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Keywords:

  • anaphylaxis;
  • mast cell;
  • oral desensitization;
  • penicillin V

Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Background:  Despite the widespread use of an acute oral desensitization procedure in patients with allergic reactions to a variety of antibiotics, the precise mechanism of this procedure is poorly understood.

Objective:  To investigate the mechanisms underlying acute oral desensitization to antibiotics.

Methods:  Using a murine model of active systemic anaphylaxis to penicillin V (Pen V), mice previously sensitized to Pen V were desensitized by oral feeding of Pen V. The dose was doubled every 15 min and five feedings were given. The achievement of acute oral desensitization was evaluated by induction of active systemic and active cutaneous anaphylaxis, and by measuring the plasma levels of platelet-activating factor and histamine. Antigen-specific serum IgE antibody (Ab) levels were determined by passive cutaneous anaphylaxis.

Results:  Mice fed more than 3 mg of cumulative dose of Pen V were completely protected from fatal systemic anaphylactic reaction and the desensitized state lasted approximately 1 h. Antigen-specific mast cell desensitization, but not hapten inhibition, consumption of IgE Abs, or depletion of mast cell mediators, occurred during acute oral desensitization.

Conclusions:  Acute oral desensitization to Pen V occurred in the mice, and antigen-specific mast cell desensitization was associated with the underlying mechanism for oral desensitization.

Abbreviations:
ACA

active cutaneous anaphylaxis

Ab

antibody

BSA

bovine serum albumin

i.p.

intraperitoneally

OVA

ovalbumin

PAF

platelet-activating factor

Pen V

penicillin V

PCA

passive cutaneous anaphylaxis

Immediate hypersensitivity reactions to antibiotics ranging from urticaria to fatal anaphylaxis are a major obstacle that limits their use. Since a desensitization was introduced into the clinical field during the 1960s based on the oral administration of increasing doses of β-lactam drugs to desensitized skin test-positive patients prior to full-dose antibiotic therapy (1), desensitization has become a popular technique employed for patients who have infections for which the antibiotic is essential or highly desirable despite the risk of allergic reactions (2–11). The oral route of desensitization appears safer, easier and less expensive than parenteral route (12).

The proposed hypothesis explaining the mechanisms underlying acute oral desensitization are consumption of IgE in immune complexes, hapten inhibition, mediator depletion from mast cells and basophils, and antigen-specific mast cell desensitization (13–15). However, the precise mechanisms are yet to be elucidated, probably because of the lack of a suitable experimental model.

We have previously developed a murine model of IgE-dependent penicillin V (Pen V)-induced active systemic anaphylaxis, which is 100% fatal in C57BL/6 mice (16). In this study, we established a murine model of acute oral desensitization to Pen V, and investigated the mechanism(s) underlying oral desensitization. It was determined that oral desensitization to Pen V was associated with antigen-specific mast cell desensitization.

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Animals

Specific pathogen-free female C57BL/6 mice were obtained from the Korean Institute of Chemistry Technology (Daejeon, Korea), and were kept in our animal facility for at least 2 weeks before use. All mice were used at 8–10 weeks of age. All experimental animals used in this study were under the protocol approved by the Institutional Animal Care and Use Committee of the Chonbuk National University Medical School.

Reagents

Penicillin V, ovalbumin (OVA) (grade V) and bovine serum albumin (BSA) (fraction V) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Bordetella pertussis was obtained from the National Institute of Health (Seoul, Korea).

Pen V-protein conjugates

Conjugates (Pen V-OVA and Pen V-BSA) were prepared as described previously (16, 17).

Induction of active systemic anaphylaxis to Pen V

Mice were sensitized intraperitoneally (i.p.) with 500 μg of Pen V-OVA conjugates plus 2 × 109B. pertussis and 1.0 mg of alum, and were challenged i.v. with 100 μg of Pen V-BSA conjugates 3 weeks later (16, 17). After the challenge injections were administered, the mice were observed for 1 h, and signs of shock and the number of deaths were recorded.

