Naunyn-Schmiedeberg's Arch. Pharmacol. 273, 414--421 (1972) �9 by Springer-Verlag 1972

Properties of the Histamine Stores Affected in the Anaphylactic and Anaphylatoxin Shock

of the Guinea Pig I I . Effect of P r e t r e a t m e n t w i th A n t i g e n *

G. GA~B~ and K. D. F~I~DB~G

Institnt fiir Pharmakologie und Toxikologie der Universitgt GSttingen

Received February 23, 1972

Summary. 1. Sensitized guinea pigs were protected with antihistamine and slowly injected (within 3 rain) with antigen. 50~ of the animals survived. After rapid injection of the same antigen dose ( ~ 20 mg/kg ovalbumin) all animals died.

2. The lung histamine content was decreased to about half of the normal vs in animals which died during the treatment as well as in surviving guinea pigs. 1 week later the lung histamine content was again within the normal range.

3. During this period an additional anaphylactle shock resulted only in a small reduction of the overall lethality (about 20~ Although no antihistamine had been given the animals died significantly later. Plasma histamine levels were depressed, sometimes to about 1/10 of the values found normally in anaphylaetic shock. The lung histamine content did increase from 2 to 5 f~g per g tissue. All parameters measured were normal i week after the antigen infusion.

4. During the same period of time the lethality of an anaphylatoxin shock was not reduced. Plasma histamine levels were diminished whereas the histamine content of the hmgs did increase. As compared to an anaphylatoxin shock in un- pretreated animals not exposed to antigen plasma histamine levels were in the normal range after 1 day. At this moment lung histamine content was still reduced by the antigen pretreatment.

5. We assume that the guinea pig lung contains two different histamine pools, one having a short half-life (less 1 day) and being depleted in anaphylatoxin shock, the other recovering more slowly (within 1 week}, and being released in addition in anaphylaxis. The first one corresponds well with the properties of the non-mastcell-store described by Brodie et al. (1966), the latter seems to be identical with the typical mastcell-pool.

Key words: Histamine -- Anaphylactie Shock -- An~phytatoxin Shock.

The lung his tamine content in the guinea pig shows only a short lasting decrease after adminis t ra t ion of anaphy la tox in (Friedberg a nd Poppe, 1967; Garbe and Friedbe~g, 1972), which is known to be a very

* A preliminary report of this study was presented during the 12. Frfihjahrs- tagung (21.--24. 3. 1971) of the Deutsche Pharmakologisehe Gesellschaft in Mainz (Garbe and Friedberg, 1971).

Properties of the Histamine Stores. II 415

strong histamine liberator in the guinea pig. Surprisingly this decrease had already disappeared 1 h after the injection. Anaphylactie shock in the guinea pig is also accompanied by a strong liberation of histamine. But recovery of the lung histamine content could not yet be studied, because all animals died after administration of a large dose of antigen. We found that under an antihistamine cover and a slow intravenous injection of a large dose of antigen, about 50 ~ of the animals survived. We studied the recovery of the histamine pool in the lung and the changes of plasma and lung histamine after injection of a second antigen dose or anaphylatoxin.

The methods used in this study were the same as in the previous paper (Garbe and Friedberg, 1972).

Results

A. Slow Injection of Antigen First we studied whether there are differences in the rate of survival

ff the time course of the antigen injection was varied. We tried several kinds of slow i.v. injection under mepyramine (100 ~g/kg) (10 or 200/0 of the antigen dose 0.5 rain prior to the rapid injection of the rest; or infusion within 0.6, 6 or 10 min). 5[o noteworthy changes of the survival rate could be observed (45--600/0; mean: 54~ 104 animals). There- fore an infusion time of 3 min was chosen for the experiments. The rate of survival was 51 ~ (243 animals). During the antigen infusion normal signs of shock were observed: respiratory distress, bristling up of hair, later succumbing position and death. The autopsy revealed the typical signs of the protracted shock: dilatation of the right ventricle and atrium together with congestion of the intestine.

