Persistent organic pollutants and biomarkers of diabetes risk in a cohort of Great Lakes sport caught fish consumers.

BACKGROUND: Exposure to persistent organic pollutants (POPs) is associated with increased diabetes risk, although the mechanism of action is not well delineated. METHODS: We investigated established diabetes biomarkers that could implicate potential mechanistic pathways, including C-reactive protein (CRP), a marker of systemic inflammation; gamma glutamyl transferase (GGT), a liver enzyme associated with oxidative stress; and adiponectin, an adipokine modulating glucose regulation and fatty acid oxidation. These biomarkers as well as hemoglobin A1c (HA1c), and POPs [polychlorinated biphenyls (PCBs), p,p-dichlorodiphenyldichloroethylene (DDE) and polybrominated diphenyl ethers (PBDEs)] were measured in a cohort of Great Lakes sport caught fish (GLSCF) consumers. We examined associations of POPs and fish consumption with HA1c and incident diabetes, and evaluated mediation and moderation by the diabetes biomarkers. RESULTS: Odds of incident diabetes were elevated with exposure to DDE and PCBs. DDE and PCB 118 were positively, and fish meals were inversely, associated with HA1c. CRP was inversely associated with saltwater and total fish meals, particularly in persons with higher adiposity, but did not mediate the associations of fish meals with HA1c. There were few associations of POPs with adiponectin, CRP and GGT, with the exception of positive associations of PCB 118 with GGT, PBDEs with GGT in older persons, and PBDEs with adiponectin. Adiponectin, CRP and GGT did not mediate associations of DDE and PCBs with HA1c or incident diabetes. However, the association of DDE with HA1c was stronger in persons with higher CRP, GGT and BMI, and lower adiponectin, while the association of PCB 118 with HA1c was stronger in persons with higher GGT. CONCLUSIONS: These findings suggest that adiponectin, CRP and GGT did not mediate effects of POPs on diabetes or HA1c. However, POPs may have stronger effects on blood glucose in persons at higher risk for diabetes.

Uterine leiomyomata in a cohort of Great Lakes sport fish consumers.

Diet and endocrine disrupting persistent organic pollutants (POPs) have been associated with gynecologic conditions including uterine leiomyomata (UL), endometriosis, and ovarian cysts. Great Lakes sport fish consumption is a source of exposure to POPs such as p,p'-diphenyldichloroethene (DDE) and polychlorinated biphenyls (PCBs). This study was designed to examine retrospectively the effects Great Lakes sport fish consumption on the incidence of UL and to examine the effects of DDE and PCB serum levels on prevalent UL in women participating in the Great Lakes Fish Consumption Study. We hypothesized that associations of exposures with UL would be modified by breastfeeding status. Years of sport fish consumption, demographic, health, and reproductive data were assessed by survey. In a subgroup, serum was collected and tested for DDE and PCB levels. Effects of years of Great Lakes sport fish and sport fish consumption were modeled using time-dependent Cox proportional hazards regression and effects of POP exposures on UL were modeled using multiple logistic regression. Years of sport fish consumption were associated with UL, with an incidence rate ratio of 1.2 (95% CI 1.0-1.3) for each 10-year increment of fish consumption. Summary measures of POP exposures in the overall group were not associated with UL. In the subgroup of women who never breastfed and in whom PCB measurements were available, however, UL was significantly associated with PCBs and groupings of estrogenic, antiestrogenic, and dioxin-like PCBs. These findings support the possibility that PCB exposures from fish consumption may increase the risk of UL and highlight the importance of additional studies exploring biologic pathways by which they could be acting.

Associations of PCBS, dioxins and furans with follicle-stimulating hormone and luteinizing hormone in postmenopausal women: National Health and Nutrition Examination Survey 1999-2002.

