Marc Peters-Golden

Influence of body mass index on the response to asthma controller agents

The incidence of asthma has been positively associated with obesity. Asthma comprises diverse “phenotypes” reflecting heterogeneity in a number of characteristics, including response to therapy. The present authors examined whether body mass index (BMI) influenced the response to placebo, as well as to two asthma controller medications. A post hoc analysis was performed, pooling data from four double-blind, placebo-controlled studies randomising 3,073 moderate asthmatic adults to montelukast (n = 1,439), beclomethasone (n = 894) or placebo (n = 740). The primary end point was asthma control days; other end points were forced expiratory volume in one second, β-agonist use and nocturnal awakening. Analyses were conducted using BMI classification into normal (<25.0 kg·m−2; 52% of patients), overweight (25–29.9 kg·m−2; 32%) and obese (≥30.0 kg·m−2; 16%) categories, as well as BMI as a continuous variable. The treatment groups were balanced for BMI, demographic characteristics and parameters of asthma control. The placebo response for all end points was generally lower with increasing BMI. Similarly, the response to the inhaled corticosteroid decreased, whereas the response to the leukotriene antagonist remained stable. In conclusion, post hoc data from the present study suggested that body mass index may influence the natural history of asthma control (as reflected by response to placebo) and may differentially influence response to the two active agents, warranting explicit testing in future prospective studies.

Cultured lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis have a diminished capacity to synthesize prostaglandin E2 and to express cyclooxygenase-2.

Prostaglandin E2 (PGE2) inhibits fibroblast proliferation and collagen synthesis. In this study, we compared lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis (F-IPF) and from patients undergoing resectional surgery for lung cancer (F-nl) with respect to their capacity for PGE2 synthesis and their expression and regulation of cyclooxygenase (COX) proteins. Basal COX activity, assessed by quantitating immunoreactive PGE2 synthesized from arachidonic acid, was twofold less (P < 0.05) in F-IPF than F-nl. In F-nl, incubation with the agonists PMA, LPS, or IL-1 increased COX activity and protein expression of the inducible form of COX, COX-2, and these responses were inhibited by coincubation with dexamethasone. By contrast, F-IPF failed to demonstrate increases in COX-2 protein expression or COX activity in response to these agonists. Under conditions of maximal induction, COX activity in F-IPF was sixfold less than that in F-nl (P < 0.05). Our data indicate that F-IPF have a striking defect in their capacity to synthesize the antiinflammatory and antifibrogenic molecule PGE2, apparently because of a diminished induction of COX-2 protein. This reduction in the endogenous capacity of F-IPF to down-regulate their function via PGE2 may contribute to the inflammatory and fibrogenic response in IPF. Moreover, we believe that this represents the first description of a defect in COX-2 expression in association with a human disease.

Prostaglandin E2 Inhibits Alveolar Macrophage Phagocytosis through an E-Prostanoid 2 Receptor-Mediated Increase in Intracellular Cyclic AMP

Prostaglandin E2 is a potent lipid mediator of inflammation that effects changes in cell functions through ligation of four distinct G protein-coupled receptors (E-prostanoid (EP)1, EP2, EP3, and EP4). During pneumonia, PGE2 production is enhanced. In the present study, we sought to assess the effect of endogenously produced and exogenously added PGE2 on FcRγ-mediated phagocytosis of bacterial pathogens by alveolar macrophages (AMs), which are critical participants in lung innate immunity. We also sought to characterize the EP receptor signaling pathways responsible for these effects. PGE2 (1–1000 nM) dose-dependently suppressed the phagocytosis by rat AMs of IgG-opsonized erythrocytes, immune serum-opsonized Klebsiella pneumoniae, and IgG-opsonized Escherichia coli. Conversely, phagocytosis was stimulated by pretreatment with the cyclooxygenase inhibitor indomethacin. PGE2 suppression of phagocytosis was associated with enhanced intracellular cAMP production. Experiments using both forskolin (adenylate cyclase activator) and rolipram (phosphodiesterase IV inhibitor) confirmed the inhibitory effect of cAMP stimulation. Immunoblot analysis of rat AMs identified expression of only EP2 and EP3 receptors. The selective EP2 agonist butaprost, but neither the EP1/EP3 agonist sulprostone nor the EP4-selective agonist ONO-AE1-329, mimicked the effects of PGE2 on phagocytosis and cAMP stimulation. Additionally, the EP2 antagonist AH-6809 abrogated the inhibitory effects of both PGE2 and butaprost. We confirmed the specificity of our results by showing that AMs from EP2-deficient mice were resistant to the inhibitory effects of PGE2. Our data support a negative regulatory role for PGE2 on the antimicrobial activity of AMs, which has important implications for future efforts to prevent and treat bacterial pneumonia.

