PDE4 inhibitors activate a mitochondrial apoptotic pathway in chronic lymphocytic leukemia cells that is regulated by protein phosphatase 2A

Chronic lymphocytic leukemia (CLL) cells, but not peripheral blood T cells, undergo apoptosis following treatment with inhibitors of type 4 cyclic nucleotide phosphodiesterase (PDE4), a process that correlates dose dependently with elevation of adenosine 3',5'-cyclic monophosphate (cAMP) in leukemic cells. We show that treatment of CLL cells with rolipram, a prototypic PDE4 inhibitor, and forskolin, an adenylate cyclase activator, induces mitochondrial depolarization, release of cytochrome c into the cytosol, caspase-9 and -3 activation, and cleavage of poly(adenosine diphosphate [ADP]-ribose)polymerase. Inhibitors of caspase-9, but not caspase-8, block rolipram/forskolin-induced CLL apoptosis. In a subset of CLL patients, B-cell lymphoma 2 (Bcl-2)-associated death promoter homolog (Bad), a proapoptotic Bcl-2 family member that when phosphorylated on specific serine residues is sequestered in the cytosol by 14-3-3, was dephosphorylated at Ser112 following rolipram/forskolin treatment of leukemic cells. Rolipram/forskolin treatment also induced Bad to accumulate in CLL heavy-membrane fractions, consistent with Bad translocation to mitochondria. To determine the mechanism for rolipram/forskolin-induced Bad dephosphorylation, we examined CLL phosphatase activity. Rolipram/forskolin treatment augmented protein phosphatase 2A (PP2A) activity, as well as levels of immunoreactive PP2A catalytic subunit. Treatment of CLL cells with a concentration of okadaic acid (5 nM) that selectively inhibits PP2A, reduced both rolipram/forskolin-induced mitochondrial cytochrome c release and mitochondrial depolarization. Okadaic acid restored Bad Ser112 phosphorylation and Bad association with 14-3-3 in rolipram/forskolin-treated CLL cells. These results suggest that PDE4 inhibitors may induce CLL apoptosis by activating PP2A-induced dephosphorylation of proapoptotic BH3-only Bcl-2 family members such as Bad.     

Modulation of TNF and GM-CSF release from dispersed human nasal polyp cells and human whole blood by inhibitors of different PDE isoenzymes and glucocorticoids

The aim of this study was to investigate the role of the inhibitors of different PDE isoenzymes (PDE 1-5) on the production of two pro-inflammatory cytokines - tumor necrosis factor alpha (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Two in vitro models were used to compare the antiinflammatory properties of PDE inhibitors with that of glucocorticoids. The effect on TNF release from diluted human blood following lipopolysaccharide (LPS from Salmonella abortus equi) stimulation as well as the GM-CSF and TNF release from human nasal polyp cells following allergic stimulation were investigated. Both models proofed to be well suited for the characterisation of the antiinflammatory properties of new chemical entities.In diluted human blood and dispersed human nasal polyp cells the induced TNF release was most potently suppressed by selective PDE4 inhibitors. Amrinone and milrinone, selective PDE3 inhibitors, suppressed TNF secretion to a lesser extent. The effects of theophylline (unspecific PDE inhibitor), vinpocetine (PDE1 inhibitor), EHNA (PDE2 inhibitor) and the PDE5 inhibitors zaprinast and E 4021 were weak. In human blood, the tested glucocorticoids beclomethasone, dexamethasone and fluticasone inhibited the LPS induced TNF release potently in a concentration dependent manner, whereas in dispersed human nasal polyp cells, the effect of the glucocorticoids on allergically induced TNF release, with the exception of dexamethasone, was much less pronounced. Glucocorticoids were the most potent inhibitors of GM-CSF release and the effect correlates well with the affinity to the glucocorticoid receptor. The selective PDE 4 inhibitors, and to a certain extent the PDE3 inhibitors amrinone and milrinone, reduced the GM-CSF release in a concentration dependent manner. In all investigations selective PDE4 inhibitors reduced TNF release to a much higher degree (4-10 fold) than GM-CSF release.     

