Functional analysis of macrophage hybridomas. I. Production and initial characterization

A series of macrophage hybridomas were generated by fusion of splenic adherent cells with P388D1 tumor cells. Forty-two cell lines were established, and each was cloned by limiting dilution. Six clones that exemplified the spectrum of macrophage heterogeneity were selected for further analysis. Qualitative and quantitative differences in phenotype and functional activity were noted. Some clones constitutively expressed Ia antigens, whereas others only expressed detectable levels of Ia after lymphokine activation. The level of antigen-presenting activity generally correlated with the level of Ia expression. Furthermore, interclonal differences were noted in the levels of receptor-mediated phagocytosis and IL 1 secretion. Generally, the hybridoma clones maintained stable phenotypic and functional properties during approximately 1 yr of continuous in vitro culture. These cloned hybridoma cell lines represent a useful resource to analyze macrophage biology and to dissect structure and function relationships.     

Inhibition of lymphoid cell growth by a lipid-like component of macrophage hybridoma cells

Previous studies have shown that plasma membranes of murine lymphocytes and lymphoid tumor cells can reversibly inhibit the growth of both normal and transformed lymphocytes. The inhibitor can be extracted with organic solvents and has properties consistent with it being a lipid or lipid-like component of the membrane. This report identifies a series of cloned macrophage hybridoma cell lines, obtained by fusion of splenic adherent cells and the P388D1 line, which have very high levels of lipid-like growth-inhibitory molecules. Furthermore, a survey of seven cloned lines indicated that the macrophages fell into two distinct groups with regard to their level of growth-inhibitory activity. Group 1 lines had little or no inhibitory activity when cells were examined for their effect on a B lymphocyte proliferative response. Organic extracts from these macrophages had inhibitory activity (on a per cell basis) comparable to that seen with extracts of the P388D1 parental cell line and lymphoid tumor cells. In contrast, relatively low numbers of Group 2 macrophages could profoundly inhibit B macrophage proliferation. The growth-inhibitory activity was quantitatively recovered in organic extracts of the macrophages. Although the precise nature of the lipid moiety remains undefined, the data argue against the involvement of oxidized cholesterol. These findings indicate that lipid-like inhibitors of cell growth are present and functional in these macrophage cell lines. In addition, the results demonstrate that the inhibitory activity found in plasma membranes and liposomes is present and active in the membranes of intact cells, which is in contrast to the possibility that the inhibitor is an artifact generated during subcellular fractionation. Thus, the inhibitor is likely to have a physiologic role in growth control and in macrophage-mediated immunoregulation, probably acting via a mechanism involving cell-cell contact.     

Serological characterization of macrophage hybridomas: identification of an interferon-gamma-inducible surface marker

Macrophage hybridoma clones prepared by fusion of splenic adherent cells with P388D1 tumor cells have previously been shown to be heterogeneous with respect to function at the clonal level. In this study the macrophage clones were phenotypically characterized by indirect RIA using a battery of rat MAbs to murine myeloid and lymphoid cell surface markers. All macrophage clones expressed the common leukocyte antigen T200 and the Mac-1 alpha and beta chains. Markers which were differentially expressed among the clones included class II antigens and the antigens detected by MAbs MIV 55, MIV 38, and 14G8. The antigens detected by the latter three MAbs were referred to as MBR-1, -2 and -3, respectively. Functional heterogeneity did not correlate with phenotypic heterogeneity among the macrophage clones. Treatment of macrophage clones with IFN-gamma resulted in a significant increase in the expression of class II antigens and induced the expression of MBR antigens on some clones which were constitutively negative for these markers. The clonal distribution and induction patterns of class II antigen as compared to MBR antigen indicated that regulation of expression of these markers was independent. In addition, the clonal distribution and induction pattern of MBR antigens, along with competitive binding studies using radiolabeled MIV 38 and 14G8 MAbs, suggested that the three MBR antigens were similar or closely associated molecules.     

