Interplay of cysteinyl leukotrienes and TGF-ß in the activation of hepatic stellate cells from Schistosoma mansoni granulomas.

Hepatic stellate cells (HSCs) have a critical role in liver physiology, and in the pathogenesis of liver inflammation and fibrosis. Here, we investigated the interplay between leukotrienes (LT) and TGF-ß in the activation mechanisms of HSCs from schistosomal granulomas (GR-HSCs). First, we demonstrated that GR-HSCs express 5-lipoxygenase (5-LO), as detected by immunolocalization in whole cells and confirmed in cell lysates through western blotting and by mRNA expression through RT-PCR. Moreover, mRNA expression of 5-LO activating protein (FLAP) and LTC(4)-synthase was also documented, indicating that GR-HSCs have the molecular machinery required for LT synthesis. Morphological analysis of osmium and Oil-Red O-stained HSC revealed large numbers of small lipid droplets (also known as lipid bodies). We observed co-localization of lipid droplet protein marker (ADRP) and 5-LO by immunofluorescence microscopy. We demonstrated that GR-HSCs were able to spontaneously release cysteinyl-LTs (CysLTs), but not LTB(4,) into culture supernatants. CysLT production was highly enhanced after TGF-ß-stimulation. Moreover, the 5-LO inhibitor zileuton and 5-LO gene deletion were able to inhibit the TGF-ß-stimulated proliferation of GR-HSCs, suggesting a role for LTs in HSC activation. Here, we extend the immunoregulatory function of HSC by demonstrating that HSC from liver granulomas of schistosome-infected mouse are able to release Cys-LTs in a TGF-ß-regulated manner, potentially impacting pathogenesis and liver fibrosis in schistosomiasis.

Vesicle-mediated secretion of human eosinophil granule-derived major basic protein.

Major basic protein (MBP), the predominant cationic protein of human eosinophil specific granules, is stored within crystalloid cores of these granules. Secretion of MBP contributes to the immunopathogenesis of varied diseases. Prior electron microscopy (EM) of eosinophils in sites of inflammation noted losses of granule cores in the absence of granule exocytosis and suggested that eosinophil granule proteins might be released through piecemeal degranulation (PMD), a secretory process mediated by transport vesicles. Because release of eosinophil granule-derived MBP through PMD has not been studied, we evaluated secretion of this cationic protein by human eosinophils. Intracellular localizations of MBP were studied within nonstimulated and eotaxin-stimulated human eosinophils by both immunofluorescence and a pre-embedding immunonanogold EM method that enables optimal epitope preservation and antigen access to membrane microdomains. In parallel, quantification of transport vesicles was assessed in eosinophils from a patient with hypereosinophilic syndrome (HES). Our data demonstrate vesicular trafficking of MBP within eotaxin-stimulated eosinophils. Vesicular compartments, previously implicated in transport from granules to the plasma membrane, including large vesiculotubular carriers termed eosinophil sombrero vesicles (EoSVs), were found to contain MBP. These secretory compartments were significantly increased in numbers within HES eosinophils. Moreover, in addition to granule-stored MBP, even unstimulated eosinophils contained appreciable amounts of MBP within secretory vesicles, as evidenced by immunonanogold EM and immunofluorescent colocalizations of MBP and CD63. These data suggest that eosinophil MBP, with its multiple extracellular activities, can be mobilized from granules by PMD into secretory vesicles and both granule- and secretory vesicle-stored pools of MBP are available for agonist-elicited secretion of MBP from human eosinophils. The recognition of PMD as a secretory process to release MBP is important to understand the pathological basis of allergic and other eosinophil-associated inflammatory diseases.

Human eosinophils secrete preformed, granule-stored interleukin-4 through distinct vesicular compartments.

