P-selectin Ligand Research Articles

Tissue-Specific Induction of CCR6 and Nrp1 During Early CD4+ T Cell Differentiation.

Upon differentiation, T cells acquire tissue-specific homing properties allowing efficient targeting of effector T cells into distinct inflamed organs. Priming of T cells within skin-draining, peripheral lymph nodes (pLNs) leads to the expression of E- and P-selectin ligands, which facilitate migration into inflamed skin, whereas activation within gut-draining, mesenteric LNs (mLNs) results in induction of chemokine receptor CCR9 and integrin α4β7, both required for migration of effector T cells into mucosal tissues. In addition to the local tissue microenvironment, both organ-specific dendritic cells and LN-resident stromal cells are critical factors to shape T cell migration properties. Here, we identify two additional homing-related molecules, CCR6 and Neuropilin-1 (Nrp1), upregulated in T cells early during differentiation solely in pLNs, but not mLNs. Surprisingly, intestinal inflammation resulted in an ameliorated induction of CCR6 and Nrp1 in pLNs, suggesting that a local inflammation within the gut can systemically alter T cell differentiation. Finally, transplantation of mLNs to a skin-draining environment revealed that LN stromal cells also contribute to efficient CCR6 induction in pLNs. Collectively, these findings identify further aspects of early T cell differentiation within skin-draining pLNs, which could be utilized to further develop tailored and highly specialized vaccination strategies.

Core 2 ß1,6-N-acetylglucosaminyltransferase-I, crucial for P-selectin ligand expression is controlled by a distal enhancer regulated by STAT4 and T-bet in CD4+ T helper cells 1.

P-selectin ligands (P-ligs) support the recruitment of lymphocytes into inflamed tissues. Binding to P-selectin is mediated by oligosaccharide groups synthesized by means of several glycosyltransferases including core 2 ß1,6-N-acetylglucosaminyltransferase-I (C2GlcNAcT-I), encoded by the gene Gcnt1. Using Gcnt1(-/-) Th1 cells, we show that C2GlcNAcT-I is crucial for inflammatory T cell homing in vivo. To understand the molecular regulation of Gcnt1 in CD4(+) T helper cells, we performed ChIP-on-chip experiments across the Gcnt1 locus assessing the chromatin structure in P-lig-expressing versus non-expressing CD4(+) T cells. This identified a distal region about 20kb upstream of the promoter where the presence of a H3K27me3 mark correlated with Gcnt1 repression. This region possessed IL-12-dependent enhancer activity in reporter assays, in accordance with preferential IL-12-dependent induction of Gcnt1 in vitro. STAT4 and T-bet cooperated in control of the enhancer activity. Deficiency in either one resulted in drastically reduced Gcnt1 mRNA expression in differentiated Th1 cells. While both STAT4 and T-bet were bound to the enhancer early after activation only T-bet binding persisted throughout the expansion phase after TCR signal cessation. This suggests sequential action of STAT4 and T-bet at the enhancer. In summary, we show that Gcnt1 transcription and subsequent P-lig induction in Th1 cells is governed by binding of STAT4 and T-bet to a distal enhancer and further regulated by epigenetic marks such as H3K27me3.

Abstract 4944: Detection of functional P-selectin ligands expressed on colon cancer tissue using a novel flow-based assay

