Inhibition of macrophage-induced antigen-specific T lymphocyte proliferation by poly I:C: strain and antigen independence

We have previously demonstrated that IFN-alpha/beta, poly I:C (an inducer of IFN-alpha/beta), and IFN-gamma can inhibit the ability of KLH-pulsed peritoneal macrophages to induce proliferation of syngeneic, KLH immune T lymphocytes in CBA/J mice. In this study, we show that this IFN-induced immunosuppression is not restricted to CBA/J (H-2k) mice but is also seen in BALB/cJ (H-2d) mice. A similar inhibition of proliferation is observed with the KLH-specific T cell hybridoma BDK, 100, which requires KLH-pulsed macrophages for optimum proliferation and IL-2 production. The immunosuppression produced by IFN was also independent of the antigen employed. Inhibition of T lymphocyte proliferation was observed when casein, instead of KLH, was used to immunize T cells and to pulse peritoneal macrophages in vivo. Utilizing KLH and casein, the antigen specificity of the inhibition was demonstrated. Therefore, the inhibition by the IFN-inducer poly I:C of macrophage-induced, antigen-specific T cell proliferation is not limited by H-2 type of the mice or to one antigen.     

雷公藤红素抑制成人T细胞白血病细胞增殖及机制

本研究旨在分析雷公藤红素对成人T细胞白血病细胞增殖、凋亡的影响,并探讨其分子机制。使用不同浓度的雷公藤红素溶液处理多种成人T细胞白血病细胞株,通过四唑盐比色法(MTT)、克隆形成实验检测细胞的增殖情况;Annexin V/PI双染检测细胞凋亡情况;最后通过Western blotting及双荧光素酶报告基因技术探究雷公藤红素抑制成人T细胞白血病细胞生长的调控机制。结果表明雷公藤红素能显著抑制成人T细胞白血病细胞增殖并诱导其凋亡,随着雷公藤红素浓度的增加Bax/Bcl-2蛋白比率明显升高,凋亡途径中Caspase-3/7蛋白也随之被切割活化,同时病毒编码的癌蛋白Tax的表达也明显受到抑制。以上结果表明,雷公藤红素通过调控Bcl-2家族蛋白,激活了Caspase途径诱导细胞凋亡,并通过抑制病毒关键蛋白Tax的表达,从而有效抑制了成人T细胞白血病细胞的增殖。该研究为临床应用雷公藤红素治疗成人T细胞白血病提供了实验依据。     

Inhibition of antigen-induced T-cell proliferation by antibodies to endogenous AKR-MuLV

Lymph node cells from BALB/c mice immunized with ovalbumin or human γ-globulin were restimulated in vitro with these antigens and assayed for antigen-induced proliferation. The proliferative response was shown to be antigen specific and T cell dependent. A rabbit antiserum to envelope and core proteins of AKR murine leukemia virus was found to inhibit antigen-induced T-cell proliferation. The IgG fraction and F(ab′)₂ fragments of the antiserum were also inhibitory. The inhibition occurred after the initial step of antigen-T cell interaction and viral absorption studies showed the inhibition to be specific for anti-AKR virus antibodies. A hypothesis for the mechanism of inhibition is discussed in relation to a functional role for endogenous murine leukemia virus.     

Inhibition of antigen-specific T cell proliferation and cytokine production by protein kinase A type I

cAMP inhibits biochemical events leading to T cell activation by triggering of an inhibitory protein kinase A (PKA)-C-terminal Src kinase pathway assembled in lipid rafts. In this study, we demonstrate that activation of PKA type I by Sp-8-bromo-cAMPS (a cAMP agonist) has profound inhibitory effects on Ag-specific immune responses in peripheral effector T cells. Activation of PKA type I inhibits both cytokine production and proliferative responses in both CD4(+) and CD8(+) T cells in a concentration-dependent manner. The observed effects of cAMP appeared to occur endogenously in T cells and were not dependent on APC. The inhibition of responses was not due to apoptosis of specific T cells and was reversible by a PKA type I-selective cAMP antagonist. This supports the notion of PKA type I as a key enzyme in the negative regulation of immune responses and a potential target for inhibiting autoreactive T cells.     

