Induction of late cutaneous reactions by skin-blister fluid from allergen-tested and normal skin

W. Dorsch,

M.D., and J. Ring, M.D., Ph.D. Munich.

West German>

Ten healthy \wlunteers (ji\*e otopic. ji1.e nonutopic) ond screen pori~~r~t.s s@rin~ jfwtt trllrrgic bronchial asthma and rhinitislconjunctivitis us well as shwving duo1 reactions qfier intradermal or bronchial ullergen challenge were inwstigoted. UsinK the suction blister technique. NY ohtuined skin-blister fluid (SBF) from dual skin reuction~ 30. 60. 180. und 300 min ufter allergen injection und from normal untested skin. The biologic, ucri,,ityi O] SBF \\u.s t~~stetl by intrudermul reinjection of the fluid into the donor. SBF tuken jtiorn dual .rkin reuctions 30 or 60 min qfter allergen injection produced lute cutuneons reuctions (LCRs) quite similur IO those induct4 b! the allergen. SBF tuken from LCR ureas IX0 or 300 min ufter untigen testing hud much \twker c>ffects. similar to SBF from untested skin. A possible wntent of‘ ullergcn cstruct in SBF ./born allergen-tested skin ureus 1~0s WI responsible for the observed eflects us tlemon.struted in passive cutuneouJ onaphvluris experiments in monkeys. High do.w.s of SBF from ~oltestc~~ skin were uhle IO induce LCRs similur to but weuker rhun LCRs produced b! SBF tulwrt ut euriy phases jborn duul skin reaction.s. Similar volrtmes of uutolo~ous hepurin-plusmu or wrrtm did not induce LCRs. II is concluded thut during the initiul phu.se of duo1 skin reuctiwr.\, ./irctor.s ure @med that ore uble IO induce LCRs. The generotiorr of thew mec1iutor.s yeem.\ IO he c~trrr.sed(11 Icw.st in purr by the extru\~u.sution of plu.smu.

Late cutaneous reactions (LCRs) were first observed by Prausnitz and Kiistner’ in their original work &scribing the transferability of immediate type hypersensitivity. LCRs usually occur after intense immediate reaction&” and consist of erythema, edema hyperthermia, tenderness, and hyperalgesia, reaching maximum after 4 to 8 hr and disappearing usualltl within 12 to 24 hr.‘. ‘-6 The biphasic course of immediate and late cutaneous as well as bronchial reactions is sometimes called a dual reaction.j-’ The pathogenesis of this phenomenon is not yet clear. Some authors’, ‘). I0 have proposed a type III reaction according to Coombs and Gell; others have discussed a similarity to cutaneous basophil anaphylaxis.” Antibodies of the IgE class are primarily involved”. ‘; furthermore, there is preliminary evidence of an involvement of the coagulation system.‘* In a previous study, we have shown that inhibitors

of the cyclooxygenase pathway of prostaglandin synthesis are able to reduce the intensity of erythema during LCRs6 without influencing the immediate wheal-and-flare response. It was the aim of this study to investigate skin blister fluid (SBF) taken from positive immediate and late cutaneous reactions as well as from normal skin with regard to its potency in inducing LCRs. MATE?@ALAMO-S

Humrn

Seven patients suffering from allergic asthma (aged 18 to 38 yr, six male and one female) with dual skin as well as dual bronchial reactions after allergen testing and ten control subjects (aged 20 to 38 yr, eight male and two female) were investigated after informed consent was obtained. Five of the IO control subjects had a positive history of atopy (hay fever. allergic rhinitis and conjunctivitis. urticaria).

Induction From tlx Kinderpoliklinik and Dermatology Ludwtg-Maximilians University. Received for publication March 21. 1980. Accepkd for publication Sept. 9, 1980.

