Functional disruption of alpha4 integrin mobilizes bone marrow-derived endothelial progenitors and augments ischemic neovascularization

The cell surface receptor alpha4 integrin plays a critical role in the homing, engraftment, and maintenance of hematopoietic progenitor cells (HPCs) in the bone marrow (BM). Down-regulation or functional blockade of alpha4 integrin or its ligand vascular cell adhesion molecule-1 mobilizes long-term HPCs. We investigated the role of alpha4 integrin in the mobilization and homing of BM endothelial progenitor cells (EPCs). EPCs with endothelial colony-forming activity in the BM are exclusively alpha4 integrin-expressing cells. In vivo, a single dose of anti-alpha4 integrin antibody resulted in increased circulating EPC counts for 3 d. In hindlimb ischemia and myocardial infarction, systemically administered anti-alpha4 integrin antibody increased recruitment and incorporation of BM EPCs in newly formed vasculature and improved functional blood flow recovery and tissue preservation. Interestingly, BM EPCs that had been preblocked with anti-alpha4 integrin ex vivo or collected from alpha4 integrin-deficient mice incorporated as well as control cells into the neovasculature in ischemic sites, suggesting that alpha4 integrin may be dispensable or play a redundant role in EPC homing to ischemic tissue. These data indicate that functional disruption of alpha4 integrin may represent a potential angiogenic therapy for ischemic disease by increasing the available circulating supply of EPCs.     

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.     

Hematopoietic Progenitor Cell Rolling in Bone Marrow Microvessels: Parallel Contributions by Endothelial Selectins and Vascular Cell Adhesion Molecule 1

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

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.     

Development of CD8alpha/alpha and CD8alpha/beta T cells in major histocompatibility complex class I-deficient mice.

Peripheral CD8(+) T cells mainly use CD8alpha/beta, and their development is mainly dependent on the major histocompatibility complex (MHC) class I proteins K(b) and D(b) in H-2(b) mice. In this report, we have shown that the development of CD8alpha/beta TCR-alpha/beta cells in lymphoid organs as well as in intestinal intraepithelial lymphocytes (iIELs) is dependent on the MHC class I K(b) and D(b) proteins. In contrast, TCR-alpha/beta CD8alpha/alpha cells are found mainly in iIELs, and their numbers are unaffected in K(b)D(b) double knockout mice. Most of the TCR-gamma/delta cells in the iIELs also bear CD8alpha/alpha, and they are also unaffected in K(b)D(b) -/- mice. In beta2-microglobulin (beta2m)-deficient mice, all of the TCR-alpha/beta CD8alpha/alpha and CD8alpha/beta T cells disappear, but TCR-gamma/delta cells are unaffected by the absence of beta2m.     

胰高血糖素样肽-2对急性胰腺炎小鼠肠道淋巴细胞归巢影响的实验研究

目的 探讨胰高血糖素样肽-2(GLP-2)对急性胰腺炎(AP)小鼠肠道淋巴细胞归巢受体整合素a4β7和归巢配体肠道黏膜地址素细胞黏附分子-1(MAdCAM-1)表达的影响.方法 将96只小鼠随机分为AP组、GLP-2组、对照组,每组32只.运用雨蛙素联合脂多糖腹腔注射制备小鼠AP模型.GLP-2组于制模后15 min开始腹腔注射250μg/kg GLP-2,每12 h 1次,连用3 d;对照组给予等量生理盐水.分别于制模后6、12、24、48 h处死小鼠后取标本,流式细胞仪检测外周血中整合素a4β7的阳性淋巴细胞数,免疫组化法测定末端回肠和Peyer结中MAdCAM-1的表达.结果 AP组小鼠制模后各时间点外周血中整合素a4β7阳性淋巴细胞数及末端回肠和Peyer结中MAdCAM-1表达均较对照组明显降低(P均＜0.05);应用GLP-2后整合素a4β7阳性淋巴细胞数及MAdCAM-1表达均较AP组明显升高(P均＜0.05),但仍低于对照组(P均>0.05).结论 GLP-2能改善AP小鼠整合素a4β7和MAdCAM-1的表达,促进肠道淋巴细胞归巢,从而改善AP时的肠道免疫功能.     

