Biomedical subjects

E C Butcher

Publications and source records attributed to E C Butcher.

At least 91 records · Page 5Linked to original sources

PubMed1996

Biology of chemokine and classical chemoattractant receptors: differential requirements for adhesion-triggering versus chemotactic responses in lymphoid cells.

Several chemoattractant receptors can support agonist-induced, integrin-dependent arrest of rolling neutrophils in inflamed venules in vivo, as well as subsequent crawling into tissues. It has been hypothesized that receptors of the Galpha(i)-linked chemoattractant subfamilies, especially receptors for chemokines, may mediate parallel activation-dependent arrest of homing lymphocyte subsets. However, although several chemokines can attract subsets of B or T cells, robust chemoattractant triggering of resting lymphocyte adhesion to vascular ligands has not been observed. To study the biology of individual leukocyte chemoattractant receptors in a defined lymphoid environment, mouse L1/2 pre-B cells and/or human Jurkat T cells were transfected with alpha (IL-8 receptor A) or beta (MIP-1alpha/CC-CKR-1) chemokine receptors, or with the classical chemoattractant C5a (C5aR) or formyl peptide receptors (fPR). All receptors supported robust agonist-dependent alpha4beta1 integrin-mediated adhesion of lymphocytes to VCAM-1. L1/2 cells cotransfected with fPR and beta7 integrin were also induced to bind MAdCAM-1, suggesting common mechanisms coupling chemoattractant receptors to activation of distinct integrins. Adhesion was rapid but transient, with spontaneous reversion to unstimulated levels within 5 min after peak binding. When observed under flow conditions, alpha4beta1-mediated arrest occurred within seconds after initiation of contact and rolling of IL-8RA transfectants on VCAM-1/IL-8 co-coated surface; and arrest reversed spontaneously after a mean of 5 min with a return to rolling behavior. Each of the receptors also conferred agonist-specific chemotaxis; however, whereas strong adhesion required simultaneous occupancy of many receptors with maximal responses above the Kd, chemotaxis in each case was suppressed at high agonist concentrations. The findings indicate that alpha and beta chemokine as well as classical chemoattractant receptors can trigger robust adhesion as well as directed migration of lymphoid cells, but that the requirements for and kinetics of adhesion triggering and chemotaxis are distinct, thus permitting their independent regulation. They suggest that the discordance between proadhesive and chemoattractant responses of circulating lymphocytes to many chemokines may reflect quantitative aspects of receptor expression and/or coupling rather than qualitative differences in receptor signaling.

Animals↗

PubMed1996

ICAM-1, VCAM-1, and MAdCAM-1 are expressed on choroid plexus epithelium but not endothelium and mediate binding of lymphocytes in vitro.

The expression of cell adhesion molecules (CAMs) in the choroid plexus was studied in normal brain and during experimental autoimmune encephalomyelitis (EAE) in the SJL/J mouse during inflammation induced by intracerebral injection of killed Corynebacterium parvum in the C3H/He mouse. Both ICAM-1 and VCAM-1, but not MAdCAM-1, were constitutively expressed on choroid plexus epithelium but not on the fenestrated capillary endothelial cells within the choroid plexus. During EAE, we observed an up-regulation of ICAM-1 and VCAM-1 and de novo expression of MAdCAM-1 on choroid plexus epithelial cells. In contrast, endothelial cells in the choroid plexus were not induced to express any of the investigated CAMs. In in situ hybridization analysis we demonstrated that ICAM-1, VCAM-1, and MAdCAM-1 were locally synthesized and that the amount of their mRNAs increased in the inflamed choroid plexus. In vitro, primary choroid plexus epithelial cells could be induced to express ICAM-1, VCAM-1, and MAdCAM-1 on their surface after treatment with proinflammatory cytokines such as tumor necrosis factor-alpha, interleukin-1, interferon-gamma, and lipopolysaccharide. To investigate the functional status of the expressed CAMs we performed Stamper-Woodruff binding assays on frozen sections of inflamed and naive brains. ICAM-1, VCAM-1, and MAdCAM-1 expressed in choroid plexus epithelial cells mediated binding of lymphocytes via their known ligands LFA-1 and alpha4-integrin, respectively. The expression of ICAM-1, VCAM-1, and MAdCAM-1 on choroid plexus epithelial cells together with the lack of their expression on the fenestrated choroid plexus endothelium raises the possibility that the epithelial blood-cerebrospinal-fluid barrier plays an important role in the immunosurveillance of the central nervous system.

