Allergen induced histamine release and immunoreactive-leukotriene C4 generation from leukocytes in mite sensitive asthmatic patients

Leukocytes from mite sensitive asthmatic patient were challenged with the allergen and the supernatant was assayed for histamine and immunoreactive-leukotriene C₄ (i-LTC₄). The release of histamine was quantitated by an automated fluorometric technique and i-LTC₄ was determined using a commercial radioimmunoassay kit. The results of analysis of the supernatant by high speed liquid chromatography, together with observations of modulation of the formation by agents, indicated that i-LTC₄ consisted of LTC₄ with a little amount of LTD₄. i-LTC₄ was generated as a result of basophil activation but not derived from the other cells such as monocytes and eosinophils. Allergen induced a concentration-dependent release of histamine and i-LTC₄ and the maximal release of histamine and i-LTC₄ occurred at the same dose of the allergen. At optimal concentration of the allergen, basophils produced 20.4 ± 17.9 ng of i-LTC₄/10⁶-cells (mean ± S.D., n=39) and histamine release was 55.6 ± 20.1% of total histamine. There was a significant correlation in the capacity of leukocytes to release histamine and i-LTC₄ (r=0.47, p<0.01). We found a correlation between maximal histamine release or cell sensitivity, allergen concentration for 50% histamine release, and a ratio of specific IgE to mite to total IgE in the serum, but the amount of i-LTC₄ failed to correlate significantly with the ratio. The releasability and the cell sensitivity of asthmatic patients' cells to the allergen for histamine release paralleled the severity to symptoms, but this correlation was not significant in i-LTC₄ generation.     

Measurement of immunoreactive leukotriene C4 in blood of asthmatic children

Peptide leukotriene (LT) such as LTC4, LTD4, LTE4 have been considered to be major mediators of immediate type hypersensitivity reaction such as asthma. We have developed a rapid and simple extraction method using a Sep-Pak C18 cartridge for the measurement of LTC4 by radioimmunoassay (i-LTC4). In this extraction method, 91% LTC4 was recovered in a final methanol fraction. The identity was confirmed by the recovery test and by the dilution method. The amount of i-LTC4 in plasma from asthmatic patients was determined by radioimmunoassay after the extraction. The order of the plasma level of i-LTC4 was; severe asthma greater than slight or moderate asthma greater than asthmatic patient without attack greater than healthy adult. The highest level of LTC4 was 0.27 +/- 0.11 pmol/ml in severe asthmatic plasma.     

Human basophil releasability. III. Genetic control of human basophil releasability

Basophil releasability implies that, in addition to the surface density of IgE molecules, biochemical events determine the capacity to release chemical mediators in response to activating stimuli. We studied the IgE (anti-IgE)-mediated and non-IgE-mediated (f-met peptide and the Ca2+ ionophore A23187) releasability of human basophils obtained from 14 monozygotic (MZ) (ages 25.7 +/- 13.3 yr; mean +/- SDM) and 13 dizygotic (DZ) twin pairs (ages 20.4 +/- 9.9 yr). A significant intrapair correlation coefficient of the maximal percent of anti-IgE-induced histamine release was found in the MZ, whereas no significant correlation was found in the DZ. The mean intrapair variance of anti-IgE-induced histamine release in MZ (VMZ) and in DZ (VDZ) gave an F value equal to 3.84 (p less than 0.01) and a heritability (H) index of 0.74. Similar findings were obtained with respect to the sensitivity to a standard concentration (10(-1) micrograms/ml) of anti-IgE. No correlation between serum IgE level and anti-IgE-induced histamine release was found in either MZ or DZ. A significant intrapair correlation coefficient of f-met peptide-induced histamine release was found in both the MZ and the DZ. The difference between MZ and DZ was not significant. The VMZ and the VDZ of the f-met peptide-induced histamine release gave an F value of 1.52 (NS) and an H value of 0.34. The intrapair correlation coefficient of A23187-induced release was significant in MZ and not significant in DZ. The mean intrapair variance of A23187-induced histamine release gave an F value of 2.33 (NS) and an H index of 0.57. Similar findings were obtained by using suboptimal (3 X 10(-1) micrograms/ml) concentrations of A23187. There was no correlation between the sensitivity of basophils to release in response to anti-IgE and their response to f-met peptide or A23187, in either the MZ or the DZ. We conclude that the ability of basophils to respond to anti-IgE and A23187 is influenced by genetic factors.     

