Expression of mucosal homing receptor alpha4beta7 by circulating CD4+ cells with memory for intestinal rotavirus.

The integrin alpha4beta7 mediates lymphocyte binding to mucosal addressin cell adhesion molecule-1, and its expression defines lymphocytes capable of trafficking through the intestines and the intestinal lymphoid tissues. We examined the ability of discrete alpha4beta7(hi) and alpha4beta7- subsets of circulating memory phenotype (CD45RA-) CD4+ T cells to proliferate in response to rotavirus, a ubiquitous intestinal pathogen. alpha4beta7(hi) memory (CD45RA-) CD4+ T cells displayed much greater reactivity to rotavirus than alpha4beta7- memory or naive (CD45RA+) CD4+ T cells. In contrast, alpha4beta7- memory cells were the predominant population responsive to mumps antigen after intramuscular vaccination. Our results are consistent with the conclusion that natural rotavirus infection, an enteric pathogen, results in a specific circulating memory CD4+ response that is largely limited to the gut-homing alpha4beta7+ subpopulation. This phenotype is not shared with memory cells elicited by intramuscular immunization (shown here) or by skin contact allergens. The results support the hypothesis that gut trafficking memory CD4+ T cells comprise cellular memory for intestinal antigens and suggest that regulated expression of alpha4beta7 helps target and segregate intestinal versus systemic immune response.     

Modulation of L-selectin ligand expression during an immune response accompanying tumorigenesis in transgenic mice.

Immune surveillance depends on lymphocyte access to tissue. Lymphocytes emigrate from blood when adhesion receptors such as L-selectin and the alpha 4 beta 7 integrin on these cells bind to ligands expressed on venular endothelium. Among transgenic mouse lines expressing an oncoprotein (Tag) in islet beta cells, some recognize Tag as nonself. In these mice, Tag expression elicits both beta cell hyperplasia with subsequent progression to tumors and lymphocytic infiltration. Endothelial ligands for L-selectin and alpha 4 beta 7 were upregulated in infiltrated islets in these transgenic mice. These ligands were not expressed in tumors, which were devoid of lymphocytic infiltration. In contrast, the adhesion molecules PECAM-1, ICAM-1, and VCAM-1 were expressed on endothelium in both noninfiltrated tumors and infiltrated islets. Thus, upregulation of expression of endothelial ligands for L-selectin and alpha 4 beta 7 may contribute to autoimmune infiltration. Repression of expression of these same ligands may be involved in the failure of tumor immunity.     

Vascular addressins are induced on islet vessels during insulitis in nonobese diabetic mice and are involved in lymphoid cell binding to islet endothelium.

In the nonobese diabetic (NOD) mouse, lymphocytic and monocytic infiltration of the pancreatic islets leads to beta cell destruction. To investigate the mechanisms by which lymphocytes enter the NOD pancreas, pancreata were immunostained using monoclonal antibodies to a variety of adhesion molecules known to be involved in lymphocyte binding to vascular endothelium, an initial step in the migration of lymphocytes from blood into organized lymphoid and inflamed tissues. These adhesion molecules include: lymphocyte homing receptors involved in tissue-selective binding of lymphocytes to peripheral lymph node (L-selectin) or mucosal lymphoid tissue (LPAM-1, alpha 4 beta 7-integrin) high-endothelial venules (HEV); and HEV ligands peripheral vascular addressin (PNAd) and mucosal vascular addressin (MAdCAM-1). In NOD pancreata, alpha 4 beta 7 is expressed on most infiltrating cells at all stages of insulitis, whereas L-selectin expression is more pronounced on cells in the islets at later stages. During the development of insulitis, MAdCAM-1 and to a lesser extent PNAd became detectable on vascular endothelium adjacent to and within the inflamed islets. The Stamper-Woodruff in vitro assay was used to examine lymphoid cell binding to such vessels. These functional assays show that both the mucosal (MAdCAM-1/alpha 4 beta 7) and the peripheral (PNAd/L-selectin) recognition systems are involved in this binding. Our findings demonstrate that expression of peripheral and mucosal vascular addressins is induced on endothelium in inflamed islets in NOD pancreas, and that these addressins participate in binding lymphoid cells via their homing receptors. This suggests that these adhesion molecules play a role in the pathogenesis of diabetes in these mice by being involved in the migration of lymphocytes from blood into the inflamed pancreas.     

