GBM Derived Gangliosides Induce T Cell Apoptosis through Activation of the Caspase Cascade Involving Both the Extrinsic and the Intrinsic Pathway.

Previously we demonstrated that human glioblastoma cell lines induce apoptosis in peripheral blood T cells through partial involvement of secreted gangliosides. Here we show that GBM-derived gangliosides induce apoptosis through involvement of the TNF receptor and activation of the caspase cascade. Culturing T lymphocytes with GBM cell line derived gangliosides (10-20 µg/ml) demonstrated increased ROS production as early as 18 hrs as indicated by increased uptake of the dye H2DCFDA while western blotting demonstrated mitochondrial damage as evident by cleavage of Bid to t-Bid and by the release of cytochrome-c into the cytosol. Within 48-72 hrs apoptosis was evident by nuclear blebbing, trypan blue positivity and annexinV/7AAD staining. GBM-ganglioside induced activation of the effector caspase-3 along with both initiator caspases (-9 and -8) in T cells while both the caspase-8 and -9 inhibitors were equally effective in blocking apoptosis (60% protection) confirming the role of caspases in the apoptotic process. Ganglioside-induced T cell apoptosis did not involve production of TNF-α since anti-human TNFα antibody was unable to protect T cells from nuclear blebbing and subsequent cell death. However, confocal microscopy demonstrated co-localization of GM2 ganglioside with the TNF receptor and co-immunoprecipitation experiments showed recruitment of death domains FADD and TRADD with the TNF receptor post ganglioside treatment, suggesting direct interaction of gangliosides with the TNF receptor. Further confirmation of the interaction between GM2 and TNFR1 was obtained from confocal microscopy data with wild type and TNFR1 KO (TALEN mediated) Jurkat cells, which clearly demonstrated co-localization of GM2 and TNFR1 in the wild type cells but not in the TNFR1 KO clones. Thus, GBM-ganglioside can mediate T cell apoptosis by interacting with the TNF receptor followed by activation of both the extrinsic and the intrinsic pathway of caspases.

Myeloid derived suppressor cell infiltration of murine and human gliomas is associated with reduction of tumor infiltrating lymphocytes.

Myeloid derived suppressor cells (MDSCs) are bone marrow derived cells with immunosuppressive properties. We have shown previously that MDSCs numbers are elevated in the circulation of GBM patients and that they produce reversible T cell dysfunction. Here, we evaluated whether MDSCs infiltrate human GBM tissues, and whether a commonly used mouse model of GBM reproduces the biology of MDSCs that is observed in patients. We evaluated tumor specimens from patients with newly diagnosed GBM. We harvested and evaluated normal brain, tumors and hematopoietic tissues from control, vehicle and sunitinib-treated mice. In human GBM tumors, MDSCs represented 5.4 ± 1.8 % of total cells. The majority of MDSCs (CD33+HLADR-) were lineage negative (CD14-CD15-), followed by granulocytic (CD15+CD14-) and monocytic (CD15-CD14+) subtypes. In murine GBM tumors, MDSCs were 8.06 ± 0.78 % of total cells, of which more were monocytic (M-MDSC, CD11b+ Gr1-low) than granulocytic (G-MDSC, CD11b+ Gr1-high). Treatment with the tyrosine kinase inhibitor sunitinib decreased the infiltration of both granulocytic and monocytic MDSCs in murine GBM tumors. In the hematopoietic tissues, circulating G-MDSC blood levels were reduced after sunitinib treatment. In tumors, both CD3(+) and CD4(+) T cell counts increased following sunitinib treatment (p ≤ 0.001). Total T cell proliferation (p < 0.001) and interferon gamma production (p = 0.004) were increased in the spleens of sunitinib treated mice. Sunitinib-treated mice survived longer than vehicle-treated mice (p = 0.002). MDSCs are present in both human and mouse GBM tumors. Sunitinib may have an immunostimulatory effect, as its use is associated with a reduction in G-MDSCs and improvement in anti-tumor immune function.

