Interleukin-1 receptor-like proteins synthesized by translation, in vitro, of immunopurified polysomal messenger RNA

Interleukin-1 (IL-1) receptors can be solubilized from murine cell surfaces and immunoprecipitated with a xenogeneic rat antiserum raised in this laboratory. We demonstrated first that this antiserum contains antibodies directed against IL-1 receptors. We have now successfully used this antiserum as a reagent to immunopurify polysomes along with their messenger RNA from a murine leukemic cell line known to express relatively high levels of IL-1 receptors. The immunoselected mRNA was translated into proteins in vitro. The translation products contained an IL-1 binding protein which could specifically bind to immobilized IL-1 but not to other immobilized ligands such as interleukin-2 or tumor necrosis factor-alpha. The translation products which bound to IL-1 could be acid-eluted from the immobilized ligand, and the proteins released could still specifically bind to IL-1 in a receptor-ligand binding reaction. The eluted IL-1 binding proteins, as well as soluble receptor-ligand complexes derived from them, could also be immunoprecipitated with the xenogeneic rat antiserum. The xenogeneic rat antiserum could, furthermore, immunoprecipitate the IL-1 binding proteins from the translated products before ligand was added. The residual translated products no longer interacted with IL-1. We conclude that our antiserum contains antibodies that recognize determinants expressed on the following proteins: on nascent chains of IL-1 binding proteins; on soluble translated IL-1 binding proteins; on soluble complexes of IL-1 binding proteins that had been cross-linked with IL-1 ligand; and on cell surface-associated IL-1 receptors. The translated and unprocessed IL-1 binding proteins have a molecular mass of approximately 52,000-56,000 daltons.     

Monospecific and common glycoprotein ligands for E- and P-selectin on myeloid cells

E- and P-selectin are inducible cell adhesion molecules on endothelial cells, which function as Ca(2+)-dependent lectins and mediate the binding of neutrophils and monocytes. We have recently identified a 150- kD glycoprotein ligand for E-selectin on mouse myeloid cells, using a recombinant antibody-like form of mouse E-selectin. Here, we report that this ligand does not bind to an analogous P-selectin fusion protein. Instead, the chimeric P-selectin-IgG protein recognizes a 160- kD glycoprotein on the mouse neutrophil progenitor 32D cl 3, on mature mouse neutrophils and on human HL60 cells. The binding is Ca(2+)- dependent and requires the presence of sialic acid on the ligand. This P-selectin-ligand is not recognized by E-selectin. Removal of N-linked carbohydrate side chains from the 150-kD and the 160-kD monospecific selectin ligands abolishes the binding of both ligands to the respective selectin. Treatment of HL60 cells with Peptide: N- glycosidase F inhibited cell binding to P- and E-selectin. In addition, glycoproteins of 230 and 130 kD were found on mature mouse neutrophils, which bound both to E- and P-selectin in a Ca(2+)-dependent fashion. The signals detected for these ligands were 15-20-fold weaker than those for the monospecific ligands. Both proteins were heavily sialylated and selectin-binding was blocked by removal of sialic acid, but not by removal of N-linked carbohydrates. Our data reveal that E- and P-selectin recognize two categories of glycoprotein ligands: one type requires N-linked carbohydrates for binding and is monospecific for each of the two selectins and the other type binds independent of N- linked carbohydrates and is common for both endothelial selectins.     

The kinetics of S phase entry by FMP2.1: effect of IL-3 and GM-CSF receptor expression and ligand affinity

