Coordinated regulation of lymph node vascular-stromal growth first by CD11c+ cells and then by T and B cells

Lymph node blood vessels play important roles in the support and trafficking of immune cells. The blood vasculature is a component of the vascular-stromal compartment that also includes the lymphatic vasculature and fibroblastic reticular cells (FRCs). During immune responses as lymph nodes swell, the blood vasculature undergoes a rapid proliferative growth that is initially dependent on CD11c(+) cells and vascular endothelial growth factor (VEGF) but is independent of lymphocytes. The lymphatic vasculature grows with similar kinetics and VEGF dependence, suggesting coregulation of blood and lymphatic vascular growth, but lymphatic growth has been shown to be B cell dependent. In this article, we show that blood vascular, lymphatic, and FRC growth are coordinately regulated and identify two distinct phases of vascular-stromal growth--an initiation phase, characterized by upregulated vascular-stromal proliferation, and a subsequent expansion phase. The initiation phase is CD11c(+) cell dependent and T/B cell independent, whereas the expansion phase is dependent on B and T cells together. Using CCR7(-/-) mice and selective depletion of migratory skin dendritic cells, we show that endogenous skin-derived dendritic cells are not important during the initiation phase and uncover a modest regulatory role for CCR7. Finally, we show that FRC VEGF expression is upregulated during initiation and that dendritic cells can stimulate increased fibroblastic VEGF, suggesting the scenario that lymph node-resident CD11c(+) cells orchestrate the initiation of blood and lymphatic vascular growth in part by stimulating FRCs to upregulate VEGF. These results illustrate how the lymph node microenvironment is shaped by the cells it supports.     

Synchrony of high endothelial venules and lymphatic vessels revealed by immunization

The mature phenotype of peripheral lymph node (LN) high endothelial venules (HEVs), defined as MAdCAM-1(low) PNAd(high) LTbetaR(high) HEC-6ST(high), is dependent on signaling through the lymphotoxin-beta receptor (LTbetaR). Plasticity of PLN HEVs during immunization with oxazolone was apparent as a reversion to an immature phenotype (MAdCAM-1(high) PNAd(low) LTbetaR(low) HEC-6ST(low)) followed by recovery to the mature phenotype. The recovery was dependent on B cells and was inhibited by LTbetaR-Ig treatment. Concurrent with HEV reversion, at day 4 following oxazolone or OVA immunization, reduced accumulation of Evans blue dye and newly activated DCs in the draining LNs revealed a temporary afferent lymphatic vessel (LV) functional insufficiency. T cell priming to a second Ag was temporarily inhibited. At day 7, lymphangiogenesis peaked in both the skin and draining LN, and afferent LV function was restored at the same time as HEV phenotype recovery. This process was delayed in the absence of B cells. LV and HEV both express the LTbetaR. During lymphangiogenesis in the draining LN, HEV, and LV were directly apposed; some vessels appeared to express both PNAd and LYVE-1. Pretreatment with LTbetaR-Ig drastically reduced the number of PNAd+ LYVE-1+ vessels, suggesting a reduction in LV and HEV cross-talk. The concordance in time and function and the close physical contact between LVs and HEVs in the remodeling process after immunization indicate that the two vascular systems are in synchrony and engage in cross-talk through B cells and LTbetaR.     

Osteoblasts stimulated with pulsed electromagnetic fields increase HUVEC proliferation via a VEGF‐A independent mechanism

The clinically beneficial effect of low frequency pulsed electromagnetic fields (ELF‐PEMF) on bone healing has been described, but the exact mechanism of action remains unclear. A recent study suggests that there is a direct autocrine mitogenic effect of ELF‐PEMF on angiogenesis. The hypothesis of this study is that ELF‐PEMF also has an indirect effect on angiogenesis by manipulation of vascular endothelial growth factor (VEGF)‐A‐based paracrine intercellular communication with neighboring osteoblasts. Conditioned media experiments measured fetal rat calvarial cell (FRC) and human umbilical vein endothelial cell (HUVEC) proliferation using tritiated thymidine uptake. We demonstrate that ELF‐PEMF (15 Hz, 1.8 mT, for 8 h) has an indirect effect on the proliferation rate of both endothelial cells and osteoblasts in vitro by altering paracrine mediators. Conditioned media from osteoblast cells stimulated with ELF‐PEMF increased endothelial proliferation 54‐fold, whereas media from endothelial cells stimulated with ELF‐PEMF did not affect osteoblast proliferation. We examined the role of the pro‐angiogenic mediator VEGF‐A in the mitogenic effect of ELF‐PEMF‐stimulated osteoblast media on endothelial cells. The production of VEGF‐A by FRC as measured by ELISA was not changed by exposure to PEMF, and blocking experiments demonstrated that the ELF‐PEMF‐induced osteoblast‐derived endothelial mitogen observed in these studies was not VEGF‐A, but some other soluble angiogenic mediator. Bioelectromagnetics 30:189–197, 2009. © 2008 Wiley‐Liss, Inc.     

