Goat Anti-mouse IgD Research Articles

Protective role of mouse IgG1 in cryoglobulinaemia; insights from an animal model and relevance to human pathology.

Strait et al. described a novel mouse model of cryoglobulinaemia by challenging mice deficient in the immunoglobulin (Ig)G1 subclass (γ1(-) mice) with goat anti-mouse IgD [5]. The phenotype of wild-type mice was not remarkable, whereas γ1(-) mice developed IgG3 anti-goat IgG cryoglobulins as well as severe and lethal glomerulonephritis. Renal phenotype could not be rescued in γ1(-) mice by the deletion of C3, fragment crystalline γ receptor (FcγR) or J chain. On the other hand, early injection of IgG1, IgG2a or IgG2b inhibited the pathogenic effects of IgG3 in an antigen-dependent manner even in the absence of the FcγRIIb, an anti-inflammatory receptor. The authors concluded that the pathogenic role of IgG3 and the protective characteristic of IgG1 in this model were not explained by their abilities to bind to FcRs or effector molecules but are rather due to structural discrepancies enhancing the precipitation properties/solubility of IgG3/IgG1-containing immune complexes. The present article aims to discuss the current knowledge on IgG biology and the properties of IgGs explaining their differential propensity to acquire cryoglobulin activity.

The Mertk receptor tyrosine kinase promotes T–B interaction stimulated by IgD B-cell receptor cross-linking

The Mertk receptor tyrosine kinase facilitates macrophage and DC apoptotic-cell clearance and regulates immune tolerance. Mertk may also contribute to B-cell activation, because Mertk-KO mice fail to develop autoantibodies when allo-activated by T cells. We investigated this possibility with a well-characterized model in which injection of mice with goat anti-IgD antibody causes membrane IgD cross-linking that induces T-independent B cell activation and antigen presentation to T cells. Goat anti-mouse IgD antibody-injected C57BL/6 Mertk-KO mice had normal initial B cell activation and proliferation, but significantly lower T cell activation and proliferation, as well as lower IgE and IgG anti-goat IgG responses, as compared to C57BL/6 WT controls. B cell antigen processing, analyzed by evaluating B cell fluorescence following injection of monoclonal anti-IgD antibody labeled with biotin or FITC, was comparable between Mertk-KO mice and WT mice. IgD Ab primed B cells from Mertk-KO mice exhibited significantly lower ability in activating memory T cells isolated from WT mice injected with the same antigen 10 days before. These observations suggest that Mertk expression is required for optimal B-cell antigen presentation, which is, in turn, required in this model for optimal T cell activation and subsequent T cell-dependent B cell differentiation.

Modulation of immunoglobulin production by invariant Vα19-Jα33 TCR-bearing cells.

We have previously shown that invariant Vα19-Jα33 TCR+ (Vα19i T) cells suppress the disease progress in some models for organ specific autoimmune diseases and type IV allergy that deteriorate along with decline to excess in Th1- or Th17- immunity. In this study, we examined the effects of over-generation of Vα19i T cells on the Th2-controlled immunoglobulin isotype production in the models for type I allergy. IgE production by invariant Vα19-Jα33 TCR transgenic (Tg) mice was suppressed compared with that by non-Tg controls following administration with goat anti-mouse IgD antiserum or OVA, while IgG2a production was not influenced by the introduction of the transgene into the recipients. IgE production by wild type mice was similarly reduced when they were subjected to adoptive transfer with invariant Vα19-Jα33 TCR Tg+ but not Tg− cells prior to immunization. Furthermore, the suppression of IgE production by these recipients was enhanced when they were previously administered with a Vα19i T cell activator, one of the modified α-mannosyl ceramides. In summary, it is suggested that Vα19i T cells have potential to participate in the homeostasis of immunity and that they suppress disease progression resulting from not only Th1- but also Th2- immunity excess.

