Malcolm Shepherd

IL-33 Amplifies the Polarization of Alternatively Activated Macrophages That Contribute to Airway Inflammation

Alternatively activated macrophages (AAM) play a crucial role in type 2 immunity. Mice deficient in ST2, a receptor for the latest member of the IL-1 family, IL-33, have impaired type 2 immune responses. We therefore reasoned that IL-33/ST2 signaling may be involved in the differentiation and activation of AAM during airway inflammation. We report here that IL-33 changed the quiescent phenotype of alveolar macrophages toward an AAM phenotype that expressed mannose receptor, IL-4Rα, and produced high levels of CCL24 and CCL17 in an IL-13-dependent manner during IL-33-induced airway inflammation. Neutralization of AAM-derived CCL24 led to an amelioration of IL-33-induced eosinophilia in the lungs. Moreover, depletion of alveolar macrophages reduced IL-33-induced airway inflammation. Additionally, the attenuated OVA-induced airway inflammation in ST2−/− mice was associated with a decrease in AAM differentiation. In vitro, IL-33 amplified IL-13-induced polarization of alveolar- and bone marrow-derived macrophage toward an AAM phenotype by increasing the expression of arginase I, Ym1, as well as the production of CCL24 and CCL17. IL-13/IL-4Rα signaling was crucial for IL-33-driven AAM amplification by inducing the expression of ST2L. Finally, we showed that IL-33 was more abundantly expressed in the lung epithelial cells of asthma patients than those from healthy controls, suggesting that IL-33 may be involved in lung macrophage activation in clinical asthma. Taken together, we demonstrate here that IL-33/ST2 plays a significant role in the amplification of AAM polarization and chemokine production which contribute to innate and Ag-induced airway inflammation.

Remodelling of the PDE4 cAMP phosphodiesterase isoform profile upon monocyte-macrophage differentiation of human U937 cells

Monocytes and macrophages provide key targets for the action of novel anti‐inflammatory therapeutics targeted at inhibition of PDE4 cAMP‐specific phosphodiesterases. PDE4 enzymes provide the dominant cAMP phosphodiesterase activity in U937 human monocytic cells. Differentiation of U937 monocytic cells to a macrophage‐like phenotype causes a marked reduction in total cellular PDE4 activity. Monocytic U937 cells express the long PDE4A4, PDE4D5 and PDE4D3 isoforms plus the short PDE4B2 isoform. Differentiation of U937 cells to a macrophage‐like phenotype causes a marked downregulation of PDE4D3 and PDE4D5, elicits a marked upregulation of PDE4B2 and induces the novel PDE4A10 long isoform. Comparable patterns are found in human peripheral blood monocytes and macrophages differentiated from them. Immunopurification of PDE4 subfamilies identifies long PDE4D isoforms as providing the major PDE4 activity in U937 monocytic cells. In U937 macrophage‐like cells, the activity of the short PDE4B2 isoform predominates. No indication of either the expression or induction of PDE4C was evident. Activation of ERK exerts an inhibitory effect on total PDE4 activity in monocytic U937 cells, where the activity of long PDE4 isoforms predominates. The effect of ERK activation is switched to one of overall stimulation of total PDE4 activity in macrophage U937 cells, where the activity of the short PDE4B2 isoform predominates. The profound differentiation‐induced changes in PDE4 isoform profile identified here suggests that the development of inhibitors specific for particular PDE4 isoforms may allow for selective effects on monocytes and macrophages to be achieved.

Ion channel regulation of intracellular calcium and airway smooth muscle function

Airway hyper-responsiveness associated with asthma is mediated by airway smooth muscle cells (SMCs) and has a complicated etiology involving increases in cell contraction and proliferation and the secretion of inflammatory mediators. Although these pathological changes are diverse, a common feature associated with their regulation is a change in intracellular Ca(2+) concentration ([Ca(2+)](i)). Because the [Ca(2+)](i) itself is a function of the activity and expression of a variety of ion channels, in both the plasma membrane and sarcoplasmic reticulum of the SMC, the modification of this ion channel activity may predispose airway SMCs to hyper-responsiveness. Our objective is to review how ion channels determine the [Ca(2+)](i) and influence the function of airway SMCs and emphasize the potential of ion channels as sites for therapeutic approaches to asthma.

The role of mast cells and their mediators in reproduction, pregnancy and labour

BACKGROUND Mast cells (MCs) are the classical mediators of allergy, however, their importance in the development of innate and adaptive immune responses is increasingly being recognized. Herein, the present MC literature is summarized, with particular focus on studies of MCs in the endometrium and myometrium, and their involvement in fertility, implantation, pregnancy and labour. METHODS Recent developments in MC biology were identified by systematic searches of PubMed, Medline and Google Scholar from 2000 to November 2009. To specifically examine the role of MCs in fertility and pregnancy, we then performed a systematic review of English literature cited in the PubMed, Medline and Google Scholar databases, but extended the search period, from 1980 to January 2010 RESULTS MCs can respond to immunoglobulin E-independent innate immune stimuli and are present within the endometrium, with activation and release of mediators occurring prior to menstruation and in association with endometriosis. With respect to pregnancy, MCs are redundant during blastocyst implantation and although their mediators can induce myometrial contractility, there is no epidemiological link of preterm birth with allergy, suggesting a non-essential role or robust regulation. In males, MCs are present in the testes and are increased in oligo- and azoospermia, with MC mediators directly suppressing sperm motility in a potentially reversible manner. CONCLUSIONS MCs are prevalent in the female and male reproductive tract. However, whether MCs are absolutely required for a successful pregnancy or are fundamental to reproductive pathology, and thereby a therapeutic target, remains to be determined.

