Inhibition of the Contact Reaction to Dinitrochlorobenzene by Intravenous Injection of Dinitrobenzene Sulfonate in Guinea Pigs Sensitized to Dinitrochlorobenzene

INHIBITION OF THE CONTACT REACTION TO DINITROCHLOROBENZENE BY INTRAVENOUS INJECTION OF DINITROBENZENE SULFONATE IN GUINEA PIGS SENSITIZED TO DINITROCHLOROBENZENE* J. R. FREY,t M.D., A. L. DE WECK4 M.D. AND H. (iELEICKt

To influence the state of hypersensitivity in

been revised by Kligman (28, 29) who concludes

contact-type sensitization, two kinds of ap-

that a partial and temporary hyposensitization may be achieved after oral or intramuscular ad-

proaches have been followed: on one hand, already sensitized individuals were treated with the causal agent, in an attempt to diminish or abolish their enhanced reactivity; on the other,

ministration of the allergen for 5 to 9 months but

that severe systemic and local reactions occur, when the allergen is given during the acute phase

normal individuals were pretreated with the of the dermatitis. causal agent in order to prevent them from In experiments with guinea pigs Stratton (40, reacting to a subsequent sensitizing contact with 41) obtained a certain "skin protection" for at least six months after giving whole extracts of poison oak subcutaneously, intramuscularly or

the same substance. The first of these technics ("specific desensitiza-

tion") was used as early as in 1895 by J. orally.

The other approach, i.e. the pretrcating of Jadassohn, when he postulated, even before Richet's and von Pirquet's fundamental dis- normal individuals with the causative agent in coveries, that sensitization could be the basic order to prevent subsequent sensitization was mechanism of eczcmatous dermatitis due to first carried out by Sulzberger (42). In his reexternal agents. Desensitization of individuals markable experiments, in which the studies of with contact dermatitis due to drugs has been W. Frei (13, 14) were confirmed and continued, performed with variable results by progressive one of the projected aims (desensitization of systemic or local application of the drug (20, 26, 35). It has been claimed too (22, 21, 32, 5) that in cases of occupational dermatitis, desensitiza-

animals previously sensitized to arsphcnamine by an intracardial injection of a high dose of this

tion may occur by repeated re-exposure to the

achieved, but sensitization was prevented, when

causative agent. In animals sensitized experimentally to DNCB,

the intracardial injection was given 24 hours after the sensitizing contact. This sequence of

drug administered 14 days later) was not

benzylchloride and picrylchloride, Landsteiner events, which theoretically would not have been and Jacobs (30) could show, that by repeated expected, seems to be characteristic for arsphensubcutaneous injection of a conjugate of these amine and does not occur, as will be shown, with compounds with guinea pig serum, the con- other chemical compounds. comitantly existing sensitivity of the immediate Experiments designed to prevent sensitization type disappeared, but not the contact sensitivity to simple chemical compounds seem not to have of the delayed type. W. Jadassohn and co- been pursued until 1943, when Chase (6) by "a workers (27, 24, 25) could considerably reduce fortunate rediscovery" demonstrated that feedthe level of sensitivity of guinea pigs previously ing the allergenic chemical (DNCB) prior to sensitized to DNCB by the daily application of inducing sensitization, resulted in only weak skin this compound on the animal's nipple for 150 test reactions, and some animals were entirely consecutive days. In hypersensitivity of plant origin, desensitiza-

unresponsive (7, 8). Similar results were obtained by Baer and cotion has been extensively tried with variable workers (1, 2) with feeding pregnant guinea pigs results. This subject which has been inaugurated DNCB or picryl chloride and subsequently sen-

by McNair (33) and Shelmire (38) has recently

sitizing their offspring. Later, this group of investigators (23) could obtain a significant reduc-

Received for publication January 10, 1963. *

From the Medical Research Department of F. Hoffmann-La Roche & Co. Ltd.,t Basic, Swit-

tion of the susceptibility to sensitization to

Inselspital, University of Bern, Switzerland.

