0022 · 202 >.: / 84 / 8:102·009 1 $0200/ 0 T H E .J OURNA L OF I NVESTIr.ATI VE DERMATOLOr;y. 1\:3 :91 - 9.,. 1984 Copy ri ght Ie 1984 hy The Williams & Wilkins Co.

Vol. 83. No.2 Printed in U. S .A .

Induction of Contact Sensitivity to a Broad Variety of Allergens with Haptenized Macrophages

MARY VON BLOMBERG-VAN DER FLIER, B.Sc., RIK J. SC HEPER, PH.D. , G IJS H. BOERRI GTER, B.Sc ., AND LADISLAV POLAK, M.D.

Department of Pathology. Free Un iuersity Hospital. Amsterda m . The Neth erlands, and F. Hoffmann -La Roche & Co. Ltd., Centra/ Research Units, Basel, Switze rland

U sing guinea pigs an analysis could be made of various aspects of contact sensitivity (CS) induced by subcuta­neou s injection of syngeneic haptenized macrophages (oil-induced peritone~l ~xu~ate cells, P~C) as compared t o epicutaneous sensItizatIOn . Very lIttle PEC-bound hapten (dinitrochl.o~ob~nzene, or oxazolone) is nee~e.d for optimum sensItIzatIOn. Nevertheless, both sensIti ­zation methods induce a state of CS that may last for over 6 months, give rise to hapten-specific antibodies with a similar isotype distribution, and show suscepti­bility to cyclophosphamide pretreatment. In addition, time courses and microscopic appearance of skin test reactions after either way of sensitization are identical.

CS to a broad variety of physicochemically different antigens, including nickel, penicillin, and acrylates, is readily induced by syngeneic PEC, haptenized following a standardized procedure. As Freund's complete adju­vant is known to cause serious side e ffects like ulcera­tion and long-lasting granuloma formation, immuniza­t ion with haptenized PEC should now be considered as a clean and e ffective alternative in experimental CS studies.

When a lle rge ns e ncounter t he immune system in such a way that Lange rhans cells (LC) a re bypassed, specific unrespon ­siveness is often induced rather than delayed a lle rgic reactivity [l,2J. S ince it is known that substant ial a mounts of a lle rge n leak t hrough t he epidermis fo llowing com monly used epicuta­neouS a pp li cation procedures, it might be of importance for both expe rimenta l a nd screenin g purposes to develop sensit i­zatio n procedures in which no a lle rgen molecules escape t he effect ive presentat ion pathway . This might be ach ieved by s.c. immunization with a llergen-bearing presentator cells .

P r eparatio n of LC is tec hnica lly compli cated and t ime-con ­s uming. Appreciation of functional s imila rities between the epidermal LC a nd oi l-induced perito neal mac rop hages [3,4J led

Manuscript received December 9, 1983; accepted for publication Ap ril 6, 1984. .

Re print requests to: B. M. E. von Blomberg-va n der Fher, Depart­me nt of Pathology, Free University Hospita l, de Boelelaan 111 7, 1007 MB Amste rdam, The Nethe rlands.

Abbreviations: BSA: bovine serum albumin CS: contact sensit ivity DNCB: 2,4-dinitroc hlorobeIlZene DNFB: 2.4 -dinitroOuorobenzene DNP: dinitrophenyl E LISA: enzyme-lin ked immunosorbent assay FCA: Freund's complete adjuvant HBBS: Hanks' balanced salt solu t ion LC: Langerhans cell (s) m0: macrophage NCS: newborn calf se rum OXA: 4 -ethoxymethylene· 2-phenyloxazolone PBS: phosp hate- buffered sa line PEC: peri toneal exudate ce ll (s) P iCI: picryl chloride

91

to t he use of a ll e rgen-bearin g macrophages (ml'l) for t he induc­t ion o f con t.act se ns itiv ity (CS). Unti l now, only strong a lle rgens like 2,4-d ini trochlorobenzene (DNCB) (5) a nd picry l chlo ride (P iCl) [2,4 ,6,7 J have been used in such protocols a nd , in spite o f t he fact t hat CS in guinea pigs is conside red as t he most re leva nt a nim a l m ode l for huma n a lle rgic co ntact dermatit is 18,9], most of t his work was done in mice [2,4,6,7).