Oral desensitization protocol

Human oral desensitization is started with a low dose and is doubled every 15 min. This process continues until a considerable cumulative dose is achieved with an elapsed time of 3–4 h (2–4). A similar protocol was employed as follows. Three weeks after sensitization, the mice were orally administered 0.64 mg/0.1 ml of the initial dose of Pen V, unless otherwise stated, via a stainless gavage needle. The dose was doubled every 15 min, and a cumulative dose of 20 mg was administered after five feedings. Mice were challenged i.v. with 100 μg of Pen V-BSA 15 min after the last feeding. Control sensitized mice were given saline.

Determination of Pen V-specific IgE

Antigen-specific serum IgE levels were determined by passive cutaneous anaphylaxis (PCA) reaction as described previously (16, 17).

Determination of plasma platelet-activating factor (PAF) and histamine

The postchallenge blood was taken from the heart which was cut open, and was mixed with a 0.1 vol. of 3.8% ice-chilled citrate solution, and centrifuged immediately using an Eppendorf microfuge (Eppendorf, Hamburg, Germany). The plasma was stored at −20°C until use. Plasma PAF and histamine levels were determined as described previously (17).

Active cutaneous anaphylaxis (ACA)

Mice were sensitized i.p. with 500 μg of Pen V-OVA conjugate plus 2 × 109B. pertussis, and 1.0 mg of alum. Three weeks after sensitization, 20 μl of Pen V-BSA solution (1 μg/ml) was injected intradermally into the shaved backs of the mice. Immediately thereafter, 0.1 ml of 1% Evan's blue dye was injected i.v. The mice were killed 10 min later and punch biopsies were obtained, minced, and extracted three times with hot formamide (80°C, 3 h) (18). Pooled samples from tissue sites were centrifuged and absorbance at 610 nm was measured. A610 values were converted to micrograms of Evan's blue dye based on a standard curve of dilutions of Evan's blue dye in formamide.

Statistical analysis

Data are expressed as mean ± SD. Statistical comparison was performed using one-way anova followed by the Fisher's exact test. Significant differences between the groups were determined using the unpaired Student's t-test. P < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Induction of acute oral desensitization to Pen V

Three weeks after sensitization, groups of mice were given varying initial doses of Pen V. The doses were doubled every 15 min and five feedings were given to reach the cumulative doses indicated before a challenge injection. None of the mice developed anaphylactic symptoms during the desensitization procedure. All mice were challenged 15 min after the last feeding. Of the saline-fed control mice, 100% died of shock 15–20 min after the challenge injections, whereas the mice fed more than 3 mg of cumulative dose of Pen V were completely protected from death (Table 1). No signs of shock, except for slight itching, were observed in these mice. Oral desensitization could also be achieved by only a single feeding of more than 10 mg of Pen V (data not shown), which suggests that the frequency of feeding is not a critical factor in inducing oral desensitization. It is imperative that the exact duration of the desensitization state be determined. Groups of mice previously sensitized to Pen V were subjected to oral desensitization with 10 mg of cumulative dose of Pen V, and were challenged 15, 30, 45, and 60 min after the completion of oral intake of Pen V. Within 60 min, 100% of the challenged mice showed fatal reactions, which indicates that the effect of such treatment is temporary (Table 2). Taken together, these data demonstrate that acute oral desensitization to Pen V was easily achieved in mice, although the desensitized state did not last for a long period of time. In the subsequent experiment, a protocol consisting of feeding 10 mg of cumulative Pen V by five feedings and a 15-min interval between the last oral feeding and injection of challenge was employed.

Table 1.   Achievement of acute oral desensitization to pen V
Cumulative dose of Pen V* (mg)Mortality† (no. of dead/total)
  1. *Three weeks after sensitization, oral desensitization was started with varying initial doses of Pen V. The doses were doubled every 15 min and five feedings were given to reach the cumulative doses indicated. Mice were challenged i.v. with 100 μg of Pen V-BSA 15 min after the last feeding.

