N-glycan structures and N-glycosylation sites of mouse soluble intercellular adhesion molecule-1 revealed by MALDI-TOF and FTICR mass spectrometry

Intercellular adhesion molecule-1 (ICAM-1) is a heavily N-glycosylated transmembrane protein comprising five extracellular Ig-like domains. The soluble isoform of ICAM-1 (sICAM-1), consisting of its extracellular part, is elevated in the cerebrospinal fluid of patients with severe brain trauma. In mouse astrocytes, recombinant mouse sICAM-1 induces the production of the CXC chemokine macrophage inflammatory protein-2 (MIP-2). MIP-2 induction is glycosylation dependent, as it is strongly enhanced when sICAM-1 carries sialylated, complex-type N-glycans as synthesized by wild-type Chinese hamster ovary (CHO) cells. The present study was aimed at elucidating the N-glycosylation of mouse sICAM-1 expressed in wild-type CHO cells with regard to sialylation, N-glycan profile, and N-glycosylation sites. Ion-exchange chromatography and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) of the released N-glycans showed that sICAM-1 mostly carried di- and trisialylated complex-type N-glycans with or without one fucose. In some sialylated N-glycans, one N-acetylneuraminic acid was replaced by N-glycolylneuraminic acid, and approximately 4% carried a higher number of sialic acid residues than of antennae. The N-glycosylation sites of mouse sICAM-1 were analyzed by MALDI-Fourier transform ion cyclotron resonance (FTICR)-MS and nanoLC-ESI-FTICR-MS of tryptic digests of mouse sICAM-1 expressed in the Lec1 mutant of CHO cells. All nine consensus sequences for N-glycosylation were found to be glycosylated. These results show that the N-glycans that enhance the MIP-2-inducing activity of mouse sICAM-1 are mostly di- and trisialylated complex-type N-glycans including a small fraction carrying more sialic acid residues than antennae and that the nine N-glycosylation sites of mouse sICAM-1 are all glycosylated.     

Mouse large can modify complex N- and mucin O-glycans on alpha-dystroglycan to induce laminin binding

The human LARGE gene encodes a protein with two putative glycosyltransferase domains and is required for the generation of functional alpha-dystroglycan (alpha-DG). Monoclonal antibodies IIH6 and VIA4-1 recognize the functional glycan epitopes of alpha-DG that are necessary for binding to laminin and other ligands. Overexpression of full-length mouse Large generated functionally glycosylated alpha-DG in Pro(-5) Chinese hamster ovary (CHO) cells, and the amount was increased by co-expression of protein:O-mannosyl N-acetylglucosaminyltransferase 1. However, functional alpha-DG represented only a small fraction of the alpha-DG synthesized by CHO cells or expressed from an alpha-DG construct. To identify features of the glycan epitopes induced by Large, the production of functionally glycosylated alpha-DG was investigated in several CHO glycosylation mutants. Mutants with defective transfer of sialic acid (Lec2), galactose (Lec8), or fucose (Lec13) to glycoconjugates, and the Lec15 mutant that cannot synthesize O-mannose glycans, all produced functionally glycosylated alpha-DG upon overexpression of Large. Laminin binding and the alpha-DG glycan epitopes were enhanced in Lec2 and Lec8 cells. In Lec15 cells, functional alpha-DG was increased by co-expression of core 2 N-acetylglucosaminyltransferase 1 with Large. Treatment with N-glycanase markedly reduced functionally glycosylated alpha-DG in Lec2 and Lec8 cells. The combined data provide evidence that Large does not transfer to Gal, Fuc, or sialic acid on alpha-DG nor induce the transfer of these sugars to alpha-DG. In addition, the data suggest that human LARGE may restore functional alpha-DG to muscle cells from patients with defective synthesis of O-mannose glycans via the modification of N-glycans and/or mucin O-glycans on alpha-DG.     

