Complex formation of CD23/surface immunoglobulin and CD23/CD81/MHC class II on an EBV-transformed human B cell line and inferable role of tetraspanin

CD23, a low-affinity IgE receptor, is a type II transmembrane protein having a C-type lectin domain and it associates noncovalently with MHC class II on B cells. The results of our immunoprecipitation analysis suggest that CD23 co-exists with at least two additional molecules, surface immunoglobulin (sIg) and CD81 (and/or CD9), on the cell surface of L-KT9 cells (an Epstein-Barr virus (EBV)-transformed human B cell line). When both CD23 and sIg molecules were stimulated simultaneously by the corresponding antibodies, a large increase in CD81 in the immunoprecipitation was observed as compared with the case of stimulation by only one antibody. Simultaneous stimulation by anti-CD23 and anti-Ig may mimic the situation of B cells stimulated by an antigen/IgE complex. In addition, a large increase in MHC class II in the immunoprecipitation was also observed by cross-linking of CD23 with anti-CD23 and its second antibody as compared with the case of stimulation by anti-CD23 alone. The cross-linking of CD23 with anti-CD23 and its antibody may mimic the situation of B cells stimulated by an IgE/antigen/IgE complex. Therefore, the complex formation among CD23, sIg, MHC class II, and CD81 on the cell surface of L-KT9 cells by the antigen/IgE or IgE/antigen/IgE complex is most likely to be closely related to B cell regulatory events by signaling through sIg or MHC class II. Tetraspanins such as CD81 and CD9 are thought to be involved in the formation and the preservation of various different membrane complexes consisting of several functional proteins.     

Two peptides from CD23, including the inverse RGD sequence and its related peptide, interact with the MHC class II molecule

The human CD23 molecule (low affinity receptor for IgE) has a C-type lectin domain, a reversed Arg-Gly-Asp (RGD) sequence near the C-terminus, and an "RGD-binding inhibitory peptide" at the root of the N-sugar chain. Three peptides were synthesized to determine their functions, i.e., #1, including an inverse RGD sequence near the C-terminus; #2, RGD-binding inhibitory peptides in the gpIIIa chain of platelet integrin gpIIb/IIIa; and #3, the inverse sequence located at the root of the N-sugar chain of CD23 which has homology to peptide 2. Among the three peptide, only peptide 3 inhibited aggregation of L-KT9 cells. Isotope-labeled peptides 1 and 3 bound to MHC class II molecules but peptide 1 did not bind to CD23 molecules. Peptide 3 showed a higher affinity to MHC class II than did peptide 1. Both peptides in CD23, therefore, seem to have interesting and important functions in relation to MHC class II molecules and also to CD23 molecules when CD23 on EBV-transformed B cells acts as a lectin in homotypic cell aggregation. The physiological function of CD23 was discussed from an evolutional point of view.     

Threshold effects on the lectin-mediated aggregation of synthetic glycolipid-containing liposomes

Cholesterol analogs containing sugar residues linked by spacer groups to the cholesterol O can be incorporated into egg yolk lecithin small unilamellar liposomes. The synthetic glycolipid analogs distribute evenly on both sides of the bilayer. These liposomes are aggregated by the appropriate lectin. For example, when the sugar residue is a β-galactoside the liposomes are aggregated by ricin and when it is an α-mannoside they are aggregated by Con A. The lectin-mediated aggregation of these liposomes is reversed by the addition of the appropriate sugar. The rates but not the extents of aggregation of these liposomes are highly sensitive to the amount of glycolipid incorporated. Below approximately 5% glycolipid incorporation the rate of the lectin-mediated aggregation of these liposomes is exceedingly slow, whereas above this level rapid aggregation proceeds. At all concentrations studied the synthetic glycolipids are incorporated in a unimodal fashion so that the observed threshold effects cannot be based on possible differences in the manner in which the glycolipids are incorporated at different concentrations. This conclusion is based on (1) studies with galactose oxidase that show that the percentage of galactose oxidation in a liposome prepared from a galactosyl-containing glycolipid is independent of glycolipid concentration, and (2) studies on the aggregation of liposomes containing mixed glycolipids in which the glycolipids are shown to behave independently. The importance of a critical density of membrane-bound receptors in order for aggregation to occur is discussed.     

Aggregation of sponge cells. Function of a lectin in its homologous biological system.