The human peripheral lymph node vascular addressin is a ligand for LECAM-1, the peripheral lymph node homing receptor

The trafficking of lymphocytes from the blood and into lymphoid organs is controlled by tissue-selective lymphocyte interactions with specialized endothelial cells lining post capillary venules, in particular the high endothelial venules (HEV) found in lymphoid tissues and sites of chronic inflammation. Lymphocyte interactions with HEV are mediated in part by lymphocyte homing receptors and tissue-specific HEV determinants, the vascular addressins. A peripheral lymph node addressin (PNAd) has been detected immunohistologically in mouse and man by monoclonal antibody MECA-79, which inhibits lymphocyte homing to lymph nodes and lymphocyte binding to lymph node and tonsillar HEV. The human MECA-79 antigen, PNAd, is molecularly distinct from the 65-kD mucosal vascular addressin. The most abundant iodinated species by SDS-PAGE is 105 kD. When affinity isolated and immobilized on glass slides, MECA-79 immunoisolated material binds human and mouse lymphocytes avidly in a calcium dependent manner. Binding is blocked by mAb MECA-79, by antibodies against mouse or human LECAM-1 (the peripheral lymph node homing receptor, the MEL-14 antigen, LAM-1), and by treatment of PNAd with neuraminidase. Expression of LECAM-1 cDNA confers PNAd binding ability on a transfected B cell line. We conclude that LECAM-1 mediates lymphocyte binding to PNAd, an interaction that involves the lectin activity of LECAM-1 and carbohydrate determinants on the addressin.     

Conserved signaling through vascular endothelial growth (VEGF) receptor family members in murine lymphatic endothelial cells

Lymphatic vessels guide interstitial fluid, modulate immune responses by regulating leukocyte and antigen trafficking to lymph nodes, and in a cancer setting enable tumor cells to track to regional lymph nodes. The aim of the study was to determine whether primary murine lymphatic endothelial cells (mLECs) show conserved vascular endothelial growth factor (VEGF) signaling pathways with human LECs (hLECs). LECs were successfully isolated from murine dermis and prostate. Similar to hLECs, vascular endothelial growth factor (VEGF) family ligands activated MAPK and pAkt intracellular signaling pathways in mLECs. We describe a robust protocol for isolation of mLECs which, by harnessing the power of transgenic and knockout mouse models, will be a useful tool to study how LEC phenotype contributes to alterations in lymphatic vessel formation and function.     

I kappa B kinase complex alpha kinase activity controls chemokine and high endothelial venule gene expression in lymph nodes and nasal-associated lymphoid tissue

The lymphotoxin (LT) beta receptor plays a critical role in secondary lymphoid organogenesis and the classical and alternative NF-kappaB pathways have been implicated in this process. IKKalpha is a key molecule for the activation of the alternative NF-kappaB pathway. However, its precise role and target genes in secondary lymphoid organogenesis remain unknown, particularly with regard to high endothelial venules (HEV). In this study, we show that IKKalpha(AA) mutant mice, who lack inducible kinase activity, have hypocellular lymph nodes (LN) and nasal-associated lymphoid (NALT) tissue characterized by marked defects in microarchitecture and HEV. In addition, IKKalpha(AA) LNs showed reduced lymphoid chemokine CCL19, CCL21, and CXCL13 expression. IKKalpha(AA) LN- and NALT-HEV were abnormal in appearance with reduced expression of peripheral node addressin (PNAd) explained by a severe reduction in the HEV-associated proteins, glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), and high endothelial cell sulfotransferase, a PNAd-generating enzyme that is a target of LTalphabeta. In this study, analysis of LTbeta(-/-) mice identifies GlyCAM-1 as another LTbeta-dependent gene. In contrast, TNFRI(-/-) mice, which lose classical NF-kappaB pathway activity but retain alternative NF-kappaB pathway activity, showed relatively normal GlyCAM-1 and HEC-6ST expression in LN-HEV. In addition, in this communication, it is demonstrated that LTbetaR is prominently expressed on LN- and NALT-HEV. Thus, these data reveal a critical role for IKKalpha in LN and NALT development, identify GlyCAM-1 and high endothelial cell sulfotransferase as new IKKalpha-dependent target genes, and suggest that LTbetaR signaling on HEV can regulate HEV-specific gene expression.     