Abstract The presence of functional P-selectin ligands is well-documented for human colon cancer cell lines, but not in situ on human colon carcinoma tissue. Presently, immunostaining with antibodies is used to detect critical components of selectin ligands, e.g., sialofucosylated moieties. However, this static biochemical tissue analysis (SBTA) cannot ascertain if a potential selectin ligand is able to mediate (rolling) adhesion. Due to the immense difficulty in detecting functional selectin ligands using traditional methods, we have developed a flow-based assay known as dynamic biochemical tissue analysis (DBTA) for detecting functional selectin ligands expressed on human tissue. DBTA using P-selectin microspheres was performed on colon cancer tissue sections from multiple cases, in conjunction with SBTA using P-selectin, antibodies against purported selectin ligand carbohydrate moieties sLeX and sLeA (HECA-452, CSLEX-1, and KM-231), and antibodies against peptide structures of putative P-selectin ligands (CD24, CD44, and PSGL-1). Examination of serial sections, in the same regions of tissue displaying DBTA probe adhesion, revealed significant detection inconsistencies with SBTA. Subsequently, due to the well-documented force-dependency of selectin ligands, DBTA was conducted with microspheres coated with either HECA-452, CSLEX-1, or KM-231 to determine the effect of applied force on the detection capabilities of these antibodies. Analysis of signet ring cell colon carcinoma tissue revealed microspheres coated with HECA-452, CSLEX-1, or KM-231 antibodies all displayed significantly lower amounts of adhesion than the DBTA P-selectin microspheres. Interestingly, although microspheres coated with these antibodies adhered to signet ring cell carcinoma tissue, these DBTA probes did not interact with all regions of tissue that displayed adhesion with P-selectin microspheres. Specificity of interaction was validated using corresponding isotype control coated microspheres. Taken together, these results show DBTA with P-selectin coated microspheres is able to unequivocally detect functional P-selectin ligands, in contrast to SBTA (immunostaining) and DBTA using microspheres coated with antibodies. In summary, DBTA using P-selectin coated microspheres is able to detect functional P-selectin ligands expressed on colon cancer tissue, data that may provide valuable diagnostic and prognostic information for malignant tumors. Citation Format: Eric W. Martin, Ramiro Malgor, Vicente A. Resto, Douglas J. Goetz, Monica M. Burdick. Detection of functional P-selectin ligands expressed on colon cancer tissue using a novel flow-based assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4944.

Rapid and label-free microfluidic neutrophil purification and phenotyping in diabetes mellitus.

Advanced management of dysmetabolic syndromes such as diabetes will benefit from a timely mechanistic insight enabling personalized medicine approaches. Herein, we present a rapid microfluidic neutrophil sorting and functional phenotyping strategy for type 2 diabetes mellitus (T2DM) patients using small blood volumes (fingerprick ~100 μL). The developed inertial microfluidics technology enables single-step neutrophil isolation (>90% purity) without immuno-labeling and sorted neutrophils are used to characterize their rolling behavior on E-selectin, a critical step in leukocyte recruitment during inflammation. The integrated microfluidics testing methodology facilitates high throughput single-cell quantification of neutrophil rolling to detect subtle differences in speed distribution. Higher rolling speed was observed in T2DM patients (P < 0.01) which strongly correlated with neutrophil activation, rolling ligand P-selectin glycoprotein ligand 1 (PSGL-1) expression, as well as established cardiovascular risk factors (cholesterol, high-sensitive C-reactive protein (CRP) and HbA1c). Rolling phenotype can be modulated by common disease risk modifiers (metformin and pravastatin). Receiver operating characteristics (ROC) and principal component analysis (PCA) revealed neutrophil rolling as an important functional phenotype in T2DM diagnostics. These results suggest a new point-of-care testing methodology, and neutrophil rolling speed as a functional biomarker for rapid profiling of dysmetabolic subjects in clinical and patient-oriented settings.