Lymphocyte homing to bronchus-associated lymphoid tissue (BALT) is mediated by L-selectin/PNAd,alpha4beta1 integrin/VCAM-1, and LFA-1 adhesion pathways

Bronchus-associated lymphoid tissue (BALT) participates in airway immune responses. However, little is known about the lymphocyte-endothelial adhesion cascades that recruit lymphocytes from blood into BALT. We show that high endothelial venules (HEVs) in BALT express substantial levels of VCAM-1, in marked contrast to HEVs in other secondary lymphoid tissues. BALT HEVs also express the L-selectin ligand PNAd. Anti-L-selectin, anti-PNAd, and anti-LFA-1 mAbs almost completely block the homing of B and T lymphocytes into BALT, whereas anti-alpha4 integrin and anti-VCAM-1 mAbs inhibit homing by nearly 40%. alpha4beta7 integrin and MAdCAM-1 are not involved. Importantly, we found that mAbs against alpha4 integrin and VCAM-1 significantly block the migration of total T cells (80% memory phenotype) but not naive T and B cells to BALT. These results suggest that an adhesion cascade, which includes L-selectin/PNAd, alpha4beta1 integrin/VCAM-1, and LFA-1, targets specific lymphocyte subsets to BALT. This high level of involvement of alpha4beta1 integrin/VCAM-1 is unique among secondary lymphoid tissues, and may help unify lymphocyte migration pathways and immune responses in BALT and other bronchopulmonary tissues.     

Aberrant activation of integrin alpha4beta7 suppresses lymphocyte migration to the gut

Integrin adhesion molecules mediate lymphocyte migration and homing to normal and inflamed tissues. While the ligand-binding activity of integrins is known to be modulated by conformational changes, little is known about how the appropriate balance of integrin adhesiveness is maintained in order to optimize the migratory capacity of lymphocytes in vivo. In this study we examined the regulation of the gut homing receptor alpha4beta7 integrin by manipulating at the germline level an integrin regulatory domain known as adjacent to metal ion-dependent adhesion site (ADMIDAS). ADMIDAS normally serves to raise the activation threshold of alpha4beta7, thereby stabilizing it in the default nonadhesive state. Lymphocytes from knockin beta7 (D146A) mice, which harbor a disrupted ADMIDAS, not only expressed an alpha4beta7 integrin that persistently adhered to mucosal addressin cell adhesion molecule-1 (MAdCAM-1), but also exhibited perturbed cell migration along MAdCAM-1 substrates resulting from improper de-adhesion of the lymphocyte trailing edge. In vivo, aberrantly activated alpha4beta7 enhanced adhesion to Peyer's patch venules, but suppressed lymphocyte homing to the gut, diminishing the capacity of T cells to induce colitis. Our results underscore the importance of a proper balance in the adhesion and de-adhesion of the alpha4beta7 integrin, both for lymphocyte trafficking to the gut and for colitis progression.     

Dual binding capacity of mucosal immunoblasts to mucosal and synovial endothelium in humans: dissection of the molecular mechanisms

