Andrés Hidalgo

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.

Alloantigen-presenting plasmacytoid dendritic cells mediate tolerance to vascularized grafts.

The induction of alloantigen-specific unresponsiveness remains an elusive goal in organ transplantation. Here we identify plasmacytoid dendritic cells (pDCs) as phagocytic antigen-presenting cells essential for tolerance to vascularized cardiac allografts. Tolerizing pDCs acquired alloantigen in the allograft and then moved through the blood to home to peripheral lymph nodes. In the lymph node, alloantigen-presenting pDCs induced the generation of CCR4+CD4+CD25+Foxp3+ regulatory T cells (Treg cells). Depletion of pDCs or prevention of pDC lymph node homing inhibited peripheral Treg cell development and tolerance induction, whereas adoptive transfer of tolerized pDCs induced Treg cell development and prolonged graft survival. Thus, alloantigen-presenting pDCs home to the lymph nodes in tolerogenic conditions, where they mediate alloantigen-specific Treg cell development and allograft tolerance.

Bone marrow CD169 + macrophages promote the retention of hematopoietic stem and progenitor cells in the mesenchymal stem cell niche

Hematopoietic stem cells (HSCs) reside in specialized bone marrow (BM) niches regulated by the sympathetic nervous system (SNS). Here, we have examined whether mononuclear phagocytes modulate the HSC niche. We defined three populations of BM mononuclear phagocytes that include Gr-1hi monocytes (MOs), Gr-1lo MOs, and macrophages (MΦ) based on differential expression of Gr-1, CD115, F4/80, and CD169. Using MO and MΦ conditional depletion models, we found that reductions in BM mononuclear phagocytes led to reduced BM CXCL12 levels, the selective down-regulation of HSC retention genes in Nestin+ niche cells, and egress of HSCs/progenitors to the bloodstream. Furthermore, specific depletion of CD169+ MΦ, which spares BM MOs, was sufficient to induce HSC/progenitor egress. MΦ depletion also enhanced mobilization induced by a CXCR4 antagonist or granulocyte colony-stimulating factor. These results highlight two antagonistic, tightly balanced pathways that regulate maintenance of HSCs/progenitors in the niche during homeostasis, in which MΦ cross talk with the Nestin+ niche cell promotes retention, and in contrast, SNS signals enhance egress. Thus, strategies that target BM MΦ hold the potential to augment stem cell yields in patients that mobilize HSCs/progenitors poorly.

Leukocyte ligands for endothelial selectins: specialized glycoconjugates that mediate rolling and signaling under flow

Reversible interactions of glycoconjugates on leukocytes with P- and E-selectin on endothelial cells mediate tethering and rolling of leukocytes in inflamed vascular beds, the first step in their recruitment to sites of injury. Although selectin ligands on hematopoietic precursors have been identified, here we review evidence that PSGL-1, CD44, and ESL-1 on mature leukocytes are physiologic glycoprotein ligands for endothelial selectins. Each ligand has specialized adhesive functions during tethering and rolling. Furthermore, PSGL-1 and CD44 induce signals that activate the β2 integrin LFA-1 and promote slow rolling, whereas ESL-1 induces signals that activate the β2 integrin Mac-1 in adherent neutrophils. We also review evidence for glycolipids, CD43, L-selectin, and other glycoconjugates as potential physiologic ligands for endothelial selectins on neutrophils or lymphocytes. Although the physiologic characterization of these ligands has been obtained in mice, we also note reported similarities and differences with human selectin ligands.