FMP2.1, a cloned cell line which has morphological characteristics of mast/basophil cells, requires either interleukin 3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) for both survival and proliferation. IL-3 and GM-CSF were equally effective as proliferative stimuli. FMP2.1 cells were sensitive to growth factor stimulation in the G1 phase, which has a duration of 9.5 h. G1 cells were selected from FMP2.1 in log phase growth on the basis of Hoechst 33324 staining using a fluorescence activated cell sorter (FACS). It was found that G1 phase cells had to be exposed to either IL-3 or GM-CSF for approximately 1 h for cells to enter S (greater than 20%); without growth factor, FMP2.1 remained in G1 unable to progress into S. Receptor expression was analyzed to further understand this rapid activation of FMP2.1 into cycle. Autoradiography using either 125I-IL-3 or 125I-GM-CSF showed that most cells express both receptor types. In the presence of saturating concentrations of IL-3, FMP2.1 have a relatively high number of IL-3 receptors (42,000/cell) compared to other cell lines (e.g., 32D cl23; 13,000 receptors/cell), and far outnumber GM-CSF receptors on the same cells (600 receptors/cell). Although average IL-3 receptor expression differed for FMP2.1- and IL-3-dependent 32D cl23, the concentration-dependent proliferative response to IL-3 was essentially identical for both cell types. Scatchard plot analysis for 125I-IL-3 and 125I-GM-CSF binding to FMP2.1 cells at 4 degrees C revealed a single type of binding site for both ligands, with dissociation constants (Kd) of approximately 1 nM for GM-CSF and 8 pM for IL-3. The relatively high affinity IL-3 binding to a large number of available IL-3 receptors was associated with a shallow dose response of the FMP2.1 cells to IL-3, compared to the steep GM-CSF dose response which was mediated through fewer receptor sites of relatively low affinity. Mitogenic stimulation of G1 phase cells was observed with either IL-3 or GM-CSF, and appeared to be unaffected by differences in receptor number or binding affinity.     

Identification of a glycoprotein ligand for E-selectin on mouse myeloid cells

E-selectin is an inducible endothelial cell adhesion molecule for neutrophils which functions as a Ca(2+)-dependent lectin. Using a recombinant, antibody-like form of mouse E-selectin, we have searched for glycoprotein ligands on mouse neutrophils and the neutrophil progenitor cell line 32D cl 3. We have identified a 150-kD glycoprotein as the only protein which could be affinity-isolated with soluble E- selectin from [35S]methionine/[35S]cysteine-labeled 32D cl 3 cells. Binding of this protein was strictly Ca(2+)-dependent, was blocked by a cell adhesion-blocking mAb against mouse E-selectin, and required the presence of sialic acid on the 150-kD ligand. This glycoprotein was also affinity-isolated from mature neutrophils, in addition to a minor component at 250 kD, but could not be isolated from several other non- myeloid cell lines. The 150-kD glycoprotein was the only protein from 32D cl 3 cells, which was detectable by silver-staining after a one- step affinity-isolation.     

The construction and characterization of a biologically active recombinant IL-2 containing a lysine-rich C-terminal extension

A polylysine extended gibbon IL-2 (IL-2-L) was constructed by the addition of a lysine-rich oligopeptide, Gly3-(Lys-Lys-Asp)3-Leu-Glu to the C terminus of gibbon IL-2 by using rDNA technology. The bioactivity of the hybrid molecule was comparable to that of normal human rIL-2 in assays measuring T cell proliferation, and in generation of lymphokine-activated killer cells from PBL. Furthermore, the addition of the lysine-rich segment made the molecule more amenable to the chemical derivatization of amino groups. After biotinylation, IL-2-L retained a greater proportion of its bioactivity than normal rIL-2. In addition, biotinylated IL-2-L was capable of simultaneously binding to cell surface IL-2R and streptavidin. Thus, the addition of the lysine-rich oligopeptide facilitated the generation of an active form of biotinylated IL-2 which acts as a bridge between IL-2R-positive cells and avidin-coupled reagents and affinity supports. Such a selective means of labeling and binding IL-2-responsive cells may have great practical utility for IL-2-based immunotherapy.     

Epidermal growth factor and betacellulin mediate signal transduction through co-expressed ErbB2 and ErbB3 receptors.