Effects of hydrocortisone and aminophylline on plasma leukotriene C4 levels in patients during an asthmatic attack

To study the role of leukotriene C4(LTC4) and the effect of hydrocortisone and aminophylline on plasma LTC4 levels in patients with asthmatic attacks, we measured LTC4 in plasma of 18 asthmatics during a wheezing attack and of 7 normal subjects. Blood samples were obtained before and after treatment with aminophylline and/or hydrocortisone injections. We extracted LTC4 using a Sep-Pak C18 cartridge for the measurement of LTC4 by radioimmunoassay. The plasma levels of immunoreactive LTC4 (i-LTC4) of the normal subjects were 142 +/- 25 pg/ml (n = 7), while those of nonatopic type asthmatic patients with wheezing attacks were 208 +/- 68 pg/ml (n = 15) (p less than 0.01). Before and after treatment with both hydrocortisone succinate (100 mg) and aminophylline (250 mg), 6 asthmatic patients with wheezing attacks had a mean plasma level of i-LTC4 181 +/- 24 and 132 +/- 18 pg/ml (p less than 0.01), respectively. On the other hand, the treatment with aminophylline 250 mg alone increased the i-LTC4 levels from 178 +/- 19 pg/mg to 213 +/- 16 pg/mg (n = 6)(p less than 0.05), while treatment with hydrocortisone succinate 100 mg decreased the i-LTC4 level 0.05 from 284 +/- 99 pg/ml to 249 +/- 85 pg/ml (n = 4)(p less than 0.05). In conclusion, the present study shows that the i-LTC4 level in venous blood of patients with asthmatic attacks is decreased significantly by treatment with hydrocortisone succinate.     

Domestic exposure to fungi and total serum IgE levels in asthmatic children

We measured the number of airborne, viable fungi and house dust mite (HDM) allergen levels in the homes of a group of asthmatic children. Blood samples were drawn and the amounts of total and specific serum IgE were determined. The association between the number of fungal colonies, dust mite allergen exposure, and specific and total IgE was evaluated. The number of viable airborne fungi was high (20,543 CFU/m(3)) in those investigated houses. Der p1 concentrations on child's mattress exceeding 2 microg/g were found in 78.6% of the houses. A quantitative dose-response relationship was demonstrated between the exposure to viable, airborne molds and the amount of total IgE (r = 0.4399 and P = .0249) and the level was further increased in children with coexposure to viable fungi and HDM.     

Effect of indomethacin on allergen-induced asthmatic responses

Previous studies have suggested that inhibition of the cyclooxygenase pathway of arachidonic acid metabolism may suppress the late asthmatic responses to inhaled allergen. Both human and animal studies have suggested that prostanoids may also be involved in increases in airway responsiveness after ozone and allergen. We studied seven atopic subjects, who had a dual asthmatic response to inhaled allergen, during a control period and then after pretreatment with indomethacin (50 mg) or placebo twice daily for 2 days, administered in a randomized, double-blind manner. Indomethacin had no significant effect on the base-line airway responsiveness to histamine (P = 0.22) or the allergen-induced early or late asthmatic response (P = 0.49). However, indomethacin inhibited the increase in airway responsiveness (express as histamine PC20) after allergen inhalation. The log difference in preallergen to postallergen histamine PC20 was 0.49 +/- 0.08 (SE) during the control period, 0.46 +/- 0.08 (SE) after placebo (P = 0.81), and 0.22 +/- 0.10 (SE) after indomethacin (P = 0.02). Although indomethacin is useful for examining the role of cyclooxygenase products in asthmatic responses, it should not be considered in the treatment of asthma. We conclude that cyclooxygenase products are not significant mediators of allergen-induced early or late asthmatic responses but are involved in the pathogenesis of airway hyperresponsiveness after allergen inhalation.     