A biochemical and functional assessment of monocyte phosphodiesterase activity in healthy and asthmatic subjects

The aim of this study was to investigate whether cyclic adenosine 3'5-monophosphate (cAMP) phosphodiesterase (PDE) activity is altered in monocytes from mild asthmatic subjects. Total cAMP PDE activity (pmol/min per mg protein) was significantly greater in homogenates prepared from monocytes from asthmatic subjects (68.3 +/- 7.0, n=9) compared to healthy individuals (46.3 +/- 3.3, n=14, P<0.05). The PDE inhibitors siguazodan (PDE3-selective), rolipram (PDE4-selective) and theophylline (non-selective) produced a concentration-dependent inhibition of cAMP PDE activity in homogenates from monocytes from normal and asthmatic subjects. However, siguazodan produced significantly greater (P<0.05), and rolipram significantly less (P<0.05), inhibition of total cAMP PDE activity in monocytes from asthmatics (n=4) than from healthy individuals (n=5). cAMP PDE activity was inhibited with equal potency by theophylline in monocytes from healthy and asthmatic subjects. We also investigated the functional consequences of the changes in PDE activity in mononuclear cells obtained from asthmatic subjects. There was no significant difference in the ability of PDE4 inhibitors to attenuate TNF alpha release from monocytes obtained from asthmatic compared with healthy subjects (P>0.05). Despite a significant increase in the biochemical activity of PDE3 in monocytes from asthmatic subjects, the PDE3 inhibitor siguazodan, failed to significantly reduce TNF alpha release from human monocytes. Thus, total cAMP PDE activity is increased in monocytes taken from mild asymptomatic asthmatics compared to healthy subjects and is reflected by an increase in the proportion of PDE3 and a decrease in the proportion of PDE4. This augmented enzyme activity was not associated with an alteration in the ability of PDE4 inhibitors to attenuate mononuclear cell function from asthmatics compared to healthy individuals.     

Long-term culture of human CD34(+) progenitors with FLT3-ligand, thrombopoietin, and stem cell factor induces extensive amplification of a CD34(-)CD14(-) and a CD34(-)CD14(+) dendritic cell precursor

Current in vitro culture systems allow the generation of human dendritic cells (DCs), but the output of mature cells remains modest. This contrasts with the extensive amplification of hematopoietic progenitors achieved when culturing CD34(+) cells with FLT3-ligand and thrombopoietin. To test whether such cultures contained DC precursors, CD34(+) cord blood cells were incubated with the above cytokines, inducing on the mean a 250-fold and a 16,600-fold increase in total cell number after 4 and 8 weeks, respectively. The addition of stem cell factor induced a further fivefold increase in proliferation. The majority of the cells produced were CD34(-)CD1a- CD14(+) (p14(+)) and CD34(-)CD1a-CD14(-) (p14(-)) and did not display the morphology, surface markers, or allostimulatory capacity of DC. When cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), both subsets differentiated without further proliferation into immature (CD1a+, CD14(-), CD83(-)) macropinocytic DC. Mature (CD1a+, CD14(-), CD83(+)) DCs with high allostimulatory activity were generated if such cultures were supplemented with tumor necrosis factor-alpha (TNF). In addition, p14(-) cells generated CD14(+) cells with GM-CSF and TNF, which in turn, differentiated into DC when exposed to GM-CSF and IL-4. Similar results were obtained with frozen DC precursors and also when using pooled human serum AB+ instead of bovine serum, emphasizing that this system using CD34(+) cells may improve future prospects for immunotherapy.     

