B cells establish, but do not maintain, long-lived murine anti-peanut IgE(a).

BACKGROUND: Peanut allergy (PNA) has been reported to be transferred to tolerant recipients through organ and bone marrow (BM) transplantation. The roles T and B cells play in establishing, and the roles B cell subsets play in maintaining lifelong anti-peanut IgE levels are unknown. OBJECTIVES: To determine the cellular requirements for the transfer of murine PNA and to determine the role CD20(+) cells play in maintaining long-lived anti-peanut IgE levels. METHODS: We developed a novel adoptive transfer model to investigate the cellular requirements for transferring murine PNA. We also treated peanut-allergic (PA) mice with anti-CD20 antibody and measured IgE levels throughout treatment. RESULTS: Purified B220(+) cells from PA splenocytes and purified CD4(+) cells from naïve (NA) splenocytes are the minimal requirements for the adoptive transfer of PNA. Prolonged treatment of allergic mice with anti-CD20 antibody results in significant depletion of B cell subsets but does not affect anti-peanut IgE levels, symptoms, or numbers of IgE antibody secreting cells (ASCs) in the BM. Adoptive transfer of BM and spleen cells from allergic donors treated with anti-CD20 antibody does not result in the transfer of PNA in NA recipients, demonstrating that anti-CD20 antibody treatment depletes B cells capable of differentiating into peanut-specific IgE ASCs. CONCLUSIONS AND CLINICAL RELEVANCE: Peanut allergy can be established in a NA hosts with B220(+) cells from PA donors and CD4(+) cells from peanut-NA donors. However, long-term depletion of B220(+) cells with anti-CD20 antibody does not affect anti-peanut IgE levels. These results highlight a novel role for B cells in the development of PNA and provide evidence that long-lived anti-peanut IgE levels may be maintained by long-lived ASCs.

Epitope analysis of Ara h 2 and Ara h 6: characteristic patterns of IgE-binding fingerprints among individuals with similar clinical histories.

BACKGROUND: Ara h 2 and Ara h 6 are moderately homologous and highly potent peanut allergens. OBJECTIVE: To identify IgE-binding linear epitopes of Ara h 6, compare them to those of Ara h 2, and to stratify binding based on clinical histories. METHODS: Thirty highly peanut-allergic subjects were stratified by clinical history. Sera were diluted to contain the same amount of anti-peanut IgE. IgE binding to overlapping 20-mer peptides of Ara h 2 and Ara h 6 was assessed using microarrays. RESULTS: Each subject had a unique IgE-binding fingerprint to peptides; these data were coalesced into epitope binding. IgE from subjects with a history of more severe reactions (n = 19) had a smaller frequency of binding events (BEs) for both Ara h 2 (52 BEs of 152 (19X8epitopes) possible BEs and Ara h 6 (13 BEs of 133 (19X7 epitopes) possible BEs) compared to IgE from those with milder histories (n = 11) (Ara h 2: 47 BEs of 88 (11X8 epitopes) possible BEs, P < 0.01; Ara h 6: 25 BEs of 77 (11X7 epitopes) possible BEs, P < 0.001). Using an unsupervised hierarchal cluster analysis, subjects with similar histories tended to cluster. We have tentatively identified a high-risk pattern of binding to peptides of Ara h 2 and Ara h 6, predominantly in subjects with a history of more severe reactions (OR = 12.6; 95% CI: 2.0-79.5; P < 0.01). CONCLUSIONS AND CLINICAL RELEVANCE: IgE from patients with more severe clinical histories recognize fewer linear epitopes of Ara h 2 and Ara h 6 than do subjects with milder reactions and bind these epitopes in characteristic patterns. Close examination of IgE binding to epitopes of Ara h 2 and Ara h 6 may have prognostic value.

Vitamin E, γ-tocopherol, diminishes ex vivo basophil response to dust mite allergen.

Epidemiologic studies suggest that dietary vitamin E is a candidate intervention for atopic disease. We used in vitro and ex vivo exposures to test the hypothesis that the most common dietary isoform of vitamin E, γ-tocopherol (γT), could suppress FcεRI-mediated basophil activation. Rat basophilic leukemia (RBL)-SX38 cells that express human FcεRI were treated with or without γT, followed by stimulation with α-IgE. In the ex vivo study, 20 Der f 1-allergic volunteers consumed a γT-enriched supplement for 7 days. Their basophils were challenged ex vivo with α-IgE and graded doses of Der f 1 before and after the supplementation period. γt treatment of RBL-SX38 cells significantly reduced basophil degranulation and de novo TH2 cytokine production. Daily consumption of a γT-rich supplement by dust mite-allergic volunteers reduced basophil activation after ex vivo dust mite challenge. Vitamin E supplements rich in γT may be useful adjuncts in decreasing atopic disease.