Department

of the

Reprint requests to: Dr. W. Dorsch, Kinderpoliklinik der Universith Miinchen. Pettenkoferstrape 88, D-8000 Miinchen 2. West Gertnany 0091~6749/81/020117+07$00.70/0

??I 1981 The C. V. Mosby

subjecb

of ths skin blisters

Skin blisters were produced over a l4-mm-diameter area by continuous suction (-60 mm Hg) over 2 to 3 hr. Gcnerally, vesicles tirst appeared after about 90 min and a clear blister formed within the next half hour containing appmximately 300 to 500 ~1 of a clear fluid. When SBF was obtained over sites of dual skin reactions. Co.

Vol. 67, No. 2, pp. 117-123

118

J. ALLERGY

Dorsch and Ring

FIG. 1. Kinetics of allergen testing in concentration (first column of graphs), also listed.-

CLIN. IMMUNOL. FEBRUARY 1981

skin reactions (diameter of erythema in millimeters) after intracutaneous seven patients suffering from allergic bronchial asthma. Allergen in original column of graphs) produced large LCRs, and in a 1 : 100 dilution (second only weak LCRs in two patients. Responsible allergens and RAST results are

was applied over a period of 1 to 2 hr; when vesicles first appeared, allergen injection was performed intradermally under the vacuum area and suction was continued. SBF was collected 30, 60, 180, and 300 min after allergen injection.

a vacuum

Skin testing Unless mentioned otherwise, 50 ~1 of test solution (allergen extract, SBF, human heparin-plasma, human serum, or saline) were injected strictly intradermally. In human subjects only autologous SBF serum or plasma was used for skin tests. The reaction was read after 20 min, 1, 2, 3, 4, 6, 8, 10, 12, and 24 hr. The skin reaction was quantitated as diameter (in millimeters) of erythema. Other parameters registered were edema, pain, itching, hyperalgesia, hyperthermia, and burning sensation.

SBF SBF was drawn into tuberculin syringes (Pharma-Plast), and stored frozen at -40” C (for reinjection studies in hu-

mans) or cooled at 4” C (for monkey skin test) until used. When stored at 4” C, clotting of SBF was observed. Fresh SBF was examined under the microscope; electrophoresis was performed.

Passive cutaneous monkeys

anaphylaxis

(PCA) in

In order to examine a possible allergen content of SBF drawn from allergen-tested skin, PCA with SBF’” was performed in monkeys (Macaca fascioluris, male, weighing 4,800 gm). Monkey skin areas were passively sensitized with human serum containing high titers of IgE antibodies against mixed grass pollens, cat hair, or Dermatophagoides pteronyssinus, respectively. Twenty-four hours later, 2 ml of 1% Evans blue were injected intravenously and skin testing with SBF taken at 30 min, 2 or 6 hr after antigen injection was performed in presensitized and non-presensitized skin areas. Presensitized skin areas tested with allergen in different concentrations served as positive controls. Allergen extracts were the same as used for skin testing in the

VOLU’PE 67 NUMBF R 2

Late cutaneous

t i mr

afttr

ct

injr

ion

FIG.2. Kinetics of skin reactions (diameter of erythema

Rdballugosorbent RAW Klinikum

test (RAST)

was done using Phadebas test kits. Dr. X. Baur. Grophadem. performed the RAST analysis.

statiais Statlitical evaluation was done by calculating the mean values md standard error of the mean. The statistical significar,.e between means was calculated with Student’s t test.

RENNTS Awn-induced with uxs

skin reactions

in patients

Fig. 1 shows the kinetics of dual skin reactions after &lergen skin testing in seven patients suffering from a*lergic asthma. The list contains the responsible

119

f haurs 1

in millimeters) induced injection of 50 ~1 SBF drawn over allergen-induced dual skin reactions in the shown in Fig. 1 (patients No. 1 to 7, from top to bottom row). SBF drawn intervals after allergen testing was compared. The last column shows results SBF drawn over normal untested skin. human subjects. The results were read as diameter (in millimeter:.) of blueing 30 min after intradermal injection of the test m*erial.