The relative importance of T cell subsets in immunity and immunopathology of airborne Mycobacterium tuberculosis infection in mice

Wild-type (WT) and targeted-mutant mice incapable of making alphabeta T cells, gammadelta T cells, class I major histocompatibility complex (MHC), class II MHC, interferon (IFN)-gamma, or inducible nitric oxide synthase (NOS2), were infected with Mycobacterium tuberculosis (Mtb) by aerosol, and monitored over time for their ability to (a) control infection, (b) develop histopathology at sites of infection, and (c) survive. WT mice acquired the ability to control and to hold infection at a stationary level from day 20 on. This was associated with the development of a macrophage-dominated alveolitis at sites of infection, with increased synthesis of IFN-gamma and NOS2 mRNA, and with an median survival time (MST) of 258.5 d. In the absence of alphabeta T cells, Mtb grew progressively and rapidly to induce a necrotic, neutrophil-dominated lung pathology that killed mice with an MST of 48 d. In the absence of CD4-mediated immunity (class II(-/-) mice), progressive bacterial growth continued in the lungs and in other organs beyond day 20, resulting in an MST of 77 d. By contrast, in the absence of CD8 T cell-mediated immunity, lung infection was controlled at a 1 log higher stationary level that induced a similar histopathologic response to that of WT mice, and resulted in an MST of 232 d.     

The linkage of innate to adaptive immunity via maturing dendritic cells in vivo requires CD40 ligation in addition to antigen presentation and CD80/86 costimulation

Dendritic cell (DC) maturation is an innate response that leads to adaptive immunity to coadministered proteins. To begin to identify underlying mechanisms in intact lymphoid tissues, we studied alpha-galactosylceramide. This glycolipid activates innate Valpha14(+) natural killer T cell (NKT) lymphocytes, which drive DC maturation and T cell responses to ovalbumin antigen. Hours after giving glycolipid i.v., tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma were released primarily by DCs. These cytokines induced rapid surface remodeling of DCs, including increased CD80/86 costimulatory molecules. Surprisingly, DCs from CD40(-/-) and CD40L(-/-) mice did not elicit CD4(+) and CD8(+) T cell immunity, even though the DCs exhibited presented ovalbumin on major histocompatibility complex class I and II products and expressed high levels of CD80/86. Likewise, an injection of TNF-alpha up-regulated CD80/86 on DCs, but CD40 was required for immunity. CD40 was needed for DC interleukin (IL)-12 production, but IL-12p40(-/-) mice generated normal ovalbumin-specific responses. Therefore, the link between innate and adaptive immunity via splenic DCs and innate NKT cells has several components under distinct controls: antigen presentation in the steady state, increases in costimulatory molecules dependent on inflammatory cytokines, and a distinct CD40/CD40L signal that functions together with antigen presentation ("signal one") and costimulation ("signal two") to generate functioning CD4(+) T helper cell 1 and CD8(+) cytolytic T lymphocytes.     

The biological activity of natural and mutant pTalpha alleles

beta selection is a major checkpoint in early thymocyte differentiation, mediated by successful expression of the pre-T cell receptor (TCR) comprising the TCRbeta chain, CD3 proteins, and a surrogate TCRalpha chain, pTalpha. The mechanism of action of the pre-TCR is unresolved. In humans and mice, the pTalpha gene encodes two RNAs, pTalpha(a), and a substantially truncated form, pTalpha(b). This study shows that both are biologically active in their capacity to rescue multiple thymocyte defects in pTalpha(-/-) mice. Further active alleles of pTalpha include one that lacks both the major ectodomain and much of the long cytoplasmic tail (which is unique among antigen receptor chains), and another in which the cytoplasmic tail is substituted with the short tail of TCR Calpha. Thus, very little of the pTalpha chain is required for function. These data support a hypothesis that the primary role of pTalpha is to stabilize the pre-TCR, and that much of the conserved structure of pTalpha probably plays a critical regulatory role.     

