Studies on immunomodulatory properties of isoniazid. II. Effect of isoniazid on interleukin 2 production and interleukin 2-receptor expression

The effect of isoniazid on interleukin 2 production and interleukin 2-receptor expression by phytohemagglutinin-stimulated human T cells was investigated. High concentrations of the drug decreased interleukin 2 production while low doses (10(-5)-10(-6) M) produced a slight increase in interleukin 2 production. Isoniazid did not affect the expression of interleukin 2-receptors on the surface of T cells, except a slight decrease in cells exposed to high levels of the drug.     

Epidermal growth factor and thyrotropin-releasing hormone act similarly on a clonal pituitary cell strain. Modulation of hormone production and inhbition of cell proliferation

GH(4)C(1) cells are a clonal strain of rat pituitary cells that synthesize and secrete prolactin and growth hormone. Chronic treatment (longer than 24 h) of GH(4)C(1) cells with epidermal growth factor (EGF) (10(-8) M) decreased by 30-40 percent both the rate of cell proliferation and the plateau density reached by cultures. Inhibition of cell proliferation was accompanied by a change in cellular morphology from a spherical appearance to an elongated flattened shape and by a 40-60 percent increase in cell volume. These actions of EGF were qualitatively similar to those of the hypothalamic tripeptide thyrotropin-releasing hormone (TRH) (10(-7) M) which decreased the rate of cell proliferation by 10-20 percent and caused a 15 percent increase in cell volume. The presence of supramaximal concentrations of both EGF (10(-8)M) and TRH (10(-7)M) resulted in greater effects on cell volume and cell multiplication than either peptide alone. EGF also altered hormone production by GH(4)C(1) cells in the same manner as TRH. Treatment of cultures with 10(-8) M EGF for 2-6 d increased prolactin synthesis five- to ninefold compared to a two- to threefold stimulation by 10(-7) M TRH. Growth hormone production by the same cultures was inhibited 40 percent by EGF and 15 percent by TRH. The half- maximal effect of EGF to increase prolactin synthesis, decrease growth hormone production, and inhibit cell proliferation occurred at a concentration of 5 x 10 (-11) M. Insulin and multiplication stimulating activity, two other growth factors tested, did not alter cell proliferation, cell morphology, or hormone production by GH(4)C(1) cells, indicating the specificity of the EGF effect. Fibroblast growth factor, however, had effects similar to those of EGF and TRH. Of five pituitary cell strains tested, all but one responded to chronic EGF treatment with specifically altered hormone production. Acute chronic EGF treatment with specifically altered hormone production. Acute treatment (30 min) of GH(4)C(1) cells with 10(-8) M EGF caused a 30 percent enhancement of prolactin release compared to a greater than twofold increase caused by 10(-7) M TRH. Therefore, although EGF and TRH have qualitatively similar effects on GH(4)C(1) cells, their powers to affect hormone release acutely or hormone synthesis and cell proliferation chronically are distinct.     

Maintenance of cell proliferation and steroidogenesis in cultured human fetal adrenal cells chronically exposed to adrenocorticotropic hormone: rationalization of in vitro and in vivo findings

Previous studies indicated that acute exposure of adrenal cells to adrenocorticotropic hormone (ACTH) markedly stimulates steroidogenic capacity in vitro but also inhibits cell proliferation. However, in vivo, ACTH is known to stimulate adrenal cell growth. To address this discrepancy, we determined the effect of long-term (9-11 days) continuous or intermittent exposure to ACTH on human fetal adrenal cell proliferation and steroidogenesis. Adrenal glands from fetuses 18-22 wk gestation were studied. Fetal zone cells were plated either on plastic or on an extracellular matrix (ECM) in the presence and absence of basic fibroblast growth factor (bFGF) (0.5 ng/ml) and 1 or 10 nM ACTH. As determined by cell counting, bFGF stimulated cell proliferation during 9 days in culture. In the presence of bFGF, the average doubling time decreased from 44 to 30 h on plastic and from 37 to 26 h on ECM. Under these conditions, ACTH did not inhibit cell proliferation. Proliferation of fetal adrenal corticosteroid-producing cells in the ACTH-treated cultures also was assessed by histochemical staining for 3 beta-hydroxysteroid dehydrogenase (3 beta HSD). The number of positive cells increased more than 4-fold between Days 5 and 9 in culture. Continuous treatment with 1 nM ACTH increased dehydroepiandrosterone sulfate (DHAS) production 5- to 10-fold during the first 5 days in culture. Thereafter, the stimulated hormone production decreased over time, although there was still a difference of almost 100-fold between the control and ACTH-treated cultures at the end of 9 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of follicle-stimulating hormone and 17beta-estradiol on proliferation of chicken embryonic ovarian germ cells in culture