BACKGROUND: The general population is exposed to the group of endocrine disrupting chemicals persistent organic pollutants (POPs), that includes polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). OBJECTIVES: The aim of this research was to evaluate the associations of serum levels of PCB, PCDD, and PCDF congeners with follicle stimulating hormone (FSH) and luteinizing hormone (LH) in postmenopausal women not taking exogenous hormones. We hypothesized that associations of POPs with these gonadotropins could be modified by factors affecting endogenous hormones. METHODS: Cross-sectional analyses were conducted on data from 89 postmenopausal women using data from the National Health and Nutrition Examination Survey (NHANES). POPs were summarized based on classification schemes thought to reflect toxicological properties. Associations of POPs and gonadotropin hormones were modeled with multivariable regression models. When evidence of interaction was found, conditional effects were estimated. RESULTS: We found inverse associations of LH, but not FSH, with exposure to anti-estrogenic and/or dioxin-like POPs, but not with non dioxin-like PCBs. A doubling of dioxin-like toxic equivalents (TEQs) was associated with a decrease in LH of 11.9% (95% CI = -21.3%, -1.4%, p = 0.03). Inverse associations were enhanced by potential effect modifiers related to both direct and indirect estrogenicity, including obesity and the obesity-related condition inflammation. CONCLUSIONS: These investigations support a pattern of endocrine-disrupting effects by dioxin-like POPs among postmenopausal women, especially those with conditions related to peripheral estrogenicity.

Asthma morbidity in adult Chicago public housing residents.

BACKGROUND: Residents of public housing can experience socioeconomic disadvantages, inadequate access to health care, and particularly substandard indoor air quality due to inadequate building maintenance. OBJECTIVE: This study investigates demographic, medical management, severity, and household factors associated with asthma-related emergency department visits and hospitalizations. METHODS: A total of 103 adult participants with asthma from four Chicago housing developments completed surveys and underwent household inspections. RESULTS: Using stepwise multivariate logistic regression, we identified independent predictors of asthma-related emergency department visits: asthma controller medication use, not keeping an asthma-related doctor's appointment, and frequent nocturnal wheeze episodes. Using stepwise multivariate logistic regression, we identified independent predictors of asthma-related hospitalizations: peeling paint, plaster, or wallpaper, environmental tobacco smoke, written action plan for an asthma-related doctor or emergency department visit, and frequent nocturnal wheeze episodes. CONCLUSIONS: In multivariate models, factors related to clinical severity and asthma management were related to both emergency department visits and hospitalizations while household conditions were related only to hospitalizations. Interventions to address both asthma management and household environmental triggers may be needed to reduce asthma morbidity in low-income populations.

IL-5-induced integrin adhesion of human eosinophils caused by ERK1/2-mediated activation of cPLA2.

We examined the mechanism by which interleukin (IL)-5 causes beta(2)-integrin adhesion of human eosinophils. IL-5 caused time-dependent activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38alpha in eosinophils as detected by their phosphorylation. Preincubation of eosinophils with U0126, a mitogen-activated protein kinase/ERK kinase inhibitor, suppressed IL-5-induced activation of cytosolic phospholipase A(2) (cPLA(2)) and eosinophil adhesion, and p38 inhibition by SB203580 had neither effect. ERK1/2 phosphorylation and eosinophil adhesion were blocked by inhibition of the src-family tyrosine kinase, Janus tyrosine kinase (JAK)2, or phosphoinositide-3 kinase (PI3K). Coimmunoprecipitation assay demonstrated that Lyn, a src-family tyrosine kinase, was constitutively associated with PI3K. Inhibition of src-tyrosine kinase but not JAK2 suppressed PI3K activation. Our data suggest that IL-5 induces beta(2)-integrin adhesion of human eosinophils by regulation of cPLA(2) activation caused by ERK1/2 phosphorylation. This phosphorylation results from activation of PI3K and protein tyrosine kinases. We also find that src-family tyrosine kinase, possibly Lyn, is the upstream kinase causing PI3K activation.

Quantitation of secretory group V phospholipase A(2) in human tissues by sandwich enzyme-linked immunosorbent assay.