Leptin-Deficient Mice Exhibit Impaired Host Defense in Gram-Negative Pneumonia

Leptin is an adipocyte-derived hormone that is secreted in correlation with total body lipid stores. Serum leptin levels are lowered by the loss of body fat mass that would accompany starvation and malnutrition. Recently, leptin has been shown to modulate innate immune responses such as macrophage phagocytosis and cytokine synthesis in vitro. To determine whether leptin plays a role in the innate host response against Gram-negative pneumonia in vivo, we compared the responses of leptin-deficient and wild-type mice following an intratracheal challenge of Klebsiella pneumoniae. Following K. pneumoniae administration, we observed increased leptin levels in serum, bronchoalveolar lavage fluid, and whole lung homogenates. In a survival study, leptin-deficient mice, as compared with wild-type mice, exhibited increased mortality following K. pneumoniae administration. The increased susceptibility to K. pneumoniae in the leptin-deficient mice was associated with reduced bacterial clearance and defective alveolar macrophage phagocytosis in vitro. The exogenous addition of very high levels of leptin (500 ng/ml) restored the defect in alveolar macrophage phagocytosis of K. pneumoniae in vitro. While there were no differences between wild-type and leptin-deficient mice in lung homogenate cytokines TNF-α, IL-12, or macrophage-inflammatory protein-2 after K. pneumoniae administration, leukotriene synthesis in lung macrophages from leptin-deficient mice was reduced. Leukotriene production was restored by the addition of exogenous leptin (500 ng/ml) to macrophages in vitro. This study demonstrates for the first time that leptin-deficient mice display impaired host defense in bacterial pneumonia that may be due to a defect in alveolar macrophage phagocytosis and leukotriene synthesis.

Microsomal Prostaglandin E Synthase Is Regulated by Proinflammatory Cytokines and Glucocorticoids in Primary Rheumatoid Synovial Cells

The selective induction of PGE2 synthesis in inflammation suggests that a PGE synthase may be linked to an inducible pathway for PG synthesis. We examined the expression of the recently cloned inducible microsomal PGE synthase (mPGES) in synoviocytes from patients with rheumatoid arthritis, its modulation by cytokines and dexamethasone, and its linkage to the inducible cyclooxygenase-2. Northern blot analysis showed that IL-1β or TNF-α treatment induces mPGES mRNA from very low levels at baseline to maximum levels at 24 h. IL-1β-induced mPGES mRNA was inhibited by dexamethasone in a dose-dependent fashion. Western blot analysis demonstrated that mPGES protein was induced by IL-1β, and maximum expression was sustained for up to 72 h. There was a coordinated up-regulation of cyclooxygenase-2 protein, although peak expression was earlier. Differential Western blot analysis of the microsomal and the cytosolic fractions revealed that the induced expression of mPGES protein was limited to the microsomal fraction. The detected mPGES protein was catalytically functional as indicated by a 3-fold increase of PGES activity in synoviocytes following treatment with IL-1β; this increased synthase activity was limited to the microsomal fraction. In summary, these data demonstrate an induction of mPGES in rheumatoid synoviocytes by proinflammatory cytokines. This novel pathway may be a target for therapeutic intervention for patients with arthritis.

Arachidonic Acid Is Preferentially Metabolized by Cyclooxygenase-2 to Prostacyclin and Prostaglandin E2

The two cyclooxygenase isoforms, cyclooxygenase-1 and cyclooxygenase-2, both metabolize arachidonic acid to prostaglandin H2, which is subsequently processed by downstream enzymes to the various prostanoids. In the present study, we asked if the two isoforms differ in the profile of prostanoids that ultimately arise from their action on arachidonic acid. Resident peritoneal macrophages contained only cyclooxygenase-1 and synthesized (from either endogenous or exogenous arachidonic acid) a balance of four major prostanoids: prostacyclin, thromboxane A2, prostaglandin D2, and 12-hydroxyheptadecatrienoic acid. Prostaglandin E2 was a minor fifth product, although these cells efficiently converted exogenous prostaglandin H2 to prostaglandin E2. By contrast, induction of cyclooxygenase-2 with lipopol- ysaccharide resulted in the preferential production of prostacyclin and prostaglandin E2. This shift in product profile was accentuated if cyclooxygenase-1 was permanently inactivated with aspirin before cyclooxygenase-2 induction. The conversion of exogenous prostaglandin H2 to prostaglandin E2 was only modestly increased by lipopolysaccharide treatment. Thus, cyclooxygenase-2 induction leads to a shift in arachidonic acid metabolism from the production of several prostanoids with diverse effects as mediated by cyclooxygenase-1 to the preferential synthesis of two prostanoids, prostacyclin and prostaglandin E2, which evoke common effects at the cellular level.

Leukotrienes: underappreciated mediators of innate immune responses.

