Active Cutaneous Anaphylaxis Research Articles

Enhanced delayed-type hypersensitivity and diminished immediate-type hypersensitivity in mice lacking the inducible VPAC(2) receptor for vasoactive intestinal peptide.

Vasoactive intestinal peptide (VIP) and its G protein-coupled receptors, VPAC(1)R and VPAC(2)R, are prominent in the immune system and regulate many aspects of T cell-dependent immunity. In mouse T cells, VPAC(1)R is expressed constitutively, whereas VPAC(2)R is induced by immune stimuli. VPAC(2)R-null (VPAC(2)R(-/-)) mice on a C57BL/6 background are shown here to have normal basic immune characteristics, including serum Ig concentrations, blood levels of all leukocytes, and spleen number of total T cells (CD3(+)) and T cells bearing CD4, CD8, and CD28. Hapten-evoked cutaneous delayed-type hypersensitivity (DTH) was significantly enhanced in VPAC(2)R-null mice compared with age- and sex-matched wild-type mice. In contrast, generation of IgE anti-hapten antibodies and active cutaneous anaphylaxis were > or =70% lower in VPAC(2)R-null mice than in wild-type controls. Cytokine production by splenic CD4(+) T cells, stimulated with adherent anti-CD3 plus anti-CD28 antibodies, revealed higher levels of IL-2 (mean = 3-fold) and IFN-gamma (mean = 3-fold), and lower levels of IL-4 (mean = one-fifth) in VPAC(2)R-null mice than wild-type controls. Loss of VIP-VPAC(2)R maintenance of the normal ratio of Th2/Th1 cytokines thus leads to a state of enhanced DTH and depressed immediate-type hypersensitivity, which may alter both host defense and susceptibility to immune-mediated diseases.

Upon Prolonged Allergen Exposure IL-4 and IL-4Rα Knockout Mice Produce Specific IgE Leading to Anaphylaxis

Background: IL-4 and IL-13 are key regulators in atopic disorders and both signal through the receptor chain IL-4Rα. IL-4 and IL-13 are also the only cytokines known to induce class switching to IgE. We sought to compare allergen-specific IgE responses and allergic reactivity of wild-type (wt) mice with IL-4^–/– and IL-4Rα^–/– mice, which lack both IL-4 and IL-13 functions. Methods: BALB/c wt, IL-4^–/– and IL-4Rα^–/– mice were immunized with ovalbumin intranasally or intraperitoneally and specific antibody titers were measured by ELISA. Bronchoalveolar lavage fluids and lung tissue were analyzed cytologically and histologically. Allergic reactivity was determined by active cutaneous anaphylaxis and anaphylactic shock. Results: wt mice immunized intranasally or intraperitoneally showed high titers of specific IgE 3 and 6 weeks after primary sensitization, resulting in cutaneous anaphylaxis and anaphylactic shock upon challenge. Intranasal sensitization resulted in airway eosinophilia and goblet cell metaplasia. In contrast, IL-4^–/– and IL-4Rα^–/– mice showed no specific IgE after 3 weeks, but produced high titers after 6 weeks. At this time cutaneous anaphylaxis and anaphylactic shock could be induced as in wt mice, but lung pathology was absent. Conclusions: We conclude that upon long-term allergen exposure, alternative switch mechanisms independent of IL-4 and IL-4Rα may induce IgE but not asthma-like lung pathology. This may be relevant for the development of allergic disease, since long-term allergen exposure is a frequent condition during allergic sensitization.

Increased severity of local and systemic anaphylactic reactions in gp49B1-deficient mice.

gp49B1 is an immunoglobulin (Ig) superfamily member that inhibits FcstraightepsilonRI-induced mast cell activation when the two receptors are coligated with antibodies in vitro. The critical question of in vivo function of gp49B1 is now addressed in gene-disrupted mice. gp49B1-deficient mice exhibited a significantly increased sensitivity to IgE-dependent passive cutaneous anaphylaxis as assessed by greater tissue swelling and mast cell degranulation in situ. Importantly, by the same criteria, the absence of gp49B1 also resulted in a lower threshold for antigen challenge in active cutaneous anaphylaxis, in which the antigen-specific antibody levels were comparable in gp49B1-deficient and sufficient mice. Moreover, the absence of gp49B1 resulted in a significantly greater and faster death rate in active systemic anaphylaxis. These results indicate that gp49B1 innately dampens adaptive immediate hypersensitivity responses by suppressing mast cell activation in vivo. In addition, this study provides a new concept and target for regulation of allergic disease susceptibility and severity.