I n the present paper a n a na lysis has been made of t he state of CS induced by haptenized m l'l in guinea pigs. A number of aspects have been studied , about which up till now little is known: (1) macroscopic a nd mic roscopic appea ra nce of con tact s kin test reactions, (2) duration of co ntact hypersensitivity, (3) t he induction of a lle rgen -spec ifi c antibodies, a nd (4) suscepti­bi li ty to cyclophosphamide pretreatment.

Fina lly, it was investigated whether s.c. injection of a llergen ­bearin g peri toneal exudate ce lls (PEC) can be considered as a ge nera l method for induction of CS to a broad variety of physicoc hemically differen t a llergens including nickel, penicil ­lin , a nd ac rylates.

MATERIALS AND METHODS

AnimaL~

Inbred strain 13 gu inea pigs of eit her sex, bred at the Department of Experimental Medicine (Head Dr. H. A. Brouwer, Free University, Amsterdam), weigh ing 250-350 g, were used.

Preparation of Haptenized Macrophages

PEC were harvested in Hanks' balanced salt solut ion (HBSS) 4 days after the i.p. injection of 12 ml liquid paraffi n oil , and washed twice. PEC (3 X 106/ ml HBSS) were then incubated at 37°C for 30 min in the presence of a max imum, nontox ic dose of allergen (Table I) . [n order to fac ili tate standardi zed a llergenicity testing of physicochemi ­cally different substa nces, as a rule, for each allergen, half of t he PEC was treated with allergen from an ethanol stock solut ion, the other half from a saline stock solu tion. Din itroOuorobenzene (DNFB), however, was used from an ethanol stock solution only. Stock solutions of 40 t imes the final allergen concentration (T able I) were son icated if the a llergen was not clearly solved, and then added to t he PEC suspensions. After incubation as desc ribed above, both PEC suspensions were pooled, centrifuged, and washed twice. Viabili ty of allergen-treated m0 was determined by t rypan blue exclusion and always exceeded 80%.

Immunization Procedures

Guinea pi gs received tot.a lly 3 x 10' (un less otherwise stated) hap ­teni zed macrophages (± 4. 10' PEC) in 1 ml of HBSS by s.c. injection into ears, footpads and Oanks. In some experiments cyclophosphamide (250 mg per kg) (Enxodan, Asta-Werke AG, Brackwede, F.R.G.) was given intraperi to neally 3 days before sensitiza tion.

Other groups of guinea pigs were sensitized by epicutaneous appli­cation of 100}.l l of a 4% solu t. ion of DNCB (Merck , Darmstadt, F.R.G .) in ethanol or of 100 }.II of a 5% so lution of OXA (4-ethoxymethylene-2-phenyloxazolone, BDH , Poole, U.K.) in ethanol. At least 5 guinea pigs were used per group.

Skin Testing

Twelve to 14 days after sensit ization, gui nea pigs were shaved and depilated wi th Bioderma (Sch reuder, Soest, The Netherlands). Skin tests were performed eit her by open epicuta neous app lication of 20 }.I I of' the allergen or by patch test.ing, using 100 III of an allergen solu t ion

92 VO N BLOMBERG-V AN OER FLIER ET AL Vo l. 83, No.2

TABL E I. Alle rgens

Alle rgens Source PEe

incubation (mM ) Method

Skin test

Dose Solvent

NiSO. ·6H20 4 -Ethoxymethylene-2-

phenyloxazolone (OXA)

Penicillin G Formaldehyde 2.4 -Oi n it rofl uoroben zene

(ONFB) 2,4-Dinit rochlorobenzene

(DNCB) K 2Cr20 7 Fluorescein isothiocyanate

(FITC) Picryl chloride n-Butyl acrylate

Methyl methacrylate n-Butyl methacrylate 2- Hydroxyethyl methacrylate

Merck, Darmstadt, F.RG. BDH, Poole, U.K.

Sigma, St. Louis, Mi ssour i Brocasef, Maa rssen, Holland Fluka, A. G. Buchs, Switzerland

BDH, Poole, U.K.

Merck, Darmstadt , F .RG. Merck, Darmstadt, F. R.G.

BDH, Poole, U.K. Merck, Darmstadt, F.RG.