Myeloid-derived suppressor cell accumulation and function in patients with newly diagnosed glioblastoma.

To assess the accumulation of myeloid-derived suppressor cells (MDSCs) in the peripheral blood of patients with glioma and to define their heterogeneity and their immunosuppressive function. Peripheral blood mononuclear cells (PBMCs) from healthy control subjects and from patients with newly diagnosed glioma were stimulated with anti-CD3/anti-CD28 and T cells assessed for intracellular expression of interferon (IFN)-γ. Antibody staining of PBMCs from glioma patients and healthy donors (CD33, HLADR, CD15, and CD14) followed by 4-color flow cytometry analysis-defined MDSC levels in the peripheral blood. To assess the role of MDSCs in suppressing T cell IFNγ production, PBMCs were depleted of MDSCs using anti-CD33 and anti-CD15 antibody-coated beads prior to T cell stimulation. Enzyme-linked immunosorbent assays were used to assess plasma arginase activity and the level of granulocyte colony-stimulating factor (G-CSF). Patients with glioblastoma have increased MDSC counts (CD33+HLADR-) in their blood that are composed of neutrophilic (CD15(+); >60%), lineage-negative (CD15(-)CD14(-); 31%), and monocytic (CD14(+); 6%) subsets. After stimulation, T cells from patients with glioblastoma had suppressed IFN-γ production when compared with healthy, age-matched donor T cells. Removal of MDSCs from the PBMCs with anti-CD33/CD15-coated beads significantly restored T cell function. Significant increases in arginase activity and G-CSF levels were observed in plasma specimens obtained from patients with glioblastoma. The accumulation of MDSCs in peripheral blood in patients with glioma likely promotes T cell immune suppression that is observed in this patient population. Increased plasma levels of arginase and G-CSF may relate to MDSC suppressor function and MDSC expansion, respectively, in patients with glioma.

IL-8 is a mediator of NF-κB induced invasion by gliomas.

Glioblastoma (GBM) is the most common and deadly form of primary brain tumor with a median survival of eleven months, despite use of extensive chemotherapy, radiotherapy and surgery. We have previously shown that nuclear factor-kappa B (NF-κB) is aberrantly expressed in GBM tumors and primary cell lines derived from tumor tissue. Here we show that IL-8, a chemokine is also aberrantly expressed by GBM cell lines and expression of IL-8 is in large part, attributable to the aberrant activation of NF-κB. We hypothesized that invasiveness of GBM cells is driven at least in part by aberrantly expressed IL-8. In support of the hypothesis we found that treatment of glioma cells with an IL-8 neutralizing antibody markedly decreased their invasiveness compared to cells treated with control IgG or left untreated. Furthermore, downregulation of IL-8 protein production with use of IL-8 targeted siRNA also resulted in decreased invasion in matrigel. We next investigated the presence of IL-8 receptors by FACS analysis and found that GBM cells (U87, U251, D54 and LN229) only express CXCR1 but not CXCR2. Treatment of U87 cells with a blocking CXCR1 antibody reduced their invasion through matrigel. Finally, we found that addition of exogenous IL-8, following downregulation of NF-κB which results in loss of endogenous IL-8 production, incompletely restored tumor cell invasion. Our data indicate that IL-8 is necessary but not solely responsible for glioma cell invasion and mediates its effect in an autocrine manner.

p300- and Myc-mediated regulation of glioblastoma multiforme cell differentiation.