The effects of follicle-stimulating hormone (FSH) and 17beta-estradiol (E2) on chicken ovarian germ cell proliferation were evaluated through a germ-somatic cell coculture model. Ovarian cells from the left ovaries of 18-day-old chicken embryos were cultured in serum-free McCoy's 5A medium at 39 degrees C and challenged with FSH (0.25-1.0 IU/mL) or E2 (10(-8)-10(-5) M) alone and in combination for 48 h. The number of germ cells was counted, and the proliferating cells were immunolocalized by a specific antibody against proliferating cell nuclear antigen (PCNA). The labeling index (LI) was determined for germ cells. Results revealed that germ cells could survive and kept proliferating under support of somatic cells. Germ cells were localized by expression of a specific antibody for stem cell factor receptor c-kit. Both FSH (0.25-1.0 IU/mL) and E2 (10(-7)-10(-5) M) alone induced a marked increase in germ cell number (P<0.05), and PCNA-LI of germ cells was greater in FSH-treated groups (0.25-1.0 IU/mL) and E2-treated groups (10(-8)-10(-5) M), compared with vehicle-treated group (P<0.05). Furthermore, FSH manifested a synergistic effect with E2 (10(-6)-10(-5) M) in stimulating germ cell proliferation. These results indicate that FSH might interact with estrogen to promote ovarian germ cell proliferation in embryonic chickens near hatching.     

Sequential study on the influence of adriamycin on cell proliferation and ultrastructure of cultured mammary tumor cells

The time-dependent cytocidal and growth inhibitory effects of Adriamycin (ADM) on monolayer cultures of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumor cells were analyzed. The inhibitory effect on cell proliferation was assessed by colony formation in soft agar. Growth inhibition and [3H]thymidine labeling indices clearly demonstrate a dose-dependent antimitotic and cytotoxic effect of the drug. At low concentrations (10(-9)-10(-8) M), 90-100% of cells survived 24-hr exposure. At a higher concentration (10(-5) M), 75-80% of cells survived after 8-hr exposure; by 72 hr only 20-30% of the cells remained. Autoradiographic examination of the pulse-labeled cultures demonstrated no change in the proportion of cells in S-phase during the first 4 hr of treatment. Subsequently DNA synthesis was completely abolished and remained inhibited for the duration of the experiment (72 hr). Clonogenic assay revealed a complete arrest of growth in cells exposed to 10(-5) M ADM and greater than 60% inhibition of cell proliferation at 10(-7) M. Ultrastructural changes were not observed in cells during the first 4 hr of treatment; however, after 8 hr most surviving cells exhibited alterations in nuclear chromatin. The surviving cells showed mitochondrial degeneration, myelin body formation, and vacuolization of the endoplasmic reticulum. This study shows the potential usefulness of the primary culture system in drug evaluation. In addition, serial observation of the effects of ADM revealed a cell subpopulation of the primary culture with differential sensitivity to the drug.     

Modulatory effect of zinc on the proliferative response of murine spleen cells to polyclonal T cell mitogens

To study the effect of zinc on the proliferative response to polyclonal T cell mitogens, spleen cells from C57BL/6 mice were cultured with or without ZnCl2 and stimulated with graded doses of concanavalin A or phytohemagglutinin. Addition of 10(-4) M ZnCl2 inhibited proliferation whereas 10(-5) to 10(-6) M ZnCl2 did not modify the response to suboptimal doses of mitogen but increased DNA synthesis in cultures stimulated with high doses of mitogen (10 or 20 micrograms/ml of concanavalin A and 10 or 25 microliters/ml of phytohemagglutinin) which are supraoptimal for C57BL/6 mice, and inhibited proliferation in cultures of spleen cells from animals of this strain, low responder to T cell mitogens. In contrast, supplementation with ZnCl2 did not enhance the response to mitogen of spleen cells from high responder BALB/c mice. The enhancing effects of ZnCl2 on the proliferative response of C57BL/6 cells were not observed following depletion of adherent cells or in cultures supplemented with 5 X 10(-5) M 2-mercaptoethanol, both conditions capable of abrogating the inhibitory effect of high mitogen doses on the response of C57BL/6 cells.     