reactions

by intracutaneous same patients as at different time after injection of

allergens and the corresponding RAST results. All but one patient showed very high titers (+ + + + or + + +) of allergen-specific IgE antibodies. In the majority of the patients the skin reactions after 8 hr were still more intense than the immediate whealand-flare reaction. Low allergen concentrations (origproduced only inal concentration. I : 100 diluted) weak LCRs in two patients. Biologic activity reactions

of SBF from dual skin

The biologic activity of SBF from allergen-induced dual skin reactions taken at different time intervals after allergen injection was investigated by intraderma1 testing of autologous SBF in the contralateral forearm (patients listed in Fig. 1) Fig. 2 shows the skin-test reactions induced by 50 ~1 SBF. Maximal LCR induction was observed after injection of SBF drawn 30 and 60 min after allergen injection. SBF

J ALLERGY

120 Dorsch and Ring

Total

plasma

protein:

3.7 gm/dl 0%

Prealbumin: Albumin:

70%

Alpha-l

globulin:

3%

Alpha-2

globulin:

3%

Beta

CLIN. IMMUNOL. FEBRUARY 1981

6%

globulin:

Gamma globulin:

188

FIG. 3. Electrophoretic

analysis of SBF (patient No. 3). Similar patterns were observed in other patients and healthy volunteers.

TABLE I. Effect of SBF on PCA reactions monkeys

(Macaca

Skin reaction Control

SBF (30 min)* SBF (120 min)* SBF (300 min)*

TABLE II. Results of PCA in monkeys (Macaca fasciolarisj with intracutaneous allergen injection

in

fasciolaris)

skin

11.2 * 2.3 10.6 ? 1.4 8.4 k 1.3

Presensitization with against:

(mm) in monkeys Presensitired

skin

8.5 * 2.0 8.0 k 2.2 7.7 2 I.9

Skin reactions were measuredas diameter of blueing (mm). Mean values * SEM of eight test sites are presented.Presensitization was done with human serumcontaining high titers of IgE against grasspollens, cat fur, and dust mite. SBF drawn at different time intervals after skin testing was compared. *Time (min) after injection.

drawn at later intervals produced weaker effects similar to SBF from normal untreated skin (right column).

Allergen dilution

Cat fur

Undiluted

15

1: 10 I:25 I : 100 1:250 Saline control Histamine control

13 10 0 0 0 25

Grass pollens

16 12 2 0 0 0 25

antiserum

Dust mite

13 10 0 0 0 0 35

Skin reactions were measuredas diameter of blueing (mm). Presensitization of skin-test sites was done with human serum containing high titers of specific IgE antibodies (same sera as in Table I). Allergen extracts used were prick solutions from Bencard Allergy Service.

SBF Under the microscope SBF showed no cellular contamination, particularly no platelets. The electrophoretie analysis, showing a similar pattern to lymph fluid,14 is depicted in Fig. 3. Evaluation of allergen PCA in monkeys

content

of SBF by

PCA was used to evaluate a possible allergen content of SBF. In Table I the effects of SBF in normal and presensitized monkey skin areas are shown. Presensitization was done with human serum from patients with high concentrations of IgE antibodies against the specific allergens (mixed grass pollens, cat fur, dust mite). There was no significant difference between presensitized and normal skin areas. In particular, the diameter of blueing was never increased

(as one might expect in the case of measurable allergen contamination) by SBF but decreased in several experiments; this difference, however, was not significant. There was no difference between SBF drawn at 30 min and that drawn at a later period after allergen injections in patients in the PCA experiments. Table II shows the positive control of PCA with intracutaneous allergen testing. There was a clear dose dependence of blueing showing that the IgE antibodies used for presensitization in the abovementioned experiments were active. At an allergen dilution of 1: 100, allergen activity was no longer demonstrable in the PCA model. Thus a possible allergen content of SBF was not demonstrable in our test system, which was sensitive down to a 1: 100 dilution.

