J. Kelley Bentley

The Calmodulin-dependent Phosphodiesterase Gene PDE1C Encodes Several Functionally Different Splice Variants in a Tissue-specific Manner

We report here the identification of cDNAs for three new mouse PDE1C splice variants and the characterization of their kinetics, regulation by Ca2+, sensitivities to inhibitors, and tissue/cellular expression patterns. Sequence analysis indicated that these three cDNAs (PDE1C1, PDE1C4, and PDE1C5), together with our previously reported PDE1C2 and PDE1C3, are alternative splice products of the PDE1C gene. The results from RNase protection analysis and in situ hybridization indicated that the expression of the different PDE1C splice variants is differentially regulated in a tissue/cell-specific manner. Particularly, high levels of PDE1C mRNAs were found in the olfactory epithelium, testis, and several regions of mouse brain such as cerebellar granule cells. All of these splice variants have similar kinetic properties, showing high affinities and approximately the same relative Vmax values for both cAMP and cGMP. However, they responded to Ca2+ stimulation differently. In addition, they show different sensitivities to the calmodulin-dependent phosphodiesterase inhibitors, KS505a and SCH51866. Substrate competition experiments suggested the presence of only one catalytic site on these PDE1C isozymes for both cAMP and cGMP. In summary, these findings suggest that the PDE1C gene undergoes tissue-specific alternative splicing that generates structurally and functionally diverse gene products.

Periostin promotes fibrosis and predicts progression in patients with idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease without effective therapeutics. Periostin has been reported to be elevated in IPF patients relative to controls, but its sources and mechanisms of action remain unclear. We confirm excess periostin in lungs of IPF patients and show that IPF fibroblasts produce periostin. Blood was obtained from 54 IPF patients (all but 1 with 48 wk of follow-up). We show that periostin levels predict clinical progression at 48 wk (hazard ratio = 1.47, 95% confidence interval = 1.03-2.10, P < 0.05). Monocytes and fibrocytes are sources of periostin in circulation in IPF patients. Previous studies suggest that periostin may regulate the inflammatory phase of bleomycin-induced lung injury, but periostin effects during the fibroproliferative phase of the disease are unknown. Wild-type and periostin-deficient (periostin(-/-)) mice were anesthetized and challenged with bleomycin. Wild-type mice were injected with bleomycin and then treated with OC-20 Ab (which blocks periostin and integrin interactions) or control Ab during the fibroproliferative phase of disease, and fibrosis and survival were assessed. Periostin expression was upregulated quickly after treatment with bleomycin and remained elevated. Periostin(-/-) mice were protected from bleomycin-induced fibrosis. Instillation of OC-20 during the fibroproliferative phase improved survival and limited collagen deposition. Chimeric mouse studies suggest that hematopoietic and structural sources of periostin contribute to lung fibrogenesis. Periostin was upregulated by transforming growth factor-β in lung mesenchymal cells, and periostin promoted extracellular matrix deposition, mesenchymal cell proliferation, and wound closure. Thus periostin plays a vital role in late stages of pulmonary fibrosis and is a potential biomarker for disease progression and a target for therapeutic intervention.

H. influenzae potentiates airway epithelial cell responses to rhinovirus by increasing ICAM-1 and TLR3 expression

Rhinovirus (RV) is an important trigger of chronic obstructive pulmonary disease (COPD) exacerbations. In addition, respiratory viruses are more likely to be isolated in patients with a history of frequent exacerbations, suggesting that these patients are more susceptible to viral infection. To examine potential mechanisms for cooperative effects between bacterial and viral infection in COPD, we studied the responses of cultured human airway epithelial cells to nontypeable Hemophilus influenzae and RV. In both 16HBE14o‐ and primary mucociliary‐differentiated cells, preincubation with H. influenzae enhanced RV serotype 39‐induced protein expression of interleukin (IL)‐8, epithelial‐derived neutrophil attractant‐78, and growth‐related oncogene‐α. H. influenzae infection also increased the binding of RV39 to cultured cells, as well as expression of intercellular adhesion molecule (ICAM)‐1 and Toll‐like receptor (TLR)‐3, receptors for RV and dsRNA, respectively. Neutralizing antibody against tumor necrosis factor‐ inhibited IL‐8 expression induced by H. influenzae and RV39. Finally, siRNA against TLR3 attenuated RV‐induced IL‐8 expression. We conclude that H. influenzae infection increases airway epithelial cell ICAM‐1 and TLR3 expression, leading to enhanced binding of RV and a potentiation of RV‐induced chemokine release. These data provide a cellular mechanism by which H. influenzae infection may increase the susceptibility of COPD patients to RV‐induced exacerbations.—Sajjan, U. S., Jia, Y., Newcomb, D. C., Bentley, J. K., Lukacs, N. W., LiPuma, J. J., Hershenson, M. B. H. influenzae potentiates airway epithelial cell responses to rhinovirus by increasing ICAM‐1 and TLR3 expression FASEB J. 20, E1419 –E1429 (2006)