Basophils and Macrophages both Contribute to IgG-Mediated Anaphylaxis (36.7)

Abstract Studies of anaphylaxis in mice revealed the importance of an alternative pathway that depends on IgG, FcγRIII, and platelet activating factor (PAF). Initial reports identified macrophages (MP) as the critical PAF source, but more recent publications identify the critical source as basophils (BA). Because these studies differed in mouse strain and experimental model used, we evaluated whether either influences the relative importance of BA vs. MP. Studies in which BA are depleted with anti-CD200R3 mAb (Ba103) and MP are depleted with clodronate liposomes or desensitized with gadolinium now show that both mouse strain and anaphylaxis model strongly influence the relative importance of BA and MP. MP depletion always has a much greater effect on anaphylaxis in BALB/c than C57BL/6 mice, while the reverse is true for BA depletion. MP are also relatively more important when mice are sensitized to goat IgG by immunization with goat anti-mouse IgD Ab (GaMD) and challenged with goat IgG than when mice are primed passively with mouse IgG1 anti-TNP mAb and challenged with TNP-ovalbumin, or when unprimed or GaMD-primed mice are challenged with anti-FcγRIII mAb (2.4G2). In addition, C57BL/6 mice are considerably more sensitive than BALB/c mice to PAF. Taken together, these results suggest that increased PAF secretion by FcγRIII-activated MP in BALB/c mice may compensate for the reduced sensitivity of this strain to PAF.

IL-13 Mediated Lung Effects Are Exclusively Dependent On IL-13Ralpha1 (CD213a)

Interleukin (IL) -13 is a potent activator of inflammation and fibrosis during type-2 inflammation such as asthma, tissue remodeling and parasite immunity. However, to date whether IL-13Ralpha1 is the only (or main) IL-13 receptor in the lung has not been determined. To test the hypothesis that IL-13 receptor (R) alpha1 mediates IL-13-induced functions we report the generation of IL-13Ralpha1 deficient mice by Velocigene technology and the characterization of baseline immune phenotype of these mice. Furthermore, the effects of intratracheal delivery of IL-13 to IL-13Ralpha1-/- mice were assessed. In addition, the role of IL-13Ralpha1 was examined in an OVA-induced model of experimental asthma that included global microarray analysis (Affymetrix) of the allergen challenged lungs. IL-13Ralpha1-/- mice displayed markedly decreased serum IgE although IL-4 signaling components (i.e. serum IL-4, IL-4Ralpha) were intact. Nevertheless, these mice generated a polarized Th2 reaction in response to T cell dependent antigens (goat anti-mouse IgD and OVA). In response to IL-13, IL-13Ralpha1-/- mice displayed no goblet cell hyperplasia, mucus production as well as no CCL2, CCL11, CCL17, CCL24 and TGF-beta induction. In response to OVA challenge, IL-13Ralpha1-/- mouse displayed no observable mucus production, and chemokine induction, yet they had preserved leukocyte recruitment. Global transcriptome analysis identified IL-13Ralpha1-dependent and -independent genes in the OVA-induced asthma model that mechanistically explained the observed phenotype. These results demonstrate a definite role for IL-13Ralpha1 in mediating IL-13-induced lung pathology. Furthermore, they provide the basis for generating IL-13Ralpha1-based blocking agents for future therapeutic intervention.

IgG-Blocking Antibodies Inhibit IgE-Mediated Anaphylaxis in Vivo Through Both Antigen Interception and FcγRIIb Cross-linking