VEGF promotes tumorigenesis and angiogenesis of human glioblastoma stem cells

There is increasing evidence for the presence of cancer stem cells (CSCs) in malignant brain tumors, and these CSCs may play a pivotal role in tumor initiation, growth, and recurrence. Vascular endothelial growth factor (VEGF) promotes the proliferation of vascular endothelial cells (VECs) and the neurogenesis of neural stem cells. Using CSCs derived from human glioblastomas and a retrovirus expressing VEGF, we examined the effects of VEGF on the properties of CSCs in vitro and in vivo. Although VEGF did not affect the property of CSCs in vitro, the injection of mouse brains with VEGF-expressing CSCs led to the massive expansion of vascular-rich GBM, tumor-associated hemorrhage, and high morbidity, suggesting that VEGF promoted tumorigenesis via angiogenesis. These results revealed that VEGF induced the proliferation of VEC in the vascular-rich tumor environment, the so-called stem cell niche.     

Endothelial barrier disruption by VEGF-mediated Src activity potentiates tumor cell extravasation and metastasis

VEGF is unique among angiogenic growth factors because it disrupts endothelial barrier function. Therefore, we considered whether this property of VEGF might contribute to tumor cell extravasation and metastasis. To test this, mice lacking the Src family kinases Src or Yes, which maintain endothelial barrier function in the presence of VEGF, were injected intravenously with VEGF-expressing tumor cells. We found a dramatic reduction in tumor cell extravasation in lungs or livers of mice lacking Src or Yes. At the molecular level, VEGF compromises the endothelial barrier by disrupting a VE-cadherin-beta-catenin complex in lung endothelium from wild-type, but not Yes-deficient, mice. Disrupting the endothelial barrier directly with anti-VE-cadherin both amplifies metastasis in normal mice and overcomes the genetic resistance in Yes-deficient mice. Pharmacological blockade of VEGF, VEGFR-2, or Src stabilizes endothelial barrier function and suppresses tumor cell extravasation in vivo. Therefore, disrupting Src signaling preserves host endothelial barrier function providing a novel host-targeted approach to control metastatic disease.     

Identification of select lymphocyte homing molecules and vascular addressins in lymphotoxin-alpha deficient mice

The transmigration of lymphocytes across vascular endothelium is a critical step for the localization of lymphocytes to lymph nodes in both naive and immune reactive states. Mice deficient in lymphotoxin-alpha (LT-alpha) lack peripheral and gut associated lymph nodes. Lymphocyte function and homing ability are reported to be normal in these mice yet information regarding cell adhesion molecules and counterpart vascular addressins is lacking. The phenotype of peripheral lymphocytes from LT-alpha deficient mice was investigated by the use of fluorescent activated cell sorting and immunohistochemistry. No difference was detected in the splenocyte and tissue expression of L-selectin, alpha4beta7 or its individual integrin components, mucosal addressin cell adhesion molecule (MAdCAM-1), intracellular adhesion molecule (ICAM-1), peripheral node addressin (PNAd), or platelet/endothelial cell adhesion molecule (PECAM-1) between wild-type and LT-alpha deficient mice. Therefore, impaired expression of these lymphocyte homing and vascular addressin molecules is apparently not included in the phenotype of the LT-alpha deficient mouse.     

VEGF in biological control

Vascular endothelial growth factor A (VEGF-A) belongs to a family of heparin binding growth factors that include VEGF-B, VEGF-C, VEGF-D, and placental-like growth factor (PLGF). First discovered for its ability to regulate vascular endothelial cell permeability, VEGF is a well-known angiogenic factor that is important for vascular development and maintenance in all mammalian organs. The development of molecular tools and pharmacological agents to selectively inhibit VEGF function and block angiogenesis and/or vascular permeability has led to great promise in the treatment of various cancers, macular degeneration, and wound healing. However, VEGF is also important in animals for the regulation of angiogenesis, stem cell and monocyte/macrophage recruitment, maintenance of kidney and lung barrier functions and neuroprotection. In addition to its role in regulating endothelial cell proliferation, migration, and cell survival, VEGF receptors are also located on many non-endothelial cells and act through autrocrine pathways to regulate cell survival and function. The following review will discuss the role of VEGF in physiological angiogenesis as well as its role in non-angiogenic processes that take place in adult organs.     

