Normal Homing Research Articles

Hematopoietic Progenitor Cells Mobilize to the Site of Injury After Trauma and Hemorrhagic Shock in Rats

Trauma and hemorrhagic shock (T/HS) has been demonstrated to result in bone marrow (BM) suppression and the release of hematopoietic progenitor cells (HPC) into the peripheral blood in both human beings and experimental animals. HPC have also been identified in numerous end organs after T/HS and the ongoing loss of progenitor cells from the BM may play a role in posttraumatic BM suppression. We investigated the hypothesis that HPC will specifically migrate to sites of tissue trauma and that this process is exacerbated by hemorrhagic shock (HS). Sprague-Dawley rats (250-400 g) sustaining a unilateral lung contusion (LC) secondary to a blast wave of a percussive nail gun, were subjected to either HS (MAP 40-45 mm Hg for 45 minutes) or sham shock (SS). Animals were killed at 3 hours, 3 days, and 7 days after resuscitation and the right and left lungs from each animal were processed separately and the uninjured left lung served as a control for comparison with the contused right lung. BM mononuclear cells from each individual lung and the femurs were isolated and plated (2 x 10) in duplicate for granulocyte-macrophage colony-forming units (CFU-GM), erythroid colony-forming units (CFU-E), and erythroid burst-forming units (BFU-E) colony growth. At 3 hours, LC resulted in a significant increase in progenitor colonies able to be grown from the injured lung compared with from the uninjured lung (CFU-GM: 11 +/- 1 vs. 5 +/- 2, CFU-E: 12 +/- 7 vs. 5 +/- 3, BFU-E: 7 +/- 1 vs. 3 +/- 1 colonies per 10 BM mononuclear cells; all p < 0.05). HS resulted in a significant increase of the number of colonies of all three cell types in both the uninjured and the contused lung (all p < 0.05). At day 3 after HS, BM progenitor growth remained suppressed whereas the number of cells recoverable from the lung returned toward baseline. By day 7, hematopoietic progenitor cell growth in the BM and the number of those cells able to be grown from the lung returned to levels observed in unmanipulated rats. Unilateral LC results in the rapid mobilization of a significant number of HPC from the BM to the site of injury. BM function is maintained under this condition. The addition of HS increases HPC mobilization from the BM and sequestration at the site of injury as well as decreasing BM HPC growth. We postulate that the accumulation of progenitor cells in the injured tissue combined with an alteration of normal BM homing, as exemplified by the decrease in progenitor cells from the lung without restoration of BM function, plays a role in posttraumatic BM suppression. The mechanism of shock-mediated mobilization from the BM and the exact role and fate of these cells at the site of injury requires further investigation.

Dendritic cell expression of the transcription factor T-bet regulates mast cell progenitor homing to mucosal tissue

The transcription factor T-bet was identified in CD4+ T cells, and it controls interferon γ production and T helper type 1 cell differentiation. T-bet is expressed in certain other leukocytes, and we recently showed (Lord, G.M., R.M. Rao, H. Choe, B.M. Sullivan, A.H. Lichtman, F.W. Luscinskas, and L.H. Glimcher. 2005. Blood. 106:3432–3439) that it regulates T cell trafficking. We examined whether T-bet influences homing of mast cell progenitors (MCp) to peripheral tissues. Surprisingly, we found that MCp homing to the lung or small intestine in T-bet−/− mice is reduced. This is reproduced in adhesion studies using bone marrow–derived MCs (BMMCs) from T-bet−/− mice, which showed diminished adhesion to mucosal addresin cellular adhesion molecule–1 (MAdCAM-1) and vascular cell adhesion molecule–1 (VCAM-1), endothelial ligands required for MCp intestinal homing. MCp, their precursors, and BMMCs do not express T-bet, suggesting that T-bet plays an indirect role in homing. However, adoptive transfer experiments revealed that T-bet expression by BM cells is required for MCp homing to the intestine. Furthermore, transfer of WT BM-derived dendritic cells (DCs) to T-bet−/− mice restores normal MCp intestinal homing in vivo and MCp adhesion to MAdCAM-1 and VCAM-1 in vitro. Nonetheless, T-bet−/− mice respond vigorously to intestinal infection with Trichinella spiralis, eliminating a role for T-bet in MC recruitment to sites of infection and their activation and function. Therefore, remarkably, T-bet expression by DCs indirectly controls MCp homing to mucosal tissues.