  2. †Data represent the number of dead/total mice from three separate experiments.

10 0/15
 3 0/15
 1 8/15
 0.315/15
Table 2.   Acute oral desensitization does not last longer than 1 h
Interval (min) between the last oral feeding and challenge*Mortality† (no. of dead/total)
  1. *Cumulative dose (10 mg) of Pen V was given by five feedings, and challenge injections were given at the time indicated.

  2. †Data represent the number of dead/total mice from three separate experiments.

150/15
300/15
455/15
6015/15

Studies on the mechanisms of acute oral desensitization

We examined four proposed hypotheses for human oral desensitization, including hapten inhibition, consumption of IgE, mediator depletion, and antigen-specific mast cell desensitization.

  • 1
    Hapten inhibition. If hapten inhibition is associated with acute oral desensitization, the anaphylactic reaction to Pen V would not be elicited or markedly diminished in the presence of a large amount of free antibiotic. However, challenge injection together with up to 10 mg of free Pen V did not prevent the fatal reaction (Table 3). This observation put forth an argument against hapten inhibition as the mechanism of oral desensitization.
  • 2
    Consumption of IgE. To investigate the possibility of IgE consumption, Pen V-sensitized mice were partially bled 1 h before the initiation of the oral desensitization procedure and 15 min after cessation of the procedure, and Pen V-specific-IgE levels were then compared by PCA. The finding that there were no differences in Pen V-specific serum IgE levels between these two time points (Fig. 1) indicates that a temporary diminution of Pen V-specific serum IgE level did not occur during acute oral desensitization.
  • 3
    Mediator depletion. It is possible that slow absorption of orally administered Pen V from the gut can cause mast cells to continuously release chemical mediators, resulting in the depletion of mediators. To test this possibility, mice sensitized with Pen V-OVA were subjected to oral desensitization to Pen V, followed by a challenge with OVA. If mediator depletion occurs during oral desensitization, neither anaphylactic reactions to OVA nor elevation in plasma chemical mediators such as PAF, which is critical for fatal murine anaphylaxis (17, 19, 20) and histamine would occur in Pen V-fed mice. As shown in Table 4, fatal reactions to OVA occurred in 100% of mice, and the release of mast cell mediators was not altered by the desensitization procedure, thus also arguing against mediator depletion.
  • 4
    Mast cell desensitization. We first examined this possibility by measuring the effect of oral feeding of Pen V on plasma histamine and PAF levels in response to challenge injections. Oral feeding of Pen V prevented an increase in plasma levels of both PAF and histamine in a time-dependent manner; 15–30 min after the last feeding of Pen V, the histamine and PAF levels were 10–15% of that of the sensitized control group, and then were rapidly restored to 90% of the control level after 60 min (Fig. 2A), the time of the reappearance of 100% fatal reaction. The degree of mast cell degranulation was determined by ACA. ACA also showed similar kinetics to those of plasma levels of PAF and histamine: it was markedly diminished 15–30 min after oral feeding of Pen V, but was restored to a significant level at 60 min (Fig. 2B). Taken together, these data indicate that oral intake of Pen V resulted in the development of temporary mast cell desensitization.
Table 3.   Free Pen V dose not prevent fatal anaphylaxis
Challenge*Mortality† (no. of dead/total)
  1. *The Pen V-OVA-sensitized mice were challenged with or without 10 mg of free Pen V.

  2. †Data represent the number of dead/total mice from two separate experiments.

Pen V-BSA10/10
Pen V-BSA + Pen V (10 mg)10/10
image

Figure 1.  No changes in serum levels of penicillin V-specific IgE before and after oral desensitization. Three or five weeks after sensitization, the mice were partially bled 1 h before the initiation of oral desensitization and 15 min after cessation of the procedure, and Pen V-specific-IgE levels were compared by passive cutaneous anaphylaxis. Data represent mean ± SD of three separate experiments (n = 5 per group).

