Release Of GM-CSF Research Articles

Macrolide antibiotics modulate ERK phosphorylation and IL-8 and GM-CSF production by human bronchial epithelial cells

Macrolide antibiotics decrease proinflammatory cytokine production in airway cells from subjects with chronic airway inflammation. However, in subjects with chronic obstructive pulmonary disease, short-term azithromycin (AZM) therapy causes a transient early increase in the blood neutrophil oxidative burst followed by a decrease in inflammatory markers with longer administration. We studied the effects of clarithromycin (CAM) and AZM on proinflammatory cytokine production from normal human bronchial epithelial (NHBE) cells. CAM decreased IL-8 over the first 6 h and then significantly increased interleukin (IL)-8 at 12-72 h after exposure (P < 0.0001). AZM also increased IL-8 at 24 and 48 h, and CAM increased granulocyte-macrophage colony-stimulating factor at 48 h. In the presence of LPS, both CAM and AZM dose-dependently increased IL-8 secretion over 24 h, but after 5 days of exposure to 10 microg/ml CAM there is suppression of IL-8 (P < 0.001). PD-98059, an inhibitor of MAP kinase/ERK kinase, inhibited CAM-induced IL-8 (P < 0.0001) and GM-CSF (P < 0.01) release. The p38 MAP kinase inhibitor SB-203580 increased CAM-induced IL-8 release (P < 0.001), and the c-jun NH2-terminal kinase inhibitor SP-600125 had no effect on IL-8. At 120 min and 6 h, CAM increased phospho-ERK1/2 (pERK) but not phospho-p38 or phospho-JNK. Over the first 90 min, CAM at 10 microg/ml inhibited pERK and then increased pERK in parallel with measured IL-8 secretion. After daily CAM exposure for 5 days, both IL-8 and pERK returned to baseline. The p38 MAP kinase inhibitor, SB-203580 increased ERK phosphorylation and IL-8 secretion. These results suggest that macrolide antibiotics can differentially modulate proinflammatory cytokine secretion in NHBE cells, in part through ERK.

Modulation of GM-CSF release by enantiomers of β-agonists in human airway smooth muscle

beta 2 -Adrenergic receptor agonists can reduce the release of GM-CSF by human airway smooth muscle cells (HASMCs). These effects are considered anti-inflammatory and are ascribed to the activity of the (R)-enantiomer within the racemate of the agonist. However, the effect of the (S)-enantiomer on GM-CSF release, once thought to be inert, has not been extensively explored. Objective We hypothesized that the (S)-enantiomer may counter the effects of the (R)-enantiomer, potentially increasing GM-CSF release. Therefore, the effects of administration of individual and combined enantiomers on GM-CSF release were examined. Cultured HASMCs were stimulated with IL-1beta, TNF-alpha, and IFN-gamma and treated with (R)-enantiomers and (S)-enantiomers of albuterol and formoterol, with and without propranolol and ICI-118,551, and in combination with dexamethasone. GM-CSF in the resulting conditioned media was assessed by ELISA. (R)-enantiomers significantly reduced GM-CSF release by as much as 41% ( P < .05), which was reversible with propranolol. In contrast, (S)-enantiomers significantly increased GM-CSF release by as much as 34% ( P < .05) over release with no drug, and by 25% to 40% ( P < .05) when added with (R)-enantiomers. The decremental effect of dexamethasone was amplified by (R)-enantiomers but inhibited by (S)-enantiomers. Both propranolol and ICI-118,551 alone increased GM-CSF release in a concentration-dependent fashion, similar to (S)-enantiomers. We conclude that GM-CSF release by HASMC is downregulated by (R)-enantiomers and enhanced by (S)-enantiomers. The reversal of (R)-enantiomer and dexamethasone effects by the (S)-enantiomer suggests suppression of their anti-inflammatory effects, perhaps through an antagonistic mechanism similar to propranolol.