Contribution of Endothelial Selectins and α4 Integrins to Eosinophil Trafficking in Allergic and Nonallergic Inflammatory Reactions in Skin

AbstractThe role of endothelial selectins in mediating eosinophil recruitment was assessed using the trafficking of 111In-labeled blood eosinophils in mouse skin. An intradermal injection of chemoattractants (leukotriene B4, macrophage inflammatory protein-1α, and eotaxin) resulted in a rapid accumulation of 111In eosinophils that was reduced 49 to 91% by anti-P-selectin mAb. An anti-E-selectin mAb was ineffective, although a combined E- and P-selectin blockade resulted in >95% inhibition of all responses. The accumulation of a pulse of 111In eosinophils at sites of active cutaneous anaphylaxis (ACA) at 4 to 8 h and at 20 to 24 h after Ag challenge was completely dependent upon E- and P-selectin in combination, but not in isolation. In contrast, at 20 to 24 h after Ag challenge in a delayed-type hypersensitivity (DTH) reaction in skin, 111In eosinophil accumulation was largely independent of endothelial selectins, even when L-selectin was also blocked. An anti-α4 integrin mAb significantly reduced 111In eosinophil trafficking in both allergic reactions but was slightly more effective in the DTH reaction compared with the ACA reaction. These results show that P-selectin and to a lesser extent E-selectin mediate eosinophil recruitment in skin in acute inflammatory reactions. In allergic, late-onset inflammatory reactions, neither P- nor E-selectin alone are sufficient to mediate eosinophil accumulation; when combined, they are essential for trafficking in ACA but are less important in the DTH reaction. Whether α4 integrin-based strategies will be more effective than selectin-based strategies at inhibiting eosinophil recruitment in human disease remains to be determined.

Simple spectrophotometric analysis of passive and active ear cutaneous anaphylaxis in the mouse

Homologous passive cutaneous anaphylaxis (PCA) and active cutaneous anaphylaxis (ACA) to ovalbumin and DNP-hapten were studied in the ears of female BALB/c mice by means of assessing Evans blue dye leakage. For the quantitative evaluation of PCA and ACA, a hand-held spectrophotometer and the conventional colorimetric method were used to detect the amount of extravasated dye. The value of ΔE*ab (a numerical expression of color) obtained with the hand-held spectrophotometer and the amount of extravasated dye showed a good correlation. In the mouse ear, the sensitivity of PCA reaction was comparable to that of PCA in the rat, and ΔE*ab in the PCA and ACA reactions correlated well with the dilutions of sera and of the antigen, respectively. Thus, using a hand-held spectrophotometer is a simple, quantitative and sensitive method for ascertaining the extent of immediate-type hypersensitivity in the mouse.

Antigenicity tests of a new antineoplastic agent S-1

The antigenicity was tested of a new antineoplastic agent S-1 (a combination of tegafur (FT), CDHP and potassium oxonate (Oxo)) in mice and guinea pigs. 1. Male BALB/c or C3H/He mice were sensitized with S-1, CDHP, Oxo, and conjugates of CDHP (or Oxo) and human serum albumin (HSA). S-1 was administered by oral gavage, and the other compounds were administered intraperitoneally with adjuvant (alum). In the heterologous passive cutaneous anaphylaxis (PCA) test, using Sprague-Dawley rats as recipients, no IgE antibodies against S-1, CDHP, or Oxo were detected to any serum obtained from the sensitized mice, and no eliciting antigenicities were seen for CDHP or Oxo. 2. Male Hartley guinea pigs were sensitized with S-1, CDHP, Oxo, and conjugates of CDHP (or Oxo) and HSA. S-1 was administered by oral gavage, and the other compounds were administered subcutaneously with Freund's complete adjuvant (FCA). The homologous PCA test, active systemic anaphylaxis test, and passive hemagglutination test showed no production of antibodies against S-1, CDHP, or Oxo in any sensitized guinea pig, and no eliciting antigenicities for CDHP or Oxo. 3. Female Hartley guinea pigs were sensitized with S-1 subcutaneously with FCA. The active cutaneous anaphylaxis test revealed that S-1 did not induce cell-mediated delayed type hypersensitivity. 4. These results indicated that S-1, Oxo, and CDHP were not antigenic in mice and guinea pigs.