A membranous form of ICAM-1 on exosomes efficiently blocks leukocyte adhesion to activated endothelial cells

While intercellular adhesion molecule-1 (ICAM-1) is a transmembrane protein, two types of extracellular ICAM-1 have been detected in cell culture supernatants as well as in the serum: a soluble form of ICAM-1 (sICAM-1) and a membranous form of ICAM-1 (mICAM-1) associated with exosomes. Previous observations have demonstrated that sICAM-1 cannot exert potent immune modulatory activity due to its low affinity for leukocyte function-associated antigen-1 (LFA-1) or membrane attack complex-1. In this report, we initially observed that human cancer cells shed mICAM-1(+)-exosomes but were devoid of vascular cell adhesion molecule-1 and E-selectin. We demonstrate that mICAM-1 on exosomes retained its topology similar to that of cell surface ICAM-1, and could bind to leukocytes. In addition, we show that exosomal mICAM-1 exhibits potent anti-leukocyte adhesion activity to tumor necrosis factor-α-activated endothelial cells compared to that of sICAM-1. Taken together with previous findings, our results indicate that mICAM-1 on exosomes exhibits potent immune modulatory activity.     

Lymphatic endothelial murine chloride channel calcium-activated 1 is a ligand for leukocyte LFA-1 and Mac-1

The lymphatic circulation mediates drainage of fluid and cells from the periphery through lymph nodes, facilitating immune detection of lymph-borne foreign Ags. The 10.1.1 mAb recognizes a lymphatic endothelial Ag, in this study purified by Ab-affinity chromatography. SDS-PAGE and mass spectrometry identified murine chloride channel calcium-activated 1 (mCLCA1) as the 10.1.1 Ag, a 90-kDa cell-surface protein expressed in lymphatic endothelium and stromal cells of spleen and thymus. The 10.1.1 Ab-affinity chromatography also purified LFA-1, an integrin that mediates leukocyte adhesion to endothelium. This mCLCA1-LFA-1 interaction has functional consequences, as lymphocyte adhesion to lymphatic endothelium was blocked by 10.1.1 Ab bound to endotheliumor by LFA-1 Ab bound to lymphocytes. Lymphocyte adhesion was increased by cytokine treatment of lymphatic endothelium in association with increased expression of ICAM-1, an endothelial surface protein that is also a ligand for LFA-1. By contrast, mCLCA1 expression and the relative contribution of mCLCA1 to lymphocyte adhesion were unaffected by cytokine activation, demonstrating that mCLCA1 and ICAM-1 interactions with LFA-1 are differentially regulated. mCLCA1 also bound to the LFA-1-related Mac-1 integrin that is preferentially expressed on leukocytes. mCLCA1-mediated adhesion of Mac-1- or LFA-1-expressing leukocytes to lymphatic vessels and lymph node lymphatic sinuses provides a target for investigation of lymphatic involvement in leukocyte adhesion and trafficking during the immune response.     

Functional sialylated O-glycan to platelet aggregation on Aggrus (T1alpha/Podoplanin) molecules expressed in Chinese hamster ovary cells

Aggrus, also called T1alpha and podoplanin, is a novel platelet aggregation-inducing factor that is expressed in various carcinoma cells. Aggrus/T1alpha/podoplanin is known to be expressed in lung type I alveolar cells or lymphatic endothelial cells. However, its physiological role has not been clarified. To assess the attribution of glycosylation to Aggrus platelet aggregation activity, recombinant molecules were stably expressed in a series of Chinese hamster ovary (CHO) cell mutants, N-glycan-deficient Lec1, CMP-sialic acid transporter-deficient Lec2, and UDP-galactose transporter-deficient Lec8. A new anti-human Aggrus monoclonal antibody, YM-1, was established to detect the expression of human Aggrus on these CHO cell mutants. Aggrus on Lec1 cells induced platelet aggregation, but those on Lec2 and Lec8 cells did not. Further, the glycans on Aggrus were analyzed by lectin blotting. Aggrus expressed in CHO and Lec1 cells showed Wheat-germ agglutinin, Jacalin, and Vicia villosa lectin bindings. Lectin blotting results indicated that sialylated core 1 structures, sialic acid plus Galbeta1,3GalNAc-Ser/Thr, were critical for the platelet aggregation activity. This oligosaccharide structure is known as tumor-associated antigen, which is potentially related to the metastasis process of cancer cells.     