Multiple β1 Integrins Mediate Enhancement of Human Airway Smooth Muscle Cytokine Secretion by Fibronectin and Type I Collagen

Altered airway smooth muscle (ASM) function and enrichment of the extracellular matrix (ECM) with interstitial collagen and fibronectin are major pathological features of airway remodeling in asthma. We have previously shown that these ECM components confer enhanced ASM proliferation in vitro, but their action on its newly characterized secretory function is unknown. Here, we examined the effects of fibronectin and collagen types I, III, and V on IL-1beta-dependent secretory responses of human ASM cells, and characterized the involvement of specific integrins. Cytokine production (eotaxin, RANTES, and GM-CSF) was evaluated by ELISA, RT-PCR, and flow cytometry. Function-blocking integrin mAbs and RGD (Arg-Gly-Asp)-blocking peptides were used to identify integrin involvement. IL-1beta-dependent release of eotaxin, RANTES, and GM-CSF was enhanced by fibronectin and by fibrillar and monomeric type I collagen, with similar changes in mRNA abundance. Collagen types III and V had no effect on eotaxin or RANTES release but did modulate GM-CSF. Analogous changes in intracellular cytokine accumulation were found, but in <25% of the total ASM cell population. Function-blocking Ab and RGD peptide studies revealed that alpha2beta1, alpha5beta1, alphavbeta1, and alphavbeta3 integrins were required for up-regulation of IL-1beta-dependent ASM secretory responses by fibronectin, while alpha2beta1 was an important transducer for type I collagen. Thus, fibronectin and type I collagen enhance IL-1beta-dependent ASM secretory responses through a beta1 integrin-dependent mechanism. Enhancement of cytokine release from ASM by these ECM components may contribute to airway wall inflammation and remodeling in asthma.

Eotaxin release from human airway smooth muscle cells treated with albuterol enantiomers

RATIONALE: Eotaxin is a Th2 cytokine that functions as a chemo-attractant for eosinophils. In previous experiments, (R)- and (S)-albuterol showed differing effects on release of GM-CSF from human airway smooth muscle cells (HASMC), another important eosinophil-modulating cytokine. Therefore, we evaluated the effects of albuterol enantiomers on eotaxin release from HASMC, to further determine the scope of their effects on pro-inflammatory cytokine release.

The Met-196 → Arg Variation of Human Tumor Necrosis Factor Receptor 2 (TNFR2) Affects TNF-α-induced Apoptosis by Impaired NF-κB Signaling and Target Gene Expression

Tumor necrosis factor-alpha (TNF-alpha)-induced signaling is pivotally involved in the pathogenesis of chronic inflammatory diseases. A polymorphism in the TNF receptor 2 (TNFR2) gene resulting in a juxtamembrane inversion from methionine (TNFR2(196MET)) to arginine (TNFR2(196ARG)) has been genetically associated with an increased risk for systemic lupus erythematosus and familial rheumatoid arthritis. Albeit the mutation does not affect the TNF binding kinetics of TNFR2, the present study provides evidence that the mutation results in a significantly lower capability to induce TNFR2-mediated NF-kappaB activation. Pretriggering of TNFR2 with a receptor-specific mutein leads to an enhancement of TNFR1-induced apoptosis, which is further increased in cells carrying the TNFR2(196ARG) variant. A diminished induction of NF-kappaB-dependent target genes conveying either anti-apoptotic or pro-inflammatory functions, such as cIAP1, TRAF1, IL-6, or IL-8 is observed. The mutated form TNFR2(196ARG) shows a reduction of inducible TRAF2 recruitment upon TNF-alpha stimulation. The findings suggest a common molecular mechanism for the involvement of the TNFR2(196ARG) variant in the etiopathogenesis of different chronic inflammatory disorders.