We determined the histamine content of the lung in 30 animals which died after the antigen infusion (Fig. 1). The lung histamine was signifi- cantly decreased, indicating that a strong histamine release had taken place. The same loss in lung histamine content was found in animals which showed only moderate signs of shock and which were killed 15 min after the antigen infusion.

B. Recovery el the Lung Histamine Content Animals were killed various periods after the antigen infusion. As

shown in Fig. 1 the lung histamine content is decreased to about 50 ~ immediately after the antigen application and recovers slowly within the first week.

C. Second Antigen Injection I t was to be studied whether the slow injection of the antigen modified

a successive anaphylactie shock elicited by rapid injection of the same antigen dose.

416 G. Garbe and K.D. Friedberg:

30

His p g l ~

20

I0

Ill

I normal

II +

<5 r V V

l -I-

/0

15 rain 6-7

<5 r ~s [ V V V -"

-t-

I to I]O IO to

24 h 2 4 7

T

I . L - - .

L

to Io io

14 21 28 I o

35 42 d

Fig. 1. l~eeovery of the lung histamine content (2 4- s2) after slow i.v. antigen infusion ( ~ 20 mg/kg ovMbumin). [] = animals which died after pretreatment

Table 1. Lethality quotient 100 rain aJter antigen injection in antigen pretreated guinea pigs

• s~5 t-Test

1. shock 21.3 • 0,7 -- 30 rain 8.0 4- 2.8 < 0.00I

6--7 h 10.6 • 3.1 < 0.01 1 d 8.1 4- 1.9 < 0.001 2 d 15.6 :~ 2.8 < 0.1 4 d 16.1 :~ 2.1 < 0.05 7 d 20.9 4- 1.5 < 0.9

14 d 17.3 5- 0.9 < 0.01 28 d 20.1 4- 2.0 < 0.6

1. Lethality. After r ap id ant igen in jec t ion into u n p r e t r e a t e d an imals all ~nimMs d ied in acu te shock. This fac t is documen ted b y ~ high l e tha l i t y quot ien t (Table 1). The p r e t r e a t m e n t wi th ant igen reduced the overal l l e tha l i t y only to a smal l ex ten t (about 20 ~ ) bu t the surv iva l t ime is increased (Table 1).

2. Plasma Histamine Level. The anaphy lae t i e h i s tamine release was reduced to abou t 1/1 o b y p r e t r e a t m e n t wi th ant igen (Fig. 2). 7 to 14 days la.ter i t was agMn res to red to normal i ty .

Properties of the Histamine Stores. I I 417

3 His

pg/ml

t o t o ('N v~

C5 "4 `4 "4 C~ ~

I. shock 30min 6-7h I

tt~

(:5 "W c~

T

2

_[.

to q~

c5

I ! l I0 I~ 4 7 14 d

Fig.2. Plasma histamine levels (2 • s2) after antigen injection (~ 20 mg/kg ovalbumin) in antigen pretreated guinea pigs

25- His

]uglg

20-

15-

o T_ ~ I~1,oI I,ol,ol I. shock 15 30

rain

I,ol,ol I,oM I,o1,ol H,ol I,ol,ol I,ol,ol I,ol,ol 6 -7 1 2 4 7 14 28 h d

Fig.3. Lung histamine content (2 • s2) in antigen pretreated animals r--3 and after a second antigen injection (~_~ 20 mg/kg ovalbumin)

3. Lung Histamine Content. Surprisingly enough the second antigen injection did not cause an additional loss of histamine f rom the lung. A small bu t not significant increase was observed during the first week

418 G. Garbe and K. D. Friedberg:

1.5 His

pg /rnl

1.0

0.5

.2[

20

30rain. I

Fig. 4

15

n - - 5 5 I,o1,1 I,o1,ol I,o1,1 c 2 7d c 30rn[n 1

10

0

Fig. 5

Iii H,I 2

1,01,1 7d

Fig. 4. Plasma histamine level (2 d: s2) in anaphylatoxin shock in antigen-pretreated guinea pigs. C anaphylatoxin shock in untreated animals

Fig.5. Lung histamine content (2 • s2) in antigen-pretreated guinea pigs ~ and after anaphylatoxin shock in those animals

(Fig. 3). Analysis of variance (0.5 h - -4 days) showed a parallel shift, which was significant (p < 0.01).