SHIP deficiency enhances HSC proliferation and survival but compromises homing and repopulation

AbstractThe SH2 domain–containing inositol 5′-phosphatase-1 (SHIP) has the potential to modulate multiple signaling pathways downstream of receptors that impact hematopoietic stem cell (HSC) biology. Therefore, we postulated that SHIP might play an important role in HSC homeostasis and function. Consistent with this hypothesis, HSC proliferation and numbers are increased in SHIP–/– mice. Despite expansion of the compartment, SHIP–/– HSCs exhibit reduced capacity for long-term repopulation. Interestingly, we observe that SHIP–/– stem/progenitor cells home inefficiently to bone marrow (BM), and consistent with this finding, have reduced surface levels of both CXCR4 and vascular cell adhesion marker-1 (VCAM-1). These studies demonstrate that SHIP is critical for normal HSC function, homeostasis, and homing.

Lack of α4 integrin expression in stem cells restricts competitive function and self-renewal activity

AbstractAlpha4 integrin or VLA4 (CD49d/CD29) is a multitask molecule with wide expression within and outside the hematopoietic system. Because targeted ablation of α4 integrin leads to embryonic lethality, to study its effects on adult hematopoiesis, we used animals with conditional excision of α4 integrin (α4Δ/Δ) in hematopoietic cells. In such animals, we previously documented weakened bone marrow retention of progenitor cells during homeostasis and impaired homing and short-term engraftment after transplantation. In the present study we show that long-term repopulating cells lacking α4 integrins display a competitive disadvantage in hematopoietic reconstitution compared to normal competitors. Although initial dominance of α4+ competitors is due to their better homing and proliferative expansion early after transplantation, a progressive decline in contribution of α4Δ/Δ hematopoiesis is compatible with neither normal homing nor normal function of α4Δ/Δ hematopoietic stem cells (HSCs) in post-homing hematopoiesis. In the absence of α4+ competitor cells, α4Δ/Δ HSCs can establish long-term hematopoiesis in primary recipients, however, some resurgence of host hematopoiesis is evident, and it becomes dominant in secondary transplants, so that no survivors with exclusively α4Δ/Δ cells are seen in tertiary transplants. Collectively, our data provide compelling evidence that under regenerative stress α4 integrin assumes a greater importance than for maintenance of steady-state hematopoiesis.

Use of lipophilic near-infrared dye in whole-body optical imaging of hematopoietic cell homing

We develop an optical whole-body imaging technique for monitoring normal and leukemic hematopoietic cell homing in vivo. A recently developed near-infrared (NIR) lipophilic carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR) is used to safely and directly label the membranes of human leukemic Pre-B ALL G2 cell lines as well as primary murine lymphocytes and erythrocytes. DiR has absorption and fluorescence maxima at 750 and 782 nm, respectively, which corresponds to low light absorption and autofluorescence in living tissues. This allows us to obtain a significant signal with very low background level. A charge-coupled device (CCD)-based imager is used for noninvasive whole-body imaging of DiR-labeled cell homing in intact animals. This powerful technique can potentially visualize any cell type without use of specific antibodies conjugated with NIR fluorescent tag or loading cells with transporter-delivered NIR fluorophores. Thus, in vivo imaging based on NIR lipophilic carbocyanine dyes in combination with advanced optical techniques may serve as a powerful alternative or complementation to other small animal imaging methods.

Cytokine Exposure Changes the Hierarchy of Molecular Pathway Usage in Bone Marrow Homing.