Human Rhinovirus 1B Exposure Induces Phosphatidylinositol 3-Kinase-dependent Airway Inflammation in Mice

RATIONALE Infection with rhinovirus (RV) triggers exacerbations of asthma and chronic obstructive lung disease. OBJECTIVES We sought to develop a mouse model of RV employing RV1B, a minor group serotype that binds to the low-density lipoprotein receptor. METHODS C57BL/6 mice were inoculated intranasally with RV1B, replication-deficient ultraviolet (UV)-irradiated RV1B, or RV39, a major group virus. MEASUREMENTS AND MAIN RESULTS Viral RNA was present in the lungs of RV1B-treated mice, but not in those exposed to UV-irradiated RV1B or RV39. Lung homogenates of RV-treated mice contained infectious RV 4 days after inoculation. RV1B exposure induced neutrophilic and lymphocytic airway inflammation, as well as increased lung expression of KC, macrophage-inflammatory protein-2, and IFN-alpha and IFN-beta. RV1B-exposed mice showed airway hyperresponsiveness 1 and 4 days after inoculation. UV-irradiated RV1B induced modest neutrophilic airway inflammation and hyperresponsiveness 1 day after exposure. Both RV1B and UV-irradiated RV1B, but not RV39, increased lung phosphorylation of Akt. Confocal immunofluorescence showed colocalization of RV1B and phospho-Akt in the airway epithelium. Finally, pretreatment with the phosphatidylinositol 3-kinase inhibitor LY294002 attenuated chemokine production and neutrophil infiltration. CONCLUSIONS We conclude that RV1B induces airway inflammation in vivo. Evidence is presented that viral replication occurs in vivo and is required for maximal responses. On the other hand, viral replication was not required for a subset of RV-induced responses, including neutrophilic inflammation, airway hyperresponsiveness, and Akt phosphorylation. Finally, phosphatidylinositol 3-kinase/Akt signaling is required for maximal RV1B-induced airway neutrophilic inflammation, likely via its essential role in virus internalization.

Phosphatidylinositol 3-Kinase Is Required for Rhinovirus-induced Airway Epithelial Cell Interleukin-8 Expression

Rhinovirus (RV) is a common cause of asthma exacerbations. The signaling mechanisms regulating RV-induced airway epithelial cell responses have not been well studied. We examined the role of phosphatidylinositol (PI) 3-kinase in RV-induced interleukin (IL)-8 expression. Infection of 16HBE14o- human bronchial epithelial cells with RV39 induced rapid activation of PI 3-kinase and phosphorylation of Akt, a downstream effector of PI 3-kinase. RV39 also colocalized with cit-Akt-PH, a citrogen-tagged fluorescent fusion protein encoding the pleckstrin homology domain of Akt, indicating that 3-phosphorylated PI accumulates at the site of RV infection. Inhibition of PI 3-kinase and Akt attenuated RV39-induced NF-κB transactivation and IL-8 expression. Inhibition of PI 3-kinase also blocked internalization of labeled RV39 into 16HBE14o- cells, suggesting that the requirement of PI 3-kinase for RV39-induced IL-8 expression, at least in part, relates to its role in viral endocytosis.

MDA5 and TLR3 initiate pro-inflammatory signaling pathways leading to rhinovirus-induced airways inflammation and hyperresponsiveness.

Rhinovirus (RV), a single-stranded RNA picornavirus, is the most frequent cause of asthma exacerbations. We previously demonstrated in human bronchial epithelial cells that melanoma differentiation-associated gene (MDA)-5 and the adaptor protein for Toll-like receptor (TLR)-3 are each required for maximal RV1B-induced interferon (IFN) responses. However, in vivo, the overall airway response to viral infection likely represents a coordinated response integrating both antiviral and pro-inflammatory pathways. We examined the airway responses of MDA5- and TLR3-deficient mice to infection with RV1B, a minor group virus which replicates in mouse lungs. MDA5 null mice showed a delayed type I IFN and attenuated type III IFN response to RV1B infection, leading to a transient increase in viral titer. TLR3 null mice showed normal IFN responses and unchanged viral titers. Further, RV-infected MDA5 and TLR3 null mice showed reduced lung inflammatory responses and reduced airways responsiveness. Finally, RV-infected MDA5 null mice with allergic airways disease showed lower viral titers despite deficient IFN responses, and allergic MDA5 and TLR3 null mice each showed decreased RV-induced airway inflammatory and contractile responses. These results suggest that, in the context of RV infection, binding of viral dsRNA to MDA5 and TLR3 initiates pro-inflammatory signaling pathways leading to airways inflammation and hyperresponsiveness.