Functional analysis of cloned macrophage hybridomas. IV. Induction and inhibition of mixed lymphocyte responses

A series of macrophage (M phi) hybridomas were generated by fusion of drug-marked P388D1 (H-2d) tumor cells with CKB (H-2k) splenic adherent cells. The ability of this panel of cloned M phi hybridomas expressing various levels of surface Ia antigens to induce allogeneic mixed lymphocytes responses (MLR) was examined. All MLR stimulatory M phi hybridomas expressed surface Ia antigens. However, some Ia+ and all Ia- M phi hybridomas were unable to induce vigorous MLR responses. Furthermore, even after induction of surface Ia antigen expression with Con A supernatants (Con A Sn) or purified interferon-gamma, the nonstimulatory M phi hybridomas remained ineffective at inducing strong MLR proliferative responses. Furthermore, addition of the latter M phi hybridoma clones (both with and without Con A Sn treatment) to conventional MLR cultures resulted in inhibition of MLR responses. The series of inhibitory M phi hybridomas secreted normal levels of IL 1 upon stimulation with lipopolysaccharide. After surface Ia induction with Con A Sn, the inhibitory M phi hybridomas could stimulate secretion of IL 2 and expression of IL 2 receptors. Moreover, although they inhibited conventional MLR responses, IL 2 production and IL 2 receptor expression were not significantly inhibited. Addition of these M phi hybridomas 24 to 48 hr after initiation of MLR response also inhibited MLR proliferation. The results indicated that the group of inhibitory M phi hybridomas can inhibit MLR responses after IL 2 secretion and acquisition of IL 2 receptors. Finally, this inhibitory activity has been maintained during 1 yr of continuous in vitro culture, and the hybridomas represent a stable "homogeneous" subpopulation of inhibitory macrophages. Thus, the inhibitory phenotype appears to reflect arrest at a distinct differentiation stage.     

Functional analysis of cloned macrophage hybridomas. V. Induction of suppressor T cell responses

It has been suggested that macrophage-like accessory cells are involved in suppressor T cell (Ts) induction. To further analyze this issue, we obtained several cloned macrophage hybridoma cell lines by somatic cell fusion of the macrophage tumor P388D1 of DBA/2 (H-2d) origin with splenic adherent cells of CKB mice (H-2k). Several cloned lines displayed the serological and functional characteristics of macrophages. We evaluated the ability of these hybridomas to induce third order or effector Ts (Ts3) to suppress the contact sensitivity response against the hapten 4-hydroxy-3-nitrophenyl acetyl (NP). In contrast to the parental P388D1 and two other macrophage hybridomas, one macrophage hybridoma clone, termed 63, when conjugated with NP, induced Ts3, which suppressed contact sensitivity responses against NP but not DNFB, showing that the Ts3 were antigen specific. Macrophage hybridoma 63 could specifically induce Ts3 activity in either H-2k, H-2d, or H-2k/H-2d heterozygous hosts. Thus, macrophage hybridoma 63 functionally expressed major histocompatibility complex-related restricting determinants, and the fusion with cells from a H-2k macrophage donor caused a functional complementation of H-2d-related, Ts-inducing elements. The genetic restriction governing induction of Ts3 was controlled by genes that mapped to I-J region. Furthermore, NP-conjugated macrophage hybridoma 63 could serve as a target for elicitation of suppressor responses after administration of I-Jk, but not I-Jb, restricted suppressor factor. The data suggest that macrophage hybridomas represent a means to dissect heterogeneity within the macrophage population. The data also imply that the I-J determinants expressed on macrophages represent a ligand for the antigen receptor of Ts.     

Accessory cell function in a Con A response: role of Ia and interleukin 1

Accessory cell (A-cell) function in a Con A response was analyzed. Irradiated P388D1 cells efficiently induced a proliferative response to Con A of T cells purified from spleen cells, whereas paraformaldehyde-fixed P388D1 cells failed to serve as A cells. Although IL-1 containing culture supernatant (SN) of a macrophage hybridoma induced the Con A response of the T-cell preparations, the depletion of Ia+ cells by the treatment with anti-Ia antibody and complement abrogated the response in the presence of IL-1. Fixed P388D1 cells and the hybridoma SN synergized in the reconstitution of the response. A 15,000-Da fraction of the hybridoma SN or human recombinant IL-1 alpha was able to substitute the hybridoma SN for the response. The reconstitution of the response by IL-1 and fixed P388D1 cells was inhibited by the addition of monoclonal anti-Ia antibody. These results indicate that IL-1 or fixed P388D1 cell does not exert a sufficient signal by itself and both of them are required for the reconstitution of a Con A response of highly purified T cells, and that Ia on fixed P388D1 cells play an important role.     