Complete Identification of E-Selectin Ligands on Neutrophils Reveals Distinct Functions of PSGL-1, ESL-1, and CD44

The selectins and their ligands are required for leukocyte extravasation during inflammation. Several glycoproteins have been suggested to bind to E-selectin in vitro, but the complete identification of its physiological ligands has remained elusive. Here, we showed that E-selectin ligand-1 (ESL-1), P-selectin glycoprotein ligand-1 (PSGL-1), and CD44 encompassed all endothelial-selectin ligand activity on neutrophils by using gene- and RNA-targeted loss of function. PSGL-1 played a major role in the initial leukocyte capture, whereas ESL-1 was critical for converting initial tethers into steady slow rolling. CD44 controlled rolling velocity and mediated E-selectin-dependent redistribution of PSGL-1 and L-selectin to a major pole on slowly rolling leukocytes through p38 signaling. These results suggest distinct and dynamic contributions of these three glycoproteins in selectin-mediated neutrophil adhesion and signaling.

Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury

Selectins and their ligands mediate leukocyte rolling, allowing interactions with chemokines that lead to integrin activation and arrest. Here we show that E-selectin is crucial for generating a secondary wave of activating signals, transduced specifically by E-selectin ligand-1, that induces polarized, activated αMβ2 integrin clusters at the leading edge of crawling neutrophils, allowing capture of circulating erythrocytes or platelets. In a humanized mouse model of sickle cell disease, the capture of erythrocytes by αMβ2 microdomains leads to acute lethal vascular occlusions. In a model of transfusion-related acute lung injury, polarized neutrophils capture circulating platelets, resulting in the generation of oxidative species that produce vascular damage and lung injury. Inactivation of E-selectin or αMβ2 prevents tissue injury in both inflammatory models, suggesting broad implications of this paradigm in thromboinflammatory diseases. These results indicate that endothelial selectins can influence neutrophil behavior beyond its canonical rolling step through delayed, organ-damaging, polarized activation.

Neutrophils scan for activated platelets to initiate inflammation

Immune and inflammatory responses require leukocytes to migrate within and through the vasculature, a process that is facilitated by their capacity to switch to a polarized morphology with an asymmetric distribution of receptors. We report that neutrophil polarization within activated venules served to organize a protruding domain that engaged activated platelets present in the bloodstream. The selectin ligand PSGL-1 transduced signals emanating from these interactions, resulting in the redistribution of receptors that drive neutrophil migration. Consequently, neutrophils unable to polarize or to transduce signals through PSGL-1 displayed aberrant crawling, and blockade of this domain protected mice against thromboinflammatory injury. These results reveal that recruited neutrophils scan for activated platelets, and they suggest that the neutrophils’ bipolarity allows the integration of signals present at both the endothelium and the circulation before inflammation proceeds. Blood neutrophil cells sample platelets in the bloodstream before infiltrating surrounding tissue. A two-cell collaboration for inflammation Immune cells called neutrophils are first responders to infection. Neutrophils move within and through blood vessels to get to sites of infection quickly. Sreeramkumar et al. found that mouse neutrophils rely on platelets to help find such sites. Neutrophils extended protrusions into blood vessels. When these protrusions came into contact with platelets, the neutrophils migrated into the surrounding tissue to carry out their inflammatory functions. Preventing these neutrophilplatelet interactions alleviated collateral inflammatory damage to tissues in several injury models in mice. Science, this issue p. 1234

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, α(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.

Neutrophil mobilization via plerixafor-mediated CXCR4 inhibition arises from lung demargination and blockade of neutrophil homing to the bone marrow

The CXCR4 antagonist plerixafor augments frequency of circulating neutrophils via release from the lung and prevents neutrophil homing to the bone marrow.

Imaging receptor microdomains on leukocyte subsets in live mice

We present a simple method to identify the recruitment of leukocyte subsets and determine concurrent surface-receptor clustering in live mice. We show that CD45+ F4/80− Gr-1+ neutrophils are robustly recruited in surgery-activated cremasteric venules, whereas adherent CD45+ B220+ B lymphocytes were dominant in bone marrow venules. Most adherent Gr-1+ leukocytes are not firmly stationary but actively migrate on TNF-α–activated cremasteric venular endothelium and exhibit marked polarization of surface PSGL-1, but not LFA-1, to the trailing edge.