Suppress IgE Responses Research Articles

Substance P (subP) and Minocycline Suppress Induction of Human Ragweed Specific Memory IgE Responses By Different Mechanisms

RationaleWe reported that CD4+ and CD8+CD60+ T cells and 6 cytokines, including IL-4, were required for ragweed specific (RS) memory IgE responses by PBMC of ragweed sensitized humans, and that minocycline strongly suppressed T cell expression of phosphorylated p38 MAP kinase (p38MAPK) and IL-4, while preventing induction of RS memory IgE responses. Others previously reported that CD4+ T cells and IL-4 were required for human IgE responses and that p38MAPK is associated with IL-4. We hypothesize that subP, previously shown to suppress murine memory IgE responses, will prevent induction of human RS memory IgE responses.MethodsPBMC were obtained from ragweed sensitized humans (n=4-6) and CD4+IL-4+ and CD8+CD60+IL-4+ T cell numbers determined ± incubation with PMA/ionophore. The effect of subP ± PMA/ionophore on these cells, on CD4+p38MAPK+ T cells, and on RS memory IgE responses were determined (flow cytometry, ELISA).ResultsCD4+IL-4+ T cells were 5% of CD4+ T cells; CD8+CD60+IL4+ T cells were >99% of CD8+CD60+ T cells. SubP did not suppress CD4+IL-4+ or CD8+CD60+IL-4+ T cells. Nevertheless, subP suppressed T cell p38MAPK (30-70%) and induction of RS memory IgE responses. As reported, minocycline strongly suppressed IL-4, T cell p38MAPK and RS memory IgE responses.ConclusionsSubP and minocycline appear to suppress RS memory IgE responses by different mechanisms. SubP did not suppress IL-4 but minocycline did. Both suppressed IgE responses. It is possible that subP suppresses events occurring downstream of IL-4. Further, it is possible that subP suppresses other cytokines required for memory IgE responses. RationaleWe reported that CD4+ and CD8+CD60+ T cells and 6 cytokines, including IL-4, were required for ragweed specific (RS) memory IgE responses by PBMC of ragweed sensitized humans, and that minocycline strongly suppressed T cell expression of phosphorylated p38 MAP kinase (p38MAPK) and IL-4, while preventing induction of RS memory IgE responses. Others previously reported that CD4+ T cells and IL-4 were required for human IgE responses and that p38MAPK is associated with IL-4. We hypothesize that subP, previously shown to suppress murine memory IgE responses, will prevent induction of human RS memory IgE responses. We reported that CD4+ and CD8+CD60+ T cells and 6 cytokines, including IL-4, were required for ragweed specific (RS) memory IgE responses by PBMC of ragweed sensitized humans, and that minocycline strongly suppressed T cell expression of phosphorylated p38 MAP kinase (p38MAPK) and IL-4, while preventing induction of RS memory IgE responses. Others previously reported that CD4+ T cells and IL-4 were required for human IgE responses and that p38MAPK is associated with IL-4. We hypothesize that subP, previously shown to suppress murine memory IgE responses, will prevent induction of human RS memory IgE responses. MethodsPBMC were obtained from ragweed sensitized humans (n=4-6) and CD4+IL-4+ and CD8+CD60+IL-4+ T cell numbers determined ± incubation with PMA/ionophore. The effect of subP ± PMA/ionophore on these cells, on CD4+p38MAPK+ T cells, and on RS memory IgE responses were determined (flow cytometry, ELISA). PBMC were obtained from ragweed sensitized humans (n=4-6) and CD4+IL-4+ and CD8+CD60+IL-4+ T cell numbers determined ± incubation with PMA/ionophore. The effect of subP ± PMA/ionophore on these cells, on CD4+p38MAPK+ T cells, and on RS memory IgE responses were determined (flow cytometry, ELISA). ResultsCD4+IL-4+ T cells were 5% of CD4+ T cells; CD8+CD60+IL4+ T cells were >99% of CD8+CD60+ T cells. SubP did not suppress CD4+IL-4+ or CD8+CD60+IL-4+ T cells. Nevertheless, subP suppressed T cell p38MAPK (30-70%) and induction of RS memory IgE responses. As reported, minocycline strongly suppressed IL-4, T cell p38MAPK and RS memory IgE responses. CD4+IL-4+ T cells were 5% of CD4+ T cells; CD8+CD60+IL4+ T cells were >99% of CD8+CD60+ T cells. SubP did not suppress CD4+IL-4+ or CD8+CD60+IL-4+ T cells. Nevertheless, subP suppressed T cell p38MAPK (30-70%) and induction of RS memory IgE responses. As reported, minocycline strongly suppressed IL-4, T cell p38MAPK and RS memory IgE responses. ConclusionsSubP and minocycline appear to suppress RS memory IgE responses by different mechanisms. SubP did not suppress IL-4 but minocycline did. Both suppressed IgE responses. It is possible that subP suppresses events occurring downstream of IL-4. Further, it is possible that subP suppresses other cytokines required for memory IgE responses. SubP and minocycline appear to suppress RS memory IgE responses by different mechanisms. SubP did not suppress IL-4 but minocycline did. Both suppressed IgE responses. It is possible that subP suppresses events occurring downstream of IL-4. Further, it is possible that subP suppresses other cytokines required for memory IgE responses.