Interleukin-3 (IL-3)-dependent murine 32D cells do not detectably express epidermal growth factor receptors (EGFRs) and do not proliferate in response to EGF, heregulin (HRG) or other known EGF-like ligands. Here, we report that EGF specifically binds to and can be crosslinked to 32D transfectants co-expressing ErbB2 and ErbB3 (32D.E2/E3), but not to transfectants expressing either ErbB2 or ErbB3 individually. [125I]EGF-crosslinked species detected in 32D. E2/E3 cells were displaced by HRG and betacellulin (BTC) but not by other EGF-like ligands that were analyzed. EGF, BTC and HRG also induced receptor tyrosine phosphorylation, activation of downstream signaling molecules and proliferation of 32D.E2/E3 cells. 32D transfectants were also generated which expressed an ErbB3-EGFR chimera alone (32D.E3-E1) or in combination with ErbB2 (32D. E2/E3-E1). While HRG stimulation of 32D.E3-E1 cells resulted in DNA synthesis and receptor phosphorylation, EGF and BTC were inactive. However, EGF and BTC were as effective as HRG in mediating signaling when ErbB2 was co-expressed with the chimera in the 32D.E2/E3-E1 transfectant. These results provide evidence that ErbB2/ErbB3 binding sites for EGF and BTC are formed by a previously undescribed mechanism that requires co-expression of two distinct receptors. Additional data utilizing MDA MB134 human breast carcinoma cells, which naturally express ErbB2 and ErbB3 in the absence of EGFRs, supported the results obtained employing 32D cells and suggest that EGF and BTC may contribute to the progression of carcinomas that co-express ErbB2 and ErbB3.     

Phosphorylation of the beta-galactoside-binding protein galectin-3 modulates binding to its ligands

The beta-galactoside-binding protein galectin-3 has pleiotropic biological functions and has been implicated in cell growth, differentiation, adhesion, RNA processing, apoptosis, and malignant transformation. Galectin-3 may be phosphorylated at N-terminal Ser(6), but the role of phosphorylation in determining interactions of this endogenous lectin with its ligands remains to be elucidated. We therefore studied the effect of phosphorylation on binding of galectin-3 to two of its reported ligands, laminin and purified colon cancer mucin. Human recombinant galectin-3 was phosphorylated in vitro by casein kinase I, and separated from the native species by isoelectric focusing for use in solid phase binding assays. Non-phosphorylated galectin-3 bound to laminin and asialomucin in a dose-dependent manner with half-maximal binding at 1.5 microg/ml. Phosphorylation reduced saturation binding to each ligand by >85%. Ligand binding could be fully restored by dephosphorylation with protein phosphatase type 1. Mutation of galectin-3 at Ser(6) (Ser to Glu) did not alter galectin ligand binding. Metabolic labeling or separation by isoelectric focusing confirmed the presence of phosphorylated galectin-3 species in vivo in the cytosol of human colon cancer cells from which ligand mucin was purified. Phosphorylation significantly reduces the interaction of galectin-3 with its ligands. The process by which phosphorylation modulates protein-carbohydrate interactions has important implications for understanding the biological functions of this protein, and may serve as an "on/off" switch for its sugar binding capabilities.     

Molecular events associated with CD4-mediated Down-regulation of LFA-1-dependent adhesion.

We have previously shown that CD4 ligand binding inhibits LFA-1-dependent adhesion between CD4+ T cells and B cells in a p56(lck)- and phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. In this work, downstream events associated with adhesion inhibition have been investigated. By using HUT78 T cell lines, CD4 ligands were shown to induce a dissociation of LFA-1 from cytohesin, a cytoplasmic protein known to bind LFA-1 and to enhance the affinity/avidity of LFA-1 for its ligand ICAM-1. A dissociation of PI3-kinase from cytohesin is also observed. In parallel, we have found that CD4 ligand binding induced a redistribution of PI3-kinase and of the tyrosine phosphatase SHP-2 to the membrane and induced a transient formation of protein interactions including PI3-kinase; an adaptor protein, Gab2; SHP-2; and a SH2 domain-containing inositol phosphatase, SHIP. By using antisense oligonucleotides or transfection of transdominant mutants, down-regulation of adhesion was shown to require the Gab2/PI3-kinase association and the expression of SHIP and SHP-2. We therefore propose that CD4 ligands, by inducing these molecular associations, lead to sustained local high levels of D-3 phospholipids and possibly regulate the cytohesin/LFA-1 association.     

Visualization of intracellular trafficking: use of biotinylated ligands in conjunction with avidin-gold colloids

A simple, sensitive method to visualize the binding and internalization of protein ligands by cells in culture is described. A biotinylated toxin was used as ligand, and succinoylated avidin adsorbed onto 5.2 nm gold sols was the electron-dense marker. This method affords direct localization of proteins that are on the cell surface or intracellular without need for techniques that alter membrane integrity.     

