David C. Cottell

Lipoxins, Aspirin-Triggered Epi-Lipoxins, Lipoxin Stable Analogues, and the Resolution of Inflammation: Stimulation of Macrophage Phagocytosis of Apoptotic Neutrophils In Vivo

Lipoxins (LX) are eicosanoids with antiinflammatory activity in glomerulonephritis (GN) and inflammatory diseases, hypersensitivity, and ischemia reperfusion injury. It has been demonstrated that LXA(4) stimulates non-phlogistic phagocytosis of apoptotic polymorphonuclear neutrophils (PMN) by monocyte-derived macrophages (Mphi) in vitro, suggesting a role for LX as endogenous pro-resolution lipid mediators. It is here reported that LXA(4), LXB(4), the aspirin-triggered LX (ATL) epimer, 15-epi-LXB(4), and a stable synthetic analogue 15(R/S)-methyl-LXA(4) stimulate phagocytosis of exogenously administered excess apoptotic PMN by macrophages (M phi) in vivo in a classic model of acute inflammation, namely thioglycollate-induced peritonitis. Significant enhancement of phagocytosis in vivo was observed with 15-min exposure to LX and with intraperitoneal doses of LXA(4), LXB(4), 15(R/S)-methyl-LXA(4), and 15-epi-LXB(4) of 2.5 to 10 micro g/kg. Non-phlogistic LX-stimulated phagocytosis by M phi was sensitive to inhibition of PKC and PI 3-kinase and associated with increased production of transforming growth factor-beta(1) (TGF-beta(1)). LX-stimulated phagocytosis was not inhibited by phosphatidylserine receptor (PSR) antisera and was abolished by prior exposure of M phi to beta 1,3-glucan, suggesting a novel M phi-PMN recognition mechanism. Interestingly, the recently described peptide agonists of the LXA(4) receptor (MYFINITL and LESIFRSLLFRVM) stimulated phagocytosis through a process associated with increased TGF-beta(1) release. These data provide the first demonstration that LXA(4), LXB(4), ATL, and LX stable analogues rapidly promote M phi phagocytosis of PMN in vivo and support a role for LX as rapidly acting, pro-resolution signals in inflammation. Engagement of the LXR by LX generated during cell-cell interactions in inflammation and by endogenous LXR peptide agonists released from distressed cells may be an important stimulus for clearance of apoptotic cells and may be amenable to pharmacologic mimicry for therapeutic gain.

The role of HIF-1α in transcriptional regulation of the proximal tubular epithelial cell response to hypoxia

Epithelial cells of the kidney represent a primary target for hypoxic injury in ischemic acute renal failure (ARF); however, the underlying transcriptional mechanism(s) remain undefined. In this study, human proximal tubular epithelial cells (HK-2) exposed to hypoxia in vitro demonstrated a non-lethal but dysfunctional phenotype, closely reflective of the epithelial pathobiology of ARF. HK-2 cells exposed to hypoxia demonstrated increased paracellular permeability, decreased proliferation, loss of tight junctional integrity, and significant actin disassembly in the absence of cell death. Microarray analysis of transcriptomic changes underlying this response identified a distinct cohort of 48 genes with a closely shared hypoxia-dependent expression profile. Within this hypoxia-sensitive cluster were genes identified previously as hypoxia-inducible factor-1 (HIF-1)-dependent (e.g. vascular endothelial growth factor and adrenomedullin) as well as genes not previously known to be hypoxia-responsive (e.g. stanniocalcin 2). In hypoxia, HIF-1 bound to evolutionarily conserved hypoxia-response elements (HRE) in the promoters of these genes as well as to the HRE consensus motif. A further subset of these genes, not associated with transcriptional regulation by HIF-1, was also present, suggesting alternative HIF-1-independent pathways. Overexpression of HIF-1α in normoxia induced the expression of a significant number of the hypoxia-dependent genes; however, it did not induce the pathophysiologic epithelial response. In summary, hypoxia-elicited alterations in renal proximal tubular epithelial cells in vitro closely resemble the epithelial pathophysiology of ARF. Our data indicate that although this event may rely heavily on HIF-1-dependent gene transcription, it is likely that separate hypoxia-dependent transcriptional regulators also play a role.