Novel Mechanisms Of Immune Modulation By Alpha-1-Antitrypsin

S U N D A Y 484 CD4+ and CD8+ T Cells Of Allergic Humans Express Increased Phosphorylated p38 MAP Kinase (p38MAPK), Substance P Suppresses T Cell Expression Of p38MAPK and Memory IgE Responses Bryan McCarthy, BS, Jonathan I. Silverberg, MD, PhD, MPH, Seto M. Chice, MS, Ankuri Desai, BS, Dr. Tamar A. Smith-Norowitz, PhD, Mark Stewart, MD, PhD, Dr. Rauno Joks, MD, Helen G. Durkin, PhD; Center for Allergy and Asthma Research at SUNY Downstate, Brooklyn, NY, Center for Allergy and Asthma Research, State University of New York Downstate Medical Center, Brooklyn, NY. RATIONALE: Magnetic/electrical stimulation of IgE+ human/rat left TPO cortex increases serum/blood substance P (2 hr) and CD4+/CD8+ T cell numbers (4 hr), while suppressing IgE responses (4 days). Thymus supplied T cells to blood because thymectomy or cutting of spinal cord at T2 (thymic innervation level) abrogated their appearance. Substance P suppresses specific murine IgE responses in vivo/vitro, and a relationship between p38MAPK and IL-4 has been reported. The effect of substance P on p38MAPK expression by human lymphocyte subsets and memory IgE responses has not been studied. METHODS: Distributions of p38MAPK+ lymphocytes (CD4+,CD8+,CD19+,CD16+) in PBMC obtained from ragweed sensitized IgE+ humans were determined6 15-30 min incubation with PMA6 substance P (flow cytometry). In addition, PBMC were cultured for 0-12 days 6 ragweed antigen 6 substance P and IgE levels in supernatants determined (ELISA). RESULTS: Before PMA stimulation, virtually no p38MAPK+ T cells (CD4+, CD8+), B or NK cells were detected in PBMC (<5%). After PMA, virtually all T cells, and CD4+ T cells which had been purified from PBMC, were p38MAPK+ (90-98%), with no change in p38MAPK+ B or NK cells.When PBMCor purified CD4+ T cells were incubatedwith PMA + substance P, p38MAPK+ CD4+ and CD8+ T cell numbers were suppressed (50-75%) When PBMC were cultured with ragweed antigen, peak IgE responses were induced on day 8; inclusion of substance P in culture prevented induction of these responses on all days. CONCLUSIONS: Taken together, our results indicate that brain can suppress IgE responses by releasing substance P.