Cloning and expression of Der f 6, a serine protease allergen from the house dust mite, Dermatophagoides farinae.

House dust mite allergen is thought to be a major cause of asthma. Characterization of these allergen molecules is therefore an important step for the development of effective diagnostic and therapeutic agents against mite-associated allergic disorders. Here we report molecular cloning and expression of the group 6 (chymotrypsin-like) allergen from the house dust mite, Dermatophagoides farinae. Sequencing analysis indicates that cloned cDNA, designated Der f 6, encodes a 279 amino acid polypeptide which conserves a primary structure characteristic for chymotrypsin-like serine proteases found in mammals. Recombinant Der f 6 expressed in Escherichia coli bound IgE in a pool made of 20 sera, and induced histamine release from patients' peripheral blood cells.     

Antigen-specific T cell tolerance down-regulates mast cell responses in vivo

Fel d I is the major cat allergen that induces asthma and allergic rhinitis in humans. To investigate the mechanism of allergic responses to this allergen, a mouse model was developed. Mice sensitized to chain 1 of Fel d I exhibited T cell responses, B cell responses, and mast cell responses when challenged with the protein. Subcutaneous injections of peptides containing the dominant T cell epitopes of the allergen induced T cell tolerance in presensitized mice. When challenged with the allergen intratracheally, these tolerized mice produced a decreased amount of histamine in vivo. The decrease in histamine release was not solely dependent on the reduction of allergen-specific IgE. These data show that mast cell activity in mice with an ongoing sensitivity to allergen can be regulated through peptide-induced T cell tolerance.     

Purification and immunological characterization of a 12-kDa allergen from Epicoccum purpurascens

Exposure to Epicoccum purpurascens is implicated in respiratory allergies and asthma. Several allergens of clinical importance were identified in Epicoccum extract (EE), but only one allergen has been isolated and characterized. In the present study, a 12-kDa allergen was isolated from an Epicoccum spore-mycelial extract by concanavalin-A sepharose, reverse-phase hydrophobic and gel filtration chromatography. The purified protein was recognized as a single 12-kDa allergen on immunoblot with a serum pool of Epicoccum- sensitive patients. Of the 94 respiratory allergy patients tested intradermally, 17 showed marked positive skin reactions to EE and 12 of them reacted with the 12-kDa protein, indicating a diagnostic sensitivity of 70%. More than 80% patients' sera showed immunoglobulin E (IgE) reactivity to the purified protein in enzyme-linked immunosorbent assay and immunoblot, identifying it as a major allergen. Preincubation of pooled serum with the protein led to inhibition of IgE binding to solid-phase-bound EE (effective concentration 50%=180 ng). Twelve of the 17 serum samples showed significant basophil histamine release upon stimulation with purified protein. The protein induced significant proliferation of peripheral blood mononuclear cells of 13 patients. A high level of interleukin-4 in the culture supernatant of these cells indicated induction of a T-helper type 2 response. The purified 12-kDa protein is a clinically relevant allergen and has potential for the diagnosis and therapy of Epicoccum allergies.     

Unlocking the allergenic structure of the major house dust mite allergen der f 2 by elimination of key intramolecular interactions

We report on the structural background of the remarkable reduction of allergenicity in engineering of the major house dust mite allergen Der f 2. Disruption of intramolecular disulfide bonds in Der f 2 caused extensive conformational change that was monitored by circular dichroism and gel-filtration analysis. The degree of conformational change correlated well with the degree of reductions in the capacity to bind IgE and to induce histamine release from basophils in mite-allergic patients. Loosening the rigid tertiary structure by elimination of key intramolecular interactions is an effective strategy to reduce the number of high affinity IgE epitopes of allergen vaccine.     