We have developed a sensitive sandwich ELISA (sELISA) for quantitative determination of group V phospholipase A(2) (gVPLA(2)). This assay utilizes three monoclonal antibodies (mAbs) directed against human gVPLA(2) (MCL-1B7, MCL-2A5, and MCL-3G1), which recognize specifically different epitopes of gVPLA(2). A mixture of MCL-1B7 and MCL-2A5 was used as the capture mAb, and MCL-3G1 as the detector mAb; purified human gVPLA(2) was used as the standard protein. The limit of detection of the sELISA is 2 ng/ml; the intra- and inter-coefficients of variation were 4.97+/-0.81% and 8.42+/-3.4%. The validity of the sELISA was assured by the recovery of exogenous recombinant gVPLA(2), which was 99.7% to 102%, and demonstration of noninterference of the gVPLA(2) assay by a high concentrations of other protein from murine lung and heart. To assess the usefulness of this sELISA for tissue measurements, the amount of gVPLA(2) in cultured human epithelial cells and isolated human eosinophils was determined. Total gVPLA(2) mass in epithelial cells was 2.83+/-0.33 ng/10(7) cells; gVPLA(2) was not detected in eosinophils. The presence of high concentration of gVPLA(2) in epithelial cells was confirmed by immunoprecipitation/Western blot analysis and by flow cytometry. This assay allows for convenient differentiation between the highly homologous 14-kDa secretory PLA(2)s, gVPLA(2), gIIaPLA(2), gIbPLA(2) and gXPLA(2), and accurate quantitation of gVPLA(2) in biological samples.

Structural determinants of blockade of eosinophil activation, adhesion and secretion by synthetic analogs of phomactin.

We examined the structural determinants of phomactin analogs to assess their efficacy as antagonist of PAF. Six analogs of phomactin were synthesized to determine their inhibitory effects on adhesion, superoxide release, leukotriene C4 (LTC4) synthesis and [3H]PAF binding in human eosinophils. Phomactin analogs inhibited both PAF- and IL-5-induced eosinophil adhesion. Analog A, which bears an alkene moiety between C-1 and C-14, a ketone at the C-2 position, and an alkyne moiety between C-3 and C-4, had the greatest anti-adhesive effect. Change of the alkene between C-1 and C-14 to an alkane (analog I) decreased the anti-adhesive effect by 2.5-4 fold, while substitution of ketone by hydroxyl (analog G) at the C-2 position caused an 11-fold decrease in the anti-adhesive effect. Substitution of the alkyne moiety between C-3 and C-4 by an alkene (B and E) or alkane (D) blocked completely the anti-adhesive effect. Analogs A and I completely blocked superoxide release from eosinophils caused by phorbol-12-myristate-13-acetate or PAF and LTC4-release caused by fMLP plus cytochalasin B. Change of the alkyne moiety between C-3 and C-4 to an alkene (B and E) or alkane (D) blocked completely these inhibitory effects of phomactin. Analog A decreased the maximal binding of [3H]PAF binding to eosinophils without change of the apparent dissociation constant. We conclude that phomactin analogs are specific non-competitive PAF antagonists and have exceptional efficacy in inhibiting adhesion, metabolic activity and leukotriene secretion in human eosinophils. We further define the structural alterations in the phomactin molecule that regulate its inhibitory functions.

Epidemiology of asthma in the United States.

Incidence and prevalence rates of asthma can vary greatly according to population and location. The National Heart and Blood Institute of the National Institutes of Health defines asthma as a common chronic disorder of the airways that involves a complex interaction of airflow obstruction, bronchial hyperresponsiveness, and an underlying inflammation. This article uses the most common definitions and diagnostic methods for asthma. In 2009 there were 2.1 million asthma-related emergency department (ED) visits. ED visits lend an opportunity for providers to identify and intervene in the care of patients whose asthma is poorly controlled.

Transcellular secretion of group V phospholipase A2 from epithelium induces beta 2-integrin-mediated adhesion and synthesis of leukotriene C4 in eosinophils.

