Passive sensitization of skin and lung by guinea-pig immunoglobulins

Guinea-pig γ₁- and γ₂-globulins have been purified by preparative electrophoresis followed by chromatography. No γ₁-globulin was detectable in purified γ₂-globulin, but purified γ₁-globulin always contained fast γ₂-globulin. Normal guinea-pig serum contained much less γ₁-globulin than immune serum. Antisera prepared against normal guinea-pig serum did not contain useful amounts of antibody specific for γ₁-globulin.

Guinea-pig lung tissue was sensitized by very low concentrations of guinea-pig γ₁-globulin (of the order of 6×10^-10 molar) but γ₂-globulin antibodies were almost inactive. No evidence was found that the trace of activity in γ₂-globulin was not due to very slight contamination with γ₁-globulin antibodies.

The finding that γ₁-globulin antibodies are far more potent than γ₂-globulin antibodies in sensitizing skin has been confirmed, but several lines of evidence suggest that γ₂-globulin antibodies may also have weak activity. Thus quantitative passive cutaneous anaphylaxis (PCA) tests showed that whenever the γ₂-globulin fraction contained antibody it appeared far more potent relative to γ₁-globulin than when the same proteins were tested on lung tissue. The PCA activity of moderate amounts of purified γ₂-globulin antibodies disappeared faster than the skin sensitization produced by small amounts of γ₁-globulin antibodies, and the γ₂-globulin preparations did not contain enough γ₁-globulin impurity to account for their PCA activity. No inhibition of skin responses was observed with the largest doses of antigen tested.

The most plausible explanation of these results is that, under the conditions of our experiments, γ₂-globulin antibody had weak PCA activity. Objections to this hypothesis are discussed. The PCA activity of γ₂-globulin antibody probably involves a mechanism different from that of the sensitization produced by the highly potent γ₁-globulin antibody.

     

Passive sensitization and histamine release of basophils

This study had two purposes. First, to examine a possible functional heterogeneity of IgE regulating basophil histamine release and the effect of using two different donor cells for passive sensitization experiments. Second, to investigate basophils not releasing histamine to anti-IgE by stimulating protein kinase C with the addition of the phorbol-ester, TPA. In consecutive experiments responding donor basophils were passively sensitized with plasma from non-responding subjects. Thus, the first set of experiments included passive sensitization of acid treated donor basophils from one atopic and one non-atopic patient with plasma from 29 children with exogenous asthma to grass pollen, cat dander, or dust mites. Different secretagogues (anti-IgE, Concanavalin A, and N-formyl-methionyl-leucyl-phenylalanine) induced different histamine release responses due to a cellular property of the basophils not related to the type of IgE bound to the cell membrane. It was demonstrated that the allergen-induced histamine release did not depend on the extract or type of IgE when the biological activity of each extract and serum-specific IgE levels were similar. However, the atopic donor cells released significantly (P less than 0.05) more histamine than non-atopic donor cells. Thus, histamine release depends on the type of secretagogues and a cellular property which is maybe influenced by the presence of serum factors and a certain type of IgE in the serum of atopics. The second set of experiments included 10 patients (6 atopics and 4 non-atopics) with non-histamine releasing basophils. In the presence of 10 ng/ml TPA, however, seven of 10 patients released histamine at anti-IgE challenge. Three months later two additional patients became responsive in the presence of TPA. By passive sensitization of responding donor basophils the non-responding patients were shown to possess functionally intact IgE. Thus, the discrepancies sometimes observed between clinical symptoms, serological IgE-antibody measurements and histamine release testing in allergic patients may be related to a cellular property of basophils.     

Comparison of mouse strain skin sensitivities to anaphylactic mediators and susceptibility to passive cutaneous anaphylactic reactions.

CFW, ICR, and C57Bl/6J mouse strains were examined and compared for their levels of skin sensitivity to histamine and mellitin (a potent mast cell degranulator) and for their susceptibilities to immunoglobulin E-induced passive cutaneous anaphylactic (PCA) reactions. ICR mice were found to exhibit the highest level of sensitivity to histamine and mellitin, whereas C57Bl/6J exhibited the least. CFW mice proved to be the best PCA recipients, whereas ICR mice were the poorest. On the basis of this evidence, no direct correlation is indicated between level of sensitivity to anaphylactic mediators and degree of susceptibility to immunoglobulin E-induced PCA reactions.     