Functional analysis of cloned macrophage hybridomas. VI. Differential ability to induce immunity or suppression

We previously screened a series of macrophage hybridomas derived from fusion of P388D1 (H-2d) tumor cells with CKB (H-2k) splenic adherent cells for their ability to induce I-J restricted Ts cell responses. One Ia+ macrophage clone (63) consistently induced Ag-specific, I-J-restricted Ts. To evaluate whether macrophage hybridoma 63 also induced delayed-type hypersensitivity (DTH) immunity, mice were immunized with hapten-coupled macrophage hybridoma cells. Hapten-coupled splenic adherent cells and control macrophage hybridomas induced significant primary DTH responses, whereas hapten-coupled macrophage 63 induced little or no immunity when injected into H-2 compatible hosts. However, macrophage hybridoma 63 specifically activated I-Ak, I-Ad, or I-Ed restricted T cell hybridomas/clones, in vitro in the presence of appropriate Ag. Three different strategies designed to eliminate suppressor cell activity were successfully used to demonstrate that hapten-coupled macrophage 63 could also induce in vivo immunity. First, after immunization with hapten-coupled macrophages, mice were treated with cyclophosphamide. Second, macrophage 63 was treated with anti-IJ idiotype antibody before 4-hydroxy-3-nitrophenyl acetyl hapten (NP) coupling. Finally, haptenated macrophages were injected into I-A compatible but I-J incompatible recipients. These protocols are known to inhibit the induction of Ts activity, thus these results indirectly suggest that there is stimultaneous generation of Ts activity in vivo. The latter hypothesis was tested in adoptive transfer experiments. Transfer of lymph node cells from NP-63 primed B10.BR (H-2k) mice induced immunity in naive 4R animals, whereas the same number of immune cells suppressed NP-induced DTH responses in 5R mice. The combined results indicate that a cloned macrophage line can activate both Th and Ts cells. Macrophages which induce Ts activity may be responsible for maintaining the balance of immunity vs suppression. The data support the hypothesis that IJ interacting molecules (IJ-IM) expressed on macrophages are critical for induction of suppressor cell activity.     

Adherent Ia+ murine cell lines with characteristics of dendritic cells. II. Characteristics of I region-restricted antigen presentation

The ability of an adherent Ia+, interleukin 1+ (IL-1) tumor cell line (P388AD) to present turkey gamma-globulin (TGG) to primed T lymphocytes was demonstrated and compared with normal antigen-presenting cells (APC) found in mouse spleen. P388AD tumor cells presented TGG to long-term cultures of TGG-reactive T cells (LTTC) and to lymph node-derived T cells which were enriched on nylon wool columns and subsequently depleted of endogenous antigen-presenting cells with anti-Ia antisera and complement. MHC-restricted antigen presentation by P388AD was observed when long-term cultures of TGG-reactive T cells were used as the responding T-cell population. Furthermore, antisera directed against I-region determinants expressed on the P388AD tumor cells inhibited TGG-specific T-cell proliferation in a dose-related fashion, suggesting a functional role for the tumor cell-associated Ia molecules. The kinetics of antigen presentation to LTTC by P388AD were similar to the kinetics observed for splenic APC, although the magnitude of the proliferative response to LTTC to TGG was generally lower when antigen (Ag) was presented by the tumor cells compared to splenic antigen-presenting cells (APC). However, the magnitude of T-cell proliferation of immune lymph node (LN) T cells was comparable when Ag was presented on tumor cells or splenic APC. Several experiments suggested that Ag uptake and/or processing may be less effective in P388AD tumor cells as compared to normal splenic APC. A nonadherent Ia+, IL-1- tumor cell line (P388NA), which was isolated from the same parental tumor as P388AD, was also tested for the ability to present Ag to primed T lymphocytes and Ag-reactive LTTC. In contrast, to P388AD, the nonadherent tumor cell failed to present TGG under identical culture conditions even though Ia molecules were expressed on the tumor cells and Ag uptake had occurred. However, the defect in Ag presentation by P388NA could be corrected if an exogenous source of purified interleukin 1 was supplied to the cultures. A unique opportunity thus exists with both the P388AD and P388NA tumor cell lines to decipher some of the molecular interactions leading to T-cell proliferation during antigen presentation.     