Very late activation antigen 4-vascular cell adhesion molecule 1 interaction is involved in the formation of erythroblastic islands

Erythroblastic islands are anatomical units consisting of a central macrophage surrounded by erythroblasts. We studied the adhesion molecules involved in the formation of these structures. Central macrophages of erythroblastic islands isolated from the spleens of phlebotomized mice were clearly stained for vascular cell adhesion molecule 1 (VCAM-1). The surrounding erythroblasts of the erythroblastic islands strongly expressed the alpha 4 integrin of very late activation antigen 4 (VLA-4: alpha 4 beta 1 integrin), the counter receptor of VCAM-1, whereas most reticulocytes and erythrocytes did not. Both monoclonal antibodies (mAbs) against alpha 4 integrin and VCAM-1 disrupted the erythroblastic islands cultured in the presence of erythropoietin. Moreover, adhesion of splenic erythroblasts to tumor necrosis factor alpha-stimulated mouse splenic endothelial cells, which showed high expression of VCAM-1 but not intercellular adhesion molecule 1, was inhibited by the anti-VCAM-1 and anti-alpha 4 mAbs. These findings suggest that VLA-4-VCAM-1 interaction plays a crucial role in the formation of erythroblastic islands.     

Insertion of a C in the exon 28 of integrin alphaIIb gene leading to a frameshift mutation is responsible for Glanzmann thrombasthenia in a Japanese case.

BACKGROUND: Glanzmann thrombasthenia (GT) is a hereditary bleeding disorder caused by a qualitative or quantitative defect in the integrin alphaIIbbeta3. OBJECTIVE: Our objective is to identify the gene mutation that resulted in GT. PATIENTS AND METHODS: The patient was a 66-year-old male with a history of frequent bleeding. The expression levels of the integrin proteins in the platelets were determined by flow cytometry and Western blot analysis. The sequences of genomic DNA and mRNA encoding for alphaIIb and beta3 were analyzed by the dye-terminator cycle sequencing method. For transfection experiments, expression vectors encoding for wild-type alphaIIb, mutated alphaIIb, beta3, green fluorescent protein (GFP) fusion wild-type alphaIIb, GFP fusion mutated alphaIIb and DsRed fusion beta3 were constructed. These vectors were transfected to COS-7 cells, and the expression levels were determined. RESULTS: The alphaIIb protein was remarkably reduced in the patient's platelets, and gene analysis showed that the patient possessed compound heterozygous mutations in the alphaIIb gene. One was a C --> G substitution at the splice acceptor site (- 3) of exon 26 (CAG -->GAG) and the other was the insertion of an additional C at the region including six C bases between 2911 and 2916 in exon 28 (InsC). Transfection experiments using COS-7 cells showed that alphaIIb containing InsC had expressed and formed a complex with beta3, but had not been transported to the Golgi apparatus. CONCLUSIONS: In the present study the novel mutation InsC, leading to a frameshift that affects the transmembrane domain and the cytoplasmic tail, was found to be responsible for GT.     

Generation of interferon alpha-producing predendritic cell (Pre-DC)2 from human CD34(+) hematopoietic stem cells

Upon viral stimulation, the natural interferon (IFN)-alpha/beta-producing cells (IPCs; also known as pre-dendritic cells (DCs 2) in human blood and peripheral lymphoid tissues rapidly produce huge amounts of IFN-alpha/beta. After performing this innate antiviral immune response, IPCs can differentiate into DCs and strongly stimulate T cell-mediated adaptive immune responses. Using four-color immunofluorescence flow cytometry, we have mapped the developmental pathway of pre-DC2/IPCs from CD34(+) hematopoietic stem cells in human fetal liver, bone marrow, and cord blood. At least four developmental stages were identified, including CD34(++)CD45RA(-) early progenitor cells, CD34(++)CD45RA(+) late progenitor cells, CD34(+)CD45RA(++)CD4(+)interleukin (IL)-3Ralpha(++) pro-DC2, and CD34(-)CD45RA(++) CD4(+)IL-3Ralpha(++) pre-DC2/IPCs. Pro-DC2s have already acquired the capacity to produce large amounts of IFN-alpha/beta upon viral stimulation and to differentiate into DCs in culture with IL-3 and CD40 ligand. CD34(++)CD45RA(-) early progenitor cells did not have the capacity to produce large amounts of IFN-alpha/beta in response to viral stimulation; however, they can be induced to undergo proliferation and differentiation into IPCs/pre-DC2 in culture with FLT3 ligand.     

Evaluation of the role of platelet integrins in fibronectin-dependent spreading and adhesion.