Tumorigenic potential of glioblastoma multiforme (GBM) cells is, in part, attributable to their undifferentiated (neural stem cell-like) phenotype. Astrocytic differentiation of GBM cells is associated with transcriptional induction of Glial Fibrillary Acidic Protein (GFAP) and repression of Nestin, whereas the reciprocal transcription program operates in undifferentiated GBM cells. The molecular mechanisms underlying the regulation of these transcription programs remain elusive. Here, we show that the transcriptional co-activator p300 was expressed in GBM tumors and cell lines and acted as an activator of the GFAP gene and a repressor of the Nestin gene. On the other hand, Myc (formerly known as c-Myc overrode these p300 functions by repressing the GFAP gene and inducing the Nestin gene in GBM cells. Moreover, RNAi-mediated inhibition of p300 expression significantly enhanced the invasion potential of GBM cells in vitro. Taken together, these data suggest that dedifferentiated/undifferentiated GBM cells are more invasive than differentiated GBM cells. Because invasion is a major cause of GBM morbidity, differentiation therapy may improve the clinical outcome.

Stat3 activation is required for the growth of U87 cell-derived tumours in mice.

Previously we reported that Stat3 is persistently activated in GBM tumours and derived cell lines. Hypoxia, necrosis and neo-angiogenesis are hallmarks of GBM. To unfold the contribution of activated Stat3 to the growth of GBM, we generated human GBM cell line (U87)-derived stable clones expressing a dominant negative mutant (DN)-Stat3 in a hypoxia-inducible manner, and examined their tumour-forming potentials in immune-compromised mice. We found that the parental and vector control cell-derived tumours grew steadily, whereas DN-Stat3-expressing clone-derived tumours failed to grow beyond 2mm of thickness in mouse flanks. This blockade of tumour growth was associated with induction of tumour cell apoptosis and suppression of tumour angiogenesis. Consistent with this, mice bearing orthotopically implanted DN-Stat3-expressing clones survived significantly longer than the control mice. These data suggest that activated Stat3 is required for the growth of GBM, and that targeting Stat3 may intervene with the growth of GBM.

Aberrant constitutive activation of nuclear factor kappaB in glioblastoma multiforme drives invasive phenotype.

Several recent studies have shown that aberrant constitutive activation of nuclear factor kappaB (NF-kappaB) is present in a variety of cancers including gliomas. NF-kappaB is known to play important roles in the physiological regulation of diverse cellular processes such as inflammation, growth and immunity. In contrast, aberrant activation of this latent transcription factor promotes cancer cell migration, invasion and resistance to chemotherapy. Here we show by electro-mobility shift assay (EMSA) and immuno-staining that constitutive NF-kappaB activation is present in various malignant glioma cell lines as well as in primary cultures derived from tumor tissue. This activation was not serum dependent and it led to high IL-8 gene transcription and protein production. Over-expression of an I-kappaB super-repressor (I-kappaB SR) transgene completely blocked constitutive NF-kappaB activation, nuclear localization and transcription of some but not all NF-kappaB regulated genes indicating that NF-kappaB signaling in glioma cells is I-kappaB dependent. Surprisingly, over-expression of IkappaBSR did not have any effect on the transcription levels of anti-apoptotic genes in these glioma cultures and cell lines. Down-regulation of NF-kappaB activation reduced invasion of glioma cells through matrigel. Collectively these data suggest that aberrant constitutive activation of NF-kappaB in glioblastoma cells promotes their invasive phenotype. Interruption of this aberrant NF-kappaB activity may help reduce the spread of this infiltrative tumor.

Depressed peroxisome proliferator-activated receptor gamma (PPargamma) is indicative of severe pulmonary sarcoidosis: possible involvement of interferon gamma (IFN-gamma).

