B. Dugas

CD23 Regulates monocyte activation through a novel interaction with the adhesion molecules CD11b-CD18 and CD11c-CD18

CD23 is expressed on a variety of haemopoietic cells and displays pleiotropic activities in vitro. We report that in addition to CD21 and IgE, CD23 interacts specifically with the CD11b and CD11c, the alpha chains of the beta 2 integrin adhesion molecule complexes CD11b-CD18 and CD11c-CD18, on monocytes. Full-length recombinant CD23 incorporated into fluorescent liposomes was shown to bind to COS cells transfected with cDNA encoding either CD11b-CD18 or CD11c-CD18 but not with CD11a-CD18. The interaction was specifically inhibited by anti-CD11b or anti-CD11c, respectively, and by anti-CD23 MAbs. The functional significance of this ligand pairing was demonstrated by triggering CD11b and CD11c on monocytes with either recombinant CD23 or anti-CD11b and anti-CD11c MAbs to cause a marked increase in nitrite-oxidative products and pro-inflammatory cytokines (IL-1 beta, IL-6, and TNF alpha). These CD23-mediated activities were decreased by Fab fragments of MAbs to CD11b, CD11c, and CD23. These results demonstrate that CD11b and CD11c are receptors for CD23 and that this novel ligand pairing regulates important activities of monocytes.

Involvement of cyclic AMP and nitric oxide in immunoglobulin E-dependent activation of Fc epsilon RII/CD23+ normal human keratinocytes.

Epidermal keratinocytes (EK) are exposed to multiple inflammatory stimuli and paracrine factors secreted by various dermal cells (lymphocytes, mast cells, macrophages, fibroblasts) during wounding, cutaneous allergy, and infections. We have previously demonstrated that after stimulation with interleukin 4 or interferon-gamma, human EK express the low-affinity receptor for IgE (Fc epsilon RII/CD23) on their surface. In the present study, we showed that the ligation of CD23 by IgE/anti-IgE immune complexes or specific monoclonal antibody induces a dose-dependent release of interleukin 6 and tumor necrosis factor-alpha from EK. CD23-ligation activates the nitric oxide-dependent pathway, as demonstrated by the high levels of nitrites released in cell supernatants, and the accumulation of intracellular cyclic nucleotides in EK. These second messengers are required for IgE-dependent stimulation of cytokine production by these cells, inasmuch as this is completely abolished by the use of cAMP or nitric oxide synthase antagonists. Human epithelial keratinocytes may thus participate in IgE-mediated immune responses, through their ability to express functional CD23 antigen.

Ligation of CD23 activates soluble guanylate cyclase in human monocytes via an L-arginine-dependent mechanism.

Transduction through FcR2/CD23 was analyzed in normal human monocytes using immunoglobulin E (IgE)‐anti‐IgE immune complexes (IgE ICs) and monoclonal antibodies (mAbs) to CD23. Anti‐CD23 mAb and IgE IC triggered a time‐dependent increase in cGMP and cAMP in interleukin‐4–preincubated (CD23+) but not in unstimulated (CD23‐) monocytes. Maximal cGMP and cAMP accumulations were observed 10 and 20 min, respectively, after the onset of CD23 ligation. The increase in cGMP was inhibited with N ω‐monomethyl‐l‐arginine (L‐NMMA), which also partially affected cAMP accumulation. Addition of an anti‐CD23 mAb Fab fragment inhibited the IgE IC– and the anti‐CD23 mAb–induced cGMP and cAMP accumulation, confirming the engagement of CD23. In addition, IgE IC and anti‐CD23 mAb induced, at least in some donors, a production of nitrite that was inhibited in the presence of L‐NMMA. Taken together, these findings suggest a possible involvement of the nitric oxide synthase pathway in IgE IC–mediated activation of CD23+ monocytes. J. Leukoc. Biol. 57: 160–167; 1995.

Characterization of a constitutive type III nitric oxide synthase in human U937 monocytic cells: stimulation by soluble CD23

The soluble cleavage fragment of the low‐affinity immunoglobulin E (IgE) receptor/CD23 (sCD23 25 000 MW) and antibodies directed against their receptors on monocytes, CD11b and CD11c, stimulate the production of nitric oxide (NO) by these cells and we have suggested that the enzyme involved could be related to the endothelial constitutive type III nitric oxide synthase (ecNOS). In the present work, we have analysed the characteristic properties of this NOS isoform in the model of the human promonocytic cells U937 By reverse‐transcription polymerase chain reaction (RT‐PCR), the presence of an mRNA coding for type III NOS was found in U937 cells and the corresponding protein was detected by immunofluorescence in permeabilized cells with a specific anti‐ecNOS monoclonal antibody (mAb). Membrane extracts displayed a NOS activity dependent on the presence of calcium and calmodulin in the reaction medium and that was abrogated in the presence of EGTA. Recombinant soluble CD23 (25 000 MW) was found to trigger an NO‐dependent cGMP accumulation in these cells, which was abrogated by calcium chelators and inhibitors of the calcium/calmodulin complex. Moreover, sCD23 elicited a transient augmentation of intracytoplasmic free calcium concentration [ Ca2+]i that was dependent on the presence of calcium in the external buffer and was prevented in the presence of EGTA, indicating that it was due to a calcium influx. In conclusion, human promonocytic cells such as U937 exhibit a functional type III NOS that can be stimulated by calcium‐raising agents, such as sCD23

Role of leukotriene B4 in the interleukin-4-induced human mononuclear phagocyte activation.