Making sense of the diverse ligand recognition by NKG2D

NKG2D recognizes multiple diverse ligands. Despite recent efforts in determining the crystal structures of NKG2D-ligand complexes, the principle governing this receptor-ligand recognition and hence the criteria for identifying unknown ligands of NKG2D remain central issues to be resolved. Here we compared the molecular recognition between NKG2D and three of the known ligands, UL16 binding protein (ULBP), MHC class I-like molecule, and retinoic acid early inducible gene as observed in the ligand-complexed crystal structures. The comparison shows that while the receptor uses a common interface region to bind the three diverse ligands, each ligand forms a distinct, but overlapping, set of hydrogen bonds, hydrophobic interactions, and salt bridges, illustrating the underlying principle of NKG2D-ligand recognition being the conservation in overall shape complementarity and binding energy while permitting variation in ligand sequence through induced fit recognition. To further test this hypothesis and to distinguish between diverse recognition and promiscuous ligand binding, four ULBP3 interface mutations, H21A, E76A, R82M, and D169A, were generated to each disrupt a single hydrogen bond or salt bridge. All mutant ULBP3 displayed reduced receptor binding, suggesting a specific, rather than promiscuous, receptor-ligand recognition. Mutants with severe loss of binding affect the receptor interactions that are mostly buried. Finally, a receptor-ligand recognition algorithm was developed to assist the identification of diverse NKG2D ligands based on evaluating the potential hydrogen bonds, hydrophobic interactions, and salt bridges at the receptor-ligand interface.     

Hydrogen/deuterium exchange reveals distinct agonist/partial agonist receptor dynamics within vitamin D receptor/retinoid X receptor heterodimer

Regulation of nuclear receptor (NR) activity is driven by alterations in the conformational dynamics of the receptor upon ligand binding. Previously, we demonstrated that hydrogen/deuterium exchange (HDX) can be applied to determine novel mechanism of action of PPARγ ligands and in predicting tissue specificity of selective estrogen receptor modulators. Here, we applied HDX to probe the conformational dynamics of the ligand binding domain (LBD) of the vitamin D receptor (VDR) upon binding its natural ligand 1α,25-dihydroxyvitamin D3 (1,25D3), and two analogs, alfacalcidol and ED-71. Comparison of HDX profiles from ligands in complex with the LBD with full-length receptor bound to its cognate receptor retinoid X receptor (RXR) revealed unique receptor dynamics that could not be inferred from static crystal structures. These results demonstrate that ligands modulate the dynamics of the heterodimer interface as well as provide insight into the role of AF-2 dynamics in the action of VDR partial agonists.     

Computational model for effects of ligand/receptor binding properties on interleukin-2 trafficking dynamics and T cell proliferation response

Multisubunit cytokine receptors such as the heterotrimeric receptor for interleukin-2 (IL-2) are ubiquitous in hematopoeitic cell types of importance in biotechnology and are crucial regulators of cell proliferation and differentiation behavior. Dynamics of cytokine/receptor endocytic trafficking can significantly impact cell responses through effects of receptor down-regulation and ligand depletion, and in turn are governed by ligand/receptor binding properties. We describe here a computational model for trafficking dynamics of the IL-2 receptor (IL-2R) system, which is able to predict T cell proliferation responses to IL-2. This model comprises kinetic equations describing binding, internalization, and postendocytic sorting of IL-2 and IL-2R, including an experimentally derived dependence of cell proliferation rate on these properties. Computational results from this model predict that IL-2 depletion can be reduced by decreasing its binding affinity for the IL-2R betagamma subunit relative to the alpha subunit at endosomal pH, as a result of enhanced ligand sorting to recycling vis-à-vis degradation, and that an IL-2 analogue with such altered binding properties should exhibit increased potency for stimulating the T cell proliferation response. These results are in agreement with our recent experimental findings for the IL-2 analogue termed 2D1 [Fallon, E. M. et al. J. Biol. Chem. 2000, 275, 6790-6797]. Thus, this type of model may enable prediction of beneficial cytokine/receptor binding properties to aid development of molecular design criteria for improvements in applications such as in vivo cytokine therapies and in vitro hematopoietic cell bioreactors.     