intraperitoneally injected with DNCB during

zerland and the Department of Dermatologyj

DNCB in the offspring of guinea pigs which were

189

190

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Some groups of animals were sensitized by intrapregnancy. This actively acquired tolerance appeared to be specific since it did not extend to dermal injection in the neck of 0.1 ml of Freund's adjuvant, containing 0.05% DNCB, given on 3 citraconic anhydride, as immunologically un- alternate days (total amount of DNCB = 150 ag).

related compound. Still unresolved is the question of whether the

type of experiments commonly designated as "desensitization" and those envisaging unresponsiveness (which may be closely related to what is

called Immuno-Paralysis and Immuno-Toler-

ance) are based on a similar immunologic mechanism (9, 36).

b) DNBSO3: Intradermal injection of 0.1 ml of a

20% aqueous solution, given on 3 alternate days (total amount = 0.06 g). c) CPT: Intradermal injection of 3 )< 0.1 ml of a

20% aqueous solution given on alternate days (total amount = 0.06 g).

d) Some animals were injected simultaneously with DNCB and CPT given in Freund's adjuvant in order to obtain sensitization to both immunologically unrelated substances on the same animal.

In previous experiments (16) attempting de- 0.1 ml of Freund's adjuvant containing 0.05% sensitization, we have observed in guinea pigs DNCB and 10% CPT was injected intradermally sensitized to DNCB a not reproducible lowering on 3 alternate days. of the level of sensitivity after massive epicutane-

ous contacts with the compound on extensive skin areas.

Recently (17) we demonstrated that the contact type of hypersensitivity of guinea pigs to DNCB, as manifested by the epicutaneous test, can be partially and specifically inhibited by an injection of DNCB given in a vein of the hind leg 6 hours before performing the challenging test. This inhibitory effect was temporary, and the animals regained their original level of sensitivity a few days after the injection. In the present paper, experiments are described

in which not only a partial but also complete inhibition of the hypersensitivity was obtained

Skin Testing

DNCB: The tests were performed by epicu-

taneous application with the aid of a micropipette on the flank of 0.025 ml of 0.09, 0.05 and 0.03 per cent solutions of DNCB in acetone over three skin

areas of 2 cm'. The total amounts of DNCB applied were 22.5, 12.5 and 7.5 jig.

These and the other test solutions described

below are primarily non-irritating. DNBSO,: Epicutaneous application of 0.025 ml of 20, 6 and 2 per cent aqueous solutions as described above (total amounts 5 mg, 1.5 mg and 0.5 mg respectively). The reactions were stronger when the test solutions contained 10% "Emulgen* or 2% lauryl sulfate as wetting agents. CPT: Epicutaneous application of 0.025 ml of aqueous solutions of 10, 3 and 1 per cent. (Total

in one third of the animals injected intravenously amounts 2.5 mg, 0.75mg and 0.25mg respectively.) The test reactions were read 24 hours after apwith the immunologically related sodium dinitro- plication of the solution. Their intensity was benzenesulfonate (DNBSO3). The greager solu- evaluated as follows: bility and lower toxicity of this substance made 0.5: isolated red spots 1: diffuse slight redness possible the administration of larger amounts 2: marked redness and slight swelling than DNCB. 3: deep redness and considerable swelling The sum of the readings obtained with the three MATERIALS AND METHODS

different test concentrations of each drug gives Reagent grade 2:4 dinitro-1-chlorobenzene some numerical estimate of the hypersensitivity (DNCB) from Merck, Darmstadt; 2:4 dinitro- level of the animal tested which may be used to benzenesulforiic acid sodium salt (DNBSO3) from determine the average sensitivity of a group of Eastman Kodak, Rochester, N.Y. and a-2-chlor- animals. Details of the method of testing and its 9-(3-dimethyl-aminopropylidefle) thioxanthene hydrochloride, chiorprothixene hydrochloride (CPT) from Hoffmann-La Roche Inc. Base!.

Freund's adjuvant was prepared according to the original procedure (15). White semi-inbred guinea pigs of both sexes

quantitative evaluation have been published elsewhere (18, 19). Animals sensitized by one single application of DNCB have a relatively low sensitivity level (3.4 to 2.8) whereas animals sensitized

by injection of DNCB and Freund's adjuvant exhibit a higher sensitivity level (6.6 to 3.8).

weighing 400—500 gm were used throughout.

