Immunology, 1964, 7, 700.

The Mechanism of Anaphylaxis

II. THE ROLE OF 'MAST CELL SENSITIZING' ANTIBODY
AND DELAYED HYPERSENSITIVITY IN RAT ANAPHYLAXIS

I. MOTA*

Division of Biological and Medical Research,

Argonne National Laboratory,
Argonne, Illinois

(Received 22nd January 1964)

Summary. Both immediate and delayed cutaneous reactions were found to be
present in rats actively sensitized by the injection of antigen plus Bordetella pertussis
organisms. Delayed hypersensitivity preceded the appearance of 'mast cell sensitizing' antibody (MCSAb) in those animals which were only fully susceptible to
anaphylaxis when both delayed and immediate reactions had reached their maximum. Passive anaphylaxis with MCSAb was fatal only when transferred to actively
sensitized rats at a time when the animals had delayed cutaneous reactions but
were not yet fully susceptible to anaphylaxis. It is suggested that anaphylaxis in
rats is due to an additive or synergistic effect between immediate and delayed

hypersensitivities.
INTRODUCTION
The preceding paper (Mota, 1964) showed that rats immunized under given conditions
produced a thermolabile antibody causing mast cell damage upon reaction with antigen.
The antibody was termed 'mast cell sensitizing' antibody (MCSAb). Injection of Bordetella
pertussis, in addition to the antigen, proved particularly effective in stimulating the production of this antibody. Although there was little room for doubt that B. pertussis acted
through an adjuvant effect, resulting in increased production of MCSAb, it was not
established that this antibody alone could produce the severe and frequently fatal anaphylaxis observed in rats sensitized with B. pertussis. The present paper reports the results of
investigations undertaken to examine the role ofMCSAb in rat anaphylaxis.
MATERIAL AND METHODS
Animals, proteins, drugs and techniques, with the exceptions noted below, were the
same as described in the previous paper (Mota, 1964).

Production of Active Cutaneous Anaphylaxis (ACA)

ACA reactions were induced in actively sensitized rats known to contain serum MCSAb
by intravenous injection of 2-5 mg. Evans blue in 1 ml. saline followed immediately by
* Present address: Department of Histology, Medical School, University of Sao Paulo, Sao Paulo, Brazil.

700

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Mechanism of Anaphylaxis
intradermal injection of 0-1 ml. saline (0-85 per cent NaCI) containing small graded doses
of the specific antigen. The animals were killed 25-30 minutes after antigen injections and
the lesions were measured in the inner side of the skin with a transparent ruler. Control
reactions were obtained by intradermal injection of 0-1 ml. saline containing antigen in
nonsensitized rats and 0-1 ml. saline in sensitized animals.

Technique for Obtaining Serum and Testing for MCSAb

Small samples of serum were obtained from sensitized animals by puncture of the
ophthalmic venous plexus according to the technique described by Halpern and Pacaud
(1951). The presence of MCSAb in these samples was determined by passive cutaneous
anaphylaxis (PCA) allowing a 72 hour interval between antiserum and antigen injections.
RESULTS
(5HT) IN ACA
If MCSAb were responsible for all systemic anaphylaxis in rats, the findings in the
preceding paper that PCA induced with MCSAb can be completely prevented by
pretreatment with an antihistamine [mepyramine (pyrilamine maleate, U.S.P.)] and an
anti-5HT, [2-bromo-D-lysergic acid diethylamide (BOL-148)] would be at variance with
previous observations that neither antihistamines nor anti-5HT drugs nor both together
protected rats from anaphylactic death (see Mota, 1963 for references). Therefore, it
seemed necessary to find out whether ACA reactions induced in rats sensitized with
antigen plus B. pertussis could also be abolished by mepyramine and BOL-148. ACA reactions were induced by intradermal injection of 0'1 ml. saline containing 0-2, 0.1, 0 05,
0-025 or 0'01 mg. ovalbumin (Ea) in the dorsal skin of sensitized control animals and
sensitized animals pretreated with mepyramine plus BOL-148. When the reactions were
read 30 minutes later, all sensitized control rats had lesions that varied in diameter from
10 to 22 mm., whereas the animals pretreated with mepyramine and BOL-148 showed
no visible reaction.
ROLE OF HISTAMINE AND 5-HYDROXYTRYPTAMINE