Secretion of interleukin-4 (IL-4) by leukocytes is important for varied immune responses including allergic inflammation. Within eosinophils, unlike lymphocytes, IL-4 is stored in granules (termed specific granules) and can be rapidly released by brefeldin A (BFA)-inhibitable mechanisms upon stimulation with eotaxin, a chemokine that activates eosinophils. In studying eotaxin-elicited IL-4 secretion, we identified at the ultrastructural level distinct vesicular IL-4 transport mechanisms. Interleukin-4 traffics from granules via two vesicular compartments, large vesiculotubular carriers, which we term eosinophil sombrero vesicles (EoSV), and small classical spherical vesicles. These two vesicles may represent alternative pathways for transport to the plasma membrane. Loci of both secreted IL-4 and IL-4-loaded vesicles were imaged at the plasma membranes by a novel EliCell assay using a fluoronanogold probe. Three dimensional electron tomographic reconstructions revealed EoSVs to be folded, flattened and elongated tubules with substantial membrane surfaces. As documented with quantitative electron microscopy, eotaxin-induced significant formation of EoSVs while BFA pretreatment suppressed eotaxin-elicited EoSVs. Electron tomography showed that both EoSVs and small vesicles interact with and arise from granules in response to stimulation. Thus, this intracellular vesicular system mediates the rapid mobilization and secretion of preformed IL-4 by activated eosinophils. These findings, highlighting the participation of large tubular carriers, provide new insights into vesicular trafficking of cytokines.

Intragranular vesiculotubular compartments are involved in piecemeal degranulation by activated human eosinophils.

Eosinophils, leukocytes involved in allergic, inflammatory and immunoregulatory responses, have a distinct capacity to rapidly secrete preformed granule-stored proteins through piecemeal degranulation (PMD), a secretion process based on vesicular transport of proteins from within granules for extracellular release. Eosinophil-specific granules contain cytokines and cationic proteins, such as major basic protein (MBP). We evaluated structural mechanisms responsible for mobilizing proteins from within eosinophil granules. Human eosinophils stimulated for 30-60 min with eotaxin, regulated on activation, normal, T-cell expressed and secreted (RANTES) or platelet activating factor exhibited ultrastructural features of PMD (e.g. losses of granule contents) and extensive vesiculotubular networks within emptying granules. Brefeldin A inhibited granule emptying and collapsed intragranular vesiculotubular networks. By immunonanogold ultrastructural labelings, CD63, a tetraspanin membrane protein, was localized within granules and on vesicles outside of granules, and mobilization of MBP into vesicles within and extending from granules was demonstrated. Electron tomography with three dimension reconstructions revealed granule internal membranes to constitute an elaborate tubular network able to sequester and relocate granule products upon stimulation. We provide new insights into PMD and identify eosinophil specific granules as organelles whose internal tubulovesicular networks are important for the capacity of eosinophils to secrete, by vesicular transport, their content of preformed and granule-stored cytokines and cationic proteins.

EliCell assay for the detection of released cytokines from eosinophils.

Eosinophils contain several preformed cytokines within their specific granules. Therefore, without requiring them in de novo synthesis of cytokines, eosinophils can release quantities of granule-derived cytokines by highly regulated mechanisms. However, eosinophil "degranulation" is poorly understood, in part, because available methodologies did not appear appropriate for analyzing vesicular mobilization and transport of eosinophil granular contents. The EliCell assay is a microscopic methodology substantially modified from other techniques employed to detect cytokine release (i.e., ELISPOT). The method is a dual antibody capture/detection system in which viable eosinophils are incubated in a solid streptavidin-conjugated agarose matrix, which contains a biotinylated capture antibody against the cytokine of interest. Released cytokine is detected around non-permeabilized eosinophils with a separate fluorochrome-labeled detection antibody. Thus, the EliCell system captures and detects extracellular cytokines at the site of their release from eosinophils. As examples, we have used EliCell essays to detect the selective release of either IL-4 or IL-12 cytokines found preformed in eosinophils-from eotaxin- or anti-CD9-stimulated eosinophils, respectively. With appropriate pairs of antibodies, any preformed cytokine found into eosinophil granules could be studied and the mechanisms of their secretion evaluated by using the EliCell assay.