The biological role of sulfatides

Sulfatides (3-O-sulfogalactosylceramides, sulfated galactocerebrosides, SM4) are esters of sulfuric acid with galactosylceramides. These acidic glycosphingolipids, present at the external leaflet of the plasma membrane, are synthesized by a variety of mammalian cells. They are especially abundant in the myelin sheath of oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system. Studies using cerebroside galactosyltransferase-deficient mice revealed that sulfatides are responsible for proper structure and functioning of myelin. Large amounts of sulfatides are also found in the kidney, gastrointestinal tract, islets of Langerhans, and membranes of erythrocytes, thrombocytes and granulocytes. They are ligands for numerous proteins, but in most cases the biological role of such interactions is poorly understood. A notable exception is their binding by P- and L-selectins. Platelet sulfatides are major ligands for P-selectin, and this interaction is critical for the formation of stable platelet aggregates. Sulfatides also bind to chemokines, and seem to play a role in regulation of cytokine expression in human lymphocytes and monocytes. Aberrant metabolism of sulfatides, could cause several important human diseases. In this article, we describe the changes in sulfatide expression associated with such nervous disorders as metachromatic leukodystrophy (MLD), Parkinson's disease and Alzheimer's disease, and several types of cancer, e.g. colon cancer, kidney cancer, and ovarian cancer. We also discuss the involvement of sulfatides in cancer progression, diabetes and autoimmune and immune disorders such as multiple sclerosis. This acidic glycosphingolipids seem to play an important role in pathogenesis of infectious diseases, serving as receptors for binding various bacteria and viruses.

Targeting Selectins and Their Ligands in Cancer.

Aberrant glycosylation is a hallmark of cancer cells with increased evidence pointing to a role in tumor progression. In particular, aberrant sialylation of glycoproteins and glycolipids has been linked to increased immune cell evasion, drug evasion, drug resistance, tumor invasiveness, and vascular dissemination, leading to metastases. Hypersialylation of cancer cells is largely the result of overexpression of sialyltransferases (STs). Differentially, humans express twenty different STs in a tissue-specific manner, each of which catalyzes the attachment of sialic acids via different glycosidic linkages (α2-3, α2-6, or α2-8) to the underlying glycan chain. One important mechanism whereby overexpression of STs contributes to an enhanced metastatic phenotype is via the generation of selectin ligands. Selectin ligand function requires the expression of sialyl-Lewis X and its structural isomer sialyl-Lewis A, which are synthesized by the combined action of alpha α1-3-fucosyltransferases, α2-3-sialyltransferases, β1-4-galactosyltranferases, and N-acetyl-β-glucosaminyltransferases. The α2-3-sialyltransferases ST3Gal4 and ST3Gal6 are critical to the generation of functional E- and P-selectin ligands and overexpression of these STs have been linked to increased risk of metastatic disease in solid tumors and poor outcome in multiple myeloma. Thus, targeting selectins and their ligands as well as the enzymes involved in their generation, in particular STs, could be beneficial to many cancer patients. Potential strategies include ST inhibition and the use of selectin antagonists, such as glycomimetic drugs and antibodies. Here, we review ongoing efforts to optimize the potency and selectivity of ST inhibitors, including the potential for targeted delivery approaches, as well as evaluate the potential utility of selectin inhibitors, which are now in early clinical development.

Guided Homing of Cells in Multi-Photon Microfabricated Bioscaffolds.

Tissues contain exquisite vascular microstructures, and patterns of chemical cues for directing cell migration, homing, and differentiation for organ development and function. 3D microfabrication by multi-photon photolithography is a flexible, high-resolution tool for generating 3D bioscaffolds. However, the combined fabrication of scaffold microstructure simultaneously with patterning of cues to create both geometrically and chemically defined microenvironments remains to be demonstrated. This study presents a high-speed method for micron-resolution fabrication of scaffold microstructure and patterning of protein cues simultaneously using native scaffold materials. By the simultaneous microfabrication of arbitrary microvasculature geometries, and patterning selected regions of the microvasculature with the homing ligand P-selectin, this study demonstrates adhesion, rolling, and selective homing of cells in defined 3D regions. This novel ability to generate high-resolution geometries replete with patterned cues at high speed enables the construction of biomimetic microenvironments for complex 3D assays of cellular behavior.

The Suramin Derivative NF449 Interacts with the 5-fold Vertex of the Enterovirus A71 Capsid to Prevent Virus Attachment to PSGL-1 and Heparan Sulfate.