Inhibition of B lymphocyte activation by interferon-gamma

Helper/inducer T cell clones specific for protein antigens and class II MHC determinants consist of two nonoverlapping subsets. One (called Th1) secretes IL 2 and IFN-gamma and the other (Th2) produces BSF1 upon stimulation with antigen or polyclonal activators. By using hapten-binding normal B cells and the B lymphoma line WEHI-279 as assays for B cell helper (maturation) factors, we have shown that Th2 clone supernatants (SN) induce differentiation to antibody secretion, whereas Th1 SN do not. The failure of Th1 SN to activate B cells is due to inhibitory effects of IFN-gamma, because it can be reversed by a neutralizing monoclonal antibody specific for IFN-gamma. Thus, in the presence of this antibody, even Th1 SN stimulate B cell maturation maximally. Conversely, recombinant IFN-gamma inhibits proliferation and differentiation of B cells induced by active Th2 SN. These results demonstrate that IFN-gamma is a potent inhibitor of B lymphocyte activation and can be distinguished from growth and maturation-inducing helper factors that are produced by both subsets of helper T cells.     

Defective T-cell activation by Mycoplasma arthritidis mitogen is restored by interferon-gamma

A mitogen derived from supernatants of Mycoplasma arthritidis (MAS) has been shown to induce both T-cell activation and the production of interferon-gamma (IFN-gamma). MAS-induced response required the presence of accessory cells and is under the genetic control associated with the major histocompatibility complex (MHC). We found that recombinant IFN-gamma restored the proliferative response to MAS mitogen in unreactive mice strains, including H-2b and H-2s haplotypes. We postulated that these T-cells fail to respond since they lack part of the I-E molecules on their accessory cells. Our data suggest that interferon-gamma may be able not only to increase the levels of Ia antigens but also to promote the appearance of MHC products that are not usually present on the cell surface. Since Ia antigens have a central role on T-cell activation, we examined the effect of the level of IFN-gamma on MAS-induced T-cell activation. We analyzed the acquisition of responsiveness to IL-2 and IL-2 activity in cells pretreated with IFN-gamma and found that both the steps of T-cell activation were restored to the MAS mitogen in the unreactive mice strains by IFN-gamma.     

Inhibition of in vitro angiogenesis by lymphotoxin and interferon-gamma

The effects of lymphotoxin (LT) and interferon (IFN)-gamma on the capillary formation was examined using an in vitro angiogenesis model system. Both LT and IFN-gamma inhibited the capillary formation in a dose dependent manner. To elucidate the mode of action, effects of the lymphokines on endothelial and myofibroblastic cells were studied. We found that the lymphokines inhibited not only the growth of endothelial cells but also the production of collagen by myofibroblastic cells. These results suggest that the pleiotropic effects of the lymphokines on different types of cells might result in the inhibition of the capillary formation.     

Inhibition of cell proliferation by glycerol

The effect of glycerol on proliferation of BHK, CHO, HBL, MCF-7, and human glioma cells was studied. Cell proliferation was significantly decreased in all the cell lines at glycerol concentrations of 2-4% in the culture medium. The inhibition was dose-dependent, complete suppression of proliferation occurring at a glycerol concentration of 4% for the MCF-7 cell line and 6-8% for the BHK, CHO and human glioma cells. Studies on [3H]thymidine incorporation correlate with the effect on cell proliferation. The viability of the cells was not significantly affected until higher concentrations of glycerol (12% +) were present. Recovery studies with BHK cells indicated that replacement of the glycerol medium with glycerol-free medium resulted in full recovery following exposure to 4% glycerol and only partial recovery (65%) of proliferation rate following exposure to 10-12% glycerol. It is concluded that glycerol, a substance that is normally present in tissues, can serve as a potent inhibitor of cell proliferation.     