VOLUh E 67 NUMI!‘:R 2

Late cutaneous

reactions

121

Ho. 4. Kinetics of skin reactions (diameter of erythema in millimeters) induced by intracu&maaus injection of SBF drawn over normal skin in healthy volunteers (n = IO). Three different do&$ of SBF are compared. Mean values * SD are shown.

m

fram

untreated

normal

skin

Re;injection of SBF drawn over untreated normal skin from volunteers led to measurable LCR formation BS shown in Fig. 4. There was a clear dose rice: the injection of 250 ~1 produced sigdew nificaMly larger LCRs than 50 ~1. There was no significrtglt difference in the LCR size induced by SBF between atopic (n = 5) and nonatopic individuals (n = 5). In Table III the intensity of SBF-induced LCRI in 10 volunteers is shown by evaluating several objenive and subjective criteria such as edema, hypert&Ma, burning, pain, and itching. Generally, immdiately after the injection of SBF there was a bum&g sensation that disappeared within a few minutes. Edema formation, pain, hyperthermia, and hyperalgesia usually developed from the second or third

hour onward, reached a maximum at 6 to 10 hr, and subsided within 24 hr. These symptoms were most pronounced in the 25Oq.A test sites. T&e injection ,of 250 ~1 of autologous serum or heparia-plasma did not produce LCRs in any of the tested persuns. Edema formation in normals showed a high v&&ifi$y, being in the average clearly weaker than in patients suffering from LCRs after allergen testing. DISCUSSiON Our results show that injection of SW obtained from skin areas with allergen-induced &al skin reactions leads to inflammatory processes similar to the original skin reaction. The responsible factors are found in maximal concentration duting the etily phase of allergen-induced skin reactions; SIZF drawn

122 Dorsch and Ring

TABLE III. Kinetics

of clinical

J. ALLERGY

symptoms

of LCRs in 10 normal

healthy

CLIN. IMMUNOL. FEBRUARY 1981

volunteers*

Time after injection Volunteer

O-5 min

2 hr

1

Burning pain

2 3

Burning pain 0

Massive edema, hyperthermia Mild edema 0

4i5 6 7t

Burning pain 0 Mild burning Burning pain

Slight edema Slight edema 0 0

8t 9t

Mild burning Mild burning

lot

Mild burning

Minimal edema Hyperthermia, massive edema, and pain Massive edema

6 hr

8-24 hr

Hyperalgesia

Hyperalgesia

Mild edema Slight edema, hyperthermia Slight edema Slight edema Moderate edema Massive edema, hyperthermia, hyperalgesia 0 Mild edema

0 Hyperthermia 0 0 Hyperalgesia Edema, hyperalgesia

Hyperthermia

0 0 0

*For erythema see Fig. 4. (Maximal symptoms occurred in the 150- and 250-~1 test sites.) tHistory of atopy. from test areas 3 or 5 hr after allergen injection had

similar effects to SBF from untreated (non-allergentested) skin. The mechanism of the described phenomena cannot be explained by this study; however, several observations might help to elucidate the pathogenesis of LCRs. Of course, one might speculate that diluted allergen is still present in SBF and thus represents the initial causative agent in producing LCRs. Our monkey experiments, however, in connection with the results of patients’ skin-test titrations, show that allergen content of SBF is negligible with regard to induction of LCRs. SBF from allergen-induced dual skin reactions never caused an increased blueing in monkey skin areas presensitized with allergenspecific IgE compared with nonpresensitized areas. We conclude that allergen in SBF drawn from dual skin reactions is diluted at least 1 : 100. Furthermore, only l/ 10 (50 ~1) of the total SBF (500 ~1) was used in our skin-test experiments, and thus possible allergen content was already reduced by a factor of 10. In a similar manner, diluted allergen extracts caused only weak LCRs in two patients. Obviously, the simple exudation of plasma into normal skin by vacuum suction leads to the formation of factors able to induce LCRs. These factors have similar, but weaker, effects than inflammatory mediators formed in the early phase (i.e., within the first half hour after allergen injection) of dual skin reactions. The nature of these factors is not known, but we can say from our experiments that SBF contained no cellular elements, particularly no platelets. There was an electrophoretic

