Evidence of selective packaging and different α-granule subtypes in canine platelets.

Platelets are circulating megakaryocytic cytoplasmic fragments that are required to maintain vascular integrity and prevent haemorrhage. During activation, platelets release hundreds of bioactive proteins, mainly by secretion of α-granule content. These proteins include von Willebrand factor (vWF) and fibrinogen, major adhesive proteins involved in haemostasis. Human and mouse platelet α-granules are packaged selectively to contain different molecules and platelets can differentially release these proteins on specific receptor activation. Confocal laser scanning microscopy was used to analyse vWF and fibrinogen localization within canine platelet α-granules. In resting canine platelets, the majority of platelet vWF and fibrinogen is located in separate α-granule populations. These findings provide evidence that canine platelets contain different α-granule subtypes, suggesting that selective protein packaging occurs during canine platelet ontology.

The platelet interior revisited: electron tomography reveals tubular alpha-granule subtypes.

We have used (cryo) electron tomography to provide a 3-dimensional (3D) map of the intracellular membrane organization of human platelets at high spatial resolution. Our study shows that the open canalicular system and dense tubular system are highly intertwined and form close associations in specialized membrane regions. 3D reconstructions of individual alpha-granules revealed large heterogeneity in their membrane organization. On the basis of their divergent morphology, we categorized alpha-granules into the following subtypes: spherical granules with electron-dense and electron-lucent zone containing 12-nm von Willebrand factor tubules, subtypes containing a multitude of luminal vesicles, 50-nm-wide tubular organelles, and a population with 18.4-nm crystalline cross-striations. Low-dose (cryo) electron tomography and 3D reconstruction of whole vitrified platelets confirmed the existence of long tubular granules with a remarkably curved architecture. Immunoelectron microscopy confirmed that these extended structures represent alpha-granule subtypes. Tubular alpha-granules represent approximately 16% of the total alpha-granule population and are detected in approximately half of the platelet population. They express membrane-bound proteins GLUT3 and alphaIIb-beta3 integrin and contain abundant fibrinogen and albumin but low levels of beta-thromboglobulin and no von Willebrand factor. Our 3D study demonstrates that, besides the existence of morphologically different alpha-granule subtypes, high spatial segregation of cargo exists within individual alpha-granules.

CXCR1/2 ligands induce p38 MAPK-dependent translocation and release of opioid peptides from primary granules in vitro and in vivo.

Polymorphonuclear leukocytes (PMN) can release opioid peptides which bind to opioid receptors on sensory neurons and inhibit inflammatory pain. This release can be triggered by chemokine receptor 1/2 (CXCR1/2) ligands. Our aim was to identify the granule subpopulation containing opioid peptides and to assess whether MAPK mediate the CXCR1/2 ligand-induced release of these peptides. Using double immunofluorescence confocal microscopy, we showed that beta-endorphin (END) and Met-enkephalin (ENK) were colocalized with the primary (azurophil) granule markers CD63 and myeloperoxidase (MPO) within PMN. END and ENK release triggered by a CXCR1/2 ligand in vitro was dependent on the presence of cytochalasin B (CyB) and on p38 MAPK, but not on p42/44 MAPK. In addition, translocation of END and ENK containing primary granules to submembranous regions of the cell was abolished by the p38 MAPK inhibitor SB203580. In vivo CXCL2/3 reduced pain in rats with complete Freund's adjuvant (CFA)-induced hindpaw inflammation. This effect was attenuated by intraplantar (i.pl.) antibodies against END and ENK and by i.pl. p38 MAPK inhibitor treatment. Taken together, these findings indicate that END and ENK are contained in primary granules of PMN, and that CXCR1/2 ligands induce p38-dependent translocation and release of these opioid peptides to inhibit inflammatory pain.

The platelet release reaction: granules' constituents, secretion and functions.

Although anucleated, blood platelets are highly organized cells rich in different types of organelles. Three specific granule populations store different types of constituents, some of which are at high concentrations. Platelets thus transport some specific compounds through the whole body. During circulation, platelets are reactive to various stimuli and release the materials stored in the specific granules. This 'release reaction' is an important step of primary haemostasis. Energy and messengers required for platelet reactivity are provided by mitochondria and the dense tubular system. Each granule population has specific properties concerning both the structure and the role played by the released constituents. Dense granules contain small non-protein molecules that are secreted to recruit other platelets. alpha-Granules contain large adhesive and healing proteins. Lysosomes contain hydrolases able to eliminate the circulating platelet aggregate. The extrusion of storage granules' content to the platelet's environment occurs according to regulated secretion events: movements of granules, apposition and fusion of granule and plasma membranes. Typical platelet disorders resulting from a storage granule abnormality are referred to as a storage pool defect and are characterized by a prolonged bleeding time.