The innate immune surfactant protein (SP)-D plays a protective role in ozone (O3)-induced airway inflammation in mice

RATIONALE: The pulmonary surfactant protein SP-D may be an an important modulator of immune and inflammatory responses in the lung. O3 is a common air pollutant associated with exacerbation of asthma and chronic obstructive pulmonary disease. We hypothesized that SP-D plays a protective role the inflammatory response to acute ozone exposure. METHODS: C57BL/6 wild type mice were exposed to 3.0 ppm O3 for 6h. 2, 6, 12, 24, 48 and 72h post-exposure, lung function was assessed by whole body plethysmography. Broncho-alveolar lavage (BAL) cellular influx was quantified using cytospin preparations and inflammatory cytokine/chemokine and SP-D levels were measured by ELISA. The role of SP-D was investigated in 6-8 weeks old SP-D-/- and wild-type mice exposed to 0.5 ppm ozone for 24 hours. RESULTS: O3 exposure elicited a time-dependent increase in airway responsiveness, neutrophil, lymphocyte and macrophage influx with a peak 12-24h post-O3. These were preceded by release of IL-6, IL-12 IL-17, GM-CSF and KC, 2-6h after O3 treatment. Significant elevation of SP-D levels in the BAL (p<0.001, n=6) occurred in parallel with resolution of airway inflammation 48h post O3. O3 exposure of SP-D deficient mice resulted in a significantly greater number of neutrophils in the BAL and a delayed resolution of inflammation in comparison with wild-type controls (p<0.05, n=6). CONCLUSIONS: Elevation of SP-D levels in the lung is associated with resolution of the inflammatory changes after O3-induced acute oxidative stress. Absence of SP-D in gene deficient mice enhances and prolongs neutrophilia in the lung following ozone exposure.

Inhibitory effects of budesonide, desloratadine and dexamethasone on cytokine release from human mast cell line (HMC-1)

To determine the inhibitory potency of budesonide on interleukin (IL)-4, 6 and 8, GM-CSF and TNF-alpha release from the human mast cell line (HMC-1) in comparison with the systemic glucocorticosteroid, dexamethasone, and H(1) antagonist, desloratadine. HMC-1 was stimulated with 25 ng/ml phorbol 12- myristate 13-acetate (PMA) and 2.5 x 10(-7) M ionomycin (A23187) for 6, 12 and 24 h in both the presence and absence of 10(-6)-10(-10) M concentrations of the test drugs. Culture supernatants were collected and assayed by ELISAs. HMC-1 produced substantial amounts of GM-CSF and IL-8 and smaller amounts of TNF-alpha, IL-4 and IL-6 after being stimulated with PMA together with A23187. Budesonide and dexamethasone had potent inhibitory effects and desloratadine had modest inhibitory effects on the release of these cytokines. Budesonide was more potent than dexamethasone at most concentrations and time points. IL-4 was the cytokine which was most susceptible to inhibition by the three tested drugs. The inhibitory effects, in some cases, were time- and concentration-dependent. Budesonide had a potent inhibitory effect on cytokine release from HMC-1. Its potency was greater than that of both dexamethasone and desloratadine.

H1 histamine receptor mediates inflammatory responses in human keratinocytes

Keratinocytes participate in initiation and amplification of T-cell-mediated skin diseases. During these disorders, histamine can be released from both residents skin cells and immigrating leukocytes, and can affect the functions of dendritic cells, monocytes, and lymphocytes. Little information is available on the effects of histamine on keratinocytes. To examine the presence of functional H1 receptors on human keratinocytes and the capacity of histamine to modulate the expression of inflammatory molecules in these cells. Primary cultures of resting and cytokine-activated keratinocytes from healthy subjects were analyzed for histamine H1 receptor expression and the production of inflammatory mediators after exposure to histamine receptor agonists and antagonists. Cultured keratinocytes constitutively expressed the H1 receptor mRNA and protein, which was not influenced by IFN-gamma, TNF-alpha, or IL-4. H1 but not H2 agonists induced calcium fluxes in keratinocytes. Treatment of keratinocytes with histamine (10 -7 to 10 -4 mol/L) or beta-histine increased the IFN-gamma-induced expression of membrane intercellular adhesion molecule 1 and MHC class I but not MHC class II molecules. Moreover, H1 stimulation promoted basal CC chemokine ligand (CCL)-5/RANTES and GM-CSF secretion and augmented IFN-gamma-induced release of the chemokines CCL2/monocyte chemoattractant protein 1, CCL5/RANTES, CCL20/macrophage inflammatory protein 3alpha, and CXC chemokine ligand 10/IFN-induced protein of 10 kd, as well as GM-CSF. Administration of the H1 antihistamine levocetirizine, but not of the H2 antihistamine cimetidine, abolished histamine-dependent expression of all of the investigated proinflammatory molecules in a dose-dependent manner (0.01-10 mumol/L). Levocetirizine at higher doses also reduced intercellular adhesion molecule 1, CCL5/RANTES, and GM-CSF release induced solely by IFN-gamma. Histamine exerts proinflammatory effects on keratinocytes via the H1 receptor, and these effects are efficiently inhibited by levocetirizine.

