Our laboratory has identified IgE-dependent histamine releasing factors (HRF) from a variety of sources including nasal lavages obtained during the late phase of IgE-dependent reactions (LPR), blister fluids during the LPR, human lung macrophage and mononuclear cell supernatants and supernatants from die like cell line, U937. Only certain allergic donors' basophils respond to these a functional heterogeneity of the IgE molecule. We term the IgE that interacts with HRF as IgE+. Our first working hypothesis is that these IgE-dependent HRFs are responsible for stimulation of basophils during the LPR in those subjects who have IgE+. Preliminary purification results reveal that crude HRF is composed of a heterogeneous group of molecules. Using the U937 cell line supernatants in order to pursue structural and functional studies, we have developed a purification scheme involving Sephadex G75, anion exchange and chromatography, preparative isoelectric focusing, reverse phase HPLC and IgM anti-HRF affinity column. We plan to scale up this purification scheme by using large volumes of the supernatant from the cell line. HRF will be run on SDS gels, transblotted onto Problott and sequenced. Once sequence information is obtained, the gene for HRF will be cloned using anchored PCR techniques and the protein will be expressed. With recombinant HRF material, an IgG monoclonal antibody to HRF will be made and an ELISA specific for HRF established in order to examine the kinetics of the appearance of HRF during late phase reactions. We will utilize the in vivo antigen challenge models available to us in the skin, nose and airways for these studies. A second major hypothesis is that the heterogeneity of the IgE molecule, as defined by interaction with HRF, influences the severity of human allergic disease. We will attempt to understand the structural basis of this heterogeneity and relate it to in vivo situations. To accomplish this, we will extend our preliminary studies which reveal that using recombinant IL-4 and hydrocortisone in vitro. Since we have evidence that this functional heterogeneity is determined by differential glycosylation we will manipulate the in vitro cultures with carbohydrate processing glycosylation of the in vitro product with enzymes such as N-glycanase. We also plan to perform site-specific carbohydrate analysis of the IgE+ and the IgE- molecules. We Will attempt to raise monoclonal antibodies to discriminate between IgE+ and IgE-. Such antibodies would be useful determining the presence and/or the amount of IgE+ in various allergic disease states. Using the in vitro model of IgE synthesis, we will also determine whether immunotherapy, or other interventions such as administration of glucocorticoids influence IgE glycosylation in vivo, in collaboration with members of our Division who routinely study patients with rhinitis and asthma.
| Kleine-Tebbe, J; Hamilton, R G; Roebber, M et al. (1995) Purification of immunoglobulin E (IgE) antibodies from sera with high IgE titers. J Immunol Methods 179:153-64 |
