Type 1 vs. type 2 cytokine secretion in vitro and its regulation by hydrocortisone in humans subjected to 120-day anti-orthostatic bed-rest regime

Head-Down Bed-Rest (HDBR) mimics some of the physiological stress effects of microgravity. Six healthy volunteers were subjected to bed-rest for 120 days. Blood samples were collected one month before (PRE), on day 110 of HDBR (DAY 110), and on the 7th day after bed-rest regime ends (POST). Distribution of T-cell subsets, NK-, B-cells and monocytes was assessed in the whole blood. Distribution of cytokine secreting T-cells was assessed in PMA/ionomycin cell culture. Peripheral Blood Mononuclear Cells (PBMC) and whole blood cells (WB) were activated with a combination of PHA and LPS to assess cytokine secretion. In addition, PHA/LPS activated cell cultures were treated with 10(-6) M of hydrocortisone (HCS) in order to study stress-induced alterations in the cortisol-sensitivity of immunocytes. Results from HCS culture were compared to non-treated control cultures. Stress factors of HDBR affect immune responsiveness and immune-endocrine homeostatic interrelations in vitro as follow: 1) alter expression of surface receptor to IL-2 (CD25) by CD4+ and CD8+ T-cell subsets in PHA/LPS activated PBMC culture; 2) alter distribution of IL-2 and/or IFN-gamma producing CD4+ and CD8+ T-cells in PMA/ionomycin activated culture; 3) significantly affect secretion of IL-2, IFN-gamma, and IL-4, but not IL-10 and soluble IL-2 receptor alpha in PHA/LPS activated PBMC culture; 4) shift Type 1 vs. Type 2 cytokine balance in PHA/LPS activated culture toward to Type 1 response; 5) in vitro treatment with hydrocortisone unequally modulate expression of CD25 on CD4+, and CD8+ T-cells, as well as secretion of Type 1 and Type 2 cytokines in PHA/LPS activated PBMC culture during bed-rest regime; 6) assessment of immune profile depends from the cellular and humoral milieu of cell culture.     

The expression of p18INK4 and p27kip1 cyclin-dependent kinase inhibitors is regulated differently during human B cell differentiation

Cell cycle progression is under the control of cyclin-dependent kinases (cdks), the activity of which is dependent on the expression of specific cdk inhibitors. In this paper we report that the two cdk inhibitors, p27(Kip1) and p18(INK4c), are differently expressed and control different steps of human B lymphocyte activation. Resting B cells contain large amounts of p27(Kip1) and no p18(INK4c). In vitro stimulation by Staphylococcus aureus Cowan 1 strain or CD40 ligand associated with IL-10 and IL-2 induces a rapid decrease in p27(Kip1) expression combined with cell cycle entry and progression. In contrast, in vitro Ig production correlates with specific expression of p18(INK4c) and early G(1) arrest. This G(1) arrest is associated with inhibition of cyclin D3/cdk6-mediated retinoblastoma protein phosphorylation by p18(INK4c). A similar contrasting pattern of p18(INK4c) and p27(Kip1) expression is observed both in B cells activated in vivo and in various leukemic cells. Expression of p18(INK4c) was also detected in various Ig-secreting cell lines in which both maximum Ig secretion and specific p18(INK4c) expression were observed during the G(1) phase. Our study shows that p27(Kip1) and p18(INK4c) have different roles in B cell activation; p27(Kip1) is involved in the control of cell cycle entry, and p18(INK4c) is involved in the subsequent early G(1) arrest necessary for terminal B lymphocyte differentiation.     

Rapamycin blocks cell cycle progression of activated T cells prior to events characteristic of the middle to late G1 phase of the cycle