pattern similar to that of lymph fluid. We know that SBF contains proteins and lipids in a distribution similar to peripheral lymph fluid.‘l The colloid osmotic pressure as well as the electrolyte content of SBF is comparable to plasma. lj The exudation of plasmaas caused in our test system by negative pressureactivates the clotting system. It is known that the coagulation system is involved in the development of LCRs: several authors observed fibrin deposits in sites of LCRs.12 The exudation of plasma leads to activation of the coagulation system as well as of the kinin system.“j Kallikrein has been detected in SBF from both allergen-tested and nontested skin areas.* The role of kinins in generating LCRs is still obscure. Regarding the close interrelation between kininforming enzymes and prostaglandin metabolism, a possible role of prostaglandins as mediators of the LCR should be kept in mind.20-25 It is of special interest in this context that the erythema of LCRs can be reduced by topical indomethacin, as shown previously.6 Although IgE antibodies seem to be primarily responsible in triggering allergen-induced LCRs,“, A. “3 l2 histamine is most likely not the principal mediator of this type of inflammation. It is not possible to induce LCRs in patients with isolated immediate skin reactions by adding histamine to allergen extracts.” However, mast cell degranulation, if sufficiently intense, is followed by LCR-like phenomena even in healthy skin-as, for example, after application of compound 48/80, an allergen*Dorsch W, Geiger R: Unpublished results.

VOLU \‘E 67 NUMSi R 2

Late

indelendent mast cell degranulator’-as well as by F(ab~! fragments of anti-IgE antibodies (reverse anaphylaxis).” Prior inhalation of disodium cromoglycate (DSCG), an inhibitor of mast cell degranulation, prevent: late bronchial reactions after allergen inhalation. :‘;. “; When DSCG is given after the immediate bronchial reaction. late bronchial reaction can no longctr be suppressed.“’ El :ept for specific conditions such as bronchopulmomtry aspergillosis.; there is little evidence for immum! complex reactions playing an important part in the rathogenesis of LCRs.:‘. I. *. ‘I. 26 Cutaneous baso;,hil anaphylaxis ‘I has a histologic picture distinctl ; different from LCR.“” Tl.~:se findings, together with our data, lead to the hypcahesis that during the early phase of an immediate t;.pe reaction-induced either by IgE or by direct masi cell degranulation-factors are formed that can initi;; e LCRs. It seems likely that the exudation of plasrla itself, as caused during the immediate reaction and produced in our test system by negative pressure, may iead at least in part to the generation of mediators responsible for the development of LCRs. A better understanding of the underlying mechanism might be helpful in further elucidating the pathc!genesis of long-lasting asthmatic conditions. Our model might be suitable to identify the responsible n-ediator substances as well as to test the efficacy of nc*ti therapeutic agents in allergic diseases. bf.

wish

Pulm
to thank Section,

Dr. First

X.

Baur and Prof. Dr. Fruhmann,

Internal

Medical

Clinic,

Klinikum

Gro+dem.

University of Munich, for providing clinical data c’n some patients (results of RAST and bronchial provoca:l.)n tests). REFEUENCES I. F”lusmtr C. Kiismer H: i’.cntralbl Baktetiol [B] 2. I:~nemom L. Poothullil l:;ctors which influence 3.

4.

5.

6. 7

Studien tiber die uberempfindlichkeit. 86:160. 1921. J. Dolovich J, Hargreave FE, Day RP: the late cutaneous allergic reactions. J !b I ERG) CL.IN IMMUNOI. 55112, 1975. Da&)vich J. Hargreave FE, Chalmers R. Shier KJ. Gauldie I: 1.: re cutaneous allergic responses in isolated IgE-dependent r;.rction\. J ALI.ERGY Ct.IN IMMUNOI. 52:38, 1973. C. lley GO. Gleich GJ, Jordon RE. Schroeter AL: The late p’ase of the immediate wheal and flare skin reaction. J Clin Invest S&408. 1976. Robertson DG. Kerigan AT. Hargreave FE, Chalmers R, I:~~~lovich J: Late asthmatic responses induced by ragweed poll. I allergen. J ALI F.R(IY CLIN IMMUNOI. S&244, 1974. Iijrsch W. Baur X: The effect of topical indomethacin on ’ ergen induced dual rkin reactions. Allergy 35503. 1980. ;‘-pys J: Non-immediate type of hypersensitivity in bronchial jh hma. 01 Yamamura Y. editor: Allergology. Proceedings of th: Eighth International Congress of Allergy. Tokyo, 1973. ti. cerpta Medlca International Congress Series No. 323, p. X4.