Platelet Activation Induces Cell-Surface Immunoreactive Tissue Factor Expression, Which Is Modulated Differently by Antiplatelet Drugs

Objective—Tissue factor (TF) is the main activator of the coagulation cascade occurring in physiologic and pathologic conditions. Recent data suggest that human platelets might contain TF that is possibly derived from leukocytes. In this study, we investigated whether intraplatelet TF can be exposed on the membrane by platelet agonists. The modulation of this process by antiplatelet drugs has been evaluated as well. Methods and Results—Flow cytometric analysis of unstimulated platelets showed a small amount of membrane-associated immunoreactive TF (irTF) in whole blood, platelet-rich plasma, and washed platelets isolated from healthy subjects. ADP, thrombin receptor-activating peptide, and epinephrine significantly increased functionally active, membrane-associated irTF. ADP induced irTF exposure in a concentration- and time-dependent fashion. Agonist-induced irTF expression was completely inhibited by iloprost but not by aspirin. Interestingly, glycoprotein IIb/IIIa antagonists did not inhibit but rather potentiated the stimulatory effect of ADP on platelet irTF expression. Real-time polymerase chain reaction experiments showed detectable amounts of TF mRNA in unstimulated platelets. Conclusions—These findings indicate that platelet agonists and antiplatelet drugs might modulate platelet-associated irTF expression. Regulated TF expression establishes the potential for a previously unrecognized role for platelets in sustaining thrombus formation and growth via coagulation-mediated mechanisms.

FPR2/ALX receptor expression and internalization are critical for lipoxin A4 and annexin-derived peptide-stimulated phagocytosis

Lipoxins (LXs) are endogenously produced eicosanoids with well‐described anti‐inflammatory and proresolution activities, stimulating nonphlogistic phagocytosis of apoptotic cells by macrophages. LXA4 and the glucocorticoid‐derived annexin A1 peptide (Ac2–26) bind to a common G‐proteincoupled receptor, termed FPR2/ALX. However, direct evidence of the involvement of FPR2/ALX in the anti‐inflammatory and proresolution activity of LXA4 is still to be investigated. Here we describe FPR2/ALX trafficking in response to LXA4 and Ac2–26 stimulation. We have transfected cells with HA‐tagged FPR2/ALX and studied receptor trafficking in unstimulated, LXA4 (1–10 nM)‐ and Ac2–26 (30 µM)‐treated cells using multiple approaches that include immunofluorescent confocal microscopy, immunogold labeling of cryosections, and ELISA and investigated receptor trafficking in agonist‐stimulated phagocytosis. We conclude that PKC‐dependent internalization of FPR2/ALX is required for phagocytosis. Using bone marrow‐derived macrophages (BMDMs) from mice in which the FPR2/ALX ortholog Fpr2 had been deleted, we observed the nonredundant function for this receptor in LXA4 and Ac2–26 stimulated phagocytosis of apoptotic neutrophils. LXA4 stimulated phagocytosis 1.7‐fold above basal (P< 0.001) by BMDMs from wild‐type mice, whereas no effect was found on BMDMs from Fpr2‐/‐ mice. Similarly, Ac2–26 stimulates phagocytosis by BMDMs from wild‐type mice 1.5‐fold above basal (P<0.05). However, Ac2–26 failed to stimulate phagocytosis by BMDMs isolated from Fpr2‐/‐ mice relative to vehicle. These data reveal novel and complex mechanisms of the FPR2/ALX receptor trafficking and functionality in the resolution of inflammation.—Maderna, P., Cottell, D. C., Toivonen, T., Dufton, N., Dalli, J., Perretti, M., Godson, C. FPR2/ALX receptor expression and internalization are critical for lipoxin A4 and annexinderived peptide‐stimulated phagocytosis. FASEB J. 24, 4240–4249 (2010). www.fasebj.org

Genetic determinants of hyaloid and retinal vasculature in zebrafish

BackgroundThe retinal vasculature is a capillary network of blood vessels that nourishes the inner retina of most mammals. Developmental abnormalities or microvascular complications in the retinal vasculature result in severe human eye diseases that lead to blindness. To exploit the advantages of zebrafish for genetic, developmental and pharmacological studies of retinal vasculature, we characterised the intraocular vasculature in zebrafish.ResultsWe show a detailed morphological and developmental analysis of the retinal blood supply in zebrafish. Similar to the transient hyaloid vasculature in mammalian embryos, vessels are first found attached to the zebrafish lens at 2.5 days post fertilisation. These vessels progressively lose contact with the lens and by 30 days post fertilisation adhere to the inner limiting membrane of the juvenile retina. Ultrastructure analysis shows these vessels to exhibit distinctive hallmarks of mammalian retinal vasculature. For example, smooth muscle actin-expressing pericytes are ensheathed by the basal lamina of the blood vessel, and vesicle vacuolar organelles (VVO), subcellular mediators of vessel-retinal nourishment, are present. Finally, we identify 9 genes with cell membrane, extracellular matrix and unknown identity that are necessary for zebrafish hyaloid and retinal vasculature development.ConclusionZebrafish have a retinal blood supply with a characteristic developmental and adult morphology. Abnormalities of these intraocular vessels are easily observed, enabling application of genetic and chemical approaches in zebrafish to identify molecular regulators of hyaloid and retinal vasculature in development and disease.

