Increase In Granulocyte-macrophage Colony-stimulating Factor Research Articles

Efficacy of TurmiZn, a Metallic Complex of Curcuminoids-Tetrahydrocurcumin and Zinc on Bioavailability, Antioxidant, and Cytokine Modulation Capability

Complexes of curcumin with metals have shown much-improved stability, solubility, antioxidant capability, and efficacy when compared to curcumin. The present research investigates the relative bioavailability, antioxidant, and ability to inhibit inflammatory cytokine production of a curcuminoid metal chelation complex of tetrahydrocurcumin-zinc-curcuminoid termed TurmiZn. In vitro uptake assay using pig intestinal epithelial cells showed that TurmiZn has an ~3-fold increase (p ≤ 0.01) in uptake compared to curcumin and a ~2-fold increase (p ≤ 0.01) over tetrahydrocurcumin (THC). In a chicken model, an oral 1-g dose of TurmiZn showed a ~2.5-fold increase of a specific metabolite peak compared to curcumin (p = 0.004) and a ~3-fold increase compared to THC (p = 0.001). Oral doses (5 g/Kg) of TurmiZn in rats also showed the presence of curcumin and THC metabolites in plasma, indicating bioavailability across cell membranes in animals. Determination of the antioxidant activity by a 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scavenging assay indicated that TurmiZn was about 13x better (p ≤ 0.0001) than curcumin and about 4X better (p ≤ 0.0001) than THC, in reducing free radicals. In vitro experiments further showed significant (p ≤ 0.01) reductions of lipopolysaccharide (LPS)-induced proinflammatory cytokines such as interleukin (IL) IL-6, IL-8, IL-15, IL-18, and tumor necrosis factor (TNF)-alpha, while showing a significant (p ≤ 0.01) increase of granulocyte-macrophage colony-stimulating factor (GM-CSF) in dog kidney cells. In vivo cytokine modulations were also observed when TurmiZn was fed for 6 weeks to newborn chickens. TurmiZn reduced IL-1 and IL-6, but significantly reduced (p ≤ 0.01) IL-10 levels while there was a concurrent significant (p = 0.02) increase in interferon gamma compared to controls. Overall, these results indicate that TurmiZn has better bioavailability and antioxidant capability than curcumin or THC and has the ability to significantly modulate cytokine levels. Thus, TurmiZn could be an excellent candidate for a novel ingredient that can be incorporated into food and supplements to help overall health during the aging process.

In an in-vitro model using human fetal membranes, α-lipoic acid inhibits inflammation induced fetal membrane weakening

IntroductionWe established an in-vitro model for the study of human fetal membrane (FM) weakening leading to pPROM. In this model, granulocyte-macrophage colony-stimulating factor (GM-CSF) is a critical intermediate for both tumor necrosis factor-α (TNF; modeling infection/inflammation) and thrombin (modeling decidual bleeding/abruption)-induced weakening. Thus, inhibitors of FM weakening can be categorized as targeting GM-CSF production, GM-CSF downstream action, or both. Most progestogens inhibit both, except 17-α hydroxyprogesterone caproate which inhibits FM weakening at only one point, GM-CSF production. α-lipoic acid (LA), an over-the-counter dietary supplement, has also been previously shown to inhibit TNF and thrombin induced FM weakening. ObjectiveTo determine the point of action of LA inhibition of FM weakening. MethodsFM fragments were mounted in Transwell inserts and preincubated with/without LA/24 h, then with/without addition of TNF, thrombin or GM-CSF. After 48 h, medium was assayed for GM-CSF, and FM fragments were rupture-strength tested. ResultsTNF and thrombin both weakened FM and increased GM-CSF levels. GM-CSF also weakened FM. LA inhibited both TNF and thrombin induced FM weakening and concomitantly inhibited the increase in GM-CSF in a concentration-dependent manner. In addition, LA inhibited GM-CSF induced FM weakening in a concentration dependent manner. ConclusionsLA blocks TNF and thrombin induced FM weakening at two points, inhibiting both GM-CSF production and downstream action. Thus, we speculate that LA may be a potential standalone therapeutic agent, or supplement to current therapy for prevention of pPROM related spontaneous preterm birth, if preclinical studies to examine feasibility and safety during pregnancy are successfully accomplished.