Efficient bone marrow (BM) homing is a prerequisite for successful engraftment of transplanted hematopoietic progenitor cells (HPC). Contradictory conclusions about the dependence of homing on SDF-1/CXCR4 have clouded our understanding of its participation in homing and cooperation with other defined molecular pathways. Based on the principle that homing is the coordinated outcome of several pathways, we sought to unravel cooperative and compensatory molecular mechanisms guiding BM homing of BM-HPC. BM homing of fresh BM-HPC does not require SDF-1/CXCR4 or other Gi-protein signals, since fresh BM-HPC rendered either SDF-1 unresponsive (by AMD3100, evaluated 3h after transplantation) or Gi-signaling refractory (by Pertussis toxin (PTX), evaluated 18h after transplantation) homed similarly to untreated control BM-HPC (0.56±0.03% vs. 0.60±0.01% of injected CFU-C recovered per femur (−/+AMD3100 @3h); 0.89±0.08% vs. 0.80±0.07% (−/+PTX @18h)). This was likely due to compensation by α4-integrin interacting with VCAM-1: a contribution of SDF-1/CXCR4- or Gi-protein-mediated signals to BM homing became apparent when inhibition was exerted on α4−/− HPC, significantly augmenting the intrinsic homing defect of α4−/− HPC (0.31±0.02% vs. 0.11±0.03% (−/+AMD3100 @3h); 0.52±0.08% vs. 0.02±0.01% (−/+PTX @18h) (p<0.005)). Similar conclusions were revealed when VCAM-1 deficient hosts were used as recipients of PTX-treated BM-HPC (p<0.005 compared to VCAM-1−/− recipients of untreated control BM-HPC), but not when E/P-selectin−/− recipients were used. Thus Gi-signals do not synergistically interact with E/P-selectins, although α4 integrins do so for the homing of fresh BM-HPC (p<0.005 compared with α4−/− to WT or WT to E/P−/− transplantation). When cytokine incubated BM cells were used for transplantation (SCF, 100 ng/mL for 16–18 h) the hierarchy of molecular pathway usage in homing was shifted towards a dominance of SDF-1/CXCR4 and other Gi-signals (0.60±0.05% vs. 0.37±0.08% (−/+AMD3100 @3h, p<0.05); 0.74±0.05% vs. 0.16±0.02% (−/+PTX @18h, p<0.005)), with only partial compensation through α4/VCAM-1 and endothelial selectins. Of note, expression of α4-integrin on short-term SCF-incubated HPC was not appreciably different from untreated controls. Moreover, SCF+PTX-treated α4−/− HPC homing was virtually abrogated (p<0.05 compared to similarly treated WT HPC). E/P−/− recipients supported normal homing of SCF-incubated HPC, but homing of SCF+PTX-incubated HPC in E/P-selectin−/− recipients was further decreased (0.02±0.00%, p<0.005). Additional homing studies confirmed that these findings likewise apply to SCF-incubated c-kit+lin− cells, a population enriched for stem cells, and that this functional phenotype is not specific to SCF, since it was observed for TPO±PTX incubated BM-HPC. These studies suggest a flexible hierarchy of cooperating homing pathways, in which dominant players are repositioned with changing cytokine milieu, and possibly source of HPC.

Cell biology of conidial anastomosis tubes in Neurospora crassa.

Although hyphal fusion has been well documented in mature colonies of filamentous fungi, it has been little studied during colony establishment. Here we show that specialized hyphae, called conidial anastomosis tubes (CATs), are produced by all types of conidia and by conidial germ tubes of Neurospora crassa. The CAT is shown to be a cellular element that is morphologically and physiologically distinct from a germ tube and under separate genetic control. In contrast to germ tubes, CATs are thinner, shorter, lack branches, exhibit determinate growth, and home toward each other. Evidence for an extracellular CAT inducer derived from conidia was obtained because CAT formation was reduced at low conidial concentrations. A cr-1 mutant lacking cyclic AMP (cAMP) produced CATs, indicating that the inducer is not cAMP. Evidence that the transduction of the CAT inducer signal involves a putative transmembrane protein (HAM-2) and the MAK-2 and NRC-1 proteins of a mitogen-activated protein kinase signaling pathway was obtained because ham-2, mak-2, and nrc-1 mutants lacked CATs. Optical tweezers were used in a novel experimental assay to micromanipulate whole conidia and germlings to analyze chemoattraction between CATs during homing. Strains of the same and opposite mating type were shown to home toward each other. The cr-1 mutant also underwent normal homing, indicating that cAMP is not the chemoattractant. ham-2, mak-2, and nrc-1 macroconidia did not attract CATs of the wild type. Fusion between CATs of opposite mating types was partially inhibited, providing evidence of non-self-recognition prior to fusion. Microtubules and nuclei passed through fused CATs.