De novo design, synthesis, and in vitro activity of LFA-1 antagonists based on a bicyclic[5.5]hydantoin scaffold

LFA-1 (leukocyte function-associated antigen-1), is a member of the beta(2)-integrin family and is expressed on all leukocytes. The LFA-1/ICAM interaction promotes tight adhesion between activated leukocytes and the endothelium, as well as between T cells and antigen-presenting cells. Evidence from both animal models and clinical trials provides support for LFA-1 as a target in several different inflammatory diseases. This paper describes the de novo design, synthesis and in vitro activity of LFA-1 antagonists based on a bicyclic[5.5]hydantoin scaffold.     

Binding of the integrin Mac-1 (CD11b/CD18) to the third immunoglobulin-like domain of ICAM-1 (CD54) and its regulation by glycosylation.

Both the integrins LFA-1 and Mac-1 bind to ICAM-1, an immunoglobulin superfamily member. Previously, we localized the binding sites of LFA-1 and the major group of human rhinoviruses to the first NH2-terminal immunoglobulin-like domain of ICAM-1. Here, we show that the binding site on ICAM-1 for Mac-1 is unexpectedly distinct from that for LFA-1 and maps to the third NH2-terminal immunoglobulin-like domain. These findings provide a function for the tandem duplication of immunoglobulin-like domains in ICAM-1 and have implications for other immunoglobulin superfamily members. Mutations at two sites in the third domain that destroy consensus sequences for N-linked glycosylation enhance binding to purified Mac-1. Agents that interfere with carbohydrate processing provide evidence that the size of the N-linked oligosaccharide side chains on ICAM-1 affects binding to Mac-1 but not to LFA-1. Thus, we suggest that the extent of glycosylation on ICAM-1 may regulate adhesion to LFA-1 or Mac-1 in vivo.     

The production of macrophage inflammatory protein-2 induced by soluble intercellular adhesion molecule-1 in mouse astrocytes is mediated by src tyrosine kinases and p42/44 mitogen-activated protein kinase

Severe traumatic brain injury stimulates the release of soluble intercellular adhesion molecule-1 (sICAM-1) into CSF. Studies in cultured mouse astrocytes suggest that sICAM-1 induces the production of macrophage inflammatory protein-2 (MIP-2). In the present study, we investigated the underlying mechanisms for MIP-2 induction. sICAM-1 induced MIP-2 in astrocytes lacking membrane-bound ICAM-1, indicating that its action is due to heterophilic binding to an undescribed receptor rather than homophilic binding to surface ICAM-1. Signal transduction may be mediated by src tyrosine kinases, as the src tyrosine kinase inhibitors herbimycin A and PP2 abolished MIP-2 induction by sICAM-1. Phosphorylation of p42/44 mitogen-activated protein kinase (MAPK), but not of p38 MAPK, occurred further downstream, as evidenced by western blot analysis combined with the use of herbimycin A and specific MAPK inhibitors. By contrast, induction of MIP-2 by tumour necrosis factor-alpha (TNF-alpha) involved both p42/44 MAPK and p38 MAPK. Following stimulation with either sICAM-1 or TNF-alpha, astrocyte supernatants promoted chemotaxis of human neutrophils and incubation of these supernatants with anti-MIP-2 antibodies more efficiently suppressed the migration induced by sICAM-1 than by TNF-alpha. These results show that sICAM-1 induces the production of biologically active MIP-2 in astrocytes by heterophilic binding to an undefined receptor and activation of src tyrosine kinases and p42/44 MAPK.     