The 1st step initiation essential for allergen‐specific IgE antibody production upon the 2nd step: Induction of non‐specific IgE+ small B cells containing secondly‐sensitized allergen‐specific ones in mice firstly‐sensitized with an allergen

There was a significant amount of non‐specific, but not of allergen (e.g., papain, mite feces and four kinds of pollen)‐specific, IgE antibodies (Abs) in the sera of normal mice. An i.n. injection of each allergen without adjuvant into mice caused an increase in total IgE Ab titers with a similar time course in the serum. However, the stage of initiation of allergy varied from allergen to allergen. Submandibular lymph node cells from normal mice contained papain‐, but not mite feces‐ or pollen‐specific IgE⁺ cells and an i.n. injection of papain induced papain‐specific IgE Abs in the serum. In contrast, one (i.n.) or two (i.n. and s.c) injections of mite feces induced neither mite feces‐specific IgE⁺ cells in the lymph nodes nor mite feces‐specific IgE Abs in the serum. I.n. sensitization with cedar pollen induced cedar pollen‐specific IgE⁺ small B cells in the lymph nodes on Day 10, when non‐specific IgE Ab titers reached a peak in the serum, implying induction of related allergen‐specific IgE⁺ small cells as well. In fact, a second (s.c.) injection of ragweed (or cedar) pollen into mice sensitized i.n. once with cedar (or ragweed) pollen, but not with mite feces, induced a large amount of ragweed (or cedar) pollen‐specific IgE Abs in the serum. These results indicate that when firstly‐sensitized non‐specific IgE⁺ small B cells in mouse lymph nodes include some secondly‐sensitized allergen‐specific ones, mice produce IgE Abs specific for the secondly‐injected allergen.

     

Neutrophils and mast cells. Comparison of neutrophil-derived histamine-releasing activity with other histamine-releasing factors

Human neutrophil-derived histamine-releasing activity (HRA-N) was partially purified and found to contain a heat-stable 1400 to 2300-Da fraction which caused human basophils and rat basophil leukemia cells (RBL) to degranulate. The capacity of HRA-N to activate basophils was not related to the gender or atopic status of the basophil donor, but was related to anti-IgE responsiveness. Several lines of evidence suggest that HRA-N and anti-IgE induce histamine release through distinctly different mechanisms: 1) the time course of HRA-N- and anti-IgE-induced RBL histamine release are different; 2) HRA-N causes histamine release from RBL with and without surface-bound IgE; 3) lactic acid stripping of IgE from human basophils reduces anti-IgE-induced histamine release, but has no consistent effect on HRA-N-induced histamine release; and 4) passive sensitization of lactic acid-stripped basophils with IgE restores anti-IgE-induced histamine release but not HRA-N-induced histamine release. Several histamine-releasing factors (HRF) were compared with HRA-N. Human nasal HRF (HRF-NW, crude and partially purified fractions of 15 to 30, 3.5 to 9, and less than 3.5 kDa), like HRA-N, caused equal histamine release from both native and IgE-sensitized RBL. However, only the 15- to 30-kDa fraction caused histamine release from human basophils in the doses tested. Mononuclear cell HRF (HRF-M, crude and a partially purified 25 kDa Mr fraction) and platelet HRF (HRF-P, crude preparation) failed to cause histamine release from either native or IgE-sensitized RBL but caused 30 +/- 5.5% and 20 +/- 10% net histamine release from human basophils, respectively. HRA-N and HRF-NW were both stable to boiling. These data, taken together, suggest that the capacity of HRA-N to induce RBL and human basophil histamine release and of HRF-NW to stimulate RBL histamine release is independent of IgE. The data further suggest that HRA-N and HRF-NW can be distinguished by size, and that they both differ from mononuclear cell HRF and platelet HRF. Thus, it appears that inflammatory cells generate a family of distinct HRF.     

Molecular characterization and allergenic activity of Lyc e 2 (beta-fructofuranosidase), a glycosylated allergen of tomato.