PHA activation of encephalitogenic T cells: in vitro line selection overcomes splenic suppression

In this study we compared myelin basic protein (MBP) and phytohemagglutinin (PHA) for their ability to induce proliferation and experimental autoimmune encephalomyelitis (EAE) transfer activity in mixed cell cultures obtained from spleen and lymph nodes versus highly selected MBP-specific T cell lines and clones. Established MBP-specific cells derived initially from immune lymph nodes attained both proliferative and EAE-transfer activities after in vitro activation with either MBP or PHA. In contrast, PHA was unable to induce immune spleen cells to transfer EAE, in spite of its potent mitogenic activity. On the basis of these results, we evaluated the in vitro proliferation and differentiation responses of MBP-specific T cells during the line selection process using cells derived from both immune lymph node and immune spleen. During the initial selection process with MBP, proliferation of MBP-specific T cell precursors from immunized spleen populations was reduced relative to lymph node cells. After antigen-dependent selection the encephalitogenic cells from either organ exhibited identical in vitro response characteristics. Freshly isolated immune spleen cells were potent suppressors of MBP-specific T cell proliferation suggesting that the in vitro differences between the two organs was due to splenic suppression of the encephalitogenic cells.     

Tumor lymphangiogenesis and melanoma metastasis

Malignant melanomas of the skin primarily metastasize to lymph nodes, and the detection of sentinel lymph node metastases serves as an important prognostic parameter. There is now compelling evidence that melanomas can induce lymphangiogenesis (growth of lymphatic vessels), mainly at the tumor-stroma interface, and that the level of tumor lymphangiogenesis is correlated with the incidence of sentinel lymph node metastases and with disease-free survival. Thus, tumor lymphangiogenesis can serve as a novel prognostic predictor in melanoma. Vascular endothelial growth factor (VEGF)-C, released by melanoma cells and by tumor-associated macrophages, likely represents the major lymphangiogenic factor in melanoma, although other members of the VEGF family might also be involved. The recent discovery that tumors can induce a premetastatic niche, by inducing lymphatic vessel growth in sentinel lymph nodes even before metastasis, and that lymph node lymphangiogenesis enhances metastatic spread, indicates that activated lymphatic vessels represent novel targets for the detection and/or therapy of melanoma metastases.     

Vascular endothelial growth factor can signal through platelet-derived growth factor receptors

Vascular endothelial growth factor (VEGF-A) is a crucial stimulator of vascular cell migration and proliferation. Using bone marrow-derived human adult mesenchymal stem cells (MSCs) that did not express VEGF receptors, we provide evidence that VEGF-A can stimulate platelet-derived growth factor receptors (PDGFRs), thereby regulating MSC migration and proliferation. VEGF-A binds to both PDGFRalpha and PDGFRbeta and induces tyrosine phosphorylation that, when inhibited, results in attenuation of VEGF-A-induced MSC migration and proliferation. This mechanism was also shown to mediate human dermal fibroblast (HDF) migration. VEGF-A/PDGFR signaling has the potential to regulate vascular cell recruitment and proliferation during tissue regeneration and disease.     

Vascular endothelial growth factor-D is a key molecule that enhances lymphatic metastasis of soft tissue sarcomas

