Interleukin-1α Activity in Necrotic Endothelial Cells Is Controlled by Caspase-1 Cleavage of Interleukin-1 Receptor-2: IMPLICATIONS FOR ALLOGRAFT REJECTION*

Inflammation is a key instigator of the immune responses that drive atherosclerosis and allograft rejection. IL-1α, a powerful cytokine that activates both innate and adaptive immunity, induces vessel inflammation after release from necrotic vascular smooth muscle cells (VSMCs). Similarly, IL-1α released from endothelial cells (ECs) damaged during transplant drives allograft rejection. However, IL-1α requires cleavage for full cytokine activity, and what controls cleavage in necrotic ECs is currently unknown. We find that ECs have very low levels of IL-1α activity upon necrosis. However, TNFα or IL-1 induces significant levels of active IL-1α in EC necrotic lysates without alteration in protein levels. Increased activity requires cleavage of IL-1α by calpain to the more active mature form. Immunofluorescence and proximity ligation assays show that IL-1α associates with interleukin-1 receptor-2, and this association is decreased by TNFα or IL-1 and requires caspase activity. Thus, TNFα or IL-1 treatment of ECs leads to caspase proteolytic activity that cleaves interleukin-1 receptor-2, allowing IL-1α dissociation and subsequent processing by calpain. Importantly, ECs could be primed by IL-1α from adjacent damaged VSMCs, and necrotic ECs could activate neighboring normal ECs and VSMCs, causing them to release inflammatory cytokines and up-regulate adhesion molecules, thus amplifying inflammation. These data unravel the molecular mechanisms and interplay between damaged ECs and VSMCs that lead to activation of IL-1α and, thus, initiation of adaptive responses that cause graft rejection.     

Structure of the accessory reproductive glands of the male migratory grasshopper,Melanoplus sanguinipes

The accessory reproductive glands of Melanoplus sanguinipes comprise two bilateral masses of 16 tubules each, distinguishable in sexually mature insects as four white, ten short hyaline, one long hyaline, and a seminal vesicle. Over most of its length, the wall of each tubule consists of a simple glandular epithelium resting on a basal lamina, surrounded by a thin layer of circular muscle. However, near the junction with the ejaculatory duct, the wall of each tubule has a much thickened circular muscle layer and squamous or cuboidal epithelium, the region serving to regulate movement of secretion into the ejaculatory duct. Interdigitation of adjacent epithelial cells is common, and several kinds of specialized junctions occur. In the glandular region, all epithelial cells appear the same and may be flattened, cuboidal, or columnar depending on the tubule type. Except for those of the seminal vesicle, the glandular epithelial cells share ultrastructural features typical of cells engaged in the synthesis of protein for export. Despite these general similarities, in most instances subtle differences occur in the cellular ultrastructure of the epithelia of each tubule and in the appearance of their luminal secretions, suggesting that the tubules are functionally specialized.     

A new method for cell permeabilization reveals a cytosolic protein requirement for Ca2+ -activated secretion in GH3 pituitary cells

Ca2+ is a major regulator of exocytosis in secretory cells, however, the biochemical mechanisms underlying regulation remain to be identified. To render the secretory apparatus accessible for biochemical studies, we have developed a cell permeabilization method (cell cracking) which utilizes mechanical shear. GH3 pituitary cells subjected to cracking were permeable to macromolecules but retained a normal cytoplasmic ultrastructure including secretory granules. Incubation of the permeable cells at 30-37 degrees C with 0.1-1.0 microM Ca2+ and millimolar MgATP resulted in the release of the secretory proteins, prolactin (PRL) and a proteoglycan, but not lysosomal enzymes. Extensively washed permeable cells were incapable of releasing PRL in response to Ca2+ and MgATP addition. However, addition of cytosol was found to restore Ca2+-activated, MgATP-dependent PRL release. The cytosolic factor responsible for activity was thermolabile and protease sensitive. The protein was partially purified, and its molecular mass was estimated to be equivalent to that of a globular protein of 200-350 kDa by molecular sieve chromatography. Inhibitors of calmodulin or protein kinase C (trifluroperazine, calmidazolium, H-7) failed to inhibit Ca2+-activated PRL release, and the required cytosolic protein could not be replaced by purified calmodulin, calmodulin-dependent protein kinase II, protein kinase C, or calpactin I. Further purification and characterization of the cytosolic protein should reveal the nature of biochemical events involved in regulated secretory exocytosis.     

Identifying Space Use at Foraging Arena Scale within the Home Ranges of Large Herbivores

An intermediate spatiotemporal scale of food procurement by large herbivores is evident within annual or seasonal home ranges. It takes the form of settlement periods spanning several days or weeks during which foraging activity is confined to spatially discrete foraging arenas, separated by roaming interludes. Extended by areas occupied for other activities, these foraging arenas contribute towards generating the home range structure. We delineated and compared the foraging arenas exploited by two African large herbivores, sable antelope (a ruminant) and plains zebra (a non-ruminant), using GPS-derived movement data. We developed a novel approach to specifically delineate foraging arenas based on local change points in distance relative to adjoining clusters of locations, and compared its output with modifications of two published methods developed for home range estimation and residence time estimation respectively. We compared how these herbivore species responded to seasonal variation in food resources and how they differed in their spatial patterns of resource utilization. Sable antelope herds tended to concentrate their space use locally, while zebra herds moved more opportunistically over a wider set of foraging arenas. The amalgamated extent of the foraging arenas exploited by sable herds amounted to 12-30 km², compared with 22-100 km² for the zebra herds. Half-day displacement distances differed between settlement periods and roaming interludes, and zebra herds generally shifted further over 12h than sable herds. Foraging arenas of sable herds tended to be smaller than those of zebra, and were occupied for period twice as long, and hence exploited more intensively in days spent per unit area than the foraging arenas of zebra. For sable both the intensity of utilization of foraging arenas and proportion of days spent in foraging arenas relative to roaming interludes declined as food resources diminished seasonally, while zebra showed no seasonal variation in these metrics. Identifying patterns of space use at foraging arena scale helps reveal mechanisms generating the home range extent, and in turn the local population density. Thereby it helps forge links between behavioural ecology, movement ecology and population ecology.