Prevention and treatment of allergic inflammation by an Fcγ-Der f2 fusion protein in a murine model of dust mite-induced asthma

The immunoglobulin E (IgE) high-affinity receptor FcεRI expressed on mast cells and basophils plays a critical role in triggering allergic disease. The co-aggregation of the FcεRI and FcγRIIb receptors is inhibitory to FcεRI signaling and holds great potential for the treatment of IgE-mediated allergies. In China, Dermatophagoides farinae is a common anaphylaxis trigger. Therefore, in this study, the FcγRIIb-mediated immunomodulating activity of recombinant Fcγ-Der f2 fusion protein was tested in a Der f2-allergic murine model. Following the treatment, bronchoalveolar lavage fluid (BALF) was collected to measure the expression of several Th1/Th2-type cytokines (IL-5, TNF-α, IL-12p70, IL-4, IL-10, IFN-γ and IL-18) and histamine, while blood was used to detect the specific IgE and IgG-types anti-Der f2 antibodies, for measurement. In contrast to the saline-treated allergic mice, the levels of Der f2-specific IgE, cytokines and histamine were lowered in the Fcγ-Der f2-treated allergic mice, in addition to the rare inflammatory cell infiltration in the airways and blood vessels revealed by histopathological examination. The recombinant Fcγ-Der f2 protein was demonstrated to function as an effective immunotherapeutic agent, suggesting that chimeric human Fcγ-allergen proteins could be used in the development of antigen-specific immunotherapy for human allergic diseases.     

An immunoglobulin E-reactive chimeric human immunoglobulin G1 anti-idiotype inhibits basophil degranulation through cross-linking of FcɛRI with FcγRIIb

Background IgE binds to mast cells and basophils via its high‐affinity receptor, Fc?RI, and cross‐linking of Fc?RI‐bound IgE molecules by allergen leads to the release of allergic mediators characteristic of type I hypersensitivity reactions. Previous work has shown that cross‐linking of Fc?RI with FcγRIIb, an ITIM‐containing IgG receptor, leads to inhibition of basophil triggering. 2G10, a chimeric human IgG1 anti‐idiotype, has broad reactivity with human IgE and as such has the potential to bind simultaneously to Fc?RI‐bound IgE, via its Fab regions, and the negative regulatory receptor, FcγRIIb, via its Fc region. Objective To assess the ability of human 2G10 to inhibit anti‐IgE and allergen‐driven basophil degranulation through cross‐linking of Fc?RI‐bound IgE with FcγRIIb. Methods 2G10 was assessed for its ability to bind to FcγRIIb on transfected cells and on purified basophils. In the basophil degranulation assay, basophils were purified from peripheral blood of atopic individuals and activated with either anti‐IgE or the house dust mite allergen Der p 1, in the presence or absence of human 2G10. Basophil activation was quantified by analysis of CD63 and CD203c expression on the cell surface, and IL‐4 expression intracellularly, using flow cytometery. Results Human 2G10 was able to bind to FcγRIIb on transfected cells and on purified basophils, and induce a dose‐dependent inhibition of both anti‐IgE and Der p 1‐driven degranulation of basophils. Conclusion The inhibition of basophil degranulation by the human IgG1 anti‐idiotype 2G10 highlights the therapeutic potential of IgE‐reactive IgG antibodies in restoring basophil integrity through recruitment of the inhibitory receptor FcγRIIb.     

Suppression of mast cell degranulation through a dual-targeting tandem IgE-IgG Fc domain biologic engineered to bind with high affinity to FcγRIIb