Cigarette smoke inhibits lipopolysaccharide-induced production of inflammatory cytokines by suppressing the activation of activator protein-1 in bronchial epithelial cells.

Chronic smoking is characterized by immunosuppressive changes in the airways, leading to chronic colonization with bacteria, which in turn may contribute to the chronic obstructive pulmonary disease. The mechanisms causing this immunosuppression, however, are poorly characterized. This study evaluated whether cigarette smoke can inhibit endotoxin (LPS)-induced inflammatory cytokine production in bronchial epithelial cells and, if so, what the mechanisms are behind this effect. Pretreatment with cigarette smoke extract (CSE) concentration dependently inhibited the LPS-induced GM-CSF and IL-8 protein release, which was accompanied by decreased expression of mRNA in human bronchial epithelial cells (Beas-2B). The increase of neutrophil chemotaxis induced by conditioned medium from LPS-treated Beas-2B cells was also suppressed by CSE. In addition, the activity of LPS-induced transcription factor AP-1, but not NF-kappaB, was down-regulated by CSE. Notably, at the concentrations used, CSE had no effect on number or viability of Beas-2B cells. These data indicate that cigarette smoke possesses immunosuppressive properties by down-regulating the bacterial pathogen-induced neutrophil-mobilizing cytokine production via suppression of AP-1 activation in the airways. Hence, this study suggests a novel mechanism by which cigarette smoke may contribute to chronic colonization and chronic obstructive pulmonary disease in smokers.

IL-17 and IL-17F modulate GM-CSF production by lung microvascular endothelial cells stimulated with IL-1β and/or TNF-α

In this study, we investigated the roles of CD4 T cell cytokines IL-17 and IL-17F in GM-CSF production from lung microvascular endothelial cells (LMVECs). While a wide range of doses of IL-17 or IL-17F alone did not induce GM-CSF release from LMVECs, IL-17 had an enhancing effect on macrophage-derived IL-1β- and TNF-α-induced GM-CSF mRNA expression and production, whereas IL-17F had an enhancing effect on IL-1β-induced GM-CSF production, but a marked inhibitory effect on TNF-α-induced secretion. GM-CSF production was further enhanced with the combination of three cytokines IL-1β, TNF-α and IL-17 or IL-17F. Additionally, when Th1 or Th2 cytokine was combined with IL-1β or TNF-α, both Th1 and Th2 cytokines had a modest stimulatory effect on TNF-α-induced GM-CSF production, whereas IL-4 and IFN-γ profoundly attenuated IL-1β-induced secretion. Moreover, the regulation by IL-17 plus Th1 or Th2 cytokine of GM-CSF production from LMVECs treated with IL-1β or TNF-α was dependent on the concentration of IL-17. Our findings indicate that IL-17 and IL-17F play a differential regulatory role in GM-CSF production by LMVECs stimulated with IL-1β and/or TNF-α, which is sensitive to Th1 and Th2 cytokine modulation.