Inhibition by Trypanosoma cruzi of interferon-gamma production by mitogen-stimulated mouse spleen cells

Infection by Trypanosoma cruzi is accompanied by severe immunosuppression during the acute period. As part of our studies, to define the alterations caused by Trypanosoma cruzi in lymphocyte function, we examined in this work the interferon-gamma (IFN-gamma)-producing capacity of mitogen-stimulated mouse spleen and human peripheral blood mononuclear cells in the presence or absence of blood forms of the parasite. Co-culture of phytohaemagglutinin- or concanavalin A-stimulated spleen cells from normal mice with T. cruzi significantly decreased the levels of IFN-gamma activity found in the supernatants at 48 or 72 h. In contrast, human peripheral blood mononuclear cells, though suppressed by T. cruzi in their capacity to proliferate upon mitogenic stimulation, showed no significant decrease in IFN-gamma production. The addition of exogenous IFN-gamma did not reverse the suppressive effect of T. cruzi on either mouse or human cells. These results revealed, for the first time, the ability of T. cruzi to impair IFN-gamma production by activated mouse lymphocytes. The lack of restoration by exogenous IFN-gamma suggested that the reduced levels of this lymphokine were not, at least by themselves, the causative factor of reduced lymphoproliferation.     

Inhibition of mouse macrophage degradation of acetyl-low density lipoprotein by interferon-gamma

In vitro, metabolism of modified forms of low density lipoprotein (LDL) by macrophages via the acetyl-LDL receptor pathway promotes the massive cellular accumulation of lipid. It has been postulated that in vivo this contributes to foam cell formation in the atherosclerotic lesion. Recent studies have shown that arterial wall cells in vitro can secrete a number of cytokines, several of which have been reported to modulate macrophage cell function. Thus, cytokines have the potential to modulate the acetyl-LDL receptor pathway and to influence the rate of foam cell generation. To study the regulation of this pathway by cytokines, the effect of cytokines on the degradation of acetyl-LDL protein by mouse peritoneal macrophages was examined. Initially, supernatant from stimulated lymphocytes was used as a source of cytokines. Macrophages preincubated with supernatants obtained after the stimulation of T-cell helper type 1 (Th1) clone HDK-1 or BALB/c spleen cells degraded acetyl-LDL at a slower rate, whereas supernatant from stimulated T-cell helper type 2 (Th2) clone D-10 had no effect. Comparison of the lymphokine profiles showed that spleen and HDK-1 cells secreted several lymphokines in common including significant levels of interferon-gamma. Interferon-gamma was then directly shown to be inhibitory; an anti-interferon-gamma monoclonal antibody blocked the HDK-1-mediated inhibition by 70% and the addition of recombinant interferon-gamma (IFN-gamma) to macrophages inhibited the specific degradation of acetyl-LDL in a dose- and time-dependent manner with a maximum suppression to approximately 40% of control. The inhibition was not accompanied by an increase in the amount of cell-associated acetyl-LDL and was not due to cell death nor could it be accounted for by the presence of endotoxin. To study the mechanism of the inhibition, the effects of IFN-gamma on the itinerary of acetyl-LDL and its receptor were examined. IFN-gamma decreased specific acetyl-LDL binding only to a small degree, and the rate of lysosome-mediated degradation was not affected. The principal alteration was in the rate of transport to the lysosome which was markedly slowed. Since the receptors eventually returned to the surface to maintain a steady state, and there was not an increase in cell-associated lipoprotein, there must be other changes in the itinerary that were not identified with the techniques used. Thus, the receptor cycle is being regulated at a discrete point. IFN-gamma also suppressed the LDL receptor pathway in macrophages, but this pathway was not affected by IFN-gamma in mouse fibroblasts.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of ras-mediated cell proliferation by benzyloxybenzaldehyde