8. Zetterstriim subtilisin

0: Dual skin test rcactionb and A.cpr,gi//u.s

8:77. 1978. 9. Turner-Warwick IO.

I I.

12. 13.

14.

15. 16.

17. 18.

cutaneous

firmigclfu.c

M: Immunology

reactions

123

and serum antIho&!> extrac!\

Chn

of the lung. Lrjndon.

to

Allerg! lY7R.

Edward Arnold. p. 53. DeWeck A: lmmunologische Grundlagen und iunktionelle Pathologic der allergischen Hautreaktioncn. II, Werner M. Ruppert V. editors: Praktische Allergicdiapnostth. Stuttgart. 1974, Thieme. p. 71. Askenase PW: Role of basophils. mast cell\ and \asoamines in hypersensnikity reactions with a delayed time c transfer of immediate type hypenensitivit) from man to other primate>. J Clin Invest 4l:I40. IYhS. Vermeer BJ. Reman FC. van Gent CM: The derrrmination ol lipids and protems in buction blister Ilund. J Inbest Dermatol 73:303. 1979. Bork K: Physikalische Crundlagen der Blasenhlldung. Arch Dermatol Res 263:91, 1978. Ratnoff OD: A tangled web. The mterdependencr of mechanisms of blood clotting. tibrinolysis. immunity and inflammation. Thromb Diath Haemorrh (suppl.) 45: 109. 197 I Ratnoff OD: Mediators of inflammation. J At I I.HI,~ CI IN I\IMI;NOI 58:438. 1977. Brocklehunt WE: Role of kmins and prostaglandins in

inflammation. Proc Roy Sot Med 64~4. I971 19. Hadding L’: Komplement. Gerinnungsfaktoren und Kinine in ihrer funktionellen Verkniipfung. Z Immunitaetsforsch Immunobiol [ Suppl] 2:14. 1977. 20. Austen KF. Orange RP: Bronchial asthma: The possible role of the chemical mediators of immediate hypersensitivity in the pathogenesi\ of subacute disease. Am Rc\ Re\pir Dis 112:4?3. 1975. 21. Goldyne ME. Winkelmann RK, Ryan RJ: Prostaglandin acttvIty in human cutaneous inflammation: Detection by radioimmunoascay. Prostaglandins 4~737, 1973. 22. Greaves .MW. Sondergaard J. McDonald-Gibson W: Kecovery of prostaglandins in human cutaneous inflammation. Br Med J 2:25x. 1971. 23. Soter NA. Austen KF: The diversity ot mast cell-derlked mediator>: Implications for acute. subacute and chronic cutaneous Inflammatory dlsorden. J Inve\t Dermatol 67:? 1.7. 1976. 24. Strandberg K. Math6 AA. Yen S: Release 01 hstamme and formation of prostaglandins in human lung tissue and rat mast cells. Int Arch Allergy Appl Immunol 53520. 1977 25. Vane JR: Prostaglandins as mediators of inflammation. Ad\ Prostaglandin Thromboxane Res 2:791. 1976. 26. Booij-Noord H. Orie NGM, deVries K: lmmedlate and late bronchial obstructive reactions to inhalauon of house dust and protective effects of disodium cromoglycate and prednisolone. J AI I ERGY CLIS IMMuNOL 48:344. 1971 27. Dorsch W. Baur X. Emslander HP. Fruhmann G: Zu Pathopenese und Therapie der allergeninduziertsn verztigetten Bronchialobstruktion. Prax F’neumonol 34~461. 1980. 28. Richerson HB. Rajtora DW. Penick GD. Dick FR. Yoo TJ. Kammermeyer JK. Anuras JS: Cutancouh and nasal allergic responses in ragweed hay fever: Lack of clinical and histopathologic correlations with late phase reaction\ J Al.I.ERGt Cl.lN I?.rMr~wl. 64:66. 1979.