The 2S albumin allergens of Arachis hypogaea, Ara h 2 and Ara h 6, are the major elicitors of anaphylaxis and can effectively desensitize peanut-allergic mice.

BACKGROUND: Ara h 2 and Ara h 6, co-purified together in a 13-25 kD fraction (Ara h 2/6; 20 kD fraction) on gel filtration chromatography, account for the majority of effector activity in a crude peanut extract (CPE) when assayed with RBL SX-38 cells sensitized with IgE from human peanut allergic sera. OBJECTIVES: To determine if Ara h 2/6 are the primary peanut allergens responsible for allergic reactions in vivo and to determine if Ara h 2/6 would be sufficient to prevent allergic reactions to a complete CPE. METHODS: An oral sensitization mouse model of peanut allergy was used to assess the activity of Ara h 2/6 (20 kD) and CPE without the 20 kD fraction (CPE w/o 20 kD) for allergic provocation challenge and immunotherapy. The activity of these preparations was also tested in an assay of histamine release from human basophils in whole blood. RESULTS: Compared with mice challenged with control CPE, mice challenged with CPE w/o 20 kD experienced reduced symptoms (P < 0.05) and a smaller decrease in body temperature (P < 0.01). Results with the basophil histamine release assay corroborated these findings (P < 0.01). The mouse model was also used to administer Ara h 2/6 (20 kD) in an immunotherapy protocol, in which peanut-allergic mice treated with the 20 kD fraction experienced significantly reduced symptoms, changes in body temperature, and mast cell protease (MMCP-1) release compared with placebo (P < 0.01 for all parameters). Importantly, immunotherapy with the 20 kD fraction was just as effective as treatment with CPE, whereas CPE w/o 20 kD was significantly less effective for higher dose peanut challenges. CONCLUSIONS AND CLINICAL RELEVANCE: Ara h 2/6 are the most potent peanut allergens in vivo and can be used to desensitize peanut-allergic mice. These results have potential implications for clinical research in the areas of diagnosis and immunotherapy for peanut allergy.

Effector activity of peanut allergens: a critical role for Ara h 2, Ara h 6, and their variants.

RATIONALE: An important property of allergens is their ability to cross-link IgE and activate mast cells and basophils. The effector activity of peanut allergens has not been well characterized. METHODS: Crude extracts of fresh peanut flour were fractionated by gel filtration. Effector function was assayed by measuring degranulation of RBL SX-38 cells sensitized with IgE from individual sera and from pools of sera of peanut-allergic donors. RESULTS: Following gel filtration, 75 +/- 7% of the applied protein and 76 +/- 16% (n=3) of the applied activity (assayed with a pool of 11 sera) were recovered in the resultant fractions. The majority (85 +/- 2%; n=3) of the recovered activity resided in a fraction with a theoretical average molecular weight of approximately 20 kDa and a range of 13-25 kDa. When all the individual fractions were recombined, the measured activity was similar to that of the original extract [140 +/- 43% when measured with a pool of serum (n=2) and 66 +/- 7% when measured with individual sera (n=4)]; when all individual fractions excluding the 20 kDa fraction were recombined, the measured activity was only 8 +/- 2% (n=2) of the original extract when assayed with the serum pool and 10 +/- 4% (n=3) when assayed with the individual sera. Two-dimensional gel electrophoresis of this biologically active fraction revealed >60 protein spots. Analysis of 50 of the most prominent spots by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry and of the full mixture by automated tandem mass spectrometry coupled to online capillary liquid chromatography revealed that >97% of the protein mass consisted of Ara h 2.0101, Ara h 2.0201, Ara h 6 isoforms, and variants of these proteins. CONCLUSIONS: Ara h 2 and Ara h 6 account for the majority of the effector activity found in a crude peanut extract.

Contribution of Ara h 2 to peanut-specific, immunoglobulin E-mediated, cell activation.