PRESENCE OF A DELAYED CUTANEOUS REACTION

The above results appeared indeed to indicate a discrepancy between the effect of
antagonists of histamine and 5HT in systemic anaphylaxis and the effect of these same
drugs in ACA. However, when a group of actively sensitized rats pretreated with BOL- 148
and mepyramine, and challenged without producing any visible reaction in the injection
sites at 30 minutes, was examined 4 hours later, a faint but definite blue colour was seen
around the injection sites. The reaction increased with time to form a blue ring surrounding a hard and deep nodular lesion which was still present 24 hours later, and in some
animals even 48 hours later, by which time a central necrotic area had developed. Because
of the very low level of precipitating antibody in the sera of these animals and the slow
development of the lesions, they were considered as delayed cutaneous reactions. It seemed
clear then that these animals had a mixed cutaneous reaction in which the first immediate
type hypersensitivity reaction was followed by a delayed-type reaction and the effect of
mepyramine and BOL-148 was to suppress the first reaction without interfering with the
second.

702

I. Mota

THE EFFECT OF MEPYRAMINE AND BOL-148 ON PCA AND ACA INDUCED SIMULTANEOUSLY IN
THE SAME ANIMAL

In the face of these results more experiments were needed to eliminate the possibility
that similar reactions could occur after passive sensitization with rat antiserum taken
from animals giving delayed cutaneous reactions and having MCSAb in their sera.
Accordingly, rats actively sensitized to Ea were prepared for PCA by injecting 0-1 ml. of
three different rat antisera containing MCSAb to human serum albumin (HSA) in one
side of the dorsal skin. Seventy-two hours later the animals were injected intravenously
with 1 ml. saline containing excess HSA and Evans blue, and immediately afterwards
they were injected in three sites on the non-injected side of the dorsal skin with 0.1 ml.
saline containing 0-01, 0-1 and 0-2 mg. Ea. One group was given no other treatment;
another group was pretreated with mepyramine and BOL-148. The results of these experiments showed that, 30 minutes after challenge, both PCA and ACA reactions were
present in the control animals; in the animals pretreated with mepyramine and BOL-148
no reaction could be detected. This was verified by external inspection of the injection
sites and by killing some of the animals and observing the lesions on the inner side of the
skin. The remaining animals were then observed after 6, 12 and 24 hours. Deep indurated
lesions were detected at 6, 12 and 24 hours in the sites prepared for ACA, but at no time
did the sites prepared for PCA show accumulation of dye or any other visible reaction.
ROLE OF MCSAb AND DELAYED HYPERSENSITIVITY IN RAT ANAPHYLAXIS

The results of the previous experiments suggested that anaphylaxis in rats is the result
of a mixed reaction in which both immediate and delayed hypersensitivity are present.
Experiments were then planned to examine the role of each of these factors in systemic
anaphylaxis in the rat.
Passive Systemic Anaphylaxis with MCSAb
To assess the role of MCSAb in systemic anaphylaxis, passive systemic anaphylaxis was
induced with MCSAb. Ten rats were injected via the tail vein on each of two consecutive
days with 5 ml. of rat antiserum containing MCSAb to Ea. Seventy-two hours after the
first injection they were injected intravenously with 10 mg. Ea. One to 2 minutes after
antigen injection all animals showed signs of anaphylaxis such as difficulty in breathing,
weakness and hyperaemia of ears and feet. Symptoms increased with time, and at 30
minutes the animals were prostrate and cyanotic. However, these symptoms subsided
within 1 hour and all the animals had recovered by 2 hours after antigen. Six hours later
the animals were killed, and the skin was fixed and stained as described elsewhere
(Mota, 1957). Microscopic observation of the subcutaneous tissue of these animals showed
generalized mast cell disruption.
TIME COURSE OF APPEARANCE OF DELAYED HYPERSENSITIVITY, MCSAb AND ANAPHYLAXIS IN
ACTIVELY SENSITIZED RATS

The time course of appearance of MCSAb and delayed hypersensitivity was then
studied in an attempt to establish the relative importance of these two factors in systemic
anaphylaxis in rats. A large number of animals was injected with 0-25 ml. B. pertussis
containing 0-25 mg. Ea in the dorsal skin of each foot. At 3, 4, 5, 6, 8 and 12 days groups

703
Mechanism o Anaphylaxis
of animals were injected either intradermally, with 0-1 ml. saline containing 0-01 or 0-2
mg. Ea, for cutaneous reactions, or intravenously, with 10 mg. Ea, for systemic reactions.
In all instances the animals were bled before antigen injection and their sera were tested
for MCSAb. The results of this experiment are shown in Table 1. It can be seen that 4 and
TABLE 1
OF DELAYED CUTANEOUS REACTIONS, MCSAb AND ANAPHYLAXIS IN RATS SENSITIZED WITH ANTIGEN PLuS B. pertussis

DEVELOPMENT

Days after
sensitizing
injection

cutaneous
reaction

3
4
5
6
8
12

0/5
2/5
5/6
5/5
7/8
10/10

Delayed

Ssei

MCSAb

Ssanaphyais

0/10
0/10
0/12
11/15
16/18
20/20

0/5
0/5
0/6
10/10 (3 died)
10/10 (8 died)
10/10 (all died)

In each column the first figure represents the number ofanimals giving a
positive test and the second figure the total number of animals tested.