1,25-Dihydroxyvitamin D(3) inhibits dendritic cell differentiation and maturation in vitro

OBJECTIVE: Because of its potent immunosuppressive properties in vitro as well as in vivo, we studied the effect of 1,25-dihydroxyvitamin D(3) (calcitriol) on differentiation, maturation, and function of dendritic cells (DC). MATERIALS AND METHODS: Monocyte-derived DCs were generated with GM-CSF plus IL-4, and maturation was induced by a 2-day exposure to TNFalpha. DCs were derived from CD34(+) progenitors using SCF plus GM-CSF plus TNFalpha. For differentiation studies, cells were exposed to calcitriol at concentrations of 10(-)(9)- 10(-7) M at days 0, 6, and 8, respectively. The obtained cell populations were evaluated by morphology, phenotype, and function. RESULTS: When added at day 0, calcitriol blocked DC differentiation from monocytes and inhibited the generation of CD1a(+) cells from progenitor cells while increasing CD14(+) cells. Exposure of immature DCs to calcitriol at day 6 resulted in a loss of the DC-characteristic surface molecule CD1a, downregulation of the costimulatory molecules CD40 and CD80, and MHC class II expression, whereas the monocyte/macrophage marker CD14 was clearly reinduced. In addition, calcitriol hindered TNFalpha-induced DC maturation, which is usually accompanied with induction of CD83 expression and upregulation of costimulatory molecules. In contrast, the mature CD83(+) DCs remained CD1a(+)CD14(-) when exposed to calcitriol. The capacity of cytokine-treated cells to stimulate allogeneic and autologous T cells and to take up soluble antigen was inhibited by calcitriol. CONCLUSION: The potent suppression of DC differentiation, the reversal of DC phenotype, and function in immature DCs, as well as the inhibition of DC maturation by calcitriol, may explain some of its immunosuppressive properties.     

Inhibition of airway hyperresponsiveness and pulmonary inflammation by roflumilast and other PDE4 inhibitors

Roflumilast is an oral, once-daily phosphodiesterase 4 (PDE4) inhibitor with anti-inflammatory activity. We compared the anti-inflammatory effects of roflumilast with those of PDE4 inhibitors rolipram, piclamilast, and cilomilast in ovalbumin (OVA)-sensitized and challenged Brown-Norway rats. Animals were treated orally 1h before OVA challenge with roflumilast (0.3, 1.0, and 3.0mg/kg), rolipram (0.8, 2.8, and 8.3mg/kg), piclamilast (10.0, 20.0, and 30.0mg/kg), or cilomilast (10.3, 34.3, and 103.0mg/kg). Airway hyperresponsiveness (AHR) against adenosine was investigated by measuring airway resistance 200min after OVA challenge. Subsequently, neutrophil influx and tumor necrosis factor-alpha (TNF-alpha) release in the lungs were determined by bronchoalveolar lavage. Direct bronchodilation at the time point of AHR assessment by PDE4 inhibitors was examined in serotonin-challenged animals. Evaluation of neutropenic animals or treatment with anti-TNF-alpha antibody revealed that AHR was independent of neutrophil accumulation or TNF-alpha release. Roflumilast (50% inhibitory dose [ID(50)]=1.5mg/kg) inhibited AHR 3-, 16-, and 27-fold more potently than rolipram, piclamilast, and cilomilast, respectively. Likewise, roflumilast was a more potent inhibitor of neutrophil influx (ID(50)=0.9mg/kg) than rolipram (ID(50)=6.9mg/kg), piclamilast (ID(50)=28.1mg/kg), or cilomilast (ID(50)=37.7mg/kg). Roflumilast, rolipram, and piclamilast-but not cilomilast-suppressed OVA-induced TNF-alpha release in a dose-dependent manner. Roflumilast (ID(50)=0.9mg/kg) exhibited 9- and 23-fold more potent inhibition of TNF-alpha release than rolipram and piclamilast, respectively. Roflumilast did not inhibit serotonin-induced bronchoconstriction 4.5h after administration, suggesting that inhibition of AHR by roflumilast results from anti-inflammatory, not bronchodilatory, effects. This study suggests that roflumilast has anti-inflammatory action and provides rationale for the investigation of roflumilast in asthmatic patients.     