Effect of prostaglandin E2 on the gamma-interferon induction of antigen-presenting ability in P388D1 cells and on IL-2 production by T-cell hybridomas

The effect of prostaglandin E2 on the gamma-interferon (IFN-gamma)-mediated induction of Ia expression and antigen-presenting activity in macrophage cell lines was studied. Using a lymphokine preparation obtained from the T-cell hybridoma FS7-20.6.18 (known to produce interferon) to induce the expression of Ia in P388D1 cells, the influence of PGE2 on this phenomenon was studied. Screening of the cell cultures by indirect immunofluorescence using an anti-I-Ad monoclonal antibody confirmed the inhibitory effect of PGE2 in the induction of I-Ad. However, the inhibition of the antigen-presenting ability of these cells, as measured by their capacity to stimulate interleukin 2 (IL-2) production by antigen-specific, I-region-restricted (Ag/I) T-cell hybridomas, was more difficult to demonstrate and was only evident when using low concentrations of Ia-inducing lymphokines or when using "low avidity" T-cell hybridomas. The latter were distinguished by the limited response (in the form of IL-2 production) that was observed when they were tested with P388D1 cells that had been induced with IFN-gamma. By contrast, PGE2 had profound inhibitory effects on the ability of T-cell hybridomas to secrete IL-2 in response to Ag/I or concanavalin A. These results suggest that although PGE2 inhibits the full induction of Ia on macrophages, it has little effect on the induction of Ag/I presentation by the same cells, probably because most T cells require relatively low levels of Ia on the surface of presenting cells. T-cell responses to Ag/I are inhibited, however, because of the effects of PGE2 on the T cells themselves.     

Regulation of colony-stimulating factor 1-dependent macrophage precursor proliferation by type beta transforming growth factor

Transforming growth factor (TGF) type beta, a potent growth modulator, has recently been shown to inhibit the proliferation and function of several types of immune cells. This report investigates the effect of human platelet purified TGF-beta on CSF-1-induced proliferation in liquid cultures. We used two cell types to study TGF-beta effects, bone marrow precursors and a c-myc partially transformed CSF-1-dependent macrophage cell line designated BMM-8. We found that CSF-1-dependent proliferation of both cell types was strongly inhibited by TGF-beta in a dose-dependent manner. Approximately 1.6 and 8 pM TGF-beta inhibited 50% of CSF-1 proliferation of the bone marrow precursors and BMM-8, respectively. Inhibition appeared to be reversible, as bone marrow and BMM-8 cells proliferated in response to CSF-1 after preincubation of the cells in TGF-beta. Interestingly, inhibition of hematopoietic cells was observed only after a lag period of 24 to 48 h after onset of cultures. TGF-beta inhibition was partially diminished when increasing amounts of CSF-1 were added to the cultures. TGF-beta inhibition did not involve secondary inhibitory factors such as IFN or PG, both of which have been previously shown to suppress CSF responsiveness. Finally, flow cytometric analysis of the cell cycle indicated that within 48 h, TGF-beta-treated BMM-8 cells were prevented from entering S phase. These results suggest that TGF-beta may play an important role in the negative regulation of macrophage production.     

Inductive effect of recombinant human interleukin-1 alpha and beta on differentiation of macrophage-like tumor cell line P388D1

We used the mouse monocyte/macrophage-like tumor cell line P388D1 to test whether or not interleukin-1 (IL-1) stimulates differentiation of monocyte/macrophage progenitors. Incubation of these cells with recombinant human interleukin-1 (rhIL-1) alpha and beta resulted in their increased adherence, stimulation of nonspecific esterase activity, and increased Fc rosette formation. rhIL-1s inhibited cell growth and stimulated Fc rosette formation in a dose-dependent fashion. The cell growth inhibition due to rhIL-1s depended on the concentration of serum in culture medium. Synergism between rhIL-1 and calcium ionophore A23187 was found for the cell growth inhibition and Fc rosette formation. The presence of ethylene glycol bis- (beta-aminoethyl ether) N,N,N,N,-tetraacetic acid(EGTA) in the medium abolished the stimulatory effect of rhIL-1 on Fc rosette formation of the cell line. These results demonstrate that rhIL-1s are a potent inducer of the differentiation of the macrophage-like tumor cell line P388D1.     