Merck, Darmstadt, F .R G. Koch-Light, Colnbrook, U.K. Merck, Darmstadt, F. R G.

per patch (Leucotest R, Beiersdorf, Hamburg, F .RG.). For test concen­trations see Table I. Patches were removed afte r 20 h. Skin tests were evaluated at 24 , 48, and 72 h according to t he following scheme: equivocal or no reaction = 0; pink, spots = 0.5; red, confluent = 1.0; intense red, swollen = 2.0.

Histology

Biopsy specimens measuring 4 mm in diameter were cut under ether anesthesia from the center of t he skin test reactions and from the cont ralate ral normal skin . Biopsies were fixed and embedded in Epon as described by Dvorak et aJ [12). Several widely spaced l -l1m sections were stained with Giemsa (BDH , Poole, U.K.) 1:10 diluted in 0.067 M

phosphate buffer, pH 7.6, for 90 min at 60 · C. Different ial cell counts were performed in at least 10 microscopic,

200 ~m -diamete r fi elds (0.3 mm' total area) in t he uppermost papillary derm is [13], and numbers of cells present in normal skin were sub­t racted.

Enzyme- Linked l mmunosorbent Assay (E LISA) for Detection of dinit rophe nyl (DN P)- and OXA -Speci{ic A ntibodies

Bovine serum albumin (BSA, BDH , Poole, U.K. ) was dinit rophen­ylated as described by Lit tle and E isen [14]. Conjugation of OXA to BSA was performed as described by Askenase and Asherson [15]. The resulting preparations contained 35 groups of DNP and 27 groups of OXA per molecule BSA, respectively.

Flat-bottomed microti te r plates (Nunc-1mmunoplate 1, Nunc, Ros­kilde Denmark) were coated with 100 ~l DN P-BSA (0 .2 ~g/ml) or OXA-BSA (2 ~g/ml) in 0.1 M carbonate buffer, pH 9.6, wi th 0.02% NaN~ fo r 2 h at 37· C. The plates were rinsed 5 t imes with deionized water containing 0.05% Tween-20 (Brocacef, Maarssen, The Nether­lands) followed by an incubation with phosphate-buffered saline (PBS) conta ining 25 % newborn calf serum (NCS, GIBCO Europe, Glasgow, U.K.) for 30 min at 37· C. After 5 washings, serial dilutions (starting 1/ 2 ) of gu inea pig serum in PBS conta ining 10% NCS were added and incubated for 30 min at 37· C. Bound IgG" IgG 2 , and IgM hapten­specific antibodies were detected as described previously [16].

RESULTS

Induction of Contact Sensitivity by DNP-Modified PEC

In Fig 1 it is demonstrated t hat dinit rophenylated P E e (DNP-m 0) administered s.c . a re capable of inducing CS to D N CB. Sen sit iza tion is clearly dose-dependent, the minimal number of DNP-m0 required to induce CS be ing 1 x 10(;, whereas t he response becam e nearly plateau when more tha n 1 x 10 7 m0 were used .

By us ing radioactively labeled hap ten([ I4C]DNCB ) we were able to dem onstrate t hat 5 Jl.g of hap ten were bound to 1 x 107

DNP-P E C . T his a mount of hapte n is seve ra l orders of magni ­tude lower than t he a m ount needed to reach plateau sensit iza -

0.75 0.50

30.00 25.00

0.05

1.00 0.15

0.Q4 3.90

50.00 7.00

38.00

~ .:; ..... 'iii c (I) (J)

..... 0 CIl

C 0 0 -0

(I) (I) ... 01 (I)

c

Patch Open

Patch Patch

Open

Patc h Open

Open Open

Open Open Open

1.0

0.5

o

5.0 mg 0.8 mg

24 .0 mg 2.0

25, 13, 8 ~g

0. 1 mg 0.06 mg

100, 50, 25 I1 g 8.0 mg

20.0 mg 28.0 mg 26.0 mg

Sa line Ethanol 96%

Saline Saline

Acetone

Saline Acetone/di-n-

butylphtalate 1/ 1 [10) Acetone Arachis oil / methyl

ethyl ketone 1/ 2 (aramek, [11 /l

Aramek [11] Aramek [11] Aramek [11J

DNP-Mcp FIG 1. Induction of DNCB contac t sensit ivi ty with different num­

bers of s.c. injected DNP-PEC. Skin tests were performed 2 weeks after sensitization. T wenty-four-hour readings a re shown. The mean degree of contact sensit ivity ± SO was calculated for each group consisting of at least 5 gu inea pigs. Skin test reactions in unsensit ized cont rol guinea pigs are represented by the hatched area.

