Identification of a Cell-Surface Glycoprotein Mediating Adhesion in Human Granulocytes

Previous studies have shown that monoclonal antibody 60.3 reacting with a surface antigen common to human leukocytes inhibits phorbol ester-induced adhesion among blood mononuclear cells and precipitates from these cells three surface polypeptides with apparent molecular weights of 90,000, 130,000 and 160,000. Now we report that the same antibody, either as purified IgG or Fab fragments, also inhibits the extensive adhesion among granulocytes induced by phorbol ester. Inhibition of cell aggregation was not observed with monoclonal antibodies to C3b receptor, common leukocyte antigen T200, C3bi receptor, brain granulocyte-T lymphocyte antigen, IgG Fc receptor, class I transplantation antigen, or a granulocyte-specific antigen. Intercellular adhesion induced by either the chemotactic tripeptide N-formylmethionyl-leucyl-phenylalanine (FMLP) or the ionophore A23187 was also inhibited by antibody 60.3. However, this antibody did not affect phorbol ester-induced superoxide (O2-) generation or lysozyme release. Two major surface glycopolypeptides with apparent molecular weights of 92,000 and 155,000 were immunoprecipitated from granulocytes. Dissociation of the protein complexes obtained from blood mononuclear cells and granulocytes indicated the presence of the epitope on the 90,000-92,000 molecular-weight components. It is thus concluded that the smallest glycopolypeptides mediate adhesion in human granulocytes and mononuclear leukocytes.     

CD1 1b/CD18-Dependent Polymorphonuclear Leucocyte Interaction with Matrix Proteins in Adhesion and Migration

Adhesion of human polymorphonuclear leucocytes (PMN) stimulated with phorbol myristate acetate (PMA) to plastic dishes coated with the matrix proteins laminin (LM), libronectin (FN), collagen type I (CI) or collagen type IV (CIV) was inhibited by the monoclonal antibody 60.3 (MoAb 60.3; anti-CD18). The highest inhibitory effect was seen on adhesion to CI. PMN adhesion to CI was also effectively inhibited by Mol (anti-CD11b) but this antibody had only a minor effect on attachment of PMN to the other matrix proteins. In other experiments MoAb 60.3 inhibited LTB₄-induccd migration of PMN through polycarbonate filters (3 μm pores) coated with LM, FN, CI or CIV, with the most pronounced effect on migration through those filters coated with CI. By contrast, the antibody Mol had no effect on migration through any of the protein-coated filters tested. The results in this study suggest that the CD18 epitope, recognized by 60.3, mediates both adhesion and migration of PMN while theepitopeonCDl 1b recognized by the antibody Mol is restricted to adhesion. The results also indicate that CD11b/CD18 is the major receptor on human PMN for CI while interaction with LM, FN and CIV may in addition involve other mechanisms.     

A monoclonal antibody inhibiting leucocyte adhesion blocks induction of IL-2 production but not IL-2 receptor expression.

Monoclonal antibody 60.3 defines the leucocyte antigen CD 18 and recognizes a cell surface glycoprotein with an apparent molecular weight (MW) of 90,000 expressed by most human peripheral blood and bone marrow cells. This antibody can, among other things, block phorbol ester-induced adhesion among human mononuclear leucocytes. We show in this study that phorbol esters alone can induce peripheral blood mononuclear cells (PBL) to secrete interleukin-2 (IL-2) and that the IL-2-dependent cell line CTLL can be used for measuring this lymphokine without influence of the phorbol esters themselves. These findings make it possible to analyse the capacity of antibody 60.3 to interfere with IL-2 production and receptor expression by phorbol ester or phytohaemagglutinin (PHA)-treated human PBL. A significant positive correlation between blockage of induced cell aggregation by antibody 60.3 and reduction in IL-2 release was observed. The addition of interleukin-1 (IL-1) restored IL-2 secretion in PHA-treated, but not in 4-beta-phorbol 12, 13-dibutyrate [P(Bu)2]-treated, cells in the presence of this antibody. In parallel, IL-2 receptor expression was determined by immunofluorescence using biotinylated anti-IL-2 receptor (Tac) antibodies. FACS analysis showed that IL-2 receptor expression was unaffected by antibody 60.3, whereas DNA synthesis of the same P(Bu)2-treated PBL was inhibited. However, addition of external recombinant IL-2 overcame this proliferation blockade. These results indicate that a cell-to-cell adhesion step is necessary for the production of IL-2, but not for the expression of its receptor on both PHA- and P(Bu)2-treated human PBL.     