BACKGROUND: Ara h 2 is a potent peanut allergen but its contribution to the ability of a crude peanut extract (CPE) to cross-link IgE and activate mast cells has not been rigorously evaluated. OBJECTIVE: To measure the contribution that Ara h 2 makes to the effector function of a CPE. METHODS: Ara h 2 was specifically removed from a CPE as demonstrated by immunoblots, 2D gels, and an inhibitory ELISA. Functional assays of sham-treated and Ara h 2-depleted CPEs were performed with RBL SX-38 cells sensitized with IgE from highly peanut-allergic subjects and with naturally sensitized basophils. RESULTS: Depletion of approximately 99% of the Ara h 2 from the CPE led to an increase in the concentration of the CPE necessary to give 50% of maximal degranulation (EC50) of the SX-38 cells following sensitization with sera that contain anti-Ara h 2 IgE. Assays with a pool of 10 sera showed a small but significant increase in the EC50 following depletion of Ara h 2 (1.65+/-0.15-fold; P<0.05) and assays of seven individual sera showed a similar increase in the average EC50 (1.7+/-0.2-fold; P<0.02). The percent of the anti-peanut IgE that binds Ara h 2 correlated with an increase in the EC50 of the CPE following depletion of Ara h 2 (r=0.83; P<0.02). On the other hand, data from three of these patients studied with a basophil histamine release assay did not show a significant effect of depletion of Ara h 2. CONCLUSION: Based on its ability to cross-link IgE effectively, Ara h 2 is clearly an important peanut allergen. Its ability to cross-link IgE effectively from a specific serum is related to the proportion of anti-Ara h 2 in that serum but Ara h 2 does not account for a majority of the effector activity of the CPE for any of the sera studied.

In vitro activity of MEKK2 and MEKK3 in detergents is a function of a valine to serine difference in the catalytic domain.

MEKK2 and MEKK3 are mitogen-activated protein kinase kinase kinases (MAP3 kinases) of 70 and 71 kDa respectively that are markedly homologous (94%) in their kinase domains. Both MEKK2 and MEKK3 are able to activate the Jun kinase pathway in vivo. However, following routine immunoprecipitation in Triton X-100, MEKK2 but not MEKK3 is able to effectively phosphorylate both SEK-1 and MEK-1 and to undergo autophosphorylation. Unexpectedly, both MEKK2 and MEKK3 are functional in an in vitro kinase assay when cells are solubilized with the closely related detergent, NP-40. Given the high homology between these kinases, we set out to relate this differential sensitivity to Triton X-100 to differences in primary structure. A set of chimeric molecules were generated and the loss of activity in Triton X-100 mapped to kinase domain II/III and specifically to serine 390 of MEKK3 and valine 384 of MEKK2, residues immediately N-terminal to the active site lysine. Mutation of serine 390 of MEKK3 to a valine (as is found in MEKK2) conferred catalytic activity to MEKK3 in Triton X-100 whereas the reciprocal alteration of valine 384 of MEKK2 to a serine conferred lack of activity in Triton X-100 to MEKK2. Search of the protein database identified only three kinases, MEKK3, Pbs2p and Dd-PKI, with a serine or threonine at this site. The presence of a serine or threonine adjacent to the active site lysine in protein kinases is rare and, in MEKK3, results in detergent instability.

Isoforms of Jun kinase are differentially expressed and activated in human monocyte/macrophage (THP-1) cells.

Ten isoforms of c-jun N-terminal kinase (JNK) have been described that arise by differential mRNA splicing of three genes. In that the relative expression and function of these different JNK proteins in human monocytic cells is not known, we have examined the JNK isoforms in THP-1 monocyte/macrophage cells. Differentiation of THP-1 cells by exposure to 10(-8) M PMA for 42-48 h enhances cellular responses to LPS, including enhanced activation of total JNK activity and increased phosphorylation of p54 JNK as well as p46 JNK. Examination of JNK proteins on Western blots reveals a predominance of p46 JNK1 and p54 JNK2 proteins. Clearing of lysates by immunoprecipitation of JNK1(99% effective) removes 46% of the JNK enzymatic activity (p < 0.01), whereas clearing of JNK1 plus JNK2 (70% effective) depletes the sample of 72% of the JNK activity (p < 0.01). Further analysis, undertaken with real-time RT-PCR, revealed that 98% of the JNK messages code for three isoforms: JNK1beta1, JNK2alpha1, and JNK2alpha2. The p54 JNK that is phosphorylated in LPS-stimulated, PMA-differentiated THP-1 cells is most likely JNK2alpha2 because 97% of the p54 JNK-encoding messages code for JNK2alpha2. By analogous reasoning, the p46 JNKs that are not heavily phosphorylated, but account for approximately half of the N-terminal c-jun kinase enzymatic activity, are most likely either JNK1beta1 or JNK2alpha1 because they account for 98% of the messages that can code for 46kDa JNKS:

Measuring degranulation of mast cells.