Studies on lymph node metastasis of soft tissue sarcomas are insufficient because of its rarity. In this study, we examined the expressions of vascular endothelial growth factor (VEGF)-C and VEGF-D in soft tissue sarcomas metastasized to lymph nodes. In addition, the effects of the two molecules on the barrier function of a lymphatic endothelial cell monolayer against sarcoma cells were analyzed. We examined 7 patients who had soft tissue sarcomas with lymph node metastases and who had undergone neither chemotherapy nor radiotherapy before lymphadenectomy. Immunohistochemistry revealed that 2 of 7 sarcomas that metastasized to lymph nodes expressed VEGF-C both in primary and metastatic lesions. On the other hand, VEGF-D expression was detected in 4 of 7 primary and 7 of 7 metastatic lesions, respectively. Interestingly, 3 cases that showed no VEGF-D expression at primary sites expressed VEGF-D in metastatic lesions. Recombinant VEGF-C at 10(-8) and VEGF-D at 10(-7)and 10(-8)g/ml significantly increased the random motility of lymphatic endothelial cells compared with controls. VEGF-D significantly increased the migration of sarcoma cells through lymphatic endothelial monolayers. The fact that VEGF-D induced the migration of fibrosarcomas through the lymphatic endothelial monolayer is the probable reason for the strong relationship between VEGF-D expression and lymph node metastasis in soft tissue sarcomas. The important propensities of this molecule for the increase of lymph node metastases are not only lymphangiogenesis but also down-regulation of the barrier function of lymphatic endothelial monolayers, which facilitates sarcoma cells entering the lymphatic circulation.     

Upregulation of FIk-1 by bFGF via the ERK pathway is essential for VEGF-mediated promotion of neural stem cell proliferation

Neural stem cells (NSCs) constitute the cellular basis for embryonic brain development and neurogenesis.The processis regulated by NSC niche including neighbor cells such as vascular and glial cells.Since both vascular and glial cellssecrete vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF),we assessed the effect ofVEGF and bFGF on NSC proliferation using nearly homogeneous NSCs that were differentiated from mouse embryonicstem cells.VEGF alone did not have any significant effect.When bFGF was added,however,VEGF stimulated NSCproliferation in a dose-dependent manner,and this stimulation was inhibited by ZM323881,a VEGF receptor (Flk-1)-specific inhibitor.Interestingly,ZM323881 also inhibited cell proliferation in the absence of exogenous VEGF,suggestingthat VEGF autocrine plays a role in the proliferation of NSCs.The stimulatory effect of VEGF on NSC proliferationdepends on bFGF,which is likely due to the fact that expression of Flk-1 was upregulated by bFGF via phosphoryla-tion of ERK1/2.Collectively,this study may provide insight into the mechanisms by which mieroenvironmental nichesignals regulate NSCs.     

Upregulation of Flk-1 by bFGF via the ERK pathway is essential for VEGF-mediated promotion of neural stem cell proliferation

Neural stem cells （NSCs） constitute the cellular basis for embryonic brain development and neurogenesis. The process is regulated by NSC niche including neighbor cells such as vascular and glial cells. Since both vascular and glial cells secrete vascular endothelial growth factor （VEGF） and basic fibroblast growth factor （bFGF）, we assessed the effect of VEGF and bFGF on NSC proliferation using nearly homogeneous NSCs that were differentiated from mouse embryonic stem cells. VEGF alone did not have any significant effect. When bFGF was added, however, VEGF stimulated NSC proliferation in a dose-dependent manner, and this stimulation was inhibited by ZM323881, a VEGF receptor （Flk-1）- specific inhibitor. Interestingly, ZM323881 also inhibited cell proliferation in the absence of exogenous VEGF, suggesting that VEGF autocrine plays a role in the proliferation of NSCs. The stimulatory effect of VEGF on NSC proliferation depends on bFGF, which is likely due to the fact that expression of Flk-1 was upregulated by bFGF via phosphorylation of ERK1/2. Collectively, this study may provide insight into the mechanisms by which microenvironmental niche signals regulate NSCs.     

Hypoxia regulates VEGF expression and cellular proliferation by osteoblasts in vitro.

Numerous studies have demonstrated the critical role of angiogenesis for successful osteogenesis during endochondral ossification and fracture repair. Vascular endothelial growth factor (VEGF), a potent endothelial cell-specific cytokine, has been shown to be mitogenic and chemotactic for endothelial cells in vitro and angiogenic in many in vivo models. Based on previous work that (1) VEGF is up-regulated during membranous fracture healing, (2) the fracture site contains a hypoxic gradient, (3) VEGF is up-regulated in a variety of cells in response to hypoxia, and (4) VEGF is expressed by isolated osteoblasts in vitro stimulated by other fracture cytokines, the hypothesis that hypoxia may regulate the expression of VEGF by osteoblasts was formulated. This hypothesis was tested in a series of in vitro studies in which VEGF mRNA and protein expression was assessed after exposure of osteoblast-like cells to hypoxic stimuli. In addition, the effects of a hypoxic microenvironment on osteoblast proliferation and differentiation in vitro was analyzed. These results demonstrate that hypoxia does, indeed, regulate expression of VEGF in osteoblast-like cells in a dose-dependent fashion. In addition, it is demonstrated that hypoxia results in decreased cellular proliferation, decreased expression of proliferating cell nuclear antigen, and increased alkaline phosphatase (a marker of osteoblast differentiation). Taken together, these data suggest that osteoblasts, through the expression of VEGF, may be in part responsible for angiogenesis and the resultant increased blood flow to fractured bone segments. In addition, these data provide evidence that osteoblasts have oxygen-sensing mechanisms and that decreased oxygen tension can regulate gene expression, cellular proliferation, and cellular differentiation.     

