Suppression of human natural and antibody-dependent cytotoxicity by soluble factors from unstimulated normal lymphocytes

Serum-free culture supernatants of unstimulated normal human peripheral blood mononuclear cells contain soluble suppressor factor(s) (SSF) that significantly inhibit natural (NK) and antibody-dependent cellular cytotoxic (ADCC) activities of allogenic lymphocytes against a variety of target cells. Lymphocytes precultured with increasing concentrations of SSF showed a dose-dependent suppressive effect on these cytotoxic functions that was optimal at a concentration of 20% volume/volume. Adherent cells were not required for the production of SSF. Suppression was evident even at higher effector: target cell ratios and the inhibition was not reversed by washing lymphocytes. SSF was not itself cytotoxic, was stable at 56 degrees C, and its suppressive effect was maximal after 72 hr of incubation with effector lymphocytes. Initial estimate of the molecular weight of SSF by ultra-filtration was less than 20,000 daltons. Gel filtration of SSF on Sephacryl S-200 resulted in the elution of two peaks of activity; one in the region between markers of 13,700 and 25,000 daltons, and the other less than 13,700 daltons. Both fractions demonstrated significant suppressive activity on NK and ADCC functions of allogenic lymphocytes. SSF inhibition of NK activity could be partially reversed by incubating lymphocytes for 1 hr with human leukocyte interferon (IF) and almost completely reversed after 24 hr of IF treatment. A few selected monosaccharides (alpha-methyl-D-mannoside, L-fucose and L-rhamnose) showed a dose-dependent blocking effect on SSF activity, which suggests that SSF may act via receptor sites recognized by these sugars. As demonstrated for other lymphocyte functions, NK and ADCC activities may also be modulated by SSF elaborated by normal PBL.     

Noncytolytic inhibition of X4 virus by bulk CD8(+) cells from human immunodeficiency virus type 1 (HIV-1)-infected persons and HIV-1-specific cytotoxic T lymphocytes is not mediated by beta-chemokines

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) mediate immunologic selection pressure by both cytolytic and noncytolytic mechanisms. Non cytolytic mechanisms include the release of beta-chemokines blocking entry of R5 HIV-1 strains. In addition, CD8(+) cells inhibit X4 virus isolates via release of as yet poorly characterized soluble factors. To further characterize these factors, we performed detailed analysis of CTL as well as bulk CD8(+) T lymphocytes from six HIV-1-infected individuals and from six HIV-1-seronegative individuals. Kinetic studies revealed that secreted suppressive activities of HIV-1-specific CTL and bulk CD8(+) T lymphocytes from all HIV-1-infected persons are significantly higher than that of supernatants from seronegative controls. The suppressive activity could be blocked by monensin and brefeldin A, was heat labile, and appeared in a pattern different from that of secretion of chemokines (MDC, I-309, MIP-1alpha, MIP-1beta, and RANTES), cytokines (gamma interferon, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor), and interleukins (interleukin-13 and interleukin-16). This suppression activity was characterized by molecular size exclusion centrifugation and involves a suppressive activity of >50 kDa which could be bound to heparin and a nonbinding inhibitory activity of <50 kDa. Our data provide a functional link between CD8(+) cells and CTL in the noncytolytic inhibition of HIV-1 and suggest that suppression of X4 virus is mediated through proteins. The sizes of the proteins, their affinity for heparin, and the pattern of release indicate that these molecules are not chemokines.     