In an in-vitro model using human fetal membranes, 17-α hydroxyprogesterone caproate is not an optimal progestogen for inhibition of fetal membrane weakening

The progestogen 17-α hydroxyprogesterone caproate (17-OHPC) is 1 of only 2 agents recommended for clinical use in the prevention of spontaneous preterm delivery, and studies of its efficacy have been conflicting. We have developed an in-vitro model to study the fetal membrane weakening process that leads to rupture in preterm premature rupture of the fetal membranes (pPROM). Inflammation/infection associated with tumor necrosis factor-α (TNF-α) induction and decidual bleeding/abruption associated thrombin release are leading causes of preterm premature rupture of the fetal membranes. Both agents (TNF-α and thrombin) cause fetal membrane weakening in the model system. Furthermore, granulocyte-macrophage colony-stimulating factor (GM-CSF) is a critical intermediate for both TNF-α and thrombin-induced fetal membrane weakening. In a previous report, we demonstrated that 3 progestogens, progesterone, 17-alpha hydroxyprogesterone (17-OHP), and medroxyprogesterone acetate (MPA), each inhibit both TNF-α- and thrombin-induced fetal membrane weakening at 2 distinct points of the fetal membrane weakening pathway. Each block both the production of and the downstream action of the critical intermediate granulocyte-macrophage colony-stimulating factor. The objective of the study was to characterize the inhibitory effects of 17-OHPC on TNF-α- and thrombin-induced fetal membrane weakening invitro. Full-thickness human fetal membrane fragments from uncomplicated term repeat cesarean deliveries were mounted in 2.5cm Transwell inserts and cultured with/without 17-alpha hydroxyprogesterone caproate (10-9 to 10-7 M). After 24 hours, medium (supernatant) was removed and replaced with/without the addition of tumor necrosis factor-alpha (20 ng/mL) or thrombin (10 U/mL) or granulocyte-macrophage colony-stimulating factor (200 ng/mL). After 48 hours of culture, medium from the maternal side compartment of the model was assayed for granulocyte-macrophage colony-stimulating factor and the fetal membrane fragments were rupture strength tested. Tumor necrosis factor-alpha and thrombin both weakened fetal membranes (43% and 62%, respectively) and increased granulocyte-macrophage colony-stimulating factor levels (3.7- and 5.9-fold, respectively). Pretreatment with 17-alpha hydroxyprogesterone caproate inhibited both tumor necrosis factor-alpha- and thrombin-induced fetal membrane weakening and concomitantly inhibited the induced increase in granulocyte-macrophage colony-stimulating factor in a concentration-dependent manner. However, contrary to our prior reports regarding progesterone and other progestogens, 17-alpha hydroxyprogesterone caproate did not also inhibit granulocyte-macrophage colony-stimulating factor-induced fetal membrane weakening. 17-Alpha hydroxyprogesterone caproate blocks tumor necrosis factor-alpha- and thrombin-induced fetal membrane weakening by inhibiting the production of granulocyte-macrophage colony-stimulating factor. However, 17-alpha hydroxyprogesterone caproate did not also inhibit granulocyte-macrophage colony-stimulating factor-induced weakening. We speculate that progestogens other than 17-alpha hydroxyprogesterone caproate may be more efficacious in preventing preterm premature rupture of the fetal membranes-related spontaneous preterm birth.

Ocular surface cytokine profile in chronic Stevens-Johnson syndrome and its response to mucous membrane grafting for lid margin keratinisation

BackgroundTo study the tear cytokine and the conjunctival and oral mucosal marker profile in chronic ocular Stevens-Johnson syndrome (SJS) and their alteration following mucous membrane grafting (MMG) for lid margin...

Progesterone inhibits in vitro fetal membrane weakening

Inflammation/infection and abruption are leading causes of preterm premature rupture of the membranes. Recently, we identified granulocyte-macrophage colony-stimulating factor (GM-CSF) as a critical mediator of both tumor necrosis factor-α- (TNF; modeling inflammation) and thrombin-induced (modeling abruption) weakening of the fetal membranes. We found that (1) TNF and thrombin both induced GM-CSF in the choriodecidua, (2) blockade of GM-CSF action with neutralizing antibodies inhibited both TNF- and thrombin-induced fetal membrane weakening, and (3) GM-CSF alone induced fetal membrane weakening. GM-CSF is thus part of an overlap of the inflammation and abruption-induced fetal membrane weakening pathways. The effects of progesterone analogs on the pathways by which fetal membranes are weakened have not been investigated. We examined the effects of progesterone, medroxyprogesterone acetate (MPA) and 17α-hydroxyprogesterone (HP) on TNF- and thrombin-induced fetal membrane weakening. Full-thickness fetal membranes from uncomplicated term repeat cesarean deliveries were mounted in Transwell inserts in Minimum Essential Medium alpha and incubated at 37°C in 5% CO2. The choriodecidua side of the fetal membrane fragments were preincubated with progesterone, MPA, HP, or vehicle for 24 hours. Fetal membranes were then exposed to TNF, thrombin, or GM-CSF on the choriodecidua side for an additional 48 hours. The fetal membrane tissues were then strength tested, and medium from the choriodecidua and amnion compartments was assayed for GM-CSF content. TNF and thrombin both weakened fetal membranes and elevated media GM-CSF levels on the choriodecidua side of the fetal membrane. Pretreatment with progesterone, MPA, or HP inhibited both TNF- and thrombin-induced fetal membrane weakening and also inhibited the induced increase in GM-CSF. GM-CSF decreased fetal membrane rupture strength by 68%, which was inhibited by progestogen pretreatment with a potency order: progesterone <MPA <HP. Progestogen pretreatment blocks TNF- and thrombin-induced fetal membrane weakening by inhibiting both the production and action of GM-CSF. These findings are consistent with the administration of progestogens in the prevention of preterm premature rupture of the membranes.

