The Functional Role of TLR9 in Human Platelets

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 366-366
Author(s):  
Jonathan Noah Thon ◽  
Christian G. Peters ◽  
Rukhsana Aslam ◽  
Jesse Rowley ◽  
Andrew S. Weyrich ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Type Iv Collagen ◽  
Surface Expression ◽  
Dense Granule ◽  
Immunogold Electron Microscopy ◽  
Cell Periphery ◽  
Severe Thrombocytopenia ◽  
Viral Dna ◽  
Type Iv ◽  
Type C

Abstract Abstract 366 Human and murine platelets (PLTs) variably express toll-like receptors (TLRs), which link the innate and adaptive immune responses during infectious inflammation and atherosclerotic vascular disease. This is perhaps best exemplified by the observation that severe thrombocytopenia is associated with sepsis. While recent phenotypic and functional studies have addressed the roles of TLRs 2 and 4 in human/murine PLTs, which are primarily extracellular receptors, very little is known about the cellular localization, expression, and physiological significance of intracellular TLR9. Here we show that TLR9 localizes to unique granular compartments along the cell periphery (underlying the plasma membrane) in human PLTs. While thought to reside exclusively in the endosomes of monocytes, macrophages, and plasmacytoid dendritic cells, TLR9 expression is significantly enhanced in PLTs following incubation with thombin, ADP, PMA, CRP and type IV collagen, suggesting activation-mediated granule release. Surprisingly, TLR9 surface expression did not coincide with CD62P and CD61 expression levels upon PLT activation (which were not increased following type IV collagen incubation), and TLR9 was shown not to co-localize with known alpha-granule (fibrinogen, CD62P, PDGF-B, VEGF, CD42a, CD42b), dense granule (serotonin), endosomal (M6P, syntaxin-13), or lysosomal (LAMP-1) proteins. Immunogold electron microscopy revealed that TLR9 was expressed instead in a novel intracellular electron-dense granule (T-granule) that is mostly distributed along the plasma membrane. Incubation of resting PLTs with synthetic unmethylated Type C CpG dinucleotides (characteristic of bacterial/viral DNA) resulted in increased TLR9 surface expression, followed by increased Type C CpG sequestration and CD62P surface expression over 20 minutes. Interestingly, when TLR9 surface expression was specifically upregulated by pre-incubating PLTs with type IV collagen, subsequent incubation with Type C CpG resulted in considerably increased Type C CpG sequestration, CD62P surface expression, and PLT aggregation within 30 seconds of addition. These results imply that PLTs must be primed to express TLR9 on their surface prior to signal transduction through TLR9, and provide a mechanism for PLT regulation of the immune response to infection in human whole blood by sequestering bacterial/viral DNA and marking themselves for clearance. Taken together, this paper; (1) tracks TLR9 to a new intracellular compartment in PLTs, (2) describes a novel mechanism of TLR9 signaling, and (3) reveals a unique role for human PLTs as mediators of innate immunity at sites of vascular damage. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Initiation of Assembly of the Cell Envelope Barrier Structure of Stratified Squamous Epithelia

1999 ◽  
Vol 10 (12) ◽  
pp. 4247-4261 ◽  
Author(s):  
Peter M. Steinert ◽  
Lyuben N. Marekov
Keyword(s):  
Plasma Membrane ◽  
Cultured Cells ◽  
Cell Envelope ◽  
Immunogold Electron Microscopy ◽  
Epidermal Keratinocytes ◽  
Cross Linking ◽  
Cell Periphery ◽  
Human Epidermal Keratinocytes ◽  
Squamous Epithelia ◽  
D Cells

