Human placental Hofbauer cells promote mitogen induced T cell proliferation

Hofbauer cells are a major cell type of the human placental villous core particularly numerous at the beginning of pregnancy. In the present study we have investigated whether or not Hofbauer cells could subserve the function of accessory cells for colony formation by phytohemagglutinin stimulated allogeneic T cells. Results showed that Hofbauer cells are capable to play an accessory role on T cell proliferation probably due to the release of interleukin 1-like soluble factors.     

Thymus involution induced by 5-azacytidine

Hypomethylation of DNA, which can be achieved by incorporation of 5-azacytidine, has been correlated with derepression of genes. In order to examine thein vivo effects of 5-azacytidine on organ development and differentiation, young rats were treated with the drug. There was an almost complete reduction of thymus and a marked reduction of spleen weight, while other organs, including testes were only marginally affected. Control experiments with cytosine-arabinoside suggest that treatment with an inhibitor of DNA replicationper se is not responsible for the very rapid thymus involution triggered by 5-azacytidine in rats. In spite of the drastic reduction of thymus and spleen weight, lymphocytes of these organs were not impaired in their response to the T cell mitogen Concanavalin A.     

Heparan sulphate inhibition of cell proliferation induced by TGFbeta and PDGF

The effect of glycosaminoglycans (GAGs) on the proliferation of smooth muscle cells (SMC) and fibroblasts was assessed by culturing cells with or without GAGs. Porcine heparan sulphate (HS) inhibited proliferation in a dose dependent manner. At 167 mug/ml of HS this reached 88% and 72% inhibition of SMC and fibroblast growth, respectively. Pig and beef mucosal heparins also blocked proliferation, but to a lesser extent. In contrast, beef lung heparin, chondroitin sulphate, and dermatan sulphate failed to block growth factor induced proliferation. Continuous presence of HS was not required, suggesting that the inhibitory effects resulted from a direct effect on the cell rather than an interaction of the GAG with growth factors. The mechanism by which GAGs inhibit proliferation will be addressed in future studies.     

Segmentation of human chromosomes induced by 5-ACR (5-azacytidine)

Summary The 5-ACR (5-azacytidine) introduced in human lymphocyte cultures induces a lack or a delay of condensation of some chromosome segements corresponding to the G-bands. The resulting R-banding is very similar to that obtained with a 7-h treatment by BrdU, although the segmentation may be much stronger (pulverization) with high doses. However, the 5-ACR does not induce chromatid asymmetry, as BrdU does. This constitutes a new argument for considering that the segmentation and the asymmetry of chromatids depend, at least partly, on two different mechanisms, where proteins are probably involved. Another effect of 5-ACR is to increase chromosome associations by satellites, secondary constrictions, and telomeric regions.     

Regulatory interactions governing the proliferation of T cell subsets stimulated with pokeweed mitogen

Serial phenotyping of human peripheral blood mononuclear cells (PBMC) cultured with pokeweed mitogen (PWM) demonstrated an excess of T8+ cells after stimulation. Preferential expansion of the T8+ cell compartment was a result of T8+ cell blast transformation while T4+ cells generated fewer blasts and tended to remain as small resting cells. When the proliferative behavior of T cell subsets in PWM-stimulated PBMC with physiologic proportions of T4+ and T8+ cells was compared with that of cultures depleted of T4+ or T8+ cells, two levels of regulation of proliferation were found: without T4+ cell help, T8+ cells were unable to divide; however, in the presence of T4+ cells, PWM-stimulated T8+ cells became potent feedback inhibitors of T4+ cell proliferation. The mechanism of suppression by PWM-activated T8+ cells of T4+ cell proliferation, not only to PWM, but also to tetanus toxoid, was pursued by measuring decreased interleukin 2 (IL2) recovery from cultures containing suppressors. Although passive absorption of IL2 by PWM-activated cells could contribute to the suppression of fresh proliferative responses, as shown directly with isolated T4+ cells induced by PWM to express IL2 receptors, a much more profound suppression was mediated by PWM-activated T8+ cells. The regulation of proliferative responses of helper and suppressor T cell subsets may determine the magnitude of their subsequent interactions and thus control the ultimate outcome of in vivo physiologic and pathologic immune responses.     

