L1 adhesion molecule on human lymphocytes and monocytes: expression and involvement in binding to αvβ3 integrin

The L1 adhesion molecule is a member of the immunoglobulin (Ig) superfamily initially identified in the nervous system which contains six Ig-like domains. Besides the known L1-L1 homotypic interaction, L1 was recently shown to bind to very late antigen (VLA)-5 in the mouse and αvβ3 in the human. The sixth Ig domain is critical for this function. We now demonstrate that human CD4⁺ peripheral blood T lymphocytes, monocytes and B lymphocytes, but not CD8⁺ T lymphocytes, express L1. When compared to the expression of CD31, another ligand for αvβ3 on T lymphocytes, only a small proportion of cells were CD31⁺L1⁺ double positive. L1 was also detected on the surface of human monocytic and lymphoid tumor lines and was shown to have a molecular mass of ∼220 kDa, similar to the molecule present on neuroblastoma cells. The function of the sixth Ig domain of human L1 as an integrin ligand was also investigated. Using an RGD-containing peptide derived from the sixth Ig domain as well as a fusion protein of the sixth Ig domain of L1 and the Fc portion of human IgG1 (6.L1-Fc), we demonstrated the binding of human MED-B1 (αvβ3^hi, α5β1^lo) tumor cells and this binding was blocked by αv-specific mAb. In contrast, human Nalm-6 cells (αvβ3^lo, α5β1^hi) did not bind to the 6.L1-Fc fusion protein. MED-B1 cells could also be stained with the 6.L1-Fc fusion protein. Our results suggest that human L1 binds predominantly to αvβ3 and that its presence on leukocytes could be important for adhesion and migration.     

Dissecting the role of integrin subunits α2 and β3 in rotavirus cell entry by RNA silencing

Several cell surface molecules have been implicated in rotavirus cell entry, however, their individual relevance during this process is unknown. In this work, the expression of integrins α2, β2, and αvβ3, the heat shock cognate protein 70, and of ganglioside GM1 in different cell lines of human and simian origin was correlated with the infectivity of four rotavirus strains. We observed that different combinations of receptor expression correlated with the infectivity of rotavirus strains, suggesting that the participation of several receptors is important for rotavirus infection. To characterize the relevance of integrins α2 and αvβ3 in more detail, their expression was silenced using RNA interference. About 80% decrease in the cell content of integrins resulted in 15–30% decrease of infectivity of strains RRV and Wa when measured by a focus-forming assay, while there was no decrease of infectivity when measured by flow cytometry in integrin-deficient cells. Altogether these data suggest that integrins α2 and αvβ3 do not play a major role in the rotavirus entry process.     

Developmental expression of the αIELβ7 integrin on T cell receptor γδ and T cell receptor αβ T cells

A novel monoclonal antibody, 2E7, was shown by immunoprecipitation to be reactive with the α_IELβ7 integrin and was employed to analyze the expression of this integrin in lymphocyte subsets and during T cell ontogeny. In adult lymph nodes, α_IEL was expressed at low levels by 40–70% of CD8⁺ T cells and < 5% of CD4⁺ T cells. However, virtually all intestinal intraepithelial lymphocytes and ?20% of lamina propria CD4⁺ T cells were 2E7⁺, indicating a preferential expression of this integrin on mucosal T cells. Examination of α_IEL integrin expression during thymus ontogeny revealed that ?3–5% of fetal or adult thymocytes were 2E7⁺. Interestingly, early in fetal thymus ontogeny, ?40% of 2E7⁺ cells expressed T cell receptor (TcR)-γδ and this subset persisted through birth. A developmental switch occurred such that 2E7⁺ TcR^? CD4^?8⁺ cells detected on fetal day 19 were followed by 2E7⁺ TcR-αβ CD4^?8⁺ cells in the neonatal thymus. The latter population persisted throughout thymus ontogeny into adulthood. Interestingly, a subset of TcR-γδ Vγ3⁺ day 16 fetal thymocyte dendritic epidermal cell (DEC) precursors were 2E7⁺, but all mature DEC expressed high levels of α_IEL integrin, suggesting that the α_IEL integrin was acquired late in DEC maturation. This possibility was strenghthened by immunohistochemical localization of the majority of 2E7⁺ γδ and αβ T cells to the medullary regions of the thymus. Overall, the results demonstrate a developmentally ordered expression pattern of the α_IELβ₇ integrin that suggests a common function for this integrin during TcR-γδ and -αβ CD4^?8⁺ T cell thymocyte development or perhaps in effector functions for these subsets.     