Adhesion of Subsets of Human Blood Mononuclear Cells to Endothelial Cells In Vitro, as Quantified by Flow Cytometry

Binding of leucocytes to endothelial cells (EC) is essential as an initial step in inflammatory responses. We present a rapid, non-radioactive method to measure adhesion of human lymphoid cells to EC using flow cytometry. Freshly isolated peripheral blood mononuclear cells (PBMC) were allowed to adhere to EC grown in 24-well plates. Non-adhering cells were removed, after which adhering cells and EC were dissociated using trypsin/EDTA. These samples were subsequently analysed by flow cytometry, using scatter properties to distinguish between adhering cells and EC. The ratio of the number of adhering leucocytes and EC was calculated to quantify adhesion. Results of the flow cytometric adhesion assay were comparable to those obtained with a conventional adhesion assay using chromium-labelled cells. We additionally show that by using the flow cytometric adhesion assay, adhesion of lymphocytes and monocytes present within the adhering PBMC can be quantified simultaneously. As a model, the contribution of LFA-1 (CD11a/CD18) and ICAM-1 (CD54) in adhesion of PBMC to EC was studied. It was found that adhesion of lymphocytes and monocytes is regulated differently by phorbol ester and that the relative contribution of LFA-1 and ICAM-1 differs for both cell types.     

Effects of inflammatory cytokines and phorbol esters on the adhesion of U937 cells, a human monocyte-like cell line, to endothelial cell monolayers and extracellular matrix proteins.

The accumulation of mononuclear phagocytes at sites of chronic inflammation is dependent on an increase in the rate of extravasation of blood-borne monocytes through the vascular endothelium into the connective tissue. Once the monocytes have emigrated into the connective tissue, they may differentiate into tissue macrophages, presumably following interactions with extracellular matrix proteins. To study these processes, we tested the effects of cytokines and phorbol esters on the adhesion of U937 cells, a human monocyte-like cell line, to cultured endothelial cells (EC) and to matrix proteins. In the absence of cytokines, very few of the U937 cells adhered to EC (5% or less in most experiments). When EC were pretreated for optimal periods of time (4-8 hr) with recombinant interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumor necrosis factor-alpha (TNF alpha), or lymphotoxin (LT; also known as TNF-beta), 35-85% of the U937 cells were able to bind. Interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) did not stimulate U937-EC binding, even though IFN-gamma was shown to increase EC adhesiveness for T lymphocytes. Phorbol esters also greatly stimulated U937-EC adhesion but, in this case, the increase was due to an action on the U937 cells. A monoclonal antibody (MAb), 60.3, against the CD11/CD18 family of leukocyte adhesion molecules partially inhibited the adhesion of untreated and phorbol ester-treated U937 cells to noncytokine-treated EC. However, that MAb had no effect on U937 cell binding to TNF-alpha-treated EC. Thus U937 cells use both CD11/CD18-dependent and -independent mechanisms to adhere to EC. In the absence of stimulating agents, only a small proportion of the U937 cells (2-20%) adhered to fibronectin (FN), and almost none bound to either laminin (LN) or gelatin (denatured type I collagen). In the presence of phorbol esters, a much larger proportion of the U937 cells adhered to FN, with only slight increases in the proportion of cells which bound to LN or gelatin. Additional adhesion assays performed in the presence of a pentapeptide containing the amino acid sequence arg-gly-asp (RGD), which is part of one of the cell-binding domains of FN, demonstrated that the RGD-containing peptide almost totally blocked the phorbol ester-induced adhesion of U937 cells to FN. In contrast, the peptide had no inhibitory effect on the phorbol ester-induced binding of U937 cells to EC.     