Mast cells and basophils play a central role in allergy, asthma, and anaphylaxis, as well as in non-allergic inflammatory, neurological and autoimmune diseases. Allergen-mediated cross-linking of IgE bound to FcεRI leads to cellular activation, and the low-affinity Fc receptor FcγRIIb is a key inhibitor of subsequent degranulation. FcγRIIb, when coengaged with FcεRI via allergen bound to IgE, stimulates ITIM domain-mediated inhibitory signaling that efficiently suppresses mast cell and basophil activation. To assess the therapeutic potential of directed coengagement of FcεRI and FcγRIIb in the absence of FcεRI crosslinking, we developed a fusion protein comprising the coupled Fc domains of murine IgE and human IgG1. As a key functional component of this tandem Fcε-Fcγ biologic, we engineered its IgG1 Fc domain to bind to human FcγRIIb with 100-fold enhanced affinity relative to native IgG1 Fc. Using mast cells from mice transgenic for human FcγRIIb, we show that this tandem Fc binds with high affinity to murine FcεRI and human FcγRIIb on mast cells, triggers phosphorylation of FcγRIIb, and inhibits FcεRI-dependent calcium mobilization. Control tandem Fc biologics containing a native IgG1 Fc domain or lacking binding to Fcγ receptors were markedly less active, demonstrating that the affinity-optimized tandem Fc can inhibit degranulation through stimulation of FcγRIIb signaling as well as through competition with allergen-IgE immune complex for FcεRI binding. We propose that in the context of a fully human tandem Fc biologic, high-affinity coengagement of FcεRI and FcγRIIb has potential as a novel therapy for allergy and other mast cell and basophil-mediated pathologies.     

Fcγ receptor II dependency of enhanced presentation of major histocompatibility complex class II peptides by a B cell lymphoma

Here we show that the B cell lymphoma A20.292 is capable of enhanced antigen presentation to CD4⁺ T cells in the presence of specific antibodies. This enhancement was inhibited by anti-Fcγ receptor (R) antibodies, suggesting that it might be due to preferential uptake of the antigen/antibody complex through the FcγRII receptor. However, immunoprecipitation studies revealed that the FcR of A20.292 cells was of the B cell type, FcγRIIb1, which is not thought to be able to internalize antigen/antibody complexes via clathrin-coated pits. It was considered unlikely that A20.292 had an altered form of the B cell FcγR (RIIb1) receptor that enabled internalization, since similar enhancing effects were also observed using an FcγRII^? cell line that had been transfected with FcγRIIb1. To reconcile these findings with the expression of FcγRIIb1, it is postulated that immune complexes are concentrated on the cell surface by the FcγRIIb1 and are thus available for preferential uptake by random fluid-phase endocytosis. This results in more efficient generation of the epitopes recognized by these T cell hybridomas.     

人sTNFR II-IgG Fc融合蛋白在毕赤酵母菌中的表达及其产物分析

目的:在毕赤酵母菌中表达sTNFR II-IgG Fc融合蛋白。检测融合蛋白是否形成二聚体,并对其N-糖链进行分析。方法:从人淋巴细胞中提取总RNA,经RT-PCR扩增目的基因sTNFRII与IgG Fc,然后利用重叠延伸PCR得到嵌合体基因sTNFRII-IgG Fc,并将其插入pPIC9载体中。以重组载体转化巴斯德毕赤酵母菌中进行克隆与表达。采用ELISA筛选高表达sTNFR II-IgG Fc融合蛋白的重组毕赤酵母菌株,对融合蛋白通过还原和非还原SDS-PAGE分析其二聚体结构,采用L929细胞检测融合蛋白抗TNF-α的生物学活性,最后利用荧光辅助糖电泳(FACE)分析融合蛋白的N-糖链。结果:成功地建立了可分泌表达sTNFR II-IgG Fc融合蛋白的重组酵母菌株,摇瓶培养后融合蛋白的表达量为2mg/L。SDS-PAGE和Western blot表明,该融合蛋白能自然形成二聚体结构;可抑制TNF-α对L929细胞的杀伤作用,其中和5×104U/LTNF-α的EC50为170μg/L。FACE分析融合蛋白N-糖链的大小为11~13个糖残基。结论:在毕赤酵母菌中成功地表达了sTNFR II-IgG Fc融合蛋白,为在毕赤酵母菌中表达其他的Fc融合蛋白和抗体提供了参考。     

Insect venom immunotherapy induces interleukin-10 production and a Th2-to-Th1 shift,and changes surface marker expression in venom-allergic subjects