We examined the mechanism by which secretory group V phospholipase A(2) (gVPLA(2)) secreted from stimulated epithelial cells activates eosinophil adhesion to ICAM-1 surrogate protein and secretion of leukotriene (LT)C(4). Exogenous human group V PLA(2) (hVPLA(2)) caused an increase in surface CD11b expression and focal clustering of this integrin, which corresponded to increased beta(2) integrin-mediated adhesion. Human IIaPLA(2), a close homolog of hVPLA(2), or W31A, an inactive mutant of hVPLA(2), did not affect these responses. Exogenous lysophosphatidylcholine but not arachidonic acid mimicked the beta(2) integrin-mediated adhesion caused by hVPLA(2) activation. Inhibition of hVPLA(2) with MCL-3G1, a mAb against gVPLA(2), or with LY311727, a global secretory phospholipase A(2) (PLA(2)) inhibitor, attenuated the activity of hVPLA(2); trifluoromethylketone, an inhibitor of cytosolic group IVA PLA(2) (gIVA-PLA(2)), had no inhibitory effect on hVPLA(2)-mediated adhesion. Activation of beta(2) integrin-dependent adhesion by hVPLA(2) did not cause ERK1/2 activation and was independent of gIVA-PLA(2) phosphorylation. In other studies, eosinophils cocultured with epithelial cells were stimulated with FMLP/cytochalasin B (FMLP/B) and/or endothelin-1 (ET-1) before LTC(4) assay. FMLP/B alone caused release of LTC(4) from eosinophils, which was augmented by coculture with epithelial cells activated with ET-1. Addition of MCL-3G1 to cocultured cells caused approximately 50% inhibition of LTC(4) secretion elicited by ET-1, which was blocked further by trifluoromethylketone. Our data indicate that hVPLA(2) causes focal clustering of CD11b and beta(2) integrin adhesion by a novel mechanism that is independent of arachidonic acid synthesis and gIVA-PLA(2) activation. We also demonstrate that gVPLA(2), endogenously secreted from activated epithelial cells, promotes secretion of LTC(4) in cocultured eosinophils.

Regulation of interleukin-5-induced beta2-integrin adhesion of human eosinophils by phosphoinositide 3-kinase.

We examined the role of phosphoinositide 3-kinase (PI3K) in integrin-mediated eosinophil adhesion. Deltap85, a dominant-negative form of the class IA PI3K adaptor subunit, was fused to an HIV-TAT protein transduction domain (TAT-Deltap85). Recombinant TAT-Deltap85 inhibited interleukin (IL)-5-stimulated phosphorylation of protein kinase B, a downstream target of PI3K. beta(2)-Integrin-dependent adhesion caused by IL-5 to the plated intracellular adhesion molecule-1 surrogate, bovine serum albumin, was inhibited by TAT-Deltap85 in a concentration-dependent manner. Similarly, two PI3K inhibitors, wortmannin and LY294002, blocked eosinophil adhesion to plated bovine serum albumin. By contrast, beta(1)-integrin-mediated eosinophil adhesion to vascular cell adhesion moelcule-1 was not blocked by TAT-Deltap85, wortmannin, or LY294002. Rottlerin, a protein kinase C (PKC)-delta inhibitor, also blocked beta(2)-integrin adhesion of eosinophils caused by IL-5, whereas beta(1) adhesion to vascular cell adhesion molecule-1 was not affected. IL-5 caused translocation of PKCdelta from the cytosol to cell membrane; inhibition of PI3K by wortmannin blocked translocation of PKCdelta. Western blot analysis demonstrated that extracellular signal-regulated kinase phosphorylation, a critical intermediary in adhesion elicited by IL-5, was blocked by inhibition of either PI3K or PKC-delta. These data suggest that extracellular signal-regulated kinase-mediated adhesion of beta(2)-integrin caused by IL-5 is mediated in human eosinophils by a class IA PI3K through activation of a PKCdelta pathway.