Functional analysis of α1β1 integrin in human natural killer cells

Upon activation with interleukin (IL)-2 human natural killer (NK) cells acquire on their surface the α1β1 and α2β1 integrins and down-regulate the expression of α6β1. By employing α1β1-specific monoclonal antibody (mAb) HP-2B6, characterized in our laboratory, we examined the functional role of the α1β1 integrin in NK cells. Treatment with HP-2B6 mAb partially interfered with attachment of cultured NK cells to type I collagen, and combined with an anti-α2β1 (TEA 1/41) mAb, it completely abrogated cell adhesion to this extracelular matrix protein. In contrast, NK cell attachment to laminin was completely blocked by the anti-β1 LIA 1/2 mAb, but was unaffected by α1 and α2-specific mAb; as α3β1 and α6β1 were undetectable, the data indicate that the α1β1 integrin binding sites for type I collagen and laminin are different. Incubation with anti-α1 HP-2B6 or its F(ab')₂ fragments specifically induced a rapid homotypic aggregation of NK cells that was dependent on active metabolism, an intact cytoskeleton and the presence of divalent cations (Ca²⁺ and Mg²⁺); homotypic cell adhesion was selectively blocked by anti-CD18, CD11a or CD54 mAb. In addition, stimulation of cultured NK cells with the anti-α1 HP-2B6 enhanced TNF-α production and induced tyrosine phosphorylation of a 110-kDa protein. Pretreatment with specific inhibitors of protein tyrosine kinase (PTK) activity (tyrphostin 25 and herbimycin A) completely abrogated the functional effects induced by the anti-α1 HP-2B6 mAb. Our data show that ligation of the α1β1 integrin positively modulates IL-2-activated NK cell function via a PTK-dependent pathway.     

Expression of integrin subunits correlates with differentiation of epithelial cell lineages in developing human gastric mucosa

Laminins may play important roles in gastric gland development due to the differential localization of their α chains in human fetal fundic (oxyntic) mucosa. To extend this hypothesis, the current investigation was undertaken to compare the anatomical and cellular distribution of epithelial integrin subunits with those of laminin α chains in the human stomach at different ages (8–22 weeks of gestation) using indirect immunofluorescence. In the body, fundus and antrum regions, the β1 and α6 subunits were associated with the entire epithelium at all developmental stages in the same way as laminin chains (α1/α5) detected with 4C7 monoclonal antibody. By contrast, the α3 and β4 subunits of α3β1 and α6β4 integrins together with the laminin α3-chain were concentrated in the surface and foveolus compartments composed of differentiating mucous cells. Most importantly, the α2 integrin subunit was expressed in a rather complex pattern: (1) it was located at the base and at cell-cell boundaries of surface/foveolar epithelium, (2) was specifically repressed in differentiating parietal cells, and (3) its expression increased in maturing glands, where it became concentrated at the basal pole of epithelial cells simultaneously exhibiting a strong reactivity for laminin-2 (α2-chain). Taken altogether, our observations provide new evidence for the implication of different laminins and their receptors in the development of all human gastric epithelial lineages, surface mucous cells or glandular cells. The coordinated expression of α2 and α3 integrin subunits as well as the cellular re-distribution of α2β1 integrin likely represent key events for the differentiation of glandular secretory cell types, especially maturing chief cells responsible for the synthesis/secretion of gastric digestive enzymes in fundic-type glands. Accepted: 26 March 2000     

Over-expression of integrin β3 can partially overcome the defect of integrin β3 signaling in transglutaminase 2 null macrophages

Transglutaminase 2 (TG2) is a protein crosslinking enzyme with many additional biological functions. We have previously shown that in TG2^−/− mice the in vivo clearance of apoptotic cells is defective leading to autoimmunity. TG2 contributes to the formation of phagocytic portals by binding to both integrin β₃, a known phagocytic receptor, and its bridging molecule, MFG-E8. In TG2 null macrophages integrin β₃ cannot accumulate around the apoptotic cells and its signaling is impaired. In the present study we describe a subline of TG2 null mice, in which a compensatory increase in integrin β₃ expression, which resulted alone in a high receptor concentration around the apoptotic cells without the requirement for accumulation, partially corrected the defect in integrin β₃ signaling. Our data provide a proof for the concept that the function of TG2 is to stabilize accumulated integrin β₃ concentration in the phagocytic cup.     