Rabbit leukocyte adhesion molecules CD11/CD18 and their participation in acute and delayed inflammatory responses and leukocyte distribution in vivo

In humans the glycoprotein complexes CD11/CD18 mediate leukocyte adhesion to cells. Mouse monoclonal antibodies (mAb) 60.3, 7E4, and IB4 to human CD18, found to cross-react with rabbit white blood cells, were used to identify the antigen in rabbit cells and to study adherence of rabbit leukocytes in vitro and in vivo. These antibodies labeled almost all unfractionated rabbit blood leukocytes and immunoprecipitated surface glycopolypeptides with apparent molecular weights of 85,000 and 150,000 from these cells. Adhesion of purified rabbit polymorphonuclear cells (PMNs) to cultured vascular endothelial cells in the presence of phorbol ester was blocked by the antibodies in a dose-dependent manner. The acute inflammatory response characterized by local accumulation of PMNs and concomitant plasma extravasation following intradermal injections of zymosan-activated serum (ZAS) in rabbits was inhibited in animals pretreated intravenously with anti-CD18 mAb. Intravital microscopy of the rabbit tenuissimus muscle demonstrated that anti-CD18 mAb. Intravital microscopy of the rabbit tenuissimus muscle demonstrated that anti-CD18 treatment specifically blocked the adhesion of activated leukocytes to the venular endothelium and thereby the subsequent diapedesis of these cells into the extravascular space. The lymphocyte-dependent tissue swelling resulting from a delayed-type hypersensitivity reaction in the rabbit ear was partially inhibited by anti-CD18 mAb. Systemic anti-CD18 treatment induced a pronounced increase in the number of circulating mononuclear and polymorphonuclear cells with a maximum at 24 hr after injection of the antibody. It is concluded that GP150/GP85 is the rabbit homologue of human CD11/CD18, and that leukocyte-cell adhesion mediated by these glycoprotein complexes participates in acute and delayed inflammatory responses and leukocyte distribution in vivo.     

Identification of a Cell Surface Protein Complex Mediating Phorbol Ester-Induced Adhesion (Binding) among Human Mononuclear Leukocytes

Phorbol esters rapidly induce aggregation of human mononuclear leukocytes in vitro. Previous studies have indicated that cell surface proteins are involved. We report now that the monoclonal antibody 60.3, either as purified IgG or as Fab' fragments, to an antigen common to leukocytes completely inhibited the phorbol ester-induced intercellular adhesion (binding). No inhibition of cell aggregation was observed with monoclonal antibodies to common leukocyte antigen T 200, T-cell-associated antigen, monocyte-granulocyte antigen, brain granulocyte-T-lymphocyte antigen, transferrin receptor, mature T-cell antigens (mol.wt either 67,000 or 19,000/29,000), T helper/inducer cell antigen, sheep erythrocyte receptor, class I or class II antigens, or T cytotoxic/suppressor cell antigen. The antibody 60.3 did not inhibit stimulation of the cells since the characteristic phorbol ester-induced morphological changes and phorbol ester-enhanced cap formation of membrane glycoproteins were readily observed. Two major cell surface polypeptides with apparent molecular weights of 90,000 and 160,000 were immunoprecipitated. We conclude that this protein complex, or at least one of its components, mediates adhesion among mononuclear leukocytes.     

Discrimination of human CD4 T cell clones based on their reactivity with antigen-presenting T cells.

In this report, we describe the discrimination of human T cell clones based on their reactivity with activated T cells as antigen-presenting cells (APC). CD4+ T cell clones specific for peptide P30 of tetanus toxin (amino acids 947-967) and restricted to the DP4 molecule were established and tested for proliferation to peptide presented either by peripheral blood mononuclear cells (PBMC), Epstein-Barr virus (EBV)-transformed B cells or major histocompatibility complex (MHC) class II-expressing T cells. We found two sets of T cell clones: one set proliferated to peptide presentation by PBMC, EBV-transformed B cell lines (EBV-B cells) and MHC class II+ T cells (termed T-responder clones), while the other set of clones was only stimulated to proliferate, if the peptide was presented by PBMC or EBV-B cells, but not by T cells (T-nonresponder clones). Nevertheless, these T-nonresponder clones recognized P30 also on T cells, as revealed by Ca2+ influx. The discrimination of the clones was not due to different avidities of the T cell receptors (TcR) of individual clones for the MHC-peptide complex as T-responder and T-nonresponder clones had similar dose-response curves to P30 presented by fixed EBV-B cell lines. Addition of cytokines [interleukin (IL)-1, IL-2, IL-4 and interferon gamma] did not change the proliferative response of the clones, which was consistent throughout an observation period of greater than 4 months. T-nonresponder clones, exposed to P30 on MHC class II-expressing T cells, became not anergic, as they could be restimulated by P30 presented on EBV-B cells. The measurement of a panel of T cell activation markers and adhesion molecules on T-responder and T-nonresponder clones revealed a higher expression of the CD28 molecule on the T-nonresponder clones. The data suggest that freshly cloned T cells can be differentiated by peptide presentation on classical (PBMC, EBV-B cells) or non-classical APC (class II+ T cells), and that this discrimination is further underlined by different levels of adhesion molecules.     