Lymphocytes continuously migrate throughout the body in search of antigens. Virgin lymphocytes recirculate freely between the blood and different lymphatic organs, whereas immunoblasts extravasate preferentially into sites similar to those where they initially responded to antigen. Tissue-specific extravasation of lymphocytes is largely controlled by distinct lymphocyte surface receptors that mediate lymphocyte binding to high endothelial venules (HEV). In the present study, the molecular mechanisms determining the specificity of human mucosal (lamina propria) lymphocyte binding to different endothelial recognition systems were analyzed. Mucosal immunoblasts adhered five times better than small mucosal lymphocytes to mucosal HEV. Importantly, mucosal immunoblasts also bound to synovial HEV almost as efficiently as to mucosal HEV, but they did not adhere to peripheral lymph node HEV. To study the impact of different homing- associated molecules in this dual endothelial binding, we used a gut- derived T cell line and freshly isolated mucosal immunoblasts. Both cell types expressed integrins alpha 4, beta 1, beta 7, and lymphocyte function associated antigen 1 (LFA-1), and were CD44 positive, but practically L-selectin negative. Binding of mucosal immunoblasts to mucosal HEV was almost completely abolished by pretreatment with anti- beta 7 monoclonal antibodies, but it was independent of alpha 4/beta 1 function. In contrast, alpha 4/beta 1 partially mediated immunoblast adherence to synovial HEV, whereas alpha 4/beta 7 had only a minor role in adherence of blasts at this site. CD44 and LFA-1 contributed to HEV- binding both in mucosa and synovium. Taken together, this is the first report that demonstrates a critical role for alpha 4/beta 7 in the binding of gut lymphocytes to mucosal venules in humans. Moreover, a hitherto unknown interaction between mucosal effector cells and synovial endothelial cells was shown to be only partially mediated by the currently known homing receptors. The dual endothelial binding capacity of mucosal blasts may help to explain the pathogenesis of reactive arthritis not uncommonly associated with inflammatory and infectious bowel disease.     

B lymphocyte binding to E- and P-selectins is mediated through the de novo expression of carbohydrates on in vitro and in vivo activated human B cells.

Cell adhesion to endothelium regulates the trafficking and recruitment of leukocytes towards lymphoid organs and sites of inflammation. This phenomenon is mediated by the expression of a number of adhesion molecules on both the endothelium and circulating cells. Activation of endothelial cells (EC) with different stimuli induces the expression of several adhesion molecules (E- and P-selectins, ICAM-1, VCAM-1), involved in their interaction with circulating cells. In this report, we have studied the binding of nonactivated and activated B cells to purified E- and P-selectins. Activated but not resting B cells were able to interact with both selectins. This binding capacity of activated B cells paralleled the induction of different carbohydrate epitopes (Lewisx, sialyl-Lewisx, CD57 and CDw65) as well as other molecules bearing these or related epitopes in myeloid cells (L-selectin, alpha L beta 2 and alpha X beta 2 integrins, and CD35) involved in the interaction of different cell types with selectins. B cells infiltrating inflamed tissues like in Hashimoto's thyroiditis, also expressed these selectin-binding carbohydrates in parallel with the expression of E-selectin by surrounding follicular dendritic cells. Moreover, the crosslinking of these selectin-binding epitopes resulted in an increased binding of B cells to different integrin ligands. Thus, in addition to the involvement of integrins, E- and P-selectins could play an important role in the interaction of B lymphocytes with the endothelium during B cell extravasation into lymphoid tissues and inflammatory foci as well as in their organization into lymphoid organs.     

Tissue infiltration in a CD8 lymphocytosis syndrome associated with human immunodeficiency virus-1 infection has the phenotypic appearance of an antigenically driven response.