Antigen presentation to T cell hybridomas by a macrophage cell line: an inducible function

We have investigated the ability of an Ia-, nonantigen-presenting macrophage tumor cell line, P388D, (H-2d), to present antigen to T cell hybridomas after incubation in a lymphokine-containing preparation. P388D, cells were incubated in microtiter wells with various concentrations of Con A-stimulated spleen cell supernatants. Antigen-specific stimulation of H-2d-restricted, KLH-specific T cell hybridomas was observed by P388D1 incubated with SUP.P388D1 cells incubated for 3 days in medium or control SUP did not present antigen. In addition, no stimulation of T hybridomas was seen by P388D1 in the inhibited by the appropriate monoclonal anti-Ia reagents. These results demonstrate that a macrophage tumor cell line can be induced to present antigen and provides for large numbers of readily available, homogeneous macrophages for studying the cellular biochemical requirements for antigen processing and presentation.     

A "lymphokine-like" soluble product that induces proliferation and maturation of B cells appears in the serum-free supernatant of a T cell hybridoma as a consequence of mycoplasmal contamination

The serum-free supernatant of a cloned murine T cell hybridoma supports the proliferation and maturation to Ig secretion of purified B cells (mu+ cells) from BALB/c nu/nu mice, but has no effect on the proliferation of nylon wool-selected BALB/c nu/+ splenic T cells. Although the supernatant activates B cells without co-stimulation, it synergizes with anti-mu for the proliferative response. The induction of B cell proliferation and maturation to Ig secretion is directly related to contamination of the hybridoma by Mycoplasma hyorhinis. Hybridoma cells freed of mycoplasma by detergent treatment fail to produce active supernatant, and reinfection of the treated cells reconstitutes the activity. Furthermore, deliberate infection of a mycoplasma-free unrelated T cell hybridoma, as well as the monocytic cell line P388D1, results in the production of supernatants with B cell proliferating activity. Mycoplasma organisms isolated from the supernatant induce B cell proliferation without subsequent maturation to Ig secretion. Gel filtration chromatography of the supernatant from mycoplasma-contaminated hybridoma cells yields two peaks of activity. The first peak, found at the exclusion limit of the gel, results in B cell proliferation without maturation and may be attributed to mycoplasma organisms. The second peak (average m.w. 90,000) results in B cell proliferation as well as differentiation to Ig secretion. A "lymphokine-like" soluble product released by Mycoplasma hyorhinis is most likely responsible for this B cell activation, because fractionation of the supernatant from deliberately contaminated P388D1 cells gives essentially the same results, and gel filtration of mycoplasma-free supernatants does not generate any active fractions. The possibility should be considered that mycoplasma-derived soluble products may be among the many factors controlling in vitro B cell growth and maturation.     

Growth-inhibitory activity of lymphoid cell plasma membranes. I. Inhibition of lymphocyte and lymphoid tumor cell growth

Membranes isolated from normal spleen cells or lymphoid tumor cells were found to inhibit in vitro growth of several murine tumor cell lines including a B cell hybridoma, a thymoma, and a mastocytoma. 50% inhibition occurred at membrane protein concentrations of 60-100 micrograms/ml. A similar concentration dependence was found for inhibition of [3H]-thymidine incorporation by tumor cells and for the lipopolysaccharide-induced mitogenic response of normal spleen cells. The inhibitory activity co-purified with the plasma membrane upon fractionation of crude membranes. Membrane solubilization with deoxycholate followed by dialysis to remove the detergent gave good recovery of inhibitory activity in the resulting reconstituted membranes. Membrane-mediated growth inhibition resulted from a decreased rate of proliferation and not from increased cell death. A toxic effect of the membranes was further ruled out by the finding that increasing the fetal calf serum content of the medium could substantially reverse the growth inhibition. Thus, the plasma membrane of lymphoid cells contains a component that can slow or stop the growth of cells in culture. This membrane component may have a role in cell contact-mediated regulation of growth.     