The effects of rapamycin (RAP) on cell cycle progression of human T cells stimulated with PHA were examined. Cell cycle analysis showed that the RNA content of cells stimulated with PHA in the presence of RAP was similar to that of control T cells stimulated with PHA for 12–24 hr in the absence of the drug. This level was substantially higher than that seen in cells stimulated in the presence of cyclosporin A (CsA), an immunosuppressant known to block cell cycle progression at an early point in the cycle. However, the point in the cell cycle at which RAP acted appeared to be well before the G1/S transition, which occurs about 30–36 hr after stimulation with PHA. In an attempt to further localize the point in the cell cycle where arrest occurred, a set of key regulatory events leading to the G1/S boundary were examined, including p110^Rb phosphorylation, which occurred at least 6 hr prior to DNA synthesis, p34^cdc2 synthesis, and cyclin A synthesis. In control cultures, p110^Rb phosphorylation was detected within 24 hr of PHA stimulation; p34^cdc2 and cyclin A synthesis were detected within 30 hr. Addition of RAP to the cultures inhibited each of these events. In contrast, early events, including c-fos, IL-2, and IL-4 mRNAs expression, and IL-2 receptor (p55) expression, were only marginally affected, if at all, in PHA-stimulated T cells. Furthermore, the inhibition of cell proliferation by RAP could not be overcome by addition of exogenous IL-2. These results indicate that RAP blocks cell cycle progression of activated T cells after IL-2/IL-2 receptor interaction but prior to p110^Rb phosphorylation and other key regulatory events signaling G₁/S transition. © 1993 Wiley-Liss, Inc.     

IFN-gamma inhibits human airway smooth muscle cell proliferation by modulating the E2F-1/Rb pathway

Elucidating the factors that inhibit the increase in airway smooth muscle (ASM) mass may be of therapeutic benefit in asthma. Here, we investigated whether interferon-gamma (IFN-gamma), a potent inducer of growth arrest in various cell types, regulates mitogen-induced ASM cell proliferation. IFN-gamma (1-100 U/ml) was found to markedly decrease both DNA synthesis and ASM cell number induced by the mitogens epidermal growth factor (EGF) and thrombin. Interestingly, IFN-gamma had no effect on mitogen-induced activation of three major mitogenic signaling pathways, phosphatidylinositol 3-kinase, p70(S6k), or mitogen-activated protein kinases. Mitogen-induced expression of cell cycle regulator cyclin D1 was increased by IFN-gamma, whereas no effect was observed on degradation of p27(Kip1). Expression array analysis of 23 cell cycle-related genes showed that IFN-gamma inhibited EGF-induced increases in E2F-1 expression, whereas induction of c-myc, cyclin D2, Egr-1, and mdm2 were unaffected. Induction of E2F-1 protein and Rb hyperphosphorylation after mitogen stimulation was also suppressed by IFN-gamma. In addition, IFN-gamma decreased activation of cdk2 and expression of cyclin E, upstream signaling molecules responsible for Rb hyperphosphorylation in the late G1 phase. IFN-gamma also increased levels of IFI 16 protein, whose mouse homolog p202 has been associated with growth inhibition. Together, our data indicate that IFN-gamma is an effective inhibitor of ASM cell proliferation by blocking transition from G1-to-S phase by acting at two different levels: modulation of cdk2/cyclin E activation and inhibition of E2F-1 gene expression.     

TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling

Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressed cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence.     

IL-12 inhibition of endothelial cell functions and angiogenesis depends on lymphocyte-endothelial cell cross-talk

In vivo IL-12-dependent tumor inhibition rests on the ability of IL-12 to activate a CD8-mediated cytotoxicity, inhibit angiogenesis, and cause vascular injury. Although in vivo studies have shown that such inhibition stems from complex interactions of immune cells and the production of IFN-gamma and other downstream angiostatic chemokines, the mechanisms involved are still poorly defined. Here we show that IL-12 activates an anti-angiogenic program in Con A-activated mouse spleen cells (activated spc) or human PBMC (activated PBMC). The soluble factors they release in its presence arrest the cycle of endothelial cells (EC), inhibit in vitro angiogenesis, negatively modulate the production of matrix metalloproteinase-9, and the ability of EC to adhere to vitronectin and up-regulate ICAM-1 and VCAM-1 expression. These effects do not require direct cell-cell contact, yet result from continuous interaction between activated lymphoid cells and EC. We used neutralizing Abs to show that the IFN-inducible protein-10 and monokine-induced by IFN-gamma chemokines are pivotal in inducing these effects. Experiments with nu/nu mice, nonobese diabetic-SCID mice, or activated spc enriched in specific cell subpopulations demonstrated that CD4(+), CD8(+), and NK cells are all needed to mediate the full anti-angiogenetic effect of IL-12.     

Inhibition of p53-mediated growth arrest by overexpression of cyclin-dependent kinases.

