World Allergy Forum - IgE: Bench to Bedside
IgE Receptor Signaling
Janet M. Oliver
Crosslinking the tetrameric (abg2) IgE receptor, FceRI, on RBL-2H3 mast cells and on human basophils and mast cells activates the signal-initiating kinase, Lyn, that phosphorylates tyrosines on FceRIb and g subunits, creating binding sites for the signal-propagating kinase, Syk. The recruitment and activation of Syk leads within minutes to the tyrosine phosphorylation of many signaling proteins (phospholipase Cg isoforms, phosphatidylinositol 3-kinase, Vav, Grb2, LAT, Cbl and many others), the mobilization of stored and extracellular Ca2+, the release of inflammatory mediators by degranulation and the enhancement of integrin-mediated adhesion, and within hours to cytokine production (1-3). In human basophils lacking Syk protein (the so-called "non-releaser basophils"), FceRI signaling is substantially impaired (4). The goals of this presentation are:
To show that FceRI signaling is controlled not only by which chemical species are available for interaction, but also by where the interactions occur.
To explore mechanisms responsible for the Syk deficiency of non-releaser basophils.
The Topography of FceRI signaling
We have developed a membrane sheet isolation procedure and immunogold labeling methods in order to observe the topography of receptors, kinases and signaling proteins in the TEM at high resolution and without destroying membrane integrity (5-8). We find that FceRI is loosely co-localized with Lyn in small clusters on native membrane sheets prepared from resting RBL-2H3 mast cells (5). FceRI crosslinking with multivalent antigen causes a transient tightening of the Lyn-receptor clusters (6), followed by a rapid separation of receptor from Lyn and the redistribution of Fce RI to distinct membrane patches that stain intensely with osmium (osmiophilic patches; Figure 1A) and are often bordered by coated pits (Figure 1B). Syk is strikingly recruited to the receptor-rich patches (Figure 1B). Syk in turn recruits other signaling proteins including phospholipase Cg 2 (PLCg2), Gab2, Grb2, Cbl and a portion of phosphatidylinositol 3-kinase (PI 3-kinase)(7,8). We hypothesize that the Syk-enriched receptor patches represent primary signaling domains. FceRI crosslinking causes the scaffolding protein, LAT, to redistribute into topographically distinct "rafts" that intersect, but do not mix with, receptor clusters (7,8). LAT rafts contain an additional portion of PI 3-kinase and they concentrate PLCg 1. We hypothesize that LAT rafts represent secondary signaling domains that may propagate distinct arms of the signaling cascade. Recognition that Fce RI signaling may be controlled not only by which chemical species are available for interaction, but also by where the interactions occur, and that multiple signaling domains may be present in native membranes, has potential to open entirely new approaches to modulating FceRI function in allergy and asthma.
Mechanistic Analyses of the Non-Releaser Basophil
Basophils from ~15% of unselected donors do not degranulate in response to FceRI crosslinking (9). In a screen of normal donors (non-asthmatic by self report), we linked this unresponsiveness to the absence of Syk protein from basophils, despite apparently normal levels of Syk mRNA, and showed that Syk levels are normal in other blood leukocytes from the same donors (3,10,11). We have now explored pathways of Syk protein degradation as a possible posttranslational regulatory mechanism in human basophils (12). Three cell permeable inhibitors of proteasome-mediated protein degradation, PSI, lactacystin, and ALLN, substantially increased Syk levels in releaser basophils and restored Syk expression in nonreleaser basophils. Caspase inhibitors were less effective and inhibitors of calpain-mediated protein degradation had no effect. Among other leukocytes, only naïve CD4+ T cells had more Syk after proteasome inhibitor treatment. These data provide evidence for a lineage-restricted, proteasome-dependent mechanism that contributes to Syk regulation in human basophils and may also be relevant to naïve T cells.
It is of obvious interest to know if allergic asthmatics express basophil Syk more consistently than non-atopic non-asthmatics. We are presently screening volunteers to identify a critical number of non-atopic, non-asthmatics and atopic asthmatics for studies of Syk expression and FceRI-mediated basophil degranulation. If the presence of Syk-deficient basophils is indeed associated with the absence of allergic asthma, then a molecular understanding of basophil Syk regulation may yield new treatments useful in asthmatics.
Kepley, CL, BS Wilson, and JM Oliver. 1998. Identification of the FceRI-activated tyrosine kinases Lyn, Syk and Zap-70 in human basophils. J. Allergy Clin. Immunol. 102: 304-315.
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Oliver, JM and BS Wilson. 2001. Effector roles of IgE antibodies: Targeting allergen to the high affinity IgE receptor for FceRI-dependent signaling and antigen presentation. In Inflammatory Mechanisms in Allergic Diseases. Ed. B Zweiman and LB Schwartz. Marcel Dekker, New York. pp 197-232.
Kepley, CL, L Youssef, RP Andrews, BS Wilson and JM Oliver. 1999. Syk deficiency in nonreleaser basophils. J. Allergy Clin. Immunol. 104: 279-284.
Wilson, BS, JR Pfeiffer and JM Oliver. 2000. Observing FceRI signaling from the inside of the mast cell membrane. J. Cell Biol. 149: 1131-1142.
Lara, M, E Ortega, I Pecht, JR Pfeiffer, AM Martinez, RJ Lee, Z Surviladze, BS Wilson and JM Oliver. 2001. Overcoming the signaling defect of Lyn-sequestering, signal curtailing FceRI dimers: Aggregating the dimers allows phosphorylated Fce RI to dissociate from Lyn and form signaling complexes with Syk in specialized membrane domains. J. Immunol. 167: 4329-4337.
Wilson, BS, JR Pfeiffer, Z Surviladze, EA Gaudet and JM Oliver. 2001. High resolution mapping of mast cell membranes reveals primary and secondary domains of FceRI and LAT. J. Cell Biol. 154:645-658.
Wilson, BS, JR Pfeiffer and JM Oliver. 2002. FceRI signaling observed from the inside of the mast cell membrane. Mol. Immunol. In Press.
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Kepley, CL, LYoussef, RP Andrews, BS Wilson and JM Oliver. 2000. Syk-deficient non-releaser basophils show multiple functional defects in FceRI signaling: Partial recovery of Syk expression and signaling by incubation with IL-3. J. Immunol. 165: 5913-5920.
Kepley, CL, LYoussef, RP Andrews, BS Wilson and JM Oliver. 2000. Turning off IgE-mediated signaling nature’s way Reversible Syk deficiency in non-releaser basophils. Allergy & Clinical Immunology International. 13: 11-17.
Youssef, LA, BS Wilson and JM Oliver. 2001. Proteasome-dependent regulation of Syk tyrosine kinase expression in human basophils. Submitted for publication.