Immunoregulation by mouse T-cell clones. I. Suppression and amplification of cytotoxic responses by cloned H-Y-specific cytolytic T lymphocytes

H-Y-specific and H-2Db-restricted, Lyt-1-2+ T-cell clones ( CTLL ) with graded specific cytotoxic activities on male C57BL/6 (B6) target cells ( 1E3 , ; 2C5 , ++; 2A5 , +, 3E6 , +/-) were tested for their capacity to inhibit the generation of H-Y-specific cytotoxic T lymphocytes (CTL) in vitro. Addition of irradiated lymphocytes of CTLL 1E3 and CTLL 3E6 but not those of CTLL 2A5 or CTLL 2C5 abolished the generation of CTL from in vivo primed H-Y-specific precursor cells (CTLP) when added to fresh mixed-lymphocyte cultures (MLC). Exogenous sources of T-cell growth factors (TCGF) did not overcome suppression. Rather the presence of TCGF resulted in a further enhancement of suppressive activities in CTLL 1E3 and 3E6 and the induction of similar activities in cells from CTLL 2A5 and 2C5 , which by themselves were not inhibitory. Moreover when added to similar MLC on Day 1 instead of Day 0, only irradiated cells of CTLL 3E6 but not those of the other three CTLL were suppressive. Induction of suppressive activities in H-Y-specific CTLL was independent of the appropriate male stimulator cells since it was also observed in MLC induced by irrelevant antigens (H-2, trinitrophenol). Furthermore at low cell numbers, irradiated lymphocytes from any of the CTLL consistently enhanced CTL activities generated from H-Y-specific CTLP. This augmenting activity, which was not TCGF, could be transferred by soluble mediators present in antigen-sensitized CTLL cultures. Thus, these data indicate (i) that cytotoxic effector cells can function as suppressor cells in the generation of CTL, (ii) that the cytotoxic activity of cloned CTL does not correlate with their capacity to suppress CTL responses, (iii) that the inhibition of CTL responses by CTLL is not due to simple consumption of T-cell growth factors produced in MLC, and (iv) that different CTL clones may interfere with the generation of CTL at different stages of their maturation. Moreover, the experiments suggest an antigen-independent enhancement of suppression by the interaction of CTL with lymphokines. Together with the augmenting activity evoked by cloned CTL the data provide strong evidence for the expression of multiple immunological functions by one particular subset of T cells and suggest that cytotoxic effector cells can differentially regulate the maturation and/or clonal expression of their precursor cells.     

Cathepsin D is specifically inhibited by deoxyribonucleic acids

A cathepsin D (CD) inhibitor was searched using mouse embryonic fibroblasts deficient for CD. Synthetic DNA fragments specifically inhibited CD activity in a dose-dependent manner, but not the activities of other serine or cysteine proteinases. Cathepsin E activity was also inhibited by DNA fragments when hemoglobin was used as a substrate. CD inhibition by DNA fragments appeared to be electrostatic in nature and dependent on Tm values. Moreover, CD activity was partly inhibited by exogenously ingested DNA fragments, suggesting that DNA fragments with high Tm values are potent inhibitors of CD in vitro and partly in vivo.     

Cell lines derived from human autorosette-forming cells with suppressive activity

Concanavalin-A-stimulated human T lymphocytes from healthy donors and from patients suffering from diverse immune disorders were fractionated into rosette-forming (R) and nonrosette-forming (NR) cells. The separation method is based upon the ability of the lymphocytes to bind autologous erythrocytes and form autorosettes. Long-term cultures of the R and NR subpopulations were established. The activity of the culture supernatants on the T cell proliferation of normal human phytohemagglutinin (PHA)-induced lymphocytes and of a murine, interleukin-2 (IL-2)-dependent cytotoxic T cell line (CTLL) was investigated. Only the R cell line-derived supernatants from almost all patients tested evinced potent suppressor activity, those from healthy donors less so. The suppressive function was demonstrated not to be due to a cytotoxic effect since preincubation of the PHA-induced lymphocytes and CTLL cells with the factor did not diminish their proliferative capacity. Our study indicates the existence of a competitive relationship between the suppressor factor and IL-2. We found that inhibition of the proliferation decreased with the addition of increasing quantities of exogenous IL-2. We also observed that preincubating the CTLL cells with IL-2 prior to exposing them to the suppressive factor precludes inhibition of their proliferation. Phenotypic analysis of the suppressor cell line revealed that they were comprised of a T cell population which included OKT4+ and OKT8+ cells and that 99% of the cells formed autorosettes. Preliminary purification of the suppressive factor was performed by ultrafiltration and maximal suppression was exhibited by the fraction of less than 10,000 daltons. The development of suppressor cell lines from the unique population of autologous rosette-forming cells may be very helpful in studying the immunoregulatory properties of these cells and their suppressor activity.     