The cell envelope (CE) is a specialized structure that is important for barrier function in terminally differentiated stratified squamous epithelia. The CE is formed inside the plasma membrane and becomes insoluble as a result of cross-linking of constituent proteins by isopeptide bonds formed by transglutaminases. To investigate the earliest stages of assembly of the CE, we have studied human epidermal keratinocytes induced to terminally differentiate in submerged liquid culture as a model system for epithelia in general. CEs were harvested from 2-, 3-, 5-, or 7-d cultured cells and examined by 1) immunogold electron microscopy using antibodies to known CE or other junctional proteins and 2) amino acid sequencing of cross-linked peptides derived by proteolysis of CEs. Our data document that CE assembly is initiated along the plasma membrane between desmosomes by head-to-tail and head-to-head cross-linking of involucrin to itself and to envoplakin and perhaps periplakin. Essentially only one lysine and two glutamine residues of involucrin and two glutamines of envoplakin were used initially. In CEs of 3-d cultured cells, involucrin, envoplakin, and small proline-rich proteins were physically located at desmosomes and had become cross-linked to desmoplakin, and in 5-d CEs, these three proteins had formed a continuous layer extending uniformly along the cell periphery. By this time >15 residues of involucrin were used for cross-linking. The CEs of 7-d cells contain significant amounts of the protein loricrin, typically expressed at a later stage of CE assembly. Together, these data stress the importance of juxtaposition of membranes, transglutaminases, and involucrin and envoplakin in the initiation of CE assembly of stratified squamous epithelia.

scholarly journals Cytoplasmic domain of P-selectin (CD62) contains the signal for sorting into the regulated secretory pathway.

1992 ◽  
Vol 3 (3) ◽  
pp. 309-321 ◽  
Author(s):  
M Disdier ◽  
J H Morrissey ◽  
R D Fugate ◽  
D F Bainton ◽  
R P McEver
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Secretory Pathway ◽  
Surface Expression ◽  
Cytoplasmic Domain ◽  
Immunogold Electron Microscopy ◽  
Dense Granules ◽  
Storage Granules ◽  
Regulated Secretory Pathway ◽  
Necessary And Sufficient

P-selectin (CD62), formerly called GMP-140 or PADGEM, is a membrane protein located in secretory storage granules of platelets and endothelial cells. To study the mechanisms responsible for the targeting of P-selectin to storage granules, we transfected its cDNA into COS-7 and CHO-K1 cells, which lack a regulated exocytic pathway, or into AtT20 cells, which are capable of regulated secretion. P-selectin was expressed on the plasma membrane of COS-7 and CHO-K1 cells but was concentrated in storage granules of AtT20 cells. Immunogold electron microscopy indicated that the electron-dense granules containing P-selectin in AtT20 cells also stored the endogenous soluble hormone ACTH. Activation of AtT20 cells with 8-Br-cAMP increased the surface expression of P-selectin, consistent with agonist-induced fusion of granule membranes with the plasma membrane. Deletion of the last 23 amino acids of the 35-residue cytoplasmic domain resulted in delivery of P-selectin to the plasma membrane of AtT20 cells. Replacement of the cytoplasmic tail of tissue factor, a plasma membrane protein, with the cytoplasmic domain of P-selectin redirected the chimeric molecule to granules. We conclude that the cytoplasmic domain of P-selectin is both necessary and sufficient for sorting of membrane proteins into the regulated pathway of secretion.

scholarly journals Cell adhesion to extracellular matrix regulates the life cycle of integrins.

1995 ◽  
Vol 6 (12) ◽  
pp. 1781-1791 ◽  
Author(s):  
S L Dalton ◽  
E Scharf ◽  
R Briesewitz ◽  
E E Marcantonio ◽  
R K Assoian
Keyword(s):  
Extracellular Matrix ◽  
Life Cycle ◽  
Cell Surface ◽  
Matrix Protein ◽  
Type Iv Collagen ◽  
Surface Expression ◽  
Extracellular Matrix Protein ◽  
Integrin Subunit ◽  
Type Iv ◽  
Beta 1 Integrin

The expression of alpha 5 beta 1 integrin on the surface of fibroblasts requires adhesion to substratum. We have examined the basis for this adhesion-dependent surface expression by comparing the life cycle of integrins in parallel cultures of adherent and nonadherent cells. Results of biosynthetic labeling experiments in NRK fibroblasts showed that the synthesis and biosynthetic processing of the beta 1 integrin subunit proceed in the absence of cell attachment; however, when examining the behavior of preexisting cell surface integrins, we observed that the alpha beta 1 integrins are internalized and degraded when adhesion to substratum is blocked. A kinetic analysis of integrin internalization in cycloheximide-treated NRK cells showed that each of the fibroblast integrins we examined (in both the beta 1 and beta 3 families) are lost from the cell surface after detachment from substratum. Thus, the default integrin life cycle in fibroblasts involves continuous synthesis, processing, transport to the cell surface, and internalization/degradation. Interestingly, studies with NIH-3T3 cells expressing alpha 1 beta 1 integrin showed that the loss of cell-surface alpha 5 beta 1 integrin is blocked by adhesion of cells to dishes coated with type IV collagen (a ligand for alpha 1 beta 1 integrin) as well as fibronectin. Similarly, adhesion of these cells to dishes coated with type IV collagen stabilizes the surface expression of alpha 5 beta 1 as well as alpha 1 beta 1 integrin. We propose that the adhesion of fibroblasts to extracellular matrix protein alters the integrin life cycle and permits retention of these proteins at the cell surface where they can play important roles in transmitting adhesion-dependent signals.