Expression of Cell Adhesion Molecules, their Ligands and Tumour Necrosis Factor α in the Liver of Patients with Metastatic Gastrointestinal Carcinomas

The expression of the following cell adhesion molecules, their β1 and β2 integrin ligands and the cytokine tumour necrosis factor-α (TNF-α) was investigated by light and electron microscope immunohistochemistry in the liver tissue in 20 patients with colorectal and gastric cancer also presenting with liver metastases: intercellular adhesion molecule-1 (ICAM-1), vascular endothelial adhesion molecule-1 (VCAM-1), E-selectin, leucocyte function-associated antigen-1 (LFA-1), macrophage antigen-1 (Mac-1), and very late antigen-4 (VLA-4). We have found a parallel enhancement of the adhesion molecules and of TNF-α in liver sinusoids surrounding metastases. The expression of ICAM-1 was enhanced on sinusoidal cells in all zones of the acinus. VCAM-1 immune reactivity was diffuse but less intensive in the lobule. E-selectin expression was observed in sinusoidal cells attached to metastases. In tumour metastases the expression of ICAM-1, VCAM-1, and E-selectin was visible on the tumour vascular endothelium. Tumour infiltrating host cells sowing positive immunoreactivity for ICAM-1, VCAM-1, LFA-1, Mac-1, and VLA-4 were located mainly at the boundary between liver parenchyma and the metastasis. At the ultrastructural level, ICAM-1-positive immune deposits were observed on the cellular membrane and in some transport vesicles of gastric metastatic cells. Further, the expression of all adhesion molecules was confirmed to sinusoidal endothelial cells and tumour vessels. It is concluded that the enhanced expression of adhesion molecules in liver sinusoids could be a marker for the assessment of the ability of sinusoidal endothelial cells to control the recruitment of leukocytes and monocytes to the metastatic site. They could also direct the adhesion of new circulating tumour cells to sinusoidal endothelium.     

ICAM-1 (CD54): a counter-receptor for Mac-1 (CD11b/CD18)

While the leukocyte integrin lymphocyte function-associated antigen (LFA)-1 has been demonstrated to bind intercellular adhesion molecule (ICAM)-1, results with the related Mac-1 molecule have been controversial. We have used multiple cell binding assays, purified Mac- 1 and ICAM-1, and cell lines transfected with Mac-1 and ICAM-1 cDNAs to examine the interaction of ICAM-1 with Mac-1. Stimulated human umbilical vein endothelial cells (HUVECs), which express a high surface density of ICAM-1, bind to immunoaffinity-purified Mac-1 adsorbed to artificial substrates in a manner that is inhibited by mAbs to Mac-1 and ICAM-1. Transfected murine L cells or monkey COS cells expressing human ICAM-1 bind to purified Mac-1 in a specific and dose-dependent manner; the attachment to Mac-1 is more temperature sensitive, lower in avidity, and blocked by a different series of ICAM-1 mAbs when compared to LFA-1. In a reciprocal assay, COS cells cotransfected with the alpha and beta chain cDNAs of Mac-1 or LFA-1 attach to immunoaffinity- purified ICAM-1 substrates; this adhesion is blocked by mAbs to ICAM-1 and Mac-1 or LFA-1. Two color fluorescence cell conjugate experiments show that neutrophils stimulated with fMLP bind to HUVEC stimulated with lipopolysaccharide for 24 h in an ICAM-1-, Mac-1-, and LFA-1- dependent fashion. Because cellular and purified Mac-1 interact with cellular and purified ICAM-1, we conclude that ICAM-1 is a counter receptor for Mac-1 and that this receptor pair is responsible, in part, for the adhesion between stimulated neutrophils and stimulated endothelial cells.     

Control of carbohydrate processing: the lec1A CHO mutation results in partial loss of N-acetylglucosaminyltransferase I activity.