Biochemical and functional comparisons of Mcl-1 and Bcl-2 proteins: evidence for a novel mechanism of regulating Bcl-2 family protein function

Mcl-1 is a recently described homologue of Bcl-2 whose function and biochemical characteristics remain poorly defined. Gene transfer experiments in lnterleukin-3 (IL-3)-dependent myeloid progenitor 32D.3 cells and pro-B-lymphoid FL5.12 cells demonstrated that enforced production of high levels of Mcl-1 protein failed to prolong the survival of cells when cultured in the absence of IL-3, whereas Bcl-2 did delay cell death. Mcl-1 also did not prolong the survival in vitro of 32D.3 cells that had been induced to differentiate into mature neutrophils using Granulocyte-Colony Stimulating Factor (G-CSF), whereas Bcl-2 did. 32D.3 and FL5.12 cells co-transfected with Mcl-1 and Bcl-2 displayed survival kinetics essentially identical to cells transfected with Bcl-2 alone, when cultured in the absence of IL-3, indicating that Mcl-1 neither enhances nor impairs Bcl-2 function. In contrast to the lack of effects of Mcl-1 in 32D.3 and FL5.12 cells, Mcl-1 (like Bcl-2) was able to neutralise Bax-induced cytotoxicity in yeast (S. cerevisiae). Moreover, the recombinant GST-Mcl-1 protein bound specifically to in vitro translated Bax protein, as well as to Bax protein present in detergent lysates prepared from 32D.3 and FL5.12 cells, based on in vitro binding assays. However, Mcl-1 and Bax proteins could not be co-immunoprecipitated from control and transfected 32D.3 and FL5.12 cells, whereas Bcl-2 and Bax were easily co-immunoprecipitated under the same conditions. The findings suggest that while Mcl-1 has the capacity to bind to and neutralise the cell death promoting activity of Bax, other factors such as perhaps additional proteins or undefined post-translational modifications may influence its ability to bind to Bax in vivo and thus affect its function as a cell death blocker.     

Soluble Interleukin-5 Receptor α-Chain Binding Assays: Use for Screening and Analysis of Interleukin-5 Mutants

Interleukin-5 (IL-5) is a key cytokine for the production, differentiation, and activation of eosinophils. IL-5 is a member of the four helical bundle family of cytokines, and in common with many members of the cytokine family it binds to a heterodimeric receptor composed of a ligand binding α-chain and a signal-transducing β-chain. We have established two receptor/ligand binding assays based on the extracellular domain of the receptor α-chain which we have produced as a fusion protein. One assay is based on scintillation proximity fluoromicrospheres and radiolabeled ligand and the other on detection of biotinylated ligand binding to immobilized receptor using a chemiluminescent substrate in a 96-well microtiter plate format. Both receptor binding assays have been optimized for high throughput screening for receptor antagonists. These assays were also used for analytical purposes and the binding of ligand to the receptor α-chain was compared directly to receptor binding assays performed on TF-1 cells which express the receptor αβ-heterodimer. These three assays have been used to study site-directed mutants of IL-5 to determine the important residues for interaction of the cytokine with each chain of the receptor (P. Graber et al. (1995) J. Biol. Chem. 270, 15762-15769).     

Structure of the HCMV UL16-MICB Complex Elucidates Select Binding of a Viral Immunoevasin to Diverse NKG2D Ligands

The activating immunoreceptor NKG2D promotes elimination of infected or malignant cells by cytotoxic lymphocytes through engagement of stress-induced MHC class I-related ligands. The human cytomegalovirus (HCMV)-encoded immunoevasin UL16 subverts NKG2D-mediated immune responses by retaining a select group of diverse NKG2D ligands inside the cell. We report here the crystal structure of UL16 in complex with the NKG2D ligand MICB at 1.8 Å resolution, revealing the molecular basis for the promiscuous, but highly selective, binding of UL16 to unrelated NKG2D ligands. The immunoglobulin-like UL16 protein utilizes a three-stranded β-sheet to engage the α-helical surface of the MHC class I-like MICB platform domain. Intriguingly, residues at the center of this β-sheet mimic a central binding motif employed by the structurally unrelated C-type lectin-like NKG2D to facilitate engagement of diverse NKG2D ligands. Using surface plasmon resonance, we find that UL16 binds MICB, ULBP1, and ULBP2 with similar affinities that lie in the nanomolar range (12–66 nM). The ability of UL16 to bind its ligands depends critically on the presence of a glutamine (MICB) or closely related glutamate (ULBP1 and ULBP2) at position 169. An arginine residue at this position however, as found for example in MICA or ULBP3, would cause steric clashes with UL16 residues. The inability of UL16 to bind MICA and ULBP3 can therefore be attributed to single substitutions at key NKG2D ligand locations. This indicates that selective pressure exerted by viral immunoevasins such as UL16 contributed to the diversification of NKG2D ligands.     