Defective glucocorticoid receptor nuclear translocation and altered histone acetylation patterns in glucocorticoid-resistant patients

Most chronic inflammatory diseases are well controlled by glucocorticoids. However, a minority of patients fails to respond adequately to this treatment. We wished to determine whether glucocorticoid insensitivity in a group of steroid-resistant (SR) and steroid-dependent (SD) asthmatic subjects resulted from an inability of the glucocorticoid receptor (GR) to translocate into the nucleus. Glucocorticoid receptor nuclear translocation was determined in PBMCs by immunocytochemistry and GR function measured by suppression of TNF-alpha-induced GM-CSF release and effects of dexamethasone on histone acetylation. Glucocorticoid repression of TNF-alpha-induced GM-CSF release was reduced in PBMCs from SD and SR patients. This inhibition correlated with a failure of GR to translocate into the nucleus and induce histone acetylation in response to dexamethasone. In addition, dexamethasone failed to inhibit TNF-alpha-induced histone acetyltransferase activity, which predominantly targeted histone residues lysine (K)8 and K12. However, in a subset of patients, even high levels of GR nuclear translocation failed to produce histone acetylation in response to dexamethasone. Histone H4 K5 acetylation, a marker of dexamethasone transactivation, was specifically reduced in this group. However, cells from this subset of steroid-insensitive subjects were still capable of inhibiting TNF-alpha-induced histone acetylation. We have identified a novel mechanism of glucocorticoid insensitivity in a group of SR and SD subjects. These data suggest that most patients respond to glucocorticoids according to the degree of GR nuclear translocation occurring, but some subjects with steroid resistance have a reduced response because of a failure of steroids to transactivate, rather than transrepress.

In vitro effects of native human acute myelogenous leukemia blasts on fibroblasts and osteoblasts

Bone marrow stromal cells constitute a heterogeneous population, and in the present study we investigated intercellular crosstalk via release of soluble mediators between native human AML blasts and fibroblasts/osteoblasts. Coculture of nonleukemic stromal cells and AML blasts separated by a semipermeable membrane decreased proliferation of the fibroblast line HFL1, and the inhibition was maintained when HFL1 and AML cells were cultured in direct contact. A similar inhibitory effect was observed for osteoblastic sarcoma cell lines (Cal72, SJSA-1) and normal osteoblasts. GM-CSF was released by both nonleukemic cells and a subset of AML blast populations, and increased levels of GM-CSF were detected in AML cocultures with fibroblasts and osteoblastic sarcoma cells when testing AML cell populations with constitutive GM-CSF release. Furthermore, constitutive IL-1beta secretion by AML blasts was detected only for a subset of patients, whereas relatively high levels of IL-1RA were observed for all patients; coculture of AML blasts with HFL1 fibroblasts and osteoblastic sarcoma cells increased IL-1beta levels for patients with constitutive IL-1beta secretion, whereas IL-1RA levels were slightly decreased but still generally higher than IL-1beta levels (tested only for HFL1 fibroblasts). The bidirectional crosstalk between AML blasts and stromal cells with increased release of AML growth factors may be important in leukemogenesis, whereas the decreased stromal cell proliferation combined with the persistent release of IL-1RA may in addition inhibit remaining normal hematopoiesis and thereby contribute to bone marrow failure in AML.

15d-PGJ 2 inhibits oxidized LDL-induced macrophage proliferation by inhibition of GM-CSF production via inactivation of NF-κB

Macrophage-derived foam cells play an important role in atherosclerotic lesions. Oxidized low-density lipoprotein (Ox-LDL) induces macrophage proliferation via production of GM-CSF in vitro. This study investigated the effects of 15-deoxy-Δ 12,14-prostaglandin J 2 (15d-PGJ 2), a natural ligand for peroxisome proliferator-activated receptor γ, on macrophage proliferation. Mouse peritoneal macrophages and RAW264.7 cells were used for proliferation study and reporter gene assay, respectively. Twenty microgram per milliliter of Ox-LDL induced [ 3H]thymidine incorporation in mouse peritoneal macrophages, and 15d-PGJ 2 inhibited Ox-LDL-induced [ 3H]thymidine incorporation in a dose-dependent manner. Ox-LDL increased GM-CSF release and GM-CSF mRNA expression, and activated GM-CSF gene promoter, all of which were prevented by 15d-PGJ 2 or 2-cyclopenten-1-one, a cyclopentenone ring of 15d-PGJ 2. The suppression of GM-CSF promoter activity by 15d-PGJ 2 and 2-cyclopenten-1-one was mediated through reduction of NF-κB binding to GM-CSF promoter. These results suggest that 15d-PGJ 2 inhibits Ox-LDL-induced macrophage proliferation through suppression of GM-CSF production via NF-κB inactivation.