Antigenicity study on montirelin hydrate (NS-3)

An antigenicity study of montirelin hydrate (NS-3), a new drug for the treatment of disturbance of consciousness, was conducted in Hartley guinea pigs. Animals were sensitized twice by subcutaneous injections of montirelin hydrate in combination with Freund's complete adjuvant and twice by intramuscular injections of montirelin hydrate alone. Montirelin hydrate was found to be negative in the homologous passive cutaneous anaphylaxis test carried out with sera from the sensitized guinea pigs. Negative results were also obtained in the active cutaneous anaphylaxis test and the active systemic anaphylaxis test in guinea pigs sensitized with montilerin hydrate. These results show that montirelin hydrate has no antigenicity under the present experimental conditions.

Antigenicity studies of the aqueous extract of red ginseng in guinea pigs

The antigenicity of the aqueous extract of red ginseng (ARG) was evaluated using the following assay procedures: 1. active systemic anaphylaxis (ASA) in guinea pigs, 2. active cutaneous anaphylaxis (ACA) in guinea pigs, 3. passive cutaneous anaphylaxis (PCA) in guinea pigs with serum from guinea pigs sensitized with ARG and 4. passive hemagglutination (PHA) with serum from guinea pigs sensitized with ARG. These results suggest that ARG has no antigenicity under the conditions used. And the dose levels of ARG employed in the present experiment were confirmed not to suppress immune reactions.

Antigenicity study of paclitaxel

The antigenic property of paclitaxel was examined using its protein mixtures (paclitaxel + OVA, paclitaxel + GSA, paclitaxel + RSA) in guinea pigs and mice in comparison with ovalbumin (OVA) and the protein conjugate of 4-aminoantipriyne (AAP). The following results were obtained: 1. When guinea pigs were sensitized with paclitaxel or paclitaxel + OVA emulsified with Freund's complete adjuvant, none of active systemic anaphylaxis (ASA), passive cutaneous anaphylaxis (PCA) and Schultz-Dale reaction were induced by challenge with paclitaxel or paclitaxel + GSA (guinea pig serum albumin). In the observation of active cutaneous anaphylaxis (ACA), no changes were observed in animals treated with paclitaxel alone as a sensitizing and/or a challenging antigen under the condition where slight delayed type hypersensitivity was elicited in animals sensitized with paclitaxel + OVA and challenged with paclitaxel + GSA. 2. When mice were sensitized with paclitaxel or paclitaxel + OVA adsorbed to alum. sera of these animals revealed a negative reaction in PCA using rats by challenge with paclitaxel or paclitaxel + RSA (rat serum albumin). 3. Protein bindings of paclitaxel with the above albumins were more than 40%. As shown above, paclitaxel was considered not to possess antigenic property under the experimental condition. In addition, the dose levels of paclitaxel employed in the present experiment were confirmed not to suppress the immune response to OVA.

Active Cutaneous Anaphylaxis (ACA) in the Mouse Ear

Active cutaneous anaphylaxis (ACA) was studied in the ear of female BALB/c mice. Mice were immunized with ovalbumin in the presence of aluminium hydroxide gel or complete Freund’s adjuvant (CFA). Two weeks after the immunization, ACA was elicited in the mouse ear by injecting 10 μl of antigen solution intradermally into the ear lobe. ACA was assessed by the amount of extravasated dye, which was given intravenously just after the antigen injection. Antiallergic drugs (tranilast, ketotifen and azelastine), antihistamines (chlorpheniramine, diphenhydramine and mequitazine), β-stimulants (isoproterenol and salbutamol), theophylline and glucocorticoids (hydrocortisone, prednisolone and dexamethasone) inhibited the reaction significantly. These drugs inhibited both ACA in mice immunized with alum-precipitated antigen and ACA in mice injected with CFA-emulsified antigen similarly. ACA in the mouse ear might be a useful tool for studying drugs for allergy.