Animals↗

PubMed1995

Inhibition of T cell costimulation by VCAM-1 prevents murine graft-versus-host disease across minor histocompatibility barriers.

Activation of T cells leading to graft-vs-host disease (GVHD) requires two signaling events: the Ag-specific signal generated through the engagement of the TCR/CD3 complex with antigenic peptide fragments presented by MHC molecules on APCs and the second signal provided through additional costimulatory ligands. T cells have preferential costimulatory requirements depending on their state of activation-induced maturation. In the present study, we investigated the role of the receptor-ligand pair VLA-4 (alpha 4 beta 1) and VCAM-1 in allogeneic T cell responses in vitro and in vivo. Anti-VCAM-1 mAb effectively inhibited mixed lymphocyte culture (MLC) across several major MHC barriers and secondary MLC across minor histocompatibility Ags (95.5% and 90.0% inhibition, respectively). In contrast, anti-VLA-4 mAb inhibited a CD8(+)-mediated primed CTL response in vitro by 100%, yet had little effect on proliferative responses. In the B10.D2/nSnJ-->BALB/c (both H-2d) system of GVHD, BALB/c received anti-VCAM-1, or anti-VLA-4 or controls NS-1 or Y13-259 for the first 5 wk after transplant. Anti-VLA-4 mAb delayed the onset of GVHD, but failed to reduce incidence, severity or GVHD-related mortality. In contrast, anti-VCAM-1 reduced the incidence of GVHD from 100% (18/18) in control animals to 53.3% (8/15) (p < 0.01) on day 70 post-transplant and significantly decreased GVHD-related mortality. Sixty percent (9/15) of anti-VCAM-1 recipients survived more than 180 days after transplant. Long-term engraftment of allogeneic bone marrow was documented in all transplanted mice by PCR analysis of a microsatellite region in the IL-1 beta gene.

Animals↗

PubMed1995

A central role for microvillous receptor presentation in leukocyte adhesion under flow.

Leukocyte adhesion to endothelium requires specialized mechanisms for contact initiation under flow. L-selectin (CD62L), an efficient initiator of adhesion, is clustered on the tips of leukocyte microvilli. To test whether microvillous presentation is critical for contact formation ("tethering"), we transfected lymphoid cells with chimeras of L-selectin and CD44, an adhesion molecule that is excluded from microvilli. CD44 transmembrane and intracellular (TM-IC) domains targeted the L-selectin ectodomain to the planar body, whereas L-selectin TM-IC segments conferred CD44 ectodomain clustering on microvilli. Wild-type and chimeric transfectants bound similarly to anti-ectodomain MAbs in static assays, but MAb binding under flow was much more efficient in the context of microvillous presentation. Similarly, wild-type and chimeric L-selectin possessed equivalent lectin activity, but microvillous presentation dramatically enhanced contact initiation on a native ligand. These findings demonstrate a critical role for receptor topography in leukocyte adhesion and suggest a novel regulatory mechanism of leukocyte trafficking.

Animals↗

PubMed1995

Organization, regulatory sequences, and alternatively spliced transcripts of the mucosal addressin cell adhesion molecule-1 (MAdCAM-1) gene.

The mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is expressed selectively at venular sites of lymphocyte extravasation into mucosal lymphoid tissues and lamina propria, where it directs local lymphocyte trafficking. MAdCAM-1 is a multifunctional type l transmembrane adhesion molecule comprising two distal lg domains involved in alpha 4 beta 7 integrin binding, a mucin-like region able to display L-selectin-binding carbohydrates, and a membrane-proximal lg domain homologous to lgA. We show in this work that the MAdCAM-1 gene is located on chromosome 10 and contains five exons. The signal peptide and each one of the three lg domains are encoded by a distinct exon, whereas the transmembrane, cytoplasmic tail, and 3'-untranslated region of MAdCAM-1 are combined on a single exon. The mucin-like region and the third lg domain are encoded together on exon 4. An alternatively spliced MAdCAM-1 mRNA is identified that lacks the mucin/lgA-homologous exon 4-encoded sequences. This short variant of MAdCAM-1 may be specialized to support alpha 4 beta 7-dependent adhesion strengthening, independent of carbohydrate-presenting function. Sequences 5' of the transcription start site include tandem nuclear factor-kappa B sites; AP-1, AP-2, and signal peptide-1 binding sites; and an estrogen response element. Our findings reinforce the correspondence between the multidomain structure and versatile functions of this vascular addressin, and suggest an additional level of regulation of carbohydrate-presenting capability, and thus of its importance in lectin-mediated vs alpha 4 beta 7-dependent adhesive events in lymphocyte trafficking.

Alternative Splicing↗

PubMed1995

TABS, a T cell activation antigen that induces LFA-1-dependent aggregation.

We describe here a mAb, DATK44, which induces homotypic aggregation of TK1 cells (a CD8 lymphoma). The glycoprotein recognized by DATK44 is of approximate m.w. 50 kDa and is expressed by monocytes, neutrophils, and subsets of lymphocytes, as well as on the high endothelial venule in peripheral and mesenteric lymph nodes. We named this Ag TABS (T cell activation B cell subset Ag), as TABS appears on T lymphocyte activation and is expressed at low and high levels by B cells. TABS is differentially regulated during T lymphocyte development, CD4+veCD8+ve thymocytes being TABShigh, while single positive CD4+ve and CD8+ve thymocytes are TABSdull CD4-veCD8-ve thymocytes are clearly split into dull and bright populations by the mAb. On exit from the thymus, T lymphocytes cease to express TABS, but T lymphocyte activation results in re-expression of TABS. TABS also shows tight coregulation with heat stable Ag on resting lymphocytes, but coexpression of these two molecules is lost upon lymphocyte activation. DATK44-induced aggregation of TK1 cells is temperature sensitive and blocked by pretreatment of the cells with metabolic inhibitors, genestein, dibutyl cAMP or cytochalasin B, while colchicine, staurosporin, sphingosine, okadaic acid, and W7 are without effect. DATK44-induced TK1 cell aggregation appears to be mediated by the LFA-1 pathway, as aggregation is blocked by anti-LFA-1 and anti-ICAM-1 mAbs but not by Abs capable of blocking CD44 and alpha 4 beta 7-mediated adhesion. Thus, TABS appears to be an adhesion inducer that selectively activates LFA-1-mediated lymphocyte aggregation.

Animals↗

PubMed1995

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.

Amino Acid Sequence↗

PubMed1995

Primitive human hematopoietic progenitors adhere to P-selectin (CD62P).

P-selectin was shown to bind committed human hematopoietic progenitors (colony-forming unit-granulocyte-macrophage [CFU-GM] and burst-forming unit-erythroid [BFU-E]) as identified by their expression of the CD34 antigen and by in vitro clonogenic assays. In addition, P-selectin bound all precursors (pre-CFU) of committed myeloid progenitors assayed by their ability to sustain hematopoiesis in both conventional stroma-containing and stroma-free, cytokine-dependent systems. Binding of CD34+ cells to P-selectin was temperature-independent and shear-resistant, occurred only in the presence of divalent cations, was protease sensitive, and was completely blocked by anti-P-selectin antibody. Neuraminidase treatment of CD34+ cells completely abrogated their binding to P-selectin, implying a prominent role for sialic acid in the structure and function of the P-selectin ligand on hematopoietic progenitors. Monoclonal antibodies (MoAbs) CSLEX-1 and HECA-452, which identify carbohydrate epitopes involving sialic acid, bound to 33% and 35% of CD34+ cells, respectively, and included the majority of CFU-GM and pre-CFU. Three-color flow cytometric analysis showed a precise codistribution of CSLEX-1 and HECA-452 antigens on CD34+ cells, implying recognition of the same glycoprotein antigen by the two MoAbs. Treatment of CD34+ cells with neuraminidase completely abolished binding of both MoAbs. In addition, HECA-452 partially blocked the adhesion of CD34+ cells to P-selectin. P-selectin glycoprotein ligand (PSGL-1), recently molecularly cloned from the promyelocytic leukemia cell line HL60, was expressed by CD34+ cells as determined by reverse transcription polymerase chain reaction. Combined with the functional and biochemical characteristics, these data suggest that PSGL-1 may comprise an important P-selectin ligand expressed by primitive hematopoietic cells, but do not preclude the existence of additional P-selectin ligands on these cells.