Rat fibroblasts transformed by a temperature-sensitive mutant of murine p53 undergo a reversible growth arrest in G1 at 32.5 degrees C, the temperature at which p53 adopts a wild-type conformation. The arrested cells contain inactive cyclin-dependent kinase 2 (cdk2) despite the presence of high levels of cyclin E and cdk-activating kinase activity. This is due in part to p53-dependent expression of the p2l cdk inhibitor. Upon shift to 39 degrees C, wild-type p53 is lost and cdk2 activation and pRb phosphorylation occur concomitantly with loss of p2l. This p53-mediated growth arrest can be abrogated by overexpression of cdk4 and cdk6 but not cdk2 or cyclins, leading to continuous proliferation of transfected cells in the presence of wild-type p53 and p2l. Kinase-inactive counterparts of cdk4 and cdk6 also rescue these cells from growth arrest, implicating a noncatalytic role for cdk4 and cdk6 in this resistance to p53-mediated growth arrest. Aberrant expression of these cell cycle kinases may thus result in an oncogenic interference with inhibitors of cell cycle progression.     

Hypoxia modulates cyclin and cytokine expression and inhibits peripheral mononuclear cell proliferation.

Previously, we found that hypoxia can deeply affect the production of cytokines in human peripheral mononuclear cells (PBMC). Here, we demonstrated that the cycle progression of hypoxic PBMC, cultured in the presence or not of a specific T cell activator such as phytohaemagglutinin (PHA), was delayed when compared with aerobic cultures. This delay was accompanied by a decrease of the expression of specific cyclins associated to cell cycle progression phases. Ribonuclease Protection Assay (RPA) studies reveal a decrease in the expression of cyclin A and B in PHA-stimulated PBMC kept for 40 hr under hypoxic condition (2% O(2)), when compared with aerobic cultures (20% O(2)). In concomitance, a decrease of cyclin D2 expression was present after 16 hr of hypoxic treatment. However, the decrease was transient and disappeared after 40 hr of hypoxic treatment. Furthermore, cyclin C expression was not affected by hypoxia. Hypoxia-induced cyclin modulation was accompanied by an increased synthesis of interleukin (IL)-2 and IL-4, analyzed by ELISA. By evaluating these results, it appears that hypoxia induces a growth suppressive state in mitogen-activated PBMC by inhibiting the synthesis of mitotic cyclins A and B. However hypoxic PBMC maintain their viability and capability of producing stimulatory cytokines, after mitogen treatment. This should be important in local hypoxia, usually associated with necrotic areas, in inflammation, and infections, where T lymphocyte capability of producing stimulatory cytokines is desirable.     

IL-10 inhibits human T cell proliferation and IL-2 production.

Human IL-10 has been reported previously to inhibit the secretion of IFN-gamma in PBMC. In this study, we have found that human IL-10 inhibits T cell proliferation to either mitogen or anti-CD3 mAb in the presence of accessory cells. Inhibited T cell growth by IL-10 was associated with reduced production of IFN-gamma and IL-2. Studies of T cell subset inhibition by human IL-10 showed that CD4+, CD8+, CD45RA high, and CD45RA low cells are all growth inhibited to a similar degree. Dose response experiments demonstrated that IL-10 inhibits secretion of IFN-gamma more readily than T cell proliferation to mitogen. In addition, IL-2 and IL-4 added exogenously to IL-10 suppressed T cell cultures reversed completely the inhibition of T cell proliferation, but had little or no effect on inhibition of IFN-gamma production. Thus, in addition to its previously reported biologic properties, IL-10 inhibits human T cell proliferation and IL-2 production in response to mitogen. Inhibition of IFN-gamma production by IL-10 appears to be independent of the cytokine effect of IL-2 production.     