This unit describes methods for measuring exocytosis of preformed mediators from secretory granules as an indication of IgE receptor-mediated activation of mast cells. The first basic protocol describes the measurement of biogenic amines (serotonin and histamine) secreted by activated rodent mast cells (for serotonin) or rodent and human mast cells (for histamine). The second basic and alternate protocols detail techniques for measuring the release of beta-glucuronidase, an enzyme that is synthesized by human and rodent mast cells, stored in secretory granules, and released during degranulation. Methods for assaying other enzymes released during degranulation, such as beta-hexosaminidase and tryptase, are discussed in the . These protocols can also be applied to basophils where appropriate.

A diabetogenic gene prevents T cells from receiving costimulatory signals.

T cell fate following antigen encounter is determined by several intracellular signals generated by the interaction of the T cell with an antigen-presenting cell. In the periphery activation requires T cell receptor signaling (signal one) in combination with costimulatory signals (signal two), usually provided through the cognate interaction of CD28 and B7 molecules. Provision of signal one alone to purified murine peripheral T cells in vitro induces apoptosis or anergy rather than promoting activation. These T cells can be rescued from apoptosis if they are provided with costimulation supplied, for example, by engaging the CD28 co-receptor with an anti-CD28 monoclonal antibody or by adding an exogenous source of interleukin-2. However, a majority of peripheral T cells from autoimmune, diabetes-prone Biobreeding (BB) rats exhibited different responses to these stimuli. T cells from these rats could not be rescued from apoptosis by costimulation. This was not due to the inability of BB-DP T cells to upregulate CD28 and the IL-2 receptor in response to TCR crosslinking. The failure of these costimulatory interactions to rescue BB-DP T cells segregated with the diabetes-susceptibility gene iddm1. Iddm1 in the rat causes peripheral T cell lymphopenia, which is associated with a dramatically shortened peripheral T cell life span. Our results indicate that a diabetogenic gene may contribute to autoimmunity by negating costimulatory signals important for the survival of long-lived peripheral T cells.

Production of TNF-alpha by murine bone marrow derived mast cells activated by the bacterial fimbrial protein, FimH.

Production of tumor necrosis factor alpha (TNF-alpha) by mast cells is an important aspect of host defense against gram negative bacteria. In order to define the intracellular pathways utilized by mast cells in this physiological, protective role, we have studied the production of TNF-alpha in bone marrow derived mast cells from the C3H/HeJ (LPS-insensitive) strain following exposure to bacteria expressing the fimbrial protein, FimH. Mast cells exposed to FimH produce TNF-alpha (300-1200 pg/10(6) cells) over 1-3 h compared with 1800-15,000 pg/10(6) cells produced by cells triggered via IgE/antigen. This low level of TNF-alpha production in vitro is compatible with the protective in vivo role of mast cells to produce modest amounts of TNF-alpha in contrast to the large amounts of mediators released during maximal activation. A second difference between the two signals is sensitivity to cyclosporin A (CsA). The IgE/antigen pathway is inhibited by 90-95% at 0.02 to 0.5 microM cyclosporin A whereas the FimH pathway is inhibited by only 40%. These data demonstrate that the intracellular pathway activated by FimH is different from that activated by IgE/antigen both in terms of amount of TNF-alpha produced and in sensitivity to CsA. This is the first evidence that FimH activates mast cells via a pathway that is distinct from that used by IgE/antigen.

Arachidonate-induced eicosanoid synthesis in RBL-2H3 cells: stimulation with antigen or A23187 induces prolonged activation of 5-lipoxygenase.