Macrophage antigens associated with adhesion: identification by a monoclonal antibody specific for Lewis lung carcinoma cells

A monoclonal antibody specific for Lewis lung carcinoma (3LL) cells (Mab 5B5) was found to recognize antigens expressed on murine macrophages and on a macrophage hybridoma line upon cell adhesion on plastic surfaces. These antigens were also present on the surface of murine macrophage tumor M5076 cells which develop solid tumors and metastases. The M5076 tumor cells freshly isolated from the primary tumor and from hepatic metastases strongly bound Mab 5B5 but lost this capacity after adhesion. Freshly isolated thioglycolate-elicited peritoneal mouse macrophages were not labeled by Mab 5B5; however, after 1 h of adhesion, 50% of the adherent macrophages were directly incubated with Mab 5B5 prior to harvesting by scraping. Permeabilization of peritoneal macrophages by saponin showed that the antigens recognized by Mab 5B5 were present inside the cells before adhesion. Similar results were obtained with the 2C11-12 macrophage hybridoma cells. P388D1 cells (a weakly adherent macrophage tumor cell line), HL60 cells (a human promyelocytic cell line), and human monocytes were poorly labeled without permeabilization but were strongly labeled by Mab 5B5 upon permeabilization. The specificity of the monoclonal antibody in relation to the adherence capacity of these cells is discussed.     

Modulation of EL-4 mouse lymphoma cell proliferation by macrophages and tumor related factors

It is well known that macrophages play an important role in the control of tumor growth. This control may be the result of a direct action of macrophages or mediated by several biologically active products or factors elaborated by these and other cell populations. Our studies on the proliferation of a murine T-cell lymphoma (EL-4) showed that the treatment of the ascitic fluid (from the peritoneum of EL-4 bearing mice) with carbonyl iron resulted in a depletion of phagocytes concomitant with a significant increase of [3H] thymidine uptake by EL-4 cells. Further, the growth of EL-4 cells cultured in semisolid agar was significantly inhibited by an underlayer of large quantities of macrophages both from normal and EL-4 bearing mice as well as when cultured in the presence of PGE2. The underlayer of tumor macrophages P388 D1 resulted in an increase of the EL-4 cell growth. Also, conditioned media obtained from in vitro liquid cultures of EL-4 cells and L 1210 cells (B-lymphoma) produced a remarkable inhibition of the in vitro cloning capacity and [3H] thymidine uptake by EL-4 cells. These data support the hypothesis that different factors from normal and hemopoietic tumor cells may control the tumor growth and point out that self-produced factors may modulate the proliferation of tumor cells.     

Lymphokine production by human T cell hybridomas

Human T cell hybridomas were generated by hybridization of SH9 cells, the 6-thioguanine-resistant variant of human T lymphoma Hut102-B2, with concanavalin A-stimulated human peripheral blood lymphocytes. The hybrid nature of the established cell lines was documented by the difference in D14S1 restriction fragment length polymorphism between SH9 and the hybridoma cell DNA, and by the expression of OKT11 antigen on hybrid cells. T cell growth factor, macrophage growth factor (MGF), and interferon (IFN) activities have been demonstrated in the supernatants of different hybrid cultures, but not in SH9 cell cultures. Substantial quantities of MGF were secreted by several hybrids including the L23 line. MGF activity was dose-dependent, heat-labile, and synergistic with indomethacin. High titers of IFN activity were found in the cultures of hybridoma L415 and its subclones. Neutralization with specific antisera showed the IFN synthesized by L415 clones was immune interferon (IFN-gamma). Like the parental SH9 line, all of the hybridomas producing these lymphokines exhibited a cell surface phenotype typical for helper T cells. The hybridoma system therefore shows potential for the study of various lymphokines produced by human helper T lymphocytes.     

Cellular basis of immunomodulation by cholera toxin in vitro with possible association to the adjuvant function in vivo

Cholera toxin (CT) is a potent oral immunogen that also acts as a strong mucosal adjuvant for immune responses to related as well as unrelated Ag. To elucidate the immunomodulating effects of CT at the cellular level we have examined interactions of CT with APC and with B and T lymphocytes in vitro. CT markedly stimulated the production of IL-1 from APC (mouse peritoneal macrophages or macrophage cell line P388D1) but did not induce Ia-Ag and had marginal, if any, effect in potentiating Ia Ag expression stimulated by rIFN-gamma on these cells. CT had differential effect on T cell proliferation in vitro, usually strongly inhibitory but on Con A-stimulated spleen cells during prolonged (greater than or equal to 5 days) culture or when added on day 4 or later to these cultures up to a two- to three-fold enhancement of proliferation was seen. CT-induced inhibition of T cell proliferation was associated with decreased production of IL-2 and anergy to exogenously added IL-2 despite apparently normal expression of IL-2R. Similar to what was found with T cells LPS-stimulated spleen B cells demonstrated both inhibition and enhancement of proliferation in the presence of CT: in high concentrations (greater than or equal to 10(-8) M) and early in culture (day 3) CT had a strong inhibitory effect on the proliferation of B cells, whereas later (day 6) and/or at lower CT concentrations (10(-9) to 10(-11) M) the proliferation was increased up to 10-fold. The net effect of CT treatment on Ig-production by LPS-stimulated spleen B cells was seen as an enhanced level of IgA and IgG but not IgM in culture supernatants. The differential effects of CT on the cells of the immune system observed in vitro may, singly or in combination, explain the immunostimulatory function of CT.     