Mechanism of cyclosporin A-induced immunosuppression. Cyclosporin A inhibits receptor-mediated and non-receptor-mediated lymphokine production as well as interleukin-2-induced proliferation in cloned T lymphocytes

The effects of cyclosporin A (CyA) on the activation processes of cloned murine cytotoxic T lymphocytes (CTL) have been examined. With the use of Day 7 resting cloned CTL it was possible to separate the functions of lymphokine production (macrophage-activating factor, MAF) and interleukin 2 (IL-2)-induced proliferation of these cells. The effect of CyA on each of these activities was analyzed independently. CyA was found to inhibit both receptor-mediated MAF production in response to stimulation with antigen and lectin and MAF production in response to non-receptor-mediated stimulation (by anti-Thy-1 antibodies, ionophore, and phorbol ester). Further, CyA was observed to inhibit the re-entry of these resting CTL into the cell cycle upon stimulation with IL-2. The effect of CyA on MAF production did not appear to be due to inhibition of the signal-transducing mechanism involved in this process (i.e., inositol lipid hydrolysis, calcium mobilization, and protein phosphorylation). The action of CyA on the IL-2-induced proliferation was not due to inhibition of IL-2 receptor expression or the binding of IL-2 to its receptor. Thus, CyA appeared to mediate its suppressive effects on MAF production and IL-2-induced proliferation through an action on some later step(s) in the signal pathways of these activities.     

Conservation of epidermal growth factor receptor function in the human parasitic helminth Schistosoma mansoni

The epidermal growth factor receptor (EGF-R) plays an important role in development and cell differentiation, and homologues of EGF-R have been identified in a broad range of vertebrate and invertebrate organisms. This work concerns the functional characterization of SER, the EGF-R-like molecule previously identified in the helminth parasite Schistosoma mansoni. Transactivation assays performed in epithelial Madin-Darby canine kidney cells co-transfected with SER and a Ras-responsive reporter vector indicated that SER was able to trigger a Ras/ERK pathway in response to human epidermal growth factor (EGF). These results were confirmed in Xenopus oocytes showing that human EGF induced meiosis reinitiation characterized by germinal vesicle breakdown in SER-expressing oocytes. Germinal vesicle breakdown induced by EGF was dependent on receptor kinase activity and shown to be associated with phosphorylation of SER and of downstream ERK proteins. (125)I-EGF binding experiments performed on SER-expressing oocytes revealed high affinity (2.9 x 10(-9) M) of the schistosome receptor for human EGF. Phosphorylation of the native SER protein present in S. mansoni membranes was also shown to occur upon binding of human EGF. These data demonstrate the ability of the SER schistosome receptor to be activated by vertebrate EGF ligands as well as to activate the classical ERK pathway downstream, indicating the conservation of EGF-R function in S. mansoni. Moreover, human EGF was shown to increase protein and DNA synthesis as well as protein phosphorylation in parasites, supporting the hypothesis that host EGF could regulate schistosome development. The possible role of SER as a receptor for host EGF peptides and its implication in host-parasite signaling and parasite development are discussed.     

A soluble 61-kDa protein is associated with inhibition of lectin-induced proliferation and IL-2 synthesis

