William Cushley

CD23/FcεRII: molecular multi-tasking.

CD23 is the low‐affinity receptor for immunoglobulin (Ig)E and plays important roles in the regulation of IgE responses. CD23 can be cleaved from cell surfaces to yield a range of soluble CD23 (sCD23) proteins that have pleiotropic cytokine‐like activities. The regions of CD23 responsible for interaction with many of its known ligands, including IgE, CD21, major histocompatibility complex (MHC) class II and integrins, have been identified and help to explain the structure–function relationships within the CD23 protein. Translational studies of CD23 underline its credibility as a target for therapeutic intervention strategies and illustrate its involvement in mediating therapeutic effects of antibodies directed at other targets.

The antimitotic effect of the neem terpenoid azadirachtin on cultured insect cells

When cultured insect cells (Sf9) were grown in the presence of 5 x 10(-6) M azadirachtin, there was a rapid increase in the mitotic index, with the appearance of many aberrant mitotic figures. Flow cytometry established that cells accumulated in the G2/M phase of the cell cycle, and that the effect was concentration-dependent. At 10(-8) M a period of 20 h was necessary to raise the proportion in G2/M to 42% above the control values, but at 5 x 10(-6) M more than 90% of the cells were in this phase. Azadirachtin had the same effect on C6/36 mosquito cells, but failed to affect L929 murine fibroblast cells even at a concentration of 10(-4) M over 72 h. Experiments with colchcine and taxol showed similarities of action between azadirachtin and colchicine, and azadirachtin was apparently able to displace colchicine-fluorescein from binding-sites in living insect cells. Another similarity between azdirachtin and colchicine was that both phytochemicals prevented the polymerisatrion in vitro of mammalian tubulin, although the azadirachtin was much less effective.

STUDIES ON THE SITE AND MECHANISM OF ATTACHMENT OF PHOSPHORYLCHOLINE TO A FILARIAL NEMATODE SECRETED GLYCOPROTEIN

We have recently shown that the immunomodulatory substance phosphorylcholine (PC) is covalently attached to ES-62, a major secreted protein of the filarial nematode parasite Acanthocheilonema viteae, via an N-linked glycan. Linkage of PC to N-glycans is previously unreported, and hence we have investigated the biochemical events underlying it. PC addition was found by pulse-chase experiments to be a fairly early event during intracellular transport, occurring within 40-60 min of protein synthesis. Biosynthetic labeling/immunoprecipitation experiments revealed that addition of PC to ES-62 was blocked by (i) brefeldin A, an inhibitor of trafficking of newly synthesized proteins from the endoplasmic reticulum (ER) to the Golgi, (ii) 1-deoxynorijirimycin, an inhibitor of glucosidase activity in the ER, and (iii) 1-deoxymannojirimycin, an inhibitor of mannosidase I in the cis Golgi. Swainsonine, an inhibitor of mannosidase II in the medial Golgi, did not affect PC addition. Taken together these data indicate that PC attachment is a post-ER event which is dependent on generation of an appropriate substrate during oligosaccharide processing. Furthermore, they strongly suggest that PC addition takes place in the medial Golgi and that the substrate for addition is the 3-linked branch of Man5GlcNAc3 or Man3GLcNAc3.

The CD23a and CD23b proximal promoters display different sensitivities to exogenous stimuli in B lymphocytes.

The single human CD23 gene encodes two protein products differing by six or seven amino acids in the extreme N-terminal cytoplasmic domain. The patterns of expression of CD23a and CD23b transcripts differs as a function of cell type and cell stimulation, with expression of CD23a being largely restricted to B cells and CD23b synthesis being inducible in a variety of haematopoietic cells by a range of exogenous stimuli. In this study, short defined sequences of the CD23a and CD23b proximal promoter regions were used to drive expression of exogenous reporter genes in transiently-transfected B cells exposed to a range of cellular stimuli. The CD23a promoter was activated only by IL-4, whereas the CD23b promoter was stimulated not only by IL-4, but also by stimulation with anti-μ, and anti-CD40. Deletion mutant analysis illustrated that of the two putative STAT6 binding sites present in the CD23a proximal promoter, deletion of the first site abrogated IL-4-driven transcriptional activation. Conversely, deletion of both STAT6 binding sites in the CD23b promoter was required before IL-4 sensitivity was lost. When the same CD23b promoter mutants were studied in the context of anti-CD40 and anti-μ stimulation of transfected cells, deletion of the NF-κB site abrogated anti-CD40-driven transcriptional activation, but not anti-μ-mediated effects which required additional deletion of putative AP1 sites lying close to the CD23b initiator methionine codon. The data of this report are consistent with the interpretation that the upstream regions of the CD23a and CD23b isoform coding sequences show distinct sensitivities to agents which induce CD23 protein expression at the plasma membrane, and that transcriptional activation by discrete stimuli reflects activation of particular transcriptional regulatory factors.

αvβ5 Integrin Sustains Growth of Human Pre-B Cells through an RGD-independent Interaction with a Basic Domain of the CD23 Protein

CD23 is a type II transmembrane glycoprotein synthesized by hematopoietic cells that has biological activity in both membrane-bound and freely soluble forms, acting via a number of receptors, including integrins. We demonstrate here that soluble CD23 (sCD23) sustains growth of human B cell precursors via an RGD-independent interaction with the αvβ5 integrin. The integrin recognizes a tripeptide motif in a small disulfide-bonded loop at the N terminus of the lectin head region of CD23, centered around Arg172, Lys173, and Cys174 (RKC). This RKC motif is present in all forms of sCD23 with cytokine-like activity, and cytokine activity is independent of the lectin head, an “inverse RGD” motif, and the CD21 and IgE binding sites. RKC-containing peptides derived from this region of CD23 bind αvβ5 and are biologically active. The binding and activity of these peptides is unaffected by inclusion of a short peptide containing the classic RGD sequence recognized by integrins, and, in far-Western analyses, RKC-containing peptides bind to the β subunit of the αvβ5 integrin. The interaction between αvβ5 and sCD23 indicates that integrins deliver to cells important signals initiated by soluble ligands without the requirement for interactions with RGD motifs in their common ligands. This mode of integrin signaling may not be restricted to αvβ5.