Lec1 CHO cell glycosylation mutants are defective in N-acetylglucosaminyltransferase I (GlcNAc-TI) activity and therefore cannot convert the oligomannosyl intermediate (Man5GlcNAc2Asn) into complex carbohydrates. Lec1A CHO cell mutants have been shown to belong to the same genetic complementation group but exhibit different phenotypic properties. Evidence is presented that lec1A represents a new mutation at the lec1 locus resulting in partial loss of GlcNAc-TI activity. Structural studies of the carbohydrates associated with vesicular stomatitis virus grown in Lec1A cells (Lec1A/VSV) revealed the presence of biantennary and branched complex carbohydrates as well as the processing intermediate Man5GlcNAc2Asn. By contrast, the glycopeptides from virus grown in CHO cells (CHO/VSV) possessed only fully processed complex carbohydrates, whereas those from Lec1/VSV were almost solely of the Man5GlcNAc2Asn intermediate type. Therefore, the Lec1A glycosylation phenotype appears to result from the partial processing of N-linked carbohydrates because of reduced GlcNAc-TI action on membrane glycoproteins. Genetic experiments provided evidence that lec1A is a single mutation affecting GlcNAc-TI activity. Lec1A mutants could be isolated at frequencies of 10(-5) to 10(-6) from unmutagenized CHO cell populations by single-step selection, a rate inconsistent with two mutations. In addition, segregants selected from Lec1A X parental cell hybrid populations expressed only Lec1A or related lectin-resistant phenotypes and did not include any with a Lec1 phenotype. The Lec1A mutant should be of interest for studies on the mechanisms that control carbohydrate processing in animal cells and the effects of reduced GlcNAc-TI activity on the glycosylation, translocation, and compartmentalization of cellular glycoproteins.     

Mac-1, but not LFA-1, uses intercellular adhesion molecule-1 to mediate slow leukocyte rolling in TNF-alpha-induced inflammation

We have previously shown that Mac-1 and LFA-1 play a cooperative role in slow leukocyte rolling in inflamed vessels, and that, although both have a role in leukocyte adhesion, the contribution from LFA-1 exceeds that of Mac-1. In this study, we used mice deficient in ICAM-1 (ICAM-1(null)) to study the function of ICAM-1 as an endothelial ligand for Mac-1 and LFA-1. The cremaster muscles of these mice were treated with TNF-alpha and prepared for intravital microscopy. We found that the average rolling velocity in venules was not different in ICAM-1(null) mice (4.7 micro m/s) compared with wild-type mice (5.1 micro m/s). Similarly, leukocyte adhesion efficiency in ICAM-1(null) mice (0.11 +/- 0.01 mm) was similar to that in Mac-1(-/-) (0.12 +/- 0.03 mm) mice but significantly increased compared with that in LFA-1(-/-) (0.08 +/- 0.01 mm) mice and significantly reduced from that in wild type (0.26 +/- 0.04 mm). When both LFA-1 and ICAM-1 were blocked, rolling velocity increased, and adhesion efficiency and arrest decreased. However, blocking both Mac-1 and ICAM-1 had no greater effect than either blockade alone. We conclude that endothelial ICAM-1 is the main ligand responsible for slow leukocyte rolling mediated by Mac-1, but not LFA-1.     

Soluble intercellular adhesion molecule-1 (sICAM-1): an overview

Soluble intercellular adhesion molecule-1 (sICAM-1) represents a circulating form of ICAM-1 that is constitutively expressed or is inducible on the cell surface of different cell lines. It serves as a counter-receptor for the lymphocyte function-associated antigen (LFA-1). Interaction between ICAM-1, present on endothelial cells, and LFA-1 facilitates leukocyte adhesion and migration across the endothelium. ICAM-1 and its circulating form have been implicated in the development of any number of diseases.     

Force spectroscopy of LFA-1 and its ligands, ICAM-1 and ICAM-2

Single-molecule measurements of the interaction of leukocyte function-associated antigen-1 (LFA-1), expressed on Jurkat T cells, with intercellular adhesion molecules-1 and -2 (ICAM-1 and ICAM-2) were conducted using atomic force microscopy (AFM). The force spectra (i.e., unbinding force versus loading rate) of both the LFA-1/ICAM-1 and LFA-1/ICAM-2 interactions were acquired at a loading rate range covering 3 orders of magnitude (50-60,000 pN/s) and revealed a fast loading regime and a slow loading regime. This indicates that the dissociation of both complexes involves overcoming a steep inner and a wide outer activation barrier. LFA-1 binding to ICAM-1 and ICAM-2 was strengthened in the slow loading regime by the addition of Mg(2+). Differences in the dynamic strength of the LFA-1/ICAM-1 and LFA-1/ICAM-2 interactions can be attributed to the presence of wider barriers in the ICAM-2 complex, making it more responsive to a pulling force than the ICAM-1 complex.     