Interferon-alpha and -beta inhibit the in vitro differentiation of immunocompetent human dendritic cells from CD14(+) precursors

Dendritic cell (DC) precursors and immature DC reside in epithelium where they encounter pathogens and cytokines, which stimulate their differentiation. We hypothesized that type-I interferons (IFN-alpha and -beta), cytokines that are produced early in the innate immune response against viruses and some bacteria, may influence DC differentiation and function. To examine this possibility, we used an in vitro model of DC differentiation in which initial culture of human CD14(+) monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 generates immature DC, and subsequent culture with tumor necrosis factor (TNF)-alpha drives the final development into mature DC. We found in this model that IFN-alpha/beta, added from the initiation of the culture on, significantly reduced the survival and altered the morphology and differentiation of DC. TNF-alpha-dependent maturation of IFN-beta-treated immature DC led to cells with reduced expression of CD1a, CD40, CD54, and CD80 when compared with mature DC controls. IFN-alpha/beta-treated DC further had a reduced capacity to induce naive Th-cell proliferation through allostimulation or anti-CD3 monoclonal antibody stimulation. In addition, IFN-alpha/beta-treated DC secreted less IL-12 upon stimulation with Staphylococcus aureus Cowan strain or with CD4(+) T cells, and this decrease correlated directly with their inability to support CD4(+) T-cell secretion of IFN-gamma, even though T-cell lymphotoxin production was unaffected. These findings indicate that type-I IFNs can influence the generation of acquired immune responses by modifying T-helper cell differentiation through the regulation of DC differentiation and function.     

Nonsteroidal anti-estrogens inhibit the functional differentiation of human monocyte-derived dendritic cells

Dendritic cells (DC) are professional antigen-presenting cells with a unique capacity to initiate and regulate immune responses. Immature CD1a(+) DC can be cultured from CD14(+) monocytes in the presence of interleukin (IL)-4 and granulocyte macrophage colony-stimulating factor in vitro. Results of this study show that the nonsteroidal anti-estrogens toremifene and tamoxifen inhibit this differentiation. In the presence of anti-estrogens the cells lose CD14 expression, but remain CD1a(-) and clearly have less dendritic processes than immature DC. Functionally, anti-estrogen-treated cells are inferior to immature DC in inducing proliferation of allogeneic T cells and in producing IL-12 p70 protein after CD40 ligation. The expression of the costimulatory molecules CD80 and CD86 is differentially regulated by anti-estrogens during DC differentiation. Furthermore, anti-estrogens are also able to inhibit the terminal maturation of DC. By inhibiting the functional differentiation of DC, anti-estrogens may have a role in the treatment and prevention of autoimmune diseases. (Blood. 2000;95:2875-2882)     

Galectin-9 and T cell immunoglobulin mucin-3 pathway is a therapeutic target for type 1 diabetes

Galectin-9 (Gal-9), a ligand for T cell Ig mucin-3 (Tim-3), induces apoptosis in cluster of differentiation 4 (CD4)(+) Tim-3(+) T helper 1 (T(H)1) cells via the Gal-9-Tim-3 pathway and negatively regulates T(H)1 immunity. In turn, Gal-9 activates dendritic cells (DC) to produce TNF-α, which promotes the T(H)1 response. We investigated the efficacy of Gal-9 against T(H)1-mediated autoimmune diabetes in NOD mice and compared with anti-Tim-3 monoclonal antibody (RMT3-23), which inhibited the binding between Tim-3-Ig and Gal-9 in a solid-phase binding assay. mRNA expression of Gal-9 was prominently induced by the treatment of interferon-γ in MIN6 cells, and Gal-9 was also expressed in the pancreatic β-cells in NOD mice, suggesting Gal-9 may be released from pancreatic β-cells to terminate T(H)1-mediated inflammation. Long-term injection of Gal-9 exhibits preventive efficacy for development of diabetes in NOD mice, but RMT3-23 demonstrated further prominent therapeutic potential compared with Gal-9. Gal-9 induced apoptosis of CD4(+)Tim-3(+) T(H)1 cells at the concentration of 0.2 μM, whereas RMT3-23 failed to enhance the apoptosis of CD4(+)Tim-3(+) T(H)1 cells. In contrast, Gal-9 induced TNF-α production in cultured DC in a dose-dependent manner; however, RMT3-23 inhibited Gal-9-induced TNF-α production in a dose-dependent manner. Although Gal-9 exhibited certain therapeutic potential against autoimmune diabetes by enhancing apoptosis of CD4(+)Tim-3(+) T(H)1 cells, RMT3-23 exhibited prominent therapeutic efficacy by suppressing the TNF-α production and activation of DC. Taken together, the inhibition of the Gal-9-Tim-3 pathway on DC, upstream of T(H)1 response, may be a new target for the treatment of type 1 diabetes.     