Antigens, CD↗

PubMed1995

Involvement of CD31 in lymphocyte-mediated immune responses: importance of the membrane-proximal immunoglobulin domain and identification of an inhibiting CD31 peptide.

CD31 (PECAM-1) is an immunoglobulin gene superfamily cell adhesion molecule found on vascular endothelium, platelets, and leukocytes. Lymphocyte expression of CD31 is most closely associated with the CD45RA+CD8+ naive T phenotype. CD31 has recently been shown to play a role in leukocyte egress to inflammatory sites. The mechanism of CD31 adhesion remains under investigation. Several investigators have reported evidence for a heterotypic ligand. We have previously shown that CD31 is phosphorylated with cell activation, which suggests a possible role for CD31 in cell activation events. We therefore studied the effects of CD31 antibodies on in vitro assays of lymphocyte activation. One CD31 antibody, LYP21, inhibited the mixed lymphocyte reaction (MLR) in a specific and dose-dependent fashion. An LYP21 epitope was localized to the sixth Ig domain of CD31. This peptide and a scrambled control peptide were synthesized and used to study effects of this epitope on lymphocyte activation. The CD31 peptide strongly inhibited the MLR. Because CD31 is expressed on both stimulator and responder populations, stimulator peripheral blood leukocytes and responder lymphocyte populations were separately incubated with CD31 peptide or control peptide and then washed before mixing. The CD31 peptide inhibited the MLR equally when either stimulator or responder cells were preincubated with the CD31 peptide. We further sorted responder cells into CD31-high and CD31-low populations and separately incubated these subsets with peptides. The CD31 peptide strongly inhibited MLRs, regardless of level of responder-cell CD31 expression. Examination of MLR reactions involving the CD31 peptide showed dispersed small aggregates of cells, rather than the single large aggregate observed in control MLRs. The CD31 peptide did not affect activation of lymphocytes by phorbol myristate acetate (PMA) and ionomycin. These results suggest that a surface CD31-ligand interaction may have a functional role in alloimmune lymphocyte activation and identify a functionally important domain of CD31.

Amino Acid Sequence↗

PubMed1995

alpha 4 integrins mediate lymphocyte attachment and rolling under physiologic flow.

Of the several families of adhesion receptors involved in leukocyte-endothelial cell interactions, only the selectins have been shown to initiate leukocyte interaction under physiologic shear; indeed, beta 2 (CD18) intergrins responsible for neutrophil arrest are unable to engage without prior selectin-mediated rolling. In contrast, alpha 4 (CD49d) integrins are shown here to initiate lymphocyte contract ("tethering") in vitro under shear and in the absence of a selectin contribution. The alpha 4 integrin ligands MAdCAM-1 and VCAM-1 support loose reversible interactions including rolling, as well as rapid sticking and arrest that is favored following integrin activation. Moreover, alpha 4 beta 7 mediates L-selectin (CD62L)-independent attachment of blood-borne lymphocytes to lamina propria venules in situ. Scanning electron microscopy of alpha 4 beta 7hi lymphoid cells reveals that, like L-selectin, alpha 4 beta 7 is highly concentrated on microvillous sites of initial cellular contact, whereas the beta 2 integrin LFA-1 is excluded from villi. Thus, alpha 4 but not beta 2 integrins can initiate leukocyte adhesion under flow, a capacity that may be in part a function of topographic presentation on microvilli.

