Biomedical subjects

Paul S Frenette

Publications and source records attributed to Paul S Frenette.

18 recordsLinked to original sources

PubMed2006

Identification of a radio-resistant and cycling dermal dendritic cell population in mice and men.

In this study, we explored dermal dendritic cell (DC) homeostasis in mice and humans both in the steady state and after hematopoietic cell transplantation. We discovered that dermal DCs proliferate in situ in mice and human quiescent dermis. In parabiotic mice with separate organs but shared blood circulation, the majority of dermal DCs failed to be replaced by circulating precursors for >6 mo. In lethally irradiated mice injected with donor congenic bone marrow (BM) cells, a subset of recipient DCs remained in the dermis and proliferated locally throughout life. Consistent with these findings, a large proportion of recipient dermal DCs remained in patients' skin after allogeneic hematopoietic cell transplantation, despite complete donor BM chimerism. Collectively, our results oppose the traditional view that DCs are nondividing terminally differentiated cells maintained by circulating precursors and support the new paradigm that tissue DCs have local proliferative properties that control their homeostasis in the steady state. Given the role of residual host tissue DCs in transplant immune reactions, these results suggest that dermal DC homeostasis may contribute to the development of cutaneous graft-versus-host disease in clinical transplantation.

Acute Disease↗

PubMed2006

Signals from the sympathetic nervous system regulate hematopoietic stem cell egress from bone marrow.

Hematopoietic stem and progenitor cells (HSPC), attracted by the chemokine CXCL12, reside in specific niches in the bone marrow (BM). HSPC migration out of the BM is a critical process that underlies modern clinical stem cell transplantation. Here we demonstrate that enforced HSPC egress from BM niches depends critically on the nervous system. UDP-galactose ceramide galactosyltransferase-deficient (Cgt(-/-)) mice exhibit aberrant nerve conduction and display virtually no HSPC egress from BM following granulocyte colony-stimulating factor (G-CSF) or fucoidan administration. Adrenergic tone, osteoblast function, and bone CXCL12 are dysregulated in Cgt(-/-) mice. Pharmacological or genetic ablation of adrenergic neurotransmission indicates that norepinephrine (NE) signaling controls G-CSF-induced osteoblast suppression, bone CXCL12 downregulation, and HSPC mobilization. Further, administration of a beta(2) adrenergic agonist enhances mobilization in both control and NE-deficient mice. Thus, these results indicate that the sympathetic nervous system regulates the attraction of stem cells to their niche.

Animals↗

PubMed2006

B cell-driven lymphangiogenesis in inflamed lymph nodes enhances dendritic cell mobilization.

Dendritic cell (DC) migration from the periphery to lymph nodes is regulated by the pattern of genes expressed by DCs themselves and by signals within the surrounding peripheral environment. Here, we report that DC mobilization can also be regulated by signals initiated within the downstream lymph nodes, particularly when lymph nodes enlarge as a consequence of immunization. Lymph node B lymphocytes orchestrate expansion of the lymphatic network within the immunized lymph node. This expanded network in turn supports increased DC migration from the periphery. These results reveal unique relationships between B cells, lymphatic vessels, and migratory DCs. Knowledge that DC migration from the periphery is augmented by B cell-dependent signals reveals new potential strategies to increase DC migration during vaccination.

Animals↗

PubMed2005

CD44 is a physiological E-selectin ligand on neutrophils.

The selectin family of adhesion molecules and their glycoconjugated ligands are essential for blood polymorphonuclear neutrophil (PMN) extravasation into inflammatory and infectious sites. However, E-selectin ligands on PMNs are not well characterized. We show here that CD44 immunopurified from G-CSF-differentiated 32D cells or from peripheral blood PMNs binds specifically to E-selectin. In contrast, CD44 extracted from bone marrow stromal or brain endothelial cell lines does not interact with E-selectin, suggesting cell-specific posttranslational modifications of CD44. PMN-derived CD44 binding activity is mediated by sialylated, alpha(1,3) fucosylated, N-linked glycans. CD44 enables slow leukocyte rolling on E-selectin expressed on inflamed endothelium in vivo and cooperates with P-selectin glycoprotein ligand-1 to recruit neutrophils into thioglycollate-induced peritonitis and staphylococcal enterotoxin A-injected skin pouch. CD44 extracted from human PMNs also binds to E-selectin. Moreover, we demonstrate that CD44 is hypofucosylated in PMNs from a patient with leukocyte adhesion deficiency type II, suggesting that it contributes to the syndrome. These findings thus suggest broader roles for CD44 in the innate immune response and uncover a potential new target for diseases in which selectins play a prominent role.