Differential expression and tissue compartmentalization of the inflammatory response following thermal injury

Studies have shown that both animal tissue-fixed immune cells and human peripheral blood mononuclear cell (PBMC) functions are altered after burn injury. Additional studies suggest that the burn injury-induced alterations in these divergent cell populations from different species are similar. It remains unknown, however, whether the observed changes in animal tissue-fixed immune cell function following thermal injury also occurs to a similar extent in the PBMC population. The aim of our study was to compare PBMC and tissue-fixed immune cell functions from the same animal using a murine burn model. At 7 days post-burn, mice were more susceptible to sepsis and delayed type hypersensitivity responses were suppressed. Splenocytes isolated from injured mice displayed suppressed proliferation and increased IL-10 production. In contrast, PBMC from injured mice displayed suppressed proliferation, IL-2 and IFN-gamma production. Splenic macrophage nitric oxide, PGE(2), TNF-alpha, IL-6 and IL-10 production was enhanced post-burn and IL-12 production was suppressed. PBMC from such animals displayed enhanced PGE(2) production and suppressed IL-6 and IL-12 production. These results indicate that while an immunosuppressive Th(2) phenotype (increased IL-10 and/or suppressed IL-2, IFN-gamma) was induced in both the splenic and PBMC compartments post-injury, differential expression and dimorphism in the response also exists. Thus, the assessment of only PBMC function in burn patients may not accurately reflect the patient's actual immune status at the tissue level.     

p21Cip1 and p27Kip1 act in synergy to alter the sensitivity of naive T cells to TGF-beta-mediated G1 arrest through modulation of IL-2 responsiveness

Induction of G(1) arrest by TGF-beta correlates with the regulation of p21(Cip1) and p27(Kip1), members of the Cip/Kip family of cyclin-dependent kinase inhibitors (cki). However, no definitive evidence exists that these proteins play a causal role in TGF-beta(1)-induced growth arrest in lymphocytes. In this report we show the suppression of cell cycle progression by TGF-beta is diminished in T cells from mice deficient for both p21(Cip1) and p27(Kip1) (double-knockout (DKO)) only when activated under conditions of optimal costimulation. Although there is an IL-2-dependent enhanced proliferation of CD8(+) T cells from DKO mice, TGF-beta is able to maximally suppress the proliferation of DKO T cells when activated under conditions of low costimulatory strength. We also show that the induction of p15(Ink4b) in T cells stimulated in the presence of TGF-beta is not essential, as TGF-beta also efficiently suppressed proliferation of T cells from p15(Ink4b-/-) mice. Finally, although these cki are dispensable for the suppression of T cell proliferation by TGF-beta, we now describe a Smad3-dependent down-regulation of cdk4, suggesting a potential mechanism underlying to resistance of Smad3(-/-) T cells to the induction of growth arrest by TGF-beta. In summary, the growth suppressive effects of TGF-beta in naive T cells are a function of the strength of costimulation, and alterations in the expression of cki modify the sensitivity to TGF-beta by lowering thresholds for a maximal mitogenic response.     

Cooperative regulation of the cell division cycle by the protein kinases RAF and AKT

The RAS-activated RAF-->MEK-->extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3'-kinase (PI3'-kinase)-->PDK1-->AKT signaling pathways are believed to cooperate to promote the proliferation of normal cells and the aberrant proliferation of cancer cells. To explore the mechanisms that underlie such cooperation, we have derived cells harboring conditionally active, steroid hormone-regulated forms of RAF and AKT. These cells permit the assessment of the biological and biochemical effects of activation of these protein kinases either alone or in combination with one another. Under conditions where activation of neither RAF nor AKT alone promoted S-phase progression, coactivation of both kinases elicited a robust proliferative response. Moreover, under conditions where high-level activation of RAF induced G(1) cell cycle arrest, activation of AKT bypassed the arrest and promoted S-phase progression. At the level of the cell cycle machinery, RAF and AKT cooperated to induce cyclin D1 and repress p27(Kip1) expression. Repression of p27(Kip1) was accompanied by a dramatic reduction in KIP1 mRNA and was observed in primary mouse embryo fibroblasts derived from mice either lacking SKP2 or expressing a T187A mutated form of p27(Kip1). Consistent with these observations, pharmacological inhibition of MEK or PI3'-kinase inhibited the effects of activated RAS on the expression of p27(Kip1) in NIH 3T3 fibroblasts and in a panel of bona fide human pancreatic cancer cell lines. Furthermore, we demonstrated that AKT activation led to sustained activation of cyclin/cdk2 complexes that occurred concomitantly with the removal of RAF-induced p21(Cip1) from cyclin E/cdk2 complexes. Cumulatively, these data strongly suggest that the RAF-->MEK-->ERK and PI3'K-->PDK-->AKT signaling pathways can cooperate to promote G(0)-->G(1)-->S-phase cell cycle progression in both normal and cancer cells.     