Sensitization Technic a) DNCB: Most of the animals were sensitized

by one single epicutaneous application of 0.002 ml of a 50% solution in acetone (total amount

0.001 gm) on a skin area of 0.5 cm2 located on the

neck. By such a procedure nearly 100% of the animals become sensitized (18, 19).

Inhibition of Contact Reaction Fourteen days after the sensitizing application (or injection) the animals were tested in order to evaluate their hypersensitivity level and the tests read and evaluated on the 15th day (first or "reference test"). On the 17th day after sensitization, *

Octaethyleneglycol monodecylether.

SPECIFIC INHIBITION OF CONTACT DERMATITIS

191

the animals were injected intravenously with DNBSO3 solutinn. Six hours later they were tested

again in the usual way (second or "challenge

al i._°ft_—"\. _—

test"). Additional skin tests were performed 1, 5 and 12 days after the intravenous injection. DNBSO, was given in doses of 500 and 1000 mg/kg in a vein of the hind leg (injection of 2 or 4 ml/kg respectively of a 25% aqueous solution). Under short ether anesthesia the skin covering the vein was incised, the vein penetrated directly bI

days

with the needle and the incision closed with Michel clamps. Controls

The following groups of control animals were set up, the guinea pigs being sensitized and tested

cl:,

in the same way as were the group receiving intravenous DNBSO3:

a) Intravenous saline injection + anesthesia + skin incision b) Anesthesia + skin incision c) Skin testing only ("positive controls" not

dl

:Fct1s1

days

2animaa

days

068603 iv os mgtkg

2 0

10

mentioned in our figures). Other groups were not sensitized hut had:

d) Skin testing only ("negative controls")

e) Intravenous DNBSO2 injection + skin testing. Control groups a) and b) would show the part played by the liquid injected and by the operative trauma. Group c) would show the influence of repeated skin testing on the sensitivity level. Group d) and e) should show the effect of repeated skin testing and intravenous injection of DNBSO3 on the hypersensitivity level of normal animals.

el

-

14 animals

1

3 2

20

30 days

16 animals

1

0

0

10

20

st days

Fm. 1. Inhibition of the contact reaction to DNCB by intravenous injection of DNBSO, in

guinea pigs sensitized by epicutaneous application of DNCB. Course of the average sensitivity level in a) guinea pigs sensitized by one application of DNCB on the neck. First series of tests after 14 days. Intravenous injection of 500 ing/kg DNBSO, in saline on the 16th day. Application of EXPERIMENTS AND RESULTS the challenge tests 6 hours after the intravenous Further series of tests at later intervals. 1) Cross-reactions between DNCB and DNBSO3 injection. b) control group of sensitized guinea pigs tested same intervals but receiving intravenous injecIn order to demonstrate the immunologic rela- at tion of saline alone. c) control group of sensitized tionship between DNCB and DNJISO3, various guinea pigs tested at same intervals but receiving only anesthesia and skin incision. d) control group of non-sensitized guinea pigs tested at same inter-

TABLE 1 Cross reactions between DNCB and DP'/BSO:i

vals and receiving 500 mg/kg DNBSO3 intravenously on the 16th day. e) control group of nonsensitized guinea pigs tested at same intervals.

Animals Tested with:

groups of guinea pigs were sensitized and tested DNa so,

DNCB

Animals

Sensitized with:

Tests Total no. positive of animals

DNCB DNBSO3

44

23

Tests positive

44 24

26 7 5 6 20 S

Total no.

with one or the other compound. As shown in Table I, positive skin tests could be elicited with

of animals

both substances, regardless of which one had been

32*

used for the sensitization. Higher proportions of positive tests with DNBSO2 were obtained with

7t

aqueous solutions of the same concentration

St

when wetting agents were added, as already reported by Eisen et al. (10).

16

20t St

* DNBSO2 in aqueous solution.

t DNBSO, in aqueous solution + Emulgen or lauryl sulfate.