Stromal cell-derived factor-1 binding to its chemokine receptor CXCR4 on precursor cells promotes the chemotactic recruitment, development and survival of human osteoclasts

Osteoclasts (Oc) derive from hematopoietic precursors present in the circulation and bone marrow, and they differentiate into multinucleated bone-resorbing cells in response to the dual essential signals receptor activator of NF-κB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) primarily provided by bone marrow stromal cells (BMSC) and osteoblasts (Ob). However, little is known about signals that direct Oc precursors from the circulation into bone or control their migration within the marrow. Stromal cell-derived factor-1 (SDF-1 or CXCL12) is a chemokine highly expressed by bone endothelium, BMSC, and immature Ob that is essential for the normal homing, early development, and survival of various hematopoietic progenitor cells. We investigated whether SDF-1 and its unique chemokine receptor CXCR4 were involved in regulating human Oc precursor chemotaxis, development, function, or survival. CXCR4 was highly expressed by freshly isolated human monocyte (MN) populations, in vitro generated Oc and Oc-like cells, and mature Oc isolated from human femoral bones. SDF-1 markedly stimulated the chemotactic recruitment of circulating human MN capable of generating bone-resorptive Oc, leading to a 4-fold increase in Oc formation and greater bone pit resorption after their M-CSF + RANKL induced differentiation compared to spontaneously migrating cells. SDF-1 also directly promoted early (but not later) stages of Oc development via stimulating precursor cell numbers, multinucleated cell fusion, increased cell size, and tartrate-resistant acid phosphatase (TRAP) activity in a similar, but non-additive, fashion to M-CSF + RANKL. While SDF-1 did not cause full development of bone-resorbing Oc or stimulate the resorptive function of mature Oc directly, it also did not interfere with any actions promoted by M-CSF + RANKL. In mature human Oc, SDF-1 proved equally as effective as M-CSF + RANKL for preventing Oc apoptosis induced by cytokine withdrawal. In both cases, Oc survival was accompanied by analogous rises in the mRNA ratios for anti-apoptotic Bcl-x L and Bfl-1 relative to pro-apoptotic Bax, and by marked protein suppression of the critical pro-apoptotic signal Bim. These findings demonstrate for the first time that SDF-1 chemoattracts circulating human Oc precursors capable of developing into bone-resorptive Oc, and that it can stimulate MN cell fusion and TRAP activity, mimic M-CSF + RANKL in early osteoclastogenic effects, substitute for M-CSF + RANKL in maintaining the survival of mature human Oc, and suppress Oc expression of Bim protein. Thus, high levels of SDF-1 produced by bone endothelium, BMSC, and Ob may selectively target circulating Oc precursors into bone and stimulate their marrow migration into suitable perivascular stromal sites for their early development, RANKL differentiation, and survival. Consequently, SDF-1 may be a key factor linking bone vascular cells, BMSC, Ob, and Oc in the normal homeostatic regulation of bone development and remodeling.

Gi Protein Signals Are Required for BM Homing of Hemopoietic Progenitor Cells, and Cooperate with Alpha4-Intergrin and Endothelial Selectins.