Rhinovirus Infection of Allergen-Sensitized and -Challenged Mice Induces Eotaxin Release from Functionally Polarized Macrophages

Human rhinovirus is responsible for the majority of virus-induced asthma exacerbations. To determine the immunologic mechanisms underlying rhinovirus (RV)-induced asthma exacerbations, we combined mouse models of allergic airways disease and human rhinovirus infection. We inoculated OVA-sensitized and challenged BALB/c mice with rhinovirus serotype 1B, a minor group strain capable of infecting mouse cells. Compared with sham-infected, OVA-treated mice, virus-infected mice showed increased lung infiltration with neutrophils, eosinophils and macrophages, airway cholinergic hyperresponsiveness, and increased lung expression of cytokines including eotaxin-1/CCL11, IL-4, IL-13, and IFN-γ. Administration of anti–eotaxin-1 attenuated rhinovirus-induced airway eosinophilia and responsiveness. Immunohistochemical analysis showed eotaxin-1 in the lung macrophages of virus-infected, OVA-treated mice, and confocal fluorescence microscopy revealed colocalization of rhinovirus, eotaxin-1, and IL-4 in CD68-positive cells. RV inoculation of lung macrophages from OVA-treated, but not PBS-treated, mice induced expression of eotaxin-1, IL-4, and IL-13 ex vivo. Macrophages from OVA-treated mice showed increased expression of arginase-1, Ym-1, Mgl-2, and IL-10, indicating a shift in macrophage activation status. Depletion of macrophages from OVA-sensitized and -challenged mice reduced eosinophilic inflammation and airways responsiveness following RV infection. We conclude that augmented airway eosinophilic inflammation and hyperresponsiveness in RV-infected mice with allergic airways disease is directed in part by eotaxin-1. Airway macrophages from mice with allergic airways disease demonstrate a change in activation state characterized in part by altered eotaxin and IL-4 production in response to RV infection. These data provide a new paradigm to explain RV-induced asthma exacerbations.

Neonatal rhinovirus induces mucous metaplasia and airways hyperresponsiveness through IL-25 and type 2 innate lymphoid cells.

BACKGROUND Early-life human rhinovirus infection has been linked to asthma development in high-risk infants and children. Nevertheless, the role of rhinovirus infection in the initiation of asthma remains unclear. OBJECTIVE We hypothesized that, in contrast to infection of mature BALB/c mice, neonatal infection with rhinovirus promotes an IL-25-driven type 2 response, which causes persistent mucous metaplasia and airways hyperresponsiveness. METHODS Six-day-old and 8-week-old BALB/c mice were inoculated with sham HeLa cell lysate or rhinovirus. Airway responses from 1 to 28 days after infection were assessed by using quantitative PCR, ELISA, histology, immunofluorescence microscopy, flow cytometry, and methacholine responsiveness. Selected mice were treated with a neutralizing antibody to IL-25. RESULTS Compared with mature mice, rhinovirus infection in neonatal mice increased lung IL-13 and IL-25 production, whereas IFN-γ, IL-12p40, and TNF-α expression was suppressed. In addition, the population of IL-13-secreting type 2 innate lymphoid cells (ILC2s) was expanded with rhinovirus infection in neonatal but not mature mice. ILC2s were the major cell type secreting IL-13 in neonates. Finally, anti-IL-25 neutralizing antibody attenuated ILC2 expansion, mucous hypersecretion, and airways responsiveness. CONCLUSIONS These findings suggest that early-life viral infection could contribute to asthma development by provoking age-dependent, IL-25-driven type 2 immune responses.

Regulation and function of cyclic nucleotides.

Recent work has greatly expanded our knowledge of the structure, regulation and diversity of enzymes involved in the synthesis and degradation of cyclic nucleotides. This review focuses on recent work that provides insight into the structure and function of the cyclases and phosphodiesterases that regulate cyclic nucleotide metabolism. Particular emphasis is given to the roles played by multiple isoforms of each enzyme system.

Spermatozoa contain a guanine nucleotide-binding protein ADP-ribosylated by pertussis toxin

Spermatozoa from invertebrates (sea urchin, starfish) and vertebrates (trout, guinea pig, bull, pig, human) contain a membrane-bound protein that is ADP-ribosylated by pertussis toxin but not by cholera toxin. The Mr of this protein is 39,000 in invertebrate sperm and 41,000 in mammalian sperm, but 40,000 in trout spermatozoa. The pertussis toxin substrate from sea urchin sperm copurified with [gamma-35S]GTP binding activity. Chymotryptic maps of this ADP-ribosylated protein from sea urchin sperm were the same as those of alpha-subunit of Go from rat brain. Antiserum to the beta-subunit of bovine retinal transducin bound to a sperm protein with Mr approximately 35,000. These studies are the first describing a guanine nucleotide-binding coupling protein in sperm.