Effects of follicle-stimulating hormone and 17β-estradiol on proliferation of chicken embryonic ovarian germ cells in culture

The effects of follicle-stimulating hormone (FSH) and 17beta-estradiol (E2) on chicken ovarian germ cell proliferation were evaluated through a germ-somatic cell coculture model. Ovarian cells from the left ovaries of 18-day-old chicken embryos were cultured in serum-free McCoy's 5A medium at 39 degrees C and challenged with FSH (0.25-1.0 IU/mL) or E2 (10(-8)-10(-5) M) alone and in combination for 48 h. The number of germ cells was counted, and the proliferating cells were immunolocalized by a specific antibody against proliferating cell nuclear antigen (PCNA). The labeling index (LI) was determined for germ cells. Results revealed that germ cells could survive and kept proliferating under support of somatic cells. Germ cells were localized by expression of a specific antibody for stem cell factor receptor c-kit. Both FSH (0.25-1.0 IU/mL) and E2 (10(-7)-10(-5) M) alone induced a marked increase in germ cell number (P<0.05), and PCNA-LI of germ cells was greater in FSH-treated groups (0.25-1.0 IU/mL) and E2-treated groups (10(-8)-10(-5) M), compared with vehicle-treated group (P<0.05). Furthermore, FSH manifested a synergistic effect with E2 (10(-6)-10(-5) M) in stimulating germ cell proliferation. These results indicate that FSH might interact with estrogen to promote ovarian germ cell proliferation in embryonic chickens near hatching.     

Signals involved in T cell activation. I. Phorbol esters enhance responsiveness but cannot replace intact accessory cells in the induction of mitogen-stimulated T cell proliferation

The role of accessory cells (AC) in the initiation of mitogen-induced T cell proliferation was examined by comparing the effect of intact macrophages (M phi) with that of 4-beta-phorbol 12-myristate 13-acetate (PMA). In high-density cultures, purified guinea pig T cells failed to proliferate in response to stimulation with phytohemagglutinin (PHA), concanavalin A (Con A), or PMA alone. The addition of M phi to PHA or Con A but not PMA-stimulated cultures restored T cell proliferation. The addition of PMA to high-density T cell cultures stimulated with PHA or Con A also permitted [3H]thymidine incorporation, but was less effective than intact M phi in this regard. This action of PMA was dependent on the small number of AC contaminating the T cell cultures as evidenced by the finding that PMA could not support mitogen responsiveness of T cells that had been depleted of Ia-bearing cells by planning, even when these cells were cultured at high density. When PMA was added to T cell cultures supported by optimal numbers of M phi, catalase-reversible suppression of responses was noted. Even in cultures containing catalase, PMA failed to enhance responsiveness above that supported by optimal numbers of M phi. A low-density culture system was used to examine in greater detail the possibility that PMA could completely substitute for M phi in promoting T cells activation. In low-density cultures, mitogen-induced T cell proliferation required intact M phi. PMA could not support responses even in cultures supplemented with interleukin 1-containing M phi supernatants or purified interleukin 2 alone or in combination. Similar results were found in high-density cultures of T cells depleted of Ia-bearing cells. These results support a model of T cell activation in which AC play at least two distinct roles. The initiation of the response requires a signal conveyed by an intact M phi, which cannot be provided by either a M phi supernatant factor or PMA. The response can be amplified by additional M phi or M phi supernatant factors. PMA can substitute for M phi in this regard and can provide the signal necessary for amplification of T cell proliferation supported by small numbers of intact AC.     

All-trans retinoic acid induces apoptosis in acute myeloblastic leukaemia cells

The effect of all-trans retinoic acid (ATRA) on leukaemia cell differentiation, proliferation and induction of apoptosis was studied by using autonomously growing blast cells isolated from eight patients with acute myeloblastic leukaemia (AML) either at diagnosis ( n=4) or at relapse (n=4). No morphological or functional differentiation induced by ATRA was observed in any of the cases studied. In cell cultures, inhibition of leukaemia cell growth by ATRA was obvious, especially in the case of clonogenic cells, and it was both time- and concentration-dependent. Induction of apoptosis was more difficult to achieve. The cells retained over 90% viability in suspension when the ATRA exposure at any of the concentrations studied was 48 h or less. When the time of exposure to ATRA was longer than 48 h, the viability of the cells decreased in a concentration-dependent manner. Apoptosis was observed morphologically in each of the AML cases with 10-5 to 10-8 M ATRA, if the incubation time of cells in ATRA was 72 h. The percentage of apoptotic cells increased with increasing ATRA concentrations from 12± 9% of 10-8 M ATRA to 78±12% of 10-5 M ATRA. The DNA electrophoretic method was able to detect apoptosis in all the AML samples exposed to 10-7 and 10-6 ATRA for 48 h and occasionally in cases where lower concentrations and longer exposure time were used. In conclusion, the present study shows that it is possible to induce apoptotic leukaemia cell death in vitro with ATRA in AML, and this effect is dependent on both concentration and exposure time.     