For the first time, the biological role of a lectin in the process of reaggregation of single cells from the same species (marine sponge: Geodia cydonium Jam.) is described. The galactose-specific lectin does not promote aggregation, but prevents the antiaggregation receptor from disaggregating cell clumps. Competition experiments showed that the lectin inactivates the antiaggregation receptor by binding to it, most likely via its terminal galactose residues. The lectin converts reversibly aggregation-deficient cells (carrying functional cell membrane-bound antiaggregation receptor molecules) to aggregation-susceptible cells.     

Distribution of receptor sites on large glycoprotein molecules by electron microscopy. Application to epiglycanin.

Electron microscopy was used to map the loci of immunochemically active sites on individual glycoprotein molecules. The positions of specific galactose residues and asparagine-linked carbohydrate chains containing specific mannose residues in epiglycanin, a glycoprotein of extended conformation from the surface of TA3 mouse mammary tumour cells, were observed in complexes with Ricinus communis toxin and concanavalin A respectively. The maximum number of Ricinus communis toxin molecules attached to a single epiglycanin molecule was 23, and the average number was 16. Only one concanavalin A molecule was observed attached to any epiglycanin molecule, and this at one end of the molecule, suggesting the presence of only one receptor for this lectin. By means of this new approach for mapping specific residues, evidence has been obtained that suggests microheterogeneity in epiglycanin with respect to the locations of carbohydrate chains containing receptors for Ricinus communis toxin.     

Characterization of a novel C-type lectin-like gene, LSECtin: demonstration of carbohydrate binding and expression in sinusoidal endothelial cells of liver and lymph node

A new C-type lectin-like gene encodes 293 amino acids and maps to chromosome 19p13.3 adjacent to the previously described C-type lectin genes, CD23, dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), and DC-SIGN-related protein (DC-SIGNR). The four genes form a tight cluster in an insert size of 105 kb and have analogous genomic structures. The new C-type lectin-like molecule, designated liver and lymph node sinusoidal endothelial cell C-type lectin (LSECtin), is a type II integral membrane protein of approximately 40 kDa in size with a single C-type lectin-like domain at the COOH terminus, closest in homology to DC-SIGNR, DC-SIGN, and CD23. LSECtin mRNA was only expressed in liver and lymph node among 15 human tissues tested, intriguingly neither expressed on hematopoietic cell lines nor on monocyte-derived dendritic cells (DCs). Moreover, LSECtin is expressed predominantly by sinusoidal endothelial cells of human liver and lymph node and co-expressed with DC-SIGNR. LSECtin binds to mannose, GlcNAc, and fucose in a Ca(2+)-dependent manner but not to galactose. Our results indicate that LSECtin is a novel member of a family of proteins comprising CD23, DC-SIGN, and DC-SIGNR and might function in vivo as a lectin receptor.     

Induction of the CD23/nitric oxide pathway in endothelial cells downregulates ICAM-1 expression and decreases cytoadherence of Plasmodium falciparum-infected erythrocytes

Cytoadherence of parasitized red blood cells (PRBCs) to postcapillary venules and cytokine production are clearly involved in the pathogenesis of cerebral malaria. Nitric oxide and TNF-alpha have been proposed as major effector molecules both in protective and physiopathological processes during malaria infections. Nitric oxide production has been shown to be induced by engagement of CD23 antigen. This study aimed to investigate the potential role of the CD23/nitric oxide pathway in the control of the cytoadherence of PRBCs on human endothelial cells. We demonstrate that normal human lung endothelial cells (HLECs) are able to express the low affinity receptor for IgE (Fc in RII/CD23), following cell incubation with interleukin 4 or PRBCs. Ligation of the CD23 antigen by a specific anti-CD23 monoclonal antibody at the cell surface of HLECs was found to induce iNOS mRNA and protein expression, NO release and P. falciparum killing. In addition, the specific CD23-engagement on these cells also induced a significant decrease in ICAM-1 expression, an adhesion molecule implicated in PRBCs cytoadherence. These data not only described for the first time the expression of a CD23 antigen at the cell surface of endothelial cells but also suggest a possible new regulatory mechanisms via the CD23/NO pathway during malaria infection.     