Characterization of porcine CD5 and CD5+ B cells

Evidence strongly supports a role for the lymphocyte transmembrane glycoprotein CD5 in intracellular signalling events, whereby antigen-dependent growth and differentiation signals are augmented. Apart from its role in activation-related signalling, CD5 has been regarded as a possible B cell lineage marker differentiating subsets, CD5⁺ B cells (also termed B1 cells) and conventional B cells (or B2 cells). To extend these investigations to the study of pigs, porcine B cells were examined for evidence of CD5 expression. The influence of cellular activation on CD5 expression and CD5's role in signal transduction events and lymphocyte proliferation were examined. Using an anti-porcine CD5 MoAb (b53b7), porcine B cells were shown to be heterogeneous for CD5 expression. As in other species, B lymphocyte CD5 expression is low (dull), while IgM is high (bright). Ten to 30% of pig blood B lymphocytes are CD5⁺, with the highest frequency in neonates. Anti-CD5 antibody treatment was sufficient to induce rapid but transient calcium ion flux in porcine peripheral blood lymphocytes (PBL). CD5 expression increased on PBL following treatment with phorbol myristate acetate (PMA), lipopolysaccharide (LPS), or immobilized anti-IgM. LPS, PMA, and concanavalin A (Con A) but not anti-CD5, anti-IgM, or combinations of these antibodies induced lymphocyte ³H-thymidine uptake. CD5⁺ B cells are a common constituent of porcine circulating lymphocytes and resemble B1 cells of mice, man and other species in CD5 expression, frequency and lymphoid organ distribution. Porcine CD5, like CD5 in other species, mediates signal transduction, leading to changes in intracellular calcium concentration, but this signal alone is insufficient to promote cell division. A subset of porcine B cells up-regulates CD5 expression following phorbol ester activation.     

Reduced antigen-presenting function of human Epstein-Barr virus (EBV)-B cells and monocytes after UVB radiation is accompanied by decreased expression of B7, intercellular adhesion molecule-1 (ICAM-1) and LFA-3.

In this study, the effect of ultraviolet-B (UVB) radiation on antigen-presenting function was studied, to investigate whether antigen-presenting cells (APC) are inhibited by UVB through a common mechanism. Two types of human APC were used: EBV-B cells and monocytes, and these were irradiated in vitro with single low doses of UVB (range 0-200 J/m2). Irradiation of EBV-B cells or monocytes resulted in similar dose-dependent reduction in APC function, when determined by the allogeneic mixed leucocyte reaction (MLR) or Candida albicans- or tetanus toxoid-specific T cell response. Our study shows that the reduced APC function was not likely to be caused by alterations in antigen processing or cytokine production. However, UVB-irradiated APC displayed marked changes in adhesion molecule expression. Irradiated EBV-B cells showed reduced expression of ICAM-1 (30%), LFA-3 (25%) and B7-1 (35%), while expression of HLA-DR, CD19 and LFA-1 was not affected. UVB irradiation of monocytes did result in reduction in the expression of HLA-DR (30%), LFA-3 (40%), ICAM-1 (65%) AND B7-1 and B7-3 (90%), but had no effect on CD14, LFA-1 and ICAM-3 expression. Addition of non-irradiated cells (but not the supernatant of these cells) or CD28 antibodies partly restored T cell activation, indicating that UVB-induced reduction in APC function is at least partly mediated via impairment of co-stimulatory molecule expression.     

Activation of human B cells through the CD19 surface antigen results in homotypic adhesion by LFA-1-dependent and -independent mechanisms.