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Table 4.   Mediator depletion does not occur during acute oral desensitization
Pen V feeding*Mortality (no. of dead/total)PAF (ng/ml)Histamine (μg/ml)
  1. *One group of sensitized mice was desensitized with 10 mg of cumulative dose of Pen V. All mice were challenged i.v. with 500 μg of OVA 15 min after the last feeding, and killed 2 min thereafter.

  2. †Data represent the number of dead/total mice from three separate experiments.

  3. ‡Data represent the mean ± SDs of three separate experiments (n = 5 per group).

 15/15†70 ± 22‡4.5 ± 0.8
+15/1566 ± 144.4 ± 1.0
image

Figure 2.  Time-course of plasma levels of histamine and platelet-activating factor (PAF), and active cutaneous anaphylaxis (ACA) after oral desensitization. At the time indicated after oral desensitization, mice were challenged and bled 2 min postchallenge for the measurement of plasma levels of PAF and histamine (A) and were subjected to ACA (B). Data represent mean ± SD of three separate experiments (n = 5 per group). *P < 0.01 vs control. ##P < 0.05 vs control.

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Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

An acute oral desensitization technique has been used for many years to avoid or minimize allergic reactions to antibiotics. The precise mechanisms underlying oral desensitization, however, have not been studied in detail because of the limitations of human studies. In this study, we established a murine model of acute oral desensitization to Pen V and demonstrated temporary mast cell desensitization as the mechanism responsible for the process of desensitization.

Acute oral desensitization to Pen V can be achieved easily in mice which have been previously sensitized to Pen V by oral administration of 1–10 mg of cumulative doses of Pen V, but the desensitized state was usually diminished within 1 h. Although it is difficult to make a direct comparison between the desensitized state of the human and mouse, repeated oral desensitization or use of long-acting preparations of antibiotic appears to sustain the desensitized state for a longer period of time. This simple and reproducible murine model of acute oral desensitization will provide a valuable tool with which to study the pathophysiology of acute oral desensitization to antibiotics.

The hypothesis explaining penicillin desensitization includes hapten inhibition, consumption of IgE, mast cell mediator depletion, and mast cell desensitization (13–15). Given that free drugs or univalent drug-carrier conjugates could compete with multivalent conjugates for an IgE-antigen binding site, hapten inhibition could be considered a possible mechanism for oral desensitization (21). However, simultaneous injection of a large amount of free antibiotic (up to 10 mg) together with the challenge regimen (Pen V-BSA) could not prevent fatal anaphylaxis; this finding acts as an argument against hapten inhibition as a mechanism for oral desensitization. Furthermore, we could not find any compelling evidence for IgE consumption because there were no differences in serum antibiotic-specific IgE levels measured before and after oral desensitization.

It was demonstrated that oral desensitization with Pen V did not prevent anaphylactic reactions provoked by an unrelated antigen, OVA, in mice sensitized with Pen V-OVA (Table 4). Furthermore, plasma PAF and histamine levels after challenge with OVA in the Pen V-fed group were similar to those of the control group that was not desensitized, which furthers the argument against mediator depletion. This observation correlates with data from human studies, which revealed that immediate wheal and flare skin responses provoked by histamine, compound 48/80, and unrelated antigens were not affected by the oral penicillin desensitization procedure in patients allergic to penicillin (2).

Our time-kinetic studies have revealed marked decreases in plasma levels of PAF and histamine, as well as skin mast cell degranulation in response to challenge injection after oral desensitization (Fig. 2). Additionally, the appearance and subsequent disappearance of antigen-specific mast cell desensitization were associated with the absence and presence of clinical fatal reactions respectively. These data indicate an association of antigen-specific desensitization of mast cells with acute oral desensitization. Several investigators have reported that skin-test responses to β-lactam antigens may be diminished or absent after desensitization of penicillin-allergic patients (1–3, 13, 14), further supporting the antigen-specific desensitization of mast cells in acute oral desensitization.

