Elsevier

International Immunopharmacology

Volume 7, Issue 9, September 2007, Pages 1170-1178
International Immunopharmacology

Cross-reactivity of anti-phosphorylcholine antibodies to neuromuscular blockers in a murine model of immunization

https://doi.org/10.1016/j.intimp.2007.04.011Get rights and content

Abstract

Hypersensitivity reactions to curare-like neuromuscular blocking agents (NMBA) used in anesthesiology are more frequent in females and often occur at the first exposure to these drugs. To evaluate the antibody response to a hapten sharing the same allergenic epitope with NMBA in a murine model of immunization with Nitrophenylphosphorylcholine (NPPC) coupled with Keyhole Limpet Hemocyanin (KLH). BALB/c mice from both sexes were intraperitoneally immunized with NPPC–KLH with alum and boosted twice, on 7 and 14 days. The antibodies were tested for specificity to PC and for cross-reactivity to the haptens, NPPC and PC, as well as to the NMBAs. Mice immunized with NPPC–KLH produced anti-PC antibodies mainly of IgM, IgG1 and IgG3 isotypes, at similar levels in both sexes. Different affinities for the haptens were detectable between isotypes, with anti-PC IgG3 antibody reactivity mostly related to the choline portion of the NPPC hapten. When comparing among sexes, females developed greater IgG2 affinity to the hapten than males. Cross-reactivity to NMBAs was predominant among the anti-PC IgG3 antibodies, mainly in females achieving 75% of inhibition with 16 mM of suxamethonium, while other isotypes achieved up to 30% and was absent for IgE. The investigation of antibody isotypes regarding sensitization to choline-derived structures could contribute to understanding the differential ability of females to produce antibodies that are cross-reactive with NMBAs.

Introduction

Allergic reactions to drugs are a common problem in clinical practice. Immediate reactions are those associated with greater risk. IgE-mediated reactions occur after prior patient sensitization by the drug or by a structural analogue and are named anaphylactic reactions. Anaphylactic-type reactions that occur in the perianesthetic period, despite their rarity, are associated with high mortality, being one of the main causes of death attributable to secondary reactions to drugs [1], [2]. These anaphylactic reactions to NMBAs have been reported in 1/6000–1/20,000 of general anesthesias [3]. Considerable differences in prevalence exist between countries; in France, the estimated incidence of IgE-mediated anaphylactic incidence is 1/13,000 anesthetic procedures [4].

The majority of anaphylactic reactions are thought to be IgE-mediated [5], [6]. These reactions are mostly (70% to 75%) due to the use of NMBAs and are nine times more frequent in females than in males [7]. Of note is that patients sensitive to NMBAs frequently present adverse reactions at the first exposure to these drugs and a high rate of cross-reactivity to all compounds of the group [8], [9]. The quaternary ammonia group of the choline structure functionally characterizes the NMBAs and is probably the sensitizing epitope [10]. The chemical structures of the NMBAs are at least bivalent for the quaternary ammonium epitope. They must present the capacity to cross-link the IgE antibody on sensitized mast cells and basophils in order to initiate an anaphylactic reaction. Therefore, patients who present IgE antibodies to quaternary ammonium should be considered individuals at risk when subjected to general anesthesia with NMBAs.

IgE antibodies that cross-react with the quaternary ammonium ion of NMBAs were usually evaluated by a radioimmunoassay based on p-amino-phenylphosphoryl-choline bound to agarose or morphine sulphate coupled to Sepharose [10], [11], [12]. Specific IgE antibodies to NMBAs usually react with the other drugs of the same group, remaining detectable in RAST or ELISA assays [11], [13].

The frequency of sensitization to quaternary ammonium ions in the absence of previous anesthesia is reported as 9.3% in the adult population with no identified risk factor [14]. The possibility that sensitization occurs through contact with ammonia quaternary compounds present in antiseptics like Benzalkonium chloride, Cetylpyridinium hydrochloride, or other industrial products such as cosmetics, have been reported [10], [15], [16]. These products are preferentially used by women and may explain the occurrence of reactions after the first exposure to NMBAs. Conversely, it has been suggested that the sensitization could occur by contact with self or infectious agent antigens containing choline structures [17].

Despite the existence of diagnostic tests for NMBAs, the rarity of these reactions and the lack of knowledge regarding other risk factors besides gender raise some difficulties for assessing these tests for large population screening. Improving current knowledge of the situations that determine the production of antibodies directed to phosphorylcholine analogs, associated with a presurgical questionnaire discriminating the individuals at risk, appears to be an adequate approach.

To address the profile of reactivity of anti-phosphorylcholine (PC) antibodies between males and females, a murine model of immunization with a NPPC-carrier was evaluated. The isotype profile of the antibodies and their cross-reactivity with choline derivatives, such as the NMBA, were studied.

Section snippets

Animals

BALB/c mice, male and female, between 15 and 18 weeks of age were provided by the animal facilities of the São Paulo University Medical School. Lewis rats (RT1l,l), from both sexes, between 8 and 12 weeks of age, bred in our own laboratory's animal facilities were used for passive cutaneous anaphylaxis reaction.

Hapten diazotation and protein haptenization

Initially the hapten NPPC (Sigma) was submitted to a diazotation method [18]. For haptenization with KLH, 100 mg of KLH (Sigma, St Louis MO, USA) was added to the equivalent of 80 mg of

Profile of antibody production in NPPC–KLH-immunized mice

To assess the cross-reactivity of anti-PC antibodies with the NMBAs, we first evaluated the profile of antibodies to nitrophenylphosphorylcholine (NPPC) coupled with Keyhole Limpet Hemocyanin (KLH) in immunized BALB/c mice. Fig. 1 shows that quaternary ammonium ion in the structures of PC and NPPC is also shared by the SUX structure.

Immunized BALB/c mice with NPPC–KLH preferentially induced IgM, IgG1 and IgG3 anti-PC antibody production compared to non-haptenized KLH-immunized group and

Discussion

In this study, it was demonstrated that nitrophenylphosphorylcholine–carrier protein (NPPC–KLH) immunization in mice was able to preferentially induce IgM, IgG1 and IgG3 antibody production to phosphorylcholine (PC). Comparison between the sexes revealed that female immunized mice showed greater affinity mediated by IgG3 and IgG2 antibodies to the PC hapten than males. Antibodies developed by the immunization process also showed cross-reactivity to NMBAs was exerted mainly by IgG3 antibodies,

Acknowledgments

This research was supported by FAPESP and LIM 56-HC/FMUSP.

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