Daocheng Zhu

A novel human immunoglobulin Fc gamma Fc epsilon bifunctional fusion protein inhibits Fc epsilon RI-mediated degranulation.

Human mast cells and basophils that express the high-affinity immunoglobulin E (IgE) receptor, Fcɛ receptor 1 (FcɛRI), have key roles in allergic diseases. FcɛRI cross-linking stimulates the release of allergic mediators. Mast cells and basophils co-express FcγRIIb, a low affinity receptor containing an immunoreceptor tyrosine-based inhibitory motif and whose co-aggregation with FcɛRI can block FcɛRI-mediated reactivity. Here we designed, expressed and tested the human basophil and mast-cell inhibitory function of a novel chimeric fusion protein, whose structure is γHinge-CHγ2-CHγ3-15aa linker-CHɛ2-CHɛ3-CHɛ4. This Fcγ–Fcɛ fusion protein was expressed as the predicted 140-κD dimer that reacted with anti-human ɛ- and γ-chain specific antibodies. Fcγ–Fcɛ bound to both human FcɛRI and FcγRII. It also showed dose- and time-dependent inhibition of antigen-driven IgE-mediated histamine release from fresh human basophils sensitized with IgE directed against NIP (4-hydroxy-3-iodo-5-nitrophenylacetyl). This was associated with altered Syk signaling. The fusion protein also showed increased inhibition of human anti-NP (4-hydroxy-3-nitrophenylacetyl) and anti-dansyl IgE-mediated passive cutaneous anaphylaxis in transgenic mice expressing human FcɛRIα. Our results show that this chimeric protein is able to form complexes with both FcɛRI and FcγRII, and inhibit mast-cell and basophil function. This approach, using a Fcγ–Fcɛ fusion protein to co-aggregate FcɛRI with a receptor containing an immunoreceptor tyrosine-based inhibition motif, has therapeutic potential in IgE- and FcɛRI-mediated diseases.

CD45 controls interleukin-4-mediated IgE class switch recombination in human B cells through its function as a Janus kinase phosphatase

CD45 plays a critical regulatory role in receptor signaling through its protein tyrosine phosphatase and Janus kinase (JAK) phosphatase activities. To investigate whether CD45 also plays a regulatory role in Ig class switching in human B cells, we examined the effects of CD45 triggering on Ig class switching to IgE and its relationship with CD45 JAK phosphatase activity. Anti-CD45 triggering of CD45 significantly inhibited interleukin-4 + anti-CD40-induced switch recombination in a switch recombination vector assay in stably transfected Ramos 2G6 human B cells, as well as Ig ε germ-line transcription and Sμ-Sε switch recombination in primary human B cells. These negative regulatory effects on Ig class switching were concomitant with the ability of CD45 to dephosphorylate the induced phosphorylation of JAK1, JAK3, and signal transducer and activator of transcription 6, but not on stress-activated/mitogen-activated protein kinases. We also showed that phosphorylated JAK1 and JAK3 were directly dephosphorylated by recombinant CD45 in vitro. These results indicate that CD45 is able to function as JAK phosphatase in human B cells and that this activity is directly associated with the negative regulation of the class switch recombination to IgE. CD45 may be an appropriate target drug for modulating IgE in allergic diseases.

Genetically engineered negative signaling molecules in the immunomodulation of allergic diseases

Purpose of reviewThis review summarizes current knowledge regarding the control of human mast cell and basophil signaling and recent developments using a new therapeutic platform consisting of a human bifunctional γ and ε heavy chain (Fcγ-Fcε) protein to inhibit allergic reactivity. Recent findingsCrosslinking of FcγRIIb to FcεRI on human mast cells and basophils by a genetically engineered Fcγ-Fcε protein (GE2) leads to the inhibition of mediator release upon FcεRI challenge. GE2 protein was shown to inhibit cord blood-derived mast cell and peripheral blood basophil mediator release in vitro in a dose-dependent fashion, including inhibition of human IgE reactivity to cat. IgE-driven mediator release from lung tissue was also inhibited by GE2. The mechanism of inhibition in mast cells included alterations in IgE-mediated Ca2+ mobilization, spleen tyrosine kinase phosphorylation and the formation of downstream of kinase-growth factor receptor-bound protein 2-SH2 domain-containing inositol 5-phosphatase (dok-grb2-SHIP) complexes. Proallergic effects of Langerhans like dendritic cells and B-cell IgE switching were also inhibited by GE2. In vivo, GE2 was shown to block passive cutaneous anaphylaxis driven by human IgE in mice expressing the human FcεRI and inhibit skin test reactivity to dust mite antigen in a dose-dependent manner in rhesus monkeys. SummaryThe balance between positive and negative signaling controls mast cell and basophil reactivity, which is critical in the expression of human allergic diseases. This approach using a human Fcγ-Fcε fusion protein to co-aggregate FcεRI with the FcγRII holds promise as a new therapeutic platform for the immunomodulation of allergic diseases and potentially other mast cell/basophil-dependent disease states.