Inflammatory mediators in late antigen-induced rhinitis

To investigate the mechanisms responsible for the late-phase response in patients with allergies, we measured four biochemical mediators (histamine, tosyl-L-arginine methyl ester [TAME]-esterase, kinin, and prostaglandin D2) in nasal secretions after nasal challenge with pollen antigen in 12 patients with allergy. Nine patients had an immediate response and a recurrence of symptoms 3 to 11 hours after challenge. The clinical symptoms during recurrence were accompanied by a second increase in levels of histamine, TAME--esterase, and kinin over base-line values, although kinin levels were lower than during the immediate response. In contrast, although the levels of prostaglandin D2 were significantly increased during the immediate response, they did not increase above base line during the late response. Rechallenge with allergen 11 hours after the initial provocation, however, was associated with reappearance of all four biochemical mediators, including prostaglandin D2. We conclude that the late response to nasal challenge with allergen is accompanied by a second increase in the concentrations of histamine and TAME--esterase but differs from the immediate response in the lack of prostaglandin D2 production and in the amount of kinin generated. Since histamine is released only by mast cells and basophils and prostaglandin D2 is not produced by basophils, we suggest that these cells are partly responsible for the late-phase response.     

A role for anaphylactic sensitization in tumor control

In order to evaluate the possible preventive role of an anaphylactic condition on tumor development, C57BL/6J mice were immunized or hyperimmunized by the intraperitoneal route with egg albumin in alum before the chemical induction of fibrosarcoma. Preimmunization using a protocol which elicits an optimal IgE response produced: (1) a significant reduction of tumor incidence; (2) an increase of survival time, and (3) a decrease of tumor growth rate in animals with higher IgE titers (greater than 640). On the other hand, hyperimmunized mice, which were suppressed in their anti-egg albumin IgE response, showed no changes in tumor incidence and survival time when compared to controls. Our results suggest a suppressive effect on tumor development related to the ability of the animal to mount an IgE response to antigens unrelated to the tumor.     

Capsaicin and anaphylactic reactions in the guinea-pig

The influence of capsaicin on anaphylactic reactions in the guinea-pig was studied bothin vivo andin vitro. In guinea-pigs actively sensitized with ovalbumin, Herxheimer microshock was elicited by antigen aerosol and the preconvulsion time recorded. The preconvulsion time was reduced by about 30% in animals pretreated with capsaicin (1 mg/kg) injected i.p. 30 min before antigen aerosol, whereas it remained unchanged when the drug was administered two days before aerosol treatment. Capsaicin shows a partial protective effect when the provocative aerosol was administered 3 h after the last of three doses of capsaicin (100 g/kg, i.p.), which had been injected for three consecutive days.Ileum longitudinal muscle strips were used forin vitro anaphylaxis studies. These were isolated from guinea-pigs actively sensitized with ovalbumin and histamine release evoked by antigen was measured. Preparations perfused with capsaicin (10^–6–10^–4 M) and desensitized to the drug, showed a lower anaphylactic release of histamine. This effect was dose-dependent, with the histamine release reduced by 35% at higher concentrations (10^–5–10^–4 M) of capsaicin. The mechanism of the influence of capsaicin on anaphylactic reactions is discussed briefly.     

Effect of various GABA-receptor agonists and antagonists on anaphylactic histamine release in the guinea-pig ileum

In this paper we confirm the previously reported inhibition by GABA of anaphylactic histamine release from isolated guinea-pig ileum longitudinal muscle. Moreover we report that: A) GABA-inhibition of anaphylactic histamine release is mimicked both by GABA-A and GABA-B agonists; both GABA-A and GABA-B antagonists are effective in reversing GABA's inhibitory effect; B) the effect is exerted specifically by GABA-ergic drugs: taurine and -alanine are ineffective; C) the GABA-ergic effect seems not to involve cholinergic and adrenergic transmission. It is concluded that it might be interesting to assess the clinical value of GABA-ergic drugs in allergic gut disorders.     