NF449, a sulfated compound derived from the antiparasitic drug suramin, was previously reported to inhibit infection by enterovirus A71 (EV-A71). In the current work, we found that NF449 inhibits virus attachment to target cells, and specifically blocks virus interaction with two identified receptors—the P-selectin ligand, PSGL-1, and heparan sulfate glycosaminoglycan—with no effect on virus binding to a third receptor, the scavenger receptor SCARB2. We also examined a number of commercially available suramin analogues, and newly synthesized derivatives of NF449; among these, NF110 and NM16, like NF449, inhibited virus attachment at submicromolar concentrations. PSGL-1 and heparan sulfate, but not SCARB2, are both sulfated molecules, and their interaction with EV-A71 is thought to involve positively charged capsid residues, including a conserved lysine at VP1-244, near the icosahedral 5-fold vertex. We found that mutation of VP1-244 resulted in resistance to NF449, suggesting that this residue is involved in NF449 interaction with the virus capsid. Consistent with this idea, NF449 and NF110 prevented virus interaction with monoclonal antibody MA28-7, which specifically recognizes an epitope overlapping VP1-244 at the 5-fold vertex. Based on these observations we propose that NF449 and related compounds compete with sulfated receptor molecules for a binding site at the 5-fold vertex of the EV-A71 capsid.

Abstract 335: Dynamic biochemical tissue analysis of novel P-selectin ligands expressed by colon cancer

Abstract Selectin/selectin ligand interactions have been implicated in mediating the adhesion of circulating tumor cells to distant sites during metastasis. Previous work from our lab has demonstrated the limitations of static biochemical tissue analysis (SBTA), using both selectins and antibodies against glycotopes, in detecting functional selectin ligands expressed on tissue. To address these shortcomings, we developed a novel tissue interrogation method, termed dynamic biochemical tissue analysis (DBTA), in which selectin-coated microspheres are perfused over tissues in a microfluidic device to detect functional selectin ligands in situ. In this work, DBTA using P-selectin microspheres was performed on cancer tissue sections in conjunction with SBTA using antibodies against P-selectin ligands CD24, CD44, and PSGL-1. Using DBTA, calcium-dependent selectin/selectin ligand adhesive interactions in the form of P-selectin microsphere rolling was observed on four distinct cases of colorectal cancer tissue. Examination of serial sections with SBTA in the same regions of tissue displaying P-selectin microsphere rolling revealed no detectable levels of CD24, CD44, or PSGL-1 epitopes, while only one case displayed CD44 expression that was in agreement with DBTA. However, not all regions displaying specific reactivity with the P-selectin microspheres used in DBTA were recognized with the CD44 antibody in SBTA. These results imply the presence of novel P-selectin ligands in the tissue section. Follow-up CD45 SBTA ruled out the possibility of microsphere interaction with infiltrated leukocytes, in agreement with the lack of PSGL-1 detection. Consistent with our DBTA studies using microspheres coated with E- and L-selectin on other types of cancer tissue, DBTA reveals unequivocal detection of functional P-selectin ligands by generating results distinct from SBTA. To further characterize the adhesion of P-selectin-coated microspheres to functional selectin ligands that were not identified with SBTA, a more refined method of interaction analysis that discretizes rolling into a sequence of succinct pauses (i.e., brief stationary adhesion) was used. In this method, microsphere rolling recorded at 300 frames per second was assessed using a cross-correlation tracking algorithm to obtain a robust point estimate that represents the off-rate bond breakage of the ensemble of receptor-ligand complexes that mediate microsphere adhesion to the tissue surface. This adhesion parameter lays the framework for potentially correlating the density and type of functional ligand(s) expressed with tumor stage and/or aggressiveness, furthering our understanding of aberrantly expressed functional selectin ligands as potential cancer biomarkers. Citation Format: Eric Martin, Ramiro Malgor, Douglas Goetz, Monica Burdick. Dynamic biochemical tissue analysis of novel P-selectin ligands expressed by colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 335. doi:10.1158/1538-7445.AM2015-335

Platelet activation markers overexpressed specifically in patients with aspirin-exacerbated respiratory disease