Distribution of α4β7 and αEβ7 integrins on thymocytes,intestinal epithelial lymphocytes and peripheral lymphocytes

β₇ is expressed on subsets of thymocytes, while T and B lymphocytes show heterogeneous expression of β₇. Here, we examine the phenotype of the thymocyte and lymphocyte subsets which express α₄β₇ and α_Eβ₇ using mAb against α_Eβ₇ and mAb DATK32 which recognizes a combinatorial epitope on α₄β₇. β₇⁺ thymocytes have a mature phenotype: TcR⁺, CD11a^hi CD44^hi HSA^dull. Small subsets of double-negative CD4_?CD8_?, single-positive CD4⁺ and CD8⁺ thymocytes express α₇, while double-positive CD4⁺ CD8⁺ thymocytes are β₇. However, two integrins α_Eβ₇ and α₄β₇ recognized by anti-β₇ are not expressed on an identical subpopulation of thymocytes, as β_Eα₇⁺α₄β₇^?, α_Eβ₇⁺α₄β₇⁺ and α_Eβ₇^?α₄β₇⁺ thymocyte subsets are evident. Similarly, intraepithelial lymphocytes express high levels of α_Eβ₇ but little α₄β₇. In the spleen, Peyer's patches and lymph nodes, α₄β₇ is expressed at higher levels on most B lymphocytes than on the majority of T lymphocytes, while a small subset of T lymphocytes, which includes both CD4⁺ and CD8⁺ lymphocytes, express high levels of β₇ in the form of α₄β₇ and α_Eβ₇, although, as observed with lymphocytes, not all α₄β₇^hi CD4^? lymphocytes expressed α₄β₇. The population of α₄β₇^hi CD4 lymphocytes are enriched in Peyer's patches and form subsets of the memory CD4⁺ lymphocyte population, which can be further subdivided on the basis of α_Eβ₇, L-selectin and α₄ expression. Therefore, memory CD4⁺ lymphocytes are highly heterogeneous in their expression of adhesion receptors, and presumably these subpopulations will exhibit very different trafficking properties.     

CD9, but not other tetraspans,associates with the β1 integrin precursor

The molecules of the tetraspan superfamily have two unequal extracellular domains separated by four transmembrane (TM) domains. These molecules are associated on the cell surface with each other and with other partner molecules, in particular β1 integrins. We now show that CD9 associates with the precursor of the β1 integrin (preβ1). This association is detected as early as 15 min after metabolic labeling, and the use of Brefeldin A demonstrates that it does not require Golgi modifications of either CD9 or integrin β1. The specificity of this interaction is demonstrated by the fact that other tetraspans, CD63, CD81, and CD82, do not associate with preβ1, and CD9 does not associate with immature human histocompatibility leukocyte antigen class I. In order to localize the region of CD9 responsible for the association with the β1 integrin, we have generated two reciprocal chimeric CD9/CD82 molecules with the junction localized just after the third TM region. The large extracellular loop of CD9 or the fourth TM domain, or both, appear to be sufficient to mediate an association with the mature integrin with a high efficiency, compared to CD82. By contrast, association with preβ1 requires at least two regions of the molecule. Mutation of CD9 at the consensus site of the tetraspan superfamily, localized between the second and the third TM domain, did not impair the co-precipitation of preβ1. Finally, because preβ1 associates with calnexin, we have investigated a possible association of CD9 with this chaperone molecule. CD9 associates with calnexin independently of its association with the β1 integrin, suggesting that calnexin could be involved in the processing of CD9.     

