L-selectin (CD62L), a lectin-like adhesion molecule, mediates lymphocyte homing and leukocyte accumulation at sites of inflammation. Its transmembrane (TM) and intracellular (IC) domains confer clustering of L-selectin on microvilli of resting leukocytes, which is important for L-selectin function. Following activation of protein kinase C (PKC) or calmodulin inhibition, the wild-type (WT) protein is rapidly cleaved in its membrane-proximal ectodomain. To examine whether L-selectin topography or TM/IC domains are involved in this shedding process, we used stable transfectants expressing WT L-selectin (on microvilli) or chimeric molecules consisting of the L-selectin ectodomain linked to the TM/IC domains of CD44 (excluded from microvilli) or CD31 (randomly distributed). PKC activation by PMA altered the cells' surface morphology, but did not induce a redistribution of L-selectin ectodomains. All cell lines shed ectodomains upon PMA activation in a dose-dependent fashion and with similar kinetics. Calmodulin inhibition by trifluoperazine induced shedding in both WT and chimera transfectants. At high trifluoperazine concentrations, shedding of WT L-selectin was significantly more pronounced than that of chimeric molecules. Regardless of the activating stimulus, shedding was blocked by a hydroxamate-based metalloprotease inhibitor, suggesting that ectodomain down-regulation occurred through proteolytic cleavage by identical protease(s). These results show that the recognition site(s) for PKC-induced L-selectin shedding is exclusively contained within the ectodomain; the nature of subsurface structures and surface topography are irrelevant. Shedding induced by calmodulin inhibition has two components: one requires the L-selectin TM/IC domain, and the other is independent of it.

It has been proposed that two different antigen-experienced T cell subsets may be distinguishable by their preferential ability to home to lymphoid organs (central memory cells) or nonlymphoid tissues (effector memory/effector cells). We have shown recently that murine antigen-primed CD8(+) T cells cultured in interleukin (IL)-15 (CD8(IL-15)) resemble central memory cells in phenotype and function. In contrast, primed CD8(+) T cells cultured in IL-2 (CD8(IL-2)) become cytotoxic effector cells. Here, the migratory behavior of these two subsets was investigated. Naive, CD8(IL-15) cells and, to a lesser degree, CD8(IL-2) cells localized to T cell areas in the spleen, but only naive and CD8(IL-15) cells homed to lymph nodes (LNs) and Peyer's patches. Intravital microscopy of peripheral LNs revealed that CD8(IL-15) cells, but not CD8(IL-2) cells, rolled and arrested in high endothelial venules (HEVs). Migration of CD8(IL-15) cells to LNs depended on L-selectin and required chemokines that bind CC chemokine receptor (CCR)7. Both antigen-experienced populations, but not naive T cells, responded to inflammatory chemokines and accumulated at sites of inflammation. However, CD8(IL-2) cells were 12 times more efficient in migrating to inflamed peritoneum than CD8(IL-15) cells. Furthermore, CD8(IL-15) cells proliferated rapidly upon reencounter with antigen at sites of inflammation. Thus, central memory-like CD8(IL-15) cells home avidly to lymphoid organs and moderately to sites of inflammation, where they mediate rapid recall responses, whereas CD8(IL-2) effector T cells accumulate in inflamed tissues, but are excluded from most lymphoid organs.

The integrin alphaLbeta2 has three different domains in its headpiece that have been suggested to either bind ligand or to regulate ligand binding. One of these, the inserted or I domain, has a fold similar to that of small G proteins. The I domain of the alphaM and alpha2 subunits has been crystallized in both open and closed conformations; however, the alphaL I domain has been crystallized in only the closed conformation. We hypothesized that the alphaL domain also would have an open conformation, and that this would be the ligand binding conformation. Therefore, we introduced pairs of cysteine residues to form disulfides that would lock the alphaL I domain in either the open or closed conformation. Locking the I domain open resulted in a 9,000-fold increase in affinity to intercellular adhesion molecule-1 (ICAM-1), which was reversed by disulfide reduction. By contrast, the affinity of the locked closed conformer was similar to wild type. Binding completely depended on Mg(2+). Orders of affinity were ICAM-1 > ICAM-2 > ICAM-3. The k(on), k(off), and K(D) values for the locked open I domain were within 1.5-fold of values previously determined for the alphaLbeta2 complex, showing that the I domain is sufficient for full affinity binding to ICAM-1. The locked open I domain antagonized alphaLbeta2-dependent adhesion in vitro, lymphocyte homing in vivo, and firm adhesion but not rolling on high endothelial venules. The ability to reversibly lock a protein fold in an active conformation with dramatically increased affinity opens vistas in therapeutics and proteomics.