Reduced BMPR2 expression induces GM-CSF translation and macrophage recruitment in humans and mice to exacerbate pulmonary hypertension

Idiopathic pulmonary arterial hypertension (PAH [IPAH]) is an insidious and potentially fatal disease linked to a mutation or reduced expression of bone morphogenetic protein receptor 2 (BMPR2). Because intravascular inflammatory cells are recruited in IPAH pathogenesis, we hypothesized that reduced BMPR2 enhances production of the potent chemokine granulocyte macrophage colony-stimulating factor (GM-CSF) in response to an inflammatory perturbation. When human pulmonary artery (PA) endothelial cells deficient in BMPR2 were stimulated with tumor necrosis factor (TNF), a twofold increase in GM-CSF was observed and related to enhanced messenger RNA (mRNA) translation. The mechanism was associated with disruption of stress granule formation. Specifically, loss of BMPR2 induced prolonged phospho-p38 mitogen-activated protein kinase (MAPK) in response to TNF, and this increased GADD34-PP1 phosphatase activity, dephosphorylating eukaryotic translation initiation factor (eIF2α), and derepressing GM-CSF mRNA translation. Lungs from IPAH patients versus unused donor controls revealed heightened PA expression of GM-CSF co-distributing with increased TNF and expanded populations of hematopoietic and endothelial GM-CSF receptor α (GM-CSFRα)-positive cells. Moreover, a 3-wk infusion of GM-CSF in mice increased hypoxia-induced PAH, in association with increased perivascular macrophages and muscularized distal arteries, whereas blockade of GM-CSF repressed these features. Thus, reduced BMPR2 can subvert a stress granule response, heighten GM-CSF mRNA translation, increase inflammatory cell recruitment, and exacerbate PAH.

Toll‐like receptor‐mediated eosinophil–basophil differentiation: autocrine signalling by granulocyte–macrophage colony‐stimulating factor in cord blood haematopoietic progenitors

Eosinophils are multi-functional leucocytes that play a role in inflammatory processes including allergy and infection. Although bone marrow (BM) inflammatory cells are the main source of eosinophil-basophil (Eo/B) differentiation-inducing cytokines, a recent role has been demonstrated for cytokine induction through Toll-like receptor (TLR)-mediated signalling in BM progenitors. Having previously demonstrated that cord blood (CB) progenitors induce Eo/B colony-forming units (CFU) after lipopolysaccharide (LPS) stimulation, we sought to investigate the intracellular mechanisms by which LPS induces Eo/B differentiation. Freshly isolated CD34-enriched human CB cells were stimulated with LPS (and/or pharmacological inhibitors) and assessed for alterations in haematopoietic cytokine receptor expression and signalling pathways by flow cytometry, Eo/B CFU in methylcellulose cultures, and cytokine secretion using Luminex assays. The LPS stimulation resulted in a significant increase in granulocyte-macrophage colony-stimulating factor (GM-CSF)-responsive, as opposed to interleukin-5-responsive, Eo/B CFU, which also correlated with significant increases in CD34(+) cell GM-CSFRα expression. Functionally, CB CD34(+) cells secrete abundant amounts of GM-CSF following LPS stimulation, via a p38 mitogen-activated protein kinase (MAPK)-dependent mechanism; this secretion was responsible for Eo/B CFU formation ex vivo, as shown by antibody blockade. We show for the first time that LPS stimulation of CB progenitor cells results in autocrine activation of p38 MAPK-dependent GM-CSF secretion facilitating Eo/B differentiation ex vivo. This work provides evidence that early life exposure to products of bacterial agents can modulate Eo/B differentiation, representing a novel mechanism by which progenitor cells can respond to microbial stimuli and so affect immune and inflammatory responses.

N-acetyl-B-D-glucosaminidase and inflammatory response after cardiopulmonary bypass.