[Granules of neutrophils]. / Ziarnistosci granulocytów obojetnochlonnych.

The exocytosis of cytoplasmic granules plays a most important role in the regulation of many neutrophils functions: adhesion, phagocytosis, killing of bacteria and interaction with endothelial cells. Neutrophils contain following types of granules: azurophilic (primary) granules, specific (secondary) granules, gelatinase-rich tertiary granules and secretory vesicles. Neutrophil granules may be classified on the basis of their size, morphology, density or with reference to a given protein.

The individual regulation of granule protein mRNA levels during neutrophil maturation explains the heterogeneity of neutrophil granules.

The in vivo mRNA levels for 16 granule proteins during neutrophil differentiation were determined to address the question of whether the synthesis of granule proteins is regulated individually or blockwise. RNA was extracted from peripheral blood granulocytes and three different populations of neutrophil precursors isolated from human bone marrow by Percoll density centrifugation. The mRNA levels in relation to the maturation of the cells were determined by Northern blot for the 12 matrix proteins myeloperoxidase, proteinase-3, elastase, defensin, lactoferrin, NGAL, hCAP-18, transcobalamin-I, SGP28, gelatinase, lysozyme, and serglycin and the 4 membrane proteins CD68, CD11b, N-formyl-methionyl-leucyl-phenylalanine receptor, and CD35. This panel of transcripts ensured that markers for all exocytosable organelles of the neutrophil were included in the study. A highly differentiated distribution of mRNAs for granule proteins was demonstrated that can explain the heterogeneity of the intracellular storage granules and secretory vesicles of the neutrophil. Furthermore, the individual distribution of these transcripts provides the basis for a more detailed assessment of neutrophil maturation than that obtained by morphological studies or the use of a single marker protein for azurophil, specific, and gelatinase granules.

Distinct granule populations in human neutrophils and lysosomal organelles identified by immuno-electron microscopy.

In this paper, we illustrate the fine structural localization of distinct marker proteins in the organelles of human neutrophils and outline our preferred methods for processing ultrathin cryosections for use with immunoelectron microscopy. Previous work has determined the subcellular localization of certain marker proteins within intact polymorphonuclear neutrophilic leukocytes (PMN) and PMN fractions. These are as follows: myeloperoxidase (MPO) for azurophilic granules, lactoferrin for specific/secondary granules, gelatinase for gelatinase/tertiary granules, albumin for the secretory vesicles, and HLA class I and L-selectin for the plasma membrane. In addition to analyzing the heterogeneity of the PMN granule populations, new information on the lysosomal system of this cell is reviewed and extended by the localization of the lysosome-associated membrane proteins (LAMPs) and the cation-independent mannose 6-phosphate receptor (CI-M6PR). LAMPs were absent in all identified granule populations, but were found in the membranes of vesicles, multivesicular bodies (MVB), and multilaminar compartments (MLC). We show here that MVB contain CI-M6PR whereas MLC do not. Furthermore, since MLC contain LAMPs but not the receptor, they probably correspond to the late endosome. By current criteria, the true lysosomes of the resting PMN are MVB and MLC. Finally, although azurophil granules contain acid hydrolases their membranes do not contain LAMPs and they cannot be classified as lysosomes, but rather are more similar to regulated secretory granules.

Monocyte-derived dendritic cells have a phenotype comparable to that of dermal dendritic cells and display ultrastructural granules distinct from Birbeck granules.

Most monocyte-derived dendritic cells (DC) display CD1a, like Langerhans cells (LC) and some dermal DC, but their relationship with these skin DC remains unclear. To address this issue, we studied the expression of different antigens characteristic of skin DC and of monocyte/macrophages in CD1a+ and CD1a- monocyte-derived DC. Their phenotype indicated that they may be related to dermal DC rather than to LC, i.e., they were all CD11b-positive, and 72% were Factor XIIIa-positive, but they did not express E-cadherin nor VLA-6. It is interesting that CD1a+ and CD1a-cells showed intracytoplasmic granules that were different from LC Birbeck granules. These phenotypical and ultrastructural features are comparable to those of CD14-derived DC obtained from cord blood precursors [C. Caux et al. J. Exp. Med. 184, 695-706]. These results show a close relationship between these two in vitro models, which are both related to dermal DC.

