Invasive tumor dissemination in vitro and in vivo involves the proteolytic degradation of ECM barriers. This process, however, is only incompletely attenuated by protease inhibitor–based treatment, suggesting the existence of migratory compensation strategies. In three-dimensional collagen matrices, spindle-shaped proteolytically potent HT-1080 fibrosarcoma and MDA-MB-231 carcinoma cells exhibited a constitutive mesenchymal-type movement including the coclustering of β1 integrins and MT1–matrix metalloproteinase (MMP) at fiber bindings sites and the generation of tube-like proteolytic degradation tracks. Near-total inhibition of MMPs, serine proteases, cathepsins, and other proteases, however, induced a conversion toward spherical morphology at near undiminished migration rates. Sustained protease-independent migration resulted from a flexible amoeba-like shape change, i.e., propulsive squeezing through preexisting matrix gaps and formation of constriction rings in the absence of matrix degradation, concomitant loss of clustered β1 integrins and MT1-MMP from fiber binding sites, and a diffuse cortical distribution of the actin cytoskeleton. Acquisition of protease-independent amoeboid dissemination was confirmed for HT-1080 cells injected into the mouse dermis monitored by intravital multiphoton microscopy. In conclusion, the transition from proteolytic mesenchymal toward nonproteolytic amoeboid movement highlights a supramolecular plasticity mechanism in cell migration and further represents a putative escape mechanism in tumor cell dissemination after abrogation of pericellular proteolysis.

Constitutive egress of bone marrow (BM)-resident hematopoietic stem and progenitor cells (HSPCs) into the blood is a well-established phenomenon, but the ultimate fate and functional relevance of circulating HSPCs is largely unknown. We show that mouse thoracic duct (TD) lymph contains HSPCs that possess short- and long-term multilineage reconstitution capacity. TD-derived HSPCs originate in the BM, enter the blood, and traffic to multiple peripheral organs, where they reside for at least 36 hr before entering draining lymphatics to return to the blood and, eventually, the BM. HSPC egress from extramedullary tissues into lymph depends on sphingosine-1-phosphate receptors. Migratory HSPCs proliferate within extramedullary tissues and give rise to tissue-resident myeloid cells, preferentially dendritic cells. HSPC differentiation is amplified upon exposure to Toll-like receptor agonists. Thus, HSPCs can survey peripheral organs and can foster the local production of tissue-resident innate immune cells under both steady-state conditions and in response to inflammatory signals.

After homing to lymph nodes, CD8 + T cells are primed by dendritic cells (DCs) in three phases. During phase one, T cells undergo brief serial contacts with DCs for several hours, whereas phase two is characterized by stable T cell-DC interactions. We show here that the duration of phase one and T cell activation kinetics correlated inversely with the number of complexes of cognate peptide and major histocompatibility complex (pMHC) per DC and with the density of antigen-presenting DCs per lymph node. Very few pMHC complexes were necessary for the induction of full-fledged T cell activation and effector differentiation. However, neither T cell activation nor transition to phase two occurred below a threshold antigen dose determined in part by pMHC stability. Thus, phase one permits T cells to make integrated 'measurements' of antigen dose that determine subsequent T cell participation in immune responses.The naive T cell population expresses a broad array of unique T cell antigen receptors (TCRs), each with a discrete affinity for a given complex of cognate peptide and major histocompatibility complex (pMHC). Naive T cells constantly survey and sample antigenpresenting cells (APCs) in secondary lymphoid tissues in search of rare cognate pMHC complexes 1 . Due to the diversity of the TCR repertoire, only one in 1 × 10 5 to 1 × 10 6 T cells expresses a TCR with sufficient affinity for any given antigen to transmit an activating Correspondence should be addressed to U. H.v.A. (E-mail: uva@cbr.med.harvard.edu). Note: Supplementary information is available on the Nature Immunology website. AUTHOR CONTRIBUTIONSS.E.H. designed the study, did and analyzed experiments, and wrote the manuscript; U.H.v.A designed the study and wrote the manuscript; T.R.M., I.B.M., A.P., M.P.F., B.S. and T.J. did experiments; B.L. and H.C.W. provided reagents; and M.N.A., H.Z. and A.K.C. modeled the experimental data. COMPETING INTERESTS STATEMENTThe authors declare competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/natureimmunology/.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript stimulus 2,3 . Activation of naive T cells also requires costimulatory and cytokine signals 4,5 , which are typically provided by mature dendritic cells (DCs) in secondary lymphoid tissues 6 . As they 'hunt' for their cognate antigen, naive T cells recirculate between the blood and lymph nodes and spend less than 1 d in any given secondary lymphoid tissue 1,7 . When T cells encounter antigen in the proper context, they become activated and upregulate the activation marker CD69, which causes their retention in lymph nodes 8 . The trapped cells proliferate and acquire effector functions. Effector cells egress from lymph nodes and travel to peripheral tissues to seek out cells presenting cognate antigen 9 .Although this chain of events is well establ...

We have used intravital microscopy to study physiologically perfused microvessels in murine bone marrow (BM). BM sinusoids and venules, but not adjacent bone vessels, supported rolling interactions of hematopoietic progenitor cells. Rolling did not involve L-selectin, but was partially reduced in wild-type mice treated with antibodies to P- or E-selectin and in mice that were deficient in these two selectins. Selectin-independent rolling was mediated by α4 integrins, which interacted with endothelial vascular cell adhesion molecule (VCAM)-1. Parallel contribution of the endothelial selectins and VCAM-1 is not known to direct blood cell trafficking to other noninflamed tissues. This combination of constitutively expressed adhesion molecules may thus constitute a BM-specific recruitment pathway for progenitor cells analogous to the vascular addressins that direct selective lymphocyte homing to lymphoid organs.