Suppression of Allergen‐Specific IgE in Offspring After Preconceptional Immunisation: Maternal, Paternal and Genetic Influences

Immunisation of female mice with the allergen ovalbumin (OVA) during pregnancy reduces the OVA-specific IgE response in adult offspring. To approach primary prevention strategies for allergy, we investigated to what extent genetic, paternal and maternal factors influence this suppressive effect on allergic sensitisation in offspring and investigated the possibility of pregestational immunisation. Maternal allergen immunisation reduced OVA-specific IgE levels in immunised offspring, even after maternal immunisation up to 8 weeks before conception without further allergen exposure. Immunisation of immunodeficient BALB/c severe combined immune deficiency (SCID) dams mated with wild type males did not lead to IgE suppression in offspring, indicating the importance of a functional maternal immune system. Immunisation of male mice before the relevant spermatogenesis did not cause antibody suppression in offspring. OVA-specific IgG1, presumably of maternal origin, was present in naïve offspring only from immunised dams and was associated with suppressed IgE responses after offspring immunisation. The IgE-suppressive effect of maternal immunisation was demonstrated in all three immunocompetent strains tested (NIH/OlaHsd, BALB/cA and C57BL/6 mice). In conclusion, suppression of allergen-specific IgE production in offspring could not be induced by paternal immunisation, and genetic factors were of minor importance. In contrast, we demonstrate the necessity of maternal factors, possibly allergen-specific IgG1, resulting from a functional adaptive immune response, for the IgE-suppressive effect in offspring. These maternal factors could be induced by immunisation of female mice even before conception.

Non-antibiotic properties of tetracyclines as anti-allergy and asthma drugs

All available therapies for human allergic disease target IgE mediated pathologic responses after IgE has been produced. We are developing tetracyclines as anti-allergy drugs to prevent IgE production, based on our findings that minocycline or doxycycline treatment of allergic asthmatic humans significantly improves their asthma symptoms, reduces their oral steroid requirements, and strongly suppresses their ongoing IgE responses (ELISA, mast cell mediated cutaneous late phase responses); the tetracyclines also strongly suppress peak IgE responses of BPO-KLH sensitized mice (ELISPOT assay, ELISA, skin tests). The antibiotic activity of the tetracyclines is not required for suppression of IgE responses; inclusion of minocycline or doxycycline in sterile culture prevents anti-CD40/IL-4 mediated induction of memory IgE responses by PBMC of allergic asthmatic patients (ELISA), and induction of specific memory IgE responses by spleen cells of BPO-KLH sensitized mice (ELISPOT assay, ELISA). The tetracyclines affect an epsilon specific pathway because IgM, IgG and IgA responses did not decrease. Further, in humans, DTH responses to recall antigens did not decrease. In related studies, we found that two distinct T cell subsets: CD4+CD60 negative and CD8+CD60+ (CD60 is a ganglioside) (humans) and CD4+ Asialo GM1 ganglioside negative and CD8+Asialo GM1 ganglioside+ (mice), both are required for induction of memory IgE responses. Phosphorylated (phos) p38 MAP kinase, but not phos ERK or phos JNK expression by CD4+ and CD8+, including CD8+CD60+, T cells is increased in allergic asthmatic humans, as is IL-4 and IL-10 production. The tetracyclines appear to target T cell pathways to induce suppression of IgE responses because they suppress phos p38 MAP kinase expression by both CD4+ and CD8+, including CD8+CD60+, T cell subsets, and IL-4 and IL-10, while upregulating IL-2 and IFN gamma, and suppressing IgE responses. Our finding that tetracyclines do not require antibiotic activity to suppress IgE responses opens the door to development of new tetracycline-based and other therapeutics for human allergic disease.

Two Distinct T Cell Subsets, CD4+ and CD8+CD60+, and Their Cytokines Are Required for In Vitro Induction of Human Ragweed-Specific Memory IgE Responses