The current study was carried out to elucidate the immunoregulatory changes induced by venom immunotherapy (VIT) in bee or wasp allergic subjects. All subjects included in this study had a history of severe systemic allergic reactions to stings of the respective insect as well as positive skin tests with the respective venom or venom-specific IgE in the sera. Parameters assessed in peripheral blood mononuclear cells (PBMC) before and after initiation of VIT (rush therapy reaching a maintenance dose of 100 μg venom injected subcutaneously within 1 week) were expression of CD3, CD4, CD8, CD45RA, CD45R0, interleukin (IL)-2 receptor (R)α, IL-4R, IL-12R, FcσRII, CD40, and CD40 ligand (CD40L), cells producing interferon (IFN)-γ and IL-10 after stimulation with phorbol 12-myristate 13-acetate + ionomycin in the presence of monensin measured by flow cytometry; secretion of IFN-γ, IL-4, and IL-10 measured by ELISA (IFN-γ and IL-10 were additionally measured by PCR), and proliferation after stimulation with the respective venom. Significant decreases were observed after VIT for proliferative response to venom and venom + IL-4, IL-4 secretion, FcσRII, CD40, and CD40L expression. Significant increases were observed after VIT for IFN-γ concerning the amount secreted and the number of producing cells, and IL-10. IL-10 was mainly produced by CD4⁺ cells that were negative for IFN-γ, but some double-positive (IL-10 and IFN-γ) cells were always detected. Addition of blocking anti-IL-10 antibodies, but not isotype control antibodies, prevented down-regulation of proliferation (but not IL-4 secretion) and further enhanced IFN-γ secretion after VIT. These data indicate that in insect venom allergic subjects, VIT not only induces a rapid shift in cytokine expression from Th2 to Th1 cytokines, but also leads to induction of the immunosuppressive cytokine IL-10, which may be important for the limitation of potentially harmful allergen-specific Th1 responses. The described changes in cytokine expression may be responsible for subsequent increases in allergen-specific IgG and decreases in IgE production, as well as suppressive activity observed in earlier studies.     

IL-15-IgG2b fusion protein accelerates and enhances a Th2 but not a Th1 immune response in vivo,while IL-2-IgG2b fusion protein inhibits both

We have explored how IL-15 influences Th1 or Th2 type immune response in vivo. Intraperitoneal application of an IL-15-IgG2b fusion protein (FP) to mice did neither significantly affect the footpad swelling nor the production of hemagglutinizing antibodies in a delayed type hypersensitivity reaction to sheep red blood cells. In contrast, in an established murine Th2 model of sensitization to ovalbumin (OVA), IL-15-IgG2b FP plus OVA sensitization resulted in massively accelerated and enhanced allergen-specific IgE and IgG1 antibody production. In vitro, stimulation of spleen cells from OVA-sensitized mice with OVA+IL-15 or OVA+IL-15-IgG2b resulted in a significantly enhanced IgE production. IL-4 secretion was significantly induced by IL-15 but not by IL-15-IgG2b. An IL-2-IgG2b FP with the same Fc tail as the IL-15-IgG2b FP was used as control in both models. In striking contrast to the IL-15-IgG2b FP, IL-2-IgG2b significantly inhibited the Th2 type antibody production in vivo. The current study suggests that IL-15-IgG2b may be employed as a potent accelerator and enhancer of Th2 type immune responses in vivo, while IL-2-IgG2b can suppress the latter.     

Treatment with Soluble Tumor Necrosis Factor Receptor (sTNFR):Fc/p80 Fusion Protein Ameliorates Relapsing-remitting Experimental Autoimmune Encephalomyelitis and Decreases Chemokine Expression

