. DNA damage was induced by the addition of bleomycin or the exposure of the culture to UV irradiation. DNA repair (unscheduled DNA synthesis) was determined by measuring [3H]thymidine incorporation. After UV irradiation, there was almost twice as much [3H]thymidine incorporation in cells obtained from young rats as in those obtained from old rats. Equal amounts of bleomycin resulted in substantially greater damage to DNA in cells from old rats than from young rats. For equal amounts of DNA damage there was again diminished [3H]thymidine incorporation in cells obtained from old rats. Finally equal amounts of bleomycin resulted in equal damage to DNA when the bleomycin was added to isolated rat liver nuclei from young or old rats. Bleomycin treated nuclei from young rats incorporated substantially more [3H]thymidine triphosphate (TTP) than bleomycin treated nuclei from old rats. The results indicate that hepatocytes from old rats are much more susceptible to bleomycin than hepatocytes from young rats and that the capacity for DNA repair synthesis is impaired in hepatocytes from old rats.     

Transferrin can replace serum for in vitro growth of mitogen-stimulated T lymphocytes

Activation of human T cells by mitogens was compared in cultures containing serum, human serum albumin or purified human transferrin as growth support. The mitogenic effect of the lectins leucoagglutinin, concanavalin A and Wistaria floribunda agglutinin was measured as incorporation of [3H]thymidine into the cellular DNA of the lymphocytes. Three different preparations of transferrin were all able to fully substitute serum or serum albumin as growth promotors, when present at concentrations of 10 microgram/ml or more. A small contamination of transferrin in the human serum albumin preparations used was shown to be responsible for their growth-supporting effect, while no need for the presence of albumin itself could be demonstrated.     

Catalase and Lipopolysaccharide Enhance Proliferation in the Rat Mixed Lymphocyte Reaction

Proliferation of rat spleen cells in a mixed lymphocyte culture was amplified fivefold or more in the presence of 2000 units of catalase/ml, as measured by [3H]thymidine incorporation. A similar effect was observed with 1 microgram of lipopolysaccharide (LPS)/ml. Addition of polymyxin B abrogated the promotional effect of LPS, but not that of catalase. These results indicate that the hydrogen peroxide generated by some cells in the rat spleen cell mixed lymphocyte culture suppresses the proliferative response. The demonstration that removal of plastic adherent cells (reducing the percentage of monocytes/macrophages by 75-80%) also results in a 5- to 10-fold increase in a subsequent MLR, indicates that some of the adherent cells may be the producers of hydrogen peroxide, which at higher concentrations suppresses the T-cell proliferation. The enhanced proliferation was not mainly due to increased interleukin 2 (IL-2) production, since the IL-2 concentrations of catalase and LPS-containing cultures were lower than those of control cultures.     

Lymphocyte cultures with small numbers of cells.

Prior experience with cultures of cerebrospinal fluid lymphocytes indicated a need to develop methods for culturing small numbers of cells. Peripheral blood lymphocytes (PBL) were obtained from 20 normal volunteers. Standard microcultures using 100 X 10(3) PBL/0.2 ml and cultures with 50, 25 and 12.5 X 10(3) in 0.1 ml or 0.2 ml were established in RPMI 1640 with autologous plasma. These cultures were incubated with PHA (1--30 microgram) for 3, 4 and 5 days, pulsed with [3H]thymidine and harvested. In unstimulated cultures, cpm declined linearly with decreasing cell numbers. Standard cultures (100 X 10(3) PBL/0.2 ml) had maximal PHA stimulation (80,916 +/- 6394) at day 3 with 30 microgram PHA. Other 0.2 ml cultures had lower cpm. By culturing 25 X 10(3) PBL in 0.1 ml for 3 days cpm were 82,874 +/- 6875 with 30 microgram PHA and 77,153 +/- 6022 with 15 microgram PHA and were similar to standard cultures. Similar cpm were seen with 12.5 X 10(3) PBL in 0.1 ml after 4 days with 30 micrograms of PHA (80,838 +/- 6674) and with 15 micrograms of PHA (72,860 +/- 6243), and also after 5 days with 30 micrograms of PHA (86,703 +/- 6732) and with 15 micrograms of PHA (74,066 +/- 6388). The maximal response (126,578 +/- 6580) was seen with 25 X 10(3) PBL/0.1 ml at day 4 with 30 micrograms of PHA. By decreasing culture volume to 0.1 ml and increasing time, the number of cells necessary to give PHA responses similar to standard cultures can be reduced by 75--88%.     