CD8(+)CD60(+) T cells (80-98% CD45RO(+); 20% CD23(+)) are significantly increased in the blood of serum IgE(+) ragweed-sensitized (RS) compared with serum IgE-nonatopic humans (p = 0.001). CD8(+)CD60(+) T cells of the RS patients produced IL-2, IL-4, IL-10, IL-12, IFN-alpha. and IFN-gamma, but not IL-6 or IL-13. When their PBMC were cultured with ragweed Ag (RA), peak IgE responses occurred on day 10; none was induced with non-cross-reacting or without Ag; nonatopic PBMC did not respond to any stimulant. When either CD4(+) or CD8(+)CD60(+) T cells were depleted from RS PBMC before culture with RA, no IgE responses were induced. If purified CD4(+) T cells or low numbers of CD8(+)CD60(+) T cells were added back to the depleted PBMC, IgE responses were restored. However, higher numbers of CD8(+)CD60(+) T cells totally suppressed IgE responses. Total suppression also was obtained when RS PBMC were cultured with RA and either anti-IL-2, IL-4, IL-10, IL-12, IFN-gamma (all concentrations), or IFN-alpha (low concentrations), but not anti-IL-6 or IL-13. Higher concentrations of anti-IFN-alpha potentiated IgE responses.

CD23: An overlooked regulator of allergic disease

Given the importance of immunoglobulin (Ig) E in mediating type I hypersensitivity, inhibiting IgE production would be a general way of controlling allergic disease. The low-affinity IgE receptor (FceRII or CD23) has long been proposed to be a natural regulator of IgE synthesis. In vivo research supporting this concept includes the observation that mice lacking CD23 have increased IgE production whereas mice overexpressing CD23 show strongly suppressed IgE responses. In addition, the finding that mice injected with monoclonal antibody directed against the coiled-coil stalk of CD23 have enhanced soluble CD23 release and increased IgE production demonstrates that full-length, trimeric CD23 is responsible for initiating an IgE inhibitory signal. The recent identification of ADAM10 (a disintegrin and metalloprotease) as the CD23 metalloprotease provides an alternative approach for designing therapies to combat allergic disease. Current data suggest that stabilizing cell-surface CD23 would be a natural means to decrease IgE synthesis and thus control type I hypersensitivity.

An Essential Role for IL-18 in CD8 T Cell-Mediated Suppression of IgE Responses

The ability of CD8 T cells to suppress IgE responses is well established. Previously, we demonstrated that CD8 T cells inhibit IgE responses via the induction of IL-12, which promotes Th1 and suppresses Th2 responses. In this study, we show that IL-18 also plays an essential role in IgE suppression. In vitro, IL-18 synergized with IL-12 to promote Th1/T cytotoxic 1 and inhibit Th2/T cytotoxic 2 differentiation. OVA-specific TCR transgenic (OT-I) CD8 cells induced both IL-12 and IL-18 when cultured with OVA(257-264) peptide-pulsed dendritic cells. In vivo, IL-18(-/-) mice exhibited higher IgE and IgG1 levels compared with wild-type mice after immunization with OVA/alum. Furthermore, adoptive transfer of CD8 T cells from OVA-primed mice suppressed IgE responses in OVA/alum-immunized mice, but not in IL-18(-/-) mice. IgE suppression in IL-18(-/-) mice was restored if CD8 T cells were coadoptively transferred with IL-18-competent wild-type bone marrow dendritic cell progenitors, demonstrating an essential role of IL-18 in CD8 T cell-mediated suppression of IgE responses. The data suggest that CD8 T cells induce IL-18 production during a cognate interaction with APCs that synergizes with IL-12 to promote immune deviation away from the allergic phenotype. Our data identify IL-18 induction as a potentially useful target in immunotherapy of allergic disease.

Novel 2-(Substituted phenyl)benzimidazole Derivatives with Potent Activity against IgE, Cytokines, and CD23 for the Treatment of Allergy and Asthma

The effectiveness of the injectable anti-IgE antibody omalizumab has validated IgE as an important target for allergic diseases, thus spawning the development of small-molecule IgE inhibitors. Herein, a brief SAR is described for novel phenylbenzimidazole compounds that potently suppress IgE responses. In addition to IgE, these agents inhibit other targets critical for allergic response. The profile of orally active AVP-13358, the lead compound of this series currently in clinical trials, is described.