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease with pathological and clinical similarities to the major human demyelinating disease multiple sclerosis (MS). Multiple lines of evidence in recent years implicate the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) in the pathogenesis of both EAE and MS. TNF-α cellular responses are mediated by signaling through receptors, which are expressed in two functional forms, designated according to molecular weight p55/60 and p75/80. We report a treatment trial using the extracellular domain of the p80 TNFR in a bivalent fusion construct designated soluble tumor necrosis factor receptor (sTNFR):Fc to treat EAE. sTNFR:Fc/p80, given after the onset of clinical signs, reduced the clinical deficit of the first attack of relapsing-remitting EAE (RR-EAE) and the exacerbation rate for subsequent attacks. The effect was reversible as mice treated with sTNFR:Fc/p80 reverted to an exacerbation rate and disease severity typical of placebo-treated animals after treatment was discontinued. Treatment of RR-EAE with sTNFR:Fc/p80 decreased expression of chemokines MIP-1α (Monocyte Inflammatory Protein)/CCL3, MIP-1β/CCL4 and MIP-2/CXCL1-2 in the central nervous system. This treatment trial reveals an important function of TNF in the pathogenesis of RR-EAE and propose the mechanism of beneficial action of sTNFR:Fc/p80 in this disease.     

Altered influenza virus haemagglutinin (HA)-derived peptide is potent therapy for CIA by inducing Th1 to Th2 shift

There has been an increase in interest in the use of altered peptides as antigen-specific therapeutic agents in autoimmune diseases. Here we investigated the inhibitory effect and possible mechanism of an altered influenza virus haemagglutinin (HA)-derived peptide in collagen-induced arthritis (CIA). CIA was induced in DBA/1 mice by immunisation with type II collagen (CII). Altered HA308-317, wild-type HA308-317 or irrelevant peptide was administered intranasally beginning from arthritis onset. Clinical and histological scores were assessed, and cytokine levels in the serum or supernatants from splenocytes were determined. The percentages of Th1 and Th2 cells in response to different peptides were analysed by FACS both in vivo and in vitro. Our results showed that intranasal administration of altered HA308-317 peptide significantly ameliorated CIA. The therapeutic effect of altered HA308-317 peptide was associated with a substantial decrease in production of interferon (IFN)-γ, interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, anti-CII IgG, IgG1 and IgG2a antibodies, and an markedly increase in production of IL-10 and IL-4 in serum or supernatants from splenocytes treated with altered HA308-317 peptide. The percentage of Th2 (CD4(+)IL-4(+)) cells was upregulated significantly by altered HA308-317 peptide with a decreased percentage of Th1 (T helper 1; CD4(+)INF-γ(+)) cells both in vivo and in vitro. These findings suggest that altered HA308-317 peptide might be a promising candidate for rheumatoid arthritis (RA) treatment.     

FcγRI mediates serum amyloid P inhibition of fibrocyte differentiation

Fibrotic diseases, such as cardiac and pulmonary fibrosis, have a poor prognosis with no FDA approved therapies. Monocyte-derived, fibroblast-like cells, called fibrocytes, participate in the formation of fibrotic lesions. The conserved pentraxin protein SAP inhibits fibrocyte differentiation in cell culture, and injections of SAP significantly reduce fibrosis in several animal models. SAP binds to the receptors for the Fc portion of IgG (FcγR) and has been crystallized bound to FcγRIIa (CD32a). The in vivo activity of SAP appears to be dependent on the FcRγ. We find that mutagenesis of the residues critical for SAP binding to FcγRIIa only moderately decreases the ability of SAP to inhibit fibrocyte differentiation. In murine cells, deletion of FcRγ or FcγRI (CD64) significantly reduced sensitivity to SAP. Deletion of the combination of FcγRIIb, FcγRIIIa, and FcγRIV did not significantly affect sensitivity to SAP, whereas deletion of just the inhibitory receptor FcγRIIb (CD32b) increased sensitivity to SAP. In human cells, siRNA-mediated reduction of FcRγ or FcγRI levels significantly decreased sensitivity to SAP, whereas reduction of FcγRIIb levels increased sensitivity to SAP. These observations suggest that SAP, at least in part, uses FcγRI and FcRγ to inhibit fibrocyte differentiation.     