The role of estrogens on the proliferation of human breast tumor cells (MCF-7)

The cloned human breast tumor cell line C7MCF7-173 behaved as an estrogen-dependent tumor in the nude mice. In contrast, E2 added to serum-less media did not increase the multiplication rate of these cells over the values obtained in the control cultures. Media supplemented with charcoal-dextran stripped (CD) human female serum (FHS) resulted in inhibition of cell proliferation in a concentration-dependent pattern (40% = 20% greater than 10% greater than 5% greater than 2.5%). E2 addition to all but the 2.5% CDFHS significantly increased the proliferation rate of these cells. The E2 concentration required to attain maximal proliferation rate increased as the serum concentration of the medium increased (e.g. 3 X 10(-11)M for 10% CDFHS, 3 X 10(-10)M for 40% CDFHS). E2 concentrations higher than the one needed to achieve maximal proliferation rate resulted in decreased cell yields (shut-off mechanism). Similar effects were obtained with synthetic and other natural estrogens. CD fetal bovine serum (FBS) also inhibited the proliferation of C7MCF7-173 cells; however, at similar concentration the inhibitory effect of CDFHS was more potent than the one obtained with CDFBS. The addition of "growth factors" (insulin, Epidermal Growth Factor and transferrin) and non-estrogenic steroids to 10% CDFHS failed to overcome the inhibitory effect of this serum. These results suggest that: (1) human and fetal bovine sera contain a specific inhibitor of the proliferation of E2-sensitive cells (estrocolyones), and (2) E2 promotes cell proliferation by neutralizing this inhibitor.     

Simvastatin and atorvastatin enhance gene expression of collagen type 1 and osteocalcin in primary human osteoblasts and MG-63 cultures

To clarify the mechanism of the stimulatory effect of statins on bone formation, we have assessed the effect of simvastatin and atorvastatin on osteoblast activity by analysing cell proliferation, as well as collagen, osteocalcin, and bone morphogenetic protein-2 (BMP2) gene expression in primary human osteoblast (hOB) and MG-63 cell line cultures. Explants of bone from patients without any metabolic disease under orthopedic hip procedures were used to obtain hOB. Cell cultures were established, synchronized, and different concentrations of simvastatin or atorvastatin were added (10(-9) M, 10(-8) M, 10(-7) M, 10(-6) M) during the experiment. Cell proliferation was analyzed after 24 h. Collagen polypeptide alpha1 type 1 (COL1A1) gene expression, osteocalcin, and BMP2 expression levels were quantified by real-time PCR after 24 h incubation with statins. There was a statistically significant decrease in cell proliferation related to simvastatin or atorvastatin addition at all concentrations in primary hOB compared with those not treated. A significant increase in COL1A1, osteocalcin, and BMP2 gene expression was detected when hOB cultures were treated with simvastatin or atorvastatin at different concentrations. Similar but less significant effects were found on MG-63 cells. After statin treatment we observed both an arrest of proliferation in hOB cells and an increase in collagen, osteocalcin, and BMP2 gene expression, consistent with a stimulatory effect towards mature osteoblast differentiation. These findings support the bone-forming effect of statins, probably through the BMP2 pathway.     