Differential inhibition of inducible T cell cytokine secretion by potent iron chelators

Effector functions and proliferation of T helper (Th) cells are influenced by cytokines in the environment. Th1 cells respond to a synergistic effect of interleukin-12 (IL-12) and interleukin-18 (IL-18) to secrete interferon-gamma (IFN-gamma). In contrast, Th2 cells respond to interleukin-4 (IL-4) to secrete IL-4, interleukin-13 (IL-13), interleukin-5 (IL-5), and interleukin-10 (IL-10). The authors were interested in identifying nonpeptide inhibitors of the Th1 response selective for the IL-12/IL-18-mediated secretion of IFN-gamma while leaving the IL-4-mediated Th2 cytokine secretion relatively intact. The authors established a screening protocol using human peripheral blood mononuclear cells (PBMCs) and identified the hydrazino anthranilate compound 1 as a potent inhibitor of IL-12/IL-18-mediated IFN-gamma secretion from CD3(+) cells with an IC(50) around 200 nM. The inhibitor was specific because it had virtually no effect on IL-4-mediated IL-13 release from the same population of cells. Further work established that compound 1 was a potent intracellular iron chelator that inhibited both IL-12/IL-18- and IL-4-mediated T cell proliferation. Iron chelation affects multiple cellular pathways in T cells. Thus, the IL-12/IL-18-mediated proliferation and IFN-gamma secretion are very sensitive to intracellular iron concentration. However, the IL-4-mediated IL-13 secretion does not correlate with proliferation and is partially resistant to potent iron chelation.     

Differential inhibition of sister chromatid condensation induced by 5-azadeoxycytidine in human chromosomes

The deoxycytidine analogue 5-azadeoxycytidine (5-aza-dC) induces differential inhibition of sister chromatid condensation when cells are treated with this substance for two replication cycles, as the subsequent staining of metaphase chromosomes with Giemsa shows. The bifilarly substituted chromatid is dramatically longer than the unifilar one. A percentage of the metaphases treated with 5-azad-C even show a complete undercondensation of the bifilarly substituted chromatid. The optimum conditions for inducing sister chromatid differentiation were determined. No method has been developed as yet to permit enhancement of the differential staining in 5-aza-dC-treated preparations. The interactions between 5-aza-dC and chromosomal DNA as well as the factors involved in the differential staining of sister chromatids are discussed.     

Histological study of cell death in digital malformations induced by 5-azacytidine: suppressive effect of caffeine

The present study investigated microscopically the process of 5-azacytidine (5-AC)-induced digital teratogenesis and caffeine's suppressive effect on this process. Three distinct zones of programmed cell death were observed in control and caffeine-treated embryos 3 hours after 5-AC injection: the preaxial and postaxial ectodermal regions and the central part of the mesodermal regions. 5-AC temporarily suppressed programmed cell death in the ectoderm and mesoderm 3 hours after it was injected. However, caffeine promoted programmed cell death; normal programmed cell death was observed in the limb buds of embryos whose dams were treated with 5-AC and caffeine. The percentage of total cell death in hindlimb buds of embryos treated with 5-AC and caffeine was higher than that from embryos treated with 5-AC, whereas 5-AC-induced digital malformations were reduced by post-treatment with caffeine. Cell death reached a maximum 12 hours after the injection in limb buds from 5-AC and caffeine-treated embryos and at 24 hours in the 5-AC treated embryos. Furthermore, in the 5-AC and caffeine-treated embryos, the frequency of cell deaths at 12 hours increased almost linearly with the doses of caffeine in parallel with the reduction of 5-AC-induced malformation frequency by caffeine. These results suggest that although induced cell death may be one of the factors leading to digital malformations produced by 5-AC, it is not essential, and the existence of other factors affecting the pattern formation of the limb bud is proposed.     

Role of glycosaminoglycans in the regulation of T cell proliferation induced by thymic stroma-derived T cell growth factor

The present study investigates the regulatory effects of glycosaminoglycans such as heparin and heparan sulfate on T cell proliferation induced by thymic stromal cell monolayer or its derived T cell growth factor (TCGF). A thymic stromal cell clone (MRL104.8a) supported the growth of Ag-specific, IL-2-dependent Th cell clone (9-16) in the absence of Ag and IL-2 by producing a unique TCGF designated as thymic stroma-derived T cell growth factor (TSTGF). The addition of heparin to cultures in which the growth of 9-16 Th cells was otherwise stimulated by the MRL104.8a monolayer or a semipurified sample of the TSTGF resulted in heparin dose-dependent inhibition of 9-16 Th proliferation. The dose of heparin required for inducing 50% reduction of TSTGF-induced proliferation of Th at a given cell number was found to be proportional to the magnitude of the TSTGF added to cultures, suggesting that heparin exerted its inhibitory effect by binding to the TSTGF rather than by acting on Th cells. A similar growth-inhibiting effect of heparin was observed in IL-7-dependent proliferation of pre-B cell line or Th, but not in IL-2-dependent T cell proliferation or IL-3-dependent myeloid cell proliferation. A strong affinity of TSTGF and IL-7 for heparin was confirmed by the fact that both TSTGF and IL-7 adhered to columns of heparin-agarose and were eluted by salt. When various glycosaminoglycans were tested for the heparin-like Th growth-regulatory capacity, heparan sulfate exhibited Th growth-inhibiting ability comparable to that observed for heparin. These results indicate that the activity of thymic and/or bone marrow stroma-derived lymphocyte growth factor (TSTGF/IL-7) but not of Th-producing TCGF (IL-2) is negatively regulated by heparin or heparan sulfate, which would represent major glycosaminoglycans in the extra-cellular matrix of stromal cells.     