BACKGROUND AND AIM: Recent evidence suggests that the transcription factor, PPARgamma, is an important negative regulator of inflammation. Because studies of murine adipocytes and macrophages implicate IFN-gamma, a key mediator of granuloma formation in sarcoidosis, as a PPARgamma antagonist, we investigated the relationship between PPARgamma and IFN-gamma in bronchoalveolar lavage (BAL) cells of sarcoidosis patients and healthy controls. METHODS: BAL cells were analyzed for PPARgamma and IFN-gamma mRNA expression by quantitative PCR and for PPARgamma protein by immunocytochemistry and western blotting. RESULTS: In sarcoidosis patients with severe, treatment-requiring disease, IFN-gamma was strikingly elevated and PPARgamma gene expression was deficient. In contrast, PPARgamma expression of non-severe patients was comparable to control but was still accompanied by increased IFN-gamma. By confocal microscopy, nuclear PPARgamma protein was detectable in alveolar macrophages from non-severe patients unlike previous observations of severe patients. In vitro exposure of BAL cells or purified alveolar macrophages to IFN-gamma resulted in dose-dependent repression of PPARgamma mRNA in both sarcoidosis and controls. IFN-gamma treatment also reduced PPARgamma protein in BAL lysates and nuclear PPARgamma content in control alveolar macrophages, resulting in a diffuse cytoplasmic PPARgamma distribution similar to that observed in severe sarcoidosis. CONCLUSION: These novel results indicate that IFN-gamma represses PPARgamma in human alveolar macrophages but that in sarcoidosis, PPARgamma rather than IFN-gamma levels correlate best with disease severity. Data also emphasize the complex nature of PPARgamma restorative mechanisms in alveolar macrophages exposed to an inflammatory environment containing IFN-gamma -- a potential PPARgamma antagonist.

An inverse relationship between peroxisome proliferator-activated receptor gamma and allergic airway inflammation in an allergen challenge model.

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPAR-gamma) expression has not been evaluated in bronchoalveolar lavage (BAL) cells from allergic asthmatic patients. OBJECTIVE: To determine whether inappropriate down-regulation of PPAR-gamma in alveolar macrophages may contribute to persistent airway inflammation in allergic asthma. METHODS: We used segmental allergen challenge as a model of in vivo experimental allergic asthmatic exacerbation and airway inflammation. PPAR-y gene expression was evaluated at baseline and 24 hours later in asthmatic patients and controls using real-time polymerase chain reaction. Immunofluorescence was used to determine cellular location of the PPAR-gamma protein. RESULTS: We demonstrate for the first time to our knowledge that PPAR-gamma messenger RNA and protein, which are highly expressed in alveolar macrophages of healthy individuals, are significantly reduced in asthmatic patients after segmental allergen challenge. In allergic asthmatic patients (n=9), PPAR-gamma gene expression decreased significantly from baseline to postchallenge BAL (median decrease, 45%; P = .008). Furthermore, immunofluorescence staining demonstrated that PPAR-gamma protein was associated with alveolar macrophages and not with inflammatory eosinophils and neutrophils. CONCLUSION: Results implicate down-regulation of PPAR-gamma in BAL cells as a potential factor in dysregulation of lung homeostasis in asthmatic patients. The present findings suggest that PPAR-gamma agonists could have a future role in asthma therapy and warrant further study.

Peroxisome proliferator-activated receptor gamma activity is deficient in alveolar macrophages in pulmonary sarcoidosis.

The ligand-activated transcription factor, peroxisome proliferator-activated receptor gamma (PPAR gamma), has pleiotropic effects on lipid and glucose metabolism as well as modulating immune activity. In Th1-predominant models of inflammatory bowel disease and arthritis, PPAR gamma ligands can ameliorate clinical disease severity, partly by downregulating a range of inflammatory cytokines. However, PPAR gamma has not been evaluated in chronic sarcoidosis, a disease characterized by persistent activation of Th1 immune responses in alveolar macrophages. We hypothesized that a deficiency of PPAR gamma activity contributes to ongoing inflammation in pulmonary sarcoidosis via failure to repress proinflammatory transcription factors. To address this, we studied eight patients with active sarcoidosis and nine healthy control subjects by bronchoscopy. Bronchoalveolar lavage specimens from patients revealed a striking reduction of PPAR gamma activity by electrophoretic mobility shift assay in alveolar macrophages compared with healthy control subjects, with a concomitant upregulation of nuclear factor (NF)-kappa B activity. Immunostaining and real-time polymerase chain reaction demonstrated reductions of PPAR gamma nuclear protein and gene expression. The data show for the first time that alveolar macrophages from patients with active sarcoidosis exhibit activation of NF-kappa B and deficiency of PPAR gamma. Although these results do not demonstrate a direct causal effect, they are consistent with the hypothesis that insufficient PPAR gamma activity contributes to ongoing dysregulated inflammation in pulmonary sarcoidosis by failing to suppress NF-kappa B.