Glucocorticoid-induced surface expression of annexin 1 blocks beta2-integrin adhesion of human eosinophils to intercellular adhesion molecule 1 surrogate protein.

BACKGROUND: Glucocorticoids attenuate the population of eosinophils and T lymphocytes in asthmatic airways. The decrease in airway eosinophilia is caused both by accelerated cell death and by induction of blockade of integrin adhesion. In this study, we examined the hypothesis that annexin 1 surface expression, which is upregulated by the glucocorticoid receptor, prevents integrin adhesion essential to cell migration by blocking intracellular translocation of cytosolic group IV phospholipase A2 (cPLA2). OBJECTIVE: To examine the relationship of the glucocorticoid on annexin 1 expression and the effect of blockade of annexin 1 activity on adhesion of human eosinophils in vitro. To determine the relationship between annexin 1surface expression and nuclear membrane translocation of cPLA2. METHODS: Eosinophils isolated from human peripheral blood were pretreated with fluticasone propionate (FP), and beta2-integrin adhesion was measured after stimulation with IL-5 or eotaxin. Effects of FP on cPLA2 expression, phosphorylation, and translocation were determined. The role of annexin 1 was examined by using annexin 1 blocking antibody and/or mimetic peptides. RESULTS: Fluticasone propionate decreased stimulated eosinophil adhesion and caused 4-fold increase in annexin 1 expression on the plasma membrane. Inhibition of adhesion by FP was blocked with annexin 1 blocking antibody. Annexin 1 N-terminal mimetic peptide also blocked beta2-integrin adhesion. Translocation of cPLA2 to the nuclear membrane was significantly blocked by incubation with FP. Blockade was reversed with annexin 1 blocking antibody. CONCLUSION: Blockade of beta2-integrin adhesion by glucocorticoid is regulated by annexin 1, which blocks cPLA2 translocation to nuclear membrane.

Blockade of focal clustering and active conformation in beta 2-integrin-mediated adhesion of eosinophils to intercellular adhesion molecule-1 caused by transduction of HIV TAT-dominant negative Ras.

We transduced dominant negative (dn) HIV TAT-Ras protein into mature human eosinophils to determine the signaling pathways and mechanism involved in integrin-mediated adhesion caused by cytokine, chemokine, and chemoattractant stimulation. Transduction of TAT-dnRas into nondividing eosinophils inhibited endogenous Ras activation and extracellular signal-regulated kinase (ERK) phosphorylation caused by IL-5, eotaxin-1, and fMLP. IL-5, eotaxin-1, or fMLP caused 1) change of Mac-1 to its active conformation and 2) focal clustering of Mac-1 on the eosinophil surface. TAT-dnRas or PD98059, a pharmacological mitogen-activated protein/ERK kinase inhibitor, blocked both focal surface clustering of Mac-1 and the change to active conformational structure of this integrin assessed by the mAb CBRM1/5, which binds the activation epitope. Eosinophil adhesion to the endothelial ligand ICAM-1 was correspondingly blocked by TAT-dnRas and PD98059. As a further control, we used PMA, which activates ERK phosphorylation by postmembrane receptor induction of protein kinase C, a mechanism which bypasses Ras. Neither TAT-dnRas nor PD98059 blocked eosinophil adhesion to ICAM-1, up-regulation of CBRM1/5, or focal surface clustering of Mac-1 caused by PMA. In contrast to beta(2)-integrin adhesion, neither TAT-dnRas nor PD98059 blocked the eosinophil adhesion to VCAM-1. Thus, a substantially different signaling mechanism was identified for beta(1)-integrin adhesion. We conclude that H-Ras-mediated activation of ERK is critical for beta(2)-integrin adhesion and that Ras-protein functions as the common regulator for cytokine-, chemokine-, and G-protein-coupled receptors in human eosinophils.

Blockade of LTC4 synthesis caused by additive inhibition of gIV-PLA2 phosphorylation: Effect of salmeterol and PDE4 inhibition in human eosinophils.