Virus-induced transient immune suppression and the inhibition of T cell proliferation by type I interferon

Vaccine-induced memory is necessary for protective immunity to pathogens, but many viruses induce a state of transient immune suppression that might contribute to the inability of a vaccine to elicit immunity. We evaluated here the fate of bystander T cells activated by third party cognate antigens during acute viral infections in vivo, using distinct models to track and specifically activate HY and P14 transgenic bystander CD8 T cells in vivo during acute arenavirus infections of mice. Viral infections acted as stimulatory adjuvants when bystander T cells were exposed to an inflammatory milieu and cognate antigens at the beginning of infections, but bystander CD8 T cell proliferation in response to cognate antigen was inhibited 3 to 9 days after virus infection. Reduced proliferation was not dependent on Fas-FasL- or tumor necrosis factor (TNF)-induced activation-induced cell death or on deficiencies of antigen presentation. Instead, reduced proliferation was associated with a delayed onset of division that was an intrinsic defect of T cells. Inhibition of proliferation could be simulated by exposure of T cells to the Toll-like receptor agonist and type I interferon (IFN) inducer poly(I · C). T cells lacking IFN-α/β receptors resisted both the suppressive effects of preexposure to poly(I · C) and the stimulatory effects of type I IFN, indicating that the timing of exposure to IFN can have negative or positive effects on T cell proliferation. Inhibition of T cell receptor-stimulated bystander CD8 T cell proliferation during acute viral infections may reflect the reduced ability of vaccines to elicit protective immunity when administered during an acute illness.     

Control of B cell proliferation: inhibition of responses to B cell mitogens induced by plasma cell tumors

A multitude of factors has been described that positively and negatively regulate B cell proliferation. A model system for the study of negative control of B cell function is provided by mice bearing plasmacytomas (PC-mice). In PC-mice, the primary immune response, as measured by development of antibody-forming cells (AFC), is severely suppressed. The present report specifically identifies a block in B cell proliferation as the apparent cause of this reduction in AFC production. Thus, the proliferative response of B cells from the spleens of PC-mice (PC-spleens) was significantly impaired when stimulated with four different B cell mitogens (lipopolysaccharide, Salmonella typhimurium mitogen, anti-mu conjugated to Sepharose, and 8-mercaptoguanosine). Nevertheless, the mitogen-responsiveness of these B cells was recovered when they were segregated by various methods from macrophages. These data suggest that the proliferative ability of the B cells in PC-spleens is inherently normal. In concordance with this conclusion, it was shown that suppressor cells from PC-spleens can block the proliferation of normal B cells derived from nontumor-bearing mice. This inhibition does not require direct cell contact and is mediated via soluble factors. The relevance of these results to previous studies of PC-induced immunosuppression and to the control of normal B cell proliferation is discussed.     

Multiple new phenotypes induced in 10T1/2 and 3T3 cells treated with 5-azacytidine.

Three new mesenchymal phenotypes were expressed in cultures of Swiss 3T3 and C3H/10T1/2CL8 mouse cells treated with 5-azacytidine or 5-aza-2'-deoxycytidine. These phenotypes were characterized as contractile striated muscle cells, biochemically differentiated adipocytes and chondrocytes capable of the biosynthesis of cartilage-specific proteins. The number of muscle and fat cells which appeared in treated cultures was dependent upon the concentration of 5-azacytidine used, but the chondrocyte phenotype was not expressed frequently enough for quantitation. The differentiated cell types were only observed several days or weeks after treatment with the analog, implying that cell division was obligatory for the expression of the new phenotypes. Oncogenically transformed C3H/10T1/2CL8 cells also developed muscle cells after exposure to 5-azacytidine, but at a reduced rate when compared to the parent line. Five subclones of the 10T1/2 line which were the progeny of single cells all expressed both the muscle and fat phenotypes following 5-azacytidine treatment. The effects of the analog are therefore not due to the selection of preexisting myoblasts or adipocytes in the cell populations. Rather, it is possible that 5-azacytidine, after incorporation into DNA, causes a reversion to a more pluripotential state from which the new phenotypes subsequently differentiate.