Comparison of different adjuvants of protein and DNA vaccination for the prophylaxis of IgE antibody formation

A high-molecular-weight mite allergen Der f11 that was hardly purified for immunotherapy was used to develop the DNA vaccine pDf11. We have shown that vaccination of mice with pDf11 induces Th1 responses characterized by suppression of IgE responses. In the present study, effects of different adjuvants on pDf11 were first studied. Mice receiving pDf11±CpG, bestatin, and bupivacaine had better suppression of IgE responses than those receiving pDf11±lipofectin or alum. Bestatin could greatly boost IgG2a responses. Immunomodulating effects of different adjuvants between protein and DNA vaccines were further elucidated. CpG was the best for both protein and DNA vaccines to profoundly suppress IgE responses, but alum, bestatin and lipofectin were useless for rDf11 to induce IgE inhibition. Neither did the combination of rDf11 and pDf11 have further IgE suppression. In conclusion, CpG is the unique adjuvant for the protein vaccine rDf11 to inhibit IgE responses. In contrast, the DNA vaccine pDf11±CpG, bestatin, or bupivacaine induces profound suppression of IgE responses.

Induction of specific Th1 responses and suppression of IgE antibody formation by vaccination with plasmid DNA encoding Der f 11

DNA vaccines encoding low-molecular-weight allergens have been used to prevent IgE responses. A high-molecular-weight mite allergen Der f 11 that was hardly to be purified for immunotherapy was used to develop a DNA vaccine here. Vaccination of mice with plasmid DNA encoding Df11 (pDf11) induced Th1 responses characterized by IgG2a responses and spleen cell secretion of IFN-γ. In contrast, sensitization with recombinant Der f 11 (rDf11) and alum induced Th2 responses characterized by IgE responses and spleen cell secretion of IL-4 and IL-5. Vaccination with pDf11 prevented the induction of IgE responses. Moreover, it could inhibit on-going IgE responses. The debate whether CD4 + or CD8 + T cells were the regulatory cells to inhibit IgE responses by DNA vaccination was also examined. First, sensitization of pDf11-vaccinated mice after depletion of CD8 + T cells still showed suppression of IgE responses. Secondly, adoptive transfer of either CD4- or CD8-depleted spleen cells from pDf11-vaccinated mice suppressed IgE responses. In conclusion, this is the first report to confirm the therapeutic effect of a DNA vaccine encoding a strong allergen on specific IgE responses. Both CD4 + and CD8 + T cells are crucial for the immunomodulation of IgE responses by pDf11.

Suppression of IgE antibody response in mice by a naphthalene derivative, TEI-6472

In the present report, we investigated suppressive effects of a naphthalene derivative, (7E)- N-(2-carboxyphenyl)-8-(2-naphthyl)-5,6-trans-5, 6-methano-7-octenamide L-lysine salt (TEI-6472), on in vitro and in vivo antigen-specific IgE response. Anti-trinitrophenyl (TNP) IgE response induced in vitro in TNP-keyhole limpet hemocyanin (KLH)-primed murine spleen cells was suppressed by about 60% in the presence of 10 −6 M TEI-6472. On the other hand, anti-TNP IgG1 and IgM response were not significantly suppressed under the same conditions. Proliferative responses of BALB/c spleen cells stimulated by lipopolysaccharide, concanavalin A or allogeneic spleen cells were not inhibited by TEI-6472 at 10 −6-10 −5 M. Interleukin 4 production from helper T-cell clone, D10.G4.1 was suppressed only slightly (less than 20%) at 10 −6 M TEI-6472. This compound was also effective in suppressing secondary anti-TNP IgE response in mice that were immunized twice with TNP-KLH and alum. When 10–20 mg/kg/day TEI-6472 was administered s.c. for 5 consecutive days starting from one day before the first and the second immunization, secondary anti-TNP IgE response was inhibited most strongly (40–45%). Anti-TNP IgG1 response was also inhibited but to a smaller extent (20–24%), while anti-TNP IgM response was suppressed only slightly (0–15%). These results suggest that, under appropriate conditions, TEI-6472 can suppress IgE responses more preferentially both in vitro and in vivo.