HIV-1 infection may initiate to an HLA-associated response designated diffuse infiltrative lymphocytosis syndrome, characterized by increased numbers of circulating CD8 T cells that infiltrate salivary glands, lungs, gastrointestinal tract, and kidneys. Since this response could either be an antigenically driven process induced by HIV-1 or a lymphoproliferation of cells with neoplastic or unusual features, we sought to define the phenotype of the cellular populations, the nature of tissue derangement, and the tissue localization of virus in diffuse infiltrative lymphocytosis syndrome. Circulating CD8 T cells were greatly increased while CD4 T cell numbers remained in the range found in asymptomatic seropositive persons. The majority of CD8 and CD4 T cells in both blood and tissues had the memory phenotype of CD29+ (beta 1 integrin) and CD11a+/CD18 (beta 2 integrin) expression, but lacked markers of recent activation. A proportion of the circulating CD8 T cells also expressed CD57 (Leu 7) but not other markers of natural killer cells. HIV-encoded proteins were identified in tissue macrophages located in periacinar areas of the salivary glands. CD54 (intercellular adhesion molecule-1), a ligand for the CD11a integrin, was strongly expressed on postcapillary venule endothelium within lymphoid foci, and HLA-DR molecules were found on limited regions of ductular epithelium adjacent to lymphoid aggregates. These findings suggest that (a) the visceral lymphocytic infiltration in diffuse infiltrative lymphocytosis syndrome is an antigen-driven, and MHC-determined, host immune response to an element associated with HIV-1 infection, and (b) that the specific adhesive molecule interactions mediating the cellular influx, as well as the subsequent tissue damage, reflect altered patterns of gene expression in tissues undergoing an immune response.     

Rapid acquisition of tissue-specific homing phenotypes by CD4(+) T cells activated in cutaneous or mucosal lymphoid tissues.

Effector and memory T cells can be subdivided based on their ability to traffic through peripheral tissues such as inflamed skin and intestinal lamina propria, a property controlled by expression of 'tissue-specific' adhesion and chemoattractant receptors. However, little is known about the development of these selectively homing T cell subsets, and it is unclear whether activation in cutaneous versus intestinal lymphoid organs directly results in effector/memory T cells that differentially express adhesion and chemoattractant receptors targeting them to the corresponding nonlymphoid site. We define two murine CD4(+) effector/memory T cell subsets that preferentially localize in cutaneous or intestinal lymphoid organs by their reciprocal expression of the adhesion molecules P-selectin ligand (P-lig) and alpha 4 beta 7, respectively. We show that within 2 d of systemic immunization CD4(+) T cells activated in cutaneous lymph nodes upregulate P-lig, and downregulate alpha 4 beta 7, while those responding to antigen in intestinal lymph nodes selectively express high levels of alpha 4 beta 7 and acquire responsiveness to the intestinal chemokine thymus-expressed chemokine (TECK). Thus, during an immune response, local microenvironments within cutaneous and intestinal secondary lymphoid organs differentially direct T cell expression of these adhesion and chemoattractant receptors, targeting the resulting effector T cells to the inflamed skin or intestinal lamina propria.     

A homing mechanism for bone marrow-derived progenitor cell recruitment to the neovasculature

CD34+ bone marrow-derived progenitor cells contribute to tissue repair by differentiating into endothelial cells, vascular smooth muscle cells, hematopoietic cells, and possibly other cell types. However, the mechanisms by which circulating progenitor cells home to remodeling tissues remain unclear. Here we show that integrin alpha4beta1 (VLA-4) promotes the homing of circulating progenitor cells to the alpha4beta1 ligands VCAM and cellular fibronectin, which are expressed on actively remodeling neovasculature. Progenitor cells, which express integrin alpha4beta1, homed to sites of active tumor neovascularization but not to normal nonimmune tissues. Antagonists of integrin alpha4beta1, but not other integrins, blocked the adhesion of these cells to endothelia in vitro and in vivo as well as their homing to neovasculature and outgrowth into differentiated cell types. These studies describe an adhesion event that facilitates the homing of progenitor cells to the neovasculature.     

Developmental stage, phenotype, and migration distinguish naive- and effector/memory-like CD4+ regulatory T cells

Regulatory T cells (Tregs) fulfill a central role in immune regulation. We reported previously that the integrin alphaEbeta7 discriminates distinct subsets of murine CD4+ regulatory T cells. Use of this marker has now helped to unravel a fundamental dichotomy among regulatory T cells. alphaE-CD25+ cells expressed L-selectin and CCR7, enabling recirculation through lymphoid tissues. In contrast, alphaE -positive subsets (CD25+ and CD25-) displayed an effector/memory phenotype expressing high levels of E/P-selectin-binding ligands, multiple adhesion molecules as well as receptors for inflammatory chemokines, allowing efficient migration into inflamed sites. Accordingly, alphaE -expressing cells were found to be the most potent suppressors of inflammatory processes in disease models such as antigen-induced arthritis.     

