Christopher Haslett

The human cationic host defense peptide LL‐37 mediates contrasting effects on apoptotic pathways in different primary cells of the innate immune system

The human cathelicidin LL‐37 is a cationic host defense peptide (antimicrobial peptide) expressed primarily by neutrophils and epithelial cells. This peptide, up‐regulated under conditions of inflammation, has immunomodulatory and antimicrobial functions. We demonstrate that LL‐37 is a potent inhibitor of human neutrophil apoptosis, signaling through P2X7 receptors and G‐protein‐coupled receptors other than the formyl peptide receptor‐like‐1 molecule. This process involved modulation of Mcl‐1 expression, inhibition of BID and procaspase‐3 cleavage, and the activation of phosphatidylinositol‐3 kinase but not the extracellular signal‐regulated kinase 1/2 mitogen‐activated protein kinase pathway. In contrast to the inhibition of neutrophil apoptosis, LL‐37 induced apoptosis in primary airway epithelial cells, demonstrating alternate consequences of LL‐37‐mediated modulation of apoptotic pathways in different human primary cells. We propose that these novel immunomodulatory properties of LL‐37 contribute to peptide‐mediated enhancement of innate host defenses against acute infection and are of considerable significance in the development of such peptides and their synthetic analogs as potential therapeutics for use against multiple antibiotic‐resistant infectious diseases.

Nitric oxide: a key regulator of myeloid inflammatory cell apoptosis

AbstractApoptosis of inflammatory cells is a critical event in the resolution of inflammation, as failure to undergo this form of cell death leads to increased tissue damage and exacerbation of the inflammatory response. Many factors are able to influence the rate of apoptosis in neutrophils, eosinophils, monocytes and macrophages. Among these is the signalling molecule nitric oxide (NO), which possesses both anti- and proapoptotic properties, depending on the concentration and flux of NO, and also the source from which NO is derived. This review summarises the differential effects of NO on inflammatory cell apoptosis and outlines potential mechanisms that have been proposed to explain such actions.

Expression of the developmental Sonic hedgehog (Shh) signalling pathway is up-regulated in chronic lung fibrosis and the Shh receptor patched 1 is present in circulating T lymphocytes

During pulmonary development, Sonic hedgehog (Shh) and transforming growth factor β1 (TGF‐β1) signalling both contribute to branching morphogenesis. In interstitial lung disease, the complex alveolar structure of the lung is disrupted and remodelled, which leads to fibrosis, loss of respiratory surface, morbidity, and mortality. It is well documented that TGF‐β1 is involved in fibrosis. However, little is known about Shh signalling in damaged epithelia. This study examined whether or not components of the Shh signalling pathway, as well as TGF‐β1, are expressed in human fibrotic lung disease (cryptogenic fibrosing alveolitis and bronchiectasis) and in murine experimental models of fibrotic and non‐fibrotic chronic pulmonary inflammation. Using immunohistochemistry, it was observed that Shh, like TGF‐β1, is up‐regulated in epithelial cells at sites of fibrotic disease but not non‐fibrotic inflammation. The Shh receptor patched was detected in infiltrating mononuclear cells and alveolar macrophages, as well as normal resting peripheral blood T lymphocytes. Neither Shh nor patched is expressed by hyperproliferative goblet cells in inflammatory epithelium. This study demonstrates that patched is present in human peripheral CD4 and CD8 lymphocytes at both protein and mRNA levels. Taken together, these results suggest that components of the highly conserved Shh signalling pathway may play a role in the remodelling of damaged pulmonary epithelium and that damaged epithelium and cells of the immune system may communicate via this pathway. Copyright

Kinetics of pulmonary neutrophil recruitment and clearance in a natural and spontaneously resolving model of airway inflammation

Background Neutrophil apoptosis and phagocytic clearance have been proposed as key determinants affecting the resolution of airway inflammation.

Regulation of Pulmonary and Systemic Bacterial Lipopolysaccharide Responses in Transgenic Mice Expressing Human Elafin

ABSTRACT The control of lung inflammation is of paramount importance in a variety of acute pathologies, such as pneumonia, the acute respiratory distress syndrome, and sepsis. It is becoming increasingly apparent that local innate immune responses in the lung are negatively influenced by systemic inflammation. This is thought to be due to a local deficit in cytokine responses by alveolar macrophages and neutrophils following systemic bacterial infection and the development of a septic response. Recently, using an adenovirus-based strategy which overexpresses the human elastase inhibitor elafin locally in the lung, we showed that elafin is able to prime lung innate immune responses. In this study, we generated a novel transgenic mouse strain expressing human elafin and studied its response to bacterial lipopolysaccharide (LPS) when the LPS was administered locally in the lungs and systemically. When LPS was delivered to the lungs, we found that mice expressing elafin had lower serum-to-bronchoalveolar lavage ratios of proinflammatory cytokines, including tumor necrosis factor alpha (TNF-α), macrophage inflammatory protein 2, and monocyte chemoattractant protein 1, than wild-type mice. There was a concomitant increase in inflammatory cell influx, showing that there was potential priming of innate responses in the lungs. When LPS was given systemically, the mice expressing elafin had reduced levels of serum TNF-α compared to the levels in wild-type mice. These results indicate that elafin may have a dual function, promoting up-regulation of local lung innate immunity while simultaneously down-regulating potentially unwanted systemic inflammatory responses in the circulation.

Regulation of neutrophil apoptosis and removal of apoptotic cells.