2) Inhibitory Effect of DNBS'O3 (Fig. 1, Table 2)

According to the technic described above, 59

guinea pigs were sensitized against DNCB, tested after 14 days in order to determine their

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

192

TABLE 2 Inhibition of the contact reaction to DNCB by intravenous injection of DNBSOS in guinea pigs sensitized by epicutaneous application of DNCB Intensity of the reactions to the challenge test Intensity of the Reactions (Sensitivity Level) Experiment

Animals

500 mg/kg DNBSO3 iv.

59

100%

2 mi/kg saline i.v. Anesthesia + skin incision only

0

<1

<2

<3

20 34%

24 41%

13

22%

2 3%

0 0%

34 52%

29%

28 45%

28%

66

0

1

12

100%

0%

1%

18%

62

0

2

15

100%

0%

3%

24%

3

19

17

Challenge test applied 6 hours after the intravenous injection. The intensity of the reactions represents the average value of the 3 tests per animal of each group (see Materials and Methods).

sensitivity level ("reference test") and injected way but was submitted only to anesthesia, skin intravenously with DNBSO3 at the dose of 500 incision and saline injection. A third group (c) mg/kg one day later. Six hours after the injec- was equally sensitized receiving anesthesia only. tion, they were tested with DNCB for the second Groups (d) and (e) were sensitized to DNCB or time ("challenge test"), this test being read 24 CPT only (positive controls). Group (f) was hours later. Upon injection of DNBSO3, the sensitized to CPT and injected with DNBSO3, hypersensitivity level of the animals to DNCB and finally group (g) was not sensitized but rewas markedly decreased, as shown by comparing the intensity of the reactions observed in the same

ceived DNBSO3.

animals to the same test concentrations before and after the injection.

with 500 mg/kg DNBSO3, the challenge test with

In the ten double-sensitized animals injected

DNCB was partially inhibited, the sensitivity level against CPT remaining practically uniminhibitory agent, only some diminution of the paired. The injection of DNBSO3 in animals hypersensitivity level was obtained (17). In the sensitized to CPT only did flot reduce signifipresent experiments with DNBSO3, approxi- cantly their sensitivity level to CPT. of the 59 animals showed complete Previous experiments on animals similarly mately inhibition, the challenge tests being negative with double-sensitized or sensitized to CPT only, all concentrations used. Another j!4 showed which received an intravenous injection of DNCB partial but marked inhibition, the challenge already demonstrated the specificity of the inreactions being weaker than degree 1. This con- hibitory effect (17). trasts strikingly with the two control groups d) and e) of 66 and 62 animals, where no negative 4) Flare-up Reactions (Fig. 3, Table 3) In our previous experiments using DNCB as an

reactions were found,

of them showing reac-

tions of degree 3 or stronger. In animals not sensitized previously to DNCB, the intravenous

injection of DNBSO3 did not seem to induce sensitivity.

3) Specificity of Inhibition (Fig. 2) Ten animals (group a) were sensitized simultaneously to DNCB and to CPT, and were subsequently injected with 500 mg/kg of DNBSO3. A control group (b) was sensitized in the same

In the same group of animals mentioned under 2), it was observed that the reference test became distinctly stronger 48 hours and 72 hours after the injection of DNBSO3. This occurred concomitantly with the inhibitory effect on the

challenge test, performed 6 hours after the intravenous injection. This flare-up reaction could be observed in 32 out of the 59 animals receiving DNBSO3, the remaining 27 animals of

the group having not been followed up in this

SPECIFIC INHIBITION OF CONTACT DERMATITIS

)

and could well be recognized after 24 hours. At

iv

ONCBsCPT

—-

ii 089003 is

Tianimais

is

ONCBnCPT

dl CEO

30 days

30 days

Ap

.i0.ii ii

3

20

el

days

2J i-i

—

i$0

500mg/kg ——

- —— .]L..

——

diffusely erythematous skin, the reference tests were readily observed as more erythematous and swollen areas. The evaluation of the challenge tests, however, was sometimes made difficult by the erythematous background. The generalized dermatitis was observed quite regularly in a first group of 6 animals which had

days

days

venous

injection of 500 mg/kg of DNBSO3.