Normal bone marrow (BM) contains T cells whose function and origin are poorly understood. We observed that CD8+ T cells in BM consist chiefly of CCR7+ L-selectin+ central memory cells (TCMs). Adoptively transferred TCMs accumulated more efficiently in the BM than naive and effector T cells. Intravital microscopy (IVM) showed that TCMs roll efficiently in BM microvessels via L-, P-, and E-selectin, whereas firm arrest required the VCAM-1/alpha4beta1 pathway. alpha4beta1 integrin activation did not depend on pertussis toxin (PTX)-sensitive Galphai proteins but was reduced by anti-CXCL12. In contrast, TCM diapedesis did not require CXCL12 but was blocked by PTX. After extravasation, TCMs displayed agile movement within BM cavities, remained viable, and mounted potent antigen-specific recall responses for at least two months. Thus, the BM functions as a major reservoir for TCMs by providing specific recruitment signals that act in sequence to mediate the constitutive recruitment of TCMs from the blood.

The adhesive mechanisms allowing hematopoietic progenitor cells (HPC) homing to the bone marrow (BM) after BM transplantation are poorly understood. We investigated the role of endothelial selectins and vascular cell adhesion molecule-1 (VCAM-1) in this process. Lethally irradiated recipient mice deficient in both P-and E-selectins (P͞E؊͞؊), reconstituted with minimal numbers (<5 ؋ 10 4 ) of wild-type BM cells, poorly survived the procedure compared with wild-type recipients. Excess mortality in P͞E؊͞؊ mice, after a lethal dose of irradiation, was likely caused by a defect of HPC homing. Indeed, we observed that the recruitment of HPC to the BM was reduced in P͞E؊͞؊ animals, either splenectomized or spleen-intact. Homing into the BM of P͞E؊͞؊ recipient mice was further compromised when a function-blocking VCAM-1 antibody was administered. Circulating HPC, 14 hr after transplantation, were greatly increased in P͞E؊͞؊ mice treated with anti-VCAM-1 compared with P͞E؊͞؊ mice treated with just IgG or wild-type mice treated with either anti-VCAM-1 or IgG. Our results indicate that endothelial selectins play an important role in HPC homing to the BM. Optimal recruitment of HPC after lethal doses of irradiation requires the combined action of both selectins and VCAM-1 expressed on endothelium of the BM.

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related death. The biologic processes contributing to TRALI are poorly understood. All blood products can cause TRALI, and no specific treatment is available. A "2-event model" has been proposed as the trigger. The first event may include surgery, trauma, or infection; the second involves the transfusion of antileukocyte antibodies or bioactive lipids within the blood product. Together, these events induce neutrophil activation in the lungs, causing endothelial damage and capillary leakage. Neutrophils, in response to pathogens or under stress, can release their chromatin coated with granule contents, thus forming neutrophil extracellular traps (NETs). Although protective against infection, these NETs are injurious to tissue. Here we show that NET biomarkers are present in TRALI patients' blood and that NETs are produced in vitro by primed human neutrophils when challenged with anti-HNA-3a antibodies previously implicated in TRALI. NETs are found in alveoli of mice experiencing antibody-mediated TRALI. DNase 1 inhalation prevents their alveolar accumulation and improves arterial oxygen saturation even when administered 90 minutes after TRALI onset. We suggest that NETs form in the lungs during TRALI, contribute to the disease process, and thus could be targeted to prevent or treat TRALI. IntroductionTransfusion-related acute lung injury (TRALI) is a rare but serious complication of blood transfusion that occurs within 6 hours of transfusion and is characterized by hypoxemia, respiratory distress, and pulmonary infiltrates. 1 Over the years, prevention measures have resulted in a significant reduction in cases. However, TRALI is still the leading cause of transfusion-related mortality, and its prevalence is likely underestimated; one study suggested that more than 2% of cardiac surgery patients are affected. 2 Only supportive treatment is available to the patient, including mechanical ventilation and oxygen supplementation. Many of the severe cases have been linked to the presence of antineutrophil antibodies in the transfused product. 3,4 These antibodies bind to the recipients' neutrophils, activate them, and induce sequestration in the pulmonary capillaries, resulting in tissue injury. 5 Activated neutrophils can release neutrophil extracellular traps (NETs) 6 that are composed of DNA fibers decorated with histones and antimicrobial proteins 7 originally contained in the neutrophil granules. The structure and the composition of NETs allow them to trap and prevent the spread of pathogens and also to kill Gram-negative and Gram-positive bacteria, as well as yeast. 6 NET formation follows a specific pattern characterized by histone hypercitrullination, 8 chromatin decondensation, dissolution of the granular and nuclear membranes, and cytolysis. 9 Despite NETs' beneficial antimicrobial function, 6,10 their formation at the wrong time, in the wrong place, or in the wrong amount can have a negative effect on the host. NETs and their c...

Dendritic cells (DCs) carry antigen from peripheral tissues via lymphatics to lymph nodes. We report here that differentiated DCs can also travel from the periphery into the blood. Circulating DCs migrated to the spleen, liver and lung but not lymph nodes. They also homed to the bone marrow, where they were retained better than in most other tissues. Homing of DCs to the bone marrow depended on constitutively expressed vascular cell adhesion molecule 1 and endothelial selectins in bone marrow microvessels. Two-photon intravital microscopy in bone marrow cavities showed that DCs formed stable antigen-dependent contacts with bone marrow-resident central memory T cells. Moreover, using this previously unknown migratory pathway, antigen-pulsed DCs were able to trigger central memory T cell-mediated recall responses in the bone marrow.