Addition of CD19 monoclonal antibodies (mAb) to highly purified tonsillar B cells resulted in homotypic adhesion and the formation of cell clusters. This response was completely blocked by antibody to LFA-1, indicating an LFA-1-dependent adhesion mechanism. In contrast, aggregate formation by B cells activated with phorbol myristate acetate (PMA) was only partially inhibited by anti-LFA-1 antibody, and those formed in response to PMA plus CD19 antibody were not inhibited at all, suggesting aggregation of activated B cells stimulated with CD19 antibody was LFA-1 independent. This was confirmed with B-cell lines. The pre-B-cell line Nalm-6 formed aggregates in response to CD19 antibody which were not inhibited with anti-LFA-1. In addition, CD19 antibody induced aggregate formation by an Epstein-Barr virus (EBV)-transformed B-cell line derived from an LFA-1-deficient donor. These results suggest that different adhesion molecules may operate at different stages of B-cell activation, and that CD19 may be important in cell-cell interactions involved in regulation of antibody responses.     

Blood Leucocyte Subsets of the Rat: Expression of Adhesion Molecules and Localization Within High Endothelial Venules

Although several distinct adhesion pathways are now well characterized, it is not clear whether analysis of adhesion molecule expression on leucocytes is sufficient to predict their interaction with endothelium in vivo. Therefore, in the present study this question was addressed by investigating the interaction between blood leucocyte subsets and high endothelial venules (HEV). The expression of different types of adhesion molecule (CD44, α4-integrins, LFA-1, ICAM-1, CD2 and L-selectin) on lymphocytes, NK cells, monocytes and granulocytes of rat blood was determined by flow cytometry. In the same animals the numbers of blood leucocyte subsets present in the HEV of axillary lymph nodes and Peyer's patches were analysed using immunohistology. In the HEV of both axillary lymph nodes and of Peyer's patches lymphocytes (> 10.000 per mm²), as well as small numbers of NK cells and monocytes (< 500 per mm²), were found. In contrast, granulocytes were not detected here. Lymphocytes, NK cells, monocytes and granulocytes each expressed CD44, α4-integrins, LFA-1, ICAM-1, CD2 and L-selectin in a pattern characteristic to cell type, but this did not correlate with the different ability of the leucocyte subsets to interact with the two types of HEV. In conclusion, determining the expression of CD44, α4-integrins, LFA-1, ICAM-1, CD2 and L-selectin on blood leucocytes alone is not sufficient to predict leucocyte/endothelium interaction in vivo     

A CD44 monoclonal antibody differentially regulates CD11a/CD18 binding to intercellular adhesion molecules CD54, CD102 and CD50

We have made a monoclonal anti-CD44 antibody which is able to activate the leukocyte integrin CD11a/CD18. Activated T cells strongly aggregated, and the aggregation was shown to be intercellular adhesion molecule (ICAM)-1 (CD54) and ICAM-2 (CD102) dependent. Using purified ICAM coated on plastic, only binding to ICAM-1 was increased by the CD44 antibody, whereas activation by phorbol ester increased binding to both ICAM-1 and ICAM-3. The binding to ICAM-2 was not affected by either treatment. These findings show that the CD11a/CD18 integrin can be activated in a ligand-specific manner by engagement of CD44.     

Expression of the CMRF-35 antigen, a new member of the immunoglobulin gene superfamily, is differentially regulated on leucocytes.

A new monoclonal antibody, CMRF-35, has been generated that recognized a 224 amino acid cell surface protein which is a novel member of the immunoglobulin gene superfamily. The antibody, raised against large granular lymphocytes (LGL), stains LGL, monocytes, macrophages and granulocytes but not platelets or erythrocytes. In addition, a subset of peripheral blood T lymphocytes (26.6 +/- 13.4% CD5+ cells) and B lymphocytes (13.7 +/- 6.8% CD20+ cells) stained with CMRF-35 but tonsil T and B cells were essentially negative. Expression of the CMRF-35 antigen (Ag) on different leucocyte populations was markedly influenced by stimulation of the cells with mitogens and cytokines. Activation of peripheral blood T cells with phytohaemagglutinin (PHA), or phorbol myristate acetate (PMA) and calcium ionophore (CaI) led to a decrease in the proportion of CMRF-35+ T lymphocytes. In contrast, PHA activation of tonsil T lymphocytes resulted in an increase in CMRF-35 Ag expression (47.1 +/- 1.5% CD5 cells at 6 days). An increase in CMRF-35 Ag was also seen on phorbol ester and CaI-activated tonsil B cells. No change in CMRF-35 expression on natural killer (NK) cells occurred following activation with interleukin-2 (IL-2) but the CMRF-35 Ag was down-regulated following Fc receptor stimulation. A moderate increase in CMRF-35 expression occurred during monocyte-macrophage differentiation and the expression of the Ag on monocytes was differentially regulated by interferon-gamma (IFN-gamma). This regulation of the CMRF-35 Ag on the leucocyte surface suggests that the molecule has an important function common to diverse leucocyte types.     