Surfactant blocks lipopolysaccharide signaling by inhibiting both mitogen-activated protein and IkappaB kinases in human alveolar macrophages.

Surfactant plays an important role in lung homeostasis and is also involved in maintaining innate immunity within the lung. Lipopolysaccharide (LPS) from gram-negative bacteria is known to elicit acute proinflammatory responses in lung diseases such as acute respiratory distress syndrome and pneumonia, among others. Our previous studies demonstrated that the clinically used, natural surfactant product Survanta inhibited proinflammatory cytokine secretion from LPS-stimulated human alveolar macrophages. Here we investigated the effect of Survanta on mitogen-activated protein (MAP) and IkappaB kinases. Survanta blocked LPS-induced activation of nuclear factor-kappaB, a key regulatory transcription factor involved in cytokine production, by preventing phosphorylation of IkappaBalpha, and its subsequent degradation. IkappaB is phosphorylated by specific kinases (IKK) before degradation. Survanta inhibited activity of both alpha and beta subunits of IKK, thereby delaying the phosphorylation of IkappaB. Interestingly, IKK-alpha is predominant in alveolar macrophages, whereas IKK-beta predominates in monocytes. Survanta also inhibited extracellular signal-regulated kinase and p38 MAP kinase activity induced by LPS. Data are the first to show that surfactant may regulate lung homeostasis in part by inhibiting proinflammatory cytokine production through reduction of IKK and MAP kinase activity.

Elevated IL-10 inhibits GM-CSF synthesis in pulmonary alveolar proteinosis.

Pulmonary alceolar proteinosis (PAP) is an autoimmune lung disease characterized by accumulation of surfactant material within the lung. Autoantibodies to GM-CSF as well as high levels of IL-10 are also found in the lungs in PAP. Previous studies suggest that treatment with recombinant GM-CSF is beneficial for patients with low levels of GM-CSF antibodies. The role of IL-10 in PAP, however, is unknown and the hypothesis that IL-10 may affect PAP GM-CSF synthesis has not been addressed. The current findings show that GM-CSF secretion is significantly compromised in PAP bronchoalveolar lavage (BAL) cells compared to controls, but surprisingly, GM-CSF mRNA levels are elevated. In contrast, IL-10 protein and mRNA levels are both highly elevated in PAP. In vitro analysis of GM-CSF regulation indicates that both secretion and mRNA levels are sharply reduced by IL-10 and increased by anti-IL-10 antibody. The phenomenon of elevated GM-CSF mRNA in BAL cells appears not to be due to lack of negative feedback by GM-CSF protein. Results suggest that in PAP, GM-CSF synthesis is deficient and associated with negative regulation by IL-10. Furthermore, IL-10 gene expression becomes even more elevated in patients who do not respond to recombinant GM-CSF therapy and have high anti-GM-CSF titers. Based on these observations, we hypothesize that IL-10 may be an indicator of PAP clinical response to GM-CSF therapy.