BACKGROUND: Prior investigations have demonstrated that beta(2)-adrenoceptor stimulation is ineffective in inhibiting synthesis of eicosanoids in human eosinophils. This effect has been postulated to relate to density or structural differences in the beta(2)-adrenoceptor or its coupled G-protein. However, recent reports indicate that cAMP-specific PDE4 activity in eosinophils is 10-fold that of other inflammatory cells. We postulated that selective blockade of PDE4 in eosinophils would unmask the inhibitory effect of beta(2)-adrenoceptor stimulation and that this inhibition would result from decreased phosphor-ylation of cytosolic group IV-PLA(2) (gIV-PLA(2)). OBJECTIVE: To determine (a) whether PDE4 inhibition alone with rolipram blocked secretions of arachidonic acid (AA) and leukotriene C(4) (LTC(4)) caused by activation of eosinophils with formyl-met-leu-phe plus cytochalasin B (FMLP/B), (b) to determine if PDE4 inhibition plus beta(2)-adrenoceptor agonist act additively to augment endogenous cAMP concentration, and (c) to determine the mechanism by which additive inhibition of AA and LTC(4) synthesis is regulated by cAMP. METHODS: Human eosinophils were pretreated with buffer, salmeterol or rolipram (singly or combination) before FMLP/B activation. Release of AA and LTC(4), intracellular cAMP concentration, and phosphorylation and activation of gIV-PLA(2) were determined. RESULTS: Rolipram unmasked the inhibitory effect of beta(2)-adrenoceptor stimulation with salmeterol and significantly attenuated the stimulated release of AA and subsequent LTC(4). Inhibition corresponded to increased cAMP production caused by rolipram alone or rolipram plus salmeterol and blocked proportionately the phosphorylation and activation of gIV-PLA(2) in FMLP/B-activated eosinophils. CONCLUSIONS: Inhibition of PDE4 by rolipram unmasks beta(2)-adrenergic blockade of LTC(4) synthesis caused by FMLP/B.

Activation of group IV cytosolic phospholipase A2 in human eosinophils by phosphoinositide 3-kinase through a mitogen-activated protein kinase-independent pathway.

Activation of group IV cytosolic phospholipase A(2) (gIV-PLA(2)) is the essential first step in the synthesis of inflammatory eicosanoids and in integrin-mediated adhesion of leukocytes. Prior investigations have demonstrated that phosphorylation of gIV-PLA(2) results from activation of at least two isoforms of mitogen-activated protein kinase (MAPK). We investigated the potential role of phosphoinositide 3-kinase (PI3K) in the activation of gIV-PLA(2) and the hydrolysis of membrane phosphatidylcholine in fMLP-stimulated human blood eosinophils. Transduction into eosinophils of Deltap85, a dominant negative form of class IA PI3K adaptor subunit, fused to an HIV-TAT protein transduction domain (TAT-Deltap85) concentration dependently inhibited fMLP-stimulated phosphorylation of protein kinase B, a downstream target of PI3K. FMLP caused increased arachidonic acid (AA) release and secretion of leukotriene C(4) (LTC(4)). TAT-Deltap85 and LY294002, a PI3K inhibitor, blocked the phosphorylation of gIV-PLA(2) at Ser(505) caused by fMLP, thus inhibiting gIV-PLA(2) hydrolysis and production of AA and LTC(4) in eosinophils. FMLP also caused extracellular signal-related kinases 1 and 2 and p38 MAPK phosphorylation in eosinophils; however, neither phosphorylation of extracellular signal-related kinases 1 and 2 nor p38 was inhibited by TAT-Deltap85 or LY294002. Inhibition of 1) p70 S6 kinase by rapamycin, 2) protein kinase B by Akt inhibitor, or 3) protein kinase C by Ro-31-8220, the potential downstream targets of PI3K for activation of gIV-PLA(2), had no effect on AA release or LTC(4) secretion caused by fMLP. We find that PI3K is required for gIV-PLA(2) activation and hydrolytic production of AA in activated eosinophils. Our data suggest that this essential PI3K independently activates gIV-PLA(2) through a pathway that does not involve MAPK.