5 days after the sensitizing injection delayed cutaneous reactions were already present, but
no MCSAb or systemic anaphylaxis could be detected at that time. However, on the
sixth day most of the animals gave both MCSAb and delayed cutaneous reactions, and
mild or strong shock resulted after intravenous injection of antigen. It must be noted that,
although all the animals showed signs of shock (such as difficulty in breathing and weakness) immediately after antigen injection, these signs had disappeared and the animals had
apparently recovered about 1 hour later. However, 6 hours after antigen injection the
animals were again weak, and 4 hours later, when they were observed for the last time
that day, they were all prostrate. On the following day all had recovered except for three
that were found dead. These three animals had MCSAb in the serum, but so did some
of the animals that did not die (see Table 1). On the eighth day and even more on the
twelfth day the animals had cutaneous reactions of the immediate type, followed by very
conspicuous delayed reactions which attained maximal intensity 48 hours later. On the
twelfth day all animals had MCSAb in the serum and all showed very severe and fatal
anaphylaxis.
PASSIVE SYSTEMIC ANAPHYLAXIS WITH MCSAb IN RATS WITH DELAYED HYPERSENSITIVITY

The previous experiment suggested that actively sensitized rats are fully susceptible to
anaphylaxis only when both immediate and delayed hypersensitivity are fully established.
In order to test this possibility, rats were actively sensitized by injecting Ea plus B.
pertussis, as already described. Some of the animals were then passively sensitized by
intravenous injection of 5 ml. of rat antiserum containing MCSAb to Ea (PCA titre, 100)
on the third and fourth days after the antigen injection. A control group of nonsensitized
rats was passively sensitized in this same way. Seventy-two hours after the first injection
of antiserum and 6 days after antigen injection, some of each group of animals were
injected intradermally with 0 01, 0 1 or 0-2 mg. Ea for cutaneous reactions, and some were
injected via the tail vein with 1 ml. saline containing 10 mg. Ea for systemic anaphylaxis.

704
I. Mo)ta
The results of this experiment are given in Table 2. All animals in the three groups showed
anaphylactic shock when challenged intravenously with antigen, but shock was much
more severe in the animals subjected to both active and passive sensitization, all of which
died less than 2 hours after challenge. None of the animals in the passively sensitized
group died from anaphylactic shock, and only three of ten in the actively sensitized group
died (many hours after antigen injection).
TABLE 2
THE RESPONSE OF RATS TO ACTIVE, PASSIVE, AND ACTIVE PLUS PASSIVE SENSITIZATION
Passive anaphylaxis was induced with MCSAb in actively sensitized rats at a time when cutaneous
delayed reactions were present, but the animals were not yet fully susceptible to anaphylaxis.

Time between
active sensitization and
challenge (days)

Treatment
Active sensitization with Ea

plus B. pertussis

Passive sensitization with

MCSAb to Ea
Active and passive sensitization to Ea

Time between
passive sensitization and
challenge (days)

3
6

Cutaneous

delayed
deaty

reaction

Time between
A.l*
y*

8/10

10/10 (3)

15/15 (0)

6/6 (6)

challenge
ang

death (hr.)

>10

<2

* Positive reactions/total number of animals. Figure in parentheses indicates number of deaths.

pertUssiS-TREATED RATS
The fatal anaphylaxis presented by the animals subjected to passive anaphylaxis 5 days
after receiving an injection of antigen plus B. pertussis might be interpreted as a consequence
of increased sensitivity due to treatment with B. pertussis organisms (Kind, 1958). In
order to investigate this possibility, rats were injected with B. pertussis in the same way as
described for active sensitization except for the omission of antigen. Two days later five of
these animals plus five untreated control rats were passively sensitized by intravenous
injection of 10 ml. of rat antiserum containing MCSAb to Ea. Passive anaphylaxis was
then induced by intravenous injection of 10 mg. Ea 72 hours later. Both untreated and
B. pertussis-treated animals presented very strong anaphylactic shock. Thus, pretreatment
with B. pertussis does not increase the sensitivity of the animals to passive anaphylaxis with
MCSAb, a result in agreement with results of previous experiments using precipitating
antibodies (Mota, 1962).
PASSIVE SYSTEMIC ANAPHYLAXIS IN B.