Corticosteroid Insensitivity in Smokers with Asthma: Clinical Evidence, Mechanisms, and Management

Corticosteroids are the most effective treatment for asthma, but the therapeutic response varies considerably between individuals. Several clinical studies have found that smokers with asthma are insensitive to the beneficial effects of short- to medium-term inhaled corticosteroid treatment compared with non-smokers with asthma. It is estimated that 25% of adults in most industrialized countries smoke cigarettes, and similar surveys amongst asthmatic individuals suggest that the prevalence of smoking in this grouping mirrors that found in the general population. Therefore, cigarette smoking is probably the most common cause of corticosteroid insensitivity in asthma. Cigarette smoking and asthma are also associated with poor symptom control and an accelerated rate of decline in lung function. The mechanism of corticosteroid insensitivity in smokers with asthma is currently unexplained but could be due to alterations in airway inflammatory cell phenotypes, changes in glucocorticoid receptor α/β ratio, and/or reduced histone deacetylase activity. Smoking cessation should be encouraged in all smokers with asthma. Short-term benefits include improvements in lung function and asthma control. However, the numbers of sustained quitters is disappointingly small. Additional or alternative drugs need to be identified to treat those individuals who are unable to stop smoking or who have persistent symptoms following smoking cessation.

Peripheral blood dendritic cell subtypes are significantly elevated in subjects with asthma

Background Dendritic cells (DCs) are crucial for the processing of antigens, T lymphocyte priming and the development of asthma and allergy. Smokers with asthma display altered therapeutic behaviour and a reduction in endobronchial DC CD83 expression compared with non‐smokers with asthma. No information is available on the impact of smoking on peripheral blood DC profiles.

Mast cells reside in myometrium and cervix, but are dispensable in mice for successful pregnancy and labor

Parturition is associated with myometrial and cervical inflammation. The causes and consequences of this inflammatory response are not clear. Mast cells (MCs) are important inducers of allergic and non‐allergic inflammation, and their secreted products can induce myometrial contractions. Thus, mast cell activation has been hypothesized to have a role in initiating labor and/or driving labor‐associated inflammation. We report that small numbers of MCs expressing chymase and tryptase are present in the myometrium and cervix of pregnant women. Labor did not lead to any change in mast cell abundance in these tissues, but was associated with reduced expression of the mast‐cell regulator FcεR1A, indicative of a change in mast cell properties. This coincided with contraction‐dependent myocyte production of interleukin‐10 (IL‐10), a known suppressor of FcεR1A expression. MCs were also found in the uterine horn and cervical region of pregnant C57BL/6 mice, increasing in number in the cervix, but not the myometrium, with labor. As expected, these cells were absent from mast‐cell‐deficient KitW−sh mice. Nonetheless, pregnant KitW−sh mice showed no defects in the timing of labor induction or in the upregulation of leukocyte markers during labor. Thus, MCs are present in the uterus and cervix of humans and mice, and our mouse studies suggest that they do not have a vital role in the induction of labor, or in the promotion of labor‐associated inflammation.

Functional Expression of IgG-Fc Receptors in Human Airway Smooth Muscle Cells

IgE-Fc receptors and IgG-Fc receptors are expressed on hematopoietic cells, but some evidence suggests that these receptors are also found on nonhematopoietic cells, including human airway smooth muscle (hASM) cells. Our study characterizes the expression of IgE-Fc receptors (FcεRI/CD23) and IgG-Fc receptors (FcγRs-I, -II, and -III) in cultured hASM cells by flow cytometry and Western blotting, and the functional activity of receptors was determined through quantification of cell proliferation and released cytokines. Expression of Fc receptor-linked intracellular signaling proteins and phosphorylation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 1/2 and p38(MAPK) in hASM cells was examined by Western blotting. Expression of FcεRI and CD23 was not detectable in hASM cells. However, FcγRI and FcγRII were shown to be expressed on these cells. Specific antibodies, validated using transfected cell lines, revealed that the inhibitory IgG receptor, FcγRIIb, was the most abundant Fc receptor subtype expressed. Although cross-linking FcγR with heat-aggregated γ globulin (HAGG) did not induce detectable cell stimulation, pretreating hASM cells with HAGG significantly inhibited IL-1α-induced increases in cytokine levels and basic fibroblast growth factor-induced cell proliferation. This inhibitory effect of HAGG was abrogated by preincubation of cells with an anti-FcγRIIb antigen-binding fragment (Fab). Expression of proteins involved in the canonical FcγRIIb inhibitory signaling pathway was established in hASM cells. Pretreatment of hASM cells with HAGG significantly inhibited IL-1α- and basic fibroblast growth factor-induced extracellular signal-regulated kinase 1/2 and p38(MAPK) phosphorylation. This study identifies functional expression of FcγRIIb in hASM cells, with the potential to suppress their remodeling and immunomodulatory roles.

Effectiveness of bronchial thermoplasty in severe asthma in ‘real life’ patients compared with those recruited to clinical trials in the same centre:

Published information on the effectiveness of bronchial thermoplasty (BT) for severe asthma in ‘real life’ patients is limited. We compared safety and efficacy outcomes 12 months post procedure in 10 clinic patients and 15 patients recruited to clinical trials of BT at the same centre. Baseline asthma severity was greater in the clinic group. Adverse events were similar. Clinical improvements occurred in 50% of the clinic patients compared with 73% of the research patients.

Arachidonic acid metabolites and enzyme transcripts in asthma are altered by cigarette smoking

Arachidonic acid metabolites are implicated in the pathogenesis of asthma although only limited information is available on the impact of current smoking history on these metabolites. The aim of the study was to examine the effect of smoking status on urinary, sputum, and plasma eicosanoid concentrations and relevant enzyme transcripts in asthma.