MAdCAM-1 costimulates T cell proliferation exclusively through integrin α4β7, whereas VCAM-1 and CS-1 peptide use α4β1: evidence for “remote” costimulation and induction of hyperresponsiveness to B7 molecules

We have analyzed the effects of the α⁴ integrin ligands mucosal addressin cell adhesion molecule-1 (MAdCAM-1), vascular cell adhesion molecule-1 (VCAM-1), and the fibronectin CS-1 splice variant on T cell activation. Immobilized MAdCAM-1 and VCAM-1 IgG-Fc chimeras and a fibronectin CS-1 peptide efficiently costimulate T cell proliferation when antigen presentation is mimicked by anti-CD3 antibody. VCAM-1-Fc and fibronectin CS-1, which are adhesive ligands for both the α⁴ β₁ and α⁴ β₇ integrins, medicate T cell costimulation exclusively through integrin α⁴ β₁, but not through α⁴ β₇. The inability of VCAM-1-Fc to costimulate via α⁴ β₇ suggests that cell adhesion per se is insufficient, and that exquisite recognition and activation events must be triggered. MAdCAM-1-Fc mediates costimulation exclusively via α⁴ β₇, and can both synergize with and induce hyperresponsiveness to the classical costimulator B7-2. MAdCAM-1-Fc and VCAM-1-Fc, but not B7-2, effectively costimulate when immobilized on sites spatially distant from the anti-CD3 antibody (“remote” costimulation). In vitro, the relative potencies of the CAM were VCAM-1-Fc > ICAM-1-Fc > MAdCAM-1-Fc > B7-Fc, except at high concentrations where ICAM-1 was the most potent. Features of costimulatory CAM revealed by this study have important implications for the design of immunotherapeutic vaccine strategies to combat cancer and infection.     

Recognition of E-cadherin on epithelial cells by the mucosal T cell integrin αM290β7 (αEβ7)

The integrin α_M290β7 on the surface of a T cell hybridoma, MTC-1, mediated adhesion of these cells to the mouse epithelial cell line CMT93. This interaction was critically dependent on the presence of divalent cations; Mn²⁺ strongly promoted adhesion, Ca²⁺ was ineffective and Mg²⁺ gave intermediate results. Antibodies to molecules on the surface of CMT93 cells were tested for inhibition of adhesion. One monoclonal antibody (mAb) against E-cadherin, ECCD-2, was found to have significant inhibitory activity. Other mAb to E-cadherin and antibodies to other molecules had no effect. To show that inhibition by ECCD-2 was specific for adhesion mediated by α_M290β7, MTC-1 cells were induced to adhere to CMT93 via the LFA-1/ICAM-1 pathway. For this purpose, the epithelial cells were treated with interferon-γ and tumor necrosis factor-α to induce ICAM-1 expression and, in addition, α_M290β7 on MTC-1 cells was down-regulated by culturing the cells in the absence of transforming growth factor β. Under these circumstances adhesion of MTC-1 cells to CMT93 was inhibited by an antibody to LFA-1 but not by ECCD-2. Transfection of mouse L cells with cDNA for mouse E-cadherin enabled MTC-1 cells to adhere to them through the α_M290β7 integrin; this interaction was inhibited both by ECCD-2 and by blocking antibody against the integrin. These data strongly suggest that E-cadherin is a principal ligand for α_M290β7.     

Cross talk between αvβ3 and α4β1 integrins regulates lymphocyte migration on vascular cell adhesion molecule 1

Local inflammation leads to increased expression of the vascular cell adhesion molecule (VCAM)-1 on vascular endothelium which contributes to the encapture of leukocytes from the circulating blood through the leukocyte ligand α4β1 integrin. Inflammatory vascular endothelium expresses VCAM-1 at high density. We found that the speed of locomotion of activated lymphocytes migrating along surfaces coated with recombinant VCAM-1 at a comparable density to that found on inflammatory endothelium was slow. However, lymphocytes do migrate and extravasate rapidly under inflammatory conditions, indicating that there must be mechanisms that regulate the interaction between α4β1 and VCAM-1 in vivo. Here we show that the lymphocyte αβ3 integrin and integrin-associated protein (IAP) is able to regulate this interaction. The occupancy of lymphocyte αvβ3 integrin by platelet cell adhesion molecule-1 or vitronectin regulated the speed of α4β3 integrin-dependent locomotion of lymphocytes on recombinant VCAM-1. This allowed rapid lymphocyte migration at VCAM-1 densities which are typical of inflammatory vessels. This αvβ3-mediated enhanced migration of lymphocytes via α4β1 is likely to depend on the interaction of αvβ3 integrin with the IAP. Furthermore, this motile process correlates with polarization of the actin cytoskeleton in lymphocytes. Our results suggest that cross talk between αvβ3 integrin and α4β1 integrin is a mechanism in the regulation of lymphocyte locomotion along inflammatory endothelium and subsequent transendothelial migration. This can explain how lymphocytes overcome tight adhesion to the vascular endothelium and start rapid migration along and through the endothelial lining of blood vessels into inflammatory tissue.     

