A novel murine model of pulmonary fibrosis: the role of platelets in chronic changes induced by bleomycin.

Idiopathic pulmonary fibrosis (IPF) is a disease that causes scarring and destruction of lung tissue that is ultimately fatal. There is a need to develop improved treatments for IPF. One problem with identifying novel treatments of IPF is the poor predictability of current preclinical models. Few model investigate lung function changes, rather relying on histological changes which doesn't adequately reflect the complete clinical situation. The aim of this study was to establish a novel model of pulmonary fibrosis where we could investigate changes in lung function, and histology. We have also utilised this model to investigate the role of platelets in pulmonary fibrosis as platelets have been recognised as having a broader role than just facilitating haemostasis. Lung fibrosis was induced in male C57BL6/J mice by intranasal bleomycin on Days 0, 1, 2, 5, 6 and 7. Platelets were depleted by twice-weekly administration of anti-platelet antibodies. On Day 35 mice were assessed by examining lung function, platelet infiltration into lung tissues and bronchoalveolar lavage fluid (BAL), levels of BAL Tissue growth factor (TGF)-ß levels, and the degree of fibrosis evaluated histologically. Repeated bleomycin administration caused loss of lung function associated with fibrosis assessed histologically. Platelet depletion resulted in a reduction in fibrosis and modest inhibition of lung function changes. We have established a novel model of pulmonary fibrosis that is associated with a decline in lung function similar to the clinical setting. Furthermore, platelet depletion resulted in a less severe fibrosis suggesting that targeting platelets maybe worth further investigation.

Radiolabelling and immunohistochemistry reveal platelet recruitment into lungs and platelet migration into airspaces following LPS inhalation in mice.

INTRODUCTION: Platelets are under investigation for their role in host defence and inflammatory lung diseases and have been demonstrated to be recruited to the lung. However, the mechanisms and consequences of platelet recruitment into lungs are poorly understood. We have utilised a murine model to investigate the mechanisms of platelet involvement in lung inflammation induced by intranasal administration of LPS. OBJECTIVES: Our aim was to characterise lung platelet recruitment following LPS inhalation in mice using immunohistochemistry, and non-invasive and invasive radiolabelled platelet tracking techniques. RESULTS: Intranasal administration of LPS caused an increase in lung platelet staining in lung tissue and elicited the recruitment of radiolabelled platelets into the lung. Prior to these responses in the lung, we observed an earlier decrease in blood platelet counts, temporally associated with platelet recruitment to the liver and spleen. Non-invasive measurements of thoracic radioactivity reflected changes in blood counts rather than extravascular lung platelet recruitment. However, both in situ counting of radiolabelled platelets and immunostaining for platelet surface markers showed LPS-induced increases in extravascular platelets into lung airspaces suggesting that some of the platelets recruited to the lung enter air spaces. CONCLUSIONS: Intranasal administration of LPS activates the innate immune response which includes a fall in peripheral blood platelet counts with subsequent platelet recruitment to the lung, spleen and liver, measured by immunohistochemistry and radiolabelling techniques.

Use of animal models in IPF research.

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease with a poor prognosis and limited treatment options. Many compounds have shown efficacy in preclinical models of this condition, but only pirfenidone and nintedanib have been approved for clinical use. It is widely accepted that the current animal models of IPF need to be improved and in this review we have critically evaluated the current state of play of preclinical models of IPF and discuss the challenges facing this field. The popular model of a single intratracheal (I.T.) administration of bleomycin could be adapted to provide a more progressive fibrosis as is thought to occur in humans. Furthermore, currently the majority of new drugs are investigated in preclinical models of IPF are dosed using a prophylactic dosing regimen, whereas patients are almost always treated after the fibrosis is well established. Using a therapeutic dosing regimen in preclinical models would be a better way to establish potential efficacy of new drugs. The most popular endpoints examined in pre-clinical models of IPF are histological scoring and lung collagen content. However in IPF patients imaging and lung function tests are more commonly used as end points. We propose that examining more clinically relevant endpoints in pre-clinical models could also provide give a better indication of a compound's potential efficacy on endpoints measured in patients.