Several examples suggest a relationship between in vitro migratory capacity and bone marrow (BM) homing. Pertussis toxin (PTX) is a potent inhibitor of Gi protein coupled receptor signaling and as such, it blocks hemopoietic progenitor cell (HPC) migration in vitro. However, contrary to expectation, no effects on BM homing were observed in previous studies. We therefore re-examined the effect of PTX on BM homing of HPC. We found that prolonged incubation of BM cells or fetal liver cells with PTX inhibited their BM homing in irradiated or non-irradiated recipients by >75%. A concomitant increase in circulating CFU-C in blood provided added assurance that the data were not due to non-specific toxicity. This inhibition of BM homing was of functional consequence, since PTX-treated cells provided impaired radioprotection and markedly decreased short-term engraftment. Detailed studies showed that inhibition of in vitro migration and ribosylation of Gi proteins were complete only after extended incubation with PTX, whereas short-term incubation for 1–2 h, as used in previous studies, was insufficient. In addition, the incubation of BM cells with SCF may have exaggerated the negative effect of PTX on the inhibition on BM homing. We next sought to test the basis of the residual 25% homing of PTX treated cells. As PTX did not directly inhibit hemopoietic cell adhesion, we tested homing of PTX-incubated and control BM cells in genetic models deficient in certain adhesion molecules on either the hemopoietic cells or the hemopoietic microenvironment. BM homing of PTX-treated α4integrin-/- BM cells transplanted into wild-type (WT) mice, or PTX-treated WT cells transplanted into EPselectin-/- recipients inhibited BM homing significantly stronger than either modality alone, to >90% and >95%, respectively. Of note, untreated WT cells have normal homing in EPselectin-/- mice. These data demonstrate cooperativity between Gi blockade and α4integrin or endothelial selectin blockade in inhibiting BM homing of progenitor cells. Absence of β2integrin or L-selectin in the presence or absence of PTX did not inhibit BM homing. In summary, these studies show that Gi protein signals are required for BM homing of progenitor cells. PTX is a very strong inhibitor of BM homing, suggesting that it may inhibit convergent signals from more than one mediator. We further show that the residual BM homing of migration-impaired BM cells is due to participation of α4 integrin on the transplanted cells, or to endothelial selectins on the host side.

Selectins—an emerging target for drug delivery

Selectins are multifunctional adhesion molecules that mediate the initial interactions between circulating leukocytes and cells of the endothelium. First identified over a decade ago, selectins have provided insight into areas as diverse as normal lymphocyte homing, leukocyte recruitment during inflammatory responses, carbohydrate ligand biosynthesis and adhesion-mediated signalling. Of late, selectins were introduced as targets for drug delivery in the development of new anti-inflammatory therapeutics and in anti-cancer therapy. This review will examine the selectins and their ligands with a focus on recent findings on their role in physiology and pathophysiology as well as the emerging role of selectins as targets in controlled drug delivery.

Impaired selectin-ligand biosynthesis and reduced inflammatory responses in β-1,4-galactosyltransferase-I–deficient mice

AbstractSelectins recognize ligands containing carbohydrate chains such as sialyl Lewis x (sLex) that are mainly presented at the terminus of N-acetyl lactosamine repeats on core 2 O-glycans. Several glycosyltransferases act successively to extend the N-acetyl lactosamine repeats and to synthesize sLex, and β-1,4-galactosyltransferase (β4GalT) plays a key role in these processes. Recently isolated 6 β4GalT genes are candidates, but their individual roles, including those in selectin-ligand biosynthesis, remain to be elucidated. More than 80% of the core 2 O-glycans on the leukocyte membrane glycoproteins of β4GalT-I–deficient mice lacked galactose residues in β-1,4 linkage, and soluble P-selectin binding to neutrophils and monocytes of these mice was significantly reduced, indicating an impairment of selectin-ligand biosynthesis. β4GalT-I–deficient mice exhibited blood leukocytosis but normal lymphocyte homing to peripheral lymph nodes. Acute and chronic inflammatory responses, including the contact hypersensitivity (CHS) and delayed-type hypersensitivity (DTH) responses, were suppressed, and neutrophil infiltration into inflammatory sites was largely reduced in these mice. Our results demonstrate that β4GalT-I is a major galactosyltransferase responsible for selectin-ligand biosynthesis and that inflammatory responses of β4GalT-I–deficient mice are impaired because of the defect in selectin-ligand biosynthesis.