At present, the mechanism of a temporal desensitization of mast cells is unknown. Receptor-mediated endocytosis is a mechanism by which cells can internalize macromolecular ligands from the extracellular environment (22). Internalized ligand-receptor complexes are identified as degraded, accumulated, or recycled to the plasma membrane as either unbound or ligand-bound receptors. Like many other protein membranes, cross-linking of the FcɛR triggers endocytosis of cross-linked IgE, and co-endocytosis of some monomeric IgE and some uncoupled receptors (23–25). Furuichi et al. (26) have reported that IgE-receptor complexes might normally be released from endocytosed complexes and recycled to the cell surface. Given these observations, it is possible that temporal mast cell desensitization might result from the inability of antigens to bind with their receptors because of internalization of Pen V-specific Abs during oral desensitization procedure, and subsequent recycling of the internalized Abs leads to recurrence of clinical shock symptoms.

Although we have demonstrated temporary mast cell desensitization as the mechanism for the development of acute oral desensitization in this study, it is difficult to definitively state that mast cells are the only cell population responsible for the phenomenon, because mast cells are not essential for the development of the Pen V-induced systemic fatal reaction, in which IgE plays a major role (17). There is no evidence that FcɛRI can be expressed on cells other than mast cells and basophils in the mouse (27). Furthermore, it has been reported that FcɛRI is more widely distributed on hematopoietic cells than previously suspected (28). Given these considerations, cells (e.g. basophils) other than mast cells are probably involved in the development of acute oral desensitization.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This work was supported by the Korea Science and Engineering Foundation (KOSEF) through the Research Center for Allergic Immune Diseases at Chonbuk National University Medical School (R13-2002-038-01001-0).