The alpha 4-integrin supports leukocyte rolling and adhesion in chronically inflamed postcapillary venules in vivo

A role for the alpha 4-integrin (alpha 4 beta 1 or alpha 4 beta 7), has been implicated in the recruitment of peripheral blood mononuclear cells (PBMCs) to sites of inflammation. However, the adhesive interactions (i.e., tethering, rolling, and adhesion) mediated by the alpha 4-integrin have not been characterized in vivo. The objective of this study was to establish a model wherein postcapillary venules were chronically inflamed, and then use intravital microscopy to identify the adhesive interactions mediated by the alpha 4-integrin in vivo. Between 4 and 20 d after immunization with Mycobacterium butyricum, animals developed a systemic vasculitis characterized by large increases in the numbers of rolling and adhering leukocytes within mesenteric venules. The selectins could only account for approximately 50% of the leukocyte rolling whereas the remaining cells rolled exclusively via the alpha 4-integrin. Anti-alpha 4 therapy also eliminated the increase in leukocyte adhesion observed in this model, whereas selectin therapies and an anti-CD18 (beta 2-integrin) monoclonal antibody (mAb) did not reduce adhesion. A serum against polymorphonuclear leukocytes (PMNs) was used to confirm that a significant proportion of rolling cells, and most of the adhering cells were PBMCs. Sequential treatment with anti-PMN serum and the anti-alpha 4 mAb demonstrated that alpha 4-dependent rolling was distinct from PMN rolling populations. Initial leukocyte tethering via the alpha 4- integrin could not be demonstrated in this model, whereas L-selectin did support leukocyte tethering. These data suggest that the alpha 4- integrin can mediate both rolling and adhesion in the multistep recruitment of PMBCs in vivo, and these interactions occur independently of the selectins and beta 2-integrins.     

The alpha 4 integrin chain is a ligand for alpha 4 beta 7 and alpha 4 beta 1

The heterodimeric alpha 4 integrins alpha 4 beta 7 lymphocyte Peyer's patch adhesion molecule ([LPAM]-1) and alpha 4 beta 1 (very late antigen-4) are cell surface adhesion molecules involved in lymphocyte trafficking and lymphocyte-cell and matrix interactions. Known cellular ligands include vascular cell adhesion molecule (VCAM)-1, which binds to alpha 4 beta 1 and alpha 4 beta 7, and the mucosal addressin cell adhesion molecule (MAdCAM)-1, which binds to alpha 4 beta 7. Here we show that the alpha 4 chain of these integrins can itself serve as a ligand. The alpha 4 chain, immunoaffinity purified and immobilized on glass slides, binds thymocytes and T lymphocytes. Binding exhibits divalent cation requirements and temperature sensitivity which are characteristic of integrin-mediated interactions, and is specifically inhibited by anti-alpha 4 integrin antibodies, which exert their effect at the cell surface. Cells expressing exclusively alpha 4 beta 7 (TK-1) or alpha 4 beta 1 (L1-2) both bound avidly, whereas alpha 4-negative cells did not. A soluble 34-kD alpha 4 chain fragment retained binding activity, and it inhibited lymphocyte adhesion to alpha 4 ligands. It has been shown that alpha 4 integrin binding to fibronectin involves an leucine-aspartic acid-valine (LDV) motif in the HepII/IIICS region of fibronectin (CS-1 peptide), and homologous sequences are important in binding to VCAM-1 and MAdCAM-1. Three conserved LDV motifs occur in the extracellular sequence of alpha 4. A synthetic LDV-containing alpha 4- derived oligopeptide supports alpha 4-integrin-dependent lymphocyte adhesion and blocks binding to the 34-kD alpha 4 chain fragment. Our results suggest that alpha 4 beta 7 and alpha 4 beta 1 integrins may be able to bind to the alpha 4 subunit on adjacent cells, providing a novel mechanism for alpha 4 integrin-mediated and activation-regulated lymphocyte interactions during immune responses.     