Lipoxins Induce Actin Reorganization in Monocytes and Macrophages But Not in Neutrophils: Differential Involvement of Rho GTPases

Lipoxins (LXs) are endogenously produced eicosanoids that inhibit neutrophil trafficking and stimulate nonphlogistic phagocytosis of apoptotic neutrophils by monocyte-derived macrophages. In this study we assessed the effect of LXs on cell ultrastructure and actin reorganization in human leukocytes and investigated the signaling events that subserve LX bioactivity in this context. LXA(4) (10(-9) mol/L), the stable synthetic analogues 15-(R/S)-methyl-LXA(4) and 16-phenoxy-LXA(4) (10(-11) mol/L), but not the LX precursor 15-(S)-HETE, induced marked changes in ultrastructure and rearrangement of actin in monocytes and macrophages. In contrast, LXA(4) did not modify actin distribution in neutrophils under basal conditions and after stimulation with leukotriene B(4). Blockade of Rho kinases by the inhibitor Y-27632 prevented LXA(4)-triggered actin reorganization in macrophages. To investigate the role of the specific small GTPases in LX-induced actin rearrangement we used THP-1 cells differentiated to a macrophage-like phenotype. THP-1 cells stimulated with LXs, but not with 15-(S)-HETE, showed an increase in membrane-associated RhoA and Rac by immunoblotting. Additionally, a twofold increase in Rho activity was seen in response to LXA(4). LX-induced actin rearrangement and RhoA activation were inhibited by the cell permeable cAMP analogue 8-Br-cAMP, whereas Rp-cAMP, an inhibitor of protein kinase A, mimicked the effect of LXA(4). These data demonstrate that LXs stimulate RhoA- and Rac-dependent cytoskeleton reorganization, contributing to the potential role of LXs in the resolution of inflammation.

Lipoxin A4 and Aspirin-Triggered 15-Epi-Lipoxin A4 Antagonize TNF-α-Stimulated Neutrophil-Enterocyte Interactions In Vitro and Attenuate TNF-α-Induced Chemokine Release and Colonocyte Apoptosis in Human Intestinal Mucosa Ex Vivo

Lipoxins (LXs) are lipoxygenase-derived eicosanoids and putative endogenous braking signals for inflammation in the gastrointestinal tract and other organs. Aspirin triggers the production of 15-epimers during cell-cell interaction in a cytokine-primed milieu, and aspirin-triggered 15-epi-5(S),6(R),15(S)-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid (15-epi-LXA4) may contribute to the bioactivity profile of this prototype nonsteroidal anti-inflammatory drug in vivo. We determined the effect of LXA4, 15-(R/S)-methyl-11,12-dehydro-LXA4 methyl ester (15-(R/S)-methyl-LXA4), and stable analogs of LXA4 on TNF-α-stimulated neutrophil-enterocyte interaction in vitro and TNF-α-stimulated chemokine release, changes in mucosal architecture, and enterocyte apoptosis in cytokine-activated intact human colonic mucosa ex vivo. LXA4, 15-(R/S)-epi-LXA4, and 16-phenoxy-11,12-dehydro-17,18,19,20-tetranor-LXA4 methyl ester (16-phenoxy-LXA4) inhibited TNF-α-stimulated neutrophil adherence to epithelial monolayers at nanomolar concentrations. In parallel experiments involving human colonic mucosa ex vivo, LXA4potently attenuated TNF-α-stimulated release of the C-X-C chemokine IL-8, and the C-C chemokines monocyte-chemoattractant protein-1 (MCP-1) and RANTES. Exposure of strips of normal human colonic mucosa to TNF-α induced disruption of mucosa architecture and enhanced colonocyte apoptosis via a caspase-3-independent mechanism. Prior exposure of the mucosa strips to 15-(R/S)-methyl-LXA4 attenuated TNF-α-stimulated colonocyte apoptosis and protected the mucosa against TNF-α-induced mucosal damage. In aggregate, our data demonstrate that lipoxins and aspirin-triggered 15-epi-LXA4 are potent antagonists of TNF-α-mediated neutrophil-enterocyte interactions in vitro, attenuate TNF-α-triggered chemokine release and colonocyte apoptosis, and are protective against TNF-α-induced morphological disruption in human colonic strips ex vivo. Our observations further expand the anti-inflammatory profile of these lipoxygenase-derived eicosanoids and suggest new therapeutic approaches for the treatment of inflammatory bowel disease.