Lipopolysaccharide (LPS) induced pulmonary neutrophil recruitment and platelet activation is mediated via the P2Y1 and P2Y14 receptors in mice.

Platelet activation occurs during host defence and in various inflammatory disorders. In animal models of infection and inflammation, experimental depletion of platelets leads to significantly reduced leukocyte recruitment and impaired clearance of pathogens from the lung. It is now appreciated that purinergic receptor activation is required for leukocyte activation, motility and adhesion, and platelet interactions with leukocytes can be modulated by purinergic stimulation of platelets. Here, we have investigated the role of platelet P2Y1, P2Y12, P2Y14, and P2X1 receptors on leukocyte recruitment and chemotaxis. Mice were administered either vehicle controls or selective P2Y1, P2Y12, P2Y14, or P2X1 antagonists intravenously before intranasal administration of lipopolysaccharide (LPS) to investigate the effect of these drugs on pulmonary leukocyte recruitment, peripheral platelet counts, bleeding times, and ex vivo platelet aggregation. Separately, platelets were incubated with P2Y1, P2Y12, P2X1 antagonists, or P2Y14 agonists to assess effects on platelet-induced neutrophil chemotaxis in vitro. Pulmonary neutrophil recruitment induced by intranasal LPS administration was inhibited in mice administered either with P2Y1 or P2Y14 antagonists, but not with P2Y12 or P2X1 antagonists. Furthermore, the administration of either a P2Y1 or a P2Y14 antagonist reversed the incidence of peripheral thrombocytopaenia associated with LPS exposure. Bleeding times were significantly increased in mice administered P2Y1, P2Y12, or P2X1 antagonists, whilst ex vivo platelet aggregation to ADP was significantly reduced. These haemostatic responses remained unaltered following antagonism of P2Y14. In vitro chemotaxis assays revealed direct antagonism of platelet P2Y1, but not P2Y12 or P2X1 receptors suppressed platelet-dependent neutrophil motility towards Macrophage derived chemokine (MDC, CCL22). Furthermore, the stimulation of platelets with selective P2Y14 agonists (UDP-glucose, MRS2690) resulted in significant platelet-dependent neutrophil chemotaxis. These results reveal a role for P2Y1 and P2Y14 activation of platelets following exposure to LPS, whilst haemostatic indices were unaffected by inhibition of platelet function with the P2Y14 antagonist in response to LPS.

Platelets and allergic inflammation.

Irrefutable clinical evidence demonstrates the activation of platelets in allergic diseases, including asthma, allergic rhinitis, and eczema. Indeed, experimental models of allergic disease have now shown that platelets play a fundamental role in the tissue recruitment of leucocytes following exposure to allergens. Furthermore, the extravascular presence of platelets in lungs of patients with asthma, and in animal models of allergic lung inflammation suggests that platelets may also contribute directly to allergic inflammation, through alterations in lung function, or by modulating processes involved in airway wall remodelling. Despite significant platelet activation in patients with allergic diseases, it is of note that these patients have been described as having a mild haemostastic defect, rather than an increased incidence of thrombosis. This suggests a dichotomy exists in platelet activation during inflammation compared to haemostasis, and that hitherto undiscovered platelet activation pathways might be exploited to create novel anti-inflammatory therapies without affecting the critical function of platelets in haemostasis.

Induction of platelet aggregation after a direct physical interaction with diesel exhaust particles.

