Douglas J. Taatjes

NF-κB Activation in Airways Modulates Allergic Inflammation but Not Hyperresponsiveness

Airways display robust NF-κB activation and represent targets for anti-inflammatory asthma therapies, but the functional importance of NF-κB activation in airway epithelium remains enigmatic. Therefore, transgenic mice were created in which NF-κB activation is repressed specifically in airways (CC10-IκBαSR mice). In response to inhaled Ag, transgenic mice demonstrated significantly ameliorated inflammation, reduced levels of chemokines, T cell cytokines, mucus cell metaplasia, and circulating IgE compared with littermate controls. Despite these findings, Ag-driven airways hyperresponsiveness was not attenuated in CC10-IκBαSR mice. This study clearly demonstrates that airway epithelial NF-κB activation orchestrates Ag-induced inflammation and subsequent adaptive immune responses, but does not contribute to airways hyperresponsiveness, the cardinal feature that underlies asthma.

Human Monoclonal Antiphospholipid Antibodies Disrupt the Annexin A5 Anticoagulant Crystal Shield on Phospholipid Bilayers: Evidence from Atomic Force Microscopy and Functional Assay

The antiphospholipid (aPL) syndrome is an autoimmune condition that is marked by recurrent pregnancy losses and/or systemic vascular thrombosis in patients who have antibodies against phospholipid/co-factor complexes. The mechanism(s) for pregnancy losses and thrombosis in this condition is (are) not known. Annexin A5 is a potent anticoagulant protein, expressed by placental trophoblasts and endothelial cells, that crystallizes over anionic phospholipids, shielding them from availability for coagulation reactions. We previously presented data supporting the hypothesis that aPL antibody-mediated disruption of the anticoagulant annexin A5 shield could be a thrombogenic mechanism in the aPL syndrome. However, this has remained a subject of controversy. We therefore used atomic force microscopy, a method previously used to study the crystallization of annexin A5, to image the effects of monoclonal human aPL antibodies on the crystal structure of the protein over phospholipid bilayers. In the presence of the aPL monoclonal antibodies (mAbs) and beta(2)-GPI, the major aPL co-factor, structures presumed to be aPL mAb-antigen complexes were associated with varying degrees of disruption to the annexin A5 crystallization pattern over the bilayer. In addition, measurements of prothrombinase activity on the phospholipid bilayers showed that the aPL mAbs reduced the anti-coagulant effect of annexin A5 and promoted thrombin generation. These data provide morphological evidence that support the hypothesis that aPL antibodies can disrupt annexin A5 binding to phospholipid membranes and permit increased generation of thrombin. The aPL antibody-mediated disruption of the annexin A5 anticoagulant shield may be an important prothrombotic mechanism in the aPL syndrome.

Hydroxychloroquine protects the annexin A5 anticoagulant shield from disruption by antiphospholipid antibodies: evidence for a novel effect for an old antimalarial drug

Annexin A5 (AnxA5) is a potent anticoagulant protein that crystallizes over phospholipid bilayers (PLBs), blocking their availability for coagulation reactions. Antiphospholipid antibodies disrupt AnxA5 binding, thereby accelerating coagulation reactions. This disruption may contribute to thrombosis and miscarriages in the antiphospholipid syndrome (APS). We investigated whether the antimalarial drug, hydroxychloroquine (HCQ), might affect this prothrombotic mechanism. Binding of AnxA5 to PLBs was measured with labeled AnxA5 and also imaged with atomic force microscopy. Immunoglobulin G levels, AnxA5, and plasma coagulation times were measured on cultured human umbilical vein endothelial cells and a syncytialized trophoblast cell line. AnxA5 anticoagulant activities of APS patient plasmas were also determined. HCQ reversed the effect of antiphospholipid antibodies on AnxA5 and restored AnxA5 binding to PLBs, an effect corroborated by atomic force microscopy. Similar reversals of antiphospholipid-induced abnormalities were measured on the surfaces of human umbilical vein endothelial cells and syncytialized trophoblast cell lines, wherein HCQ reduced the binding of antiphospholipid antibodies, increased cell-surface AnxA5 concentrations, and prolonged plasma coagulation to control levels. In addition, HCQ increased the AnxA5 anticoagulant activities of APS patient plasmas. In conclusion, HCQ reversed antiphospholipid-mediated disruptions of AnxA5 on PLBs and cultured cells, and in APS patient plasmas. These results support the concept of novel therapeutic approaches that address specific APS disease mechanisms.