Active and Passive Cutaneous Anaphylaxis in Wbb6f1Mouse, A Mast Cell-Deficient Strain

WBB6F1 mouse, a mast cell-deficient strain, was tested for active and passive cutaneous anaphylactic reactions. Active cutaneous anaphylaxis was not produced in mice which had been immunized for 1 to 2 weeks by an intraperitoneal injection of bovine serum albumin with either adjuvant, Freund's complete adjuvant or Bordetella pertussis organisms, even though circulatory IgE and IgG1 antibodies were raised. Passive cutaneous anaphylaxis (PCA) was also absent, when the mice had been sensitized with allogeneic IgE or IgG1 monoclonal antibodies. However, obvious PCA was produced when allogeneic or xenogeneic hyperimmune serum was employed. These findings indicate that mast cells are not necessarily needed for the production of PCA. Some mechanism quite different from the well-elucidated mechanism, i.e., IgE- or IgG1 antibody-triggered histamine release from mast cells, seems to be operative.

ANTIGENICITY STUDY OF BUSPIRONE HYDROCHLORIDE IN GUINEA PIGS AND MICE

Buspirone hydrochloride(buspirone) and buspirone-ovalbumin mixture were examined for their antigenicity in guinea pigs and mice in comparison with ovalbumin (OVA) and 2, 4-dinitrochlorobenzene (DNCB)-OVA conjugate. The results obtained were as follows: 1. When guinea pigs were sensitized with buspirone or buspirone-OVA emulsified with Freund's complete adjuvant (FCA), these animals showed negative reactions in active systemic anaphylaxis (ASA), active cutaneous anaphylaxis (ACA), passive cutaneous anaphylaxis (PCA), passive hemagglutination (PHA) and Schultz-Dale test. 2. When mice were sensitized with buspirone or buspirone-OVA adsorbed to alum, these animals revealed a negative reaction in PCA using rats. 3. As positive controls, guinea pigs were sensitized with OVA or DNCB-OVA emulsified with FCA, and mice with OVA or DNCB-OVA adsorbed to alum. As a result, these animals disclosed positive reactions in ASA, ACA, PCA, PHA and Schultz-Dale test. As shown above, buspirone was considered to possess neither antigenic nor haptenic properties.

Antigenicity tests on propiverine hydrochloride in guinea pigs and mice.

Antigenicity of propiverine hydrochloride (P-4), a newly developed drug for pollakisuria, was investigated in guinea pigs and mice. 1. Two strains of mice (BALB/c and C3H/He) showed no production of antibodies against P-4 inoculated with aluminum hydroxide gel (alum) as an adjuvant, judged by the heterologous passive cutaneous anaphylaxis (PCA) test using rats. On the other hand, antibodies against P-4-ovalbumin (OVA) conjugate inoculated with alum was definitely detected. 2. In the studies with guinea pigs, both the inoculation of P-4 alone and of P-4 with Freund's complete adjuvant (FCA) as an adjuvant did not produce positive reactions in any of homologous passive cutaneous anaphylaxis (PCA), active systemic anaphylaxis (ASA) and passive hemagglutination (PHA) tests. On the other hand, the inoculation of P-4-OVA conjugate with FCA produced positive reaction in all of PCA, ASA and PHA tests. 3. In the active cutaneous anaphylaxis (ACA) test in guinea pigs inoculated with P-4-OVA conjugate with FCA, positive reaction was produced by eliciting injection of P-4-human serum albumin (HSA). 4. In the Schultz-Dale reaction test, any animals in any groups showed no positive reaction to both eliciting antigens, P-4 and P-4-HSA. 5. These findings showed that P-4 had no antigenicity in guinea pigs and mice.

IgE antibody production and cutaneous anaphylactic reactions in rats infected with Clonorchis sinensis.