BACKGROUND: There is a proven link between exposure to traffic-derived particulate air pollution and the incidence of platelet-driven cardiovascular diseases. It is suggested that inhalation of small, nanosized particles increases cardiovascular risk via toxicological and inflammatory processes and translocation of nanoparticles into the bloodstream has been shown in experimental models. We therefore investigated the ability of diesel exhaust particles (DEP) to interact physically and functionally with platelets. METHODS: The interaction of DEP and carbon black (CB) with platelets was examined by transmission electron microscopy (TEM), whereas the functional consequences of exposure were assessed by measuring in vitro and in vivo platelet aggregation via established methods. RESULTS: Both DEP and CB were internalized and seen in proximity with the open canalicular system in platelets. DEP induced platelet aggregation in vitro whereas CB had no effect. DEP induced Ca(2+) release, dense granule secretion and surface P-selectin expression, but not toxicologic membrane disruption. Low concentrations of DEP potentiated agonist-induced platelet aggregation in vitro and in vivo. CONCLUSIONS: DEP associate physically with platelets in parallel with a Ca(2+) -mediated aggregation response displaying the conventional features of agonist-induced aggregation. The ability of DEP to enhance the aggregation response to platelet stimuli would be expected to increase the incidence of platelet-driven cardiovascular events should they be inhaled and translocate into the blood. This study provides a potential mechanism for the increased thrombotic risk associated with exposure to ambient particulate air pollution.

Defining a role for platelets in allergic inflammation.

There is now considerable evidence suggesting a role for platelets as inflammatory cells. These actions are distinct from their classically known actions performed during thrombosis and haemostasis, and include the expression of adhesion molecules and contact-dependent activation of leucocytes, the release of a plethora of inflammatory mediators, activation in cells of the adaptive immune response and the ability to migrate and undergo chemotaxis. Chronic asthma is a disease characterized by a mixed inflammatory cell pulmonary infiltrate, AHR (airways hyper-responsiveness) and tissue remodelling. Clinical data from patients suffering from asthma, allergic rhinitis and allergic dermatitis reveal changes in platelet behaviour and function during or after allergen exposure. Furthermore, mouse models of allergic inflammation demonstrate a role for intact platelets in eosinophil and lymphocyte recruitment to the lungs, a mechanism that is P-selectin (platelet selectin)-dependent. Models of chronic inflammation also reveal the participation of platelets in tissue remodelling events whereby platelet depletion was found to be more effective in suppressing airway remodelling processes than the administration of a glucocorticosteroid. This process of destruction and repair to the architecture of airway tissue is therefore perhaps enhanced by platelet activation. Recent evidence demonstrates that platelets can undergo chemotaxis and indicates an ability to migrate through inflamed tissue, where they localize to specific tissue sites. Indeed, platelets have been shown to become activated and recruited to various body compartments in direct response to allergen via IgE and this is suggestive of a link between the innate and adaptive immune responses. Thus these actions may lead to pathophysiological events that alter disease progression, since platelet depletion suppresses AHR in allergic rabbits. Further investigations into the role of platelets in inflammation may be beneficial in the search for future therapeutic targets in the treatment of asthma and allergy.

Novel uses for anti-platelet agents as anti-inflammatory drugs.

An alteration in the character and function of platelets is manifested in patients with inflammatory diseases, and these alterations have been dissociated from the well-characterized involvement of platelets in thrombosis and haemostasis. Recent evidence reveals platelet activation is sometimes critical in the development of inflammation. The mechanisms by which platelets participate in inflammation are diverse, and offer numerous opportunities for future drug intervention. There is now acceptance that platelets act as innate inflammatory cells in immune responses, with roles as sentinel cells undergoing surveillance, responding to microbial invasion, orchestrating leukocyte recruitment, and migrating through tissue, causing damage and influencing repair processes in chronic disease. Some of these processes are targeted by drugs that are being developed to target platelet participation in atherosclerosis. The actions of platelets therefore influence the pathogenesis of diverse inflammatory diseases in various body compartments, encompassing parasitic and bacterial infection, allergic inflammation (especially asthma and rhinitis), and non-atopic inflammatory conditions, for example, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis (RA), inflammatory bowel disease (IBD) and atherosclerosis. This review will first discuss the evidence for platelet activation in these various inflammatory diseases, and secondly discuss the mechanisms by which this pathogenesis occurs and the various anti-platelet agents which have been developed to combat platelet activation in atherosclerosis and their potential future use for the treatment of other inflammatory diseases.

Ligand characterization using microphysiometry.