Animals↗

PubMed2005

Sickle cell vaso-occlusion.

The vaso-occlusion model has evolved impressively over the past several decades from polymerization-based concepts to a complex, wide-ranging schema that involves multistep, heterogeneous, and interdependent interactions among sickle erythrocytes (SSRBCs), adherent leukocytes, endothelial cells, plasma proteins, and other factors. Endothelial activation, induced directly or indirectly by the proinflammatory behavior of SSRBCs, is the most likely initiating step toward vaso-occlusion. Given the complexity and dynamic relationships of the potential mechanisms leading to vaso-occlusion, further in vivo studies in relevant sickle cell animal models will most likely yield the greatest advances and promote the development of novel, more effective therapeutic strategies.

Anemia, Sickle Cell↗

PubMed2004

A novel model for lymphocytic infiltration of the thyroid gland generated by transgenic expression of the CC chemokine CCL21.

Lymphocytic infiltrates and lymphoid follicles with germinal centers are often detected in autoimmune thyroid disease (AITD), but the mechanisms underlying lymphocyte entry and organization in the thyroid remain unknown. We tested the hypothesis that CCL21, a chemokine that regulates homeostatic lymphocyte trafficking, and whose expression has been detected in AITD, is involved in the migration of lymphocytes to the thyroid. We show that transgenic mice expressing CCL21 from the thyroglobulin promoter (TGCCL21 mice) have significant lymphocytic infiltrates, which are topologically segregated into B and T cell areas. Although high endothelial venules expressing peripheral lymph node addressin were frequently observed in the thyroid tissue, lymphocyte recruitment was independent of L-selectin or lymphotoxin-alpha but required CCR7 expression. Taken together, these results indicate that CCL21 is sufficient to drive lymphocyte recruitment to the thyroid, suggest that CCL21 is involved in AITD pathogenesis, and establish TGCCL21 transgenic mice as a novel model to study the formation and function of lymphoid follicles in the thyroid.

Adoptive Transfer↗

PubMed2004

Enforced fucosylation of neonatal CD34+ cells generates selectin ligands that enhance the initial interactions with microvessels but not homing to bone marrow.

Hematopoietic progenitor/stem cell homing to the bone marrow requires the concerted action of several adhesion molecules. Endothelial P- and E-selectins play an important role in this process, but their ligands on a large subset of neonate-derived human CD34+ cells are absent, leading to a reduced ability to interact with the bone marrow (BM) microvasculature. We report here that this deficiency results from reduced alpha1,3-fucosyltransferase (FucT) expression and activity in these CD34+ cells. Incubation of CD34+ cells with recombinant human FucTVI rapidly corrected the deficiency in nonbinding CD34+ cells and further increased the density of ligands for both P- and E-selectins on all cord blood-derived CD34+ cells. Intravital microscopy studies revealed that these FucTVI-treated CD34+ cells displayed a marked enhancement in their initial interactions with the BM microvasculature, but unexpectedly, homing into the BM was not improved by FucTVI treatment. These data indicate that, although exogenous FucT enzyme activity can rapidly modulate selectin binding avidity of cord blood CD34+ cells, further studies are needed to understand how to translate a positive effect on progenitor cell adhesion in bone marrow microvessels into one that significantly influences migration and lodgement into the parenchyma.

Animals↗

PubMed2004

Integrin alpha4beta7 and its counterreceptor MAdCAM-1 contribute to hematopoietic progenitor recruitment into bone marrow following transplantation.

Previous studies have shown that alpha4beta1 (very late activation antigen-4 [VLA-4]) and vascular cell adhesion molecule-1 (VCAM-1) play a major role in hematopoietic progenitor cell (HPC) homing to bone marrow (BM). However, the antibody used to block VLA-4 function in the mouse (hybridoma clone PS/2) is not specific to VLA-4 but inhibits both alpha4beta1 and alpha4beta7 integrins. Here we have evaluated the contribution of alpha4beta7 in HPC homing to BM. Lineage(neg)Sca-1(pos)c-kit(pos) cells from adult mouse BM and the factor-dependent cell progenitor (FDCP)-mix progenitor cell line express similar levels of alpha4beta7 by flow cytometry. The alpha4beta7 complex was functional since the chemokine CXCL12 enhanced the adhesion of FDCP-mix to immobilized mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and this was completely abrogated by anti-alpha4beta7 (hybridoma clone DATK32) or anti-alpha4 integrins (PS/2). BM intravital microscopy revealed that alpha4beta7 plays a predominant role in initial tethering and rolling but not in firm adhesion of FDCP-mix cells. Using homing assays, we demonstrate that alpha4beta7 on HPCs contributes to about half of all alpha4 integrin-mediated homing activity following BM transplantation. MAdCAM-1 is likely expressed since its inhibition significantly reduced HPC homing. Although there may be other alpha4beta7 integrin ligands involved (eg, fibronectin and VCAM-1), these data thus suggest that alpha4beta7 and its counterreceptor MAdCAM-1 represent a novel adhesion pathway mediating HPC homing to BM.