An inhibitor of lectin-induced splenocyte proliferation from serum of normal chickens has been characterized. This suppressive factor, found in both serum and plasma and at concentrations as low as 3%, causes a 50% inhibition in proliferative responses to T-cell lectins of autologous and heterologous lymphoid cells. The inhibitor in serum also dramatically suppresses murine IL-2 synthesis, proliferation of murine spleen cells stimulated with PHA, and synthesis of DNA in xenogeneic-transformed mammalian lymphoblastoid cell lines. Serum does not block binding of the lectin to lymphoid cells and the suppressive activity cannot be overcome by any dose of lectin. The inhibitor of DNA synthesis is destroyed by pepsin. NH4(2)SO4 (50%) and TCA (15%) treatments both precipitate the suppressor factor, which further indicates that the suppressive factor is a protein. A 330-fold purification of the inhibitory protein from serum was obtained when boiled serum was passed over a Sepharose 6B and then a DEAE-Sephacel column which was washed at pH 5.0 and eluted with 0.2 M NaCl. SDS-PAGE with silver staining revealed a nonreduced protein with an apparent molecular weight of 61 kDa. Less than 2 micrograms of the protein thus obtained caused a 50% inhibition in the proliferation of chicken lymphoid cells to Con A. The inhibitor of DNA synthesis is therefore not cytotoxic, does not bind to Con A or to mannose or glucose residues on lymphocytes, is acid and heat stable, and is associated with a protein that has a molecular weight of 61 kDa. Since such low concentrations of this naturally occurring, proteinaceous, immunosuppressive factor cause substantial inhibition of IL-2 synthesis and proliferative activity of T cells, this protein may be a very important immunomodulator.     

R-factor from the natural strain of Salmonella derby carrying a DNA-polymerase gene

A R-factor which determines multiple stability to antibiotics (Cm, Pn, Sm) was found in a Salmonella derby strain isolated from the clinical material. The plasmid was eliminated by treatment with ethidium bromide; the DNA-polymerase activity in the antibiotic-sensitive derivatives measured under conditions optimal for DNA-polymerase I from E. coli was found to be decreased 10-50-fold. Plasmid DNA of S. derby K89 was fractionated by electrophoresis in agarose gel; individual zones I-IV were obtained, using a preparative technique. Upon transformation of S. derby K82 pol- cells, only plasmid DNA in zone II (designed as pSD Cm pol) gave Cm-resistant transformants, in which the DNA-polymerase activity decreased to the normal level. The experimental results pont to the binding of the DNA-polymerase gene to the S. derby plasmid.     

Apolipoprotein E is a biologically active constituent of the normal immunoregulatory lipoprotein, LDL-In

A normal plasma lipoprotein, termed LDL-In, has been shown to be a potent inhibitor of mitogen-driven human lymphocyte proliferation in vitro and of primary antibody responses in the mouse. To determine whether the immunoregulatory activity of LDL-In resided with the protein rather than the lipid constituents of LDL-In, one of the apoproteins of LDL-In, apoprotein E, was isolated from plasma and was analyzed for its inhibitory activity. Apoprotein E, isolated after delipidization of lipoproteins with either methyl ethyl ketone or ethanol and ethyl ether, was immunosuppressive. Furthermore, the characteristics of inhibition of cellular [3H]thymidine uptake by isolated apoprotein E were identical to those characteristics obtained with suppression by LDL-In. Inhibition by apoprotein E and LDL-In required preincubation of the cells with either apoprotein or lipoprotein for 24 hr before exposure of the cells to mitogen for maximal expression of suppressive activity, and this inhibition could not be reversed by removal of non-cell-associated inhibitor before stimulation. Neither apoprotein E or LDL-In was inhibitory when they were added to the cells after mitogen stimulation. The only difference noted between suppression by apoprotein E and LDL-In was that of dose. Compared with quantitative estimates of the apoprotein E content of LDL-In, significantly more isolated apoprotein E was required than lipoprotein-associated E for comparable levels of suppression. The potency of apoprotein E could be increased by adding it to cells in the presence of dimyristoylphosphatidylcholine/cholesterol vesicles. The data suggesting that phospholipid increased the specific activity of apoprotein E by altering its molecular dispersion was obtained from analyses of the interaction of apo E with cells, as well as the plastic culture vessels. The results suggested that the molecular dispersion and perhaps organization of isolated apoprotein E in an aqueous system is critical to its interaction with lymphocytes and subsequently its biological activity.     