D. Anaphy la tox in Shock

In addition we examined whether the histamine release by anaphyla- toxin is affected by pretreatment with antigen.

1. Lethality. For a short time (30 min) after the antigen pretreat- ment under antihistamine cover the lethality was reduced by 30 ~ of the controls as documented by a significantly decreased quotient 100: min until death. 1 day later no difference could be observed to unpretreated animals.

2. P lasma His tamine Level. Related to the lethality in the AT shock but in contrast to the anaphylactic shock the plasma histamine level was significantly reduced only 30 min after the antigen pretreatment (Fig.4). 1 day later normal values were obtained again in line with the lethality.

Properties of the Histamine Stores. II 419

3. L u n g His tamine Content. As expected from the earlier experiments lung histamine content was increased after anaphylatoxin application. However a decrease was already observed 2 days after antigen pretreat~ ment (Fig. 5).

Discussion

Regarding the strong loss of histamine in the guinea pig lung we assume that the reactions following intravenous antigen infusion are equivalent with an anaphylactic shock provoeated by rapid injection. I t is the first time that the recovery of the lung histamine content after an anaphylaetic reaction is described. In contrast to the anaphylatoxin shock the lung histamine content needs one week until normal amounts are reached again. This points to the possibility that two pharmaco- logically distinct histamine pools might be present in the lung of the guinea pig. The first one having a short half-life, is depleted by anaphyla- toxin and behaves similar to the "non-mastcell-stores" described by Brodie et al. (1966). This store seems to be able to take up histamine from the circulation (e. g. the non-mastce]l-store of the submaxillary gland of the cat, Erjavee et al., 1967). The increase in the lung histamine content after a second antigen reinjection might point into the same dh-ection. The second histamine store in the guinea pig lung has a long half-life and is in addition being depleted in anaphylaetic shock. I t is known that these "mastcell-stores" can be depleted by compound 48/80. Brodie et al. (1966) postulated that the mastcell-stores can be differen- tiated from the non-mastcell-stores with the use of compound 48/80. I t induces a histamine release rarely exeeding 30 ~ from the guinea pig lung (Feldberg and Mongar, 1954; Mongar and Schild, 1952, 1953, 1956, 1957, 1958 ; I~othschild, 1970). According to the theory of Brodie et al.

(1966) this might be a further indication that there are masteell-stores, too, in the guinea pig lung.

The first information about the existence of two distinct histamine pools in the same organ was reported by Lorenz et al. (1969). He stained sections of the gastric mueosa of the dog with toluidine blue at pit 0.3 and 4.0 by the method described by Enerbgck (1966a--d) and found both, "typical" and "atypical" mastcells. Enerb~ck (1966d) stated that "atypical" mastcells have the properties of the non-mastcell-stores described by Brodie et at. (1966).

The most striking finding after a second rapid antigen injection is the increase in the lung histamine content. In our opinion this increase cannot be explained by an increase in histamine forming capacity (I-IFC; Xahlson et al., 1966). Although the HFC 3 h after an antigen application is increased, the total enzyme activity of the guinea pig lung--as reported by Kahlson et al. (1966)--is so small, that it cannot serve as an explanation. We found the lung histamine content to be increased already in animals which died within 6 rain (~- 2.1 ~g/g tissue). Animals dying in protracted shock (about 25 rain) had a histamine increase averaging 5.2 ~g/g tissue. Because of the short time, too, this phenomenon cannot be mediated by an elevated ttFC.