Aspirin-exacerbated respiratory disease (AERD) is characterized by respiratory reactions on ingestion of COX-1 inhibitors and cysteinyl leukotriene overproduction. The hypersensitivity reaction is induced by low doses of aspirin that inhibit COX-1 in platelets. We sought to explore the role of platelets in the pathogenesis of AERD in patients under stable conditions and during an aspirin challenge test. Stable patients with AERD (n= 30), aspirin-tolerant asthma (ATA; n= 21), or idiopathic chronic eosinophilic pneumonia (n= 10) were enrolled. Platelet activation was estimated based on expression levels of P-selectin (CD62P), CD63, CD69, and GPIIb/IIIa (PAC-1) in peripheral platelets; percentages of circulating platelet-adherent leukocytes; and plasma levels of soluble P-selectin (sP-selectin) and soluble CD40 ligand (sCD40L). In the stable condition, expression of all surface markers on platelets, the percentage of platelet-adherent eosinophils, and the plasma levels of sP-selectin and sCD40L were significantly higher in patients with AERD compared with those in patients with ATA. P-selectin and CD63 expression on platelets and plasma sP-selectin and sCD40L levels were positively correlated with the percentage of platelet-adherent eosinophils. Among these markers, P-selectin expression and plasma sP-selectin levels positively correlated with urinary concentrations of leukotriene E4. Additionally, plasma sP-selectin and sCD40L levels were negatively correlated with lung function. In contrast, platelet activation markers in patients with AERD did not change during the aspirin challenge test. Peripheral platelets were activated more in patients with stable AERD compared with those in patients with stable ATA, patients with idiopathic chronic eosinophilic pneumonia, and control subjects. Platelet activation was involved in cysteinyl leukotriene overproduction and persistent airflow limitations in patients with AERD.

The Role of Endometrial Selectins and Their Ligands on Bovine Conceptus Attachment to the Uterine Epithelium During Peri-Implantation Period.

A successful pregnancy depends on the blastocyst's implantation to the maternal endometrium; however, the initial interaction between blastocyst and uterine epithelium has not been well characterized. The objectives of this study were to determine if selectins and their ligands were expressed in the bovine conceptus and/or uterus during the periattachment period and to study whether selectins were associated with conceptus attachment to the uterine epithelium. Through the RNA-sequence analysis of bovine conceptuses on Days 17, 20, and 22 (Day 0 = day of estrus), only the SELL ligand, podocalyxin (PODXL), and P-selectin (SELP) ligand, SELPLG, were found. Quantitative PCR analysis confirmed the presence of PODXL and SELPLG in these conceptuses and revealed that SELL, mRNA and protein, detected in the uterine epithelium but not in conceptuses increased during the periattachment period. In the cultured endometrial epithelial cells (EECs), SELL transcript was up-regulated when uterine flushings from Day 20 pregnant animals were placed onto these cells. SELL was also up-regulated when cultured EECs were treated with progesterone, EGF, or bFGF, but not with IFNT. In the coculture system with EECs and bovine trophoblast CT-1 cells, SELL expression in EECs was effectively reduced by its small interfering RNA; however, IFNT, a marker for CT-1 cell attachment to EECs, was not reduced, nor was a transcription factor of IFNT, CDX2. These observations suggest that the conceptus could attach to the uterine epithelium through the use of endometrial SELL and embryonic selectin ligands, possibly initiating the conceptus attachment process in the bovine species.