tion by epicuta n eous application of the a llergen (1250 Jl.g p er a nima l, [17]) or t he a m ount commonly used when immunizing wi th Freunds' complete adjuva n t (FC A, 500 Jl.g per a nimal, [5]).

Time Course and Microscopic Appearance of Skin T est Reactions and Duration of CS

Contact s kin reactivity o f guinea pigs, inject ed wi t h 3 x 10' DNP-m 0 s.c. 2 weeks ea r lie r , was measured at va rious times a fter cha llen ge . As seen in Fig 2 both magni tude a nd time course of the contact skin reactions in DNP-PEC immunized guinea pigs were t he sam e a s in epicuta neous ly sensitized

Aug. 1984 INDUCTION OF CONTACT SE NSITIVITY WITH HAPTENIZED MACROPHAGES 93

a nima ls. Essentia lly simi lar resul ts were obtained in other groupS of guinea p igs when OXA-modified P~C were compared to epicutaneously applied OXA for sensIt IzatIOn (data not s hown).

Contact skin reactions were a lso cha racteri zed by micro­scopic evaluation of the infiltrating cells . No significant differ­e nces cou ld be found in t he composit ion of t he cellular infil­t rat es (Table II) : numbers of neutrophilic and eosinophilic g ranulocytes were negligible «1 %) in a ll reactions, whereas t he numbers of basophils for eit her allergen were independen t of the mode of sensitization. As it is known t hat CS induced in guinea pigs by epicutaneous application of a llergens genera lly lasts for years [18], one group of an imals immunized with 3 x 107 DNP-modified macrophages was skin tested only 6 months later. Skin test reactivity to DNCB appeared to be still clea rly detectab le (0.7 ± 0.2, n = 8; nonimmunized control groups never exceeded 0.25).

Induction of Hapten -Specific Antibodies

It was t hen investigated whether hapten-specifi c ant ibodies, which are genera lly present after epicutaneous sensit ization, could be detected fo llowing injection of DNP-PEC or OXA­PEC (Fig 3) . No anti-DNP antibodies could be fo und 14 days after immunization with 3 x 107 DNP-m",. Injection of lOR DNP-m"" however, induced anti-DNP antibody titers compa­rable to t hose found in epicutaneously sensit ized animals.

In cont rast to DNCB, OXA, upon epicutaneous application, does not induce a detectable IgM and IgGz-antioxazolone an­tibody response, alt hough a substantial IgG , response may

o hours

FIG 2. Time courses of ONCB contact skin test reaction elicited 2 weeks after s.c. immunization wi t h 3 X 107 ONP-macrophages (_), epicutaneous sens itization with ONCB (e) or in unimmllnized controls ( .... ). The mea n degree of CS ± SO of at least 5 guinea pigs.

deve lop. Immunization with 3 x 107 OXA- modified m'" clearly induced a similar pattern: OXA-specific antibod ies were ob­served only in t he IgG , subclass in 6 out of 11 gu inea pigs tested.

Immunization with Haptenized Macrophages As a General Method for the Induction of CS

To investigate whether the injection of haptenized macro­phages can be app lied as a genera l method for t he induction of CS, we incubated PEC with 12 physicochemically different allergens, in maximum nontox ic concentrations, fo llowing a standardi zed procedure. Syngeneic guinea pigs were t hen sen ­sit ized with 3 x 107 hapten -pulsed m", (PEC) and challenged epicutaneously after 10- 14 days (see Table I) . Nonimmunized animals were tested simultaneously as controls. T he resul ts are shown in Fig 4. For comparison , data from max imization test and FCA immunization procedures as reported by Magnusson and Kligman [19], van der Walle et a l [11], and Pola k et al [20] are included in t he Table.