Animals↗

PubMed1995

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.

Carrier Proteins↗

PubMed1995

Novel mouse endothelial cell surface marker is suppressed during differentiation of the blood brain barrier.

Few markers specific for mouse endothelium exist. We describe here one such marker, MECA-32, a monoclonal antibody which shows high specificity for mouse endothelium in both embryonic and mature tissues. The MECA-32 antigen has a M(r) of 50-55 x 10(3) under reducing conditions and M(r) of 100-120 x 10(3) under nonreducing conditions. It is expressed on most endothelial cells in the embryonic and in the adult mouse, with the exception of the brain, skeletal, and cardiac muscle, where it has a more restricted distribution. In skeletal and cardiac muscle only small arterioles and venules express the MECA-32 antigen, while in the brain its expression is negatively correlated with the differentiation of the vasculature to form the blood brain barrier. Interestingly, during embryonic development the antigen occurs on the brain vasculature up to day 16 of gestation (E16), whereupon it disappears. The embryonic brain is an avascular organ anlage which is vascularized by ingrowth of external blood vessels. Differentiation of the vasculature to form the blood brain barrier occurs at approximately E16 in the mouse. This differentiation correlates with the downregulation of MECA-32 antigen expression. Between E12 and E16 MECA-32 detects most endothelial cell surfaces of the blood vessels in the brain. No MECA-32 antigen is found in the brain at E17 or any later stage of development with the exception of the vasculature of the circumventricular organs. The results suggest that MECA-32 antigen expression is temporally and spatially correlated with the development of the blood brain barrier.

Animals↗

PubMed1995

Rolling of lymphocytes and neutrophils on peripheral node addressin and subsequent arrest on ICAM-1 in shear flow.

We studied leukocyte interactions in shear flow with peripheral lymph node addressin (PNAd), a mixture of glycoproteins expressed on high endothelial venules (HEV) that is required for lymphocyte homing and has been shown to contain a ligand for L-selectin. T lymphocytes and neutrophils tether and roll on plastic-immobilized PNAd and E-selectin at 1.8 dyn/cm2 wall shear stress, but fail to interact with immobilized ICAM-1, a ligand for LFA-1 and Mac-1, at the same flow rate. Cells roll faster on PNAd than on P-selectin or E-selectin. L-selectin mAb inhibit T lymphocyte and neutrophil tethering to PNAd, but do not inhibit T lymphocyte tethering to purified E-selectin. If allowed to interact with ICAM-1 under static conditions, phorbol ester-treated T lymphocytes, but not resting T lymphocytes, are able to form stationary adhesions that withstand the detachment force generated by 36 dyn/cm2 wall shear stress. In contrast, a wall shear stress of 7.3 dyn/cm2 detaches 50% of resting T lymphocytes bound to PNAd. Incubating T lymphocytes on PNAd and ICAM-1 does not result in adhesion strengthening, suggesting that adhesion through PNAd by L-selectin does not stimulate lymphocyte LFA-1 avidity for ICAM-1. Chemoattractant stimulation of neutrophils or phorbol ester stimulation of lymphoblasts rolling on coimmobilized PNAd and ICAM-1 results in rapid arrest and firm sticking, extending the model of sequential selectin-mediated rolling and subsequent integrin-mediated firm arrest to lymphocytes and ligands expressed on HEV.

Antigens, Surface↗

PubMed1995

Distinct roles of L-selectin and integrins alpha 4 beta 7 and LFA-1 in lymphocyte homing to Peyer's patch-HEV in situ: the multistep model confirmed and refined.