Functional receptor for C3a anaphylatoxin is expressed by normal hematopoietic stem/progenitor cells, and C3a enhances their homing-related responses to SDF-1

AbstractComplement has recently been implicated in developmental pathways and noninflammatory processes. The expression of various complement components and receptors has been shown in a wide range of circulating myeloid and lymphoid cells, but their role in normal hematopoiesis and stem cell homing has not yet been investigated. We report that normal human CD34+ cells and lineage-differentiated hematopoietic progenitors express the complement anaphylatoxin C3a receptor (C3aR) and respond to C3a. Moreover, C3a, but not the biologically inactive desArg-C3a, induces calcium flux in these cells. Furthermore, we found that C3 is secreted by bone marrow stroma and that, although C3a does not influence directly the proliferation/survival of hematopoietic progenitors, it (1) potentiates the stromal cell–derived factor 1 (SDF-1)–dependent chemotaxis of human CD34+ cells and lineage-committed myeloid, erythroid, and megakaryocytic progenitors; (2) primes SDF-1–dependent trans-Matrigel migration; and (3) stimulates matrix metalloproteinase-9 secretion and very late antigen 4 (VLA-4)–mediated adhesion to vascular cell adhesion molecule 1 (VCAM-1). Furthermore, we found that murine Sca-1+ cells primed by C3a engrafted faster in lethally irradiated animals. These results indicate that normal human hematopoietic stem and progenitor cells express functional C3aR and that the C3aR-C3a axis sensitizes the responses of these cells to SDF-1 and thus may be involved in promoting their homing into the bone marrow via cross talk with the SDF–CXC chemokine receptor-4 (CXCR4) signaling axis. C3a is the first positive regulator of this axis to be identified.

Selectins: critical mediators of leukocyte recruitment

Selectins are multi-functional adhesion molecules that mediate the initial interactions between circulating leukocytes and the endothelium. First identified over a decade ago, selectins have provided insight into areas as diverse as normal lymphocyte homing, leukocyte recruitment during inflammatory responses, carbohydrate ligand biosynthesis and adhesion-mediated signalling. This review will examine the selectins and their ligands with a focus on recent findings using knockout technology as well as the emerging role of selectins as signalling molecules.

Retrotransposition of a yeast group II intron occurs by reverse splicing directly into ectopic DNA sites.

Group II introns, the presumed ancestors of nuclear pre-mRNA introns, are site-specific retroelements. In addition to "homing" to unoccupied sites in intronless alleles, group II introns transpose at low frequency to ectopic sites that resemble the normal homing site. Two general mechanisms have been proposed for group II intron transposition, one involving reverse splicing of the intron RNA directly into an ectopic DNA site, and the other involving reverse splicing into a site in RNA followed by reverse transcription and integration of the resulting cDNA by homologous recombination. Here, by using an "inverted-site" strategy, we show that the yeast mtDNA group II intron aI1 retrotransposes by reverse splicing directly into an ectopic DNA site. This same mechanism could account for other previously described ectopic transposition events in fungi and bacteria and may have contributed to the dispersal of group II introns into different genes.

Antigen-specific immune responsiveness and lymphocyte recruitment in leukocyte adhesion deficiency type II.

The leukocyte adhesion deficiency syndrome type II (LAD-II) is caused by a general defect in fucose metabolism, which leads to the absence of fucosylated sugar determinants such as the selectin ligand SLe(x). In view of the important role of selectins in lymphocyte migration and homing, we have explored the in vivo immune responsiveness and lymphocyte recruitment to the skin, in response to the neo-antigen keyhole limpet hemocyanin (KLH) in a LAD-II patient. We observed a normal priming of KLH-specific T cells as well as a strong in vivo anti-KLH antibody response, both indicative of a normal T-B cell function and collaboration. Skin biopsies from the patient's skin taken prior to antigenic challenge showed the presence of normal numbers and subsets of T cells. Upon KLH injection, a large number of T cells were found to be recruited to the site of challenge, which was paralleled by up-regulation of the endothelial adhesion molecules ICAM-1 (CD54), VCAM-1 (CD106) and E-selectin (CD62E). The recruited T cell showed a normal subset distribution but lacked cutaneous lymphocyte antigen (CLA), the cutaneous homing receptor. However, the clinical symptoms of delayed-type hypersensitivity in the patient (redness and swelling) were severely depressed compared to that in the controls. In conclusion, the LAD-II patient showed a normal T cell priming and T cell-dependent antibody response, a normal baseline skin homing, and a significant T cell recruitment to the site of KLH challenge, indicating that fucosylated sugar determinants such as SLe(x) and CLA are not strictly required for adequate immune responsiveness and (skin) homing.