Control of IgE Responses. III. IL-6 and IFN-α Are Isotype-Specific Regulators of Peak BPO-Specific IgE Antibody-Forming Cell Responses in Mice

The ability of cytokines (IL-4, IL-5, IL-6, IFN-α, IFN-γ, TNF-α, GmCSF) to regulate peak benzylpenicilloyl (BPO)-specific IgE antibody-forming cell (AFC) responses was investigated. These responses were induced in BALB/c mice by ip injection of BPO-keyhole limpet hemocyanin (BPO-KLH; 10 μg) in aluminum hydroxide gel on Days 0, 21, and 42. On Day 44, or on Days 43, 44, and 45, mice were injected sc with varying doses of cytokine or anti-cytokine antibody. On Day 46, the numbers of BPO-specific AFC (IgM, IgG1, IgE and IgA) in spleen were determined ex vivo in enzyme-linked immunosorbent spot assay. Among the cytokines tested, only IL-6 suppressed BPO-specific IgE AFC responses in an isotype-specific fashion (60-90%). However, treatment of mice with anti-IL-6 also suppressed these responses, suggesting that IL-6 can either suppress or increase peak antigen specific IgE responses, depending upon its concentration. Among the cytokines tested, only IFN-α increased BPO-specific IgE AFC responses in an isotype-specific fashion. Since treatment with anti-IFN-α suppressed these responses, it appears that IFN-α is required to maintain peak antigen-specific IgE AFC responses. IL-4 or IFN-γ nonspecifically suppressed responses of all isotypes. Treatment with anti-IL-4 also suppressed IgE responses, suggesting that this cytokine is required to maintain peak antigen specific IgE responses. Treatment with anti-IFN-γ increased IgE responses, indicating that IFN-γ suppresses peak antigen-specific IgE responses.

Ricin-Sensitive Early Activated CD8+ T Cells Suppress IgE Responses and Regulate the Production of IFN-γ and IL-4 by Splenic CD4+, CD8+ T Cells.

Immunization of rats with a bystander antigen and the toxic lectin, ricin, results in a dramatically potentiated IgE response which was only seen when ricin was administered together with antigen, or 24 h later, suggesting that the toxin was acting on cells which were activated as the result of immunization. The identity of these activated cells was investigated in radioligand bindings studies with 125I ricin. No difference was seen in the number of ricin binding sites on splenic CD4+ and CD8+ T cells from untreated rats, but the number of ricin binding sites was 13-fold higher on CD8+ but not on CD4+ T cells from immunized rats. Adoptive transfer of 1 × 108 >99% pure CD8+ T cells from animals, immunized 3 days before with antigen alone, suppressed the IgE response to antigen plus alum by over 90%. The IgE suppressive effect of these CD8+ T cells was not antigen specific, and they had no effect on the IgG antibody response. CD4+ T cells from none of these animals had any effect on the subsequent IgE of IgG antibody response. The capacity of CD4+ and CD8+ T cells stimulated with PHA or PMA and ionomycin to secrete the cytokines IL-2 and IFN-γ or to make IL-4 mRNA was also compared. CD8+ T cells were the highest producers of IFN-γ and accounted for 65% oftotal splenic IFN-γ. Immunization with ricin plus antigen reduced the amount of IFN-γ secreted by CD4+ (OX22+/--) and CD8+ T cells 5- to 10-fold. Over 95% of the IL-2 produced was derived from OX22+CD4+ T cells, and this did not change. In spleen cells from unimmunized animals, the largest amounts of IL-4 mRNA were found in CD8+ T cells. Following immunization with antigen plus ricin, total splenic IL-4 mRNA production was increased 3- to 4-fold and following boosting with ricin plus antigen was increased a further 3 to 4-fold. These results show: (i) that approximately 50% of splenic CD8+T cells are activated within24h of immunization with antigen, (ii) that adoptive transfer of these cells suppresses IgE production, (iii) that co-administration of ricin and antigen selectively depletes them, (iv) that this is associated with a massive increase in serum IgE a decreased capacity of T cells to produce IFN-γ and an increased capacity to make IL-4. These data suggest that CD8+ T cells, activated early in the immune response, play an important part in regulating cytokine and IgE production.