Interleukin‐4 (IL‐4) induced a time‐ and dose‐dependent production of leukotriene B4 (LTB4) by human resting monocytes indicating that IL‐4 induced the activation of the 5‐lipoxygenase pathway in resting human monocytes. Maximal effect was observed in the presence of 10 ng/ml IL‐4, and in kinetics experiments LTB4 production plateaued 40 min after the onset of stimulation. When stimulated for 48 hr with IL‐4, resting human monocytes expressed and released the low‐affinity receptor for IgE (CD23) and were partially inhibited in the presence of a highly non‐redox 5‐lipoxygenase inhibitor (BW B70C), suggesting that the production of LTB4 partially contributed to the IL‐4‐induced CD23 expression and release. This hypothesis was strengthened by the fact that exogenous LTB4 (10 nm) was found to increase the effect of a suboptimal dose of IL‐4 (1 ng/ml). In addition to these phenotypical changes, IL‐4 primed the phorbol‐12‐myristate‐13‐acetate (PMA)‐induced luminol‐dependent chemiluminescence response (LDCL) by normal human monocytes, this priming effect being abrogated in the presence of BW B70C. Taken together, these data indicated that IL‐4 induced the production of LTB4 by activation of the 5‐lipoxygenase pathway in human monocytes, and that the activation of this pathway could upregulate the expression and release of CD23 and the respiratory burst of these cells.

Purification of normal human bone-marrow-derived basophils.

It is well established that basophils and eosinophils share a common differentiation pathway, although the factors regulating their terminal commitment (towards one or other lineage) are not yet fully defined. Interleukin-3 (IL-3) is a major differentiation factor for both human eosinophils and basophils, yielding a mixed population composed predominantly of eosinophilic cells (65 +/- 9%; n = 4), basophils at different stages of maturity (29 +/- 6%; n = 4) and monocytes/macrophages (6 +/- 3%; n = 4), after 3-4 weeks in culture. Using a relatively rapid and simple method involving a first step of gradient density centrifugation over a Percoll gradient (d = 1.063 g/ml) and a subsequent step of adhesion on tissue-culture-treated plastic, a cell population composed of 94 +/- 5% normal basophils and their precursors, with no demonstrable mast cells, was reproducibly obtained from human hematopoietic cells cultured for 3-4 weeks in the presence of recombinant IL-3. These cells contained high levels of histamine (1.39 +/- 0.14 pg/cell) and released this mediator upon stimulation with calcium ionophore A23187 and in a dose dependent manner upon stimulation with IgE-anti IgE, demonstrating their functional capacity. This relatively simple method therefore permits the production of large quantities of pure populations of normal and functional human bone-marrow-derived-basophils.

Platelet Activating Factor Inhibits Both the Proliferation and the Differentiation of Activated B Lymphocytes in Response to Interleukin-2: A Comparison with Interleukin-4 - Mediated Inhibitory Effects

The role of the platelet activating factor in human B lymphocyte responses to Interleukin-2 was examined and compared with that of Interleukin-4 by assessing the ability of this molecule to modulate proliferation and differentiation. Highly purified B lymphocytes were prestimulated for 48 h with Staphylococcus aureus Cowan Strain I and then were cultured with Interleukin-2 alone or in combination with either Interleukin-4 or the platelet activating factor and the proliferation (after 3 days) and the immunoglobulins (IgG and IgM) production (after 7 days) were evaluated. When SAC-activated B lymphocytes were preincubated overnight with PAF (0.0001 to 1 μM) or with IL-4 (1 to 100 U/ml) both the IL-2-induced proliferation and immunoglobulins secretion were inhibited. This inhibition was not a reflection of a decreased expression of the IL-2 receptor (CD25) because this expression was not modified on SAC-activated B lymphocytes after preincubation with either PAF or IL-4. Moreover, this suppression effect was not the result of a delayed response to IL-2. The PAF-induced suppression was overcome in the presence of PAF antagonists (BN 52021 and BN 50730) but was not modified in the presence of a neutralizing anti-IL-4 antiserum. On the other hand, the IL-4 mediated suppression was totally reversed in the presence of the neutralizing anti-serum and only marginally reversed in the presence of the PAF antagonists. These results indicate that both PAF and IL-4 may exert a number of immunoregulatory actions on human B lymphocyte proliferation and differentiation. They interfere with the stimulation of activated B lymphocytes by IL-2 and could play an important immunoregulatory role in the determination of isotypic regulation in the specific humoral responses.