Sybille Lecoanet-Henchoz

Annexin V used for measuring apoptosis in the early events of cellular cytotoxicity

BACKGROUND Current cytotoxic assays, including Cr release and fluorescent assays, do not directly measure the proportion of target cells which are killed by apoptosis. Cell-mediated cytotoxicity induced by CTLs and NK cells is mainly regulated by the perforin-granzyme, the Fas ligand (Fas L), and the Tumor Necrosis Factor (TNF)-alpha pathways. Perforin generates pores in the membrane of target cells, allowing granzyme B to enter and initiate apoptosis. The other effectors, Fas L and TNF-alpha act by an apoptosis mechanism, leading to DNA fragmentation. A three color flow cytometric method to measure cell-mediated cytotoxicity induced by CTLs or NK cells is described. METHODS The fluorochromes used are: PKH-26, a stable membrane dye for the labeling of the effector cells, annexin V-FITC which allows the direct evaluation of early apoptotic cells and propidium iodide which distinguishes membrane permeabilized and late apoptotic cells. RESULTS By eliminating through gating PKH-26 positive effector cells, we obtain a direct estimation of the percentage of target cells in the early stages of apoptosis as well as the percentage of target cells dying after late apoptosis and membrane permeabilization. The cytotoxic activity of IL-2 stimulated PBL against K562, Jurkat and KYM-1 was evaluated. CONCLUSIONS This rapid and novel assay permits the discrimination of the cell death mechanisms occurring during a cytotoxic response and to precisely evaluate the contribution of apoptosis in the early phases of cell-mediated cytotoxicity.

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.

The distribution of IL-13 receptor α1 expression on B cells, T cells and monocytes and its regulation by IL-13 and IL-4

To study the expression of IL‐13 receptor α1 (IL‐13Rα1), specific monoclonal antibodies (mAb) were generated. Surface expression of the IL‐13Rα1 on B cells, monocytes and T cells was assessed by flow cytometry using these specific mAb. Among tonsillar B cells, the expression was the highest on the IgD+ CD38− B cell subpopulation which is believed to represent naive B cells. Expression was also detectable on a large fraction of the IgD−CD38− B cells but not on CD38+ B cells. Activation under conditions which promote B cell Ig class switching up‐regulated the expression of the receptor. However, the same stimuli had an opposite effect for IL‐13Rα1 expression levels on monocytes. While IL‐13Rα1 mRNA was clearly detectable in T cell preparations, no surface expression was detected. However, permeabilization of the T cells showed a clear intracellular expression of the receptor. A soluble form of the receptor was immunoprecipitated from the supernatant of activated peripheral T cells, suggesting that T cell IL‐13Rα1 might have functions unrelated to the capacity to form a type II IL‐4 / IL‐13R with IL‐4Rα.

Structure and functions of CD23.

This review summarizes recent data on CD23, a low affinity receptor for IgE (Fc epsilon RII). CD23 is the only FcR which does not belong to the immunoglobulin gene superfamily. The CD23 molecule was discovered independently as an IgE receptor on human lymphoblastoid B cells [1], as a cell surface marker expressed on Epstein-Barr-Virus-transformed B cells (EBVCS) [2] and as a B-cell activation antigen (Blast 2) [3]. CD23 was shown to be a low affinity receptor for IgE [4,5]. Similar to most FcR, soluble forms of CD23 (sCD23) are released into extracellular fluids. The soluble fragments formed by proteolytic cleavage of surface CD23 are not only capable of binding IgE (IgE binding factors) but also exhibit multiple functions that are not IgE related. These observations together with the finding that CD23 displays significant homology with Ca(2+)-dependent (C-type) animal lectins, suggested the existence of natural ligands other than IgE. The recent finding that CD23 interacts with CD21, CD11b and CD11c indicates that CD23 should be viewed not only as a low affinity IgE receptor but also as an adhesion molecule involved in cell-cell interaction. After a brief overview of the molecular structure, there follows a discussion of the biological activities ascribed to human CD23.

CD23 and B-cell activation

The past year has seen the publication of significant new findings on the regulation of CD23 expression, the precise interaction of CD23 with CD21 and its functional consequences. Moreover, new advances have been made in unravelling the biochemical network of events downstream from the triggering of CD23 in human B cells. Analyses of the properties of CD23-deficient mice have demonstrated a link between IgE regulation and CD23 in vivo.

A new role for CD23 in inflammation

Abstract CD23, is expressed on a variety of haematopoietic cell types and displays pleiotropic activities in vitro . Here Jean-Yves Bonnefoy and collegues discuss a novel interaction between CD23 and the α chains of the β 2 integrins, CD11b and CD11c, that leads to a proinflammatory pattern of macrophage activation. They describe how neutralizing antibodies to CD23 can decrease the severity of murine arthritis.