BACKGROUND: Recent studies have shown that platelet adhesion and subsequent aggregation can occur in vivo in the absence of the two principal platelets adhesive ligands, von Willebrand factor and fibrinogen. These results highlight a possible role for fibronectin in supporting thrombus formation. OBJECTIVE AND METHODS: To evaluate the platelet integrins and subsequent activation pathways associated with fibronectin-dependent platelet adhesion utilizing both human and murine platelets. RESULTS: Platelets can adhere to fibronectin via the integrin alpha(IIb)beta(3), leading to formation of lamellipodia. This is mediated through an interaction with the tenth type III domain in fibronectin. Spreading on fibronectin promotes alpha(IIb)beta(3)-mediated Ca(2+) mobilization and tyrosine phosphorylation of focal adhesion kinase and phospholipase C gamma2. In contrast, studies with blocking antibodies and mice demonstrate that alpha(5)beta(1) and alpha(v)beta(3) support adhesion and promote formation of filopodia but not lamellipodia or tyrosine phosphorylation of these proteins. Further, neither alpha(5)beta(1) nor alpha(v)beta(3) is able to induce formation of lamellipodia in the presence of platelets agonists, such as collagen-related-peptide (CRP). CONCLUSIONS: These observations demonstrate that integrins alpha(5)beta(1) and alpha(v)beta(3) support platelet adhesion and the generation of filopodia but that, in contrast to the integrin alpha(IIb)beta(3), are unable to promote formation of lamellipodia.     

The development of experimental autoimmune encephalomyelitis in the mouse requires alpha4-integrin but not alpha4beta7-integrin.

Because monoclonal antibodies (mAbs) directed against alpha4-integrin and VCAM-1 inhibit the development of experimental autoimmune encephalomyelitis (EAE) in vivo, it has been concluded that the successful therapeutic effect is due to interference with alpha4beta1/VCAM-1-mediated interaction of autoaggressive T cells with the blood-brain barrier. A possible role for alpha4beta7-integrin, or interference with other T cell mediated events during the pathogenesis of EAE, has not been considered. We have compared the effects of mAb therapy on the development of EAE in the SJL/N mouse, using a large panel of mAbs directed against alpha4, beta7, the alpha4beta7-heterodimer, and against VCAM-1. Although encephalitogenic T cells express both alpha4-integrins, mAbs directed against the alpha4beta7-heterodimer or against the beta7-subunit did not interfere with the development of EAE. In contrast, mAbs directed against alpha4 and VCAM-1 inhibited or diminished clinical or histopathological signs of EAE. Our data demonstrate for the first time that alpha4beta7 is not essential for the development of EAE. Furthermore, our in vitro studies suggest that the therapeutic effect of anti-alpha4-treatment of EAE might also be caused by inhibition of antigen-specific T cell proliferation.     

Defined alphabeta T cell receptors with distinct ligand specificities do not require those ligands to signal double negative thymocyte differentiation

During T cell development in the thymus, pre-T cell receptor (TCR) complexes signal CD4(-) CD8(-) (double negative [DN]) thymocytes to differentiate into CD4(+) CD8(+) (double positive [DP]) thymocytes, and they generate such signals without apparent ligand engagements. Although ligand-independent signaling is unusual and might be unique to the pre-TCR, it is possible that other TCR complexes such as alphabeta TCR or alphagamma TCR might also be able to signal the DN to DP transition in the absence of ligand engagement if they were expressed on DN thymocytes. Although alphagamma TCR complexes efficiently signal DN thymocyte differentiation, it is not yet certain if alphabeta TCR complexes are also capable of signaling DN thymocyte differentiation, nor is it certain if such signaling is dependent upon ligand engagement. This study has addressed these questions by expressing defined alphabeta TCR transgenes in recombination activating gene 2(-/-) pre-Talpha(-/-) double deficient mice. In such double deficient mice, the only antigen receptors that can be expressed are those encoded by the alphabeta TCR transgenes. In this way, this study definitively demonstrates that alphabeta TCR can in fact signal the DN to DP transition. In addition, this study demonstrates that transgenic alphabeta TCRs signal the DN to DP transition even in the absence of their specific MHC-peptide ligands.     

The Src family tyrosine kinase Fyn regulates natural killer T cell development.