Morphological and cytochemical determination of cell death by apoptosis

Several modes of cell death are now recognized, including necrosis, apoptosis, and autophagy. Oftentimes the distinctions between these various modes may not be apparent, although the precise mode may be physiologically important. Accordingly, it is often desirable to be able to classify the mode of cell death. Apoptosis was originally defined by structural alterations in cells observable by transmitted light and electron microscopy. Today, a wide variety of imaging and cytochemical techniques are available for the investigation of apoptosis. This review will highlight many of these methods, and provide a critique on the advantages and disadvantages associated with them for the specific identification of apoptotic cells in culture and tissues.

Group V Secretory Phospholipase A2 Promotes Atherosclerosis Evidence From Genetically Altered Mice

Objective—Group V secretory phospholipase A2 (GV sPLA2) has been detected in both human and mouse atherosclerotic lesions. This enzyme has potent hydrolytic activity towards phosphatidylcholine-containing substrates, including lipoprotein particles. Numerous studies in vitro indicate that hydrolysis of high density lipoproteins (HDL) and low density lipoproteins (LDL) by GV sPLA2 leads to the formation of atherogenic particles and potentially proinflammatory lipid mediators. However, there is no direct evidence that this enzyme promotes atherogenic processes in vivo. Methods and Results—We performed gain-of-function and loss-of-function studies to investigate the role of GV sPLA2 in atherogenesis in LDL receptor–deficient mice. Compared with control mice, animals overexpressing GV sPLA2 by retrovirus-mediated gene transfer had a 2.7 fold increase in lesion area in the ascending region of the aortic root. Increased atherosclerosis was associated with an increase in lesional collagen deposition in the same region. Mice deficient in bone marrow–derived GV sPLA2 had a 36% reduction in atherosclerosis in the aortic arch/thoracic aorta. Conclusions—Our data in mouse models provide the first in vivo evidence that GV sPLA2 contributes to atherosclerotic processes, and draw attention to this enzyme as an attractive target for the treatment of atherosclerotic disease.

The annexin A5-mediated pathogenic mechanism in the antiphospholipid syndrome: role in pregnancy losses and thrombosis.

Annexin A5 (AnxA5) binds to phospholipid bilayers, forming two-dimensional crystals that block the phospholipids from availability for coagulation enzyme reactions. Antiphospholipid (aPL) antibodies cause gaps in the ordered crystallization of AnxA5 which expose phospholipids and thereby accelerate blood coagulation reactions. The aPL antibody-mediated disruption of AnxA5 crystallization has been confirmed on artificial phospholipid bilayers and on cell membranes including endothelial cells, placental trophoblasts and platelets. Recently, we reported that hydroxychloroquine, a synthetic antimalarial drug, can reverse this antibody-mediated process through two mechanisms: (1) by inhibiting the formation of aPL IgG-β2glycoprotein I complexes; and (2) by promoting the formation of a second layer of AnxA5 crystal ‘patches’ over areas where the immune complexes had disrupted AnxA5 crystallization. In another translational application, we have developed a mechanistic assay that reports resistance to AnxA5 anticoagulant activity in plasmas of patients with aPL antibodies. AnxA5 resistance may identify a subset of aPL syndrome patients for whom this is a mechanism for pregnancy losses and thrombosis. The elucidation of aPL-mediated mechanisms for thrombosis and pregnancy complications may open new paths towards addressing this disorder with targeted treatments and mechanistic assays.