This unit describes the use of a Cytosensor microphysiometer for functional characterization of an agonist and antagonist to a G protein-coupled receptor, the muscarinic M1 receptor. Concentration-response profiles are used to calculate values for the EC50 of the response of cells to the agonist and the pA2 value for the antagonist. Support protocols describe optimization of two aspects of this procedure: the duration of ligand exposure at a given concentration and the length of recovery time between the administration of two different concentrations of ligand to minimize the impact of desensitization. The Cytosensor microphysiometer allows the measurement of receptor activation in both adherent cells, such as the M1WT3 cells used here or in suspension cultures.

Murine models of inflammation: role of CD23.

The role of IgE in eosinophil recruitment and bronchial hyperresponsiveness has been extensively studied with murine models of inflammation. Many investigators using various knockout models have clearly shown that both IgE-dependent and -independent pathways play a role in eosinophil recruitment and bronchial hyperresponsiveness after allergen challenge, illustrating the complexity of airways inflammation. The expression of this response is likely to involve many interacting pathways, and it will be a considerable challenge to determine key points within these pathways that will yield novel targets for future therapeutic strategies.

Cytokines in airway inflammation.

With over 50 potential asthma mediators, cytokines are the latest group of substances which have been investigated for their potential role in this disease. The use of murine models of allergic inflammation has facilitated the investigation of the role of individual cytokines in this response. The use of targeted gene disruption, overexpression of genes and monoclonal antibodies directed against cytokines have allowed a detailed examination of the role cytokines play in IgE production, eosinophil recruitment and bronchial hyperresponsiveness, which are the characteristic features of the asthma phenotype. Despite the introduction of this new methodology, conflicting reports relating to the role of cytokines in allergic inflammation, highlight the complexity of allergic inflammation and challenge the notion that a single cytokine can explain the asthma phenotype.

Squalamine inhibits angiogenesis and solid tumor growth in vivo and perturbs embryonic vasculature.

The novel aminosterol, squalamine, inhibits angiogenesis and tumor growth in multiple animal models. This effect is mediated, at least in part, by blocking mitogen-induced proliferation and migration of endothelial cells, thus preventing neovascularization of the tumor. Squalamine has no observable effect on unstimulated endothelial cells, is not directly cytotoxic to tumor cells, does not alter mitogen production by tumor cells, and has no obvious effects on the growth of newborn vertebrates. Squalamine was also found to have remarkable effects on the primitive vascular bed of the chick chorioallantoic membrane, which has striking similarities to tumor capillaries. Squalamine may thus be well suited for treatment of tumors and other diseases characterized by neovascularization in humans.

Nerve growth factor stimulates rapid metabolic responses in PC12 cells.

Research into the effects of nerve growth factor (NGF) has involved study of either the signal transduction process or the morphological result of growth factor treatment (cell proliferation and/or differentiation). The Cytosensor Microphysiometer, a silicon-based biosensor system that allows the continuous and real-time monitoring of extracellular acidification rate changes of cells, was used to study the response of PC12 cells to NGF. Stimulation resulted in a rapid increase in the acidification rate of cells in a concentration-dependent fashion (0.1-200 ng/ml NGF; mean effective concentration value of 153 +/- 54 pM). Inhibition of the NGF receptor-linked protein tyrosine kinase by either genistein or K252a attenuated the acidification rate response to NGF. In addition, the acidification response to NGF could be modified by inhibiting Na+/H+ exchange and, separately, glycolysis. This implicates these processes in the metabolic response of PC12 cells to NGF stimulation.

Recombinant human ciliary neurotrophic factor stimulates the metabolic activity of SH-SY5Y cells as measured by a cytosensor microphysiometer.