These animals had a high sensitivity level (5.3) to start with. On the other hand, animals sensitized by epicutaneous application of DNCB alone and

——

DNCB

5 animals

'

20

30 days

i OPESO3 iv

DtdBii3 iv

a)

500mg/kg

o

peared on the third day after the onset of the dermatitis and lasted for several days. On the

been sensitized by intradermal injection of DNCB in Freund's adjuvant and which received an intra-

uana:

COT

fl

regular (roseoliform). A generalized scaling ap-

Li

2i

10

OSCOsCPT

ONCe

the onset, it was diffuse and later became ir-

—

__—

;Tas 1 %——

193

iii mg/kg

p

2

S animals

ii

32 animals

-

- 30 days

i

FIG. 2. Specificity of the inhibitory effect of intravenously injected DNBSO3 on the contact reaction to DNCB. Course of the average sensitivity level to DNCB and CPT in: a) guinea pigs

10

ii

30

20

30

20

Ii

days

0NCB

double-sensitized by one application of DNCB and

three injections of CPT. Intravenous injection of

4 OdaCI iv

500 mg/kg DNBSO3 in saline. b) control group of

double-sensitized animals injected with saline only. c) control-group of double-sensitized animals

received only anesthesia and skin incision. d) control group of animals sensitized to DNCB only. e) control group of animals sensitized to

a

DNBSO3 in saline. g) control group of nonsensitized animals tested at same intervals and

DNCB

41 animals

CPT only. f) control group of animals sensitized to CPT only. Intravenous injection of 500 mg/kg receiving 500 mg/kg DNBSO1 intravenously on the 10th day.

respect. In control animals, the reference test

'1

ii

An Anesthesia

a) 38 animals

presented the normal vanishing course. 5) Generalized Dermatitis

In two groups of animals, the intravenous injection of DNBSO3 elicited a generalized dermatitis, characterized by an erythematous rash in-

volving the whole skin. The erythema was clearly apparent on the shaved flanks and on the glabrous skin of the ears; it could be recognized also on intact skin through the fur. The erythema appeared 5 hours after the injection of DNBSO3

days

tys

F'u. 3. Flare-up of older test sites in guinea pigs sensitized to DNCB and intravenously injected with DNBSO3. Readings on three successive

days of the reference test applied before the i.v. injection, the challenge test applied six hours after the i.v. injection, and of the additional tests. a) guinea pigs sensitized to DNCB by epicutaneous application and injected intravenously with 500 mg/kg DNBSO3 in saline. b) and c) guinea pigs sensitized in the same way but submitted to saline injection or anesthesia and skin incision only.

194

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

TABLE 3 Flare-up of older test sites after intravenous injection of DNB,803 in guinea pigs sensitized by epicutaneous

application of DNCB Intensity of the reactions to the reference test Intensity of the Reactions (Sensitivity Level) Hours altec application of the teat Experiment

No. of Animals

24

48

72

Hours after iv. injection

500 mg/kg, DNBSO3 i.v

2m1/kgsalinei.v Anesthesia+skinincisiononly

32 41 38

—

0

30

2.9 2.8 2.8

3.5 2.5 2.6

4.0 2.2 2.4

Intensity of the reactions expressed as the average of the sum of the readings at various intervals after application of the reference tests (see Materials and Methods). In most animals of the first group (i.v. injection of DNBSO3), the second series of tests (challenge test) gives negative or very low readings (see Fig. 1, Table 2). demonstrating a lower sensitivity level, as well as reactions as compared with the controls. In our non-sensitized animals, did not show any gen- previous experiments, when DNCB was used for

eralized rash upon intravenous injection of the same dose (500 mg/kg) of DNBSO3. In a second group of 6 animals sensitized by the usual epieutaneous application of DNCB and demonstrating a low sensitivity level (3.8), the intravenous injection of the high dose of 1000 mg/kg of DNBSO3 could elicit a similar derma-

intravenous injection, no complete inhibition, but only a diminution of the test reactions was obtained. This difference might be due to the

nIsCUssION

tion of circulating anti-hapten antibodies by the corresponding hapten-protein conjugates

fact that DNBSO3, because of its lower toxicity, could be given in doses of 500 and 1000 mg/kg