Investigation of the immunocompetent cells that bind early pregnancy factor and preliminary studies of the early pregnancy factor target molecule

Early pregnancy factor (EPF) is a secreted protein with immunosuppressive and growth factor properties. It has been shown to suppress the delayed-type hypersensitivity response in mice as well as acute and chronic forms of experimental autoimmune encephalomyelitis in rats and mice, respectively. In previous studies, we have demonstrated that EPF binds to a population of lymphocytes and we hypothesized that it mediates its suppressive effects by binding to CD4+ T cells. In the present study, we isolated monocytes and subpopulations of lymphocytes and labelled them with fluoresceinated EPF in order to determine which populations bind EPF. We demonstrated that EPF binds specifically to CD4+, CD8+, CD14+ (monocytes) and CD56+ NK cells but not to CD19+ B cells. The identity of the molecule(s) on the cell surface that is targeted by EPF is unknown, but as EPF is an extracellular homologue of the intracellular protein chaperonin 10 (Cpn10), we examined the possibility that the EPF receptor is a membrane-associated form of chaperonin 60 (Cpn60), the functional associate of Cpn10 within the cell. The EPF target molecule on lymphocytes was visualized by chemical cross-linking of exogenous iodinated Cpn10 to cells and probed with anti-Cpn60. The effect of anti-Cpn60 on activity in the EPF bioassay, the rosette inhibition test, was also examined. In both instances, no specific interaction of this antibody and the putative receptor was observed. It was concluded that the cell surface molecule targeted by EPF is unlikely to be a homologue of Cpn60.     

Modulation of granulomatous hypersensitivity. V. Participation of histamine receptor positive and negative lymphocytes in the granulomatous response of Schistosoma mansoni-infected mice

The possible role of histamine and histamine-receptored inflammatory cells in the granulomatous response of Schistosoma mansoni-infected mice was examined. Special staining revealed the presence of numerous mast cells, many partially degranulated within the liver granulomas. Treatment of infected mice with cimetidine (an H2 receptor antagonist) enhanced, and diphenyhydramine (an H1 receptor antagonist) decreased the granulomatous response. Fluorescein-labeled histamine-rabbit serum albumin conjugate (H-FRSA) and unlabeled conjugate (H-RSA)-coated culture plates were used to identify and isolate cells with histamine receptors. A large proportion of granuloma macrophages, lymphocytes, eosinophils, neutrophils, and splenic lymphocytes had histamine receptors. Elution of adherent cells from H-RSA-coated culture plates with H1 or H2 receptor antagonists suggested that receptors on granuloma cells were predominately H1 with some granuloma lymphocytes bearing H2-type receptors. Splenic lymphocytes from infected mice were functionally divided according to the presence or absence of histamine receptors on their cell surface. Receptor-negative lymphocytes appeared to mediate SEA-stimulated MIF production (TDH cells) and participated in the adoptive transfer of suppression of granulomas (TH cells). Whereas, TS cells appeared to have histamine receptors. Based on these data, it is inferred that lymphocytes that regulate lymphokine production (TS cells) within the granuloma may be triggered via their histamine receptors to exert suppressive activity.     

Isolation and characterization of high affinity aptamers against DNA polymerase iota

Human DNA-polymerase iota (Pol ι) is an extremely error-prone enzyme and the fidelity depends on the sequence context of the template. Using the in vitro systematic evolution of ligands by exponential enrichment (SELEX) procedure, we obtained an oligoribonucleotide with a high affinity to human Pol ι, named aptamer IKL5. We determined its dissociation constant with homogenous preparation of Pol ι and predicted its putative secondary structure. The aptamer IKL5 specifically inhibits DNA-polymerase activity of the purified enzyme Pol ι, but did not inhibit the DNA-polymerase activities of human DNA polymerases beta and kappa. IKL5 suppressed the error-prone DNA-polymerase activity of Pol ι also in cellular extracts of the tumor cell line SKOV-3. The aptamer IKL5 is useful for studies of the biological role of Pol ι and as a potential drug to suppress the increase of the activity of this enzyme in malignant cells.     