Attenuation of antigen-induced bronchospasm by fenoterol in the guinea-pig

When guinea-pigs sensitized to ovalbumin were challenged with ovalbumin, the histamine concentrations in lung tissue decreased, whereas those in tracheal and heart tissues increased during the 15 min following challenge. Fenoterol (2.5 mg/kg, i.p.), administered 30 min prior to challenge, attenuated both the decrease and the increase in lung and tracheal histamine concentrations, respectively. The challenge-induced increase in heart histamine was also prevented. In the anaesthetized guinea-pig, a marked increase in intratracheal pressure (ITP) occurred on challenge. This was of longer latency than the increase in ITP induced by histamine and was consistent with a release process. Treatment of challenged guinea-pigs with fenoterol (2.5 mg/kg, i.v.) markedly reduced the increased ITP. However, when fenoterol was administered prior to challenge, the increase in ITP was abolished. These results indicate that there is an apparent relationship between the inhibition of histamine releasein vivo by fenoterol pretreatment and the greater inhibitory effect of fenoterol on antigen-induced increases in ITP when administered prophylactically.     

Reduction of antigen-induced contraction of sensitized guinea-pig tracheal smooth muscle in vitro by calmodulin inhibitors

The effect of two calmodulin inhibitors, 1-[bis(p-chlorophenyl)methyl]-3-[2,4-dichloro-beta-(2,4-dichlorobenzylox y) phenethyl]imidazolinium chloride (R 24571) and chlorpromazine (CPZ) on antigen-induced contraction of guinea-pig tracheal smooth muscle was studied. Ketotifen, an anti-allergic compound, was used as a comparative drug. Contraction of sensitized guinea-pig trachea induced by antigen challenge was reduced by all three agents. The two calmodulin inhibitors effected relaxation of contraction of guinea-pig trachea induced by histamine and leukotriene D4 (LTD4). Ketotifen relaxed histamine-induced contraction, but hardly affected LTD4-induced tone. Contrary to chemical mediator induced tone, all examined agents had no effect on resting tone. These three agents shifted histamine- and LTD4-induced concentration-contraction curves to the right. They produced a downward displacement of the maximum, without a parallel shift in histamine- and LTD4-induced concentration-contraction curves. The two calmodulin inhibitors did not affect the antigen-induced release of histamine and SRS-A from sensitized guinea-pig lung tissue. Ketotifen slightly inhibited the release of histamine. These results suggest that R 24571 and CPZ, calmodulin inhibitors, reduced an antigen-induced contraction of sensitized guinea-pig trachea in vitro mainly by affecting the contractility of tracheal smooth muscle by chemical mediators but not by interfering with the release of mediators.     

The effects of atropine on anaphylactic shock in the guinea-pig

The effects of atropine, 2 mg/kg i.v., on anaphylactic shock were studied in guinea-pigs sensitized to ovalbumin.Atropine only moderately reduced (–31%) the increase in pulmonary resistance observed and slightly prolonged (+26%) the survival time in pretreated animals compared with controls. These effects, however, were not statistically significant. The drug temporarily improved ventilation but had no influence on haematosis.On the other hand, atropine significantly reduced the amount of histamine released (–60%) and of GMPc synthetized in the lung (–21%). The levels of AMPc and prostaglandins E₁, E₂ and F₂ remained comparable to those of control animals.These results suggest that the reflex-induced action of the cholinergic system during anaphylaxis primarily affects large-calibre airways and that the role of acetylcholine in severe reactions is moderate when compared with the direct action of other mediators.     

Passive sensitization of human airways induces mast cell degranulation and release of tryptase