The influence of alumina and ultra-high molecular weight polyethylene particles on osteoblast–osteoclast cooperation

Particle-induced macrophage activation, mainly by UHMWPE wear, has been recognized as the biological mechanism leading to periprosthetic bone resorption, which is responsible for the loosening of the total hip replacements (THR). Ceramic-on-ceramic implants have been advocated as a means of reducing wear products. Many studies investigated the effect of alumina (Al 2O 3) particles on monocytes/macrophages, but only limited information are available on their participation to bone turnover. An in vitro model was performed to investigate how Al 2O 3 and UHMWPE particles may influence the osteoblast–osteoclast interaction: human osteoblasts (HOB) were obtained from trabecular bone, while osteoclasts were derived from peripheral blood mononuclear cells (PBMC) of healthy donors. The amount of IL6, TNF α, GM-CSF, and other factors acting on the bone turnover, i.e. the ‘receptor activator of NFkB’ ligand (RANKL) and osteoprotegerin (OPG), was detected in culture medium of particle-challenged HOB (HOB-CM). The Al 2O 3 and UHMWPE particles did not affect either cell viability or TNF and GM-CSF release, while the increase in IL6 release seemed to be dependent on the particle concentration. UHMWPE increased the release of RANKL from HOB, while OPG and OPG-to-RANKL ratio were significantly inhibited. The ability of HOB-CM to promote osteoclastogenesis was tested via osteoblast/monocyte cooperation: after seven days of culture UHMWPE HOB-CM induced a large amount of multinucleated TRAP-positive giant cells, as well as significantly reduced the amount of IL6, GM-CSF and RANKL in the supernatant. With regard to the inductive effect on the osteoclastogenesis, our results show that the Al 2O 3 wear debris are less active.

Cement particles containing radio-opacifiers stimulate pro-osteolytic cytokine production from a human monocytic cell line

Proponents of the biological theory of aseptic loosening have in recent years tended to concentrate on the production and distribution of particulate ultra-high-molecular-weight polyethylene (UHMWPE) debris around the potential joint space. However, mechanical loading of cemented implants with the differing elastic moduli of metal stems, polymethylmethacrylate (PMMA) cement and bone can result in relative micromotion, implying the potential for production of metal and PMMA particles from the stem-cement interface by fretting wear. In order to investigate the production and biological reactivity of debris from this interface, PMMA and metal particulate debris was produced by sliding wear of PMMA pins containing barium sulphate and zirconium dioxide against a Vaquasheened stainless steel counterface. This debris was characterised by SEM, energy-dispersive analysis by X-ray (EDAX) and image analysis, then added to cell cultures of a human monocytic cell line, U937, and stimulation of proosteolytic cytokines measured by ELISA. Large quantities of PMMA cement debris were generated by the sliding wear of PMMA pins against Vaquasheened stainless steel plates in the method developed for this study. Both cements stimulated the release of pro-osteolytic TNFalpha from the U937 monocytic cell line, in a dose-dependent fashion. There was a trend towards greater TNFalpha release with Palacos cement than CMW cement at the same dose. Palacos particles also caused significant release of IL-6, another pro-osteolytic cytokine, while CMW did not. The particulate cement debris produced did not stimulate the release of GM-CSF or IL1beta from the U937 cells. These results may explain the cytokine pathway responsible for bone resorption caused by particulate PMMA debris. Radio-opaque additives are of value in surgical practice and clinical studies to quantify the relevance of these in vitro findings are required before the use of cement containing radio-opacifier is constrained.