Until now, only a small amount of information is available about tomato allergens. In the present study, a glycosylated allergen of tomato (Lycopersicon esculentum), Lyc e 2, was purified from tomato extract by a two-step FPLC method. The cDNA of two different isoforms of the protein, Lyc e 2.01 and Lyc e 2.02, was cloned into the bacterial expression vector pET100D. The recombinant proteins were purified by electroelution and refolded. The IgE reactivity of both the recombinant and the natural proteins was investigated with sera of patients with adverse reactions to tomato. IgE-binding to natural Lyc e 2 was completely inhibited by the pineapple stem bromelain glycopeptide MUXF (Man alpha 1-6(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3)GlcNAc). Accordingly, the nonglycosylated recombinant protein isoforms did not bind IgE of tomato allergic patients. Hence, we concluded that the IgE reactivity of the natural protein mainly depends on the glycan structure. The amino acid sequences of both isoforms of the allergen contain four possible N-glycosylation sites. By application of MALDI-TOF mass spectrometry the predominant glycan structure of the natural allergen was identified as MMXF (Man alpha 1-6(Man alpha 1-3)(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3) GlcNAc). Natural Lyc e 2, but not the recombinant protein was able to trigger histamine release from passively sensitized basophils of patients with IgE to carbohydrate determinants, demonstrating that glycan structures can be important for the biological activity of allergens.     

Influence of sensitization and allergen provocation procedures on the development of allergen-induced bronchial hyperreactivity in conscious, unrestrained guinea-pigs

The effects of different sensitization and allergen provocation regimens on the development of allergen-induced bronchial hyperreactivity (BHR) to histamine were investigated in conscious, unrestrained guinea-pigs. Similar early and late phase asthmatic reactions, BHR for inhaled histamine after the early (6 h) as well as after the late reaction (24 h), and airway inflammation were observed after a single allergen provocation in animals sensitized to produce mainly IgG or IgE antibodies, respectively. Repeating the allergen provocation in the IgE-sensitized animals after 7 days, using identical provocation conditions, resulted in a similar development of BHR to histamine inhalation. Repetition of the allergen provocation during 4 subsequent days resulted in a decreased development of BHR after each provocation, despite a significant increase in the allergen provocation dose necessary to obtain similar airway obstruction. The number of inflammatory cells in the bronchoalveolar lavage was not significantly changed after repeated provocation, when compared with a single allergen provocation. Finally, we investigated allergen-induced bronchial hyperreactivity by repetition of the sensitization procedure at day 7 and 14 (booster), followed by repeated allergen provocation twice a week for 5 weeks. Surprisingly, no BHR to histamine could be observed after either provocation, while the number of inflammatory cells in the bronchoalveolar lavage fluid after 5 weeks was enhanced compared with controls. These data indicate that both IgE and IgG sensitized guinea-pigs may develop bronchial hyperreactivity after a single allergen provocation. Repeated allergen exposure of IgE sensitized animals causes a gradual fading of the induced hyperreactivity despite the on-going presence of inflammatory cells in the airways, indicating a mechanism of reduced cellular activation.     

Nuclear magnetic resonance structure-based epitope mapping and modulation of dust mite group 13 allergen as a hypoallergen

IgE-mediated allergic response involves cross-linking of IgE bound on mast cells by specific surface epitopes of allergens. Structural studies on IgE epitopes of allergens are essential in understanding the characteristics of an allergen and for development of specific allergen immunotherapy. We have determined the structure of a group 13 dust mite allergen from Dermatophagoides farinae, Der f 13, using nuclear magnetic resonance. Sequence comparison of Der f 13 with homologous human fatty acid-binding proteins revealed unique surface charged residues on Der f 13 that may be involved in IgE binding and allergenicity. Site-directed mutagenesis and IgE binding assays have confirmed four surface charged residues on opposite sides of the protein that are involved in IgE binding. A triple mutant of Der f 13 (E41A_K63A_K91A) has been generated and found to have significantly reduced IgE binding and histamine release in skin prick tests on patients allergenic to group 13 dust mite allergens. The triple mutant is also able to induce PBMC proliferation in allergic patients with indices similar to those of wild-type Der f 13 and shift the secretion of cytokines from a Th2 to a Th1 pattern. Mouse IgG serum raised using the triple mutant is capable to block the binding of IgE from allergic patients to wild-type Der f 13, indicating potential for the triple mutant as a hypoallergen for specific immunotherapy. Findings in this study imply the importance of surface charged residues on IgE binding and allergenicity of an allergen, as was also demonstrated in other major allergens studied.     