Restricted T cell expression of IL-2/IFN-gamma mRNA in human inflammatory disease

It has been difficult to demonstrate functionally distinct T cell populations in humans on the basis of cytokine secretion. As previous investigators have examined the T cell cytokine profile from immunized animals, we examined whether Th1 or Th2 type T cells could be identified in the peripheral blood or cerebrospinal fluid (CSF) immune compartments from subjects with or without inflammatory diseases. Using limiting dilution analysis and growth with PHA and IL-2/IL-4, we directly cloned a total of 177 T cells from the peripheral blood and CSF of seven subjects, four with inflammatory disease and three control subjects, and examined the cytokine message profile after stimulation with ionomycin and PMA. We found that most clones from both the peripheral blood and CSF express IL-1, IL-2, IL-4, IFN-gamma, or TNF-alpha cytokine mRNA after activation with ionomycin and PMA. All T cell clones tested produced TNF-alpha mRNA, and all but 14 produced IFN-gamma mRNA. As reported previously, Th0 cells, which produced IFN-gamma, IL-2, IL-4, and IL-5 mRNA, were found in most subjects. In striking contrast, Th1 cells, which expressed IL-2 and IFN-gamma but not IL-4 or IL-5 mRNA, were present in both peripheral blood and CSF of subjects with inflammatory disease but not found in peripheral blood or CSF of subjects without systemic inflammation. Th2 cells, expressing IL-4 and IL-5 but not IFN-gamma or IL-2 mRNA, were not found in any subject. These data present the first evidence for Th1 T cell clones in humans that may be associated with systemic inflammation.     

Genistein Induces G2 Arrest in Malignant B Cells by Decreasing IL-10 Secretion

Chronic B cell malignancies are often chemoresistant and the development of new therapeutic modalities is a high priority. Many B cell malignancies have autocrine production of IL-10, which regulates B cell growth and differentiation. Here we demonstrated that the soy isoflavone genistein, a tyrosine kinase inhibitor, rapidly decreased IL-10 secretion followed by upregulation of IFN-gamma and inhibition of cell proliferation with predominantly G2 arrest. The antiproliferative effects of genistein could be reversed by the addition of exogenous IL-10. Genistein downregulated cdc25C and cdk1 as well as anti-apoptotic proteins survivin and Ian-5. After genistein withdrawal, the G2M arrested cells re-entered the cell cycle and underwent apoptosis, which was significantly augmented by fludarabine. We conclude that genistein can sensitize malignant B cells to the action of other chemotherapeutic agents by modulating the cytokine profile and controlling cell cycle progression.     

人外周血活化淋巴细胞survivin基因的转录激活和表达相关的信号转导通路研究

Survivin是与细胞增殖和细胞凋亡调控密切相关的重要功能蛋白质,也是肿瘤临床诊断的分子标志物及治疗研究的理想靶标.由于发现人外周血活化淋巴细胞存在Survivin蛋白的显著表达,故以植物血凝素(PHA)和IL-2共刺激培养的正常人外周血单个核细胞为实验材料,采用RT-PCR、蛋白质印迹以及细胞周期分析等实验方法,观察淋巴细胞活化与Survivin蛋白表达之间的相互关系,并利用3种激酶抑制剂探索了淋巴细胞活化所致Survivin蛋白表达相关的信号转导通路.实验结果表明：人外周血单个核细胞在刺激培养36 h前后出现survivin基因的明显转录激活和蛋白质的起始表达,表达产物的水平随培养时间的延长而增加,且具有明显的细胞周期和周期时相依赖性.JAK2的抑制剂AG490阻断细胞周期的运行,且强烈抑制survivin基因的转录激活和蛋白质表达,PI3K和MEK的抑制剂Wortmannin和PD98059也有一定程度的抑制作用.所得实验结论是：survivin基因的转录激活和蛋白质表达与淋巴细胞活化相关联,代表细胞处在增殖状态.JAK2介导的JAK-STAT信号通路是淋巴细胞活化,Survivin蛋白表达必需和最重要的信号转导通路,PI3K和MEK介导的信号通路也具有一定的影响作用.