Immunization with haptenized macrophages generally cor­related well with t he other immunizing procedures. As expected, no CS could be induced to chromium. T he guinea pig strain 13 used in t his study is known to be nonresponsive to t his allergen [20]. Few a llergens induced lower responsiveness upon hapten­PEC immunization (formalin and 2-0H-ethylmethacrylate), whereas t he induction of ni ckel hypersensitivity was very ef­fective with ni ckel-modified PEC.

S usceptibility to Cyclophosphamide Pretreatment

Since CS induced by epicutaneously applied DNCB or oxa­zo lone has been shown to be under control of cyclophospha-

,~,---... , . ONP litre -==========t= .. anti - C>!uuo&one tlt,.. ----. , l

"'1 6.

l" ~ .1'· -.

~t, •• /~ 01 G2 M . at G2 M Ig

SenS11I,atlOn ONCB e.c. , ONP- J"4:» DNP m4> ~ ac. . (ha..sn4> 4(00)19 3.10 I,d sooo pg _~

FI G 3. Induction of hapten-speci fi c ant ibodies by s.c. immunization with 3 x 107 or lOB haptenized macrophages (PEC), as compared to epicutaneolls (e.c.) sensit ization with ONCB and OXA. Both individua l and median IgG" IgG2, and IgM tite rs, as detected in ELISA-assays were assessed at 14 days afte r sensitization as well as in unsensitized controls.

TABLE II. Microscopic analysis of contact skin test reactions elicited 2 week_5 after the s.c. injection of haptenized PEe or after epicu.taneous sensitization

Sensitization" Skin test erythema> Total

cells'

3 X 107 ONP-m0 S.C. DNCB e.c. 0.8 ± 0.2 26 ± 12 400 J.lg DNCB e.c.d id 1.1 ± 0.3 38 ± 15

id 0.1 ±0. 1 6±4 3 X 107 OXA-m0 S.C. OXA e.c. 0.8 ± 0.3 47 ± 3 500 J.lg OXA e.c . id 1.0 ± 0. 3 50 ± 10

id 0.0 ± 0.1 6±2

a Groups of at least 5 guinea pigs had been sensitized 2 weeks before skin testing. > Skin test reactivity was evaluated at 24 h, a fter which sa mples were taken for histology. ' Number of cells per microscopic field. d Epicutaneously.

Skin test infilt rate

Percent Percent mononuclear basophils

88.9 ± 3.8 10.6 ± 4.5 87 .9 ± 18 11.4 ± 2.4 96.8 ± 4.0 3.0 ± 2.9 94.0 ± 19.6 5.3 ± 4.0 92.0 ± 4.2 8.2 ± 4.2 93.0 ± 3.5 3.5 ± 3.5

94 VON BLOMBERG-VAN DER FLIER ET AL

Degree of contact sensitrvity Responders

Mergen 05 10 MejHmm. FCA - imm. 2)

31 ONce 0/8 n.d .

515

PiCI 3)

818 n.d.

3) 718 "<S.

ATC 3) 617 n.d .

9/9 11 /20 (16)

9/9

\/8 0 / 8 (18)

4/5 20/20 (16

Formalin 1/ 8 15/20 (16)

515

6/6 8/8 ( 17)

216 2/8 (17)

0/6 0/8 (17)

~~ . 0 / 6 4/& (17

FIG 4. The induction of CS to different allergens by s.c. immuni ­zation with 3 x 10' haptenized macrophages (PEC). The results are expressed as mean degree of CS ± SD at 24 h after open epicutaneous challenge or 48 h after patch testing (see Table 1) in sensitized guinea pigs either with (Wl) or without (m) cyclophosphamide pretreatment or in unsensitized controls (0).

I The fraction of immunized guinea pigs showing a stronger sk in test reaction than any of the control animals.

, Results obtained from the literature. , No standardized FCA data available. These substances are gener­

al ly regarded as strong sensitizers /10,17,19J.

mide-sensitive suppressor cells [21], it was also investigated whether similar regulatory mechanisms would playa role in hapten-macrophage-induced CS. From the results presented in Fig 4 it is clear that not only CS induced by DNP-PEC, but also by nickel -PEC is markedly enhanced by cyclophosphamide pretreatment. Moreover, cyclophosphamide pretreatment al ­lowed for the development of clear CS to formalin.