Glycosyl modification facilitates homo- and hetero-oligomerization of the serotonin transporter. A specific role for sialic acid residues

The serotonin transporter (SERT) is an oligomeric glycoprotein with two sialic acid residues on each of two complex oligosaccharide molecules. In this study, we investigated the contribution of N-glycosyl modification to the structure and function of SERT in two model systems: wild-type SERT expressed in sialic acid-defective Lec4 Chinese hamster ovary (CHO) cells and a mutant form (after site-directed mutagenesis of Asn-208 and Asn-217 to Gln) of SERT, QQ, expressed in parental CHO cells. In both systems, SERT monomers required modification with both complex oligosaccharide residues to associate with each other and to function in homo-oligomeric forms. However, defects in sialylated N-glycans did not alter surface expression of the SERT protein. Furthermore, in heterologous (CHO and Lec4 cells) and endogenous (placental choriocarcinoma JAR cells) expression systems, we tested whether glycosyl modification also manipulates the hetero-oligomeric interactions of SERT, specifically with myosin IIA. SERT is phosphorylated by cGMP-dependent protein kinase G through interactions with anchoring proteins, and myosin is a protein kinase G-anchoring protein. A physical interaction between myosin and SERT was apparent; however, defects in sialylated N-glycans impaired association of SERT with myosin as well as the stimulation of the serotonin uptake function in the cGMP-dependent pathway. We propose that sialylated N-glycans provide a favorable conformation to SERT that allows the transporter to function most efficiently via its protein-protein interactions.     

Lec3 Chinese hamster ovary mutants lack UDP-N-acetylglucosamine 2-epimerase activity because of mutations in the epimerase domain of the Gne gene

Lec3 Chinese hamster ovary (CHO) cell glycosylation mutants have a defect in sialic acid biosynthesis that is shown here to be reflected most sensitively in reduced polysialic acid (PSA) on neural cell adhesion molecules. To identify the genetic origin of the phenotype, genes encoding different factors required for sialic acid biosynthesis were transfected into Lec3 cells. Only a Gne cDNA encoding UDP-GlcNAc 2-epimerase:ManNAc kinase rescued PSA synthesis. In an in vitro UDP-GlcNAc 2-epimerase assay, Lec3 cells had no detectable UDP-GlcNAc 2-epimerase activity, and Lec3 cells grown in serum-free medium were essentially devoid of sialic acid on glycoproteins. The Lec3 phenotype was rescued by exogenously added N-acetylmannosamine or mannosamine but not by the same concentrations of N-acetylglucosamine, glucosamine, glucose, or mannose. Sequencing of CHO Gne cDNAs identified a nonsense (E35stop) and a missense (G135E) mutation, respectively, in two independent Lec3 mutants. The G135E Lec3 mutant transfected with a rat Gne cDNA had restored in vitro UDP-GlcNAc 2-epimerase activity and cell surface PSA expression. Both Lec3 mutants were similarly rescued with a CHO Gne cDNA and with CHO Gne encoding the known kinase-deficient D413K mutation. However, cDNAs encoding the known epimerase-deficient mutation H132A or the new Lec3 G135E Gne mutation did not rescue the Lec3 phenotype. The G135E Gne missense mutation is a novel mechanism for inactivating UDP-GlcNAc 2-epimerase activity. Lec3 mutants with no UDP-GlcNAc 2-epimerase activity represent sensitive hosts for characterizing disease-causing mutations in the human GNE gene that give rise to sialuria, hereditary inclusion body myopathy, and Nonaka myopathy.     