Opioid peptides modulate production of interferon gamma by human mononuclear cells

The opioid neuropeptides have previously been shown to bind to and affect leukocyte function including lymphocyte proliferation, NK-cell activity, mononuclear cell chemotaxis, immunoglobulin synthesis, and lymphokine production. The effect of the opioid peptides beta-endorphin and Met-enkephalin on interferon gamma (IFN) production by concanavalin A-stimulated human mononuclear cells was examined. Both beta-endorphin and Met-enkephalin enhanced IFN production by the majority of donor mononuclear cells tested and did so at concentrations between 10(-14) and 10(-10) M. When 10(-12) M beta-endorphin or Met-enkephalin were included in concanavalin A-stimulated mononuclear cell cultures, IFN concentrations were significantly enhanced to 205 +/- 45 and 252 +/- 67% of control, respectively. Although the majority of cell preparations tested exhibited an enhanced production of IFN in response to these opioid peptides, some did not. When beta-endorphin or Met-enkephalin were utilized at 10(-11) M, 10 of 15 and 7 of 11 responded with IFN production greater than 20% above the control (untreated) level. There was not an absolute correlation between an enhanced response to beta-endorphin and Met-enkephalin, suggesting the presence of multiple receptor types on these cells for opioids. The opioid receptor antagonist, naloxone, did not significantly prevent the opiate effect. When 10(-8) M naloxone was included in cultures containing 10(-12) M beta-endorphin or Met-enkephalin no significant inhibition of the effect of either opioid on IFN production was observed.     

Studies on immunomodulatory properties of isoniazid: V. Influence of isoniazid on secretion of interleukin-1.

Isoniazid in vitro decreased interleukin-1 production by glass-adherent cells from the blood of healthy donors. The previously described modulation of T cell activation by isoniazid seems to be independent of decreased interleukin-1 secretion.     

Development of Hammondia heydorni in cultured bovine and ovine cells

Sporozoites were excysted from oocysts of Hammondia heydorni obtained from a naturally-infected dog and inoculated into monolayer cultures of bovine pulmonary artery endothelial cells (CPA), Madin-Darby bovine kidney (MDBK) cells, bovine monocytes (M617), or ovine monocytes (WOMO). Sporozoites penetrated all four cell lines and underwent asexual reproduction by endodyogeny (as determined by electron microscopy) to form cyst-like structures at four to nine days after sporozoite inoculation (DAI). At 4-10 DAI, considerably more zoites were harvested from M617 cultures (80.1 x 10(6) zoites) than from CPA (17.4 x 10(6], MDBK (47.3 x 10(6], and WOMO (53.5 x 10(6]. Little or no parasite multiplication occurred at 10-16 DAI. Zoites harvested at 7 DAI and transferred to freshly prepared cultures did not penetrate cells nor develop further.     

Dibutyryl cyclic AMP modulation of gap junctions in SW-13 human adrenal cortical tumor cells

Cultured human adrenal cortical adenocarcinoma cells (SW-13) form a confluent monolayer of epithelial-like cells when seeded into culture flasks. Following a 24-48 hr non-mitotic period, cells begin to divide and become confluent within a week after seeding at 5 X 10(4) cells/cm2. The SW-13 cells were exposed to dibutyryl cyclic AMP (DbcAMP), cyclic AMP (cAMP), sodium butyrate, and adrenocorticotropin (ACTH). The rate of SW-13 cell proliferation was measured with a DNA microfluorometric assay, as well as by procedures measuring the incorporation of 3H-thymidine. In addition, following administration of ACTH and DbcAMP, the fractional area of membrane covered by gap junctions was quantitated with freeze-fracture electron microscopic techniques. Dibutyryl cyclic AMP at a concentration of 1 X 10(-3) M decreased the growth rate of the cell population. There was a corresponding increase in the fractional area of gap junctions found on the cell membrane in 96-hr DbcAMP-treated cultures. ACTH (40 mU/ml) exposure failed to produce an increase in the fractional area of gap junctions or to alter the rate of cell proliferation. From these data it can be suggested that elevations in cAMP levels within the cell can be related to both the proliferation of gap junctions and the decrease in cell proliferation in the SW-13 tumor cell.     

Androgen-dependent expression of c-jun and c-fos in human non-transformed epithelial prostatic cells: association with cell proliferation

OBJECTIVE: To assess the effect of dihydrotestosterone (DHT) on the gene expression of C-FOS and C-JUN and on the proliferation of human non-transformed epithelial prostatic (HNTEP) cells. METHODS: Cell proliferation (MTT) and C-FOS and C-JUN mRNA expression (RT-PCR) were determined in cells treated with DHT (10(-8), 10(-10), and 10(-13)M) or with control medium. RESULTS: DHT 10(-13) M had a significant stimulatory effect on cell proliferation (p < 0.05) and C-FOS and C-JUN gene expression when compared to cells treated with higher concentrations of this hormone (10(-10) and 10(-8)M) or with the control group. CONCLUSIONS: Our data demonstrate that the increase in C-FOS and C-JUN expression and cell growth in HNTEP cells is maximal with the lowest DHT concentration (10(-13)M). These proto-oncogenes may play a role in the control of hormone responsiveness and cell proliferation in HNTEP cells.     