STRUCTURE AND DYNAMICS OF THE FUSION PORE IN LIVE CELLS

Atomic force microscopy reveal pit‐like structures typically containing three or four, ∼150nm in diameter depressions at the apical plasma membrane in live pancreatic acinar cells. Stimulation of secretion causes these depressions to dilate and return to their resting size following completion of the process. Exposure of acinar cells to cytochalasin B results in decreased depression size and a loss in stimulable secretion. It is hypothesized that depressions are the fusion pores, where membrane‐bound secretory vesicles dock and fuse to release vesicular contents. Zymogen granules, the membrane‐bound secretory vesicles in exocrine pancreas, contain the starch digesting enzyme, amylase. Using amylase‐specific immunogold labeling, localization of amylase at depressions following stimulation of secretion is demonstrated. This study confirms depressions to be the fusion pores in pancreatic acinar cells. High‐resolution images of the fusion pore in live pancreatic acinar cells reveal the structure in much greater detail than has previously been observed.

Intramural Plasminogen Activator Inhibitor Type-1 and Coronary Atherosclerosis

Altered expression of plasminogen activator inhibitor type-1 in vessel walls, reviewed here, might affect coronary atherogenesis. Upregulation might exacerbate vasculopathy by potentiating thrombosis and by inhibiting vascular smooth muscle cell migration, resulting in attenuation of thickness of elaborated fibrous caps implicated in the vulnerability of atheroma to rupture.

Mechanisms of asbestos-induced nitric oxide production by rat alveolar macrophages in inhalation and in vitro models.

To evaluate the contribution of reactive nitrogen species to inflammation by asbestos, Fischer 344 rats were exposed to crocidolite or chrysotile asbestos by inhalation to determine whether increases occurred in nitric oxide (NO.) metabolites from alveolar macrophages (AMs). AMs from animals inhaling asbestos showed significant elevations (p < .05) in nitrite/nitrate levels which were ameliorated by NG-monomethyl-L-arginine (NMMA), an inhibitor of inducible nitric oxide synthase (iNOS) activity. Temporal patterns of NO. generation from AMs correlated with neutrophil influx in bronchoalveolar lavage samples after asbestos inhalation or bleomycin instillation, another model of pulmonary fibrosis. To determine the molecular mechanisms and specificity of iNOS promoter activation by asbestos, RAW 264.7 cells, a murine macrophage-like line, and AMs isolated from control rats were exposed to crocidolite asbestos in vitro. These cells showed increases in steady-state levels of iNOS mRNA in response to asbestos and more dramatic increases in both iNOS mRNA and immunoreactive protein after addition of lipopolysaccharide (LPS). After transfection of an iNOS promoter/luciferase reporter construct, RAW 264.7 cells exposed to LPS, crocidolite asbestos and its nonfibrous analog, riebeckite, revealed increases in luciferase activity whereas cristobalite silica had no effects. Studies suggest that NO. generation may be important in cell injury and inflammation by asbestos.

Increased phosphorylated extracellular signal-regulated kinase immunoreactivity associated with proliferative and morphologic lung alterations after chrysotile asbestos inhalation in mice.

Activation of extracellular signal-regulated kinases (ERK) has been associated with the advent of asbestos-associated apoptosis and proliferation in mesothelial and alveolar epithelial cells and may be linked to the development of pulmonary fibrosis. The objective of studies here was to characterize the development of inflammation, cellular proliferation, and fibrosis in asbestos-exposed C57Bl/6 mice in relationship to patterns of ERK phosphorylation. Inflammation occurred after 10 and 20 days of asbestos exposure as evidenced by increases in total protein and neutrophils in bronchoalveolar lavage fluid. Increases in cell proliferation were observed at 30 days in bronchiolar epithelia and at 4, 14, and 30 days in the alveolar compartment of the lung. Trichrome-positive focal lesions of pulmonary fibrosis developed at 30 days in the absence of elevations in lung hydroxyproline or procollagen mRNA levels. Striking increases in ERK phosphorylation were observed within pulmonary epithelial cells at sites of developing fibrotic lesions after 14 and 30 days of inhalation. In addition to characterizing a murine inhalation model of asbestosis, we provide the first evidence showing activation of ERK signaling within lung epithelium in vivo, following inhalation of asbestos fibers.