Immunologic studies with LFA-1- and Mo1-deficient lymphocytes from a patient with recurrent bacterial infections

A patient and his parents, deficient for lymphocyte function associated antigen-1 (LFA-1) and Mo1 (OKM1), were studied with respect to leukocyte surface marker expression and functional properties. The patient had a history of severe recurrent bacterial infections. Two siblings had already died of bacterial infections. The patient's granulocytes, monocytes, and lymphocytes expressed low but detectable amounts (less than or equal to 10%) of LFA-1 and Mo1. Intracellularly, LFA-1 and Mo1 (OKM1) were detectable and LFA-1 expression was enhanced on patient T cells stimulated with phytohemagglutinin. Granulocytes and monocytes of both the patient's parents expressed markedly decreased amounts of LFA-1 and Mo1. Lymphocytes of the mother expressed 40 to 60% of the amount of LFA-1 expressed on control lymphocytes, but his father's lymphocytes showed a normal LFA-1 expression. Granulocytes of the patient and of his deceased sister showed normal phagocytosis, but they had a dysfunction in the activation of the oxidative metabolism. Functional activities mediated by patient T cells were all normal. Moreover, all lymphocyte functions, including killer (K), natural killer (NK), cytotoxic T cell activity, helper activity for in vitro immunoglobulin (Ig) production by normal B cells, and PHA-induced proliferation were inhibitable by anti-LFA-1 monoclonal antibodies. K and NK activity mediated by patient leukocytes was 100-fold more sensitive to the inhibiting effect of anti-LFA-1 antibody than K and NK activity of normal donor leukocytes. Thus, although the amount of LFA-1 expressed was strongly reduced, it was still sufficient and required for the functional activity exhibited by patient T cells. The major functional defect observed with leukocytes of the patient and his father was an apparent B cell defect. B cells of the father and of the patient failed to produce Ig in the pokeweed mitogen (PWM)-driven system. The B cells of patient and of his father only produced Ig when cultured with T cells of the father, and not with normal donor T cells or T cells of the mother, in the presence of exogenous interleukin 2 (IL 2). In addition, the father's B cells produced Ig when cocultivated with patient T cells in the IL 2-driven system. This restriction of helper T cell activity is noteworthy because PWM- and IL 2-driven Ig synthesis by normal lymphocytes show no histocompatibility requirements between cooperating T and non-T cell populations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Competition between intercellular adhesion molecule-1 and a small-molecule antagonist for a common binding site on the alphal subunit of lymphocyte function-associated antigen-1

The lymphocyte function-associated antigen-1 (LFA-1) binding of a unique class of small-molecule antagonists as represented by compound 3 was analyzed in comparison to that of soluble intercellular adhesion molecule-1 (sICAM-1) and A-286982, which respectively define direct and allosteric competitive binding sites within LFA-1's inserted (I) domain. All three molecules antagonized LFA-1 binding to ICAM-1-Immunoglobulin G fusion (ICAM-1-Ig) in a competition ELISA, but only compound 3 and sICAM-1 inhibited the binding of a fluorescein-labeled analog of compound 3 to LFA-1. Compound 3 and sICAM-1 displayed classical direct competitive binding behavior with ICAM-1. Their antagonism of LFA-1 was surmountable by both ICAM-1-Ig and a fluorescein-labeled compound 3 analog. The competition of both sICAM-1 and compound 3 with ICAM-1-Ig for LFA-1 resulted in equivalent and linear Schild plots with slopes of 1.24 and 1.26, respectively. Cross-linking studies with a photoactivated analog of compound 3 localized the high-affinity small-molecule binding site to the N-terminal 507 amino acid segment of the alpha chain of LFA-1, a region that includes the I domain. In addition, cells transfected with a variant of LFA-1 lacking this I domain showed no significant binding of a fluorescein-labeled analog of compound 3 or ICAM-1-Ig. These results demonstrate that compound 3 inhibits the LFA-1/ICAM-1 binding interaction in a directly competitive manner by binding to a high-affinity site on LFA-1. This binding site overlaps with the ICAM-1 binding site on the alpha subunit of LFA-1, which has previously been localized to the I domain.