Leukotriene B(4) (LTB(4)) is a potent chemoattractant active on multiple leukocytes, including neutrophils, macrophages, and eosinophils, and is implicated in the pathogenesis of a variety of inflammatory processes. A seven transmembrane-spanning, G protein-coupled receptor, called BLTR (LTB(4) receptor), has recently been identified as an LTB(4) receptor. To determine if BLTR is the sole receptor mediating LTB(4)-induced leukocyte activation and to determine the role of LTB(4) and BLTR in regulating leukocyte function in inflammation in vivo, we generated a BLTR-deficient mouse by targeted gene disruption. This mouse reveals that BLTR alone is responsible for LTB(4)-mediated leukocyte calcium flux, chemotaxis, and firm adhesion to endothelium in vivo. Furthermore, despite the apparent functional redundancy with other chemoattractant-receptor pairs in vitro, LTB(4) and BLTR play an important role in the recruitment and/or retention of leukocytes, particularly eosinophils, to the inflamed peritoneum in vivo. These studies demonstrate that BLTR is the key receptor that mediates LTB(4)-induced leukocyte activation and establishes a model to decipher the functional roles of BLTR and LTB(4) in vivo.

T cell homing to peripheral lymph nodes (PLNs) is defined by a multistep sequence of interactions between lymphocytes and endothelial cells in high endothelial venules (HEVs). After initial tethering and rolling via L-selectin, firm adhesion of T cells requires rapid upregulation of lymphocyte function-associated antigen 1 (LFA-1) adhesiveness by a previously unknown pathway that activates a Galpha(i)-linked receptor. Here, we used intravital microscopy of murine PLNs to study the role of thymus-derived chemotactic agent (TCA)-4 (secondary lymphoid tissue chemokine, 6Ckine, Exodus-2) in homing of adoptively transferred T cells from T-GFP mice, a transgenic strain that expresses green fluorescent protein (GFP) selectively in naive T lymphocytes (T(GFP) cells). TCA-4 was constitutively presented on the luminal surface of HEVs, where it was required for LFA-1 activation on rolling T(GFP) cells. Desensitization of the TCA-4 receptor, CC chemokine receptor 7 (CCR7), blocked T(GFP) cell adherence in wild-type HEVs, whereas desensitization to stromal cell-derived factor (SDF)-1alpha (the ligand for CXC chemokine receptor 4 [CXCR4]) did not affect T(GFP) cell behavior. TCA-4 protein was not detected on the luminal surface of PLN HEVs in plt/plt mice, which have a congenital defect in T cell homing to PLNs. Accordingly, T(GFP) cells rolled but did not arrest in plt/plt HEVs. When TCA-4 was injected intracutaneously into plt/plt mice, the chemokine entered afferent lymph vessels and accumulated in draining PLNs. 2 h after intracutaneous injection, luminal presentation of TCA-4 was detectable in a subset of HEVs, and LFA-1-mediated T(GFP) cell adhesion was restored in these vessels. We conclude that TCA-4 is both required and sufficient for LFA-1 activation on rolling T cells in PLN HEVs. This study also highlights a hitherto undocumented role for chemokines contained in afferent lymph, which may modulate leukocyte recruitment in draining PLNs.

Noninflamed skin venules support constitutive leukocyte rolling. P-selectin controls the rolling frequency, whereas E-selectin dictates rolling velocity (Vroll). Fucosylated selectin ligands are essential for all interactions, as rolling was absent in mice doubly deficient in alpha1,3-fucosyltransferase (FucT)-IV and FucT-VII. The rolling fraction was reduced in FucT-VII-/- animals but normal in FucT-IV-/- mice. However, Vroll was markedly increased in both strains. P-selectin ligands generated by FucT-VII are crucial for initial leukocyte tethering, whereas E-selectin ligands that permit maximum slowing of Vroll require simultaneous expression of FucT-IV and FucT-VII. These results demonstrate a role for FucT-IV in selectin-dependent adhesion and suggest that the endothelial selectins and FucTs have distinct but overlapping functions in the immunosurveillance of the skin.

After birth, the bone marrow (BM) is the principal site of hematopoiesis in mammals. Thus, a large number of newly formed blood cells must penetrate the wall of BM microvessels to enter the circulation. In addition, the BM appears to function as a lymphoid organ and is also part of the macrophagal system. Subsets of circulating lymphocytes and other cells of the immune system continuously home to the BM. However, neither the mechanisms of blood cell migration to and from the BM nor its precise role in the immune system are well understood. One reason for the relative paucity of data on BM physiology is the fact that normal BM is surrounded by thick cortical bone that impedes direct observation and experimental manipulation. One notable exception is the calvaria of the murine skull where hematopoietically active BM is only covered by a thin lamella of bone that is sufficiently translucent to allow a detailed in situ analysis of the BM microcirculation by epi-fluorescence microscopy. Here, we review our current knowledge of the anatomic, hemodynamic, and endothelial properties of the specialized microvascular bed within murine skull BM. In addition, we summarize recent studies on the molecular mechanisms that mediate the homing of circulating hematopoietic progenitor cells to the BM, an event that is critical for the success of BM transplantations.