Role of monocytes and eosinophils in human respiratory syncytial virus infection in vitro

RSV infection in airway epithelial cells (EC) results in production of the chemokines RANTES and MIP1α and the leukocyte differentiation factor GM-CSF. The chemokines attract monocytes and eosinophils to the site of infection, where GM-CSF may influence their function and differentiation. In turn, these inflammatory cells may limit the progression of RSV infection, as well as initiate immune responses. In the present study, the effect of monocytes and eosinophils on viral replication and infection-dependent release of EC-derived cytokines was investigated. The modulation of immune cell costimulatory molecules, CD80, CD86, CD40, and HLA-DR, and the release of the CD4 + T cell chemoattractant IL-16 were also investigated. Employing immunofluorescence techniques, monocytes and eosinophils in cocultures with infected EC were found to inhibit the spread of RSV to uninfected cells. Monocytes also had a significant effect on replication of RSV. Monocytes phagocytized the virus, while eosinophils inhibited reinfection mainly by extracellular means. The release of G-CSF and GM-CSF in the infected cultures was not significantly affected by either monocytes or eosinophils, while RANTES release was significantly decreased. The expression of CD40, CD80, CD86, and HLA-DR on monocytes, but not on eosinophils, increased in an RSV-dose-dependent manner. IL-16 release was not induced in RSV-infected EC, but was significantly increased in coculture with monocytes. These results suggest that both monocytes and eosinophils attracted to the site of RSV infection play an important role in confining infection, while RSV-exposed monocytes may be involved in promoting/polarizing immune responses to RSV.

Regulation of the interleukin-5 receptor alpha-subunit on peripheral blood eosinophils from healthy subjects.

The aim was to study in vitro regulation of the IL-5 receptor alpha (IL-5R alpha) on purified peripheral blood eosinophils from healthy subjects. The IL-5R alpha was down-regulated, in a dose-dependent manner, by recombinant IL-5 and GM-CSF, with IL-5 being most potent. This down-regulation was not induced by autocrine release of GM-CSF or IL-5, respectively. Incubation of eosinophils with cell-free peritoneal dialysis fluid (PF) collected from a patient with peritoneal fluid eosinophilia (PFE), induced up-regulation of the proportion of CD69 positive eosinophils, in parallel with down-regulation of the proportion of IL-5R alpha positive eosinophils. Experiments with neutralizing antibodies against IL-5 and GM-CSF, revealed that IL-5 was the principal cytokine responsible for the down-regulation of the IL-5R alpha. When eosinophils were incubated with PF collected from the same patient in remission or with PF collected from a newly started patient or a patient with bacterial peritonitis, less down-regulation of the IL-5R alpha was observed. In conclusion our data indicate that IL-5, as opposed to its proposed action on eosinophil progenitors, down-regulates the IL-5R alpha chain on mature eosinophils. We therefore suggest that an IL-5 driven inflammation generates an eosinophil tissue phenotype that is characterized by a low IL-5R alpha expression. These aspects of IL-5 action on IL-5R alpha expression could gain new insights into the mechanisms of specific immuno-modulatory therapies, such as anti-IL-5.

Pleural mesothelial cells modulate polymorphonuclear leukocyte apoptosis in empyema.

In bacterial empyema, the recruited polymorphonuclear leukocytes (PMN) represent important phagocytic cells involved in antibacterial defense. In this study we demonstrate that pleural fluids (PF) obtained from patients with empyema (EMP) contains significantly higher levels of granulocyte colony stimulating factor (GM-CSF), and PMN incubated in empyema (EMP) pleural fluid (PF) showed significantly less apoptosis than congestive heart failure (CHF) PF. Staphylococcus aureus-stimulated PMC released significantly (P < 0.001) higher levels of GM-CSF than resting PMC. Staphylococcus aureus-stimulated PMC (SPMC)-CM significantly (P < 0.001) inhibited PMN apoptosis. In SPMC-CM-incubated PMN the antiapoptotic gene Bcl-xL mRNA and protein expression was up-regulated; Bak mRNA and protein expression was down-regulated compared to control PMN. The active caspases activity significantly decreased. When SPMC-CM and EMP PF were immunodepleted with GM-CSF antibody, PMN apoptosis was significantly higher. The delay in apoptosis of PMN is in part attributable to the release of cytokine GM-CSF by activated PMC. These findings suggest that S. aureus-activated PMC extend PMN life span by modulating Bcl-xL and Bak gene expression and active caspases activity during acute inflammation and empyema.