A common pathway for T lymphocyte activation involving both the CD3-Ti complex and CD2 sheep erythrocyte receptor determinants

T lymphocyte activation with monoclonal antibodies directed against the CD2 (T,p50) sheep red blood cell receptor antigen and against CD3 (T,p19,29) has been investigated. Co-stimulation of purified T lymphocytes with anti-CD3 (SP34) and anti-CD2 (9-1), which detects a unique epitope on the CD2 molecule, results in T cell activation and cell proliferation. Each antibody alone is unable to mediate this effect. Co-stimulation of purified T cells with two different anti-CD2 antibodies, 9-1 and 9.6, which detect two different epitopes on the CD2 molecule, are also mitogenic. In contrast, the combination of anti-CD3 (SP34) and anti-CD2 (9.6) cannot induce T cell activation. These data suggest that the CD2 epitope defined by the 9-1 antibody is functionally important for T cell activation via the CD3/Ti complex. Furthermore, it is demonstrated that anti-CD3 (SP34) induces epitopic modulation of the CD2 molecule, resulting in enhanced expression of the CD2, 9-1 epitope. This epitope modulation of the CD2 (9-1) epitope by anti-CD3 (SP34) occurs instantaneously at 4 degrees C and in the presence of NaN3. The functional interaction between CD3 and CD2 occurs in spite of any evidence of complex formation between these two molecules. These data suggest that the T cell differentiation antigens CD3 and CD2 are jointly involved in antigen-specific T cell activation. The data are consistent with a model for antigen-specific T cell activation involving both the CD3/Ti complex and subsequent activation of the CD2 complex T cell activation by co-stimulation with anti-CD3 (SP34) and anti-CD2 (9-1) is substantially enhanced by the addition of exogenous, purified interleukin 1 (IL 1). These data would suggest that the CD2 complex, as well as the putative IL 1 receptor, are involved in separate and complementary receptor-ligand interactions, resulting in the amplification of antigen-specific T cell responses.     

Analysis of the role of leukocyte function-associated antigen-1 in activation of human influenza virus-specific T cell clones

The role of leukocyte function-associated Ag-1 (LFA-1) in intercellular adhesion is well documented. Previously, we demonstrated that the LFA-1 molecule (CD11a/CD18) can also regulate the induction of proliferation of peripheral blood T cells. In these studies, we observed opposite effects of antibodies against CD11a (LFA-1-alpha-chain) or CD18 (LFA-1-beta-chain). Here, we determined the effects of anti-CD11a and anti-CD18 mAb on proliferation of cloned influenza virus-specific T cells. Anti-CD18 mAb had similar inhibiting effects on the proliferative response of T cell clones induced by immobilized anti-CD3 mAb as it had on the response of peripheral blood T cells. In contrast to its costimulatory effect on resting peripheral blood T cells, anti-CD11a mAb did not increase the proliferation of cloned T cells. Similar differences in effects of anti-CD11a and anti-CD18 mAb were observed when proliferation of the T cell clones was induced by immobilized anti-TCR mAb. When proliferation was induced by influenza virus presented by monocytes as APC, both anti-CD11a and anti-CD18 mAb inhibited T cell proliferation. However, when EBV-transformed B cells were used as APC, neither anti-CD11a nor anti-CD18 mAb inhibited proliferation. These results demonstrate that the effects of antibodies against CD11a (LFA-1-alpha) or CD18 (LFA-1-beta) on T cell proliferation depend on 1) the stage of activation of the T cells, 2) the activation stimulus and its requirement for intercellular adhesion involving LFA-1, and 3) the type of cell used to present Ag.     

一种新型的重组单链三特异抗体的构建与表达

双特异抗体由于其缺乏共刺激信号 ,激活的T细胞往往会导致凋亡。为了更有效地杀伤肿瘤细胞 ,构建了抗卵巢癌单链×抗CD3单链×抗CD2 8单域重组三特异抗体的表达载体。利用大肠杆菌中的分子伴侣FkpA与三特异抗体共表达 ,提高了三特异抗体的在BL2 1Star菌中的可溶性原核表达。ELISA结果显示 ,可溶性的重组单链三特异抗体 (sTRI)与SK OV 3细胞的膜抗原 ,Jurkat细胞上的CD3分子以及重组CD2 8抗原均有较强的结合活性。桥连试验证明该抗体能同时与SK OV 3细胞和Jurkat细胞结合。体外杀伤试验表明该抗体能激活外周血T细胞来杀伤肿瘤细胞。形态学观察也进一步说明该抗体具有良好的结合活性以及体外杀伤活性。这种新型的重组单链三特异抗体为基于T细胞的癌症免疫治疗建立了一个新的技术平台。     