T lymphocytes express two Src tyrosine kinases, Lck and Fyn. While thymocyte and T cell subsets are largely normal in fyn(-/-) mice, animals lacking Lck have impaired T cell development. Here, it is shown that Fyn is required for the rapid burst of interleukin (IL)-4 and IL-13 synthesis, which occurs promptly after T cell receptor activation. The lack of cytokine induction in fyn mutant mice is due to a block in natural killer (NK) T cell development. Studies using bone marrow chimeras indicate that the defect behaves in a cell-autonomous manner, and the lack of NK T cells is probably not caused by inappropriate microenvironmental cues. Both NK T cells and conventional T cells express similar levels of Lck, implying that Fyn and Lck have distinct roles in regulating NK T cell ontogeny. The fyn mutation defines the first signaling molecule that is selectively required for NK T cell, but not for T lymphocyte or NK cell development.     

Insulin induces the release of vasodilator compounds from platelets by a nitric oxide-G kinase-VAMP-3-dependent pathway

Insulin-induced vasodilatation is sensitive to nitric oxide (NO) synthase (NOS) inhibitors. However, insulin is unable to relax isolated arteries or to activate endothelial NOS in endothelial cells. Since insulin can enhance platelet endothelial NOS activity, we determined whether insulin-induced vasodilatation can be attributed to a NO-dependent, platelet-mediated process.Insulin failed to relax endothelium-intact rings of porcine coronary artery. The supernatant from insulin-stimulated human platelets induced complete relaxation, which was prevented by preincubation of platelets with a NOS inhibitor, the soluble guanylyl cyclase inhibitor, NS 2028, or the G kinase inhibitor, KT 5823, and was abolished by an adenosine A2A receptor antagonist. Insulin induced the release of adenosine trisphosphate (ATP), adenosine, and serotonin from platelet-dense granules in a NO-dependent manner. This response was not detected using insulin-stimulated platelets from endothelial NOS-/- mice, although a NO donor elicited ATP release. Insulin-induced ATP release from human platelets correlated with the association of syntaxin 2 with the vesicle-associated membrane protein 3 but was not associated with the activation of alphaIIbbeta3 integrin. Thus, insulin elicits the release of vasoactive concentrations of ATP and adenosine from human platelets via a NO-G kinase-dependent signaling cascade. The mechanism of dense granule secretion involves the G kinase-dependent association of syntaxin 2 with vesicle-associated membrane protein 3.     

A novel Phe171Cys mutation in integrin alpha causes Glanzmann thrombasthenia by abrogating alphabeta complex formation.

BACKGROUND: Glanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by lack of platelet aggregation induced by most agonists. The disease is caused by mutations in either alpha(IIb)[glycoprotein (GP) IIb] or beta(3) (GPIIIa) genes that lead to a lack or dysfunction of the integrin alpha(IIb)beta(3) which serves as a fibrinogen receptor. PATIENTS: Mucocutaneous bleeding manifestations and platelet dysfunction consistent with GT were observed in three members of a Cypriot family: a 3-year-old proband, her father and her paternal uncle. OBJECTIVE: To determine the molecular basis of GT in this family and to characterize possible biochemical and structural defects. RESULTS: Analysis of the patients' platelets by fluorescence-activated cell sorting demonstrated trace amounts of beta(3), no alpha(IIb) and no alpha(IIb)beta(3) on the membrane. Sequence analysis revealed a novel T607G transversion in exon 5 of the alpha(IIb) gene predicting a Phe171Cys alteration that created a PstI recognition site. All three patients were homozygous for the mutation, the mother and paternal grandparents of the proband were heterozygous, whereas 110 healthy subjects lacked this transversion. Chinese hamster ovary cells cotransfected with cDNAs of mutated alpha(IIb) and wild-type beta(3) failed to express alpha(IIb)beta(3) as shown by immunoprecipitation and immunohistochemistry experiments. Structural analysis of the alpha(IIb)beta(3) model, which was based on the crystal structure of alpha(v)beta(3), indicated that Phe171 plays an essential role in the interface between the beta-propeller domain of alpha(IIb) and the betaA domain of beta(3). CONCLUSIONS: A novel Phe171Cys mutation in the alpha(IIb) gene of patients with GT is associated with abrogation of alpha(IIb)beta(3) complex formation.