whereas DNCB in doses larger than 10 mg/kg was not tolerated. The inhibition obtained may be considered titis in 3 animals, 2 dying shortly after the injection (1000 mg/kg is about the LD5O%) specific, i.e. not due to an impairment of the and 1 showing no dermatitis but complete in- organism's general reactivity, since the hyperhibition of the challenge tests. sensitivity against another unrelated hapten These preliminary results seem to indicate that (CPT) was not influenced by the intravenous the generalized dermatitis can be induced in injection of the DNBSO3. Because the inhibitory effect demonstrated by highly sensitized animals with relatively low doses of DNBSO3 whereas lesser sensitized DNCB and DNBSO3 is specific, it should be guinea pigs will require higher doses. The patho- possible to determine whether the inhibition is logical and microscopic findings accompanying due to the injected compounds acting as unithe appearance of the erythematous rash have functional haptens or to dinitrophenyl conjugates formed in vivo. It is well known, since the classical not yet been evaluated. experiments of Landsteiner, that the precipitaThe results of our experiments demonstrate that in guinea pigs with contact hypersensitivity to DNCB, a complete although temporary inhibition of the contact reaction can be achieved by an intravenous injection of DNBSO3. In fact in j of the animals, the challenge test became negative, another j showing definitely weaker

may be specifically inhibited by an excess of unifunctional hapten, blocking the antibody's combining sites and forming soluble hapten-antibody

complexes. A similar mechanism may explain the specific inhibition of immediate type allergic

reactions, such as anaphylaetie shock (31),

SPECIFIC INHIBITION OF CONTACT DERMATITIS

passive cutaneous anaphylaxis (34) and whealand-erythema reactions (11) by unifunctional haptens. It is quite clear that, in the case of the delayed type contact reaction, we are faced with a very different situation. Preliminary experiments, which will be reported elsewhere (43), suggest that the inhibitory effect of intravenously injected DNCB and DNBSO3 on the contact reaction to DNCB is not due to the compounds acting as haptens but to the dinitrophenyl protein conjugates formed in vivo.

195

by an epicutaneous test, could be completely inhibited for some days. The inhibition was specific as evidenced by the unchanged reactivity

to an other, immunologically unrelated substance, to which the same animals were sensitized concomitantly.

Coincident with the inhibitory effect on the challenging test, the intravenous injection of DNBSO3 brought about, in some animals, a flare-up of older test sites. In highly sensitized animals and in instances

On the other hand, the fact that the intra- of large dosage, the intravenous injection of venous injection of DNBSO3 causes both an DNBSO3 elicited a generalized dermatitis. inhibition of the contact reaction and a flare-up of old test sites might at first glance seem para-

doxical. Similarly, the fact that there is some

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429, 1958.

2. BAEE, R. L.: Remarks on prevention of aller-

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3.

in the contact reaction. Finally our experiments arc consistent with the results obtained by Sulzbcrgcr (42) in guinea pigs

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provoked a flare-up of the site of sensitization or

the site of previous—actually evanishcd—test. We could further confirm Sulzberger's observation, that in some of his animals, the intracardial injection of the compound elicited a generalized dermatitis, a phenomenon we arc noting now after intravenous injections of DNBSO3 in approximately j of our animals so treated. Such an

feeding certified food azodyes in paraphenyl-

Handbuch Haut-GeschlKr. J. Jadassohn; Erganzungswerk II, 1: 379, Berlin, Springer Verlag, 1962.

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S.

exacerbation and generalization of localized eczematous dermatitis after a subsequent oral or parenteral supply of antigen has been often described in the human (37, 3, 44, 12, 4, 39). We will consider later Sulzbergcr's findings concerning the state of unresponsiveness obtained with an intracardial injection of arsphenaminc given

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Guinea pigs with contact hypersensitivity to dinitrochlorobenzcne (DNCB) were injected intravenously with dinitrobenzene sulfonic acid sodium salt (DNBSO3) six hours before perform-

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gen mit Neosalvarsan am Tier. Salvarsan-

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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

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LANDSTEJNEII, K. AND JACOBS, J.: Studies on

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