The accumulation of neutrophils during inflammation is essential for the destruction and removal of invading microorganisms. However, for resolution of inflammation to occur, neutrophils must also be removed from the inflammatory site since these cells are capable of releasing tissue toxic molecules. Neutrophil removal has been shown to occur via apoptosis and phagocyte clearance of apoptotic cells. Therefore, manipulation of these processes is likely to be a key therapeutic strategy in the management of inflammatory disease. In this review, we examine mediators of neutrophil survival and apoptosis and the signalling pathways that regulate the balance between life and death in these cells.

Interferon gamma suppresses glucocorticoid augmentation of macrophage clearance of apoptotic cells

One of beneficial effects of glucocorticoids (GC) in inflammation may be the augmentation of macrophages capacity for phagocytosis of apoptotic cells, a process that has a central role in resolution of inflammation. Here we define the phenotype of GC‐treated monocyte‐derived macrophages, comparing to IFN‐γ‐treated and IL‐4‐treated monocyte‐derived macrophages and combinatorial treatment. Our data indicate that the cytokine microenvironment at an inflammatory site will critically determine monocyte functional capacity following treatment with GC. In particular, whilst GC exert dominant regulatory effects over IFN‐γ in terms of cell surface receptor repertoire and morphology, the acquisition of a macrophage capacity for clearance of apoptotic cells is prevented bycombined treatment. In terms of mechanism, GC augmentation of phagocytosis was reversed even when monocytes were pre‐incubated with GC for the first 24u2004h of culture, a period that is critical for induction of a highly phagocytic macrophage phenotype. These findings have important implications for the effectiveness of GC in promoting acquisition of a pro‐phagocytic macrophage phenotype in inflammatory diseases associated with high levels of IFN‐γu2009

Role of leukotrienes in the regulation of human granulocyte behaviour: dissociation between agonist-induced activation and retardation of apoptosis

Since most inflammatory mediators that stimulate granulocyte responsiveness also delay apoptosis, it is often assumed that activation and longevity are causally related. Using isolated human peripheral blood neutrophils and eosinophils, we examined this association by exploiting the proinflammatory lipid mediators, the leukotrienes (LTs), and investigated granulocyte function and apoptosis. LTB4 induced elevation of intracellular free Ca2+ concentration ([Ca2+]i), cell polarisation and retardation of neutrophil apoptosis, although the antiapoptotic effect occurred only at concentrations 300 nM. LTB4‐induced activation was attenuated by CP‐105,696, a BLT1‐specific antagonist suggesting classical LTB4 receptor BLT1 involvement. Despite demonstrating the presence of the neutrophil intracellular LTB4 receptor peroxisome‐proliferator activator receptor‐α (PPARα) in neutrophils, the selective PPARα agonist WY‐14,643 did not mimic LTB4‐induced prosurvival effects. LTB4‐induced survival, however, also appeared to be mediated by BLT1 since CP‐105,696 inhibited the LTB4‐mediated antiapoptotic effect. Furthermore, based on studies with CP‐105,696 and 5‐lipoxygenase inhibitors, lipopolysaccharide (LPS)‐, granulocyte–macrophage colony‐stimulating factor (GM‐CSF)‐, dexamethasone‐ and dibutyryl‐cAMP (db‐cAMP)‐induced delay of neutrophil apoptosis did not involve autocrine production of LTB4. Although LTB4 and LTD4 induced human eosinophil [Ca2+]i elevation and polarization, these LTs did not influence eosinophil apoptosis. Furthermore, LTB4‐ and LTD4‐induced eosinophil activation was attenuated by CP‐105,696 and the Cys‐LT1 receptor antagonist montelukast, respectively, highlighting specific receptor dependency. Thus, mediator‐triggered granulocyte activation and antiapoptotic pathways are distinct events that can be differentially regulated.

Expression of V1A and GRP receptors leads to cellular transformation and increased sensitivity to substance-P analogue-induced growth inhibition

Small-cell lung cancer (SCLC) is a particularly aggressive cancer, which metastasises early. Despite initial sensitivity to radio- and chemo-therapy, it invariably relapses, so that the 2-year survival remains less than 5%. Neuropeptides particularly arginine vasopressin (AVP) and gastrin-releasing peptide (GRP) act as autocrine and paracrine growth factors and the expression of these and their receptors are a hallmark of the disease. Substance-P analogues including [D-Arg1,D-Phe5,D-Trp7,9, Leu11]-substance-P (SP-D) and [Arg6,D-Trp7,9,NmePhe8]-substance-P (6–11) (SP-G) inhibit the growth of SCLC cells by modulating neuropeptide signalling. We show that GRP and V1A receptors expression leads to the development of a transformed phenotype. Addition of neuropeptide provides some protection from etoposide-induced cytotoxicity. Receptor expression also leads to an increased sensitivity to substance-P analogue-induced growth inhibition. We show that SP-D and SP-G act as biased agonists at GRP and V1A receptors causing blockade of Gq-mediated Ca2+ release while directing signalling to activate ERK via a pertussis toxin-sensitive pathway. This is the first description of biased agonism at V1A receptors. This unique pharmacology governs the antiproliferative properties of these agents and highlights their potential therapeutic potential for the treatment of SCLC and particularly in tumours, which have developed resistance to chemotherapy.

Regulation of granulocyte apoptosis by hemopoietic growth factors, cytokines and drugs: potential relevance to allergic inflammation.

It has become apparent that the resolution of inflammation depends on the removal of unwanted inflammatory cells, a process governed by physiological apoptosis and non-inflammatory clearance of apoptotic cells. Granulocytes are central to many of the pathophysiological consequences of uncontrolled inflammatory reactions. Hemopoietic factors and cytokines play a critical role in regulating the longevity of these cells in vitro and in vivo. Here we review the progress that has been made in the understanding of granulocyte apoptosis and the implications for immunotherapy and pharmacological strategies in the treatment of allergic inflammatory diseases for therapeutic gain.