Strait RT, Morris SC, Finkelman FD. J Clin Invest. 2006;116:833–841 PURPOSE OF THE STUDY. It has been hypothesized that at least part of the mechanism of successful allergen immunotherapy is the induction of specific immunoglobulin (Ig) G that can “block” IgE-dependent responses by competitively preventing IgE binding and/or by signaling via inhibitory FcγRIIb on basophils and mast cells, thereby downregulating FcεRI-dependent signaling. In vivo evidence of this blocking function has been lacking. The authors of this study carefully addressed whether and how specific IgG can inhibit IgE-mediated anaphylaxis by using a murine model. METHODS. The authors had previously established a model of anaphylaxis by immunizing BALB/c mice with goat anti-mouse IgD antibody (GαMD), which elicits a strong T-helper 2 response with high levels of IgE, IgG1, and mastocytosis. In this study they very cleverly modified the model by conjugating GαMD with the hapten trinitrophenyl to generate a trinitrophenyl-specific IgG and IgE response to allow for more detailed evaluation of the role of different antibody isotypes with the same epitope specificity. They used a number of tools including antibody blocking of FcγRs, FcRIII-deficient knockout mice, and pharmacologic inhibition to discriminate between IgE- and IgG-dependent anaphylaxis. Assessment of anaphylaxis was by temperature drop and hemoconcentration. IgG-trinitrophenyl antibody complexes were measured by enzyme-linked immunosorbent assay (ELISA). In some experiments, interleukin 4 secretion was measured from whole blood by ELISA. RESULTS. In immunized mice, the authors showed that IgE-dependent anaphylaxis is primarily inhibited by IgG preventing antigen-induced cross-linking of cell-associated IgE. In FcγRIII-deficient mice (ie, those capable of only IgE-dependent anaphylaxis), blockade of the FcγRIIb inhibitory receptors did not exacerbate antigen-induced anaphylaxis, and IgG–antigen complexes could be detected in whole blood within 5 minutes of antigen administration. Furthermore, animals passively sensitized with IgE in the absence of IgG reacted with 1000-fold lower doses of antigen. Epitope density was inversely correlated with threshold dose for inducing reactivity. Finally, by adding back increasing amounts of IgG to the passively IgE-sensitized animals, they showed that FcRIIb does inhibit IgE responses when IgG levels are low. CONCLUSIONS. Antigen-specific IgG does block IgE-mediated reactions in vivo by both direct competition and signaling via the inhibitory IgG receptor, FcγRIIb. REVIEWER COMMENTS. This was the first in vivo demonstration of specific IgG's ability to block IgE-dependent reactions, which has been suggested for many years by the frequently observed rise in allergen-specific IgG titer during successful immunotherapy. There are potentially important large differences between the mouse model and IgG induction in the context of allergen immunotherapy, including the fact that IgG-dependent anaphylaxis is well established in mice but uncertain to even exist in humans. It is also unclear from the article how the ratio of IgG to IgE in this model compares to the ratio of IgG and IgE specific to a particular allergen in human patients. However, the authors carefully and elegantly demonstrated the capacity and mechanistic details of IgG blocking antibody in vivo and strongly support the concept of blocking IgG in the context of immunotherapy.

Shift from IL-3- to IgE-induced IL-4 production as basophils become a predominant source of IL-4 (37.15)

Abstract We compared CD4+ T cell and basophil contributions to in vivo IL-4 production and evaluated stimuli that induce basophil IL-4 production during immune responses to a protein Ag, goat anti-mouse IgD antiserum (GaMD), and a nematode parasite. IL-4 production by basophils preceded CD4+ T cells, but CD4+ T cells produced most IL-4 secreted during the primary response to GAMD and the secondary response to Ag challenge 4–5 d after priming. In contrast, basophils produced most of the IL-4 secreted in mice challenged 12–14 d after priming. Basophil IL-4 secretion was IL-3-dependent but influenced by IgE during the primary response and IL-3- and CD4+ T cell-dependent/IgE-independent during the d 4–5 secondary response, but was IgE-dependent and IL-3/CD4+ T cell-independent during the d 12–14 secondary response. Basophil IL-4 secretion during a primary infection with Trichinella spiralis was localized to the liver and was partially IL-3- and IgE-independent. Thus, IL-3-dependent basophil IL-4 production precedes and supplements the primary T cell IL-4 response to a protein Ag; basophil IL-4 production can exceed T cell IL-4 production in animals challenged with Ag after basophils are primed by Ag-specific IgE; and helminthic parasites can stimulate basophil IL-4 secretion through a distinct mechanism.

IgG1, IgG2a, complement and FcR in immune complex disease (90.16)

Abstract Immune complexes (IC) contribute to tissue damage in some diseases but prevent it in others. We hypothesize that this contrast occurs because different immunoglobulin (Ig) isotypes involved in the IC interact differently with both stimulatory and inhibitory FcγR and complement (C). To study this we compared IgG1 (which poorly activates C and stimulatory FcγR) to other Ig isotype-mediated IC processing using a model in which wild-type (WT) mice immunized with goat anti-mouse IgD antiserum (GaMD), produce a large, predominantly IgG1 Ab response, produce large amounts of IC, and eliminate them without developing renal disease. In GaMD-immunized IgG1-deficient mice, IgG2a (which activates C and interacts with stimulatory FcγR) predominates and mice develop severe, lethal IC-mediated renal disease that can be prevented by immunization with antigen-specific IgG1. Renal disease also develops in C3-deficient, FcγRIIb-deficient and anti-FcγRII/III mAb treated WT but not in FcRγ-deficient mice immunized with GaMD. Surprisingly, GaMD-immunized IgG1-deficient mice that are also lacking both FcRγ and C3 or lacking FcRγ and treated with a C5aR antagonist still develop renal disease. Together these findings suggest that IgG1 may protect against IC disease through a C- and FcγRIIb-dependent mechanism, while non-IgG1, presumably IgG2a, may mediate IC injury through a FcRγ- and either C3 or C5aR-independent mechanism.