Increased platelet activation in subjects chronically exposed to cadmium: A pilot study

Cadmium exposure has been reported to be associated with the risk of vascular disorders. Here, we investigated platelet activity in subjects with chronic cadmium exposure. Eighteen and 15 women participated in this study as chronically cadmium-exposed and control non-exposed subjects, respectively. Plasma P-selectin and CD40 ligand (CD40L), soluble markers of platelet activation, were measured. Platelet aggregation in whole blood, P-selectin and activated glycoprotein (aGP) IIb/IIIa expression on platelets and platelet–leukocyte aggregates were determined. The levels of plasma P-selectin and CD40L increased in subjects with chronic cadmium exposure compared with control subjects. Platelet aggregation induced by adenosine diphosphate (ADP) was higher in cadmium-exposed subjects than control subjects. Cadmium-exposed subjects had higher baseline and ADP-induced aGPIIb/IIIa expression on platelets than control subjects. Platelet–neutrophil aggregates also increased in cadmium-exposed subjects. Blood cadmium correlated with ADP-induced aggregation, aGPIIb/IIIa expression and platelet–neutrophil aggregates, while urinary cadmium correlated with soluble P-selectin. However, cadmium only at high concentration (15 µM) could potentiate ADP-induced platelet activation in vitro. In conclusion, our pilot data show that cadmium-exposed subjects have increased baseline platelet activation and reactivity.

Reproducibility over time and effect of low-dose aspirin on soluble P-selectin and soluble CD40 ligand.

Platelet markers [soluble CD40 ligand (sCD40L) and soluble p selectin (sPselectin)] are associated with platelet activation and cardiovascular events. We sought to investigate the reproducibility of these markers over time and the effect of low-dose aspirin on sCD40L and sPselectin in plasma and serum. Following an overnight fast, 40 healthy volunteers had weekly phlebotomy and were administered aspirin 81 mg/day between weeks 3 and 4. Reproducibility over time was assessed by coefficient of variation (CV) and inter-class correlation coefficient. Correlation between markers was assessed using Pearson r statistic. Difference between levels pre- and post-aspirin was measured with Wilcoxon signed-rank test. Data are presented as median (interquartile range). sCD40L and sPselectin measurements were reproducible over time in plasma and serum (CV < 10 %). Measurement of sCD40L and sPselectin in plasma correlated with levels in serum before aspirin and after aspirin. There was no significant correlation between sCD40L and sPselectin. After 1-week of aspirin 81 mg/day, there was a reduction in sCD40L and sPselectin in serum and plasma, respectively. Soluble CD40L and sPselectin are independent markers that are reproducible over time in both plasma and sera and are reduced by 1-week of low-dose aspirin.

Potential Role of Soluble CD40 Ligand as Inflammatory Biomarker in Colorectal Cancer Patients

Platelet activation observed in cancer patients is associated with the release of various cytokines, including P-selectin and CD40 ligand (CD40L). We analyzed the plasma levels of sCD40L in association with adhesion molecules (sP-selectin and sVCAM-1) to check the hypothesis of a possible involvement in cancer progression.Blood samples were obtained from 59 patients with different stages of colorectal cancer (CRC) and 29 age and gender-matched control subjects. Plasma sCD40L, sP-selectin, and sVCAM-1 concentrations were measured with quantitative sandwich enzyme-linked immunoassay.All patients with CRC had significantly higher levels of sCD40L (p<0.001), sP-selectin (p<0.02), and sVCAM-1 (p<0.03), as compared to healthy subjects. The level of sCD40L significantly correlated with sP-selectin (p<0.05) in patients with distant metastases to the liver. We also observed a high negative correlation between sP-selectin and platelets count (p<0.02) in patients with lymph node metastasis. The receiver-operator curve for CRC patients indicated that the area under the curve for sCD40L was 0.915, which may indicate its high efficiency as an inflammatory marker.In our study, the sCD40L correlated with sP-selectin in patients with advanced stage of CRC, which might indicate its possible participation in metastasis formation.