Circulating lymphocytes home to the mucosal lymphoid organs, Peyer's patches (PP), through high endothelial venules (HEV). In situ analyses revealed that transfused lymph node cells (LNCs) interact with PP-HEV in a series of overlapping adhesion events: L-selectin (CD62L) > alpha 4 beta 7 initiates interaction, L-selectin and alpha 4 beta 7 both participate in rolling, and G alpha i-linked activation triggers arrest that requires both alpha 4 beta 7 and LFA-1. alpha 4 beta 7 dramatically reduces rolling velocity, and appears to be required for engagement of LFA-1. In contrast with resting LNC, preactivated LNC or alpha 4 beta 7hi lymphoma cells require only alpha 4 beta 7 for arrest in PP-HEV. The predominant PP-HEV ligand for alpha 4 beta 7 but also apparently for L-selectin is the mucosal addressin MAd-CAM-1. These results validate the concept of multimolecular adhesion/decision cascades in physiologic lymphocyte-endothelial recognition, define a novel role for alpha 4 integrins as a "bridge" between selectin and beta 2 integrin-dependent events, and reemphasize the potential for direct adhesion through preactivated alpha 4 integrins alone.

Animals↗

PubMed1995

Heat stable antigen (mouse CD24) supports myeloid cell binding to endothelial and platelet P-selectin.

P-selectin is a Ca(2+)-dependent lectin that participates in leukocyte adhesion to vascular endothelium and platelets. Myeloid cells and a subset of T lymphocytes express carbohydrate ligands at the cell surface. Previously, we suggested that heat stable antigen (HSA/mouse CD24), an extensively glycosylated cell surface molecule on many mouse cells, is a ligand for P-selectin. Here we show that HSA mediates the binding of monocytic cells and neutrophils to P-selectin. The monocytic cell lines ESb-MP and J774, peritoneal exudate cells, and bone marrow neutrophils could bind to lipopolysaccharide-activated bend3 endothelioma cells under rotation-induced shear forces and this binding was inhibited by mAb to P-selectin and HSA. Blocking was weak at room temperature but more efficient at 4 degrees C when integrin-mediated binding was decreased. Also the adhesion of neutrophils to stimulated platelets expressing P-selectin was blocked by HSA- and P-selectin-specific mAb. Latex beads coated with purified HSA from myeloid cells bound to activated endothelioma cells or platelets, and the binding was similarly blocked by mAb to P-selectin and HSA respectively. The HSA-coated beads were stained with P-selectin-IgG, very weakly with L-selectin-IgG but not with E-selectin-IgG. The staining was dependent on divalent cations and treatment with endoglycosidase F or neuraminidase indicated that sialylated N-linked glycans were recognized. The presence of these glycans was confirmed by biosynthetic labeling studies. Our data suggest that HSA, in addition to the recently identified 160 kDa glycoprotein ligand on mouse neutrophils, belongs to a group of monospecific P-selectin ligands on myeloid cells.

Animals↗

PubMed1995

Lymphocytes infiltrating the CNS during inflammation display a distinctive phenotype and bind to VCAM-1 but not to MAdCAM-1.

The nature of inflammatory lymphocytes recruited to the CNS has been studied in a model of chronic inflammation. Injection of killed Corynebacterium parvum into the cortex of the mouse brain produces a circumscribed inflammatory cellular infiltrate around the injection site, and recruited mononuclear inflammatory cells (IC) can be isolated for flow cytometric analysis. The majority of IC were T cells. In comparison with the predominant naive population of mesenteric lymph node T cells, IC T cells express much higher levels of CD44, LFA-1 and ICAM-1, and lower levels of CD45RB, features commonly associated with memory (previously activated) cells. In addition, in contrast to the L-selectin+ alpha 6-integrinlow phenotype of naive lymph node T cells, IC T cells lacked L-selectin and were alpha 6-integrin-. Mac-1, recently proposed as another marker of memory T cell differentiation, was not displayed by IC T cells, suggesting that Mac-1 expression may be heterogeneous among memory T cell subsets. A subset of mesenteric lymph node (MLN) T cells, probably representing activated T cells undergoing the naive to memory transition, but not of IC T cells, expressed high levels of alpha 6-, beta 7- and alpha E-integrin. IC and MLN naive T cells expressed comparable levels of alpha 4-integrin, but IC T cells stain poorly with anti-beta 7 mAbs and with mAb DATK 32, specific for the alpha 4 beta 7 heterodimeric lymphocyte homing receptor for the mucosal addressin MAdCAM-1, suggesting that these inflammatory cells express more alpha 4 beta 1 than alpha 4 beta 7. Consistent with this, in in vitro adhesion assays, brain IC bound better than MLN cells to the alpha 4 beta 1 integrin ligand VCAM-1 and the LFA-1 ligand ICAM-1 but adhered very poorly to the alpha 4 beta 7 ligand MAdCAM-1. These findings are consistent with and extend previous immunohistological studies of T cells in murine experimental autoimmune encephalomyelitis, and demonstrate a distinctive phenotype for lymphocytes being present in the chronically inflamed brain.