Endogenously produced IL-4 strongly enhances CD8+ T cell proliferation and accumulation in homeostatic conditions and during Th2 responses (87.24)

Abstract IL-7 and IL-15 have important stimulatory roles in CD8+ T cell homeostasis. We evaluated whether another γc-related cytokine, IL-4, also stimulates CD8+ T cell proliferation and accumulation in vivo. Wild-type (WT) mice have twice as many splenic CD8+ T cells as IL-4- or IL-4Rα-deficient mice, with similar numbers of naïve cells but ~5-fold more memory phenotype cells. WT CD8+ T cells outcompete IL-4Rα-deficient CD8+ T cells following cell transfer and SCID mice reconstituted with equal numbers of WT and IL-4Rα-deficient CD8+ T cells have twice as many splenic WT as IL-4Rα-deficient CD8+ T cells 2 weeks later. WT, but not IL-4Rα-deficient CD8+ T cells proliferate when transferred into IL-4Rα-deficient mice that have been immunized with goat anti-mouse IgD antibody or infected with Nippostrongylus brasiliensis to stimulate a Th2 response. Consistent with this, splenic CD8+ T cell accumulation is entirely IL-4- and IL-4Rα-dependent and proliferation is predominantly IL-4- and IL-4Rα-dependent in mice infected w132th Schistosoma mansoni and pulmonary CD8+ T cell proliferation is rapidly suppressed by anti-IL-4R□ mAb in a mouse model of asthma. Furthermore, even ng quantities of IL-4 stimulate CD8+ T cell proliferation and accumulation in vivo. Thus, IL-4 is a potent, biologically important stimulator of CD8+ T cell proliferation and accumulation in vivo. Supported by the US Dept of Veterans Affairs and NIH.

The combined effect of IL-4 and IL-10 suppresses the generation of, but does not change the polarity of, type-1 T cells in Histoplasma infection

Dominant type-1 cytokine production is induced in a murine model of systemic histoplasmosis. We used this model to investigate whether the presence of antagonistic cytokines during T cell priming changes the polarity of T cells in response to Histoplasma infection. Before infection with Histoplasma capsulatum, mice were injected twice with goat anti-mouse IgD antiserum (GalphaMdelta), which induced expression of dominant type-2 cytokines. At days 7 and 14 after infection, the GalphaMdelta-treated mice had suppressed IFN-gamma response and a significantly greater fungal burden in their spleens and lungs. The number of IFN-gamma-producing cells as well as the level of IFN-gamma produced per cell was greatly reduced. Not only CD4+ T cells but also CD8+ T cells were affected. The number of Histoplasma-induced IFN-gamma-producing cells was partially restored in GalphaMdelta-treated IL-4-/- and IL-10-/- mice and completely restored in IL-4-/- IL-10-/- mice. Thus, the combined effect of IL-4 and IL-10 suppressed the generation of IFN-gamma-producing cells. A longitudinal study demonstrated that as IL-4 and IL-10 decreased, the number of Histoplasma-induced IFN-gamma-producing cells rapidly increased, and fungal clearance improved, demonstrating that the presence of IL-4 and IL-10 did not permanently change the polarity of T cells.