420 G. Garbe and K.D. Friedberg:

We have to consider, too, whether the increase in lung histamine content can be performed by a deposition of leucocytes and thromboeytes in the lung. Since the antigen infusion is equivalent to an anaphylactic shock it is to be postulated that these particles are deposited already during the antigen infusion. A recovery within 30 rain bringing about an increase in histamine content of 2--5 ~g/g lung tissue by this mechanism seems therefore unlikely.

In addition to the increase of the lung histamine content plasma histamine levels are diminished to a great extent. This fact is also reflected by the lethality. Many of the animals died in protracted shock although no antitfistamine was given together with the rapid antigen injection. We do not think that this fact is brought about by a strong desensitization of the animals. All guinea pigs showed marked signs Of shock and the overall lethality was hardly reduced. I t is more likely that the main histamine pool of the guinea pig-- the lung--has become exhausted and a second antigen contact had to mobilize histamine from pools other than the lung. After one week all parameters of a following anaphylactie shock cannot be distinguished from a shock in control animals.

Another interesting point is how the anaphylatoxin shock is altered by the antigen pretreatment. A decreased plasma histamine level can only be demonstrated 0.5 h after antigen infusion. (Animals which received only antihistamine and anaphylatoxin in the same time interval showed no decrease in plasma histamine level: 1.10 ~: 0.17~zg/ml;

~ s~; n = 6. The reduced lethality 0.5 h after antigen infusion is due to the large dose of antihistamine [mepyramine maleate 100 ~zg/kg] used during antigen infusion.) 1 day after antigen pretreatment anaphylatoxin causes normal high plasma histamine levels. The lung histamine content behaves similar as in anaphy]axis but the time course is different. 2 days after antigen infusion we see a loss again in lung histamine content whereas in anaphylaetic shock still an increase occurs.

The different reactions of the plasma and lung histamine levels after AT or antigen application point to the view that AT depletes histamine from a store with a short half-life and that antigen attacks another store, too, which recovers more slowly. We think that there are similarities to mastcell- and non-mastcell-stores as described by Brodie et al. (1966).

Acknowledgement. This work was supported by grants of the Deutsche For- schungsgcmeinsch~ft.

References

:Brodie, B. B., Beavcn, M. A., Erjavec, F., Johnson, H. L. : Uptake and release of Ha-histamine. In: Mechanisms of release of biogenic amines. Proc. Wenner Gren Center Intern. Symposium Series, Vol. 5, pp. 401--415. London: Pergamon Press 1966.

:Properties of the Histamine Stores. I I 421

Enerbiick, L. : Mastcells in rat gastrointestinal mucosa. 1. Effects of fixation. Acta path. mierobiol, scand. 66, 289--302 (1966@

-- Mastcells in rat gastrointestinal mucosa. 2. Dye-binding and metachromatie properties. Acta path. microbiol, scand. 66, 303--312 (1966b).

- - Mastcells in rat gastrointestinal mucosa. 3. Reactivity towards compound 48/80. Acta path. microbiol, scan& 66, 313--322 (1966c).

- - Mastcells in rat gastrointestinal mucosa. 4. Monoamine storing capacity. Acta path. microbiol, scand. 67, 365--379 (1966d).

Erjavee, F., Beaven, hi. A., Brodie, B. B. : Uptake and release of H3-histamine in cat submaxillary gland. Fed. Proc. 26, 237--240 (1967).

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- - -- Effect of antigen and organic bases in intra-cellular histamine in guinea pig lung. J. Physiol. (Lond.) 131, 207--219 (1956).

-- -- Inhibition of the anaphylactie reaction. J. Physiol. (Lond.) 135, 301--319 (1957).

-- -- The effect of calcium and pH on the anaphylactic reaction. J. :physiol. (Lond.) 140, 272--284 (1958).

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K. D. Friedberg Insti tut fiir :pharmakologie nnd Toxikologie der Universit~t D-3400 G6ttingen, Geiststr. 9 Germany