The chemokine stromal-derived factor-1alpha (SDF-1alpha) is a chemoattractant for CD34(+) progenitor cells, in vitro and in vivo. The receptor for SDF-1alpha, CXCR-4, is a 7 transmembrane domain receptor, which is also a coreceptor for human immunodeficiency virus (HIV). Here we show that transformation of hematopoietic cell lines by BCR/ABL significantly impairs their response to SDF-1alpha. Three different hematopoietic cell lines, Ba/F3, 32Dcl3, and Mo7e, were found to express CXCR-4 and to respond to SDF-1alpha with increased migration in a transwell assay. In contrast, after transformation by the BCR/ABL oncogene, the chemotactic response to SDF-1alpha was reduced in all 3 lines. This effect was directly due to BCR/ABL, because Ba/F3 cells, in which the expression of BCR/ABL could be regulated by a tetracycline-inducible promoter, also had reduced chemotaxis to SDF-1alpha when BCR/ABL was induced. The reduced response to SDF-1alpha was not due to an inability of BCR/ABL-transformed cell lines to migrate in general, as spontaneous motility of BCR/ABL-transformed cells was increased. In mice, injection of SDF-1alpha into the spleen resulted in a transient accumulation of untransformed Ba/F3 cells, but not Ba/F3. p210(BCR/ABL) cells administered simultaneously. The mechanism may involve inhibition of CXCR-4 receptor function, because in BCR/ABL-transformed cells, CXCR-4 receptors were expressed on the cell surface, but SDF-1alpha calcium flux was inhibited. Because SDF-1alpha and CXCR-4 are felt to be involved in progenitor cell homing to marrow, the abnormality decribed here could contribute to the homing and retention defects typical of immature myeloid cells in chronic myelogenous leukemia.

The goal of this study was to determine the mechanisms by which dendritic cells (DCs) in blood could interact with endothelium, a prerequisite to extravasation into tissues. Our results indicate that DCs express both HECA-452-reactive and nonreactive isoforms of P-selectin glycoprotein ligand 1 (PSGL-1) and can tether and roll efficiently on E- and P-selectin under flow conditions in vitro. Freshly isolated blood DCs were further observed to roll continuously along noninflamed murine dermal endothelium in vivo. This interaction is strictly dependent on endothelial selectins, as shown by experiments with blocking antibodies and with E- and P-selectin-deficient mice. We hypothesize that DCs in blood are constitutively poised at the interface of blood and skin, ready to extravasate upon induction of inflammation, and we showed that cutaneous inflammation results in a rapid recruitment of DCs from the blood to tissues. We propose that this is an important and previously unappreciated element of immunosurveillance.

Adhesion receptors that are known to initiate contact (tethering) between blood-borne leukocytes and their endothelial counterreceptors are frequently concentrated on the microvilli of leukocytes. Other adhesion molecules are displayed either randomly or preferentially on the planar cell body. To determine whether ultrastructural distribution plays a role during tethering in vivo, we used pre-B cell transfectants expressing L- or E-selectin ectodomains linked to transmembrane/intracellular domains that mediated different surface distribution patterns. We analyzed the frequency and velocity of transfectant rolling in high endothelial venules of peripheral lymph nodes using an intravital microscopy model. Ectodomains on microvilli conferred a higher efficiency at initiating rolling than random distribution which, in turn, was more efficient than preferential expression on the cell body. The role of microvillous presentation was less accentuated in venules below 20 micrometers in diameter than in larger venules. In the narrow venules, tethering of cells with cell body expression may have been aided by forced margination through collision with erythrocytes. L-selectin transfected cells rolled 10-fold faster than E-selectin transfectants. Interestingly, rolling velocity histograms of cell lines expressing equivalent copy numbers of the same ectodomain were always similar, irrespective of the topographic distribution. Our data indicate that the distribution of adhesion receptors has a dramatic impact on contact initiation between leukocytes and endothelial cells, but does not play a role once rolling has been established.