Suppressor macrophages in lymphocyte proliferation of cynomolgus monkeys

The present study demonstrated the presence of cells belonging to monocyte/macrophage lineage which suppressed mitogen-induced blastogenesis of peripheral blood lymphocytes in cynomolgus monkeys. Depletion of adherent or phagocytic cells from peripheral mononuclear cells caused a substantial increase in the blastogenic response of cynomolgus monkey lymphocytes whereas the same treatment led to marked reduction rather than enhancement in human lymphocyte blastogenesis. Addition of thioglycollate-elicited peritoneal exudate adherent cells as macrophages suppressed the blastogenic response of nonadherent lymphocytes in a dose-dependent manner. The suppressive effect was observed not only in autologous but also in allogeneic macrophages to the responder lymphocytes. Treatment of macrophages with silica, carrageenan or freezing-thawing reduced their suppressive effect but there was no reduction with mitomycin C or indomethacin. No suppressive activity was detected in the cell-free supernatant of macrophages cultured in the presence or absence of mitogens for up to 4 days. From these findings, it appeared that monocyte/macrophage lineage might be responsible for the observed suppressive effect on mitogen-induced blastogenesis of cynomolgus monkey lymphocytes.     

Macrophage-mediated cytostatic activity blocks lymphoblast cell cycle progression independently in both G1 phase and S phase

Recent work has shown that macrophage-mediated cytostatic activity inhibits cell cycle traverse in G1 and/or S phase of the cell cycle without affecting late S, G2, or M phases. The present report is directed at distinguishing between such cytostatic effects on G1 phase or S phase using the accumulation of DNA polymerase alpha as a marker of G1 to S phase transition. Quiescent lymphocytes stimulated with concanavalin A undergo a semisynchronous progression from G0 to G1 to S phase with a dramatic increase in DNA polymerase alpha activity between 20 and 30 hr after stimulation. This increase in enzyme activity was inhibited, as was the accumulation of DNA, when such cells were cocultured with activated murine peritoneal macrophages during this time interval. However, if mitogen-stimulated lymphocytes were enriched for S-phase cells by centrifugal elutriation and cocultured with activated macrophages for 4-6 hr, DNA synthesis was inhibited but the already elevated DNA-polymerase activity was unaffected. Similar results were obtained when a virally transformed lymphoma cell line was substituted as the target cell in this assay. These results show that both G1 and S phase of the cycle are inhibited and suggest that inhibition of progression through the different phases may be accomplished by at least two distinct mechanisms.     

Specific inhibition of human DNA ligase adenylation by a distamycin derivative possessing antitumor activity.

The antiviral distamycin A and its phenyl mustard derivative FCE24517 possessing antitumor activity were tested for their ability to inhibit macromolecular synthesis in three human and one murine cell line. While distamycin A was poorly active in these systems, FCE24517 inhibited DNA synthesis efficiently, RNA synthesis to a lower extent and had little effect on protein synthesis. These findings suggest that the in vivo activity of FCE24517 derives from the specific inhibition of DNA synthesis. When the two drugs were tested on several enzymes involved in human DNA metabolism a strikingly similar pattern of inhibition appeared, with distamycin A being the more potent. Both drugs showed: A), no inhibitory activity against thymidine kinase and DNA primase; B), low activity against DNA topoisomerases I and II and the 3'-5' exonuclease associated with the DNA polymerase epsilon; C), high activity against DNA polymerases alpha and epsilon, uracil-DNA glycosylase and the joining activity of the replicative DNA ligase; D), the highest inhibitory activity against the AMP-dependent DNA relaxing activity of DNA ligase. The strong in vitro inhibition of several DNA enzymatic activities, including DNA ligase, do not match with the in vivo activities of the two drugs. However a unique difference was observed: only FCE24517 inhibited the DNA-independent reaction of adenylation of human DNA ligase while the adenylation reaction of T4 and E. coli DNA ligase was unaffected by either drug. It is still unclear whether these properties are relevant for modulating the killing activity of FCE24517 against proliferating cells both in culture and in vivo. Nevertheless FCE24517 is the first known molecule capable of interacting directly and specifically with human DNA ligase.     