Interleukin-4 (IL-4) enhances and soluble interleukin-4 receptor (sIL-4R) inhibits histamine release from peripheral blood basophils and mast cells in vitro and in vivo

The aim of the study was to analyse the effect of interleukin-4 (IL-4) on allergen and anti-IgE mediated histamine release from basophils and human skin mast cells and to assess whether soluble recombinant interleukin-4 receptor (sIL4R) can inhibit these effects. Anti-IgE stimulated histamine release from peripheral blood basophils and mast cells of atopic donors was enhanced after preincubation with IL-4, whereas after preincubation with sIL-4R it was inhibited. These effects were even more pronounced when samples were stimulated with a clinically relevant allergen. In IL-4 preincubated skin mast cells, there was a similar enhancement of anti-IgE stimulated histamine release, which could again be inhibited by sIL-4R. The effects of IL-4 and sIL4R were dose- and time-dependent. Mice sensitized to ovalbumin and treated with soluble recombinant murine sIL-4R showed significantly reduced immediate-type cutaneous hypersensitivity responses compared with untreated mice. These in vivo effects were IgE independent, since there were no significant differences in total and allergen specific IgE/IgG1 antibody titres between treated and untreated mice. This indicates that IL4 exerts priming effects on histamine release by effector cells of the allergic response and that these effects are potently antagonized by soluble IL-4R both in vitro and in vivo.     

Characteristics of human basophil sulfidopeptide leukotriene release: releasability defined as the ability of the basophil to respond to dimeric cross-links

Human basophils release approximately 90 pmol of LTC4/micrograms histamine when challenged with anti-IgE antibody, but donor to donor variation produces a 1000-fold range of response. There is little conversion to LTC4 to LTE4 in purified preparations of basophils, but conversion to LTE4 does occur if cell densities are high during incubation. Like histamine release, leukotriene release is calcium and temperature dependent and is complete in 20 min, with a t1/2 of approximately 8 min. The process of desensitization also ablates leukotriene release, but there is a distinct two phase process where leukotriene release is enhanced after 5 min of desensitization, whereas histamine release is inhibited and total ablation of leukotriene release occurs only after 45 min of desensitization. Human basophils respond well to stimulation with covalently cross-linked trimeric IgE myeloma but respond poorly to dimeric IgE. This differential sensitivity to the two forms of cross-linked IgE is most exaggerated in the context of leukotriene release, where dimer is 30-fold less efficacious and 100- to 1000-fold less potent than trimer on some donors' basophils. This dichotomy of response is also observed in antigen-challenged cells, where the bivalent hapten, BPO2, also poorly induces leukotriene release in accord with the fact that it predominantly induces dimeric cross-links of penicillin-specific IgE. Anti-IgE dose-response curves reveal a region of dimeric cross-link dominance that may explain the peculiar differences observed in pharmacologic studies of basophil release induced with antigen vs anti-IgE. In addition, there is a continuum of "releasability," where some donors' basophils display no response (histamine or leukotriene release) to dimeric IgE, and others' basophils are essentially equally responsive to both dimeric and trimeric IgE. This releasability difference manifests itself by conferring increased sensitivity to antigenic challenge in those donors' basophils capable of responding to dimeric cross-links such that these donors' basophils are capable of releasing histamine upon antigen challenge while possessing only 50 molecules of cell surface antigen-specific IgE; other dimer-insensitive donors' basophils require 6 to 10-fold greater IgE densities for equal histamine release.     

Bronchoalveolar mast cells in extrinsic asthma: a mechanism for the initiation of antigen specific bronchoconstriction.