Human monocytes and neutrophils store transforming growth factor-alpha in a subpopulation of cytoplasmic granules.

Transforming growth factor-alpha (TGF-alpha) exerts several effects on target cells, such as neovascularization promotion and mitogenic signalling. Using immunoelectron microscopy, we show that monocytes and neutrophils, store TGF-alpha in cytoplasmic granules. In monocytes, TGF-alpha did not colocalize with components of peroxidase-positive granules or with albumin of secretory vesicles. Furthermore, no colocalization of TGF-alpha with components of azurophilic or specific granules or secretory vesicles was observed in neutrophils. Activated monocytes and tissue-macrophages contained much less TGF-alpha-positive granules, suggesting TGF-alpha release. Western blot analysis showed a protein of 10 kD in lysates of monocytes. TGF-alpha mRNA was detected in monocytoid cells from the bone marrow by in situ hybridization. This study shows for the first time that monocytes and neutrophils contain TGF-alpha in all stages of maturation and that TGF-alpha in monocytes is stored in a large population of peroxidase-negative granules suggesting a function for these granules. Monocytes and neutrophils are important effector cells in inflammatory reactions. The present finding that these cells contain TGF-alpha might explain complications such as fibrosis and neoplastic transformation, caused by chronic inflammation.

Development of neutrophil granule diversity.

The neutrophil has intracellular stores of both membrane proteins and soluble proteins that may be incorporated into the plasma membrane and exocytosed, respectively, at different times to meet the demands for assisting the neutrophil in adhesion to endothelium (secretory vesicles), for migration through basement membranes (gelatinase granules), and for phagocytosis, killing, and digestion of microorganisms (specific granules and azurophil granules). One reason for segregating the proteins into different subpopulations of granules is that some proteins cannot exist in the same compartment (NGAL digested if present in azurophil granules). Another reason is that the content of the different granules is needed at different times and places. The background for the diversity of neutrophil granules is the timing of biosynthesis, which again most likely is the result of transcriptional control, although this is not yet proven. It is possible that the control of exocytosis is also determined by the same mechanism--a carefully controlled timing of biosynthesis of fusion proteins that ties the content of granules to the likelihood that a given stimulus will result in exocytosis of that individual granule subset.

Distribution of cortical granules in bovine oocytes classified by cumulus complex.

The present study was conducted to examine distributional changes of cortical granules (CGs) during meiotic maturation and fertilisation in vitro and the developmental ability in bovine oocytes classified by cumulus cells. The oocytes were classified by the morphology of their cumulus cell layers as follows: class A, compact and thick; class B, compact but thin; class C, naked; and class D, expanded. Some of the oocytes were stained with Lens curinalis agglutinin (LCA) before and after maturation in vitro and after insemination, and then stained with orcein to observe their nuclear stages. The others were left in culture. Distributional patterns of the CGs were classified into four types: type I, CGs distributed in clusters; type II, CGs dispersed and partly clustered; type III, all CGs dispersed; and type IV, no CGs. Most of the oocytes before culture showed a type I pattern, but this decreased after maturation culture, whereas type III increased in class A. The oocytes of class B showed similar changes while the oocytes of class C did not. In class C, many oocytes showed type I after culture, indicating that cytoplasmic maturation was not completed. In class D, 80.4% of the oocytes exhibited type III before maturation culture, indicating that their cytoplasmic maturation was different from classes A-C. And about 70% of the class D oocytes were at the nuclear stage of germinal vesicle breakdown (GVBD) before culture. The developmental rates to blastocysts in classes A-D were 28.7%, 23.1%, 0.5% and 3.4% respectively.

Tubular bodies (Weibel-Palade bodies) in endothelial cells of glioblastomas and astrocytomas.

The mean % ratio value of the number of endothelial cells with tubular bodies for the 6 cases of the glioblastomas proved to be 32.4% in the margin, which was about two times as high as that (15.7%) in the center of the tumors, showing a tendency for tubular bodies to be increased broadly in parallel with vascularization. In the 5 cases of astrocytoma, vascularization was less marked, and the mean % ratio was lower (14.8%) in all studied locations of the astrocytoma. It is suggested that the tubular bodies in the endothelial cells of microvessels of astrocytic tumors could increase in relation to microvessel proliferation and tumor malignancy.