Preferential utilisation of deoxycytidine by undifferentiated (peanut positive) tonsillar lymphocytes

Peanut agglutinin (PNA), a D-galactose specific lectin, agglutinated 10-18% of lymphocytes isolated from tonsils of 3- to 6-yr-old children. PNA+ cells were found to be mainly B lymphocytes showing a 9.6 times higher specific activity of DNA polymerase alpha compared to the PNA- cells. The specific activity of deoxycytidine kinase as well as the incorporation of [5-3H]deoxycytidine were also much higher in PNA+ cell fraction than in PNA- fraction (7.7-fold and 6-fold, respectively). On the other hand, thymidine kinase activity and [5-3H]deoxythymidine incorporation were only 3.6 and 3.9 times higher, respectively. The data presented here show a high degree of DNA synthesis and preferential utilisation of [5-3H]deoxycytidine for DNA synthesis in this undifferentiated B lymphocyte fraction.     

Further study on the humoral response of a highly-purified spleen-derived immunosuppressive peptide (SDIP).

Some biological properties of a highly-purified non-cytotoxic spleen-derived immunosuppressive peptide (SDIP) have been investigated. SDIP was shown to inhibit the primary anti-sheep red blood cell (SRBC) response at the last step of differentiation of the lymphocyte. In the present study we demonstrated that this response seemed to be T- and adherent spleen-cell dependent as no inhibition was noticed either in the response to TNP-LPS, a T-independent antigen, or in the response to SRBC in adherent spleen cell-depleted cultures. SDIP activity did not occur through an inhibition of splenocytes DNA synthesis since [3H]-thymidine ([3H]-TdR) incorporation was not modified in mitogen or allogenic stimulated cultures. Conversely, SDIP could act through a stimulation of a T-cell subset as a low specific increase of [3H]-TdR was noticed in cultured thymocytes.     

Insulin-like growth factor binding protein-1 inhibits the DNA amplification induced by insulin-like growth factor I in human granulosa-luteal cells.

In order to study the effects of insulin-like growth factor (IGF-I) and insulin-like growth factor binding protein (IGFBP-1) on human granulosa cell proliferation after in vitro fertilization, cells were obtained after oocyte retrieval and cultured in the presence or absence of graded amounts of recombinant IGF-I, purified IGFBP-1 and [3H]thymidine. Physiological concentrations of IGF-I (2-200 ng/ml) were found to stimulate [3H]thymidine incorporation into the cells in a concentration-dependent manner. Half-maximal stimulation of [3H]thymidine incorporation was obtained with 10 ng/ml exogenous IGF-I, which was chosen for suppression experiments with graded amounts of purified IGFBP-1. Suppression of IGF-stimulated thymidine incorporation was observed when 200 ng/ml or more of IGFBP-1 was added to the culture medium. The same concentration of IGFBP-1 also markedly inhibited binding of [125I]iodotyrosyl IGF-I to the cells. It is concluded that: (i) after a refractory period, granulosa cells from hyperstimulated follicles retained their mitogenic activity; (ii) IGF-I is capable of stimulating DNA amplification in granulosa cells; and (iii) IGFBP-1 inhibits the IGF-I stimulated proliferation in these cells. In view of our previous studies showing that IGFBP-1 is synthesized by the granulosa cells as they luteinize, the present results suggest that IGFBP-1 is one of the endogenous factors locally regulating the growth and differentiation of granulosa cells.     

Reduced mitogen stimulation of DNA synthesis in human lymphocytes by a human recombinant interleukin-1 receptor antagonist.