Characterization of dendritic cell differentiation pathways from cord blood CD34(+)CD7(+)CD45RA(+) hematopoietic progenitor cells

To better characterize human dendritic cells (DCs) that originate from lymphoid progenitors, the authors examined the DC differentiation pathways from a novel CD7(+)CD45RA(+) progenitor population found among cord blood CD34(+) cells. Unlike CD7(-)CD45RA(+) and CD7(+)CD45RA(-) progenitors, this population displayed high natural killer (NK) cell differentiation capacity when cultured with stem cell factor (SCF), interleukin (IL)-2, IL-7, and IL-15, attesting to its lymphoid potential. In cultures with SCF, Flt3 ligand (FL), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF)-alpha (standard condition), CD7(+)CD45RA(+) progenitors expanded less (37- vs 155-fold) but yielded 2-fold higher CD1a(+) DC percentages than CD7(-)CD45RA(+) or CD7(+)CD45RA(-) progenitors. As reported for CD34(+)CD1a(-) thymocytes, cloning experiments demonstrated that CD7(+)CD45RA(+) cells comprised bipotent NK/DC progenitors. DCs differentiated from CD7(-)CD45RA(+) and CD7(+)CD45RA(+) progenitors differed as to E-cadherin CD123, CD116, and CD127 expression, but none of these was really discriminant. Only CD7(+)CD45RA(+) or thymic progenitors differentiated into Lag(+)S100(+) Langerhans cells in the absence of exogenous transforming growth factor (TGF)-beta 1. Analysis of the DC differentiation pathways showed that CD7(+)CD45RA(+) progenitors generated CD1a(+)CD14(-) precursors that were macrophage-colony stimulating factor (M-CSF) resistant and CD1a(-)CD14(+) precursors that readily differentiated into DCs under the standard condition. Accordingly, CD7(+)CD45RA(+) progenitor-derived mature DCs produced 2- to 4-fold more IL-6, IL-12, and TNF-alpha on CD40 ligation and elicited 3- to 6-fold higher allogeneic T-lymphocyte reactivity than CD7(-)CD45RA(+) progenitor-derived DCs. Altogether, these findings provide evidence that the DCs that differentiate from cord blood CD34(+)CD7(+)CD45RA(+) progenitors represent an original population for their developmental pathways and function. (Blood. 2000;96:3748-3756)     

Role of 4-1BB (CD137) in the functional activation of cord blood CD28(-)CD8(+) T cells