An ultrastructural study of primary granule subtypes in human neutrophils.

To investigate the heterogeneity of primary neutrophil granules, neutrophils in the bone marrow of 68 patients with hematologic and non-hematologic diseases were observed by electron microscopy. In addition to typical primary granules, 3 primary granule subtypes, i.e., parallel tubular granules (PTGs), fibrillar granules (FGs), and periodic lamellar granules (PLGs), were present in neutrophils. PTGs were found in only one case of chronic neutrophilic leukemia (CNL). On the other hand, FGs and PLGs were present in various cases at varying frequencies. Three subtype granules were seen preferentially in immature neutrophils. These subtypes were positive for myeloperoxidase, showing that they represent primary granules. In addition, hybrid PTG/FG granules, hybrid PTG/PLG granules, and hybrid FG/PLG granules were seen in the PTG-positive CNL case. This shows that a close association is present between PTGs, FGs, and PLGs.

Morphometry of Rambourg-positive and Rambourg-negative beta-cell granules after culture with low and high glucose concentrations.

Dispersed islet cells from noninbred ob/ob mice were cultured for 3 days with 3 or 20 mM D-glucose and silver stained according to Rambourg et al. Two tinctorial subsets of dark and light intracellular granules were analyzed by morphometry at the ultrastructural level. The two types of granules were similar in size and shape. However, with 3 mM glucose the dark granule cores were surrounded by larger vesicles than the light granules. With 20 mM glucose, both types of granule vesicles and cores became smaller and dark-granule cores became more rounded, compared with cultures with 3 mM glucose. The higher glucose concentration also induced a marked decrease in the number (-84%) and volume density (-90%) of dark granules. In contrast, the number of light granules increased (+60%) with maintenance of their volume density. We suggest that the dark Rambourg-positive and the light Rambourg-negative beta-cell granules are functionally distinct subsets. The dark granules are probably engaged in insulin discharge. We discuss the unclear role of the light granules with a view to previously postulated heterogeneities of the insulin granule pool and their significance for exocytosis and intracellular hormone degradation.

Size distribution of neurosecretory granules in the supraoptic nucleus and the neurohypophysis of water-loaded, normal and water-deprived rats.

The profiles of the neurosecretory granules were measured in the supraoptic nucleus and in axons, swellings and endings in the neurohypophysis. This was performed after the secretion of vasopressin had been fully suppressed by water load for 1.5, 2.5 and 24 h, after the secretion had been stimulated by water deprival for 72 h and in normal rats with water ad libitum. A finite-difference algorithm was used for unfolding the size distribution of neurosecretory granules from the observed profiles, and showed a clear grouping of the neurosecretory granules into 3 size groups: small, medium-sized and large granules. Estimates of the relative volumes of the granules from these 3 groups were combined with previous results. In the supraoptic nucleus, water deprival increased the total volumes of the small and medium-sized granules, and water load decreased the volumes of the medium-sized and especially the large granules. However, in the neurohypophysis, water deprival decreased and water load increased the volume densities of the small and medium-sized, but did not influence that of the large granules. The results indicate that the medium-sized granules are vasopressinergic and the large oxytocinergic. Moreover, the results are consistent with the hypothesis that the small contain another peptide.

Immature dense granules in platelets from mice with platelet storage pool disease.

Mepacrine uptake into platelets and bone marrow megakaryocytes was analyzed to further characterize the dense granule defects in a group of seven mouse pigment mutants that have characteristics of platelet storage pool disease (SPD). In contrast to our previous studies using electron microscopy, this method revealed that all mutants had normal numbers of dense granules. However, total mepacrine uptake in all mutant platelets was significantly diminished to less than 50% of normal uptake. Also, the flashing phenomenon observed when normal dense granules are irradiated with ultraviolet light was either greatly diminished or absent when platelets of individual mutants were similarly irradiated. Therefore the principal defect in the mutant platelets is an inability to accumulate dense granule contents rather than an absence of the granules. Mepacrine uptake into megakaryocytes was indistinguishable in normal and mutant mice. This indicates the mutant dense granule defects appear either very late in megakaryocyte development or early in platelet formation in correlation with development of the mature dense granule. By standard transmission electron microscopy we have not been able to detect gross structural or subcellular abnormalities in either platelets or megakaryocytes of mutant mice. It appears all seven mutants produce immature or functionally abnormal dense granules.