Detection of sequence variants in the gene encoding the β3 chain of laminin 5 (LAMB3)

Laminin 5, a candidate gene/protein system for mutations in the junctional forms of epidermolysis bullosa (JEB), consists of three polypeptides encoded by the LAMA3, LAMB3, and LAMC2 genes. In this study, primer pairs for the amplification of the complete cDNA as well as 22 exons of the LAMB3 gene encoding the entire β3 chain of laminin 5, were established. The primers for amplification of individual exons from genomic DNA were placed at least 50 bp away from the exon-intron borders in the flanking intronic sequences. For amplification of cDNA generated by RT-PCR, eight primer pairs covering overlapping segments of mRNA were used. The amplified sequences were used to study sequence variations of the LAMB3 gene in patients with JEB and unrelated individuals using heteroduplex analysis. Nine out of 13 JEB patients examined showed heteroduplexes in at least one of the PCR products, indicating the existence of two variable alleles in their DNA. Sequence analyses revealed putative pathogenetic mutations in seven of the JEB patients, while four of the heteroduplexes resulted from polymorphisms, reflecting a single basepair substitution. The results demonstrate that this method is useful in the detection of JEB mutations, as well as polymorphisms in the LAMB3 gene. © 1995 Wiley-Liss, Inc.     

The influence of synthetic peptides derived from the laminin α1 chain on hepatocyte adhesion and gene expression

Laminin-111, a heterotrimer composed of the laminin α1, β1, and γ1 chains, has been used as a biomaterial for primary cell culture to maintain cellular functions. Our previous studies have reported that synthetic peptides derived from laminin α1 exhibit biological functions such as influencing cell adhesion, migration, angiogenesis, and tumor metastasis. In this study we screened hepatocyte attachment peptides using twenty-five biologically active peptides from laminin α1 and examined the maintenance of hepatic function on the peptides using primary rat hepatocytes. Peptide A13 (RQVFQVAYIIIKA), mouse laminin α1 chain residues 121–133, exhibited the strongest activity. Furthermore, primary hepatocytes on A13 peptide maintained expression of hepatic differentiation markers such as tyrosine aminotransferase, tryptophan-2,3-dioxygenase, and cytochrome P450. We also determined the active core sequence of A13 using systematically truncated N- and C-terminal peptides. The results indicated that the nine-amino acid sequence RQVFQVAYI was critical for A13's hepatocyte adhesion activity. However, the truncated peptides could not interact with β1-intgerin and maintain expression of hepatic differentiation markers. The amino acid sequence of A13 peptide was required for regulating hepatocyte behavior. The hepatocyte adhesive peptides can be utilized in tailoring synthetic biomaterials in order to achieve a specific cellular response.     

The α4β1 integrin in sickle cell disease

The α4β1 integrin is an adhesion receptor expressed on reticulocytes in sickle cell disease (SCD) and mediates the adhesion of these cells to sub-endothelial matrix proteins and the endothelium. In this review, we describe the mechanism of activation of the α4β1 integrin on sickle reticulocytes and discuss novel roles for this integrin in SCD as a result of this activation. We also illustrate novel therapies in SCD that may target the integrin and alleviate vaso-occlusion.     