scholarly journals T granules in human platelets function in TLR9 organization and signaling

2012 ◽  
Vol 198 (4) ◽  
pp. 561-574 ◽  
Author(s):  
Jonathan N. Thon ◽  
Christopher G. Peters ◽  
Kellie R. Machlus ◽  
Rukhsana Aslam ◽  
Jesse Rowley ◽  
...  
Keyword(s):  
Type Iv Collagen ◽  
Surface Expression ◽  
Human Platelets ◽  
Contact Activation ◽  
Atherosclerotic Vascular Disease ◽  
Adaptive Immune ◽  
Type Iv ◽  
Tubular System ◽  
Protein Disulfide ◽  
Platelets Function

Human and murine platelets (PLTs) variably express toll-like receptors (TLRs), which link the innate and adaptive immune responses during infectious inflammation and atherosclerotic vascular disease. In this paper, we show that the TLR9 transcript is specifically up-regulated during pro-PLT production and is distributed to a novel electron-dense tubular system-related compartment we have named the T granule. TLR9 colocalizes with protein disulfide isomerase and is associated with either VAMP 7 or VAMP 8, which regulates its distribution in PLTs on contact activation (spreading). Preincubation of PLTs with type IV collagen specifically increased TLR9 and CD62P surface expression and augmented oligodeoxynucleotide (ODN) sequestration and PLT clumping upon addition of bacterial/viral ODNs. Collectively, this paper (a) tracks TLR9 to a new intracellular compartment in PLTs and (b) describes a novel mechanism of TLR9 organization and signaling in human PLTs.

The use of 1nm silver enhanced gold probes for the detection of skin antigens

1990 ◽  
Vol 48 (3) ◽  
pp. 902-903
Author(s):  
J.P Cassella ◽  
H. Shimizu ◽  
A. Ishida-Yamamoto ◽  
R.A.J. Eady
Keyword(s):  
Type Iv Collagen ◽  
Freeze Substitution ◽  
Collagen Type Iv ◽  
Electron Microscopic ◽  
Normal Human Skin ◽  
Type Iv ◽  
Type Vii Collagen ◽  
Keratin Filaments ◽  
Normal Human ◽  
Phosphate Buffered Saline

1nm colloidal gold with silver enhancement has been used in conjunction with a low-temperature post-embedding (post-E) technique for the demonstration of skin antigens at both the light microscopic (LM) and electron microscopic (EM) levels.Keratin filaments and basement membrane zone (BMZ) associated antigens in normal human skin (NHS) were immunolabelled using antibodies against keratin 14, 10, and 1, the carboxy-terminus and collagenous portion of type VII collagen, type IV collagen and bullous pemphigoid antigen (BP-Ag).Fresh samples of NHS were cryoprotected in 15% glycerol, cryofixed in propane at -190°C, subjected to freeze substitution in methanol at -80°C and embedded in Lowicryl K11M at -60°C. Polymerisation of the resin was initiated under UVR at - 60°C for 48 hours and continued at room temperature for a further 48 hours. Semith in sections were air dried onto slides coated with 3-aminopropyltriethoxysilane. The following immunolabelling protocol was adopted: Primary antibody was applied for 2 hours at 37°C or overnight at 4°C. Following washing in Dulbecco’s phosphate buffered saline (PBSA) a biotinylated secondary antibody was applied for 2 hours at 37°C. The sections were further washed in PBSA and 1nm gold avidin was applied. Sections were finally washed in PBSA and silver enhanced.

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