Antigen-specific effector T cells are prerequisite to immune protection, but because of the lack of effector cell-specific markers, their generation and differentiation has been difficult to study. We report that effector cells are highly enriched in a T cell subset that can be specifically identified in transgenic (T-GFP) mice expressing green fluorescent protein (GFP) under control of the murine CD4 promoter and proximal enhancer. Consistent with previous studies of these transcriptional control elements, GFP was strongly and specifically expressed in nearly all resting and short-term activated CD4(+) and CD8(+) T cells. However, when T-GFP mice were challenged with vaccinia virus, allogeneic tumor cells, or staphylococcal enterotoxin A, the cytotoxic and IFN-gamma-producing T cells lost GFP expression. Upon T cell receptor (TCR) ligation by alphaCD3, sorted GFP(+) cells fluxed calcium and proliferated vigorously. In contrast, GFP(-) effector cells showed a diminished calcium flux and did not proliferate. Instead, they underwent apoptosis unless supplied with exogenous IL-2. By reverse transcription-PCR analysis, the GFP(-) cells up-regulated the pro-apoptotic molecule, Fas-L, and down-regulated gene expression of the proximal TCR signaling molecule, CD3zeta, and c-jun, a component of the AP-1 transcription factor. Thus, differential regulation of TCR signaling may explain the divergent responses of naïve and effector T cells to antigen stimulation.

Peripheral lymph nodes (PLN) are critical for immunologic memory formation in response to antigens that penetrate the skin. Blood-borne lymphocytes first encounter such antigens after they home to PLN through a multi-step adhesion process that is normally initiated by L-selectin (CD62L) in high endothelial venules (HEV). Since naive T cells can not enter PLN normally in L-selectin-deficient mice, a delayed type hypersensitivity response to cutaneously applied antigen cannot be mounted. In this study, we report that the administration of activated platelets into the systemic circulation of L-selectin knockout mice restores lymphocyte trafficking to PLN, and reconstitutes T cell-mediated immunity in response to a cutaneous antigen. These effects required platelet-expressed P-selectin that allows activated platelets to transiently form a bridge between lymphocytes and HEV, thereby enabling lymphocytes to undergo subsequent beta2 integrin-dependent firm adhesion. These profound effects of platelet-mediated cell-cell interactions on lymphocyte trafficking and formation of immunologic memory may impact on a variety of autoimmune and inflammatory conditions.

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 (P/E-/-), reconstituted with minimal numbers (

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 alpha4 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.

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 LFA-1 on rolling lymphocytes, but not on granulocytes. Thus, granulocytes continue to roll through the PLN, whereas LFA-1 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)-1 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.

To characterize the adhesion cascade that directs lymphocyte homing to peripheral lymph nodes (PLNs), we investigated the molecular mechanisms of lymphocyte interactions with the microvasculature of subiliac lymph nodes. We found that endogenous white blood cells and adoptively transferred lymph node lymphocytes (LNCs) tethered and rolled in postcapillary high endothelial venules (HEVs) and to a lesser extent in collecting venules. Similarly, firm arrest occurred nearly exclusively in the paracortical HEVs. Endogenous polymorphonuclear (PMNs) and mononuclear leukocytes (MNLs) attached and rolled in HEVs at similar frequencies, but only MNLs arrested suggesting that the events downstream of primary rolling interactions critically determine the specificity of lymphocyte recruitment. Antibody inhibition studies revealed that L-selectin was responsible for attachment and rolling of LNCs, and that LFA-1 was essential for sticking. LFA-1-dependent arrest was also abolished by pertussis toxin, implicating a requirement for G alpha i--protein-linked signaling. alpha 4 integrins, which play a critical role in lymphocyte homing to Peyer's Patches, made no significant contribution to attachment, rolling, or sticking in resting PLNs. Velocity analysis of interacting LNCs revealed no detectable contribution by LFA-1 to rolling. Taken together, our results suggest that lymphocyte- HEV interactions within PLNs are almost exclusively initiated by L-selectin followed by a G protein-coupled lymphocyte-specific activation event and activation-induced engagement of LFA-1. These events constitute a unique adhesion cascade that dictates the specificity of lymphocyte homing to PLNs.

We report that the cell surface mucin CD43 acts as an anti-adhesin on T lymphocytes. CD43-deficient murine lymphocytes homed significantly more frequently to secondary lymphoid organs than wild-type cells. Intravital microscopy of peripheral lymph node venules revealed that CD43-deficient lymphocytes were twice as likely to tether, roll, and stick than wild-type cells. This effect was due to CD43 interference with the homing receptor, L-selectin, and was most pronounced in venules with low L-selectin ligand density. In vitro, CD43-deficient cells tethered to L-selectin ligands more efficiently and rolled more slowly than wild-type lymphocytes. Thus, CD43 exerts a negative regulatory effect on T cell trafficking by counterbalancing L-selectin-mediated adhesion.