The CD28(-) subset of CD8(+) T cells is associated with cytotoxic T lymphocyte (CTL) effector function. We investigated a potential role for 4-1BB, a costimulatory molecule structurally related to members of the tumor necrosis factor (TNF) receptor family, in the generation and functional activation of CD28(-) CTLs by using human cord blood (CB) cells composed exclusively of naive CD8(+) T cells with few or no CD28(-) CTLs. The 4-1BB was induced preferentially on the CB CD28(-)CD8(+) T cells when CD28 down-regulation was induced by interleukin 15 (IL-15) and IL-12 stimulation. Anti-4-1BB costimulation induced dramatic phenotypic changes in the CD28(-) CTLs, including restoration of CD28 expression as well as that of memory markers such as CD45RO and CC chemokine receptor 6 (CCR6). Anti-4-1BB costimulation also promoted long-term survival of CD28(-) CTLs, which were sensitive to activation-induced cell death upon anti-CD3 stimulation. The memory-type CD28(+) CTLs induced by anti-4-1BB costimulation acquired a greatly enhanced content of granzyme B, a cytolytic mediator, and enhanced cytotoxic activity as compared with CD28(-) CTLs. Strong cytotoxicity of memory-type CTLs to a 4-1BB ligand-expressing Epstein-Barr virus (EBV)-transformed B-cell line was almost completely abrogated by 4-1BB-Fc, a soluble form of 4-1BB, suggesting involvement of 4-1BB in cytolytic processes. Taken all together, our results suggest that 4-1BB plays a role in the differentiation of effector memory CTLs.     

Poor allostimulatory function of liver plasmacytoid DC is associated with pro-apoptotic activity, dependent on regulatory T cells

BACKGROUND/AIMS: The liver is comparatively rich in plasmacytoid (p) dendritic cells (DC), - innate immune effector cells that are also thought to play key roles in the induction and regulation of adaptive immunity. METHODS: Liver and spleen pDC were purified from fms-like tyrosine kinase ligand-treated control or lipopolysaccharide-injected C57BL/10 mice. Flow cytometric and molecular biologic assays were used to characterize their function and interaction with naturally occurring regulatory T cells (Treg). RESULTS: While IL-10 production was greater for freshly isolated liver compared with splenic pDC, the former produced less bioactive IL-12p70. Moreover, liver pDC expressed a low Delta4/Jagged1 Notch ligand ratio, skewed towards T helper 2 cell differentiation/cytokine production, and promoted allogeneic CD4(+)T cell apoptosis. T cell proliferation in response to liver pDC was, however, enhanced by blocking IL-10 function at the initiation of cultures. In the absence of naturally occurring CD4(+)CD25(+) regulatory T cells, similar levels of T cell proliferation were induced by liver and spleen pDC and the pro-apoptotic activity of liver pDC was reversed. CONCLUSIONS: The inferior T cell allostimulatory activity of in vivo-stimulated liver pDC may depend on the presence and function of Treg, a property that may contribute to inherent liver tolerogenicity.     

The differential production of cytokines by human Langerhans cells and dermal CD14(+) DCs controls CTL priming

We recently reported that human epidermal Langerhans cells (LCs) are more efficient than dermal CD14(+) DCs at priming naive CD8(+) T cells into potent CTLs. We hypothesized that distinctive dendritic cell (DC) cytokine expression profiles (ie, IL-15 produced by LCs and IL-10 expressed by dermal CD14(+) DCs) might explain the observed functional difference. Blocking IL-15 during CD8(+) T-cell priming reduced T-cell proliferation by ～ 50%. These IL-15-deprived CD8(+) T cells did not acquire the phenotype of effector memory cells. They secreted less IL-2 and IFN-γ and expressed only low amounts of CD107a, granzymes and perforin, and reduced levels of the antiapoptotic protein Bcl-2. Confocal microscopy analysis showed that IL-15 is localized at the immunologic synapse of LCs and naive CD8(+) T cells. Conversely, blocking IL-10 during cocultures of dermal CD14(+) DCs and naive CD8(+) T cells enhanced the generation of effector CTLs, whereas addition of IL-10 to cultures of LCs and naive CD8(+) T cells inhibited their induction. TGF-β1 that is transcribed by dermal CD14(+) DCs further enhanced the inhibitory effect of IL-10. Thus, the respective production of IL-15 and IL-10 explains the contrasting effects of LCs and dermal CD14(+) DCs on CD8(+) T-cell priming.     