ALLOSPECIFIC T REGULATORY AND T SUPPRESSOR CELLS IN TRANSPLANTATION

O261* Aims: In previous studies we have demonstrated that allospecific CD8+CD28- FOXP3+ T suppressor (TS) and CD4+CD25+ FOXP3 T regulatory cells (TR) can be generated in vitro by multiple stimulation of human T cells with allogeneic APC. We have now explored the in vivo relevance of TS and TR in 30 recipients of heart allografts. Methods: CD8+CD28- T cells from fresh peripheral blood were obtained using CD8 isolation kit II (Miltenyi Biotech, Auburn, CA) followed by depletion of CD28+ T cells using goat anti-mouse IgD beads coupled with mAb to CD28 (Becton Dickinson). CD4+CD45RO+CD25+ T cells were obtained by depletion of purified CD4+ T cells of CD45RA+ cells followed by selection of CD25+ T cells with magnetic microbeads (Miltenyi). TS and TR were incubated with donor or control APC for 18 hours. The phenotype of TS, TR and of APC was studied by flow cytometry, RT-PCR and luciferase transcription assays. Anti-HLA class I and class II antibodies were monitored in all patients. Results: Serial studies of the phenotype displayed by T cells from transplant recipients demonstrated a progressive increase in the size of the CD8+CD28-CD27+ PRF- FOXP3+ T cell population in rejection-free patients. CD8+CD28- FOXP3+ T cells from these recipients inhibited up-regulation of CD80 and CD86 on CD40-ligated APC from the donor. This inhibitory effect was restricted to the MHC class I antigens expressed by the transplant donor. Patients’ TS also induced the upregulation of the inhibitory receptors ILT3 and ILT4 on donor APC as shown by flow cytometry as well as by trancription assays in which APC transfected with luciferase gene plasmids containing the ILT3 or ILT4 promoter were used. CD8+CD28- FOXP3+ T cells from patients in quiescence induced ILT3 and ILT4 transcription in APC sharing HLA class I antigens with the transplant donor but not in irrelevant control APC. Serial determination of the suppressor activity displayed by patient’s CD4+CD25+ FOXP3+ T cells yielded similar results i.e. they induced transcriptional activation of ILT3 and ILT4 in APC matched to the donor for HLA-DRβ1 antigens. However, while TS could be detected as early as one month post-transplantation CD4+CD25+ TR became detectable at later times (12 months or more). TS and TR were found in 21 out of 30 patients studied. Of the remaining 9 patients who showed no regulatory T cells 7 had a history of early acute rejection episodes while two remained in quiescence. Three of these 7 patients with early rejections showed evidence of CAV two years following transplantation. Taken together our data indicate that the presence of TS and TR in patients’ circulation is negatively associated with acute or chronic rejection and with the development of anti-HLA antibodies (p<0.0001). Conclusions: Serial determination of allospecific TS and TR demonstrated that quiescence is associated with the expansion of regulatory T cell populations. Study of regulatory T cells permits the identification of patients who can benefit from partial withdrawal of immunosuppressive drugs.

IgG: Blocking and mediating anaphylaxis

Rationale Mice immunized with the T-dependent Ag, goat anti-mouse IgD Ab (GaMD), develop mastocytosis and IgE and IgG anti-goat IgG (GIgG) Ab. Surprisingly, these mice develop IgG/FcγRIII-dependent anaphylaxis, but not IgE/FcϵRI/mast cell-dependent anaphylaxis, when challenged with 100 μg of GIgG. Studies were performed to explain the absence of IgE-mediated anaphylaxis. Methods To test the hypothesis that IgG Ab inhibit IgE-mediated anaphylaxis by blocking GIgG binding to mast cell-associated IgE anti-GIgG Ab, we compared the abilities of 100 vs. 10,000 μg of GIgG to induce mast cell degranulation and anaphylaxis in GaMD-immunized mice in which IgG-dependent anaphylaxis was suppressed by anti-FcγRII/RIII mAb. We also evaluated the ability of polyclonal IgG anti-TNP Ab to suppress TNP-OVA-induced anaphylaxis and mast cell degranulation in IgE anti-TNP mAb-primed mice. Results Both 100 and 10,000 μg of GIgG induced anaphylaxis in otherwise untreated GaMD-primed mice, but only the 10,000 μg dose induced mast cell degranulation (histamine and MMCP1 release) and only the 10,000 μg dose induced anaphylaxis in anti-FcγRII/RIII mAb-treated, GaMD-primed mice. Furthermore, IgG anti-TNP Ab blocked TNP-OVA-induced anaphylaxis in mice primed with IgE anti-TNP mAb. Blocking was overcome by increasing the challenge dose of TNP-OVA. Higher doses of Ag were required to induce IgG-mediated anaphylaxis than IgE-mediated anaphylaxis. Conclusions IgG Ab can block IgE-mediated anaphylaxis by competing for Ag with mast cell-bound IgE Ab and can protect against anaphylaxis induced by relatively low quantities of Ag. However, challenge with a high Ag dose may both overcome the protective effect of IgG blocking Ab and induce IgG-mediated anaphylaxis.