Preparation of neo-galactosylated liposomes and their interaction with mouse peritoneal macrophages

In order to target liposomes to cells expressing at their surface a galactose-binding site we have prepared liposomes containing new synthetic galactolipids. Neo-galactosylated liposomes were prepared by covalently coupling beta-D-1-thiogalactopyranoside residues, substituted with a hydrophilic spacer-arm and functionalized with a sulfhydryl group, to preformed large unilamelar vesicles containing 4-(p-maleimidophenyl)butyryl phosphatidylethanolamine. The vesicles, having a galactose content above a threshold value of about 5 mol%, could be aggregated with Ricinus communis agglutinin. This aggregation was reversed by addition of excess free methyl beta-D-galactopyranoside, indicating that the surface glucidic moieties of these liposomes were accessible to the lectin. Compared to the control vesicles, the neo-galactosylated liposomes (containing 15 mol% galactose) presented in vitro an increased binding to cell possessing a beta-D-galactose specific receptor, i.e. resident mouse peritoneal macrophages. At 4 degrees C, the specific binding was about 2-fold, whereas at 37 degrees C it was increased to about 4-5-fold. This differential binding was largely unaffected by serum and, interestingly was much dependent on the degree of galactosylation of the liposomes, i.e. a threshold value of 5 mol% was needed to observe an increased binding of the targeted vesicles to the macrophages.     

HIV-1 gp41 envelope residues 650-685 exposed on native virus act as a lectin to bind epithelial cell galactosyl ceramide

The initial step in the interaction between human immunodeficiency virus (HIV-1) and epithelial cells is the binding of HIV-1 envelope glycoproteins to the epithelial cell galactosyl ceramide (GalCer). Here we show that HIV-1 envelope gp41 residues 650-685 bind GalCer in a galactose-specific manner. The gp41 residues that display this lectin activity are highly conserved among HIV-1 isolates and constitute three regions: residues 650-661, which encompass a charged helix; residues 662-667, referred to as the conserved epitope ELDKWA, the epitope recognized by antibodies that neutralize HIV-1 entry in epithelial and CD4(+)-mononucleated cells; and residues 668-685, a hydrophobic Trp-rich sequence that stabilizes the structure of the galactose binding site. Similar to other galactose-specific lectins, the gp41 lectin site is active only as an oligomer. Finally the orientation of the galactose toward the gp41 lectin site appears to be controlled by the lipid microenvironment of the epithelial membrane. From the experimental data we construct a theoretical model of the interaction between gp41 and GalCer based on thermodynamic considerations. This model integrates the dynamics and the spatial organization of the viral envelope glycoproteins, GalCer organized in raft microdomains in the apical region of the epithelial cell membrane and the interfacial water. Characterization of the minimal sequence and structure of gp41 in direct interaction with GalCer may help unravel the still unknown immunogenic determinant able to elicit antibodies against ELDKWA and target of one of the rare neutralizing antibodies against gp41.     

Human peripheral blood T helper cell-induced B cell activation results in B cell surface expression of the CD23 (BLAST-2) antigen

We have developed an in vitro system to assess the early stages of B cell activation induced by peripheral blood T helper cells. Peripheral blood mononuclear cells are cultured for 16 hr with anti-CD3 monoclonal antibody (mAb), T lymphocytes are then removed by sheep red blood cell rosette depletion, and expression of the B cell surface activation antigen CD23 (BLAST-2) is assessed by indirect immunofluorescence. Anti-CD3 mAb, but not a control anti-CD5 mAb, stimulates the expression of CD23 on 20-50% of peripheral blood B cells cultured with autologous T cells. T cell subset depletion studies show that the CD4+ T cell subset is responsible for anti-CD3-mediated induction of CD23 on autologous B cells. Anti-CD3-induced, T helper cell-dependent CD23 expression is not MHC-restricted, as allogeneic combinations of T and non-T cells, cultured in the presence of anti-CD3 antibody, also result in the expression of B cell CD23. Individuals whose monocyte Fc receptors bind murine IgG1 mAb poorly fail to trigger T cell proliferation in response to murine IgG1 anti-CD3 mAb and also fail to express B cell CD23 following culture of PBMC with IgG1 anti-CD3 mAb, while the usual expression of CD23 is seen after culture with IgG2a anti-CD3 mAb. The mechanism of anti-CD3-induced B cell activation was addressed in experiments using a two-chamber culture system. While little IL-4 activity was detected in anti-CD3-stimulated culture supernatants, optimal induction of CD23 was observed when T and B cells were cultured together in a single chamber. This suggests that under physiologic conditions, in which quantities of lymphokine may be limiting, close physical contact between the anti-CD3-activated Th cell and B cell may be required for CD23 expression. The anti-CD3-induced BLAST-2 assay will facilitate the analysis of Th cell-mediated B cell activation in any individual and should permit us to separately evaluate the roles of Th cells and B cells in the impaired immunoregulation characteristic of autoimmune disorders.     