Functional interactions between the ciliopathy-associated Meckel syndrome 1 (MKS1) protein and two novel MKS1-related (MKSR) proteins.

Meckel syndrome (MKS) is a ciliopathy characterized by encephalocele, cystic renal disease, liver fibrosis and polydactyly. An identifying feature of MKS1, one of six MKS-associated proteins, is the presence of a B9 domain of unknown function. Using phylogenetic analyses, we show that this domain occurs exclusively within a family of three proteins distributed widely in ciliated organisms. Consistent with a ciliary role, all Caenorhabditis elegans B9-domain-containing proteins, MKS-1 and MKS-1-related proteins 1 and 2 (MKSR-1, MKSR-2), localize to transition zones/basal bodies of sensory cilia. Their subcellular localization is largely co-dependent, pointing to a functional relationship between the proteins. This localization is evolutionarily conserved, because the human orthologues also localize to basal bodies, as well as cilia. As reported for MKS1, disrupting human MKSR1 or MKSR2 causes ciliogenesis defects. By contrast, single, double and triple C. elegans mks/mksr mutants do not display overt defects in ciliary structure, intraflagellar transport or chemosensation. However, we find genetic interactions between all double mks/mksr mutant combinations, manifesting as an increased lifespan phenotype, which is due to abnormal insulin–IGF-I signaling. Our findings therefore demonstrate functional interactions between a novel family of proteins associated with basal bodies or cilia, providing new insights into the molecular etiology of a pleiotropic human disorder.

The effects of cadmium exposure on the cytology and function of primary cultures from rainbow trout

Cultured epidermal cells from explants of skin of rainbow trout were used to study the cytological and functional changes following sublethal exposure to cadmium stress. The aim was to develop diagnostic markers for ecotoxicology. Cultures were exposed to the pollutant for 48 h. Cell structural and cytological changes were established by light and electron microscopy. Metabolic alterations were detected by immunohistochemistry. The relation between the initiation of cellular alterations and cadmium concentrations was compared in cultures exposed in commercially‐available serum‐free and serum‐containing medium. The expression of stress proteins (metallothionein and heat shock protein) was also studied. Rainbow trout epithelial cells exposed to cadmium showed typical morphological changes indicative of cell death by apoptosis. Sublethal exposure also resulted in cellular metabolic disturbances with increased deposits of glycogen. Increased melanization was also observed. These changes appeared at lower concentrations of cadmium when cells were exposed in serum‐free media than in serum‐containing media. Cadmium induced the expression of heat shock proteins but not of metallothioneins. The results broadly confirm in vivo findings for cadmium toxicity and suggest that this in vitro technique may have applications in aquatic toxicology.

Primary Explants of Human Uroepithelium Show an Unusual Response to Low-Dose Irradiation with Cobalt-60 Gamma Rays

Recent results using very low doses of radiation have suggested that there is a hypersensitive region where cultures show an enhanced level of cell killing leading to a non-monotonic survival curve. This effect has been observed at doses below 2 Gy in mammalian systems and at much higher doses in insect cells. In this paper we report observation of the effect in primary human uroepithelial cell cultures. The effect was measured using a postirradiation proliferation assay where irradiated explants of standard size were allowed to proliferate for 14 days after exposure to 60Co gamma irradiation. By 14 days the majority of cultures derived from explants irradiated with 2-5 Gy showed little evidence of growth inhibition and cell numbers approached or even exceeded those obtained in the controls. There was, however, a significant reduction in cell number and growth rate in all cultures exposed to doses lower than 1 Gy. Oncoprotein (p53, c-myc, bcl-2, p21 ras) and EGFR expression were also measured in these cultures and were significantly increased. Morphological evidence of apoptosis was present in all irradiated cultures at 4 h after exposure, but this persisted for longer periods in cultures exposed to low doses.