Expression of integrins and basement membrane components by wound keratinocytes.

Extracellular matrix proteins and their cellular receptors, integrins, play a fundamental role in keratinocyte adhesion and migration. During wound healing, keratinocytes detach, migrate until the two epithelial sheets confront, and then regenerate the basement membrane. We examined the expression of different integrins and their putative ligands in keratinocytes during human mucosal wound healing. Migrating keratinocytes continuously expressed kalinin but not the other typical components of the basement membrane zone: type IV collagen, laminin, and type VII collagen. When the epithelial sheets confronted each other, these missing basement membrane components started to appear gradually through the entire wound area. The expression of integrin beta 1 subunit was increased in keratinocytes during migration. The beta 1-associated alpha 2 and alpha 3 subunits were expressed constantly by wound keratinocytes whereas the alpha 5 subunit was present only in keratinocytes during reepithelialization. Furthermore, migrating cells started to express alpha v-integrins which were not present in the nonaffected epithelium. All keratinocytes also expressed the alpha 6 beta 4 integrin during migration. In the migrating cells, the distribution of integrins was altered. In normal mucosa, beta 1-integrins were located mainly on the lateral plasma membrane and alpha 6 beta 4 at the basal surface of basal keratinocytes in the nonaffected tissue. In wounds, integrins were found in filopodia of migrating keratinocytes, and also surrounding cells in several cell layers of the migrating sheet. The results indicate that migrating keratinocytes, in deep human wounds enlarge their integrin repertoire. The changes in integrin expression take place concomitantly with changes in the basement membrane composition, suggesting a close interplay of these two groups of molecules during wound healing.     

The alpha4beta1 and alpha5beta1 integrins mediate engraftment of granulocyte-colony-stimulating factor-mobilized human hematopoietic progenitor cells

BACKGROUND: The alpha4beta1 and alpha5beta1 integrins are major adhesion molecules of murine and human hematopoietic progenitor cells. Granulocyte-colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells (PBPCs) are the most important source for clinical hematopoietic cell transplantation today. The contribution of alpha4beta1 and alpha5beta1 integrins to homing of PBPCs has not been studied yet. STUDY DESIGN AND METHODS: The expression of alpha4beta1 and alpha5beta1 integrins on purified human PBPCs was analyzed. Integrin function in adhesion to recombinant fibronectin and migration on fibronectin-coated transwells was assessed with fragments combining different adhesion domains and function-blocking antibodies. Finally, the function of those integrins in a transplantation model was investigated with repopulating cells of nonobese diabetic/severe combined immune-deficient (NOD/SCID) mice. RESULTS: More than 90 percent of all purified peripheral blood CD34+ cells express alpha4beta1 integrins, whereas only 10 to 15 percent express alpha5beta1. The alpha4beta1 integrin alone influences adhesion whereas alpha4beta1 and alpha5beta1 both mediate chemotaxis of clonogenic CD34+ progenitor cells on recombinant fibronectin. Importantly, antibodies against the integrins alpha4beta1 and alpha5beta1 independently reduce the repopulation of NOD/SCID mouse marrow after transplantation of human peripheral blood CD34+ cells. CONCLUSIONS: Alpha4beta1 and alpha5beta1 integrins are functional and critical adhesion receptors expressed on G-CSF-mobilized CD34+ hematopoietic blood progenitor cells with repopulating capacity mediating engraftment after transplantation.     

Requirements for leukocyte adhesion molecules in nephrotoxic nephritis.