Comparison of PDE 4 inhibitors, rolipram and SB 207499 (ariflo), in a rat model of pulmonary neutrophilia

Using a rat model of lipopolysaccharide (LPS)-induced pulmonary inflammation, the antiinflammatory activity of SB 207499 was evaluated and compared to that of the prototypic type-4 phosphodiesterase (PDE4) inhibitor, rolipram. In dose-response experiments, we found that rats exposed to 10 microg or 100 microg of intratracheal (it) LPS developed a prominent pulmonary inflammation, due to a significant increase in the number of recoverable bronchoalveolar lavage neutrophils. The pulmonary neutrophilia, provoked by the challenge of 10 microg LPS/rat, was significant at 2 h, peaked by 16 h, declined thereafter but remained elevated for up to 48 h. Additionally, the exposure of rats to 10 microg LPS caused the local pulmonary production of TNF- alpha. In contrast to the cellular influx, TNF- alpha production peaked at 2 h and rapidly declined to negligible levels by 8 h. While low levels were detected, the levels of IL-1 beta in bronchoalveolar lavage did not significantly differ from saline challenged animals. Rats pretreated with rolipram or SB 207499, displayed dose-dependent inhibition of the LPS-induced pulmonary inflammation. Nevertheless, the pulmonary production of TNF- alpha and IL-1 beta was unaffected by either SB 207499 or rolipram. When provoked with the 10 microg dose of LPS, adrenalectomized rats produced a similar 24 h induction of pulmonary neutrophilia. Pretreatment of adrenalectomized rats with the PDE4 inhibitors showed similar inhibitory results to those obtained in normal rats. In summary, we have shown, using a rat model of LPS-induced pulmonary neutrophilic inflammation, that the inhibitory activities of rolipram or SB207499 are not linked to the production of TNF- alpha or the inhibition of IL-1 beta, and occur independently of endogenous catecholamine or corticosteroid release. Copyright Academic Press.     

Inhibition of PDE4 phosphodiesterase activity induces growth suppression, apoptosis, glucocorticoid sensitivity, p53, and p21(WAF1/CIP1) proteins in human acute lymphoblastic leukemia cells

Glucocorticoids are integral to successful treatment of childhood acute lymphoblastic leukemia (ALL) and other lymphoid malignancies. A large body of data indicates that in various model systems, elevation of cyclic adenosine monophosphate (cAMP) can potentiate glucocorticoid response, although this has not been well evaluated as a potential leukemia treatment. Although cAMP analogs have been studied, little data exist regarding the potential toxicity to leukemia cells of pharmacologic elevation of cAMP levels in leukemic blasts. Using MTT assays of cell proliferation on CEM ALL cells, we found that aminophylline and other nonspecific phosphodiesterase (PDE) inhibitors suppress cell growth. This effect is replicated by the PDE4-specific PDE inhibitor rolipram, but not by specific inhibitors of the PDE1 or PDE3 classes. We found that PDE inhibitors cause increased dexamethasone sensitivity and a synergistic effect with the adenylyl cyclase activator forskolin. We observed several important cellular characteristics associated with this treatment, including elevation of cAMP, induction of p53 and p21(WAF1/CIP1) proteins, G(1) and G(2)/M cell cycle arrest, and increased apoptosis. Sensitivity to forskolin and rolipram is shared by at least 2 pediatric ALL cell lines, CEM and Reh cells. Some cell lines derived from adult-type lymphoid malignancies also show sensitivity to this treatment. These findings suggest that PDE inhibitors have therapeutic potential in human ALL and characterize the molecular mechanisms that may be involved in this response.     