Synthesis and evaluation of fluorine-substituted 1H-pyrrolo[2,3-b]pyridine derivatives for dopamine D4 receptor imaging

Seven fluorine-substituted 1H-pyrrolo[2,3-b]pyridine derivatives were synthesized based on a lead ligand, 3-[[4-(4-iodophenyl)piperazin-1-yl]-methyl]-1H-pyrrolo[2,3-b]pyridine (L-750,667) and evaluated as potential dopamine D(4) receptor imaging agents by positron emission tomography (PET). Binding affinities of these ligands for the dopamine D(2), D(3), and D(4) receptor subtypes were measured in vitro. Most ligands showed high and selective binding for the D(4) receptor. Ligand 7 had high affinity for the D(4) receptor, whereas ligands 1, 2, and 6 showed high selectivity for the D(4) receptor. LogP values were calculated for the ligands in this series and ligand 6 had the lowest lipophilicity. (18)F-labeled ligand 7 demonstrated a uniform regional brain distribution and a rapid washout in mice, probably due to nonspecific binding. Based on their in vitro binding properties and calculated logP values, ligand 6 appears to have the most promise for dopamine D(4) receptor imaging.     

The kit ligand: a cell surface molecule altered in steel mutant fibroblasts.

The c-kit proto-oncogene, the gene at the mouse W developmental locus, is one of a substantial group of genes that appear to encode cell surface receptors but for which the ligands are unknown. We have characterized the kit ligand by a generally applicable approach: the receptor extracellular domain was genetically fused to placental alkaline phosphatase, producing a soluble receptor affinity reagent with an enzyme tag that could be easily and sensitively traced. This fusion protein, APtag-KIT, was used to demonstrate a specific binding interaction (KD = 3 x 10(-8) M) with a ligand on 3T3 fibroblast lines. In situ staining showed labeling over the whole surface of the 3T3 cells, but not extending to adjacent nonexpressing cells. These findings provide direct molecular evidence that the kit ligand can exist as a cell surface protein. Binding was not detected on 3T3 fibroblasts carrying the steel (Sl) mutation, confirming the biological significance of the binding activity and demonstrating that mutations at the Sl locus affect the expression or structure of the kit ligand.     

Antibodies against the CD44 p80, lymphocyte homing receptor molecule augment human peripheral blood T cell activation

The CD44 inhibitor Lutheran [In(Lu)]-related p80 molecule has recently been shown to be identical to the Hermes-1 lymphocyte homing receptor and to the human Pgp-1 molecule. We have determined the effect of addition of CD44 antibodies to in vitro activation assays of PBMC. CD44 antibodies did not induce PBMC proliferation alone, but markedly enhanced PBMC proliferation induced by a mitogenic CD2 antibody pair or by CD3 antibody. CD44 antibody addition had no effect upon PBMC activation induced by PHA or tetanus toxoid. CD44 antibody enhancement of CD2 antibody-induced T cell activation was specific for mature T cells as thymocytes could not be activated in the presence of combinations of CD2 and CD44 antibodies. CD44 antibody enhancement of CD2-mediated T cell triggering occurred if CD44 antibody was placed either on monocytes or on T cells. In experiments with purified monocyte and T cell suspensions, CD44 antibodies A3D8 and A1G3 augmented CD2-mediated T cell activation by three mechanisms. First, CD44 antibody binding to monocytes induced monocyte IL-1 release, second, CD44 antibodies enhanced the adhesion of T cells and monocytes in CD2 antibody-stimulated cultures, and third, CD44 antibodies augmented T cell IL-2 production in response to CD2 antibodies. Thus, ligand binding to CD44 molecules on T cells and monocytes may regulate numerous events on both cell types that are important for T cell activation. Given that recent data suggest that the CD44 molecule may bind to specific ligands on endothelial cells (vascular addressin) and within the extracellular matrix (collagen, fibronectin), these data raise the possibility that binding of T cells to endothelial cells or extracellular matrix proteins may induce or up-regulate T cell activation in inflammatory sites.     