BACKGROUND: This study was designed to examine the effect of passive sensitization (PS) on human bronchial mast cells. PS with asthmatic serum induces a hyper-responsiveness to nonspecific agonists, and immunoglobulin (Ig)E binding mainly on mast cells. METHODS: Bronchi dissected out from 19 lung specimens were incubated in normal or asthmatic serum. Immunohistochemistry was performed using monoclonal antibodies (MoAbs) directed against tryptase, chymase, or c-kit. Mast cells were classified as fully granulated (type I), partly (type II) or largely degranulated (type III). Tryptase was measured in supernatant using ELISA. Contractile response was recorded in a separated set of experiments using an organ bath system. RESULTS: PS decreased both tryptase positive cells (47.9 +/- 10.0 vs. 26.7 +/- 4.8 cell/mm2, P = 0.003) and chymase positive cells (26.1 +/- 3.3 vs. 14.9 +/- 1.8 cell/mm2, P = 0.01), but did not alter the number of c-kit positive cell. PS decreased the proportion of type I (55.4 vs. 28.9%, P < 0.0001) and, concomitantly increased that of types II (23.2 vs. 41.0%, P < 0.0001) and III (21.4 vs. 30.1%, P = 0.04). Following PS, tryptase concentration significantly increased and the magnitude of histamine response, was correlated with the amount of type II mast cells. CONCLUSION: PS of human isolated bronchi induces a mast cell degranulation related to in vitro hyper-responsiveness, along with a tryptase release.     

Recovery of anaphylactic sensitivity in the guinea-pig ileum after desensitization

The recovery of anaphylactic sensitivity of the ileum after desensitization has been investigated, and the dose—response and time—response relationships described. An attempt has been made to investigate the mechanism of recovery of sensitivity.     

Comparison of the release of various mediators from atrial and ventricular tissues of sensitized guinea-pig hearts

By comparison with ventricular tissues, collagenase-dispersed cell suspensions obtained from atrial tissues of sensitized guinea-pigs showed a higher histamine content, a higher proportion of mast cells, and a higher release with antigen or antisera to IgG, IgG₁ and IgG₂ of the following mediators: histamine, thromboxane B₂ and leukotriene C₄.     

The culture of mouse bone marrow-derived mast cells (BMMC) and the anaphylactic release of chemical mediators]

Bone marrow cells from BALB/c, C3H/He and WBB6F1+/+ mice were cultured for 5 wks in the presence of the culture supernatant from prokoweed mitogen-stimulated spleen cells to assess and compare the degree of growth, proliferation and chemical mediator release of the mast cells (BMMC) derived from them. BMMC, which were positive to alcian blue staining, were found in the suspension cells on the culture of the bone marrow cells of either species of mice after 2 wk culture. The percentages of BMMC in the suspension cells were increased with time of culture, reaching more than 90% after 5 wks. No differences in the growth and proliferation rate among BMMC from these three species were observed. However, in regard to the amount of anaphylactic leukotriene (LT) and histamine release. BMMC from BALB/c mice were superior to those from other species. From the above results, subsequent experiments were executed with BMMC from BALB/c mice. There was no obvious difference in the releasability of anaphylactic mediators among BMMC obtained at any stages of the passage during 4-12 wk culture. On the other hand, although BMMC cultured for 4 and 5 wks well responded to Ca ionophore A23187 for these mediator release, those for 6 to 12 wks obviously deteriorated with prolongation of the culture. The time course of the anaphylactic release of immunoreactive (i-) LTB4, i-LTC4 and histamine from BMMC revealed that almost maximum release was reached at 10, 20 and 5 min, respectively, after antigen challenge. Several drugs including antiallergics and beta-stimulants had no effect on their release. From these results, it is suggested that present BMMC may be inadequate cells for evaluation of antiallergic drugs that can inhibit the anaphylactic mediator release, but may be useful for the research of the mechanism of the release because the cells likely release the mediators without occurrence of complicated subordinate reactions.     

Modulation of the release of 9-hydroxy-octadecadienoic acid and other fatty acid derived mediators from guinea-pig pulmonary macrophages

Non-stimulated guinea-pig pulmonary macrophages (PM) convert arachidonic acid to thromboxane B2 and 12-hydroxy-heptadecatrienoic acid, whereas linoleic acid is metabolized to two hydroxy compounds, i.e. 9-hydroxy- and 13-hydroxy-octadecadienoic acid. Coincubation of PM with immune serum (2% v/v) resulted in a profound reduction of the release of these products. This effect seemed to be due to an inhibitory action on cyclooxygenase activity. Control serum also possessed inhibitory properties towards the release of fatty acid metabolites, possibly due to an effect on phospholipase activity. Because of the radical scavenging properties of 9-hydroxy-octadecadienoic acid, the modulation of the release of this product may be an important determinant in macrophage function.