To determine the changes in activity of plasma N-acetyl-beta-D-glucosaminidase, a marker for inflammation as well as renal, pulmonary and cardiac damage and proinflammatory cytokines in patients undergoing coronary artery bypass grafting and find out the relationship between their plasma levels with clinical outcome of patients. Cross-sectional study. The Aga Khan University, Karachi, from January to June 2003. N-acetyl-beta-D-glucosaminidase (NAG) activity and concentrations of tumor necrosis factor-alpha of (TNFalpha), interleukin 6 (IL-6), interleukin 8 (IL8) and granulocyte-macrophage colony stimulating factor (GM-CSF) were monitored in plasma samples of 12 angina patients undergoing coronary artery bypass grafting (CABG), before, immediately after and 5 days post-surgical procedure. Serum glucose concentrations were also monitored in those patients. Patient's clinical condition was monitored during this time period. No significant increase was observed in plasma NAG activity (a marker of inflammation) or in plasma levels of TNFalpha, IL-6, IL-8 and GM-CSF immediately after surgery, indicating that cardiopulmonary bypass itself does not produce any significant amount of inflammation immediately after CABG. However, 5 days post surgery, there was a significant increase in plasma NAG activity (p=0.001), TNFalpha (p=0.047) and GM-CSF (p=0.045). There was no relationship between plasma NAG activity and clinical outcome because various parameters of renal, cardiac and pulmonary functions, though slightly affected, remained within the normal limits. Increased levels of NAG and TNFalpha did not affect clinical outcome. However, data suggest that NAG can be a potential marker for inflammation and end organ damage following CABG. An increase in GM-CSF on day 5 following CABG indicates enhanced body's defense mechanism against infection.

German Cockroach Proteases and Protease-Activated Receptor-2 Regulate Chemokine Production and Dendritic Cell Recruitment

We recently showed that serine proteases in German cockroach (GC) feces (frass) decreased experimental asthma through the activation of protease-activated receptor (PAR)-2. Since dendritic cells (DCs) play an important role in the initiation of asthma, we queried the role of GC frass proteases in modulating CCL20 (chemokine C-C motif ligand 20) and granulocyte macrophage colony-stimulating factor (GM-CSF) production, factors that regulate pulmonary DCs. A single exposure to GC frass resulted in a rapid, but transient, increase in GM-CSF and a steady increase in CCL20 in the airways of mice. Instillation of protease-depleted GC frass or instillation of GC frass in PAR-2-deficient mice significantly decreased chemokine release. A specific PAR-2-activating peptide was also sufficient to induce CCL20 production. To directly assess the role of the GC frass protease in chemokine release, we enriched the protease from GC frass and confirmed that the protease was sufficient to induce both GM-CSF and CCL20 production in vivo. Primary airway epithelial cells produced both GM-CSF and CCL20 in a protease- and PAR-2-dependent manner. Finally, we show a decreased percentage of myeloid DCs in the lung following allergen exposure in PAR-2-deficient mice compared to wild-type mice. However, there was no difference in GC frass uptake. Our data indicate that, through the activation of PAR-2, allergen-derived proteases are sufficient to induce CCL20 and GM-CSF production in the airways. This leads to increased recruitment and/or differentiation of myeloid DC populations in the lungs and likely plays an important role in the initiation of allergic airway responses.

Granulocyte-Macrophage Colony-Stimulating Factor–Induced Vessel Growth Restores Cerebral Blood Supply After Bilateral Carotid Artery Occlusion

Hemodynamic compromise due to occlusive cerebrovascular disease is associated with an increased stroke risk. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been suggested to stimulate collateral blood vessel growth in various models of hemodynamic compromise. The purpose of this study was to investigate the effects of GM-CSF on cerebral hemodynamics and vessel growth in a rat model of chronically impaired cerebral blood flow (CBF). Male Sprague-Dawley rats underwent sequential bilateral carotid artery occlusion (BCO) and were treated with GM-CSF or saline for 6 weeks. Sham-occluded animals served as a control group. Baseline CBF was measured by iodo[(14)C]antipyrine autoradiography, and cerebrovascular reserve capacity was assessed by laser-Doppler flowmetry after application of 20 mg/kg body weight acetazolamide. The capillary density and arterioles immunopositive for alpha-smooth muscle actin were counted on brain sections. The cerebral angioarchitecture was visualized with a latex perfusion technique. Baseline CBF as measured by iodo[(14)C]antipyrine autoradiography was not affected by BCO. The cerebrovascular reserve capacity, however, was significantly impaired 1 week after BCO. CBF and cerebrovascular reserve capacity recovered completely in GM-CSF-treated animals but not in solvent-treated animals. Histologic analysis of the hippocampus revealed integrity of the hypoxia-vulnerable neurons in all animals. The capillary density showed a very mild increase in GM-CSF-treated animals. However, the number of intraparenchymal and leptomeningeal arterioles was significantly higher in GM-CSF-treated animals than in both other groups. Long-term GM-CSF treatment in a BCO model in rats leads to restoration of impaired cerebral hemodynamics and accompanies structural changes in the resistance-vessel network.