Coordinate expression of β1 and β2 integrin “activation” epitopes during T cell responses in secondary lymphoid tissue

The monoclonal antibodies (mAb) 15/7 and 24 recognize unique activation-dependent, conformational epitopes on β1 and β 2-integrins, respectively. The expression of both of these epitopes closely correlates with the ligand binding ability of their respective integrins, and thus serves as indicators of functional integrin “activation”. Here, we have used six-parameter flow cytometry to examine the expression of these epitopes and conventional β1- and β2-integrin epitopes during human T cell activation in secondary lymphoid tissues in vivo, focusing particularly on the virgin to memory/effector cell transition. Fresh tonsil lymphocytes were stained with mAb against conventional or activation-dependent integrin epitopes, followed by staining with mAb against CD3, CD45RA, and CD45RO, thus allowing the determination of integrin epitope expression on virgin (CD3⁺) T cells (CD45RA⁺/RO^?to±), memory/effector (CD45RA^?/RO⁺⁺) T cells, and T cells undergoing the virgin to memory/effector transition: transition region-1 (T1; CD45RA^+to++/RO⁺); -2 (T2; CD45RA⁺⁺/RO⁺⁺); and -3 (T3; CD45RA⁺/RO⁺⁺). Conventional β1- and β2-integrin epitopes progressively increase during the virgin to T3 stages of the transition in tonsil, in keeping with the generally higher levels of these adhesion molecules on memory/effector vs. virgin T cells. Expression of both the β1 (15/7)-and β2 (24)-integrin activation epitopes first appears on transitional T cells, and is maintained on a relatively constant number of cells (averaging 25-30%) throughout the T1-T3 stages. These epitopes are also noted on a subset of activated memory/effector T cells. Importantly, on both transitional and activated memory/effector T cell subsets, the expression patterns of the 15/7 and 24 epitopes vs. a variety of T cell activation antigens are identical, and the expression of these epitopes relative to each other is linearly correlated, findings strongly supporting the coordinate activation of β1 and β2 integrins duringT cell activation in vivo. These results provide the first evidence of integrin activation during an in vivo immunologic response, and demonstrate the usefulness of mAb recognizing conformational epitopes and multiparameter flow cytometry in delineating the dynamic interplay of adhesion molecules during complex physiologic processes.     

Loss of α6 and β4 integrin subunits coincides with loss of basement membrane components in oral squamous cell carcinomas

In oral squamous cell carcinomas, focal or extensive loss of basement membrane components and of integrins has been reported. The purpose of this study was to investigate whether those regions of the tumour-connective tissue interface which lack laminin and type IV collagen coincide with areas of loss of the α₆ and β₄ integrin subunits on basal keratinocytes. Out of a total of 15 poor and moderately or well differentiated squamous cell carcinomas, all showed some loss or fragmentation of basement membrane proteins and in 12 the loss was coincident with loss of α₆ and/or β₄. In three cases, there was loss of basal integrin expression in areas where the basement membrane remained intact. These results provide further evidence that loss of integrins may play an important role in tumour progression and prompt us to speculate about the sequence of events leading to tumour invasion.     

Sequence analysis of rat integrin αE1 and αE2 subunits: Tissue expression reveals phenotypic similarities between intraepithelial lymphocytes and dendritic cells in lymph

The integrin α_OX-62 subunit is defined by the OX-62 monoclonal antibody that was raised against rat dendritic cells in lymph (veiled cells) and shows properties similar to those of human α_E2 that is predominantly expressed on intraepithelial lymphocytes. To clone α_OX-62, rat probes generated using primers specific for the human α_E sequence were used to screen rat T cell cDNA libraries. cDNA clones encoding two similar but not identical α subunits that are closely related to but distinct from human α_E were isolated. α_E1 is predicted to be the rat homolog of mouse α_M290 and α_E2 corresponds to rat α_OX-62. Immunofluorescence analysis revealed that mouse α_E1 and rat α_E2 are expressed in dendritic epidermal T cells in the skin, intraepithelial lymphocytes in the small intestine and in cells with a dendritic morphology present at sites where γδ T cells occur in lymphoid organs. Unexpectedly, α_E2 is co-expressed with intracellular CD3-δ and a 33-kDa CD3 chain but not the T cell receptor in veiled cells. These findings suggest that veiled cells may be derived from a lymphoid precursor. Furthermore, veiled cells show phenotypic similarities to intraepithelial lymphocytes.     