References

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  • 1
    Reisman RE, Rose NR, Witebsky E, Arbesman CE. Penicillin allergy and desensitization. J Allergy 1962;33:178187.
  • 2
    Sullivan TJ. Antigen-specific desensitization of patients allergic to penicillin. J Allergy Clin Immunol 1982;69:500508.
  • 3
    Sullivan TJ, Yecies LD, Shatz GS, Parker CW, Wedner HJ. Desensitization of patients allergic to penicillin using orally administered beta-lactam antibiotics. J Allergy Clin Immunol 1982;69:275282.
  • 4
    Wendel Jr GD, Stark BJ, Jamson RB, Molina RD, Sullivan TJ. Penicillin allergy and desensitization in serious infections during pregnancy. N Engl J Med 1985;312:12291232.
  • 5
    Borish L, Tamir R, Rosenwasser LJ. Intravenous desensitization to beta-lactam antibiotics. J Allergy Clin Immunol 1987;80:314319.
  • 6
    Papakonstantinou G, Bogner JR, Hofmeister F, Hehlmann R. Cefotaxime desensitization. Clin Investig 1993;71:165167.
  • 7
    Matz J, Borish LC, Routes JM, Rosenwasser LJ. Oral desensitization to rifampin and ethambutol in mycobacterial disease. Am J Respir Crit Care Med 1994;149:815817.
  • 8
    Rosa ML, Romeo MA, Di Felicia F, Russo G. Desensitization treatment for anaphylactoid reactions to desferrioxamine in a pediatic patient with thalassemia. J Allergy Clin Immunol 1996;97:127128.
  • 9
    Battersby NC, Patel L, David TJ. Increasing dose regimen in children with reactions to ceftazidime. Clin Exp Allergy 1995;25:12111217.
  • 10
    Essayan DM, Kagey-Sobotka A, Colarusso PJ, Lichtenstein LM, Ozols RF, King ED. Successful parenteral desensitization to paclitaxel. J Allergy Clin Immunol 1996;97:4246.
  • 11
    Villavicencio AT, Hey LA, Patel D, Bressler P. Acute cardiac and pulmonary arrest after infusion of vancomycin with subsequent desensitization. J Allergy Clin Immunol 1997;100:853854.
  • 12
    Chisholm CA, Katz VL, McDonald TL, Bowes WA Jr. Penicillin desensitization in the treatment of syphilis during pregnancy. Am J Perinatol 1997;14:553554.
  • 13
    Levine BB, Redmond AP, Voss HE, Zolov DM. Prediction of penicillin allergy by immunological tests. Ann N Y Acad Sci 1967;145:298309.
  • 14
    Pedersen-Bjergarrd J. Specific hyposensitization of patients with penicillin allergy. Acta Allergol 1969;24:333361.
  • 15
    Parker CW. Practical aspects of diagnosis and treatment of patients who are hypersensitive to drugs. In: SamterM, editor. Hypersensitivity to drugs. New York: Pergamon Press, 1972:367.
  • 16
    Park JS, Choi IH, Lee DG, Han SS, Ha TY, Lee JH et al. Anti-IL-4 monoclonal antibody prevents antibiotics-induced active fatal anaphylaxis. J Immunol 1997;158:50025006.
  • 17
    Choi IH, Shin YM, Park JS, Lee MS, Han EH, Chai OH et al. Immunoglobulin E-dependent active fatal anaphylaxis in mast cell-deficient mice. J Exp Med 1998;188:15871592.
  • 18
    Fleming TJ, Donnadieu E, Song CH, Laethem FV, Galli SJ, Kinet JP. Negative regulation of FcεRI-mediated degranulation by CD81. J Exp Med 1997;186:13071314.
  • 19
    Braquet P, Etienne A, Touvay C, Bourgain RH, Lefort J, Vargaftig BB. Involvement of platelet-activating factor in respiratory anaphylaxis, demonstrated by PAF-acether inhibitor BN 52021. Lancet 1985;1:1501.
  • 20
    Vilain B, Lagente V, Touvay C, Desquand S, Rendon J, Lefort J et al. Pharmacological control of the in vivo passive anaphylactic shock by the PAF-acether antagonist compound BN 52021. Phamacol Res Commun (Suppl.) 1986;18:119126.
  • 21
    De Weck AL, Girard JP. Specific inhibition of allergic reactions to penicillin in man by a monovalent hapten. II. Clinical studies. Int Arch Allergy 1972;42:798815.
  • 22
    Steinman RM, Mellman IS, Muller MA, Cohen ZA. Endocytosis and recycling of plasma membrane. J Cell Biol 1983;96:127.
  • 23
    Fewtrell C, Metzger H. Larger oligomers of IgE are more effective than dimers in stimulating rat basophilic cells. J Immunol 1980;125:701710.
  • 24
    Furuichi K, Rivera J, Isersky C. The fate of IgE bound to rat basophilic leukemia cells. III. Relationships between antigen-induced endocytosis and serotonin release. J Immunol 1984;133:15131520.
  • 25
    Metzger H, Alcaraz G, Hohman R, Kinet JP, Pribluda V, Quarto R. The receptor with high affinity for immunoglobulin E. Ann Rev Immunol 1986;4:419470.
  • 26
    Furuichi K, Rivera J, Buonocore LM, Isersky C. Recycling of receptor-bound IgE by rat basophilic leukemia cells. J Immunol 1986;136:10151022.
  • 27
    De Andres B, Rakasz E, Hagen M, McCormik ML, Mueller AL, Elliot D et al. Lack of Fc-epsilon receptors on murine eosinophils: implication for the functional significance of elevated IgE and eosinophils in parasitic infections. Blood 1997;89:38263836.
  • 28
    Capron M, Morita M, Woerly G, Lengrand F, Gounni AS, Delaporte E et al. Differentiation of eosinophils from cord blood cell precursors: kinetics of FcεRI and FcεRII expression. Int Arch Allergy Immunol 1997;113:4850.