Murine isograft studies of gut immunity: Recirculation and homing of mononuclear cells

BACKGROUND & AIMS: Recirculation of lymphocytes requires appropriate signals on the lymphocytes as well as the vascular endothelium. The aim of this report was to study the expression of the mucosal addressin required for Peyer's patch-selective lymphocyte homing during ontogeny of the murine intestine and investigate the role of this addressin in recirculation of mononuclear cells. METHODS: Immunohistochemistry and murine intestinal isografts were used in which the small intestine from neonatal mice is implanted subcutaneously in mice with severe combined immunodeficiency disease. RESULTS: Expression of the mucosal addressin cell adhesion molecule 1 during murine intestinal ontogeny was detected from embryonic day 15 in developing gut and in adult Peyer's patches and lamina propria. Isografted intestine developed normally; it was not exposed to luminal contents but expressed the mucosal addressin cell adhesion molecule 1. Mononuclear cells recirculated to native and isografted intestine and recirculation was inhibited by monoclonal antibody to mucosal addressin in a regionally specific manner with greatest inhibition in the distal intestine. CONCLUSIONS: The mucosal vascular addressin is expressed early in ontogeny and can be detected in lamina propria in addition to Peyer's patches of the gut. The isograft not only developed into a morphologically normal intestine but also expressed differentiation antigens required for normal lymphoid homing to gut without exposure to luminal contents or lymphocytes. (Gastroenterology 1997 Apr;112(4):1241-9)

Chemokines and T lymphocyte activation: II. Facilitation of human T cell trafficking in severe combined immunodeficiency mice.

Previous studies from this laboratory have demonstrated that the chemokines RANTES (recombinant human regulated upon activation, normally T cell expressed and presumably secreted), macrophage chemotactic peptide-1, recombinant human macrophage inflammatory protein-1 alpha (rhMIP-1 alpha) IL-8, and IP-10 are capable of inducing human T cell infiltration into the injection site of severe combined immunodeficiency (SCID) mice reconstituted with human PBL. However, the ability of these chemokines to facilitate T cell homing into various lymphoid tissues has not been examined. Initial studies focused on the ability of rhMIP-1 beta to induce human T cell infiltration into injection sites in human PBL-SCID mice. SCID mice received s.c. injections of rhMIP-1 beta or PBS (1 microgram/injection) in the hindflank for 4 h or sequential injections for 3 days. Biopsies of the MIP-1 beta injection site revealed the presence of significant mononuclear cell accumulation 72 h after injection. Immunohistologic evaluation determined that significant numbers of human CD3+ T cells were recruited in response to MIP-1 beta injections, and this infiltration could be specifically blocked by co-administration of anti-MIP-1 beta antiserum. We subsequently examined these chemokine-injected mice for the effect of trafficking of human T cells to peripheral lymphoid organs. Flow cytometric analysis of the thymus in human PBL-SCID mice revealed that treatment with rhMIP-1 beta or rhRANTES, but not platelet factor-4, resulted in improved thymic homing of the human T cells after 72 h. This trafficking effect was shown to be direct, as pretreatment of the human T cells with the chemokines in vitro also improved peripheral lymphoid trafficking of the human cells. In addition, co-injection of rhMIP-1 beta with anti-1 beta antiserum abrogated the increase in T cell homing to the thymus. These data demonstrate that MIP-1 beta and RANTES directly augment human T cell trafficking to peripheral murine lymphoid tissues. Chemokines may, therefore, under either isogeneic or xenogeneic conditions, play a role in normal lymphocyte recirculation and homing, and may be of potential clinical use in promoting immune cell trafficking and function.

Specificity of hematopoietic stem cell homing.