Requirements for leukocyte adhesion molecules as well as cytokines have been determined in the rat model of acute nephrotoxic nephritis. Proteinuria (at 24 h) and neutrophil accumulation in renal glomeruli (at 6 h) have been used as the endpoints. For full accumulation in glomeruli of neutrophils as well as full development of proteinuria, requirements have been demonstrated for TNF alpha, (but not IL-1), CD11b (but not CD11a), very late arising-4 (CD49d/CD29), and intercellular adhesion molecule-1 but not endothelial leukocyte adhesion molecule-1 (E-selectin). By immunohistochemical approaches, infusion of antibody to glomerular basement membrane induced glomerular upregulation of intercellular adhesion molecule-1, endothelial leukocyte adhesion molecule-1, and vascular adhesion molecule-1. Treatment of rats with anti-TNF alpha or soluble recombinant human TNF receptor-1 blocked this expression. Renal arterial infusion of TNF alpha induced glomerular expression of all three endothelial adhesion molecules, but infusion of IL-1 beta did not. These data suggest that, in neutrophil and complement-dependent anti-glomerular basement membrane-induced acute nephritis in rats, there are selective requirements for cytokines, beta 1 and beta 2 integrins, and endothelial adhesion molecules. These requirements contrast with those found in other vascular beds in which complement and neutrophil-induced vascular injury has been induced by deposition of immune complexes.     

Engaging CD19 or target of an antiproliferative antibody 1 on human B lymphocytes induces binding of B cells to the interfollicular stroma of human tonsils via integrin alpha 4/beta 1 and fibronectin

Adhesion of B lymphocytes within the different compartments of secondary lymphoid organs is essential for the function of the humoral immune response. It is not currently known how the temporary immobilization of B cells in distinct areas of this complex microenvironment is regulated. The present study aimed at defining B cell antigens that initiate binding of B cells to human tonsil sections in situ. Engaging the B cell antigens CD19 and target of an antiproliferative antibody 1 (TAPA-1) with monoclonal antibodies induced adhesion of these B cells to the interfollicular stroma. This binding occurred through the integrin alpha 4 beta 1 on the B cell surface and via the extracellular matrix protein fibronectin expressed in the interfollicular compartment of the tonsil. Signaling through either antigen, CD19 or TAPA-1, depended on tyrosine kinases. Binding induced by engaging CD19 required an intact cytoskeleton, whereas TAPA- 1-transmitted adhesion did not. We suggest that CD19 and TAPA-1 have a novel and unique function by regulating an alpha 4 beta 1/fibronectin- mediated binding of B cells to the interfollicular stroma of lymphoid tissues.     

Alpha(4)beta(7)/alpha(4)beta(1) dual integrin antagonists block alpha(4)beta(7)-dependent adhesion under shear flow

The alpha(4) integrin, alpha(4)beta(7), plays an important role in recruiting circulating lymphocytes to the gastrointestinal tract, where its ligand mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is preferentially expressed on high endothelial venules (HEVs). Dual antagonists of alpha(4)beta(1) and alpha(4)beta(7), N-(2,6-dichlorobenzoyl)-(L)-4-(2',6'-bis-methoxyphenyl)phenylalanine (TR14035) and N-(N-[(3,5-dichlorobenzene)sulfonyl]-2-(R)-methylpropyl)-(D)-phenylalanine (compound 1), were tested for their ability to block the binding of alpha(4)beta(7)-expressing cells to soluble ligand in suspension and under in vitro and in vivo shear flow. Compound 1 and TR14035 blocked the binding of human alpha(4)beta(7) to an (125)I-MAdCAM-Ig fusion protein with IC(50) values of 2.93 and 0.75 nM, respectively. Both compounds inhibited binding of soluble ligands to alpha(4)beta(1) or alpha(4)beta(7) on cells of human or rodent origin with similar potency. Under shear flow in vitro, TR14035 and compound 1 blocked binding of human alpha(4)beta(7)-expressing RPMI-8866 cells or murine mesenteric lymph node lymphocytes to MAdCAM-Ig with IC(50) values of 0.1 and 1 microM, respectively. Intravital microscopy was used to quantitate alpha(4)-dependent adhesion of fluorescent murine lymphocytes in Peyer's patch HEVs. When cells were prestimulated with 2 mM Mn(2+) to activate alpha(4)beta(7) binding to ligand, anti-alpha(4) monoclonal antibody (mAb) [10 mg/kg (mpk) i.v.] blocked adhesion by 95%, and anti-beta(1) mAb did not block adhesion, demonstrating that this interaction was dependent on alpha(4)beta(7). TR14035 blocked adhesion to HEVs [ED(50) of 0.01-0.1 mpk i.v.], and compound 1 blocked adhesion by 47% at 10 mpk i.v. Thus, alpha(4)beta(7)/alpha(4)beta(1) antagonists blocked alpha(4)beta(7)-dependent adhesion of lymphocytes to HEVs under both in vitro and in vivo shear flow.     