Inhibition of mediator and cytokine release from dispersed nasal polyp cells by mizolastine.

Mizolastine is a potent and selective H1-receptor antagonist with antiallergic properties; in in-vitro animal models, mizolastine was shown to inhibit 5-lipoxygenase activity and to decrease the release of leukotrienes (LT) and tumor necrosis factor-alpha (TNF-alpha). This study investigated the effects of three concentrations of mizolastine (0.1, 1.0, 10 microM) on the release of LT (LTB4 and LTC4/D4) and prostaglandin D2 (PGD2) after stimulation by anti-IgE, and on the spontaneous release of cytokines (TNF-alpha and granulocyte/macrophage-colony-stimulating factor [GM-CSF]), from dispersed cells obtained from surgically resected nasal polyps of patients with nasal polyposis. Cells from nasal polyps were obtained using enzymatic dispersion. For experiments involving the measurement of LT and PGD2, the cells were preincubated with mizolastine or its dissolution vehicle for 20 min prior to challenge with 10 microg/ml epsilon-chain specific anti-IgE for 45 min at 37 degrees C; for the cytokine release, cells were incubated with mizolastine or its dissolution vehicle for 24 h. LT and PGD2 were measured by enzyme immunoassay (EIA) and cytokines by enzyme-linked immunosorbent assay (ELISA) using commercially available kits. Mizolastine inhibited significantly and in a dose-dependent manner the release of LTB4 and TNF-alpha at all concentrations, LTC4/D4 at 10 microM, and GM-CSF from 1 microM; no effect was observed on the release of PGD2. Mizolastine inhibits the release of LT, TNF-alpha and GM-CSF in this in vitro model, which mimics closely the inflammatory cells of allergic rhinitis.

Cooperative effects of Th2 cytokines and allergen on normal and asthmatic bronchial epithelial cells.

In sensitized individuals, exposure to allergens such as Dermatophagoides pteronyssinus (Der p) causes Th2 polarization and release of cytokines, including IL-4 and IL-13. Because Der p extracts also have direct effects on epithelial cells, we hypothesized that allergen augments the effects of Th2 cytokines by promoting mediator release from the bronchial epithelium in allergic asthma. To test our hypothesis, primary bronchial epithelial cultures were grown from bronchial brushings of normal and atopic asthmatic subjects. RT-PCR showed that each culture expressed IL-4R(alpha), common gamma-chain, and IL-13R(alpha)(1), as well as IL-13R(alpha)(2), which negatively regulates IL-13 signaling; FACS analysis confirmed IL-13R(alpha)(2) protein expression. Exposure of epithelial cultures to either Der p extracts, TNF-alpha, IL-4, or IL-13 enhanced GM-CSF and IL-8 release, and this was partially suppressible by corticosteroids. Simultaneous exposure of the epithelial cultures to IL-4 or IL-13 together with Der p resulted in a further increase in cytokine release, which was at least additive. Release of TGF-alpha was also increased by TNF-alpha and combinations of IL-4, IL-13, and Der p; however, this stimulation was only significant in the asthma-derived cultures. These data suggest that, in an allergic environment, Th2 cytokines and allergen have the potential to sustain airway inflammation through a cooperative effect on cytokine release by the bronchial epithelium. Our novel finding that IL-4, IL-13, and allergen enhance release of TGF-alpha, a ligand for the epidermal growth factor receptor that stimulates fibroblast proliferation and goblet cell differentiation, provides a potential link between allergen exposure, Th2 cytokines, and airway remodelling in asthma.