Antibody production in early life supported by maternal lymphocyte factors

To examine the influence of maternal lymphocyte factors on the immune responses in offspring in early life, antibody production in neonates born to either normal or lymphocyte-deficient mothers was analyzed. Recombination activating gene (Rag)-2 +/− mouse neonates born to Rag-2 +/+, Rag-2 +/−or Rag-2 −/−mothers were injected with goat anti-mouse IgD antiserum, and IgE and IgG 1 production was evaluated. The levels of IgE and IgG 1 were higher in the pups born to Rag-2 +/+and Rag-2 +/− dams than to lymphocyte-deficient Rag-2 −/− dams. The enhanced antibody production in the former compared with the latter neonates was also found following immunization with ovalbumin or TNP-Ficoll. Thus, the presence of maternal lymphocyte factors was suggested in neonates that augmented antigen-specific antibody production in both T cell-dependent and -independent pathways. A reduction in antibody production was observed in normal neonates when they were foster-nursed by Rag-2 −/− mothers. Thus, the maternal lymphocyte factors enhancing the immune responses in newborns were shown to be present in breast-milk.

Pathways of anaphylaxis in the mouse

Background: Although anaphylaxis is classically mediated by IgE, FcϵRI, mast cells, and histamine, several rodent studies suggest that an alternative pathway involving IgG, FcγRIII, macrophages and platelets, and platelet-activating factor (PAF) may be more important in the anaphylactic response to antigen challenge. Objectives: We sought to determine the relative roles of the classical and alternative pathways of anaphylaxis in a mouse model characterized by mastocytosis and a high level of antigen-specific IgE antibody. Methods: Wild-type, IgE-deficient, FcϵRI-deficient, and mast cell–deficient mice were immunized with goat anti-mouse IgD antibody, which induces mastocytosis and a large IgE and IgG anti-goat IgG response, and then challenged 14 days later with antigen (goat IgG) or rat anti-mouse IgE mAb. Specific vasoactive mediators, cell types, Ig isotypes, or Ig receptors were blocked or eliminated before challenge in some experiments. The severity of anaphylaxis was gauged by changes in body temperature, physical activity, and mortality. Results: Equal doses of antigen or anti-IgE mAb induced similar anaphylactic responses. Anti-IgE mAb-induced anaphylaxis was FcϵRI and mast cell dependent and mediated predominantly by histamine. In contrast, neither mast cells nor platelets appeared important for antigen-induced anaphylaxis, which was FcγRIII and macrophage dependent and mediated predominantly by PAF. Conclusions: Antigen-induced anaphylaxis in the mouse proceeds primarily through the IgG, FcγRIII, macrophage, and PAF pathway, even in an experimental model that is characterized by strong mast cell and IgE responses. The presence of FcγRIII on human macrophages makes it possible that the IgG, FcγRIII, macrophage, and PAF pathway also contributes to human anaphylaxis. (J Allergy Clin Immunol 2002;109:658-68.)

CD28 dependence of T cell differentiation to IL-4 production varies with the particular type 2 immune response.

T cell differentiation to effector cell function is required for the development of a type 2 immune response. The T cell surface molecule, CD28, is widely considered to be the principal costimulatory molecule involved in T cell differentiation to effector function, including IL-4 production, although this has been difficult to directly examine in vivo. We have studied in vivo differentiation to T cell effector function during two type 2 immune responses in CD28 knockout mice: the systemic immune response to goat anti-mouse IgD Ab and the mucosal immune response following oral inoculation with the nematode parasite, Heligmosomoides polygyrus. Our results show that in C57BL/6 CD28 knockout mice elevations in IL-4 gene expression and protein secretion are blocked during the immune response to goat anti-mouse IgD, and associated increases in serum IgG1 and IgE are also inhibited to untreated control levels. In marked contrast, T cell differentiation to IL-4 production is comparable in C57BL/6 CD28 -/- and CD28 +/+ H. polygyrus-inoculated mice, and elevations in both serum IgG1 and IgE levels occur. These results indicate that the specific kind of type 2 immune response determines whether T cell differentiation to IL-4 production is CD28 dependent.