Animals↗

PubMed2004

The integrin alphaMbeta2 anchors hematopoietic progenitors in the bone marrow during enforced mobilization.

The sulfated polysaccharide fucoidan can rapidly mobilize hematopoietic progenitor cells (HPCs) and long-term repopulating stem cells from the bone marrow (BM) to the circulation. While searching for mechanisms involved in this phenomenon we found that BM myeloid cells bound to fucoidan through the integrin alphaMbeta2 (macrophage antigen-1 [Mac-1]) and L-selectin resulting in alphaMbeta2-independent release of neutrophil elastase, but inhibition of elastase activity did not impair fucoidan-induced mobilization. Mobilization of HPCs by fucoidan was enhanced in animals deficient in alphaM (alphaM-/-) compared with wild-type (alphaM+/+) animals and higher plasma levels of the chemokine CXCL12/stromal cell-derived factor-1 (SDF-1) were achieved in alphaM-/- mice by fucoidan treatment. However, in chimeric animals harboring alphaM+/+ and alphaM-/- HPCs in the BM, alphaM-/- HPCs were preferentially mobilized by fucoidan, suggesting that the enhanced mobilization is cell intrinsic and does not result from altered microenvironment. Suboptimal doses of granulocyte colony-stimulating factor (G-CSF) or cyclophosphamide (CY) also resulted in enhanced HPC mobilization in alphaM-/- mice compared with alphaM+/+ controls, but this difference was overcome when standard doses of G-CSF or CY were administered. Taken together, these data suggest that the integrin alphaMbeta2 participates in the retention of HPCs in the BM.

Animals↗

PubMed2004

Sickle cell vasoocclusion: heterotypic, multicellular aggregations driven by leukocyte adhesion.

Homozygous expression of sickle beta-globin alters the function of blood cells and the endothelium, producing a wide spectrum of clinical manifestations. Intravital microscopy studies in sickle cell mice suggest that vasoocclusion is a complex, sequential, multistep phenomenon involving (1) endothelial activation by sickle erythrocyte (SSRBC), (2) leukocyte (WBC) adhesion to the endothelium, and (3) the direct interaction between SSRBCs and adherent WBCs, which leads to reduced blood flow and tissue ischemia. Each of these steps represents a potentially useful therapeutic target. The identification of molecular determinants mediating vasoocclusion will provide new strategies for the prevention and treatment of this debilitating illness.

Anemia, Sickle Cell↗

PubMed2003

Intravenous immune globulin prevents venular vaso-occlusion in sickle cell mice by inhibiting leukocyte adhesion and the interactions between sickle erythrocytes and adherent leukocytes.

Sickle cell vaso-occlusion is a complex multistep process likely involving heterotypic interactions among sickle erythrocytes (red blood cells [RBCs]), leukocytes (white blood cells [WBCs]), and endothelial cells. Recent data using intravital microscopy in a sickle cell mouse model suggest that adherent leukocytes in postcapillary venules play a critical role in vaso-occlusion by capturing circulating sickle RBCs. In the course of studies to investigate the adhesion receptors mediating sickle RBC-WBC interactions, we found that control nonspecific immunoglobulin G (IgG) preparations displayed significant inhibitory activity. As a result, we studied the effects of commercial intravenous human immune globulin (i.v.IG) preparations and found that i.v.IG inhibits RBC-WBC interactions in cremasteric venules in a dose-dependent manner. i.v.IG of at least 200 mg/kg dramatically reduced these interactions, even after tumor necrosis factor-alpha (TNF-alpha) stimulation, and not only increased microcirculatory blood flow but also improved survival of sickle cell mice. These data raise the possibility that i.v.IG may have a beneficial effect on sickle cell-associated vaso-occlusion.