Evidence that monoclonal antibodies against CD9 antigen induce specific association between CD9 and the platelet glycoprotein IIb-IIIa complex

Monoclonal antibodies to the CD9 antigen are powerful platelet agonists. We report here the novel finding that the anti-CD9 monoclonal antibodies 50H.19 and ALB6 promote physical association between CD9 antigen and the glycoprotein IIb-IIIa complex (GPIIb-IIIa) component of the platelet fibrinogen receptor. The monoclonal antibodies do not consistently immunoprecipitate proteins other than CD9 from 125I-labeled human platelets even if the platelets are first treated with the homobifunctional cross-linking reagent dithiobis(succinimidyl propionate), indicating that CD9 antigen is not physically associated with other membrane proteins in the resting state. However, the addition of agonistic concentrations of either monoclonal antibody before cross-linking results in the coprecipitation of proteins corresponding in mobility and peptide composition to GPIIb, and GPIIIa. The association of CD9 with the GPIIb-IIIa complex is unaffected by a combination of aspirin and ADP scavengers sufficient to abrogate anti-CD9 monoclonal antibody-induced platelet aggregation, and is therefore not dependent upon thromboxane- and ADP-mediated pathways of intracellular signalling. The specificity of the association is demonstrated by the lack of other coprecipitating major proteins, by the requirement for induction by anti-CD9 monoclonal antibodies, and by the failure to promote reciprocal association with either of the anti-GPIIb-IIIa complex monoclonal antibodies P2 or HuP1-m1a.     

Angiotensin converting enzyme in cultured endothelial cells and growth medium. Relationships to enzyme from kidney and plasma

We have previously reported that cultured cells from swine aorta possess angiotensin converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) and release it into serum-free culture medium. The present work compares enzyme from these two sources, and from swine kidney and serum, with respect to antibody and lectin binding. Purified enzyme from swine kidney, and the activity in swine serum, cultured endothelial cells and culture medium bind similarly to rabbit antibodies prepared against the kidney converting enzyme. Enzyme from each of these sources was allowed to bind to an immobilized lectin (Ricinus communis), which binds to terminal galactose residues of glycoproteins. Increasing concentrations of galactose were used to remove enzyme from the lectin column and the distribution of enzyme activity in the galactose eluates was determined. The elution pattern was similar for kidney and endothelial cell enzyme, and different from the pattern found for both serum and medium enzymes. Neuraminidase treatment of either serum or medium enzyme altered the distribution of activity eluted to that found for endothelial cell or kidney enzymes. The effects of neuraminidase suggest that the difference in lectin binding between cell and medium enzyme reflects differences in the number of terminal sialic acid residues that cover galactose residues.     

Functional association of CD9 with the Fc gamma receptors in macrophages

CD9, a member of the tetraspan family of proteins, is highly expressed on macrophages. Although a clear function for the molecule has yet to be described, we have found that the anti-CD9 mAb activates mouse macrophages. The rat anti-CD9 mAb, KMC8.8, but not the F(ab')(2), induced tyrosine phosphorylation of proteins including syk and cbl and induced cell aggregation in the mouse macrophage cell line, J774, suggesting that co-cross-linking of CD9 and Fc gamma R was required for the signal. Co-cross-linking of CD9-Fc gamma R with KMC8.8 on macrophages from three different FcR-deficient mice, FcR gamma-chain(-/-), Fc gamma RIIB(-/-), and Fc gamma RIII(-/-), revealed that Fc gamma RIII is specific and crucial for syk phosphorylation. Although both KMC8.8 and the anti-Fc gamma RIIB/III mAb, 2.4G2, evoked similar phosphorylation patterns, only KMC8.8 induced cell aggregation. Additionally, KMC8.8 treatment led to reduce levels of TNF-alpha production and p42/44 extracellular signal-related kinase phosphorylation relative to 2.4G2 stimulation. Immunofluorescence staining showed that co-cross-linking of CD9-Fc gamma R with KMC8.8 induced filopodium extension before cell aggregation, which was followed by simultaneous colocalization of CD9, Fc gamma RIIB/III, Mac-1, ICAM-1, and F-actin at the cell-cell adhesion site. Moreover, KMC8.8 treatment of Fc gamma R-deficient macrophages revealed that the colocalization of CD9, Fc gamma RIII, Mac-1, and F-actin requires co-cross-linking of CD9-Fc gamma RIII, whereas co-cross-linking of CD9-Fc gamma RIIB induced the colocalization of only CD9 and Fc gamma RIIB. Our results demonstrate that co-cross-linking of CD9 and Fc gamma Rs activates macrophages; therefore, CD9 may collaborate with FcRs functioning in infection and inflammation on macrophages.     