PU.1 regulation of human alveolar macrophage differentiation requires granulocyte-macrophage colony-stimulating factor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is critically implicated in lung homeostasis in the GM-CSF knockout mouse model. These animals develop an isolated lung lesion reminiscent of pulmonary alveolar proteinosis (PAP) seen in humans. The development of the adult form of human alveolar proteinosis is not due to the absence of a GM-CSF gene or receptor defect but to the development of an anti-GM-CSF autoimmunity. The role of GM-CSF in the development of PAP is unknown. Studies in the GM-CSF knockout mouse have shown that lack of PU.1 protein expression in alveolar macrophages is correlated with decreased maturation, differentiation, and surfactant catabolism. This study investigates PU.1 expression in vitro and in vivo in human PAP alveolar macrophages as well as the regulation of PU.1 by GM-CSF. We show for the first time that PU.1 mRNA expression in PAP bronchoalveolar lavage cells is deficient compared with healthy controls. PU.1-dependent terminal differentiation markers CD32 (FCgammaII), mannose receptor, and macrophage colony-stimulating factor receptor (M-CSFR) are decreased in PAP alveolar macrophages. In vitro studies demonstrate that exogenous GMCSF treatment upregulated PU.1 and M-CSFR gene expression in PAP alveolar macrophages. Finally, in vivo studies showed that PAP patients treated with GM-CSF therapy have higher levels of PU.1 and M-CSFR expression in alveolar macrophages compared with healthy control and PAP patients before GM-CSF therapy. These observations suggest that PU.1 is critical in the terminal differentiation of human alveolar macrophages.

Peroxisome proliferator-activated receptor-gamma is deficient in alveolar macrophages from patients with alveolar proteinosis.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a ligand-activated, nuclear transcription factor that regulates genes involved in lipid and glucose metabolism, inflammation, and other pathways. The hematopoietic growth factor, granulocyte macrophage colony-stimulating factor (GM-CSF), is essential for lung homeostasis and is thought to regulate surfactant clearance, but mechanisms involved are unknown. GM-CSF is reported to stimulate PPAR-gamma, but the activation status of PPAR-gamma in human alveolar macrophages has not been defined. In pulmonary alveolar proteinosis (PAP), a rare interstitial lung disease, surfactant accumulates in alveolar airspaces, resident macrophages become engorged with lipoproteinaceous material, and GM-CSF deficiency is strongly implicated in pathogenesis. Here we show that PPAR-gamma mRNA and protein are highly expressed in alveolar macrophages of healthy control subjects but severely deficient in PAP in a cell-specific manner. Further, we show that the PPAR-gamma-regulated lipid scavenger receptor, CD36, is also deficient in PAP. PPAR-gamma and CD36 deficiency are not intrinsic to PAP alveolar macrophages, but can be upregulated by GM-CSF therapy. Moreover, GM-CSF treatment of patients with PAP fully restores PPAR-gamma to healthy control levels. Based upon these novel findings, we hypothesize that GM-CSF regulates lung homeostasis via PPAR-gamma-dependent pathways.

Circulating monocytes from patients with primary pulmonary hypertension are hyporesponsive.

Primary pulmonary hypertension (PPH) is a rare disease of unknown etiology characterized by arterial thickening and remodeling. The transcription factor NF-kappaB is responsible for the activation of several cytokines and growth factor genes reported to be associated with PPH. Our previous study showed NF-kappaB activation in alveolar macrophages from PPH patients, suggesting the presence of a localized pulmonary inflammatory response. In PPH, circulating monocyte activity has not been previously examined. The present study was undertaken to determine whether circulating monocytes also showed evidence of activation, which could suggest a systemic response to PPH injury. Results indicated that NF-kappaB activation in monocytes from PPH patients did not differ from that of healthy controls. However, mRNA expression was decreased compared to controls for NF-kappaB-regulated genes, granulocyte macrophage colony-stimulating factor, interleukin-6, macrophage inflammatory protein-1alpha (MIP-1alpha), and vascular endothelial growth factor. MIP-1alpha protein secretion from PPH monocytes was also lower than that of controls cultured with and without endotoxin. Expression of the surface activation markers HLA-DR and CD-14 were significantly reduced on monocytes from PPH patients compared to healthy controls. Toll-like receptor-4 (TLR-4) expression was significantly increased on monocytes from PPH patients while TLR-2 remained unchanged. Thus, our data are the first to show that monocytes in PPH have decreased activation and are hyporesponsive to lipopolysaccharide (LPS) stimulation. The monocyte LPS hyporesponsiveness may in part be the result of decreased CD-14 expression, since LPS responsiveness is dependent on the physical association of LPS/CD-14 complexes with TLR-4, and without this association signal transduction does not occur. These data indicate that although PPH is a localized pulmonary disorder, there are alterations in the systemic compartment. What remains unknown is how the reduced activation of monocytes in PPH is related to the pulmonary vascular lesion.