Regulation of IgE Synthesis by CD23/CD21 Interaction

At least two cell-derived signals have been shown to be necessary for the induction of immunoglobulin isotype switching in B cells. The first signal is given by either of the soluble lymphokines interleukin (IL)-4 or IL-13 which induce germline epsilon transcript expression, but alone is insufficient to trigger secretion of IgE. The second signal is provided by a physical interaction between B cells and activated T cells, basophils and mast cells, and it has been shown that the CD40/CD40L pairing is crucial for mediating IgE synthesis. In HIGM1 syndrome, which is characterized by greatly decreased levels of IgG, IgA and IgE, there are mutations in CD40L resulting in a completely non-functional extracellular domain. CD40L is therefore playing a central role in Ig switching. Amongst the numerous pairs of surface adhesion molecules, the CD23/CD21 pair seems to play a key role in the generation of IgE. The CD23 molecule is positively and negatively regulated by factors which increase or decrease IgE production, respectively. Antibodies to CD23 have been shown to inhibit IL-4-induced human IgE production in vitro and to inhibit antigen-specific IgE responses in a rat model, in an isotype-selective manner. CD23 interacts with CD21 on B cells, preferentially driving IgE production. CD23 recognises two main epitopes on the CD21 molecule. One region consists of short consensus repeat sequences (SCRs) 1-2 and the other of SCRs 5-8. In the latter region ASn370 and Asn295 are critical in the interaction with the lectin CD23. Therefore, a restricted number of cytokines and surface molecules seems to selectively regulate human IgE synthesis.

Alpha-1 antitrypsin up-regulates human B cell differentiation selectively into IgE- and IgG4-secreting cells

Numerous allergens have proteolytic activities. It has been speculated that this property may contribute to their allergenicity. Therefore, we have evaluated the effect of different physiological protease inhibitors (PI) on the regulation of human IgE synthesis. Unexpectedly, the serine PI, alpha‐1 antitrypsin, also called alpha‐1 protease inhibitor (α1PI), induced a potent and selective dose‐dependent increase of IgE and IgG4 production by human tonsillar B cells stimulated with the IgE and IgG4 switch factors, IL‐ 4 and anti‐CD40 mAb. The other serine PI tested were inefficient. Furthermore, this effect of α 1PI was accompanied by an increase in (1) germ‐line and mature ϵ mRNA transcription, (2) proliferation and (3) membrane CD23 and CD21 expression, while the expression of other molecules involved in the regulation of IgE synthesis was unchanged. Since CD23‐CD21 pairing plays a crucial role in the up‐regulation of IgE synthesis, we have tested whether blocking this interaction affected α1PI‐increased IgE production. The neutralizing anti‐CD23 mAb, Mab 25, partly reversed the IgE increase caused by α 1PI. Moreover, α 1PI potentiation of IgE synthesis was prevented by elastase, a natural substrate of α 1PI, thereby suggesting that α 1PI may inhibit endogenous B cell enzyme(s) involved in the down‐regulation of IgE synthesis. α 1PI also potentiated IgE and IgG4 production by IL‐ 4 ‐stimulated peripheral blood mononuclear cells but was not a switch factor for IgE and IgG4 as it was unable to replace IL‐ 4 or anti‐CD40 mAb in inducing IgE and IgG4 production. In conclusion, this study shows that α 1PI acts as a potent co‐stimulus for IgE and IgG4 synthesis and suggests that the equilibrium between protease/protease inhibitor participates in the control of human IgE and IgG4 synthesis.

Thiols decrease human IL-4 production and IL-4-induced immunoglobulin synthesis.

IgE IL-4 N-acetyl-L-cysteine Thiols Th2 Correspondence to: Dr. Jean-Yves Bonnefoy, Glaxo Wellcome, Geneva Biomedical Research Institute, Immunology Department, Chemin des Aulx, 14, CH–1228 Plan-les-Ouates/Geneva (Switzerland) N-acetyl-L-cysteine (NAC) is an antioxidant precursor of intracellular glutathione (GSH) [1] which is usually given in man as a mucolytic agent. Due to its ability to decrease HIV replication [2] and to restore intraand extracellular GSH levels, NAC has been recently proposed as an anti-HIV agent [2]. In vitro, NAC and GSH have been shown to act on T cells by increasing IL-2 production, synthesis and turnover of IL-2 receptors, proliferation [3, 4] ‚ cytotoxic properties [5] and resistance to apoptosis [4]. NAC and some other thiol antioxidants have also been reported to regulate the activation of some transcription factors involved in IL-2 production, such as NFκB and activator protein-1 transcription factors [6]. In view of the effects of NAC on T cells, we have evaluated whether NAC could selectively regulate Thlor Th2-derived cytokine production. We have observed that NAC and GSH dose dependently decrease human IL-4 production by PMA plus ionomycin-stimulated peripheral blood T cells and by Con A or anti-CD3 mAbstimulated ThOand Th2-like T cell clones. This effect (ranging from 0.5 to 20 mM) was also associated to a decrease of IL-4 mRNA transcription. In contrast, NAC and GSH have no effect on IFN-γ production but increase IL-2 production as well as T cell proliferation. One can speculate that thiols may affect the activation of transcription factors belonging to the NF-κB family and implicated in the transcription of IL-4 (such as NEAT) [7]. A key function of IL-4 is its ability to induce immunoglobulin (Ig)E and IgG4 production by human B cells [8]. This production requires a second primary signal given by triggering CD40 (by an anti-CD40 mAb or by CD40 ligand) [9] and is tightly controlled by the interaction of the cell surface