Ethanol Regulates Angiogenic Cytokines During Neural Development: Evidence From an in Vitro Model of Mitogen‐Withdrawal–Induced Cerebral Cortical Neuroepithelial Differentiation

Heavy alcohol consumption during pregnancy can cause significant mental retardation and brain damage. We recently showed that ethanol depletes reserve cerebral cortical stem cell capacity. Moreover, proliferating neuroepithelial cells exposed to ethanol were resistant to subsequent retinoic acid-induced differentiation. Emerging evidence suggests that cytokines play a crucial growth-promoting role in the developing neural tube. We cultured murine cortical neurosphere cultures in control or ethanol-supplemented mitogenic medium, to mimic alcohol exposure during the period of neuroepithelial proliferation. Cultures were then treated with a step-wise mitogen-withdrawal, integrin-activation model to mimic subsequent phases of neuronal migration and early differentiation. We examined the impact of alcohol exposure during neurogenesis on the secretion of inflammatory and growth-promoting cytokines. Cortical neurosphere cultures exhibit increasingly complex differentiation phenotypes in response to step-wise mitogen-withdrawal and laminin exposure. Some inflammation-modulating cytokines were secreted independent of differentiation state. However, chemotactic cytokines were specifically secreted at high levels, as a function of differentiation stage. monocyte chemotactic protein-1, vascular endothelial growth factor-A, and interleukin (IL)-10 were coordinately decreased during differentiation compared with neuroepithelial proliferation, while granulocyte macrophage-colony stimulating factor (GM-CSF) was induced during differentiation, compared with the neuroepithelial proliferation period. Ethanol exposure during the period of neuroepithelial proliferation prevented the early differentiation-induced increase in GM-CSF while inducing differentiation-associated increase in IL-12 secretion. Embryonic cerebral cortical neuroepithelial-derived precursors secrete high levels of several angiogenic and neural-growth-promoting cytokines as they differentiate into neurons. Our data collectively suggest that ethanol exposure during the period of neuroepithelial proliferation significantly disrupts cytokine signals that are required for the support of emerging neurovascular networks, and the maintenance of neural stem cell beds.

Annexin II Stimulates RANKL Expression Through MAPK

We report that AX-II, in addition to inducing GM-CSF expression, also increases membrane-bound RANKL synthesis by marrow stromal cells and does so through a previously unreported MAPK-dependent pathway. Thus, both GM-CSF and RANKL are required for AX-II stimulation of OCL formation. Annexin II (AX-II) is an autocrine/paracrine factor secreted by osteoclasts (OCLs) that stimulates human OCL formation and bone resorption in vitro by inducing bone marrow stromal cells and activated CD4+ T cells to produce granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF in turn increases OCL precursor proliferation and further enhances OCL formation. However, the induction of GM-CSF by AX-II cannot fully explain its effects on OCL formation. In this study, we tested the capacity of AX-II to induce the expression of RANKL and the corresponding signaling pathways AX-II employs in human marrow stromal cells to induce RANKL. We also showed that both GM-CSF and RANKL are required for OCL formation induced by AX-II. Real-time RT-PCR and Western blot analysis were used to detect RANKL and osteoprotegerin (OPG) mRNA and protein expression in unfractionated human bone marrow mononuclear cells stimulated with AX-II. Soluble RANKL in the conditioned medium was analyzed by ELISA. Activation of the MAPK pathway by AX-II was tested by Western blot. The effects of OPG and anti-GM-CSF on AX-II-induced OCL formation were also examined. In addition to upregulating GM-CSF mRNA, AX-II increased RANKL mRNA expression dose-dependently in unfractionated human bone marrow mononuclear cells and modestly increased soluble RANKL in unfractionated human bone marrow mononuclear cell conditioned medium. However, AX-II markedly increased membrane-bound RANKL on human bone marrow stromal cells. Treatment of marrow stromal cells with AX-II activated MAP-kinase (ERKs) and PD 98059 abolished the effect but did not block the increase in GM-CSF. Interestingly, OPG, a natural decoy receptor for RANKL, or anti-GM-CSF partially inhibited OCL formation by AX-II in human bone marrow cells, and the combination of OPG and anti-GM-CSF completely blocked AX-II-induced OCL formation. These data show that AX-II stimulates both the proliferation and differentiation of OCL precursors through production of GM-CSF and RANKL respectively.

Cytokine expression and dendritic cell density in melanoma sentinel nodes

The sentinel lymph node (SLN) is the first draining node from the area in which a tumour is located. The presence or absence of SLN micrometastasis is an important prognostic factor for melanoma. As the first dissemination route for melanoma is lymphatic and we know that the immune system plays an important role in melanoma response, we hypothesize that melanoma and its corresponding SLN should constitute an immunological unit. Small portions of 54 SLNs from 37 patients undergoing selective lymphadenectomy were subjected to quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) to quantify messenger RNA (mRNA) transcripts of the following genes: tyrosinase, telomerase, cyclooxygenase-1 (COX-1), COX-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), interferon-gamma (IFN-gamma), IL-4, IL-10 and IL-12. In addition, 11 non-sentinel lymph nodes (NSLNs) were excised from 11 of the 37 patients and the same study was performed. Immunohistochemistry with different antibodies against dendritic cells (DCs) was performed in 10 pairs of SLNs and NSLNs. Significantly higher mRNA expression of COX-2, GM-CSF, IFN-gamma and IL-10 was found in SLNs compared with NSLNs in the overall group. DCs, as labelled by S-100 and CD1a, were significantly decreased in NSLNs compared with SLNs. These data suggest that the initial increase in GM-CSF observed in SLNs could lead to the attraction of a high number of DCs to SLNs. However, the presence of certain immunosuppressive molecules, such as IL-10 and COX-2, could block their maturation and their ability to become efficient antigen presenters.