Excessive proliferation of vascular smooth muscle cells (VSMC) in the intima is an important etiologic factor in vascular proliferative disorders such as atherosclerosis and restenosis after balloon angioplasty. Therefore, control of VSMC growth may be a suitable therapeutic intervention in vascular proliferative disorders. In the present work, we have studied the 2-benzyloxybenzaldehyde (CCY1a)-mediated antiproliferative effect and its mechanisms of action. CCY1a inhibited serum-induced VSMC proliferation in a concentration-dependent manner, as demonstrated using [³H]thymidine incorporation and MTT assays; the IC₅₀ values were calculated to be 7.0 × 10^–6 and 1.2 × 10^–5 M, respectively. Furthermore, it also significantly suppressed serum-induced progression of the cell cycle, as shown by flow cytometric analysis. CCY1a as well as PD98059 almost completely abolished serum-induced activation of p42/44 mitogen-activated protein kinase (MAPK), the downstream effectors of c-fos and c-jun mRNA expression and activator protein-1 (AP-1) DNA binding activity, suggesting the central roles of these signaling cascades. Interestingly, CCY1a also effectively blocked serum-induced IB- phosphorylation, IB- degradation and nuclear factor-B (NF-B) binding activity. Based on these observations, we examined the effect of CCY1a on serum-mediated Ras activity, an upstream regulator of the above signaling events. The data demonstrated a marked inhibition of Ras activation by CCY1a. We conclude that CCY1a blocks cell proliferation via inhibition of the upstream effector of Ras and downstream events, including p42/44 MAPK activation and c-fos and c-jun mRNA expression, as well as NF-B and AP-1 DNA binding activities.     

Inhibition of endothelial cell proliferation by gamma-interferon

Endothelial cell growth factor (ECGF) is a potent polypeptide mitogen for endothelial cells and fibroblasts. The mitogenic effects of ECGF are inhibited by the lymphokine gamma-interferon (gamma-IFN) in a dose- dependent manner. Gamma-IFN also induces a unique change in endothelial cell morphology which is maximally expressed in the presence of ECGF. The antiproliferative and phenotypic modulatory effects of gamma-IFN on endothelial cells are reversible. Inhibition of ECGF-induced endothelial cell proliferation by gamma-IFN is accompanied by a concentration- and time-dependent decrease in binding of 125I-ECGF to the endothelial cell surface. Scatchard analyses of the binding data in the presence and absence of gamma-IFN demonstrate a decrease in the number of ECGF-binding sites rather than a decrease in ligand affinity for the receptor. Cross-linking experiments with disuccinimidyl suberate demonstrate a decrease in the 170,000 Mr cross-linked receptor- ligand complex. These data suggest that gamma-IFN inhibits endothelial cell proliferation by a mechanism which involves growth factor receptor modulation.     

Inhibition of pulmonary antibacterial defense by interferon-gamma during recovery from influenza infection

Secondary bacterial infection often occurs after pulmonary virus infection and is a common cause of severe disease in humans, yet the mechanisms responsible for this viral-bacterial synergy in the lung are only poorly understood. We now report that pulmonary interferon-gamma (IFN-gamma) produced during T cell responses to influenza infection in mice inhibits initial bacterial clearance from the lung by alveolar macrophages. This suppression of phagocytosis correlates with lung IFN-gamma abundance, but not viral burden, and leads to enhanced susceptibility to secondary pneumococcal infection, which can be prevented by IFN-gamma neutralization after influenza infection. Direct inoculation of IFN-gamma can mimic influenza infection and downregulate the expression of the class A scavenger receptor MARCO on alveolar macrophages. Thus, IFN-gamma, although probably facilitating induction of specific anti-influenza adaptive immunity, suppresses innate protection against extracellular bacterial pathogens in the lung.     

Inhibition of antigen-specific proliferation of type 1 murine T cell clones after stimulation with immobilized anti-CD3 monoclonal antibody