Apoptotic cells selectively suppress the Th1 cytokine interferon γ in stimulated human peripheral blood mononuclear cells and shift the Th1/Th2 balance towards Th2

Apoptotic cells are readily recognized and engulfed by phagocytes and usually do not induce inflammation or tissue damage. Furthermore, they can actively suppress a pro-inflammatory response in phagocytes: In the presence of apoptotic cells, activated monocytes/macrophages produce more of the anti-inflammatory and immunoregulatory cytokines IL-10 and TGF-β, but less of the pro-inflammatory cytokines TNFα, IL-1β and IL-12. This immunoregulatory effect is most likely mediated by several receptors on monocytes/macrophages including the thrombospondin receptor (CD36). In addition to the modulation of cytokine secretion, apoptotic cell material inhibited the expression of MHC class II molecules on the surface of monocytes/macrophages. Decreased MHC II expression appeared to be mediated predominantly by increased IL-10 secretion in a para-/autocrine manner. Here, we show that the functional modulation of antigen-presenting monocytes/macrophages by apoptotic cells also influences T cell activation and function. When human peripheral blood mononuclear cells were stimulated with recall antigens in the presence of apoptotic cells, interferonγ (IFNγ) secretion was markedly suppressed, whereas secretion of the Th2 cytokine IL-4 was not significantly altered. Hence, apoptotic cells shift the T cell cytokine secretion pattern towards a Th2-like response. This Th2 shift can largely be prevented by neutralizing IL-10, indicating an important role of this cytokine for modulating T cell cytokine secretion patterns.     

Kinetics and functional implications of Th1 and Th2 cytokine production following activation of peripheral blood mononuclear cells in primary culture

The importance of cytokine production in some disease processes is now widely recognized. To investigate temporal relationships between cytokines, we stimulated peripheral blood mononuclear cells (PBMC) in vitro using the T cell mitogen phytohemagglutinin (PHA) and various antigens chosen to induce predominantly Th1 (streptokinase: streptodornase or purified protein derivative) or Th2 (Dermatophagoides pteronyssinus, bee or wasp venom: allergens in sensitive subjects) responses. Cytokine production was measured by sensitive bioassays or enzyme-linked immunosorbent assays. Of the 30 subjects studied, 10 were normal and 20 individuals were allergic to either D. pteronyssinus (n = 10) or bee venom (n = 10) (examined before specific allergen immunotherapy). We examined the temporal profiles of a panel of cytokines produced in prmary culture. In PHA-driven cultures, cytokines were found to be sequentially produced in the order interleukin (IL)-2, IL-4, IL-5, IL-3, interferon (IFN)-γ, IL-10, IL-6, IL-12 and tumor necrosis factor (TNF)-α. The response to allergen in allergic patients was predominantly Th2 in nature, with the production of IL-4, IL-5, IL-6 and IL-10, but little or no IFN-γ. IL-2, IL-3, TNF-α and IL-12 were also produced in low amounts. The response of both atopic and normal subjects to recall bacterial antigens was predominantly Th1, with high levels of IFN-γ, IL-2 and TNF-α. The relevance of the order, amount and speed of production, characteristic kinetics (production, consumption, homeostatic regulation) and the cell source of the cytokines are discussed.     

Non-classical binding of a polyreactive α-type anti-idiotypic antibody to B cells

Detailed information on the immunological relevance of α-type anti-idiotypic antibodies is lacking after more than 30 years since Jerne postulated his Idiotypic Network Theory. The B7Y33 mutant is a mouse-human chimeric version of the B7 MAb, a polyreactive α-type anti-idiotypic antibody, generated against an anti-GM2 ganglioside IgM Ab1 antibody. It retained the unusual self-binding activity and multispecificity of the parental murine antibody, being able to recognize several anti-ganglioside IgM antibodies as well as non-immunoglobulin antigens. Previous work with the murine B7 MAb suggested that this antibody might have immunoregulatory properties, and therefore we investigated the possible interaction of B7Y33 with immune cells. We found that B7Y33 binds to human and murine B lymphocytes. Inhibition assays using flow cytometry indicated that this antibody is capable of binding the Fc γ receptor II (FcγRII). The recognition of FcγRII-expressing K562, Raji and Daudi human cell lines, together with the capability of inhibiting the binding of an anti-human FcγRII antibody to these cells, suggest that B7Y33 interacts with both the FcγRIIa and FcγRIIb isoforms. We evaluated the contribution to the binding of different surface-exposed residues at the top of the heavy chain variable region (VH) CDR loops through the construction of mutants with substitutions in the three conventional VH CDRs (HCDRs) and the "HCDR4", located in the framework 3 (HFR3). In addition, we assessed the involvement of the Fc region by performing key mutations in the CH2 domain. Furthermore, chimeric hybrid molecules were obtained by combining the B7Y33 heavy chain with unrelated light chains. Our results indicate that the multispecificity and self-binding properties of B7Y33 are not linked to its recognition of B lineage cells, and that this phenomenon occurs in a non-classical way with the participation of both the variable and constant regions of the antibody. Two possible models for this interaction are proposed, with B7Y33 binding to two FcγRIIb molecules through the Fc and Fv regions, or simultaneously to FcγRIIb and another unknown antigen on B cells. The FcγRIIb has recently received great attention as an attractive target for therapies directed to B lymphocytes. The recognition of peripheral B lymphocytes from B cell chronic lymphocytic leukemia (B-CLL) patients by B7Y33 suggests its potential application for the treatment of B cell malignancies.     