DISCUSSION

The results of the present in vivo study demonstrate the effectiveness of oil-induced PEC as antigen-presenting cells in the guinea pig. Strong CS to a broad variety of allergens could be induced by s.c. injection of hapten-pulsed PEC. Since PEC account for about 80% of m0 and since the other cells (lympho­cytes, granu locytes, and erythrocytes) are highly ineffic ient in hapten-presentation [6], it is most likely that the m0 are the actua l stimulator cell s. The small amount of cell-bound hapten required for induction of clear DNCB CS (1 x 10" m0, contain­ing only 500 ng DNP) merely underlined the high efficiency of peritoneal m¢ to present t he hapten. In an earli er report [22] these oil -induced PEC also appeared to be highly effective in vitro in triggering contact sensit ized lymphocytes .

Contact skin test reactions, elicited after immunization with

Vol. 83, No.2

haptenized macrophages, showed a similar time course and microscopic appearance as did skin test reactions after epicu­taneous sensitization. A similarity between these two sensiti­zation methods also was found with respect to the induction of hapten -specific antibodies. Immunization with sufficiently high numbers of both DNP-PEC (1 x lOR m0) and OXA-PEC (3 x 107 m¢) , induced a hapten-specific antibody response with a similar isotype distribution as found after conventional, epi­cutaneous sensitization. With DNP-PEC no specific antibody response was detectable when 3 x 107 m0 were injected, whereas the same degree of CS was induced as with 1 x 108 m¢. This observation confirms the minor role of hapten-specific antibod­ies in contact skin reactions to this sensitizer (Boerrigter et ai, unpublished observations) [23,24J.

It should be noted that those low numbers of DNP-PEC which fail to stimulate antibody-producing B cells, seem to activate cyclophosphamide-sensitive suppressor cells. This would indicate, on the one hand, that leakage of free allergen from the sensitization site, which is unlikely to occur after macrophage-binding, is not necessary for the induction of sup­pressor cells [25], and, on the other hand, that suppressor cells do not produce specific antibodies.

From the present study it is clear that not only CS to the strong allergens DNCB and OXA (21), but also to nickel and formalin, both common allergens in humans but weak sensitiz­ers in guinea pigs, is strongly potentiated by cyclophosphamide pretreatment. This suggests that regulation by cyclophospha­mide-sensitive suppressor cells represents a general feature of contact allergy in guinea pigs.

The use of hapten-pulsed m0 for the induction of CS has been limited in the past to experimental studies with few allergens like PiCI and DNCB [4-6]. The results of this study show that CS to many different allergens can be induced by injecting oil-induced PEC, haptenized according to a standard­ized procedure, into syngeneic guinea pigs. Both frequency and intensity of sensitization may still be considerably increased by using cyclophosphamide-pretreated animals and probably also by optimization of the conjugation procedure for each individ­ual allergen. As to nickel sensitization, injection of nickel­pulsed PEC appeared to be particularly effective. In our hands this nickel sensitivity was markedly stronger than could be achieved by classical methods, including multiple FCA injec­tions (data not shown).

In conclusion, our results demonstrate that CS to a broad variety of physicochemically different allergens can be induced by injection of hapten-pulsed syngeneic peritoneal exudate cells. Since the majority of allergens do not readily sensitize when applied topically, FCA has been commonly used in the past, bringing about, however, some serious side effects such as ulce ration and long-lasting granuloma formation. Therefore, immunization with haptenized PEC should be considered as an effective and clean alternative in experimental CS, even more since the increasing availability of various inbred strains of guinea pigs and mice.

We wish to thank Dr. H. B. van der Walle for his help in studying the sensitizing potential of acrylates. We thank Mr. D. J. van Romondt and Mr. J. de Groot for their valuable technical assistance, prof. Dr. J. Oort for helpful discussions, and Ylva Abraham-Dolman and W. R. Dolman for careful typing of the manuscript.

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Aug. 1984 IND UCTIO N OF CO NTACT SENSITIVITY WITH HAPTENIZED MACRO PHAGES 95

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25 . Turk JL, Parke r D: Modulation of T lymphocyte function by B lymp hocytes in delayed hypersensitivity. Int Arch Allergy Appl Immunol 49:24 1- 246, 1975