Evaluation of cytokines in nasal secretions after nasal antigen challenge: lack of influence of antihistamines

Previous studies of inflammation in allergic rhinitis using nasal irrication have been unsatisfactory because of 1) poor reproducibility; 2) the tendency of irrigation to overdilute mediators; and 3) the failure of this technique to evaluate interstitial concentrations of relevant mediators. For this study we used filter paper as a matrix to collect nasal secretions in patients undergoing nasal antigen challenge. To evaluate inflammatory mediators of allergen-induced rhinitis during a clinical trial of fexofenadine. Subjects evaluated at a referral medical center were placed on traditional dosing of fexofenadine at 60 mg, twice daily, or placebo in a double-blind, crossover fashion for 1 week before the nasal challenge. Nasal challenge was performed with nasal insufflation of either 1,000 AU timothy or 0.1 mL ragweed (1:100 wt/vol) extract outside the pollen season. Nasal secretions were collected at baseline and then at 2, 4, and 6 hours after nasal challenge. Secretions were evaluated for expression of the cellular adhesion molecule-1, tumor necrosis factor (TNF)-alpha, interleukin (IL)-4, IL-10, macrophage inflammatory protein (MIP)-1alpha, and granulocyte-macrophage colony-stimulating factor (GM-CSF) using commercially available enzyme-linked immunoadsorbent assay kits. Patients' symptom scores were evaluated during the nasal challenge. Significantly (P < 0.05) increased peak levels of TNF-alpha, IL-4, IL-10, and MIP-1alpha were detected after antigen challenge as compared with baseline levels. There was a nonsignificant trend toward an increase in GM-CSF after antigen challenge (P = 0.07). There was no difference in the peak levels of TNF-alpha, IL-4, IL-10, MIP-1alpha, or GM-CSF measured when patients were on fexofenadine versus placebo. Finally, there were no significant differences in patients' symptom scores during antigen challenge when subjects were on fexofenadine versus placebo. Collection of nasal secretions using a filter paper matrix provides a reproducible model for accurately detecting and evaluating changes in cytokine levels after nasal challenge. Cytokine levels tend to peak 3 to 4 hours after antigen challenge. Standard doses of fexofenadine do not seem to have a mitigating effect on the production of these cytokines. Symptoms of allergic rhinitis using this type of antigen challenge did not differ from treatment with fexofenadine versus placebo.

Allergen-induced release of GM-CSF and IL-8 in vitro by nasal polyp tissue from atopic subjects prolongs eosinophil survival.

Eosinophilia is a feature of nasal polyposis. The aim of this study was to determine the role of cytokines and allergen in maintaining the eosinophilic infiltrate in this condition. Polyp fragments from house dust mite (HDM)-sensitive atopic individuals and nonatopic individuals were cultured in the presence of HDM, or phytohaemagglutinin (PHA) or culture medium alone. Culture supernatants were assayed for interleukins (IL) 3, 5, and 8 and granulocyte macrophage colony stimulating factor (GM-CSF), and eosinophil survival enhancing activity (ESEA) in vitro. Significant ESEA was produced spontaneously. When polyp tissue from atopics, but not from nonatopics, was stimulated with allergen for 2 days there was a further increase in ESEA associated with a median 12 and fourfold increase in IL-8 and GM-CSF, respectively. This increased ESEA was markedly reduced with anti-GM-CSF and, to a lesser extent, anti-IL-8 blocking antibodies. When stimulated with PHA, polyp tissue from atopic subjects also produced increased ESEA, implicating possible T-cell involvement. This was associated with a small (twofold), but significant, increase in IL-8 and a less consistent increase in GM-CSF. However, anti-IL-8 or anti-GM-CSF blocking antibodies failed to reduce the ESEA in these supernatants, suggesting involvement of other mechanisms. This study suggests that in sensitized individuals, allergen may contribute to polyp eosinophilia by stimulating the production of granulocyte/macrophage colony stimulating factor and interleukin 8.

Progestins: present and future.