Information on the transmembrane signaling events and subsequent biochemical processes initiated by ciliary neurotrophic factor (CNTF) receptor activation in neurons is lacking. SH-SY5Y cells, a human neuroblastoma cell line expressing CNTF receptors, were used to study metabolic changes associated with functional ligand-receptor interactions. Real-time measurements quantifying the rate of extracellular acidification by SH-SY5Y cells (a measure of metabolic activity) were made using a silicon-based cytosensor. Application of recombinant human CNTF (rhCNTF) to resting SH-SY5Y cells increased their acidification rate in a concentration and time-dependent manner with an apparent EC50 of 60 ng/ml. Pretreatment of cells with phosphatidylinositol-specific phospholipase C (PI-PLC) prevented the CNTF, but not an NGF-stimulated increase in acidification rate. Collectively, these results demonstrate that: (1) SH-SY5Y cells express functional CNTF receptors; and (2) the initial signal transduction mechanism activated by the CNTF receptor in SH-SY5Y cells is distinct from that activated by the NGF receptor; however, both may ultimately stimulate the same downstream biochemical messengers to increase cellular metabolism.

Nicotinic acetylcholine receptor desensitization is regulated by activation-induced extracellular adenosine accumulation.

Adenosine modulation of nicotinic ACh receptor (nAChR) function was studied in primary cultures of rat skeletal muscle. Activation of the nAChR by carbachol increased extracellular adenosine concentration in a dose-dependent manner. Furthermore, carbachol activation of the nicotinic receptor resulted in a twofold increase in cAMP levels in the muscle cells. The carbachol-dependent increase in cAMP levels was inhibited by adenosine receptor antagonists as well as by nicotinic receptor antagonists. These results suggest that the increased cAMP levels were due to adenosine receptor activation by the extracellular adenosine accumulated on nAChR activation. Others have shown that desensitization of the nAChR by agonist is mediated, in part, by phosphorylation. Since we found that nicotinic cholinergic agonists also cause adenosine accumulation with concomitant cAMP increases, we determined whether the accumulated adenosine has a role in desensitization. We found that the adenosine receptor antagonist, BW1434U, significantly inhibited carbachol-induced nAChR desensitization, indicating that extracellular adenosine is involved in nAChR desensitization. Our data suggest that nAChR function is regulated via a feedback mechanism mediated by adenosine released from muscle on activation of the nAChR.

The cytosensor microphysiometer: biological applications of silicon technology.

A silicon-based device, dubbed a microphysiometer, can be used to detect and monitor the response of cells to a variety of chemical substances, especially ligands for specific plasma membrane receptors. The microphysiometer measures the rate of proton excretion from 10(4) to 10(6) cells. This article gives an overview of experiments currently being carried out with this instrument with emphasis on receptors with seven transmembrane helices and tyrosine kinase receptors. As a scientific instrument, the microphysiometer can be thought of as serving two distinct functions. In terms of detecting specific molecules, selected biological cells in this instrument serve as detectors and amplifiers. The microphysiometer can also investigate cell function and biochemistry. A major application of this instrument may prove to be screening for new receptor ligands. In this respect, the microphysiometer appears to offer significant advantages over other techniques.

Prostaglandins sensitize nociceptors in cell culture.

Using the whole cell patch clamp technique, a population of nociceptors were identified, by virtue of their small size and capsaicin responsivity. Response to capsaicin was increased following treatment with the hyperalgesic prostaglandins, PGE2 and PGI2. Treatment of the cells with the cyclic adenosine monophosphate (cAMP) analogues, 8 bromo cAMP and dibutyryl cAMP, also resulted in an increase in the capsaicin-induced currents. The effects of the cAMP analogues were greater than that produced by prostaglandin treatment. We conclude that PGE2 and PGI2 act directly on nociceptors, with cAMP as second messenger, to sensitize them to noxious stimulation.

An electrical resonance in hair cells of the amphibian papilla of the frog Rana temporaria.

Intracellular recordings were made from hair cells in the rostral- and mid-regions of an in vitro preparation of the amphibian papilla from the frog Rana temporaria. In response to small current injections the membrane potential oscillated indicating the presence of an electrical resonator within these hair cells. The properties of this resonance were similar to those of electrical resonance in hair cells of other lower vertebrates (Crawford and Fettiplace, 1981). The frequency of these oscillations depended on the position of the cell within the papilla; the oscillation frequency at the offset of the current pulse appeared to be tonotopically organized. The role of this resonance in relation to the known frequency selectivity and tonotopic organization of this auditory organ is discussed.