DISCUSSION
The difficulty of sensitizing rats so as to cause severe or fatal anaphylaxis on later
challenge with the specific antigen is well known. This holds true also for passive sensitization, injection of considerable amounts of precipitating antibody being required to
induce anaphylactic shock of medium intensity (Mota, 1962). However, in 1952 Malkiel
and Hargis showed that this resistance could easily be overcome when the animals were
inoculated with B. pertussis in addition to, and simultaneously with, the antigen. Rats
sensitized in this manner show very severe shock, which is 90-100 per cent fatal, when
they are challenged 10 or more days after sensitization. The increased sensitivity to

Mechanism of Anaphylaxis

705

histamine and 5HT, which also occurs as a result of B. pertussis inoculation, was first
thought to explain the severity of anaphylaxis in these animals, but it was later shown
that the increased sensitivity to anaphylaxis was still present at a time when hypersensitivity
to histamine and 5HT had disappeared (Munoz, Schuchardt and Verwey, 1958). Later
it was shown that a striking increase in the anaphylactic mast cell damage also resulted
after sensitization with B. pertussis (Mota, 1958). However, the observations showing that
rats sensitized in this way are not protected by previous treatment with antagonists of
histamine and 5HT or by depletion of the mast cell population with the compound 48/80
(Mota, 1963) strongly suggested that damage to the mast cells alone could not account
for anaphylactic death in this species. Nevertheless there can be little doubt that B.
pertussis has an adjuvant effect that is reflected in an increase in agglutinins (Kind, 1957),
precipitating antibodies, MCSAb and in the establishment of a delayed hypersensitivity,
as shown in this paper. The increase in the production of the usual serum precipitating
antibody does not seem to account for the severe anaphylaxis in these animals, since rats
immunized with repeated doses of antigen, and having much higher levels of precipitating
antibody than rats injected with a single injection of antigen plus B. pertussis, do not show
the severe and fatal anaphylaxis exhibited by the latter. Thus, it seems that either enhancement in the production of MCSAb, or the presence of a delayed type of hypersensitivity, or both, are left to account for anaphylaxis in this species. When the role
played by MCSAb is judged by the experimental results in this paper, it seems that the
production of this antibody alone cannot be entirely responsible for anaphylactic death
in these animals. The results showing that passive sensitization with MCSAb is only able
to mimic the first stage of ACA, and that antagonists of histamine and 5HT, although
able to prevent PCA with MCSAb, are not able to completely prevent ACA, indicated
that something not attained by passive sensitization with serum was present in actively
sensitized animals. Whether the absence of fatal anaphylaxis after passive sensitization
with MCSAb was due to a low dose of antibody or to its inability to induce all the reactions that occur in actively sensitized animals is not known. However, the fact that
generalized mast cell damage was found in the animals subjected to passive systemic
anaphylaxis suggests that the amount of antibody used was at least sufficient to sensitize
these cells.
The results of the experiments in Table 1, which show that delayed cutaneous reactions are present at a time when MCSAb had not appeared and anaphylaxis could not
be induced, together with the observation that fatal anaphylaxis could not be obtained
after passive sensitization with MCSAb, suggest that both delayed hypersensitivity and
MCSAb may be required simultaneously for the full development of anaphylaxis in this
species. The fatal anaphylaxis that results when passive sensitization with MCSAb is
conferred on actively sensitized animals at a time when they show delayed cutaneous
reactions but are not yet fully susceptible to anaphylaxis, strongly supports this idea. In
this respect it may be worth mentioning the observation that anaphylactic death in rats
usually occurs some hours after antigen injection, although a small number of animals
may die within a few minutes after challenge. Taken together, these observations suggest
that anaphylaxis in rats sensitized with antigen plus B. pertussis is probably a complex
reaction brought about by a mixture of immediate and delayed hypersensitivities. Whether
the simultaneous presence of these two types of hypersensitivity results in an additive or
synergistic effect is not known, and further investigations are required to elucidate this

point.

L Mota
ACKNOWLEDGMENTS
It is a pleasure to acknowledge the skilled technical assistance of Mrs. Marilyn Williams
and Miss Sally Vierling. This work was supported by the U.S. Atomic Energy Commission.
I am grateful to Sandoz Pharmaceuticals for a generous gift of BOL-148.

706

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