Positioning the immune system: unexpected roles for α6‐integrins

Accurate initiation and regulation of immune responses requires the dynamic recruitment and retention of lymphocytes in lymphoid tissue. It is now well accepted that the cells of the innate and adaptive immune systems reposition themselves in response to changes in the pattern of expression of chemokines and chemokine receptors. In fact, chemokines drive not only the movement of cells between and within lymphoid microenvironments, but also modify the affinity of integrins to ensure that cells recruited to a particular location remain there. The last few years have seen great advances in our understanding of the roles of individual chemokines and their receptors in regulating movement of lymphocytes in the bone marrow and peripheral lymphoid tissues and, indeed, in lymphoid organogenesis. Similar progress has been made in appreciation of the structural biology of integrins, such that mechanisms of ligand binding (the structural consequences of integrin activation) are becoming much better understood. The work of Helen Ambrose and Simon Wagner, published in this issue of Immunology1 demonstrates a striking up-regulation of α6-integrin expression by murine germinal centre (GC) B cells, suggesting an important role for α6-containing integrins in the GC.

ROLE OF PROCESSING OF N-LINKED OLIGOSACCHARIDES IN CONTROL OF IMMUNOGLOBULIN SECRETION FROM RAT HYBRIDOMAS

The effects of inhibition of N-linked oligosaccharide processing by glucosidase and mannosidase activities upon secretion of rat hybridoma IgM and IgG have been investigated. The inhibitor of glucosidase I, castanospermine, prevents conversion of the N-linked carbohydrate groups of IgM to a complex form as assessed by resistance to digestion by endo H. The rate of secretion of IgM or IgG from the treated cells is not diminished relative to controls. Similar data are obtained for studies employing the inhibitor of glucosidase I and II activities, 1-deoxynojirimycin. Inhibition of processing by the mannosidase inhibitors 1-deoxymannojirimycin and swainsonine leads to alteration of the qualitative structure of the oligosaccharide groups present on IgG molecules, but again has no effect upon the rate of export of IgG or IgM molecules from treated cells. In each case studied the mu or gamma heavy chains isolated from lysates or culture supernatants of cells treated with glucosidase inhibitors had a higher Mr than the equivalent chains isolated either from control cultures or cultures exposed to either mannosidase inhibitor.

Interaction of the plant toxin ricin with different life cycle stages of Schistosoma mansoni.

The binding of the plant toxin ricin to various life cycle stages of Schistosoma mansoni has been studied. Fluorescein isothiocyanate (FITC)-ricin exhibited binding to schistosomula and adult worms, but not to cercariae or to freshly transformed schistosomula. Binding was specifically inhibited by the presence of galactose in the medium. Analysis of protein synthesis in treated parasites illustrated that ricin failed to intoxicate schistosomula or adult worms. Indeed, fluorescence quenching studies suggested that the fluorescent toxin was confined to the outer bilayer and was not internalised.

Epstein-Barr virus nuclear antigen-1 renders lymphocytes responsive to IL-2 but not IL-15 for survival.

Epstein-Barr virus nuclear antigen-1 (EBNA-1) is the only latent protein expressed in all virus-associated tumours. It plays a critical role in viral propagation and in the replication, episomal maintenance and partitioning of the viral genome. However, its tumorigenic potential is debated. We have previously shown that lymphocytes from a tumour-prone, EBNA-1-expressing, transgenic mouse line show increased responsiveness to interleukin-2 (IL-2). It was important to determine whether this property was unique to the transgenic line or whether it is a general consequence of EBNA-1 expression in B cells. In order to distinguish between these possibilities, explanted lymphocytes from two independent transgenic mouse lines were examined. The lymphocytes from both lines showed enhanced proliferation rates compared with controls. The transgenic lymphocytes survived for extended periods in culture, dependent on the dose of IL-2, while IL-15 (the receptor of which shares the beta and gamma chain components of the IL-2 receptor) induced little effect. In accordance with this, transgenic B cells showed enhanced induction of expression of the IL-2 receptor alpha chain (CD25), which modulates affinity for the ligand. As this phenotype is evident in lymphocytes from mice of both lines, it is necessarily independent of any transgene insertion site effects and may be attributed to EBNA-1 expression. Furthermore, 10/12 tumour-bearing transgenic mice had elevated IL-2 levels in serum and 4/6 tumours were CD25 positive. IL-2 is normally produced by activated T cells in vivo; thus, chronic immune activation or modulation could elicit this unique mode of virus-infected cell survival.

Anti-immunoglobulin and anti-CD40 stimulation induces CD25 expression by resting human tonsillar B lymphocytes

In this report, the effect of ligation of a number of B-cell surface molecules upon expression of CD25, the 55-kDa inducible component of the IL-2 receptor complex found on T and B lymphocytes, is reported. IL-4 is the only cytokine apparently capable of promoting CD25 expression in human high-density quiescent tonsillar B cells; neither IL-10 nor IL-13 could induce CD25 expression. Cross-linking of the antigen receptors or CD40 with antibody elicited CD25 expression in a dose-dependent manner. Stimulation with anti-CD40 promoted CD25 expression in approximately 25% of B cells, while anti-Ig caused 80% or more of cells to become CD25+. In experiments where the stimuli were used in combination, some additive effects upon CD25 expression were noted, but no obvious synergistic effects could be detected.