Nitric oxide blocks inflammatory cytokine secretion triggered by CD23 in monocytes from allergic, asthmatic patients and healthy controls.

BACKGROUND: In allergic asthma, monocytes/macrophages may be activated to produce inflammatory cytokines through triggering of the low-affinity IgE receptor (CD23). Elevated airway levels of nitric oxide (NO) are associated with asthmatic exacerbations. Our previous work suggested that NO may function in an anti-inflammatory capacity by downregulating endotoxin-stimulated cytokine production by alveolar macrophages and matured monocytes. OBJECTIVE: The purpose of this study was to determine the effect of NO on CD23-triggered cytokine production by monocytes from asthmatic patients and healthy controls. METHODS: Monocytes were obtained from normal volunteers (n = 13) and asthmatic patients with atopy (n = 8). Monocyte cultures were treated with interleukin-4 (IL-4) and granulocyte macrophage colony-stimulating factor (GM-CSF) for 24 hours to upregulate CD23 expression. Cultures were stimulated by anti-CD23 and treated with DETA NONOate [2,2-(hydroxynitrosohydrazonon)-bis-ethanamine] releases NO in culture with t(1/2) of 20 hours at 37 degrees C for 24 hours. Cell free culture supernatants were collected and assayed by enzyme-linked immunoadsorbent assay for macrophage inflammatory protein-1-alpha (MIP-1) and IL-6. RESULTS: NO inhibits MIP-1 secretion triggered by CD23 activation of IL-4- and GM-CSF-matured monocytes (percentage of MIP-1 suppression = 52 +/- 11 of monocytes from asthmatic patients; percentage = 55 +/- 8 healthy controls). The inhibitory effect of NO was not cytokine-specific, as similar results were obtained with IL-6 (50 +/- 9% IL-6 suppression, asthmatic patients; 66 +/- 20%, healthy controls). CONCLUSIONS: The results demonstrate for the first time an inhibitory effect of NO on cytokine production stimulated by CD23 receptor activation. We suggest that NO may be upregulated as a potent anti-inflammatory agent in the asthmatic lung.

The prostacyclin analogue treprostinil blocks NFkappaB nuclear translocation in human alveolar macrophages.

Primary pulmonary hypertension (PPH) is characterized by increased pulmonary arterial pressure and vascular resistance. We and others have observed that inflammatory cytokines and infiltrates are present in the lung tissue, but the significance is uncertain. Treprostinil (TRE), a prostacyclin analogue with extended half-life and chemical stability, has shown promise in the treatment of PPH. We hypothesize that TRE might exert beneficial effects in PPH by antagonizing inflammatory cytokine production in the lung. Here we show that TRE dose-dependently inhibits inflammatory cytokine (tumor necrosis factor-alpha, interleukin-1beta, interleukin-6, and granulocyte macrophage colony-stimulating factor) secretion and gene expression by human alveolar macrophages. TRE blocks NFkappaB activation, but IkappaB-alpha phosphorylation and degradation are unaffected. Moreover, TRE does not affect the formation of the NFkappaB.DNA complex but blocks nuclear translocation of p65. These results are the first to illustrate the anti-cytokine actions of TRE in down-regulating NFkappaB, not through its inhibitory component or by direct binding but by blocking nuclear translocation. These data indicate that inflammatory mechanisms may be important in the pathogenesis of PPH and cytokine antagonism by blocking NFkappaB may contribute to the efficacy of TRE therapy in PPH.