Rats were infected orally with 50 or 100 metacercariae of Clonorchis sinensis. Anti-Clonorchis IgE antibody, determined by passive cutaneous anaphylaxis, in the serum of infected rats appeared 30-40 days after infection and persisted for at least 120 days. Total amount of IgE fixed on the mast cells, detected by reverse passive cutaneous anaphylaxis with anti-rat IgE, was significantly more in the infected rats than in uninfected rats. Worm burden at 290 days post-infection did not correlate to anti-Clonorchis IgE antibody titers, reverse passive cutaneous anaphylaxis titers, or anti-Clonorchis anaphylactic antibody on the mast cells, determined by active cutaneous anaphylaxis. Active cutaneous anaphylaxis titers interrelated to reverse passive cutaneous anaphylaxis titers, suggesting a correlation between the amount of anti-Clonorchis anaphylactic antibodies and total IgE on the mast cells. Sensitivity of passive cutaneous anaphylaxis with anti-BSA IgE antibody was significantly suppressed in Clonorchis infected rats. Moreover, anti-BSA IgE titers in the infected rats were inversely related to reverse passive cutaneous anaphylaxis titers, indicating that increase of IgE antibody on the mast cells by Clonorchis infection interfered with sensitization of anti-BSA IgE antibody.

Host immune responsiveness to the chigger, Eutrombicula cinnabaris.

Chigger infestation is often associated with severe cutaneous reactions. Mice were given four infestations with the pest chigger Eutrombicula cinnabaris, and each exposure was separated by a 14-day mite-free period. Mean duration of engorgement was nine to ten days for a first exposure and four to five days for a fourth exposure. An initial exposure did not elicit macroscopic changes at chigger attachment sites, while all third and fourth exposure animals had marked reactions consisting of erythema, epidermal thickening and serous exudation. Approximately 20% of second exposure animals had macroscopic changes at chigger feeding sites, but these reactions were much less intense than the responses of third and fourth infestation hosts. Third and fourth exposure animals had infiltrates of lymphocytes, eosinophils, basophils and neutrophils at attachment sites, with eosinophil influx the most intense. Cutaneous reactivity to chigger feeding was adoptively transferred with lymphocytes from fourth exposure animals. Passive transfer of serum from fourth infestation donors resulted in heightened reactivity to a challenge infestation. Skin testing, after a fourth infestation, with an extract of whole E. cinnabaris larvae provided evidence for Arthus and delayed type hypersensitivity responses to chigger antigens. Chigger-reactive homocytotropic antibody was not detected by skin testing and active cutaneous anaphylaxis.

Late-phase reaction in topically induced ocular anaphylaxis in the rat.

A cellular late-phase reaction is described in a rat model of topically induced ocular anaphylaxis. Rats were immunized with dinitrophenylated Ascaris suum extract and alum and were tested for active cutaneous anaphylaxis on day 13. Rats with a strong skin test response were selected for ocular challenge with di-DNP-lysine. Macroscopic observation and histologic evaluation were performed at 1, 6, and 24 h. In rats showing a moderate macroscopic ocular response at 1 h, mast cell degranulation was significantly increased at 1 h; no significant increase in eosinophils, neutrophils or lymphocytes was found in the conjunctive of these animals. In rats showing a marked macroscopic ocular response at 1 h, mast cell degranulation was significantly increased at 1 and 6 h; the number of eosinophils was significantly increased at 1 and 6 h, and of neutrophils at 6 h only. At 24 h, neutrophil and eosinophil numbers returned to baseline levels. There was no macroscopic evidence of a late-phase response in either group of animals. Our results suggest that, in keeping with earlier observations in human skin, a strong early response to antigen is required for the development of a late-phase ocular response in the rat.

The Effects of Chlorpheniramine and Antiallergic Drugs on Ascaris suum Antigen-Induced Active Cutaneous Anaphylaxis in Dogs

Effects of chlorpheniramine and two antiallergic drugs on the active cutaneous anaphylaxis (ACA) reaction induced by intradermal injection of Ascaris suum antigen in naturally sensitized dogs were investigated. Chlorpheniramine (10 mg/kg, intraduodenally (i.d.)) almost abolished the ACA reaction. NCO-650 (100 mg/kg, i.d.) had no inhibitory effect while tranilast (300 mg/kg, i.d.) showed a weak inhibitory effect. These findings show that (i) the ACA reaction is almost totally mediated by histamine and (ii) ACA reaction is considerably resistant to antiallergic drugs such as tranilast and NCO-650.

Effect of myeloma IgE injections on passive and active cutaneous anaphylaxis in rats.