Modulation of Fcγ receptor-mediated early events by the tyrosine phosphatase CD45 in primary human monocytes. Consequences for interleukin-6 production

Stimulation of primary human monocytes from several donors by cross-linking of Fcγ receptor type I (FcγRI) and FcγRII gave rise to calcium mobilization and protein tyrosine phosphorylation. These early events were not observed without cross-linking. CD45, a transmembrane tyrosine phosphatase, when co-cross-linked with either FcγRI or FcγRII, could prevent FcγRI and FcγRII-mediated calcium mobilization and protein tyrosine phosphorylation. When interleukin (IL)-6 production was measured, we noted a strong IL-6 production after activation of primary human monocytes by cross-linking of FcγRI or FcγRII. In contrast to calcium mobilization and tyrosine phosphorylation of proteins, IL-6 production was not affected by co-cross-linking of CD45 with either FcγRI or FcγRII. Interestingly, cross-linking of the CD45 itself was sufficient to induce IL-6 production. Our results show that the CD45 molecule is important in modulating early events following stimulation of primary human monocytes by cross-linking of FcγRI or FcγRII. However, triggering of CD45 alone can also induce IL-6 production, indicating that CD45 ligation itself can give signals and may have an important role in cytokine induction pathways.     

Immune complex enhances tolerogenecity of immature dendritic cells via FcγRIIb and promotes FcγRIIb-overexpressing dendritic cells to attenuate lupus

A balance of inhibitory and activating signals determines the function of dendritic cells (DCs) in the immune response, which may be regulatory or stimulatory. Defects of inhibitory receptor FcγRIIb are involved in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE), in which high levels of circulating immune complexes (IC) exist. Our previous study showed that IC/Ig can suppress TLR4-triggered inflammatory responses in macrophages via FcγRIIb. This led us to question whether IC/Ig can polarize FcγRIIb-overexpressing DCs (DC-FcγRIIb) to be tolerogenic, thus attenuating lupus progression once infused in vivo. First, we found that IC/Ig markedly inhibited LPS- or CpG-induced DC maturation, enhanced tolerogenicity of DCs via FcγRIIb, and induced massive prostaglandin E2 (PGE2) secretion from DCs, both contributing to T-cell hyporesponsiveness. Endogenous Ig and lupus-derived IC also exhibited the same effect. DC-FcγRIIb, transfected with recombinant adenovirus encoding FcγRIIb, displayed enhanced tolerogenic function and produced more PGE2 in the presence of IC, thus further inhibiting T-cell responses. Importantly, in vivo infusion with DC-FcγRIIb significantly reduced kidney damage and prolonged the survival of lupus-prone MRL/lpr mice either before or after the onset of clinic lupus. Therefore, administration of DC-FcγRIIb may be a new approach to attenuate lupus progression.     