Transendothelial chemotaxis of human αβ and γδ T lymphocytes to chemokines

Two subpopulations of human T lymphocytes expressing different antigen receptors, α / β and γ / δ, emigrate into inflamed tissues in distinctive patterns. We compared the transmigration of α / β and γ / δ T cells to C-C and C-X-C chemokines using an in vitro transendothelial chemotaxis assay. The C-C chemokines monocyte chemoattractant protein (MCP)-1, RANTES, macrophage inflammatory protein (MIP)-1α and MIP-1β stimulated similar, dose-dependent chemotaxis of purified γ / δ T cells, whereas MCP-1, RANTES, and MIP-1α pro duced greater chemotaxis of purified α / β T cells than MIP-1β. In contrast, the C-X-C chemokines interleukin (IL)-8 and interferon-γ inducible protein-10 (IP-10) did not promote chemotaxis of either α / β or γ / δ T cells. Three γ / δ T cell clones with differing CD4 and CD8 phenotypes also migrated exclusively to C-C chemokines. Phenotypic analysis of mononuclear cells that transmigrated from an input population of unfractionated peripheral blood mono nuclear cells confirmed the results with purified γ / δ T cells. Our data demonstrate that human peripheral blood α / β and γ / δ T cells can transmigrate to MCP-1, RANTES, MIP-1α, and MIP-1β, and suggest that both T lymphocyte subpopulations share the capacity to emigrate in response to C-C chemokines during inflammation.     

LPAM-1 (integrin α4β7)-ligand binding: overlapping binding sites recognizing VCAM-1, MAdCAM-1 and CS-1 are blocked by fibrinogen,a fibronectin-like polymer and RGD-like cyclic peptides

The α4 integrin LPAM-1 (α4β7) mediates lymphocyte attachment within the extracellular matrix (ECM) by adhering to the connecting segment (CS)-1 site of fibronectin (FN). Here we reveal that very late antigen (VLA)-4⁻ LPAM-1⁺ T cell lymphoma TK-1 cells bind via LPAM-1 to multiple copies of the RGD sequence engineered within an FN-like polymer. Further, the small conformationally restrained RGD-like cyclic peptides 1-adamantaneacetyl-Cys-Gly-Arg-Gly-Asp-Ser-Pro-Cys and Arg-Cys-Asp-thioproline-Cys inhibit the adhesion of TK-1 cells to immobilized CS-1 peptide, and to endothelial counterreceptors for LPAM-1, namely mucosal addressin cell adhesion molecule (MAdCAM)-1 and vascular cell adhesion molecule (VCAM)-1. Spontaneous adhesion of the VLA-4⁻ LPAM-1⁺ B lymphoma cell line RPMI 8866 to CS-1 was likewise inhibited, confirming a previously undocumented ability of LPAM-1 to recognize the RGD tripeptide. The RGD-binding site in LPAM-1 either overlaps or is identical to sites required for interaction with MAdCAM-1, VCAM-1, and the CS-1. The binding of LPAM-1 and VLA-4 to RGD-containing ligands may have relevance in vivo given that fibrinogen at physiological concentrations is able to partially block the binding of TK-1 cells to MAdCAM-1. Hence fibrinogen and other vascular RGD-containing proteins may have mild anti-inflammatory activity required for maintaining effective homeostasis, analogous to the anti-thrombogenic activity of the vascular endothelium.     

Migration of encephalitogenic CD8 T cells into the central nervous system is dependent on the α4β1‐integrin

Although CD8 T cells are key players in neuroinflammation, little is known about their trafficking cues into the central nervous system (CNS). We used a murine model of CNS autoimmunity to define the molecules involved in cytotoxic CD8 T‐cell migration into the CNS. Using a panel of mAbs, we here show that the α4β1‐integrin is essential for CD8 T‐cell interaction with CNS endothelium. We also investigated which α4β1‐integrin ligands expressed by endothelial cells are implicated. The blockade of VCAM‐1 did not protect against autoimmune encephalomyelitis, and only partly decreased the CD8⁺ T‐cell infiltration into the CNS. In addition, inhibition of junctional adhesion molecule‐B expressed by CNS endothelial cells also decreases CD8 T‐cell infiltration. CD8 T cells may use additional and possibly unidentified adhesion molecules to gain access to the CNS.