Suppression of in vivo IgE and tissue IL-4 mRNA induction by SDZ 280.636, a synthetic muramyl dipeptide derivative

Modulation of IgE isotype expression on B cells is one of the numerous effects of muramyl peptides on the regulation of the immune system. A non toxic diacyl glycerol derivative of muramyl dipeptide (MDP), in which the l-alanine is replaced by l-threonine (MDP-Threo-GDP; SDZ 280.636), is currently under investigation as lead compound for the development of an anti-allergic drug. In this report, the modulatory effect of orally administered SDZ 280.636 in a murine model on polyclonally induced IgE levels is described. In this model, mice are injected i.v. with goat anti mouse IgD (GAMD) and challenged three to four weeks later with goat IgG (GIG). Both the primary and secondary immune responses lead to an increase of serum IgE levels. We demonstrate the efficacy of this muramyl dipeptide derivative in selectively inhibiting a polyclonal IgE response in GAMD-primed, GIG challenged mice without affecting the levels of other immunoglobulin classes. It is further shown that the induction of interleukin 4 (IL-4) gene transcript levels in lymphoid organs, which is observed as a consequence of boosting GAMD pretreated mice with GIG, is selectively suppressed in gut associated lymphoid tissues (GALT) and mesenteric lymph nodes but not in spleen. In contrast, interleukin 13 (IL-13) mRNA levels are not affected by SDZ 280.636. The findings that SDZ 280.636 inhibits polyclonal IgE responses and suppresses IL-4, but not IL-13 mRNA expression point towards differences in the regulatory pathways of IL-4 and IL-13 gene transcription in lymphoid organs. Thus the mechanism of action appears to involve a specific suppression of IL-4 gene transcription in cells occurring in Peyer's patches and mesenteric lymph nodes which are among the first constituents of the immune system encountered by an orally administered drug.

Down-Regulation of Listeria Monocytogenes-specific Th1 Cytokine Response by treatment of Mice with Goat Antibody to Mouse IgD

Injection of goat anti-mouse IgD antibody (GαM IgD) to mice has been shown to induce polyclonal IgG 1 and IgE production by B cells and IL-4 production by goat Ig-specific T cells. Surface IgD crosslinking also activates function of B cells as antigen presenting cells. Although the GαM IgD treatment is a well established system for regulation of immune response against antigens that bind to B cell receptor, we found that the GαM IgD treatment also influences immune response against irrelevant bacterial antigen. The T cells from the GαM IgD-treated Listeria monocytogenes-infected mice showed increased IL-4 production and decreased IFN-γ and IL-2 production against listerial antigen compared with those from control L. monocytogenes-infected mice. Interestingly, changes were also found in antigen presenting cells in the GaM IgD-treated mice. MHC class II expression of both B cells and macrophages decreased significantly in the GαM IgD-treated mice, suggesting cytokine induced by GαM IgD-treatment may suppress MHC class II expression and modulate APC function in the GαM IgD-treated mice. In accordance with the assumption, T cells from the GαM IgD-treated mice produced high amount of IL-4 and IL-10 in in vitro culture with goat serum which contain goat Ig. These result suggest that GαIgD treatment may modulate APC function in the GαM IgD-treated mice through Th2 type cytokine(s) produced by goat Ig-specific T cells, which results in changes of Th response against irrelevant antigen.

Induction in mice of serum IgE levels after treatment with anti‐mouse IgD antibodies is preceded by differential modulation of tissue cytokine gene transcription

Injection of mice with purified goat anti-mouse IgD (GAMD) leads to an interleukin (IL)-4-dependent increase of serum IgE levels. Challenge of GAMD-primed mice with goat IgG (GIG) initiates a secondary immune response with elevated serum IgE. In this report, kinetic cytokine transcript profiles of murine lymphoid tissues in response to primary i.v. GAMD treatment, as well as GIG challenge are presented. For the first time, gene transcription patterns of the recently described cytokines IL-12 and IL-13 are shown and compared with the corresponding patterns for other cytokine genes involved in IgE regulation, i.e. IL-4, and interferon (IFN)-gamma. After GAMD injection, two groups of induction profiles were observed in spleen, mesenteric lymph nodes and Peyer's patches: while IL-4 and IL-12p35 gene transcription was strongly enhanced, IFN-gamma, IL-12p40 and IL-13 mRNA were only moderately induced. Generally, maximal mRNA induction was measured on days 3 to 4 after GAMD treatment. The data demonstrate a clear-cut difference between the IL-4 and IL-13 response on the transcriptional level although both gene products show similar biological activities. The cytokine mRNA profiles support the assumption of IL-4 playing the central role in generating an IgE response. However, they do not reflect a strict Th1 versus Th2 cytokine gene transcription pattern but rather point towards a concerted action of various, partially antagonizing cytokines with respect to the regulation of IgE synthesis. IL-12 may, possibly via stimulation of IFN-gamma synthesis, represent a counterbalancing factor in the IL-4-mediated IgE response.