Human naive CD4 T cells produce interleukin-4 at priming and acquire a Th2 phenotype upon repetitive stimulations in neutral conditions

The maturation of naive CD4 T cells into interleukin (IL)-4-producing effectors was shown to require the presence of IL-4 at priming, the cellular origin of which remains unclear. We demonstrate here that naive T cells themselves release IL-4 at very low levels that are nevertheless sufficient to promote their development into Th2-like cells. This conclusion is based on three observations: (1) highly purified human naive CD4T cells, of neonatal or adult origin, develop into Th2 effectors upon repetitive cycles of stimulation with anti-CD3 monoclonal antibody (mAb) cross-linked to CD32-B7 transfected L fibroblasts followed by IL-2 expansion; (2) IL-4 protein is readily detectable in the concentrated supernatant fluids of priming cultures performed in the presence of anti-IL-4 receptor mAb; and (3) addition of anti-IL-4 or anti-IL-4 receptor mAb at priming markedly inhibits the acquisition of IL-4- and IL-5-producing capacity while enhancing that of interferon-γ.     

Maternal allergen immunisation to prevent sensitisation in offspring: Th2-polarising adjuvants are more efficient than a Th1-polarising adjuvant in mice

Background

Preconception allergen immunization prevents neonatal allergen sensitization in mice by a complex interaction between regulatory cells/factors and antibodies. The present study assessed the influence of maternal immunization with ovalbumin (OVA) on the immune response of 3 day-old and 3 week-old offspring immunized or non-immunized with OVA and evaluated the effect of IgG treatment during fetal development or neonatal period.

Results

Maternal immunization with OVA showed increased levels of FcγRIIb expression in splenic B cells of neonates, which were maintained for up to 3 weeks and not affected by additional postnatal OVA immunization. Maternal immunization also exerted a down-modulatory effect on both IL-4 and IFN-γ-secreting T cells and IL-4 and IL-12- secreting B cells. Furthermore, immunized neonates from immunized mothers showed a marked inhibition of antigen-specifc IgE Ab production and lowered Th2/Th1 cytokine levels, whereas displaying enhanced FcγRIIb expression on B cells. These offspring also showed reduced antigen-specific proliferative response and lowered B cell responsiveness. Moreover, in vitro evaluation revealed an impairment of B cell activation upon engagement of B cell antigen receptor by IgG from OVA-immunized mice. Finally, in vivo IgG transference during pregnancy or breastfeeding revealed that maternal Ab transference was able to increase regulatory cytokines, such as IL-10, in the prenatal stage; yet only the postnatal treatment prevented neonatal sensitization. None of the IgG treatments induced immunological changes in the offspring, as it was observed for those from OVA-immunized mothers.

Conclusion

Maternal immunization upregulates the inhibitory FcγRIIb expression on offspring B cells, avoiding skewed Th2 response and development of allergy. These findings contribute to the advancement of prophylactic strategies to prevent allergic diseases in early life.     

The formyl peptide N-formyl-methionyl-leucyl-phenylalanine downregulates the expression of FcgammaRs in interferon-gamma-activated monocytes/macrophages in vitro and in vivo

N-Formyl peptides are cleavage products of bacterial and mitochondrial proteins that have pro-inflammatory activities and play an important role in antibacterial host defence. FcγRI is a receptor for the Fc portion of immunoglobulin G expressed in monocytes that mediates cytotoxicity and is upregulated by interferon-γ (IFN-γ) and interleukin-10 (IL-10). In this report, we demonstrate that N-formyl-methionyl-leucyl-phenylalanine (FMLP) downregulates the expression of FcγRI in IFN-γ-treated monocytes, but not in IL-10-treated monocytes. We determine that supernatants obtained from monocytes treated with IFN-γ and then exposed to FMLP induce the downregulation of FcγRI in naïve monocytes. This effect is abrogated by the protease inhibitors phenylmethylsulphonyl fluoride and phosphoramidon, which inhibit serine and metalloproteases, respectively. Supernatants from FMLP-treated neutrophils also induce the downregulation of FcγRI, when added to naïve monocytes. Similar observations were obtained in vivo in a mouse model of chronic inflammation. In vivo, FMLP also downregulates the expression of FcγRs in IFN-γ-activated macrophages. Our results support the existence of a new mechanism through which FMLP could modulate the activity of monocytes/macrophages during bacterial infections.