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CD23, the low-affinity IgE receptor, is up-regulated on interleukin (IL)-4-stimulated B cells and monocytes, with a concomitant increase in the release of soluble fragments of CD23 (sCD23) into the medium by proteolytic processing of the surface-bound intact CD23. The effect of inhibition of the processing of CD23 on IgE production in human and mouse cells and in a mouse model in vivo was evaluated. CD23 processing to sCD23 from RPMI 8866 (a human Epstein-Barr virus-transformed B cell line) cell membranes was inhibited by a broad-spectrum matrix-metalloprotease inhibitor, batimastat, with an IC50 of 0.15 microM. Batimastat also inhibited CD23 processing in whole RPMI 8866 cells as well as in IL-4-stimulated purified human monocytes with similar IC50. Batimastat inhibited IgE production from IL-4/anti-CD40-stimulated human tonsil B cells as well as mouse splenic B cells in a manner consistent with inhibition of CD23 processing. Release of soluble fragments of CD23 in the cell supernatants of tonsil B cells was inhibited over the concentration range of 1-10 microM batimastat and intact cell surface CD23 was increased on mouse splenic B cells in the presence of these concentrations of batimastat. IgE production of IL-4-stimulated human peripheral blood mononuclear cells was also blocked by 1-10 microM batimastat, again with comparable inhibition of sCD23 release over the same concentration range. Finally, in a mouse model of IgE production, batimastat inhibited IgE production in response to ovalbumin challenge as determined by serum IgE levels. Taken together, the data support a role of CD23 in IgE production and point to CD23 processing to sCD23 as a therapeutically relevant control point in the regulation of IgE synthesis.

When anti-idiotypic antibodies specific for idiotopes on IgE antibodies react with mast-cell-bound IgE antibody, the reaction may be expected to lead either to mediator release or to inhibition of allergen-induced release. This study was carried out to determine which would occur. Anti-idiotype antiserum (anti-Ids) was raised in syngeneic mice by immunisation with affinity-purified DNP-specific mouse monoclonal IgE. Sera from these immunised mice and from a similarly immunised rabbit inhibited the binding of the monoclonal IgE to radio-iodinated dinitrophenylated (DNP) ovalbumin, whereas a high concentration of affinity-purified rabbit antimouse epsilon-chain-specific antibody was unable to inhibit even though it was shown to bind to the monoclonal IgE. The mouse anti-Ids did not give a positive passive cutaneous anaphylactic reaction in rats sensitised with high-titre grass-pollen-specific IgE-containing antiserum, whereas it gave a positive result in rats sensitised with the DNP-specific monoclonal IgE. Rabbit antimonoclonal antiserum and rabbit anti-epsilon-chain antibody both gave positive results in these two assays. The results indicated that anti-idiotypic antibodies specific for idiotopes on IgE antibody can react and release mediators from IgE-sensitised mast cells. The significance of this in view of the increased levels of anti-idiotypes that can occur during immunotherapy is discussed.

A Gold staining allergenic band, Bet v 1, was excised from a nitrocellulose blot following SDS-PAGE of birch pollen extract. This was used to raise a polyclonal rabbit antiserum and monoclonal mouse antibodies specific only for the Bet v 1. The method offers practical advantages for the production of antibodies to individual allergens in complex extract mixtures, without laborious purification methods.

Polyacrylamide gel electrophoresis in sodium dodecyl sulphate (SDS) followed by protein staining has shown that extracts from 11 different grass pollens contained proteins with similar molecular weights to that of the allergen R7 from rye grass (Lolium perenne) pollen extract (i.e. 29,000-31,000 daltons). Western blotting and detection with polyclonal (rabbit) antibodies raised against the purified R7 (Rye I) allergen indicated that these proteins were antigenically related and their allergenic properties were demonstrated by the binding of human IgE to immunoblots. The distribution of cross-reacting antigenic determinants was further investigated by immunoblotting with 2 mouse monoclonal antibodies, R7M1 and R7M2, produced with purified R7 as the initial immunogen. The 2 monoclonal antibodies were shown to react with 'R7-like' components of grass pollen extracts other than the component from rye grass. Differences in the distribution of R7M1 and R7M2 binding were found indicating that they are directed at separate R7 epitopes.

Rye grass pollen extract was fragmented by sequential treatments with cyanogen bromide and 2-nitro-5-thiocyanobenzoic acid, and a fraction containing fragments of molecular weight greater than 10,000 Mr was isolated. The in vitro reactivity of the extract with specific IgE was extensively reduced by fragmentation. Less reduction in activity was shown either by skin testing or by inhibition of an extract-specific IgG-binding assay. Reactivity with, and ability to induce, extract-specific mouse T cells were retained by the fragment preparation, and the ability to cause transformation of lymphocytes from atopic donors was unchanged. Fragments did not induce extract-specific IgG antibody in mice, were unable to stimulate the production of T-helper cells which could collaborate in an adoptive cell-transfer system, and did not induce delayed hypersensitivity reactions in guinea pigs. The possibility that such T-cell-reactive modified allergens (T'allergoids) might be used to stimulate selectively T-cell subsets and, therefore, could be used to advantage in immunotherapy is discussed.

A conjugate of the biologically active peptide N-formyl-methionyl-leucyl-phenylalanine and rye grass pollen extract (F-MLP/rye), previously shown to react with rye grass pollen extract-specific T cells, induced the formation of allergen-specific T cells in mice. Lymph node cells prepared from mice immunized with either native extract or F-MLP/rye gave an enhanced response to unmodified rye grass pollen allergens in vitro. Syngeneic spleen macrophages were able to present the unmodified allergens to T cells obtained from both groups of mice causing their proliferation in vitro. Conjugation of the peptide into the extract brought about an extensive reduction in its reactivity with grass pollen-specific human IgE, and a loss of its ability to induce specific IgG antibody in guinea-pigs. A state of delayed hypersensitivity specific for rye grass pollen extract was produced in guinea-pigs by immunization with either the F-MLP/rye or unmodified extract. It is concluded that conjugates such as F-MLP/rye or other T' allergoids could be used as probes to investigate whether changes in T cell activity are important in immunotherapy.

Rye grass pollen extract was digested by chymotrypsin to produce fragments with a molecular weight below 10,000, as demonstrated by polyacrylamide gel electrophoresis. Chymotryptic fragments did not react with either human or mouse IgG antibodies specific for rye grass pollen, nor did they induce an antibody response in mice with specificity for the parent extract. However, macrophage-presented fragments retained the ability to react with rye-specific T cells in a lymphoproliferation assay. Furthermore, these fragments induced the development of splenocytes capable of supporting dinitrophenyl specific antibody production. This implies that the fragments still react with, and induce, rye grass pollen extract-specific T-helper cells. The possibility that such fragments might have potential for use in immunotherapy for the specific treatment of allergy is discussed.

Conjugates of rye-grass pollen extract and N-formyl-methionyl-leucyl-phenylalanine were prepared using an activated form of the tripeptide. Introduction of the peptide into the extract brought about an extensive reduction of reactivity with grass-pollen-specific IgE, as measured by RAST inhibition. Despite this loss, guinea pig alveolar macrophages and murine splenic macrophages readily presented the conjugates to T lymphocytes specific for grass pollen allergens and caused their proliferation in vitro.