The insulin-secreting and proliferative activity of established islet cells in the presence of sulfonylurea]

Exponentially growing rat islet cells (RINr) and hamster islet cells (HIT T-15) were incubated in presence of tolbutamide (10-1000 microM), gliclazide (0.1-10 microM) or glibenclamide (0.01-10 microM) for 15 hrs. Accumulation of insulin in culture medium was estimated by RIA. Effects of sulfonylureas (SU) on cell proliferation were assessed by 3H-thymidine (3H-T) incorporation into cellular DNA. All of SUs used stimulated insulin production in RIN and HIT cell cultures (with an exception of tolbutamide, which markedly suppressed insulin secretion in HIT cells at 1000 microM). 3H-T incorporation into RIN cells was elevated only in presence of gliclazide (10 microM), whereas tolbutamide at 1000 M significantly inhibited RIN cell proliferation. Gliclazide (0.1 microM) and glibenclamide (0.01-10 microM) enhanced 3H-T incorporation into HIT cells. Further detailed investigations of mechanisms of SU effects on islet cell reproduction will be of use for designing optimal strategy of hypoglycemizing therapy of diabetes mellitus.     

Behavior of confluent endothelial cells after irradiation. Modulation of wound repair by heparin and acidic fibroblast growth factor

Image analysis was used to study the repair process of a circular mechanical lesion of confluent human endothelial cells in culture after irradiation (10 Gy) prior to wounding. Coverage of denuded areas 48 and 96 h after injury of endothelial cells was identical in control and irradiated cultures, although the labeling index was lowered by 80 to 95% in irradiated cultures. The cell density of non damaged irradiated areas was decreased by 50%. When cultures were submitted to increasing doses of radiation (5.0-30 Gy), the labeling index of the cells diminished rapidly between 0 and 5.0 Gy and reached a plateau at 10 Gy. The decrease in cell density (50% of control at 96 h) was identical at each dose of radiation. Thus cell migration alone could be sufficient for the repair of the lesion, while cell proliferation would mainly maintain the original cell density. The addition of heparin to the culture medium slowed down cell migration and proliferation, but the speed of repair was identical in irradiated and non-irradiated cultures. Acidic fibroblast growth factor plus heparin accelerated equally the repair process whether the cultures were irradiated or not. In irradiated cultures the presence of acidic fibroblast growth factor and heparin maintained cell density in confluent areas at a level similar to that in non-irradiated damaged control cultures without addition of mitogens. Thus heparin and acidic fibroblast growth factor play a role in cell proliferation, in the maintenance of the cell monolayer integrity and in restoring a continuous layer by rapid cell migration and elongation after irradiation.     

Immunosuppression by spleen cells from Moloney leukemia. Comparison of the suppressive effect on antibody response and on mitogen-induced response.

The inhibitory effect of cells from leukemic spleens on the immune functions of normal lymphocytes was studied. Suppressor cells were obtained as the nonadherent fraction (NA) from splenic tumors of mice infected with MuLV-Moloney. This fraction (NA MuLV- M) contained less than 10% membrane Ig-positive (Ig+) cells, 45 to 60% theta-positive cells (theta+) and 40 to 50% naught cells (theta-, Ig-). Similarly prepared fractions from normal control spleens (NAc) containing 75 to 90% theta+cells and less than 10% Ig+ and naught cells were utilized in control cultures. Addition of the NA MuLV- M cells into cultures (Marbrook system) of normal spleen cells with sheep red blood cells suppressed the specific antibody response determined by the number of hemolytic plaque forming cells (PFC). The PFC response was significantly suppressed at a suppressor cell to responder cell ratio of 1:100, and was completely abolished at a ratio of 1:10 or higher. The control NAc fraction showed some inhibitory effect only at high suppressor to responder ratios (1:2 or 1:1). In contrast, the suppressive effect of NAMuLV-M on mitogen-induced 3H-thymidine incorporation in normal B and T cells was much weaker. Very little, if any, suppression occurred at the ratio of 1:100 or 1:10, however, about 50% decrease in DNA synthesis was observed at the ratio 1:2 or 1:1. On the basis of this differential suppressive effect, it is suggested that leukemic spleen cells can suppress the function of immunocompetent cells by more than one mechanism.