Minimal cross-linking and epitope requirements for CD40-dependent suppression of apoptosis contrast with those for promotion of the cell cycle and homotypic adhesions in human B cells

Eight different CD40 mAb shared with soluble trimeric CD40 ligand (sCD40LT) the capacity to rescue germinal center (GC) B cells from spontaneous apoptosis and to suppress antigen receptor-driven apoptosis in group I Burkitt's lymphoma cells. Three mAb (G28-5, M2 and M3) mimicked sCD40LT in its ability to promote strong homotypic adhesion in resting B cells, whereas others (EA5, BL-OGY/C4 and 5C3) failed to stimulate strong clustering. Binding studies revealed that only those mAb that promoted strong B cell clustering bound at, or near to, the CD40L binding site. While all eight mAb and sCD40LT were capable of synergizing with IL-4 or phorbol ester for promoting DNA synthesis in resting B cells, co-stimulus-independent activation of the cells into cycle through CD40 related directly to the extent of receptor cross-linking. Thus, mAb which bound outside the CD40L binding site synergized with sCD40LT for promoting DNA synthesis; maximal levels of stimulation were achieved by presenting any of the mAb on CD32 transfectants in the absence of sCD40LT or by cross-linking bound sCD40LT with a second antibody. Monomeric sCD40L, which was able to promote rescue of GC B cells from apoptosis, was unable to drive resting B cells into cycle. These studies demonstrate that CD40-dependent rescue of human B cells from apoptosis requires minimal cross-linking and is essentially epitope independent, whereas the requirements for promoting cell cycle progression and homotypic adhesion are more stringent. Possible mechanisms underlying these differences and their physiological significance are discussed.     

Analysis of antigen uptake and presentation by Epstein-Barr virus-transformed human lymphoblastoid B cells

Epstein-Barr virus-transformed human B cells (EBV-B cells), but not resting B cells or B cells activated by T cell-derived factors, have been shown to support the proliferation of tetanus toxoid (TT)-specific autologous T cell clones in response to TT antigen. The accessory cell function of EBV-B cells was compared to that of monocytes with regard to antigen uptake and processing. After an 18-h incubation period with 125I-labeled TT, the amount of radioactivity associated with the cells (approximately 50 ng/10(7) cells) and the percentage of cells containing radiolabeled material (approximately 50%) were equivalent for EBV-B cells and monocytes. Like with monocytes, EBV-B cells pulsed with TT for 18 h or more were equivalent in their capacity to induce T cell proliferation to EBV-B cells to which soluble TT was added for the duration of the culture period. The requirements for antigen uptake and presentation to T cells were similar for both EBV-B cells and monocytes. Both processes were energy dependent, inhibited by cold (4 degrees C), 2-deoxyglucose, and azide, and both required no de novo protein synthesis as they were not affected by pretreatment of the cells with the irreversible protein inhibitor pactamycin . Trypsin treatment of antigen-pulsed EBV-B cells and monocytes followed by fixation for 1 min in 0.03% paraformaldehyde completely abolished the capacity of both cell types to induce T cell proliferation. In both EBV-B cells and monocytes, antigen presentation, but not antigen uptake, was inhibited by the addition of the lysosomotropic agent chloroquine during the antigen-pulse period suggesting that the mechanisms of antigen processing are similar for both cell types. Vacuoles positive for acid phosphatase with an electron microscopic structure similar to that of lysosomes were found in EBV-B cells but not in resting B cells or B cells activated by T cell-derived factors. The present observations indicate that EBV-B cells take up antigen and process it in a fashion similar to monocytes. The presence of lysosomes appears to correlate with the capacity of B cells to present antigen.     