The progestogen 17-α hydroxyprogesterone caproate (17-OHPC) is 1 of only 2 agents recommended for clinical use in the prevention of spontaneous preterm delivery, and studies of its efficacy have been conflicting. We have developed an in-vitro model to study the fetal membrane weakening process that leads to rupture in preterm premature rupture of the fetal membranes (pPROM). Inflammation/infection associated with tumor necrosis factor-α (TNF-α) induction and decidual bleeding/abruption associated thrombin release are leading causes of preterm premature rupture of the fetal membranes. Both agents (TNF-α and thrombin) cause fetal membrane weakening in the model system. Furthermore, granulocyte-macrophage colony-stimulating factor (GM-CSF) is a critical intermediate for both TNF-α and thrombin-induced fetal membrane weakening. In a previous report, we demonstrated that 3 progestogens, progesterone, 17-alpha hydroxyprogesterone (17-OHP), and medroxyprogesterone acetate (MPA), each inhibit both TNF-α– and thrombin-induced fetal membrane weakening at 2 distinct points of the fetal membrane weakening pathway. Each block both the production of and the downstream action of the critical intermediate granulocyte-macrophage colony-stimulating factor.The objective of the study was to characterize the inhibitory effects of 17-OHPC on TNF-α– and thrombin-induced fetal membrane weakening in vitro.Full-thickness human fetal membrane fragments from uncomplicated term repeat cesarean deliveries were mounted in 2.5 cm Transwell inserts and cultured with/without 17-alpha hydroxyprogesterone caproate (10–9 to 10–7 M). After 24 hours, medium (supernatant) was removed and replaced with/without the addition of tumor necrosis factor-alpha (20 ng/mL) or thrombin (10 U/mL) or granulocyte-macrophage colony-stimulating factor (200 ng/mL). After 48 hours of culture, medium from the maternal side compartment of the model was assayed for granulocyte-macrophage colony-stimulating factor and the fetal membrane fragments were rupture strength tested.Tumor necrosis factor-alpha and thrombin both weakened fetal membranes (43% and 62%, respectively) and increased granulocyte-macrophage colony-stimulating factor levels (3.7- and 5.9-fold, respectively). Pretreatment with 17-alpha hydroxyprogesterone caproate inhibited both tumor necrosis factor-alpha– and thrombin-induced fetal membrane weakening and concomitantly inhibited the induced increase in granulocyte-macrophage colony-stimulating factor in a concentration-dependent manner. However, contrary to our prior reports regarding progesterone and other progestogens, 17-alpha hydroxyprogesterone caproate did not also inhibit granulocyte-macrophage colony-stimulating factor–induced fetal membrane weakening.17-Alpha hydroxyprogesterone caproate blocks tumor necrosis factor-alpha– and thrombin-induced fetal membrane weakening by inhibiting the production of granulocyte-macrophage colony-stimulating factor. However, 17-alpha hydroxyprogesterone caproate did not also inhibit granulocyte-macrophage colony-stimulating factor–induced weakening. We speculate that progestogens other than 17-alpha hydroxyprogesterone caproate may be more efficacious in preventing preterm premature rupture of the fetal membranes–related spontaneous preterm birth.

Differential regulation of granulocyte-macrophage colony-stimulating factor mRNA and protein expression in human thyrocytes and thyroid-derived fibroblasts by interleukin-1 alpha and tumor necrosis factor-alpha.

In this study, we provide the first report on the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) by human thyroid epithelial cells. Primary cultures of highly purified thyrocytes and thyroid-derived fibroblasts (n = 3) and three thyroid anaplastic and one largely papillary carcinoma cell lines were exposed to different potent GM-CSF stimulators, employing interleukin 1 alpha (Il-1 alpha) and tumour necrosis factor-alpha (TNF-alpha). Cytokine mRNA levels were monitored by semi-quantitative reverse transcriptase-PCR including an internal heterologous competitor fragment after 3, 6 and 18 h of culture. Culture supernatants were assayed for GM-CSF using a highly sensitive ELISA (detection limit < or = 0.5 pg/ml) after 24 h. Basal GM-CSF mRNA expression was higher in fibroblasts and SW 1736 cells compared with thyrocytes, C 634, 8505 C and HTh 74 cells. GM-CSF was spontaneously secreted by fibroblasts (means +/- S.E.M.; 43 +/- 15 pg/ml), SW 1736 (59 +/- 4 pg/ml), HTh 74 (34 +/- 4 pg/ml) and C 643 cells (12 +/- 1 pg/ml) but not by thyrocytes and 8505 C cells. Treatment with Il-1 alpha (10 U/ml) resulted in a marked increase of GM-CSF mRNA within 3 h and an increase or induction of protein expression in thyrocyte (2350 +/- 214 pg/ml), fibroblast (5242 +/- 1400 pg/ml), SW 1736 (20016 +/- 280 pg/ml) and C 643 cultures (1285 +/- 79 pg/ml). Stimulation with TNF-alpha (10 U/ml) yielded divergent results. No significant increase of GM-CSF mRNA or protein expression was found in thyrocytes although TNF-alpha receptor expression in these cells is well documented. Stimulation with TNF-alpha resulted in an increased GM-CSF production in fibroblasts (361 +/- 14 pg/ml), HTh 74 (148 +/- 51 pg/ml) and SW 1736 cultures (235 +/- 43 pg/ml). TSH (10 mU/ ml) did not stimulate GM-CSF secretion in thyrocytes and HTh 74 cells, both expressing the TSH receptor. Phorbol 12-myristate 13-acetate (10 ng/ml) enhanced GM-CSF mRNA and protein levels in all cell types investigated. Our data suggest that both thyrocytes and fibroblasts synthesize GM-CSF in response to Il-1 alpha, but only fibroblasts respond to TNF-alpha with a significant increase in GM-CSF. Anaplastic thyroid carcinomas are potential GM-CSF producers.