We studied the ability of rat basophilic leukemia (RBL-2H3) cells stimulated with either IgE/antigen or calcium ionophore, A23187, to synthesize LTC4 and PGD2 after addition of exogenous arachidonic acid. RBL-2H3 cells preferentially synthesized PGD2 in response to stimulation with low concentrations of antigen or A23187 while higher concentrations also resulted in a marked synthesis of LTC4. The synthesis of LTC4 was dependent upon initial activation of 5-lipoxygenase by IgE/antigen or A23187, since arachidonic acid lone failed to induce LTC4 synthesis. Following the addition of IgE/antigen or A23187 alone, the synthesis of PGD2 and LTC4 was essentially complete by 10 min. To determine whether a limitation of substrate precluded further eicosanoid synthesis, exogenous arachidonic acid was added to washed cells 15-145 min following the initial stimulation with IgE/antigen or A23187, PGD2 and LTC4 synthesis was resumed following the addition of arachidonic acid to washed prestimulated cells, demonstrating that the termination of eicosanoid synthesis in RBL-2H3 cells was nor caused by the inactivation of cyclooxygenase and 5-lipoxygenase. DNP-lysine was added to cells previously stimulated with IgE/antigen to stop receptor aggregation and this greatly inhibited subsequent production of LTC4 following the addition of arachidonic acid, suggesting that ongoing stimulation of Fc epsilon XsRI was required for LTC4 synthesis in this setting. These results indicate that the magnitude of a physiologic stimulus (IgE/antigen) can profoundly affect the arachidonate metabolites produced by mast cells and that the synthesis of these metabolites quickly becomes limited by substrate availability rather than the activity of cyclooxygenase or 5-lipoxygenase.

High-affinity oligonucleotide ligands to human IgE inhibit binding to Fc epsilon receptor I.

Using the systematic evolution of ligands by exponential enrichment (SELEX) method, we have identified oligonucleotides that bind to human IgE with high affinities and high specificity. These ligands were isolated from three pools of oligonucleotides, each representing 10(15) molecules: two pools contained 2'-NH2 pyrimidine-modified RNA with either 40 or 60 randomized sequence positions, and the third pool contained ssDNA with 40 randomized sequence positions. Based on sequence and structure similarities, these oligonucleotide IgE ligands were grouped into three families: 2'-NH2 RNA group A ligands are represented by the 35-nucleotide truncate IGEL1.2 (Kd = 30 nM); 2'-NH2 RNA group B ligands by the 25-nucleotide truncate IGEL2.2 (Kd = 35 nM); and the ssDNA group ligands by the 37-nucleotide truncate DI 7.4 (Kd = 10nM). Secondary structure analysis suggests G quartets for the 2'-NH2 RNA ligands, whereas the ssDNA ligands appear to form stem-loop structures. Using rat basophilic leukemia cells transfected with the human high-affinity IgE receptor Fc epsilon RI, we demonstrate that ligands IGEL1.2 and D17.4 competitively inhibit the interaction of human IgE with Fc1 epsilon RI. Furthermore, this inhibition is sufficient to dose-dependently block IgE-mediated serotonin release from cells triggered with IgE-specific Ag or anti-IgE Abs. Therefore, these oligonucleotide ligands represent a novel class of IgE inhibitors that may prove useful in the fight against allergic diseases.

ATP-dependent activation of phospholipase C by antigen, NECA, Na3VO4, and GTP-gamma-S in permeabilized RBL cell ghosts: differential augmentation by ATP, phosphoenolpyruvate and phosphocreatine.

Ghosts prepared from rat basophilic leukemia cells (RBL cell ghosts) and permeabilized with alpha-toxin from S. aureus are a simplified system for the study of Fc epsilon RI-mediated activation of phospholipase C (PLC). This activity is dependent upon ATP and magnesium, and is enhanced by the addition of another compound containing an energetic phosphate group, either phosphoenolpyruvate (PEP) or phosphocreatine (PCr). This effect appears to be specific for PEP and PCr in that other compounds with energetic phosphate bonds including fructose 1,6-bisphosphate and additional ATP are not effective. On the contrary, GTP-gamma-S, an activator of G proteins, activates PLC in the presence of ATP alone and this is not further enhanced by the addition of PEP. In addition to Fc epsilon RI and GTP-gamma-S, two other stimuli lead to enhanced activity of PLC in permeabilized RBL cell ghosts: 1) an inhibitor of tyrosine phosphatases (Na3VO4) and 2) an analog of adenosine (NECA). Data presented here extend previous results to show that activation of PLC by GTP-gamma-S is not enhanced either by the addition of PCr or by the addition of a more MgATP. Further new findings include the observations that activation of PLC by Na3VO4 is augmented by PEP and PCr in a fashion similar to that observed for Fc epsilon RI-mediated activation of PLC and that activation of PLC by NECA shows even more marked dependency on PEP than does activation by Fc epsilon RI or Na3VO4.(ABSTRACT TRUNCATED AT 250 WORDS)