Interleukin 4 and soluble CD23 as progression factors for human B lymphocytes: analysis of their interactions with agonists of the phosphoinositide "dual pathway" of signalling

Human B lymphocytes pre-activated for 24 h with a combination of phorbol dibutyrate [P(Bu)2] and ionomycin were found to provide excellent targets for assessing the detailed action of B cell progression factors. Both recombinant interleukin 4 (IL 4) and affinity-purified 25-kDa fragment of the CD23 molecule (sol-CD23) were shown to be active in this assay. While the progression activity of IL 4 was enhanced by continued co-culture with P(Bu)2, that of sol-CD23 was found to be more strictly dependent upon such a joint application with the phorbol ester. Similar requirements were observed for triggering cell-cycle progression in the pre-activated B cells when using a stimulating CD23 antibody. Ionomycin, in contrast to P(Bu)2, did not augment either IL 4 or sol-CD23 in these assays but did enhance significantly the progression activity of an anti-CDw40 antibody. When added to B cells concomitantly with, or prior to, a high dose of phorbol ester, IL 4 unexpectedly down-regulated the subsequent mitogenic response to this agent whereas, when added 24 h later, IL 4 up-regulated such stimulations. The latter sequence of additions resulted in a particularly dramatic induction of CD23 at the B cell surface, much more so than seen when B cells were incubated with either IL 4 alone or with IL 4 and P(Bu)2 together. This up-regulation of surface CD23 was, in turn, mirrored by the appearance of large amounts of the soluble form of the molecule in such cultures. The findings are discussed with reference to possible mechanisms through which IL 4 and CD23 interact to exert their multiple actions on B cell regulatory pathways.     

Phorbol ester causes down-regulation of CD11/CD18-independent neutrophil adherence to endothelium

Neutrophils adhere to interleukin-1 (IL-1)-, tumour necrosis factor (TNF)- or lipopolysaccharide (LPS)-pretreated human umbilical vein endothelial cells (HEC) by CD11/CD18-dependent and independent mechanisms. We investigated CD11/CD18-independent neutrophil adherence to LPS-pretreated HEC by: (i) pretreating neutrophils with the anti-CD18 monoclonal antibody mAb 60.3; (ii) performing assays in the absence of Mg2; or (iii) using neutrophils isolated from a patient with leucocyte adhesion deficiency (CD11/CD18-deficiency). Under each of these conditions, CD11/CD18-independent neutrophil adherence to LPS-pretreated HEC was significantly greater than adherence to untreated HEC (15-18% versus 3-7%). In each case, however, stimulation of neutrophils with phorbol ester (PMA) abolished CD11/CD18-independent adherence to LPS-pretreated HEC (less than 5% adherence). Stimulation of neutrophils with bacterial chemotactic peptide (FMLP) or calcium ionophore (A23187) likewise reduced CD18-independent adherence to LPS-pretreated HEC. PMA also inhibited CD11/CD18-independent neutrophil adherence to HEC pretreated with IL-1 or TNF (80-90% inhibition). In contrast, PMA markedly enhanced CD11/CD18-dependent adherence to untreated or LPS-treated HEC. We conclude that stimulation of neutrophils with phorbol ester or other direct agonists down-regulates the CD11/CD18-independent mechanism of neutrophil adherence to IL-1, TNF- or LPS-pretreated HEC.     

Ligation of the CD23,p45 (BLAST-2,EBVCS) antigen triggers the cell-cycle progression of activated B lymphocytes

CD23,p45 (BLAST-2,EBVCS) is a 45-kDa lineage-restricted antigen which appears on the surface of human B cells shortly after activation. A monoclonal antibody (MHM6) to CD23,p45, as well as a polyclonal rabbit antibody raised against the purified antigen were found to promote DNA synthesis in purified tonsillar B cells which had been activated with phorbol ester. Interleukin 1, which was not, by itself, stimulatory for either resting or activated B cells, significantly augmented the growth-promoting properties of MHM6. Kinetic studies indicated that while MHM6 exerted its influence in early G1, interleukin 1 acted later in the cycle just prior to the entry of cells into S phase. The findings demonstrate a role for CD23,p45 in triggering the progression of activated B lymphocytes through the G1 phase of the cell cycle. The possibility that this antigen serves as a receptor for a B cell stimulatory factor is discussed.