Human Umbilical Cord Blood‐Derived Mesenchymal Stromal Cells Display a Novel Interaction between P‐Selectin and Galectin‐1

Human multipotent mesenchymal stromal/stem cells (MSCs) have been shown to exert immunomodulatory properties that have great potential in therapies for various inflammatory and autoimmune disorders. However, intravenous delivery of these cells is followed by massive cell entrapment in the lungs and insufficient homing to target tissues or organs. In targeting to tissues, MSCs and other therapeutic cells employ similar mechanisms as leucocytes, including a cascade of rolling and adhesion steps mediated by selectins, integrins and their ligands. However, the mechanisms of MSCs homing are not well understood. We discovered that P-selectin (CD62P) binds to umbilical cord blood (UCB)-derived MSCs independently of the previously known sialyl Lewis x (sLex)-containing ligands such as P-selectin glycoprotein ligand-1 (PSGL-1, CD162). By biochemical assays, we identified galectin-1 as a novel ligand for P-selectin. Galectin-1 has previously been shown to be a key mediator of the immunosuppressive effects of human MSCs. We conclude that this novel interaction is likely to play a major role in the immunomodulatory targeting of human UCB-derived MSCs.

TIM-1 Glycoprotein Binds the Adhesion Receptor P-Selectin and Mediates T Cell Trafficking during Inflammation and Autoimmunity

Selectins play a central role in leukocyte trafficking by mediating tethering and rolling on vascular surfaces. Here we have reported that T cell immunoglobulin and mucin domain 1 (TIM-1) is a P-selectin ligand. We have shown that human and murine TIM-1 binds to P-selectin, and that TIM-1 mediates tethering and rolling of T helper 1 (Th1) and Th17, but not Th2 and regulatory T cells on P-selectin. Th1 and Th17 cells lacking the TIM-1 mucin domain showed reduced rolling in thrombin-activated mesenteric venules and inflamed brain microcirculation. Inhibition of TIM-1 had no effect on naive T cell homing, but it reduced T cell recruitment in a skin hypersensitivity model and blocked experimental autoimmune encephalomyelitis. Uniquely, the TIM-1 immunoglobulin variable domain was also required for P-selectin binding. Our data demonstrate that TIM-1 is a major P-selectin ligand with a specialized role in T cell trafficking during inflammatory responses and the induction of autoimmune disease.

Autoimmune Vitiligo Does Not Require the Ongoing Priming of Naive CD8 T Cells for Disease Progression or Associated Protection against Melanoma

Vitiligo is a CD8 T cell-mediated autoimmune disease that has been shown to promote the longevity of memory T cell responses to melanoma. However, mechanisms whereby melanocyte/melanoma Ag-specific T cell responses are perpetuated in the context of vitiligo are not well understood. These studies investigate the possible phenomenon of naive T cell priming in hosts with melanoma-initiated, self-perpetuating, autoimmune vitiligo. Using naive pmel (gp10025-33-specific) transgenic CD8 T cells, we demonstrate that autoimmune melanocyte destruction induces naive T cell proliferation in skin-draining lymph nodes, in an Ag-dependent fashion. These pmel T cells upregulate expression of CD44, P-selectin ligand, and granzyme B. However, they do not downregulate CD62L, nor do they acquire the ability to produce IFN-γ, indicating a lack of functional priming. Accordingly, adult thymectomized mice exhibit no reduction in the severity or kinetics of depigmentation or long-lived protection against melanoma, indicating that the continual priming of naive T cells is not required for vitiligo or its associated antitumor immunity. Despite this, depletion of CD4 T cells during the course of vitiligo rescues the priming of naive pmel T cells that are capable of producing IFN-γ and persisting as memory, suggesting an ongoing and dominant mechanism of suppression by regulatory T cells. This work reveals the complex regulation of self-reactive CD8 T cells in vitiligo and demonstrates the overall poorly immunogenic nature of this autoimmune disease setting.