Animals↗

PubMed1995

Soluble L-selectin (sCD62L) umbilical cord plasma levels increase with gestational age.

L-Selectin (CD62L) is a leukocyte surface membrane glycoprotein involved in extravasation and homotypic aggregation which is rapidly cleaved off after cellular activation. From culture supernatants and body fluids, soluble L-selectin (sCD62L) has been recovered with its functional activity retained. We devised a sensitive enzyme-linked immunoassay for quantitation of sCD62L which was used to measure sCD62L in umbilical cord plasma of 255 human newborns with a gestational age (GA) of 23-43 wk (median 38 wk). sCD62L levels ranged from 1.14-13.8 pmol/mL (median 7.2 pmol/mL) and showed strong correlations with GA (r = 0.71, p < 0.001), birth weight (r = 0.66, p < 0.001), and absolute neutrophil cell counts (ANC) (r = 0.62, p < 0.001) obtained from a peripheral vein within the first 6 h of life (n = 153), whereas there was a weak inverse correlation with absolute normoblast counts (r = -0.27, p < 0.001). In multiple regression analysis, only GA and ANC retained a significant association with sCD62L levels (p < 0.001). Decreased sCD62L levels were found to be associated with multiple gestation (4.8 +/- 2.4 pmol/mL versus 7.7 +/- 2.3 pmol/mL, p < 0.05) also when considering GA and ANC as covariates. In contrast, increased sCD62L levels in infants born from meconium-stained amniotic fluid, and decreased levels in newborns with acute bacterial infection could be fully attributed to differences in GA and ANC.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Infections↗

PubMed1995

L-selectin and alpha 4 beta 7 integrin homing receptor pathways mediate peripheral lymphocyte traffic to AKR mouse hyperplastic thymus.

Before the development of thymic lymphoma, AKR mice undergo a striking lymphoid hyperplasia of the thymic medulla. We have previously shown that there is a marked increase in traffic of B and T lymphocytes from the periphery into the preneoplastic, hyperplastic thymuses of these mice, in contrast to the scant traffic of such cells to normal thymuses. The traffic of lymphocytes to lymph nodes and Peyer's patches is controlled in part by the interaction of lymphocyte adhesion molecules called homing receptors with their tissue-selective endothelial ligands known as vascular addressins. We have investigated the roles of homing receptors and vascular addressins in the traffic of lymphocytes to the AKR hyperplastic thymus. We demonstrate that development of hyperplasia is accompanied by an increase in the number of thymic medullary blood vessels with high endothelial venule morphology and expression of the peripheral node addressin (PNAd) and the mucosal addressin (MAdCAM-1). In vitro and in vivo functional assays show that the addressin/homing receptor pairs PNAd/L-selectin and MAdCAM-1/alpha 4 beta 7 are involved in lymphocyte traffic to the hyperplastic thymus. These results indicate that molecular adhesion mechanisms involved in tissue-selective migration of lymphocytes to peripheral lymph node and to mucosal lymphoid tissues play a role in the recruitment of B and T lymphocytes to the AKR thymus and thus in the pathogenesis of thymic hyperplasia.

Animals↗