The ability of injected rat IgE myeloma protein IR162 to inhibit passive and active cutaneous anaphylaxis in Lewis rats was investigated. IgE injected i.p. 24 hr before the sensitization with IgE anti-ovalbumin (OVA) completely inhibited both IgE- and IgG2a-induced passive cutaneous anaphylactic (PCA) reactions at a dose (2.5 mg/100 g body weight) that resulted in peak serum concentrations of 150 micrograms IgE IR162/ml. Peak IgE IR162 serum concentrations of 20 to 60 micrograms/ml inhibited the PCA reaction in approximately 50% of the rats. Intracutaneous injection of a mixture of myeloma IgE and anti-OVA IgE in a ratio of 100:1 or more also inhibited the PCA reaction. In contrast, the PCA reaction was not inhibited by seven daily doses of IgE beginning 24 hr after passive sensitization. Likewise, the cutaneous anaphylactic reaction elicited in rats 14 days after immunization with OVA and Bordetella pertussis was not prevented by daily injections of myeloma IgE despite a 1000- to 3000-fold excess of the myeloma IgE to anti-OVA IgE serum concentration. The data demonstrate that parenteral administration of myeloma IgE inhibits the PCA reaction only when given before passive sensitization and does not prevent cutaneous anaphylaxis in actively immunized rats. Because myeloma IgE failed to inhibit anaphylactic reactions in actively immunized rats, it is questionable whether administering human IgE-derived synthetic peptides or recombinant DNA-produced IgE fragments will be able to prevent allergic diseases by blocking the IgE Fc receptors on mast cells.

Evaluation of the role of Histamine H1- and H2-receptors in cutaneous inflammation in the guinea-pig produced by histamine and mast cell degranulation.

1 The role of histamine H1- and H2-receptors in mediating the cutaneous inflammatory response produced by exogenous histamine and the release of endogenous histamine from mast cells has been investigated by a method which permits simultaneous, quantitative measurement of vasodilatation, vascular permeability and oedema formation. 2 Histamine and the selective H1-receptor agonist, 2-(2-aminoethyl) pyridine, both produced vasodilatation, increased vascular permeability and oedema formation whereas the selective H2-receptor agonist, dimaprit, produced only vasodilatation. 3 Mepyramine and cimetidine both reduced the vasodilatation response to histamine, the combination of antagonists being superior to either antagonist alone. Mepyramine (but not cimetidine) virtually abolished extravascular albumin accumulation and oedema formation. 4 Mepyramine and cimetidine both reduced the vasodilatation response produced by active cutaneous anaphylaxis and compound 48/80. However, mepyramine was less effective in reducing the vascular permeability response to mast cell degranulation than to histamine. 5 In conclusion, the vasodilator response to histamine is mediated by both H1- and H2-receptors; the permeability response to histamine is mediated solely by H1-receptors. A combination of H1- and H2-receptor antagonists appears to be more effective than either antagonist alone in reducing cutaneous inflammatory reactions involving histamine.

Immunological responses of fluke-infected and fluke-free cattle to Salmonella dublin and other antigens

Immune responses to heat-killed Brucella abortus strain 19 and to ovalbumin were compared in 15 fluke-infected and 15 fluke-free Friesian heifers. B abortus was injected 16 weeks and ovalbumin 19 weeks after the oral administration of 1000 metacercariae of Fasciola hepatica. Agglutinating antibody responses to B abortus were similar in both groups. Immediate type hypersensitivity to ovalbumin was apparently suppressed in fluke-infected animals when assessed by active and passive cutaneous anaphylaxis two weeks after sensitisation. However, when assessed by Schultz-Dale responses of intestine, in vitro, 36 weeks after sensitisation there was no difference between the groups. The heifers were subsequently given live Salmonella dublin intravenously. The fluke-infected animals which became carriers of S dublin had the most persistently elevated titres of agglutinating antibodies in their sera and the highest incidence of immediate-type hypersensitivity, as assessed by Schultz-Dale responses of intestine, but the weakest cutaneous delayed hypersensitivity reactions to S dublin. The latter might have been related to lymphopenia which developed after fluke infection. The increased susceptibility of fluke-infected cattle to S dublin cannot be attributed to impaired agglutinin responses but may result from effects on cell-mediated mechanisms.