Homing of hematopoietic stem cells (HSC) may be defined as the cells' ability to seek marrow stroma selectively, to interact with it and subsequently to lodge within it to initiate hematopoiesis. This complex process is no doubt mediated through multiple recognition/adhesion events. Homing may proceed through one of several alternative mechanisms, however, such as through physical trapping of stem cells by marrow ultrastructural elements or through the providing of a selective survival and/or proliferative advantage by marrow. A third alternative that provides for the central element of stem cell homing--its high degree of specificity--is through the action of a specific homing protein in HSC. There are data to support this latter mechanism of stem cell homing as the correct one, and the nature of this protein may be similar to that of the lymphocyte homing receptors that are lectin-like molecules. Lectin-carbohydrate interactions are known to provide enormous specificity to cell recognition processes and to participate in cellular targeting. Leukemic cells have recently been demonstrated to home to marrow stroma and proliferate in the same way as normal stem cells. Thus, identification of proteins or other adhesion molecules that participate in normal and malignant cell homing could lead to more specific recruitment regimens for tumour-free collections.

Immunohistologic and functional characterization of a vascular addressin involved in lymphocyte homing into peripheral lymph nodes.

The tissue localization or "homing" of circulating lymphocytes is directed in part by specialized vessels that define sites of lymphocyte exit from the blood. In peripheral lymph nodes, mucosal lymphoid tissues (Peyer's patches and appendix), and sites of chronic inflammation, for example, lymphocytes leave the blood by adhering to and migrating between those endothelial cells lining postcapillary high endothelial venules (HEV). Functional analyses of lymphocyte interactions with HEV have shown the lymphocytes can discriminate between HEV in different tissues, indicating that HEV express tissue-specific determinants or address signals for lymphocyte recognition. We recently described such a tissue-specific "vascular addressin" that is selectively expressed by endothelial cells supporting lymphocyte extravasation into mucosal tissues and that appears to be required for mucosa-specific lymphocyte homing (Streeter, P. R., E. L. Berg, B. N. Rouse, R. F. Bargatze, and E. C. Butcher. 1988. Nature (Lond.). 331:41-46). Here we document the existence and tissue-specific distribution of a distinct HEV differentiation antigen. Defined by monoclonal antibody MECA-79, this antigen is expressed at high levels on the lumenal surface and in the cytoplasm of HEV in peripheral lymph nodes. By contrast, although MECA-79 stains many HEV in the mucosal Peyer's patches, expression in most cases is restricted to the perivascular or ablumenal aspect of these venules. In the small intestine lamina propria, a mucosa-associated site that supports the extravasation of lymphocytes, venules do not stain with MECA-79. Finally, we demonstrate that MECA-79 blocks binding of both normal lymphocytes and a peripheral lymph node-specific lymphoma to peripheral lymph node HEV in vitro and that it also inhibits normal lymphocyte homing to peripheral lymph nodes in vivo without significantly influencing lymphocyte interactions with Peyer's patch HEV in vitro or in vivo. Thus, MECA-79 defines a novel vascular addressin involved in directing lymphocyte homing to peripheral lymph nodes.

High endothelial venule binding as a predictor of the dissemination of passaged murine lymphomas.

It has long been postulated that normal lymphocyte homing mechanisms help determine the metastatic spread of lymphoid neoplasms. The traffic of normal lymphocytes is controlled in part by the regulated expression of surface receptors for high endothelial venules (HEV), specialized venules that mediate the extravasation of circulating lymphocytes from the blood into lymphoid organs and sites of chronic inflammation. Here we have compared the in vivo growth patterns of HEV-binding vs. nonbinding murine lymphomas passaged intramuscularly into syngeneic recipients. We report that lymphomas that bind well to HEV (as assessed in a quantitative in vitro assay) disseminate widely via the blood, involving all lymph node groups symmetrically. Although both HEV-binding and nonbinding lymphomas gain access to the blood, gross involvement of lymph nodes by nonbinding lymphomas is limited to nodes draining local tumor at the site of injection, a prominent feature of these lymphomas; distant lymph nodes are not enlarged. The results suggest that the expression of functional receptors for HEV either controls the hematogenous dissemination of malignant lymphocyte populations to HEV-bearing organs, or is coregulated with factors determining this metastatic behavior. The findings support the concept that normal lymphocyte homing mechanisms are important to the spread of leukemias and lymphomas.