Neutrophil adhesion molecule expression in familial Mediterranean fever: discordance between the intravascular regulation of beta2 integrin and L-selectin expression in acute attack.

BACKGROUND: To determine the surface expression of neutrophil beta2 integrin (CD11b/CD18) and L-selectin (LS) adhesion molecules in patients with familial Mediterranean fever (FMF) and to investigate the in vitro regulation of their expression in response to chemoattractant stimuli. METHODS: Neutrophil surface expression of CD11b and LS molecules was analyzed by flow cytometry in anticoagulated whole blood drawn from FMF patients and normal controls, and the in vitro regulation of these molecules induced by the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (FMLP) was assayed. RESULTS: Patients during acute FMF attacks showed a statistically significant increased neutrophil surface CD11b compared with normal controls (mean fluorescence intensity: 22.8 +/- 13.7 vs 12.8 +/- 10.41, respectively; p = .03). There was no difference in LS expression between the groups. Neutrophils of FMF patients regulate CD11b and LS expression induced by chemoattractant (FMLP) stimulation to a degree similar to that in controls. CONCLUSIONS: beta2 Integrin is up-regulated during an acute attack of FMF in dissociation with LS expression, suggesting a unique nonchemoattractant-mediated neutrophil activation.     

VLA-4 integrin can mediate CD11/CD18-independent transendothelial migration of human monocytes.

The migration of human monocytes across unactivated and activated human umbilical vein endothelium (HUVE) in response to chemotactic factors was studied, and the adhesion molecules involved were characterized. Migration of blood monocytes or U937 cell line-derived monocytes across unactivated HUVE induced by C5a, was partially inhibited (by 75%) by mAbs (R15.7 or 60.3) to CD18 of the CD11/CD18 complex on the monocyte. However, when the HUVE was pretreated for 5 h with IL-1 alpha (0.1 ng/ml), TNF-alpha (100 U/ml), or LPS (1 ng/ml), migration induced by C5a was no longer inhibited; i.e., migration became CD18 independent. The monocyte CD18-independent migration was completely blocked by mAbs against alpha 4 or beta 1 integrin chains of VLA-4. This migration was also partially inhibited by mAbs against vascular cell adhesion molecule-1 (VCAM-1), a major counter-receptor on HUVE for VLA-4, but not by mAbs to E-selectin or intercellular adhesion molecule-1. The significant CD18-independent migration across "unactivated" HUVE was also inhibited by mAbs against alpha 4 or beta 1 chains of VLA-4, although mAbs against VCAM-1 did not inhibit under these conditions. Finally, considerable VLA-4-dependent transendothelial migration to C5a was also observed with monocytes from a patient with CD18 deficiency (leukocyte adhesion deficiency). These results suggest that (a) there is a major CD18-independent component in monocyte chemotactic factor-dependent migration across activated and unactivated endothelium; (b) that VLA-4 integrin on the monocyte has a major role in this migration; and (c) that VCAM-1 on activated endothelium functions as a counter-receptor in this process, but other ligands for VLA-4, especially on unactivated endothelium, may also be involved.