Anemia, Sickle Cell↗

PubMed2003

Macrophages, not neutrophils, infiltrate skeletal muscle in mice deficient in P/E selectins after mechanical reloading.

Our objective was to test the hypothesis that endothelial selectins, P and E selectins, are necessary for leukocyte migration after muscle injury from unloading/reloading. Mice hindlimbs were suspended for 10 days followed by reloading periods of 6 or 24 h after which the soleus muscle was dissected. Light microscopic observations showed that macrophages, but not neutrophils, were able to invade soleus muscles in mice deficient in P/E selectins (P/E-/-) during reloading periods. The recruitment efficiency of neutrophils after 6 and 24 h of reloading was minimal in P/E-/- mice relative to unloaded animals. The recruitment of macrophages in the soleus muscle was preserved in P/E-/- mice. The concentration of macrophages increased by 8.1-fold compared with unloaded muscles in double-mutant mice after 24 h of reloading. The accumulation of macrophages in reloaded muscles did not lead to fiber necrosis. Together, these findings indicate that macrophages can invade skeletal muscle through cellular mechanisms that do not involve P/E selectins during skeletal muscle reloading.

Animals↗

PubMed2003

PSGL-1 participates in E-selectin-mediated progenitor homing to bone marrow: evidence for cooperation between E-selectin ligands and alpha4 integrin.

The nature and exact function of selectin ligands involved in hematopoietic progenitor cell (HPC) homing to the bone marrow (BM) are unclear. Using murine progenitor homing assays in lethally irradiated recipients, we found that the P-selectin glycoprotein ligand-1 (PSGL-1) plays a partial role in HPC homing to the BM (a reduction of about 35% when the P-selectin binding region is blocked). Blockade of both PSGL-1 and alpha4 integrin did not further enhance the effect of anti-alpha4 integrin (a reduction of about 55%). We suspected that E-selectin ligands might contribute to the remaining homing activity. To test this hypothesis, HPC homing assays were carried out in E-selectin-deficient recipients and revealed a profound alteration in HPC homing when E-selectin and alpha4 integrin were inactivated (> 90% reduction). Competitive assays to test homing of long-term repopulating stem cells revealed a drastic reduction (> 99%) of the homed stem cell activity when both alpha4 integrin and E-selectin functions were absent. Further homing studies with PSGL-1-deficient HPCs pretreated with anti-alpha4 integrin antibody revealed that PSGL-1 contributes to approximately 60% of E-selectin ligand-mediated homing activity. Our results thus underscore a major difference between mature myeloid cells and immature stem/progenitor cells in that E-selectin ligands cooperate with alpha4 integrin rather than P-selectin ligands.

Animals↗

PubMed2003

Galactocerebrosides are required postnatally for stromal-dependent bone marrow lymphopoiesis.

Galactocerebrosides (GCs) represent a major class of glycolipids in the nervous system. Here, we show that mice lacking the key enzyme to generate GCs, UDP-galactose:ceramide galactosyltransferase (CGT(-/-)), exhibit severe postnatal atrophy of all lymphoid organs, owing to a maturational arrest before the pro-B/T cell stage. This lineage-specific defect originates from the bone marrow (BM) stroma since it is not transplantable to irradiated wild-type recipients. Remarkably, CGT(-/-) long-term B lymphoid BM cultures displayed severe deficits in the number of CD45(neg)VCAM-1(pos) stromal cells and fibronectin matrix assembly, and produced floating macrophages rather than B lymphocytes. The fibronectin network was also altered in the CGT-deficient BM parenchyma. These results point to an essential role for galactolipids in the formation of fibronectin-enriched lymphoid-specific stromal niches in the BM.

Animals↗

PubMed2002

Insights into leukocyte adhesion deficiency type 2 from a novel mutation in the GDP-fucose transporter gene.

Leukocyte adhesion deficiency type 2 (LADII) is characterized by defective selectin ligand formation, recurrent infection, and mental retardation. This rare syndrome has only been described in 2 kindreds of Middle Eastern descent who have differentially responded to exogenous fucose treatment. The molecular defect was recently ascribed to single and distinct missense mutations in a putative Golgi guanosine diphosphate (GDP)-fucose transporter. Here, we describe a patient of Brazilian origin with features of LADII. Sequencing of the GDP-fucose transporter revealed a novel single nucleotide deletion producing a shift in the open-reading frame and severe truncation of the polypeptide. Overexpression of the mutant protein in the patient's fibroblasts did not rescue fucosylation, suggesting that the deletion ablated the activity of the transporter. Administration of oral L-fucose to the patient produced molecular and clinical responses, as measured by the appearance of selectin ligands, normalization of neutrophil counts, and prevention of infectious recurrence. The lower neutrophil counts paralleled improved neutrophil interactions with activated endothelium in cremasteric venules of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. However, fucose supplementation induced autoimmune neutropenia and the appearance of H antigen on erythrocytes, albeit without evidence of intravascular hemolysis. The robust response to fucose despite a severely truncated transporter suggests alternative means to transport GDP-fucose into the Golgi complex.