Increased Platelet Binding to Circulating Monocytes in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is the most common idiopathic interstitial pneumonia and its prognosis is poor. Epidemiological evidence suggests an association of IPF with vascular disease and thrombotic tendency, which may be related to platelet activation. Platelet-monocyte adhesion in peripheral blood was examined by flow cytometry in patients with IPF (n=19), interstitial lung disease (ILD) other than IPF (n=9), and control subjects without pulmonary fibrosis (n=14). Expression of platelet activation markers P-selectin (CD62P), PSGL-1 (CD162), and CD40 ligand (CD40L) on leukocytes and platelets were studied. Plasma concentrations of soluble P-selectin and CD40L were measured by ELISA. Significantly elevated levels of platelet-monocyte binding were found in patients with IPF (35.6±4.34% [mean±SEM]) compared with patients with non-IPF ILD (23.5±3.68%) and non-ILD control subjects (16.5±2.26%; P<0.01). There was a trend towards increased divalent cation-independent platelet-monocyte binding in IPF (6.0±0.77% [mean±SEM]) compared with non-IPF ILD (4.3±1.38%) and control subjects without ILD (3.1±1.75%; P=0.058). There was no differential surface expression of platelet activation markers on subsets of leukocytes or platelets. Plasma concentrations of CD40L and soluble P-selectin did not differ between IPF and control subjects. Platelet-monocyte binding had no significant correlation with percent predicted TLco or FVC. Platelet-monocyte binding is increased in IPF, suggesting increased platelet activation. This conjugation is predominantly calcium-dependent, but there may be more calcium-independent adhesion in IPF. These findings support further research into the role of platelet activation in IPF.

Regulation of platelet responses triggered by Toll-like receptor 2 and 4 ligands is another non-genomic role of nuclear factor-kappaB

Platelets express Toll-like receptors (TLRs) that recognise molecular components of pathogens and, in nucleated cells, elicit immune responses through nuclear factor-kappaB (NF-κB) activation. We have shown that NF-κB mediates platelet activation in response to classical agonists, suggesting that this transcription factor exerts non-genomic functions in platelets. The aim of this study was to determine whether NF-κB activation is a downstream signal involved in TLR2 and 4-mediated platelet responses. Aggregation and ATP release were measured with a Lumi-aggregometer. Fibrinogen binding, P-selectin and CD40 ligand (CD40L) levels and platelet-neutrophil aggregates were measured by cytometry. I kappa B alpha (IκBα) degradation and p65 phosphorylation were determined by Western blot and von Willebrand factor (vWF) by ELISA. Platelet stimulation with Pam3CSK4 or LPS resulted in IκBα degradation and p65 phosphorylation. These responses were suppressed by TLR2 and 4 blocking and synergised by thrombin. Aggregation, fibrinogen binding and ATP and vWF release were triggered by Pam3CSK4. LPS did not induce platelet responses per se, except for vWF release, but it did potentiate thrombin-induced aggregation, fibrinogen binding and ATP secretion. Pam3CSK4, but not LPS, induced P-selectin and CD40L expression and mixed aggregate formation. All of these responses, except for CD40L expression, were inhibited in platelets treated with the NF-κB inhibitors BAY 11-7082 or Ro 106-9920. TLR2 and 4 agonists trigger platelet activation responses through NF-κB. These data show another non-genomic function of NF-κB in platelets and highlight this molecule as a potential target to prevent platelet activation in inflammatory or infectious diseases.

Strong and specific interaction of ultra small superparamagnetic iron oxide nanoparticles and human activated platelets mediated by fucoidan coating

Activated platelets play a pivotal role in cardiovascular diseases such as atherothrombosis. Therefore, strategies enabling activated platelet molecular imaging are of great interest. Herein, a chemical protocol was investigated for coating superparamagnetic iron oxide nanoparticles with low molecular weight fucoidan, a ligand of P-selectin expressed on the surface of activated platelets. The physico–chemical characterization of the obtained product demonstrated successful fucoidan coating and its potential as a T2 MRI contrast agent. The specificity and the strength of the interaction between fucoidan-coated iron oxide nanoparticles and human activated platelets was demonstrated by flow cytometry. Micromagnetophoresis experiments revealed that platelets experience magnetically-induced motion in the presence of a magnetic field gradient created by a micromagnet. Altogether, these results indicate that superparamagnetic iron oxide nanoparticles coated with low molecular weight fucoidan may represent a promising molecular imaging tool for activated platelets in human diseases.