Hypereosinophilic syndrome in Hodgkin's disease with increased granulocyte-macrophage colony-stimulating factor

We report a patient with eosinophilia accompanied by Hodgkin's disease who showed remarkable increase in granulocyte-macrophage colony-stimulating factor (GM-CSF) in plasma but no increase in interleukin-5 (IL-5). The plasma GM-CSF level normalized as eosinophilia and lymphadenopathy disappeared after chemotherapy. Immunohistochemical study with immunoperoxidase staining technique showed a positive stain in lymph node cells by monoclonal anti-GM-CSF antibody. Eosinophilia is often accompanied by Hodgkin's disease, and several cases have been reported to show high levels of plasma IL-5. To our knowledge, this is the first report to show a high level of plasma GM-CSF in Hodgkin's disease with eosinophilia.

Granulocyte-macrophage Colony Stimulating Factor (GM-CSF) and Interleukin 8 (IL-8) Production by Human Bronchial Epithelial Cells (HBEC) in Asthmatics and Controls. Lack of In Vitro Effect of Salbutamol Compared to Sodium Nedocromil

Summary: The bronchial epithelium produces cytokines that could contribute to inflammatory events in airways. In this study we determined the basal and TNFα stimulated productions of GM-CSF and IL-8 by human bronchial epithelial cells (HBEC) collected from 12 control and six asthmatic patients. Spontaneous and TNFα-induced GM-CSF or IL-8 released levels increased significantly with time. Epithelial cells from asthmatic patients spontaneously released high levels of GM-CSF (24 h), TNFα potentiated GM-CSF and IL-8 release in control subjects and only the IL-8 production in asthmatics. Nedocromil sodium, an antiinflammatory drug, and salbutamol, a beta2-agonist, are commonly used in asthma. They were evaluated on the spontaneous and TNF-induced expression of GM-CSF and IL-8 in cultured bronchial epithelial cells. Nedocromil sodium, at the concentration of 10 -6 M, reduced the TNF-induced increase in GM-CSF but not the IL-8 release. Salbutamol, at the concentration of 10 -6 or 10 -5 M, did not affect the constitutive or stimulated production of both cytokines.

Diethyldithiocarbamate Induction of Cytokine Release in Human Long-Term Bone Marrow Cultures

Abstract Diethyldithiocarbamate (DDTC) is a biochemical modulating agent that protects murine bone marrow progenitor cells from the cytotoxicity of a variety of cancer chemotherapeutic agents. However, the mechanism of this protection is not well understood. Long-term human bone marrow cultures (LTBMC) were established and at day 17 treated with 30 mumol/L DDTC for 1 hour, after which DDTC was removed and replaced with complete medium. Conditioned medium was then collected 6, 12, 24, and 48 hours later and analyzed for the presence of cytokines. A time- dependent increase in granulocyte-macrophage colony-stimulating factor (GM-CSF) (12-fold), granulocyte-CSF (G-CSF) (66-fold), interleukin (IL)- 6, (three-fold), IL-1 beta (161-fold), and tumor necrosis factor (TNF)- alpha (25-fold) was observed. The maximum increase for the factors other than TNF-alpha was at 24 to 48 hours posttreatment. However, TNF- alpha peaked as early as 6 hours post-DDTC. When conditioned medium from these cultures was tested in a granulocyte-macrophage progenitor cell (GM-CFC) assay, an increase in colony formation was observed that correlated with the increased levels of cytokines in the medium. The specificity of this effect was confirmed by the fact that the closely related congener bis(hydroxyethyl)dithiocarbamate was devoid of colony- stimulating activity. The addition of antibodies for TNF-alpha and/or IL-1 alpha following DDTC treatment did not inhibit the release of GM- CSF, G-CSF, or IL-6 from the LTBMC. These results suggest that DDTC accelerates bone marrow recovery following myelotoxic drug treatment via increased production of cytokines that are known to be essential for hematopoiesis.