Inhibition by reproterol of cAMP PDE in intact mastocytoma P-815 cells

In vitro studies in rat mastocytes and human monocytes suggested that reproterol (a selective beta(2)-adrenoceptor agonist with a theophylline moiety) exerts anti-inflammatory actions through inhibition of cyclic AMP (cAMP) PDE activity. Thus, reproterol was tested for its ability to inhibit cAMP PDE in cultured mouse mastocytoma P-815 cells. cAMP PDE activity was measured in intact cells by spectrofluorometry using the fluorescent substrate 2'-O-anthraniloyl cAMP. Reproterol was more potent than theophylline to inhibit cAMP PDE (pIC(50)=4.28+/-0.25 vs. 3.16+/-0.05). This contrasted with disrupted cells, where the PDE inhibitory potency of reproterol was low (pIC(50)=2.85+/-0.03) and similar to that of theophylline (pIC(50)=2.66+/-0.19). No cAMP PDE inhibition was found with other beta(2)-agonists tested (fenoterol, salbutamol, salmeterol and formoterol). Finally, the selective PDE inhibitors calmidazolium (100 nM), milrinone (5 microM) and rolipram (50 microM) inhibited cAMP PDE activity by approximately 20, 30 and 25% respectively. In conclusion, reproterol potently and non-specifically inhibited intracellular cAMP phosphodiesterases in intact mastocytoma cells. This can explain the previously reported beta(2)-adrenoceptor-independent anti-inflammatory actions of reproterol in vitro. Further studies are required to define the anti-inflammatory potential of reproterol in asthma.     

Codevelopment of dendritic cells along with erythroid differentiation from human CD34(+) cells by tumor necrosis factor-alpha

OBJECTIVE: Tumor necrosis factor-alpha (TNF-alpha) inhibits erythropoiesis and enhances nonerythroid colony formation. The present study examines the nature of these nonerythroid cells and investigates their physiologic role in relation to erythroid progenitor cells. MATERIALS AND METHODS: Highly purified human CD34(+) cells underwent erythroid differentiation in the presence of multiple cytokines, including stem cell factor (SCF), interleukin-3 (IL-3), and erythropoietin (EPO), with and without TNF-alpha. We enumerate colony-forming unit-erythroid (CFU-E) and glycophorin A (GPA; a specific marker for erythroid lineage) positive cells in semisolid phase as well as in liquid suspension culture. The character and roles of codeveloping nonerythroid cells in the presence of TNF-alpha were analyzed using fluorescent activating cell sorter, enzyme immunohistochemistry, and confocal microscopy. RESULTS: TNF-alpha inhibited the generation of GPA(+) cells and conversely enhanced the generation of GPA(-) cells. The GPA(-) cells were comprised of cells with excentric cell shape and were positive for HLA class I, HLA class II, CD1a, CD4, CD11c, CD14, CD40, CD80, CD83, and CD86, but not for CD3, CD8, CD19, CD20, and CD56, indicating the codevelopment of dendritic cells (DC) along with erythroid differentiation. Developing DC/DC precursors were detected within 3 days of culture. Only in the presence of TNF-alpha did CD34(+) cells proliferate by forming aggregates where both GPA(+) and CD11c(+) DC/DC precursors were present. During culture period, immature CD11c(+) DC were capable of endocytosing damaged GPA(+) cells. CONCLUSIONS: GPA(-) cells cogenerated from human CD34(+) cells during erythroid differentiation in the presence of IL-3/SCF/EPO and TNF-alpha express DC phenotypes. The CD11c(+) DC subset physically and selectively associates with developing immature erythroid cells and damaged self-GPA(+) cells and then obtains and captures self-substances.     

LTβR signaling in dendritic cells induces a type I IFN response that is required for optimal clonal expansion of CD8+ T cells

During an immune response, antigen-bearing dendritic cells (DCs) migrate to the local draining lymph node and present antigen to CD4(+) helper T cells. Antigen-activated CD4(+) T cells then up-regulate TNF superfamily members including CD40 ligand and lymphotoxin (LT)αβ. Although it is well-accepted that CD40 stimulation on DCs is required for DC licensing and cross-priming of CD8(+) T-cell responses, it is likely that other signals are integrated into a comprehensive DC activation program. Here we show that a cognate interaction between LTαβ on CD4(+) helper T cells and LTβ receptor on DCs results in unique signals that are necessary for optimal CD8(+) T-cell expansion via a type I IFN-dependent mechanism. In contrast, CD40 signaling appears to be more critical for CD8(+) T-cell IFNγ production. Therefore, different TNF family members provide integrative signals that shape the licensing potential of antigen-presenting DCs.