Ligand binding to the LFA-3 cell adhesion molecule induces IL-1 production by human thymic epithelial cells.

We have shown that human thymic epithelial (TE) cells produce IL-1 alpha, IL-1 beta, and TE cells bind to thymocytes by CD2 and LFA-1 molecules on thymocytes and LFA-3, ICAM-1 on TE cells. We investigated whether ligand binding to LFA-3 on human TE cells can modulate TE cell IL-1 production. First, we investigated the ability of human thymocytes to regulate IL-1 release by TE cells. Both autologous and allogenic emetine-treated thymocytes when cultured with TE cells augmented IL-1 release by TE cells. The augmentation of IL-1 release was cell density dependent. Inasmuch as the interaction between thymocytes and TE cells is mediated in part by CD2 molecules on thymocytes and LFA-3 molecules on TE cells we next determined the effect on IL-1 release of ligand binding (anti-LFA-3 mAb TS2/9) to TE cell surface LFA-3. Purified anti-LFA-3 mAb augmented IL-1 release in a concentration-dependent fashion. The anti-LFA-3-mediated augmentation of IL-1 release required both new protein and RNA synthesis as shown by the ability of cycloheximide and actinomycin-D to inhibit augmentation of IL-1 production by TE cells, and by direct quantitation of IL-1 alpha and IL-1 beta mRNA by Northern blot analysis. Both F(ab)'2 and Fab' fragments of anti-LFA-3 mAb augmented IL-1 alpha and IL-1 beta mRNA production, indicating that monovalent binding to cell surface LFA-3 was sufficient to provide the inducing signal. The identification of LFA-3, the cell surface ligand for thymocyte CD2 molecules, as a molecule via which TE cell-derived cytokine production may be regulated suggests a mechanism at the cell surface by which direct TE cell-thymocyte interaction might result in the triggering of local IL-1 release within the human thymic microenvironment.     

Intervesicle cross-linking with integrin alpha IIb beta 3 and cyclic-RGD-lipopeptide. A model of cell-adhesion processes

We report the synthesis of a new integrin alpha(IIb)beta(3)-specific cyclic hexapeptide that contains an Arg-Gly-Asp (RGD) sequence and is coupled to a dimyristoylthioglyceryl anchor. We demonstrate that this ligand is useful to study specific integrin binding to membrane surfaces. With the help of biotinylated analogues of the peptide, a spacer of optimal length between the peptide and lipid moieties was searched for by evaluating the binding strength with an enzyme-coupled immunosorbent assay (ELISA) and by surface plasmon resonance (SPR). It was found to be strongly dependent on the length of the spacer introduced between the biotin and peptide moieties of the ligands, which consisted either of epsilon-aminohexanoic acid (epsilonAhx) or of epsilonAhx with two additional glycine units. Best results were obtained with c[Arg-Gly-Asp-D-Phe-Lys(Biot-Ahx-Gly-Gly)-Gly-] with dissociation constants of K(D) = 0.158 microM from ELISA and K(D) = 1.1 microM from SPR measurements. The analogous lipopeptide, c[Arg-Gly-Asp-D-Phe-Lys([dimyristoyl-3-thioglyceryl-succinimido -propanoyl]Ahx-Gly-Gly)-Gly], was used as a membrane-anchored integrin ligand. It is shown by fluorescence microscopy and cryo electron microscopy that integrin reconstituted into phospholipid vesicles binds to vesicles decorated with the lipopeptide, forming regularly spaced bridges between the two kinds of vesicles. The novel integrin-specific ligand allows establishment of new model systems for systematic studies of the self-organization of integrin clusters and focal adhesion complexes.