Regulation of adhesion of AML14.3D10 cells by surface clustering of beta2-integrin caused by ERK-independent activation of cPLA2.

We examined the role of cell surface clustering of beta2-integrin caused by protein kinase C (PKC)-activated-cPLA2 in adhesion of eosinophilic AML14.3D10 (AML) cells. Phorbol 12-myristate 13-acetate (PMA) caused time- and concentration-dependent adhesion of AML cells to plated bovine serum albumin (BSA), which was blocked by anti-CD11b or anti-CD18 monoclonal antibodies (mAb) directed against beta2-integrin. Inhibition of PKC with Ro-31-8220 or rottlerin blocked PMA-induced cell adhesion in a concentration-dependent fashion. Inhibition of cytosolic phospholipase A2 (cPLA2) with trifluoromethyl ketone or methyl arachidonyl fluorophosphonate also blocked PMA-induced cell adhesion. PMA caused time-dependent p42/44 mitogen-activated protein kinase (MAPK) (ERK) phosphorylation in these cells. U0126, a MAPK/extracellular signal-regulated protein kinase kinase (MEK) inhibitor, at the concentrations that blocked PMA-induced ERK phosphorylation, had no effect on PMA stimulated AML cell adhesion. Neither p38 MAPK nor c-Jun N-terminal kinase (JNK) was phosphorylated by PMA. PMA also caused increased cPLA2 activity, which was inhibited by Ro-31-8220, but not U0126. Confocal immunofluorescence microscopy showed that PMA caused clustering of CD11b on the cell surface, which was blocked by either PKC or cPLA2 inhibition. PMA stimulation also caused up-regulation of CD11b on the AML cell surface. However, this up-regulation was not affected by cPLA2- or PKC-inhibition. Using the mAb, CBRM1/5, we also demonstrated that PMA does not induce the active conformation of CD11b/CD18. Our data indicate that PMA causes AML cell adhesion through beta2-integrin by PKC activation of cPLA2. This pathway is independent of MEK/ERK and does not require change of CD11b/CD18 to its active conformation. We find that avidity caused by integrin surface clustering - rather than conformational change or up-regulation of CD11b/CD18 - causes PMA stimulated adhesion of AML cells.

Beta2-integrin adhesion caused by eotaxin but not IL-5 is blocked by PDE-4 inhibition and beta2-adrenoceptor activation in human eosinophils.

We investigated the effect and mechanism(s) of PDE-4 treatment with concurrent beta2-adrenoceptor stimulation on human eosinophil adhesion mediated by beta2-integrin in vitro. Eosinophils were pretreated with either rolipram, a PDE-4 inhibitor, alone or combined with salmeterol, a beta2-adrenoceptor agonist, before activation with either eotaxin or IL-5. Beta2-integrin mediated adhesion was assessed as adherence to BSA, an established surrogate for ICAM-1. Rolipram caused progressive blockade (77.7 +/- 6.2%) of adhesion elicited by eotaxin. Maximal blockade of IL-5-activated adhesion by rolipram was substantially less (29.9 +/- 5.2%). Salmeterol + rolipram synergistically enhanced the blockade of eotaxin-activated adhesion. Eotaxin also caused approximately 50% increase in surface CD11b expression, which was blocked additively by rolipram + salmeterol. By contrast, CD11b upregulation caused by IL-5 was not blocked by rolipram + salmeterol. Rolipram also attenuated cPLA2 phosphorylation caused by eotaxin but did not block IL-5-induced phosphorylation. We conclude that rolipram blocks expression of CD11b and inhibits cPLA2 phosphorylation in human eosinophils, thus blocking eotaxin-induced adhesion of beta2-integrin. IL-5-induced adhesion likely utilizes a different upstream mechanism in regulation of integrin adhesion.