The effect of stimulating normal type 1 murine T cell clones with anti-CD3 antibody was examined in vitro. In the absence of accessory cells, anti-CD3 antibody immobilized on plastic plates stimulated inositol phosphate production, suboptimal proliferation, IL-2 and IL-3 production, and maximal IFN-gamma production. Addition of accessory cells augmented lymphokine production and proliferation when the effects of "high-dose suppression" were relieved by removing the T cells from the antibody-coated plates. Exposure of type 1 T cell clones to immobilized anti-CD3 antibody alone rapidly induced long-lasting proliferative unresponsiveness (anergy) to Ag stimulation that could be prevented by accessory cells. This anergic state was characterized by a lymphokine production defect, not a failure of the T cells to respond to exogenous IL-2 or to express surface Ti/CD3 complexes. In addition, anergy could not be induced in the presence of cyclosporine A. These results suggest that under certain conditions anti-CD3 antibodies may have potent immunosuppressive effects independent of Ti/CD3 modulation. Furthermore, our results support a two-signal model of type 1 T cell activation in which Ti/CD3 occupancy alone (signal 1) induces anergy, whereas Ti/CD3 occupancy in conjunction with a costimulatory signal (signal 2) induces a proliferative response.     

Nature of the antigenic complex recognized by T lymphocytes. IV. Inhibition of antigen-specific T cell proliferation by antibodies to stimulator macrophage Ia antigens.

We have previously demonstrated that nonimmune guinea pig T lymphocytes could be specifically sensitized with TNP-modified allogeneic macrophages after eliminating the alloreactive T cells with bromodeoxyuridine (BUdR) and light treatment. This procedure allowed the unique opportunity to use anti-Ia sera directed against the Ia antigens of only the stimulator macrophages or responder T cells to determine against which cell type anti-Ia would block TNP-specific stimulation. It was found that the TNP-specific DNA synthetic response of BUdR and light-treated T cells stimulated with TNP-modified allogeneic macrophages was totally eliminated by anti-Ia sera directed solely against the allogeneic stimulator macrophage. In contrast, anti-Ia sera directed only against the responder T cells had no effect on their response to TNP-modified allogeneic macrophages. These findings indicate that macrophage Ia antigens are required for efficient T cell-macrophage interactions and raise the possibility that T cell Ia antigens may not be required for collaboration with macrophages. This latter possibility was substantiated by experiments in which we show that treating T cells with anti-Ia sera and complement to remove the Ia-positive cells either before or after priming, or both, had no effect on their ability to be primed and restimulated with TNP-modified macrophages.     

Inhibition of lymphocyte proliferation by antibodies to prolactin

Recent in vivo studies have shown that treatments that decrease circulating prolactin (PRL) in rodents result in significant immunosuppression. Our attempts to demonstrate corresponding direct stimulatory effects of PRL on cultured lymphocytes were unsuccessful. However, antibodies against pituitary PRL potently inhibited both murine and human lymphocyte proliferation in response to both T and B cell mitogens. Further studies using IL 2 and IL 4 responsive cell lines (CTLL-2 and HT-2) demonstrated that the same anti-PRL antibodies inhibited the proliferative response to these cytokine growth factors. Thus, antibodies to PRL appear to block an event occurring in the G1 to GS phase transition of these cell lines, which constitutively express growth factor receptors. The inhibitory activity of anti-PRL antibodies could be adsorbed by addition of purified human PRL or by immobilized PRL on an affinity column. Antibodies to other pituitary hormones were without inhibitory effect on CTLL-2 cell proliferation. Proliferation of lymphocytes in serum-free medium was also potently inhibited by anti-PRL antibodies, suggesting that antibody effects were not due to neutralization of PRL or other factors contained in culture serum supplements. We suggest from these data that a protein with homology to PRL and recognized by these anti-PRL antibodies is produced by lymphocytes and plays a critical role in their progression through the cell cycle.     

Inhibition of lymphocyte proliferation by liver arginase.

The activities of thymidine kinase and uridine kinase (enzymes for pyrimidine salvage pathway) in phytohemagglutinin (PHA)--prestimulated lymphocytes were inhibited by arginase in a similar pattern to the inhibition on thymidine incorporation. Further study revealed that arginase did not directly affect the activities of these enzymes in the cell-free system. Thymidine kinase and uridine kinase activities of PHA-prestimulated lymphocytes were inhibited by arginase making their activities as low as that cultured in arginine-free RPMI-1640 medium. These results suggest that arginine-depletion in the culture medium is the primary mode of action of arginase on the inhibition of mitogen-stimulated lymphocyte proliferation.