Purification and characterization of a human lectin specific for penultimate galactose residues

A novel lectin has been found in human plasma. The lectin was purified by affinity chromatography using an adsorbent in which 2-O-alpha-D-glucopyranosyl-O-beta-D-galactopyranosylhydroxylysine (Glc-Gal-Hyl) was coupled to Sepharose. The molecular weight of the lectin was determined by gradient gel electrophoresis to be approximately 240,000. On polyacrylamide gel electrophoresis in sodium dodecyl sulphate, the subunit had the molecular weight of 29,500. Composition analysis has shown the lectin is a glycoprotein in which 12% of the molecule consists of carbohydrate. Native human, horse, calf, sheep, rabbit, and rat erythrocytes were agglutinated by the lectin in the presence of calcium. Glc-Gal-Hyl, N-acetylated Glc-Gal-Hyl, and stachyose inhibited the hemagglutination, whereas monosaccharides, maltose, cellobiose, lactose, raffinose, galactosylhydroxylysine, and N-acetylated galactosylhydroxylysine were not inhibitory. The lectin is strongly inhibited by the desialylated bovine erythrocyte glycoprotein, which contains galactose beta 1-3galactose beta-sequence at the nonreducing termini of the sugar chains, whereas disialylated orosomucoid did not inhibit the lectin. These results indicate that the lectin recognizes the penultimate galactose residue in a hapten molecule in contrast to usual galactose-binding proteins or galactose-specific lectins, which recognize exposed, terminal galactose residues of sugar chains.     

Evidence for an association between CD8 molecules and the T cell receptor complex on cytotoxic T cells

The T cell differentiation molecule CD8 is thought to play an important role in class I major histocompatibility complex-restricted T cell activities but the precise function of this molecule is unknown. To explore this question, we have studied several CD3+, CD8+ class I alloantigen-specific cytotoxic T lymphocyte (CTL) lines and clones. The ability of these CTL to proliferate as well as to lyse specific targets was inhibited by either anti-CD3 or anti-CD8 monoclonal antibodies. Exposure of CTL to relevant but not irrelevant target cells induced the rapid (less than 1 hr) disappearance of approximately 20 to 30% of CD3 and CD8 molecules from the cell surface. The modulation of these molecules became maximal at 6 to 12 hr and recovered thereafter in parallel. Treatment of CTL with anti-CD8 prevented alloantigen-induced modulation of CD3, and treatment with anti-CD3 blocked modulation of CD8. Incubation of CTL with the combination of anti-CD3 and goat anti-mouse Ig also resulted in modulation of CD8. In contrast, the expression of other CTL surface antigens, such as CD2 (Leu-5, T11) and HLA-DR, was not reduced by any of these manipulations. These results suggest that CD8 molecules are associated with the CD3/antigen receptor complex on the surface of CTL, and may play a direct role in antigen-induced modulation and cross-linking of the T cell receptor.     