Immunoregulatory effects of a suppressor factor from healthy pregnant women's lymphocytes after progesterone induction

Progesterone-treated pregnancy lymphocytes release an immunologic blocking factor. The mode of action of this substance was investigated. The supernatant of progesterone-treated pregnancy lymphocytes was highly suppressive of natural cytotoxicity toward human embryonic fibroblast target cells as well as of natural killer cell activity. The effect was not observed when progesterone induction was performed in the presence of RU 486, a progesterone receptor blocking agent. The factor was able to inhibit mixed lymphocyte reactions (MLRs), and transfer coculture experiments revealed that this effect was dependent on major histocompatibility complex nonspecific, nonrestricted suppressor T cells. The activation/expansion of suppressor inducer and suppressor effector T cells was further proved by fluorescence-activated cell sorter analysis of the populations from MLRs cultured in the presence of the inhibitory factor. These changes were not observed with MLRs performed in the presence of supernatants from progesterone + RU 486-treated peripheral blood lymphocytes. The inhibitory material, on the other hand, did not affect either production or function of IL-2. We conclude that in the presence of high local concentrations of progesterone, a suppressive pathway dependent on specific progesterone-CD8+ lymphocyte interaction might be established. This mechanism might play an important role in the maintenance of pregnancy.     

Inhibition of lymphocyte proliferation by detergent-solubilized mouse liver membranes

The role of phenomena analogous to fibroblast contact inhibition in lymphocyte growth regulation is controversial, although it is clear that direct cell-cell contact is vital to immunoregulation and accessory cell function. An extract of mouse liver plasma membrane proteins, referred to as suppressive liver extract (SLE), that suppresses the growth of 3T3 fibroblasts also inhibited the mitogen-induced proliferation of murine lymphocytes. A dose of 20 micrograms/ml SLE was less than 95% suppressive of proliferation in both mouse T and mouse B cells treated with a variety of mitogens. B cell growth factor, while increasing DNA synthesis overall in mitogen-stimulated B cells, did not change the extent of SLE suppression, which suggests that the SLE does not interfere with lymphocyte-growth factor interactions. In exploring a sequence of B cell activation events, we discovered that SLE had no effect on the early activation event of increased phosphatidylinositol turnover. Blastogenesis, however, was inhibited in mitogen-stimulated, SLE-treated B cells. The maximum suppressive effect was observed if the SLE was added within 8-12 h of the mitogenic stimulus. SLE did not affect the viability of cells in culture. These results point to a possible unity of regulatory mechanisms between contact inhibition in fibroblasts and the processes of immunoregulation.     

Effects of urocortin 2 and 3 on motor activity and food intake in rats

Urocortin 2 (Ucn 2) and Ucn 3 are new members of the corticotropin-releasing factor (CRF) family and bind selectively to the CRF type 2 receptor (CRF2). The effects of these peptides on behavioral changes induced by CRF were examined in rats. In a familiar environment, intracerebroventricular injection of Ucn 2 attenuated the stimulatory effect of CRF on motor activity, although it alone produced no effect. Ucn 3 suppressed motor activity and attenuated the stimulatory effect of CRF. In an open field, CRF decreased locomotion and rearing but increased grooming behavior. Ucn 2 attenuated the inhibition of locomotor activity induced by CRF without affecting other activities, such as rearing or grooming behavior. Ucn 3 had no effect on the behavioral changes induced by CRF, although it alone decreased locomotion and rearing in a manner similar to CRF. Ucn 2 was thus found to have an antagonistic effect on bi-directional motor activation induced by CRF, while Ucn 3 had a suppressive effect on motor activity. Both Ucn 2 and Ucn 3 suppressed food intake in freely-fed rats, but not immediately after injection. These results suggest that the CRF2 receptor is involved in motor suppressive effects as well as anxiolytic and anorectic effects of Ucn 2 and Ucn 3.