Bronchoalveolar lavage performed in 10 patients with extrinsic asthma and 14 controls yielded similar recoveries of fluid and cells. Mast cells and eosinophils, however, formed a greater proportion of the cells recovered from the asthmatic subjects (p less than 0.001 for mast cells; p less than 0.01 for eosinophils), the histamine content of the lavage cells being correspondingly increased (p less than 0.01). Both the percentage of mast cells and the histamine content of lavage cells were significantly inversely correlated with the forced expiratory volume in one second (FEV1; expressed as percentage of predicted) and with the ratio of FEV1 to forced vital capacity before lavage. There was also a significant inverse correlation between the concentration of histamine required to produce a 20% fall in FEV1 and the percentage of mast cells recovered (p less than 0.05). When incubated with antihuman IgE bronchoalveolar mast cells from asthmatic subjects released a significantly increased proportion of total cellular histamine than cells from control subjects at all effective doses of anti-IgE. By contrast, dose response curves for IgE dependent histamine release from peripheral blood leucocytes were similar in asthmatics and controls. Specific antigen led to release of histamine from bronchoalveolar cells and peripheral blood leucocytes of asthmatic subjects but not controls. Lying superficially within the airways, bronchoalveolar mast cells would be readily exposed to inhaled antigen and would release mediators directly on to the airway surface. Their immunological response suggests that they are likely to be important in the pathogenesis of airflow obstruction in asthma.     

Phospholipase A2 stimulation during cell secretion in rat basophilic leukemia cells

The bridging of IgE receptors on rat basophilic leukemia cells (RBL-2H3) results in a number of biochemical events that accompany histamine secretion. Prominent among these is the release of arachidonic acid from cellular phospholipids, which could be due to the activation of phospholipase enzymes. In the present experiments we studied the intracellular activation of phospholipase A2 (PLA2) during histamine release. RBL-2H3 cells were stimulated through the IgE receptor, and the homogenates were prepared and tested for phospholipase A2 activity on 1-stearoyl-2-[14C]arachidonyl-sn-3-phosphatidylcholine. The amount of activity in the homogenates was dependent on the concentration of secretagogue used to activate the cells. Under optimal conditions there was a 1.86 +/- 0.12-fold (mean +/- SEM, N = 44) increase in the activity found in homogenates of stimulated cells. Activity was present in homogenates prepared 30 sec after cell activation, was optimal between 5 and 10 min, and decreased later. In time course experiments the PLA2 activation preceded histamine release. The activation of the enzyme in the cell occurred in the presence of 10 microM EGTA in the extracellular medium, which completely inhibited release of arachidonic acid and histamine. However, the activity of the enzyme required Ca2+. The PLA2 activity in the homogenates and the extent of cell stimulation for histamine release were maximal at the same concentration of antigen, and both were blocked by the addition of a monovalent hapten. The enzyme in the homogenates was capable of cleaving arachidonic acid from different phospholipids. The production of lysophospholipids could play a critical role in histamine release from cells. These results demonstrate the activation of PLA2 enzyme in cellular homogenates during the secretory process.     

Separation of bronchoconstriction from increased ventilatory drive in a nonhuman primate model of chronic allergic asthma.

The relationship between allergen-induced ventilatory drive and bronchoconstriction was investigated in dust mite-sensitive cynomolgus macaques periodically exposed to low doses of aerosolized antigen for up to 5.5 yr. Initially, the animals responded to aerosolized dust mite allergen at a concentration of 350 arbitrary units (AU)/ml with simultaneous increases in lung resistance (RL) and respiratory rate (RR). With time, RL and RR became differentially sensitive to allergen provocation. At the end of the study period, aerosolized allergen at a concentration of 15 AU/ml doubled RR without increasing RL. When mechanically ventilated to maintain tidal volume, higher concentrations of allergen could be delivered, and RL increased. Inhaled disodium cromoglycate and intravenous diphenhydramine attenuated the increase in RR, indicating that allergen-induced release of histamine and activation of H(1) receptors mediated the response. Inhaled beta-adrenergic agonists attenuated the RR response to dust mite and to direct histamine provocation. These results demonstrate that chronic periodic allergen challenge increases the allergic sensitivity of histamine-dependent reflexes controlling ventilatory drive. Activation of these reflexes is independent of overt bronchoconstriction, but can be inhibited by beta-adrenergic agonists, indicating that beta-adrenergic agonists exert their effect independent of bronchodilation.