Inhibition of tumor cell growth by a specific 6-phosphofructo-2-kinase inhibitor, N-bromoacetylethanolamine phosphate, and its analogues

The high rate of glycolysis despite the presence of oxygen and mitochondria in tumor cells implies an important role for this process in cell division. The rate of glycolysis is assumed to be dependent on the cellular concentration of fructose 2,6-bisphosphate, the concentration of which in turn depends on a bifunctional enzyme and the ratio of this enzyme's 6-phosphofructo-2-kinase versus its fructose 2,6-bisphosphatase activities. To prove the hypothesis that inhibition of glycolysis in tumor cells by 6-phosphofructo-2-kinase inhibitors would cause inhibition of tumor cell proliferation, ten N-bromoacetylethanolamine phosphate analogues were designed, synthesized, and tested. They were screened for their activities against various human tumor cell lines to study the effects of inhibition of glycolysis on cell proliferation. The relationship between the structure of these compounds and their inhibitory activity on cell proliferation was also discussed. It was found that the activity of N-(2-methoxyethyl)-bromoacetamide, N-(2-ethoxyethyl)-bromoacetamide, and N-(3-methoxypropyl)-bromoacetamide was comparable to that of the positive control AraC. These three inhibitors showed in vivo anticancer effects in P388 transplant BDF1 mice.     

Effect of retinoic acid on the proliferation and phagocytic capability of murine macrophage-like cell lines

Retinoic acid (RA) exerted a variable degree of growth inhibitory activity on the macrophage-like cell lines P388D1, J774.2, WEHI-265, WEHI-3, and PU-5. Comparison of cell proliferation and clonal growth suggests that at concentrations of 10(-9)-10(-6) M the inhibitory activity stems from processes leading to elongation of cell cycle time and not from terminal differentiation processes. RA was shown to be a potent inducer of the development of high-phagocytic phenotypes (assessed by phagocytosis of heat-killed yeast cells) in the P388D1, J774.2, and WEHI-265 cell lines which differ substantially in their proliferative and adherence characteristics. The PU-5 and WEHI-3 cell lines were not induced by RA to express an enhanced phagocytic activity toward heat-killed yeast cells. The augmented phagocytic capability was dose dependent over a wide range of RA concentrations. In P388D1 cells, 2 X 10(-12) M RA already exerted significant phagocytosis augmentation effects, which progressively increased up to 2 X 10(-5) M RA, the highest concentration tested. Retinal, retinyl acetate, and retinol had similar effects to those of RA on both cell adherence and phagocytosis in P388D1 cells, albeit at concentrations four to six orders of magnitude higher. Optimal development of the high-phagocytic phenotype in P388D1, J774.2, and WEHI-265 cells required at least 96 hr of culture in the presence of RA; at 48 hr and 23 hr the effects were already substantial, whereas at 4 hr of exposure to RA no significant enhancement of phagocytosis could be detected. Thus both extended periods of culture in the presence of RA (more than two to three cell cycles) and high concentrations were needed for induction, in more than 90% of the cells, of the expression of a high-phagocytic phenotype. The reversion to a low-phagocytic phenotype upon removal of RA was also rather slow and required several cell cycles. In P388D1 cells RA also enhanced the phagocytosis of latex beads but had no effect on the phagocytosis of starch particles, or the extent of binding of immunoglobulin G-coated sheep red blood cells (SRBC). The expression of receptors for concanavalin A and for nonopsonized SRBC remarkably increased in RA-treated cells, as was the ability to perform Fc-receptor mediated erythrophagocytosis. Both P388D1 cells and WEHI-265 cells were induced by RA to express nitroblue tetrazolium reducing activity. The data suggest that RA induces profound changes in the functional capabilities of macrophage-like cell lines which are apparently not dependent on cessation of growth and terminal differentiation processes.