The monokine interleukin-1 is produced by monocytes/macrophages after antigen/LPS stimulation and is an important early signal for the activation of resting T cells to become antigen specific T cells. However, little is known about the regulation and inhibition of IL-1. Recently, a new monokine has been described, generated by human macrophages, called interleukin-1 receptor antagonist (IL-1ra). This new monokine adheres to IL-1 in solution and blocks IL-1 receptor binding. IL-1ra is a glycoprotein structurally similar to IL-1 beta but having no interleukin-1-like activity. Using as a model mitogen (PHA 20 micrograms/ml)-stimulated lymphocyte DNA synthesis, we found that hrIL-1ra (30 min lymphocyte pretreatment) inhibits [3H]thymidine incorporation in a dose-dependent manner. This effect is most probably due to the inhibition of endogenous IL-1, which is a very important signal for T cell activation. The inhibition was maximum at the highest hrIL-1ra concentration used (250 ng/ml). However, when hrIL-1ra was added 2 h after PHA (20 micrograms/ml), a little, if any, inhibition of lymphocyte proliferation was found. The addition of hrIL-1ra simultaneously to the cell cultures with [3H]thymidine [( 3H]TdR) 6 h before the end of culture incubation did not significantly modify the results compared to the cells treated with PHA alone, indicating no interference of hrIL-1ra on [3H]TdR lymphocyte incorporation. We also found that the antibody anti-IL-1 beta inhibits mitogen stimulated lymphocyte DNA synthesis in dose-dependent concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of [125I]deoxyuridine for semi-microevaluation of human lymphocyte response to phytohemagglutinin in vitro

[125I]5-iodo-2'-deoxyuridine ([125I]UdR) was compared with [6-3H]thymidine ([3H]TdR) in a semi-micromethod used to evaluate the response of human lymphocytes to phytohemagglutinin (PHA) in vitro. 5-Fluoro-2'-dexoyuridine (FUdR) was added to [125I]UdR in order to increase its incorporation, by preventing endogenous thymidine synthesis. The relationship between PHA concentration and the isotope uptake by lymphocytes was studied. Similar dose-response curves were found for the two precursors, with a peak at 15 microgram/ml of PHA. Nevertheless the recovery of the labeled compounds was higher when [125I]UdR together with FUdR was used.     

Macrophage-mediated killing of splenic lymphocytes in adjuvant-induced arthritis

After 72 hrs in culture, unseparated spleen cells from rats with adjuvant-induced arthritis (AA) stimulated with high concentrations of Con A (>0.63 g/ml) leaked more lactate dehydrogenase (LDH) than did either unstimulated cultures or cultures stimulated with lower concentrations of Con A. This reflects and increase in dead or dying cells at concentrations of Con A >0.63 g/ml. Since Con A did not increase LDHlleakage in macrophage-depleted cultures of AA splenocytes, the decreased viability in unseparated, Con A-stimulated cultures appears to be a macrophage-dependent phenomenon. Since the increase in LDH release at high concentrations of Con A paralleled the decrease in [³H]-thymidine incorporation, these results suggest that Con A (>0.63 g/ml) induces macrophages from AA rats to kill splenic lymphocytes in culture.     

Heparin-binding growth factors stimulate DNA synthesis in rat alveolar type II cells

The proliferation of alveolar type II cells is important for repair of the alveolar epithelium after lung injury. We have previously reported that epidermal growth factor (EGF), insulin, cholera toxin, and endothelial cell growth supplement (ECGS) stimulate DNA synthesis of rat alveolar type II cells in culture. ECGS is a crude extract from bovine neural tissue that contains heparin-binding growth factors, and in this report we have compared the effect of ECGS to purified heparin-binding growth factors. ECGS stimulated [3H]thymidine incorporation into type II cells by 3-fold with half-maximal stimulation at 50 micrograms/ml. The purified acidic, class I heparin-binding growth factors, alpha-endothelial cell growth factor (-ECGF) and beta-ECGF stimulated type II cell DNA synthesis by 10-fold and 5-fold, respectively, with half-maximal stimulation at 40 ng/ml. Acidic fibroblast growth factor (FGFa) stimulated [3H]thymidine incorporation by 16-fold with half-maximal stimulation at 20 ng/ml, whereas basic FGF (FGFb) only stimulated type II cell DNA synthesis by 3-fold. Heparin potentiates the mitogenic effect of the acidic heparin-binding growth factors for both endothelial cells and fibroblasts but was found to inhibit FGFa- and FGFb-induced [3H]thymidine incorporation in type II cells by 80% with half-maximal inhibition occurring with 0.4 micrograms/ml and 1.3 micrograms/ml, respectively. When type II cells were cultured in the absence of serum, the heparin-binding growth factors had very little effect on [3H]thymidine incorporation. Only rat high density lipoprotein (HDL), but not insulin, EGF, or transferrin, was found to act synergistically with FGFa in stimulating [3H]thymidine incorporation in type II cells cultured in serum-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)