Abnormalities, Multiple↗

PubMed2002

Primary role for adherent leukocytes in sickle cell vascular occlusion: a new paradigm.

Vascular occlusion is the major cause of morbidity and mortality in sickle cell disease but its mechanisms are poorly understood. We demonstrate by using intravital microscopy in mice expressing human sickle hemoglobin (SS) that SS red blood cells (RBCs) bind to adherent leukocytes in inflamed venules, producing vasoocclusion of cremasteric venules. SS mice deficient in P- and E-selectins, which display defective leukocyte recruitment to the vessel wall, are protected from vasoocclusion. These data uncover a previously unsuspected paradigm for the pathogenesis of sickle cell vasoocclusion in which adherent leukocytes play a direct role and suggest that drugs targeting SS RBC-leukocyte or leukocyte-endothelial interactions may prevent or treat the vascular complications of this debilitating disease.

Anemia, Sickle Cell↗

PubMed2002

Sickle cell vaso-occlusion: multistep and multicellular paradigm.

Sickle cell disease is characterized by recurrent, painful episodes and organ damage resulting from microvascular occlusion. Seminal studies performed 20 years ago revealed increased adherence of sickle erythrocytes to vascular endothelial cells. Subsequent work showed that these interactions were mediated by multiple adhesion pathways, but the relevance of these interactions has not been evaluated in vivo. Clinical data suggest that leukocytes may play a role, because leukocytosis correlates with clinical severity and early death, and administration of myeloid growth factors to patients can precipitate sickle cell crises. In addition, recent experimental data using intravital microscopy indicate that sickle erythrocytes can interact with adherent leukocytes in inflamed postcapillary and collecting venules. A novel multistep model for sickle cell vaso-occlusion is proposed in which endothelial activation is induced by sickle cells or secondary inflammatory stimuli and leads to the recruitment of adherent leukocytes. The resulting adherent leukocytes interact with circulating sickle erythrocytes, and this interaction impedes microvascular blood flow. Finally, irregularly shaped sickle cells become nonspecifically trapped, resulting in vaso-occlusion. The molecular mechanisms and requirements for the heterotypic interactions between erythrocytes and leukocytes are currently unknown and may involve further activation of adherent leukocytes or circulating erythrocytes. This model offers exciting new opportunities for therapeutic intervention and suggests a critical participation of adherent leukocytes in sickle cell vaso-occlusion.

Anemia, Sickle Cell↗

PubMed2002

Functional selectin ligands mediating human CD34(+) cell interactions with bone marrow endothelium are enhanced postnatally.

Hematopoietic progenitor cells (HPCs) can home to the bone marrow (BM) after a simple intravenous injection, but the adhesive mechanisms mediating the initial interactions of human HPCs with the BM endothelium have not been evaluated in vivo. Using fluorescence intravital microscopy and homing assays in NOD/SCID mice, we show that endothelial selectins are necessary for human adult CD34(+) cell homing, since rolling on BM endothelium and retention in the BM compartment are drastically reduced (>90%) in endothelial selectin-deficient NOD/SCID mice. Comparative analyses of CD34(+) cells collected from adults and from cord blood (CB) reveal that neonatal cells display reduced rolling fractions compared with adult CD34(+) cells obtained from peripheral blood or BM, suggesting abnormal selectin ligand function on neonatal progenitors. Flow cytometric and intravital microscopy studies suggest that this defect results from nonfunctional P-selectin ligand on a subset ( approximately 30%) of neonatal CD34(+) cells. Further analyses indicate that P-selectin glycoprotein ligand-1 (PSGL-1) is expressed in a nonfunctional form among neonatal CD34(+) cells that do not bind P-selectin and that this subset is enriched in primitive CD34(+)CD38(lo/-) progenitors. These results underscore the potential to improve homing of CB CD34(+) cells to the BM by manipulation of selectins and their ligands.

ADP-ribosyl Cyclase↗