Production and characterization of a monoclonal antibody specific for the human lymphocyte low affinity receptor for IgE: CD 23 is a low affinity receptor for IgE

In the present study a gamma 1 kappa monoclonal antibody, Mab 25, specific for the receptor for the Fc fragment of IgE on lymphocytes (Fc epsilon RL) was established. This antibody was generated after fusion of spleen cells from mice immunized with the EBV-transformed lymphoblastoid cell line RPMI 8866, which is known to express Fc epsilon RL at high density. Mab 25 inhibits strongly the binding of IgE to RPMI 8866 cells and to other Fc epsilon RL-positive EBV-transformed lymphoblastoid cell lines. A 50% inhibition of IgE binding was observed at a Mab 25 concentration of 10 ng/ml. The binding of IgE was also inhibited by Fab fragments of Mab 25, suggesting that the inhibition is not simply due to steric hindrance or to an eventual binding through its Fc portion. Mab 25 only binds to cell lines expressing Fc epsilon RL. Mab 25 immunoprecipitated a single polypeptide with an apparent m.w. of 42 Kd, pI 4.9. The membrane molecule bound to and eluted from a Mab 25 immunoabsorbent had the same apparent m.w. and pI as the Fc epsilon RL purified from an IgE immunoabsorbent. Additionally, when RPMI 8866 cell lysates were cleared with Mab 25, no Fc epsilon RL could be bound to or eluted from an IgE immunoabsorbent. Mab 25 was found to weakly bind to a minor proportion of blood (1 to 4%), tonsil (2 to 9%) and spleen (4 to 5%) mononuclear cells with a low intensity. By double fluorescence analysis, most of the Fc epsilon RL-positive cells were found to be CD 20-positive B lymphocytes. The staining pattern of Mab 25 and the biochemical characteristics of the antigen detected by Mab 25 were comparable to those of the CD 23 Mab. The four CD 23 Mab MHM 6, PL 13, HD 50, and Tü 1 were found to inhibit the binding of IgE. PL 13 was found to totally inhibit the binding of Mab 25 to RPMI 8866 cells, whereas Tü 1 and MHM 6 only partially inhibited Mab 25 binding. HD 50 was unable to block the binding of Mab 25. The finding that different CD 23/Fc epsilon RL-specific monoclonal antibodies recognizing distinct epitopes have in common the capacity of inhibiting the binding of IgE suggests that upon binding they induce a conformational alteration of the Fc epsilon RL resulting in a loss of the IgE binding capacity. In conclusion, our data demonstrate that the CD 23 antigen is a low affinity receptor for IgE on lymphocytes.     

Induction of tyrosine phosphorylation during ICAM-3 and LFA-1-mediated intercellular adhesion, and its regulation by the CD45 tyrosine phosphatase

Intercellular adhesion molecule (ICAM)-3, a recently described counter- receptor for the lymphocyte function-associated antigen (LFA)-1 integrin, appears to play an important role in the initial phase of immune response. We have previously described the involvement of ICAM-3 in the regulation of LFA-1/ICAM-1-dependent cell-cell interaction of T lymphoblasts. In this study, we further investigated the functional role of ICAM-3 in other leukocyte cell-cell interactions as well as the molecular mechanisms regulating these processes. We have found that ICAM-3 is also able to mediate LFA-1/ICAM-1-independent cell aggregation of the leukemic JM T cell line and the LFA-1/CD18-deficient HAFSA B cell line. The ICAM-3-induced cell aggregation of JM and HAFSA cells was not affected by the addition of blocking mAb specific for a number of cell adhesion molecules such as CD1 1a/CD18, ICAM-1 (CD54), CD2, LFA-3 (CD58), very late antigen alpha 4 (CD49d), and very late antigen beta 1 (CD29). Interestingly, some mAb against the leukocyte tyrosine phosphatase CD45 were able to inhibit this interaction. Moreover, they also prevented the aggregation induced on JM T cells by the proaggregatory anti-LFA-1 alpha NKI-L16 mAb. In addition, inhibitors of tyrosine kinase activity also abolished ICAM-3 and LFA-1- mediated cell aggregation. The induction of tyrosine phosphorylation through ICAM-3 and LFA-1 antigens was studied by immunofluorescence, and it was found that tyrosine-phosphorylated proteins were preferentially located at intercellular boundaries upon the induction of cell aggregation by either anti-ICAM-3 or anti-LFA-1 alpha mAb. Western blot analysis revealed that the engagement of ICAM-3 or LFA-1 with activating mAb enhanced tyrosine phosphorylation of polypeptides of 125, 70, and 38 kD on JM cells. This phenomenon was inhibited by preincubation of JM cells with those anti-CD45 mAb that prevented cell aggregation. Altogether these results indicate that CD45 tyrosine phosphatase plays a relevant role in the regulation of both intracellular signaling and cell adhesion induced through ICAM-3 and beta 2 integrins.