Leukotrienes are bronchoconstrictor and vasoactive lipid mediators that are targets in the treatment of asthma. Although they are increasingly recognized to exert broad proinflammatory effects, their role in innate immune responses is less well appreciated. These molecules are indeed synthesized by resident and recruited leukocytes during infection. Acting via cell surface G protein-coupled receptors and subsequent intracellular signaling events, they enhance leukocyte accumulation, phagocyte capacity for microbial ingestion and killing, and generation of other proinflammatory mediators. Interestingly, a variety of acquired states of immunodeficiency, such as HIV infection and malnutrition, are characterized by a relative deficiency of leukotriene synthesis. The data reviewed herein point to leukotrienes as underappreciated yet highly relevant mediators of innate immunity.

Evidence for tissue-resident mesenchymal stem cells in human adult lung from studies of transplanted allografts

The origin and turnover of connective tissue cells in adult human organs, including the lung, are not well understood. Here, studies of cells derived from human lung allografts demonstrate the presence of a multipotent mesenchymal cell population, which is locally resident in the human adult lung and has extended life span in vivo. Examination of plastic-adherent cell populations in bronchoalveolar lavage samples obtained from 76 human lung transplant recipients revealed clonal proliferation of fibroblast-like cells in 62% (106 of 172) of samples. Immunophenotyping of these isolated cells demonstrated expression of vimentin and prolyl-4-hydroxylase, indicating a mesenchymal phenotype. Multiparametric flow cytometric analyses revealed expression of cell-surface proteins, CD73, CD90, and CD105, commonly found on mesenchymal stem cells (MSCs). Hematopoietic lineage markers CD14, CD34, and CD45 were absent. Multipotency of these cells was demonstrated by their capacity to differentiate into adipocytes, chondrocytes, and osteocytes. Cytogenetic analysis of cells from 7 sex-mismatched lung transplant recipients harvested up to 11 years after transplant revealed that 97.2% +/- 2.1% expressed the sex genotype of the donor. The presence of MSCs of donor sex identity in lung allografts even years after transplantation provides what we believe to be the first evidence for connective tissue cell progenitors that reside locally within a postnatal, nonhematopoietic organ.

Cutting Edge: Macrophage Inhibition by Cyclic AMP (cAMP): Differential Roles of Protein Kinase A and Exchange Protein Directly Activated by cAMP-1

cAMP has largely inhibitory effects on components of macrophage activation, yet downstream mechanisms involved in these effects remain incompletely defined. Elevation of cAMP in alveolar macrophages (AMs) suppresses FcγR-mediated phagocytosis. We now report that protein kinase A (PKA) inhibitors (H-89, KT-5720, and myristoylated PKA inhibitory peptide 14–22) failed to prevent this suppression in rat AMs. We identified the expression of the alternative cAMP target, exchange protein directly activated by cAMP-1 (Epac-1), in human and rat AMs. Using cAMP analogs that are highly specific for PKA (N6-benzoyladenosine-3′,5′-cAMP) or Epac-1 (8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cAMP), we found that activation of Epac-1, but not PKA, dose-dependently suppressed phagocytosis. By contrast, activation of PKA, but not Epac-1, suppressed AM production of leukotriene B4 and TNF-α, whereas stimulation of either PKA or Epac-1 inhibited AM bactericidal activity and H2O2 production. These experiments now identify Epac-1 in primary macrophages, and define differential roles of Epac-1 vs PKA in the inhibitory effects of cAMP.

5-Lipoxygenase is located in the euchromatin of the nucleus in resting human alveolar macrophages and translocates to the nuclear envelope upon cell activation.

5-Lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) are two key proteins involved in the synthesis of leukotrienes (LT) from arachidonic acid. Although both alveolar macrophages (AM) and peripheral blood leukocytes (PBL) produce large amounts of LT after activation, 5-LO translocates from a soluble pool to a particulate fraction upon activation of PBL, but is contained in the particulate fraction in AM irrespective of activation. We have therefore examined the subcellular localization of 5-LO in autologous human AM and PBL collected from normal donors. While immunogold electron microscopy demonstrated little 5-LO in resting PBL, resting AM exhibited abundant 5-LO epitopes in the euchromatin region of the nucleus. The presence of substantial quantities of 5-LO in the nucleus of resting AM was verified by cell fractionation and immunoblot analysis and by indirect immunofluorescence microscopy. In both AM and PBL activated by A23187, all of the observable 5-LO immunogold labeling was found associated with the nuclear envelope. In resting cells of both types, FLAP was predominantly associated with the nuclear envelope, and its localization was not affected by activation with A23187. The effects of MK-886, which binds to FLAP, were examined in ionophore-stimulated AM and PBL. Although MK-886 inhibited LT synthesis in both cell types, it failed to prevent the translocation of 5-LO to the nuclear envelope. These results indicate that the nuclear envelope is the site at which 5-LO interacts with FLAP and arachidonic acid to catalyze LT synthesis in activated AM as well as PBL, and that in resting AM the euchromatin region of the nucleus is the predominant source of the translocated enzyme. In addition, LT synthesis is a two-step process consisting of FLAP-independent translocation of 5-LO to the nuclear envelope followed by the FLAP-dependent activation of the enzyme.