FcεRI–FcγRII Coaggregation inhibits IL-16 production from human langerhans-like dendritic cells

Abstract Langerhans-like dendritic cells (LLDC) express the high-affinity IgE receptor FceRI form that lacks the β-chain, and may play an important role in allergic inflammation via production of IL-16. Secretion of mediators by human mast cells and basophils is mediated through FceRI and is decreased by coaggregating these receptors to the low-affinity IgG receptor, FcγRII. We used a recently described human Ig fusion protein (GE2), which is composed of key portions of the human γ1 and the human e heavy chains, to investigate its ability to inhibit IL-16 production from FceRI-positive Langerhans-like dendritic cells through coaggregation of FcγRII and FceRI. Unstimulated LLDC-derived from CD14-positive monocytes from atopic donors were shown to express FcγRII, an ITIM-containing receptor, but not FceRI or FcγRIII which are activating (ITAM) receptors. When passively sensitized with antigen-specific, human IgE and then challenged with antigen, LLDC were stimulated to produce IL-16. However, when FceRI and FcγRII were coaggregated with GE2, IL-16 production was significantly inhibited. Exposure of LLDCs to GE2 alone did not induce IL-16 production. Our results further extend our studies demonstrating the ability of GE2 to inhibit FceRI-mediated responses through coaggregation with FcγRIIB and at the same time show that human LDCC can be modulated in a fashion similar to mast cells and basophils.

Inhibition of Interleukin-4-induced Class Switch Recombination by a Human Immunoglobulin Fc-Fc Chimeric Protein*

Immunoglobulin E (IgE) is important in mediating human allergic diseases. We tested the hypothesis that a human Ig Fcγ-Fcϵ bifunctional chimeric protein, GE2, would inhibit IgE class switch recombination (CSR) by co-aggregating B-cell CD32 and CD23. Indeed, GE2 directly inhibited ϵ germ-line transcription, subsequent CSR to ϵ and IgE protein production. This CSR inhibition was dependent on CD23 binding and the phosphorylation of extracellular signal-related kinase (ERK), and it was mediated via suppression of interleukin-4-induced STAT6 phosphorylation. Treatment with PD98059, a specific inhibitor of mitogen-activated protein kinase kinase 1 (MAPKK1 (MEK1)) and MEK2 reversed the ability of GE2 to decrease CSR and STAT6 phosphorylation. GE2 stimulation induced ERK phosphorylation, whereas it did not alter the phosphorylation of c-Jun N-terminal kinase or p38 MAPK. The ability of GE2 to block human isotype switching to ϵ, in addition to its already demonstrated ability to inhibit mast cell and basophil function, suggests that it will provide an important novel benefit in the treatment of IgE-mediated diseases.

Inhibition of IL-4-induced class switch recombinationby a human Ig Fcγ-Fc chimeric protein

Immunoglobulin E (IgE) is important in mediating human allergic diseases. We tested the hypothesis that a human Ig Fcγ-Fcϵ bifunctional chimeric protein, GE2, would inhibit IgE class switch recombination (CSR) by co-aggregating B-cell CD32 and CD23. Indeed, GE2 directly inhibited ϵ germ-line transcription, subsequent CSR to ϵ and IgE protein production. This CSR inhibition was dependent on CD23 binding and the phosphorylation of extracellular signal-related kinase (ERK), and it was mediated via suppression of interleukin-4-induced STAT6 phosphorylation. Treatment with PD98059, a specific inhibitor of mitogen-activated protein kinase kinase 1 (MAPKK1 (MEK1)) and MEK2 reversed the ability of GE2 to decrease CSR and STAT6 phosphorylation. GE2 stimulation induced ERK phosphorylation, whereas it did not alter the phosphorylation of c-Jun N-terminal kinase or p38 MAPK. The ability of GE2 to block human isotype switching to ϵ, in addition to its already demonstrated ability to inhibit mast cell and basophil function, suggests that it will provide an important novel benefit in the treatment of IgE-mediated diseases.