Requirement of CTLA-4 counter receptors for IL-4 but not IL-10 elevations during a primary systemic in vivo immune response.

The CD28/CTLA-4 costimulatory signal is required for TCR-mediated T cell activation resulting in increased IL-2 production in vitro, but its role in IL-4 production is unclear and few studies have examined the function of CTLA-4/CD28 in the in vivo immune response. We have examined the in vivo effects of blocking the interaction of B7 with its ligands, CTLA-4 and CD28, in an IL-4 dominant in vivo immune response to goat anti-mouse IgD. This response is characterized by elevations in serum Igs preceded by elevations in IL-2 and the Th2 cytokines: IL-4, IL-9, and IL-10. The fusion protein CTLA4-Ig administered during the in vivo immune response to goat anti-mouse IgD caused an inhibition in elevations of IL-2, IL-4, and IL-9 gene expression at both day 3 and day 6 after immunization. In contrast, IL-10 cytokine gene expression as late as day 6 after immunization was not decreased. Cell sorting analysis demonstrated that TCR-alpha beta +, CD4+ T cells were the primary source of the elevated IL-10, suggesting that T cell activation leading to IL-10 gene expression may not require CTLA-4 ligand interactions. Similarly CTLA4-Ig completely blocked elevations in the number of IL-4- but not IL-10-secreting cells, as measured by ELISPOT, in both unsorted splenic cells and sorted CD4+, TCR-alpha beta+ T cells. In situ staining of spleen sections also showed inhibition of IL-4-producing cells. These results suggest that, with the notable exception of IL-10, interaction, of B7 with its ligands is required for elevated Th2 cytokine gene expression and secretion during a primary systemic IL-4-dominant response.

Effects of IL-12 on in vivo cytokine gene expression and Ig isotype selection.

The effects of murine rIL-12 on cytokine gene expression and Ig secretion were studied in vivo. In untreated mice IL-12 enhanced IFN-gamma and IL-10 gene expression and protein secretion, reduced base line IL-3 and IL-4 gene expression, and increased serum IgG2a concentration. In mice that had been injected with goat anti-mouse IgD antibody (G alpha M delta) to induce increases in IL-3, IL-4, and IL-10 gene expression and serum IgE, IgG1, IgG2a, and IgG3 concentrations, the simultaneous injection of IL-12 enhanced IFN-gamma and IL-10 gene expression and suppressed IL-3 and IL-4 gene expression and serum IgG and IgE responses. Anti-IFN-gamma mAb neutralized most, but not all, IFN-gamma produced by mice treated with G alpha M delta and IL-12. Anti-IFN-gamma mAb enhanced IL-3 and IL-4 gene expression, did not affect IL-10 or IFN-gamma gene expression, and increased serum IgG1, IgG2a, and IgG3 levels, but had relatively little effect on serum IgE in these mice. In contrast to its effects in G alpha M delta-treated mice, IL-12 failed to inhibit the IgE response to G alpha M epsilon antibody, which stimulates mIgE+ B cells to secrete IgE. These observations demonstrate that: 1) IL-12 may limit its own effects by inducing the production of a cytokine (IL-10) that down-regulates both IL-12 production and IL-12-induced IFN-gamma production; 2) IL-12 inhibits the production of at least one cytokine, IL-3, that is not generally regarded to be strictly Th1- or Th2-associated; 3) IL-12 inhibits switching to IgE secretion to a greater extent than it inhibits switching to other Ig isotypes; and 4) the in vivo effects of IL-12 are, to a large extent, IFN-gamma-dependent.