Rac Regulates Vascular Endothelial Growth Factor Stimulated Motility

During angiogenesis endothelial cells migrate towards a chemotactic stimulus. Understanding the mechanism of endothelial cell migration is critical to the therapeutic manipulation of angiogenesis and ultimately cancer prevention. Vascular endothelial growth factor (VEGF) is a potent chemotactic stimulus of endothelial cells during angiogenesis. The endothelial cell signal transduction pathway of VEGF represents a potential target for cancer therapy, but the mechanisms of post-receptor signal transduction including the roles of rho family GTPases in regulating the cytoskeletal effects of VEGF in endothelial cells are not understood.

Here we analyze the mechanisms of cell migration in the mouse brain endothelial cell line (bEND3). Stable transfectants containing a tetracycline repressible expression vector were used to induce expression of Rac mutants. Endothelial cell haptotaxis was stimulated by constitutively active V12Rac on collagen and vitronectin coated supports, and chemotaxis was further stimulated by VEGF. Osteopontin coated supports were the most stimulatory to bEND3 haptotaxis, but VEGF was not effective in further increasing migration on osteopontin coated supports. Haptotaxis on support coated with collagen, vitronectin, and to a lesser degree osteopontin was inhibited by N17 Rac. N17 Rac expression blocked stimulation of endothelial cell chemotaxis by VEGF. As part of the chemotactic stimulation, VEGF caused a loss of actin organization at areas of cell-cell contact and increased stress fiber expression in endothelial cells which were directed towards pores in the transwell membrane. N17 Rac prevented the stimulation of cell-cell contact disruption and the stress fiber stimulation by VEGF.

These data demonstrate two pathways of regulating endothelial cell motility, one in which Rac is activated by matrix/integrin stimulation and is a crucial modulator of endothelial cell haptotaxis. The other pathway, in the presence of osteopontin, is Rac independent. VEGF stimulated chemotaxis, is critically dependent on Rac activation. Osteopontin was a potent matrix activator of motility, and perhaps one explanation for the absence of a VEGF plus osteopontin effect is that osteopontin stimulated motility was inhibitory to the Rac pathway.     

In Situ Analysis of Lymphocyte Migration to Lymph Nodes

Blood-borne lymphocytes migrate continuously to peripheral lymph nodes (PLN) and other organized lymphoid tissues where they are most likely to encounter their cognate antigen. Lymphocyte homing to PLN is a highly regulated process that occurs exclusively in specialized high endothelial venules (HEV) in the nodal paracortex. Recently, it has become possible to explore this vital aspect of peripheral immune surveillance by intravital microscopy of the subiliac lymph node microcirculation in anesthetized mice. This paper reviews technical and experimental aspects of the new model and summarizes recent advances in our understanding of the molecular mechanisms of lymphocyte homing to PLN which were derived from its use. Both lymphocytes and granulocytes initiate rolling interactions via L-selectin binding to the peripheral node addressin (PNAd) in PLN HEV. Subsequently, a G protein-coupled chemoattractant stimulus activates LEA-1 on rolling lymphocytes, but not on granulocytes. Thus. granulocytes continue to roll through the PLN, whereas LEA-I activation allows lymphocytes to arrest and emigrate into the extravascular compartment. We have also identified a second homing pathway that allows L-selectin low/(activated/memory) lymphocytes to home to PLN. P-selectin on circulating activated platelets can mediate simultaneous platelet adhesion to PNAd in HEV and to P-selectin glycoprotein ligand (PSGL)-l on lymphocytes. Through this mechanism, platelets can form a cellular bridge which can effectively substitute for the loss of L-selectin on memory cell subsets.