3. small Anim. Pract. (1971) 12, 431-443.

The immunology of allergic skin disease*

R . E. W. H A L L I W E L L

Department of Veterinary Clinical Studies, School of Veterinary Medicine, Cambridge CB3 OES

A B S T R A C T

The major immunological pathways that lead to the development of allergic skin disorders are described. The current and prospective trends in therapy are discussed in relationship to the stage in the pathogenesis at which they are effective.

I N T R O D U C T I O N

Before considering the mechanisms operating in allergic skin disease, some of the more fundamental immunological concepts will be reviewed by way ofintroduction.

An antigen can be defined as a substance which, upon entering the body, leads to the formation of antibody or of immunocompetent cells. If it is a substance which is responsible for clinical hypersensitivity or allergy, then it may be termed an allergen. Antigens are usually proteins. However, other high molecular weight substances such as fats and polysaccharides may on occasions act as antigens. It is also possible for simple chemicals to become antigenic upon combination with proteins. In this case they are termed haptens. I t is not necessary that this union should occur before presentation to the body. It can take place spontaneously with serum proteins following injection, or with epidermal proteins upon cutan- eous application.

The factors which determine whether the immune response is mediated by immunocompeteiit cells (cell mediated immunity), or by antibody (humoral immunity), or in part by both are obscure; but they include: (1) the nature of the antigen, (2) the dose and frequency of administration of the antigen, (3) the route of administration of the antigen, and (4) the species involved.

Antibodies are largely present in the y-globulin fraction of serum, and are termed immunoglobulins. The fact that, being proteins, they are antigenic when injected into other species, has led to the recognition of different classes of antibody,

* Presented to a symposium on Canine Dermatology, 14th B.S.A.V.A. Congress, London, 197 1.

43 1

43 2 R . E . W. H A L L I W E L L

viz IgG, IgM, etc., based upon differences in structure, and hence differences in antigenicity.

The reason why some individuals, and some animals, become allergic to certain substances, while others do not is poorly understood. Essentially it is related to the nature and extent of the immune response. There is evidence that in some allergic disorders this may be influenced by hereditary factors (Schwartzman, Rockey & Halliwell, 1971).

Antigens responsible for dermatological allergies may gain access to the body by one of four routes:

1. Inhalation, e.g. atopic disease of the dog, which is a pruritic condition in- duced by a hypersensitivity to pollens and other airborne protein particles (Halliwell & Schwartzman, 1971).

2. Ingestion, e.g. in the food allergies with skin manifestations. Although the intestinal wall is in general impervious to large molecular weight substances such as undigested proteins, this barrier is by no means absolute (Brunner & Walzer, 1928; Ratner & Gruehl, 1934).

3. Percutaneous, e.g. in hapten induced allergic contact dermatitis. 4. Parenteral injection, e.g. drug allergies and flea bite hypersensitivity. Just as the normal immune response of the body may be either antibody or cell

mediated, so are the allergic diseases which are of concern to dermatology. Broadly speaking, the allergic disorders can be divided into two categories :

1. Immediate hypersensitivity, which is mediated by antibody. 2. Delayed hypersensitivity, which is mediated by immunocompetent cells. One important fact about these two categories is that it is possible to transfer the

hypersensitive state from the patient to a normal homologous recipient. This can be achieved by the transference of white blood cells in the case of delayed hyper- sensitivity, and by means of serum in immediate hypersensitivities. I n this latter case the injections of serum, or fractions of serum are often given intradermally. If the site then becomes sensitized, it will respond to an injection of the allergen to which the donor was hypersensitive by the development of a wheal and ery- thema. This is the basis of the classic Prausnitz Kustner (PK) test, which has been of inestimable value in elucidating the pathogenesis of allergic disease.

I M M E D I A T E H Y P E R S E N S I T I V I T I E S

Much of the recent work on immediate hypersensitivities has been concerned with establishing the pathogenesis of atopic disease, or pollen hypersensitivity in man and the dog.

For many years it has been known that sera from atopic individuals, i.e. sufferers from allergic asthma and hay-fever would give positive PK tests upon transfer to normal recipients. In 1941 the same phenomenon was observed for the first time in a dog suffering from pruritus due to a pollen allergy (Wittich, 1941). The factor responsible for transferring the hypersensitive state was termed reagin,

T H E I M M U N O L O G Y O F A L L E R G I C S K I N D I S E A S E 433

and was not demonstrable in the sera of normal people or dogs. It is only in recent years that improved immunological techniques have enabled characteri- zation of the reagin.

Methods for separation of serum proteins are based primarily on their differing electrical charges, and differing molecular weights. If canine pollen allergic serum is subjected to preparative electrophoresis on pevikon block, the pattern indicated in Fig. 1 is obtained (Halliwell, 1971). The positive PK reactivity is seen to be primarily in the y l region, which contains the majority of the antibodies. That is to say that only the fractions indicated possess the capability of transferring the hypersensitive state to a normal recipient. This suggests immediately that reagin is immunoglobulin.

I I I I I 5 10 15 20 25

c % e ._ 2 c

U 0

Y C L

rction number

I-- Glo bu I i n b A1 bum i n + t - Y 2 ~ + ~ l + P * ~ = l

FIG. 1 . Electrophoresis of canine pollen (ragweed) allergic serum on Pevikon showing localization of the PK reactivity primarily in the yl region,

Fractionation of allergic serum according to molecular size, can be carried out with the aid of columns of Sephadex G200, which acts as a molecular sieve. The largest molecules are eluted first, and the smallest ones last. The elution pattern is recorded in Fig. 2. Until very recently only three classes of immunoglobulin had been described in the dog viz IgG, which provides the bulk of the antibody, IgM, the largest and most efficient antibody, and IgA, the prime responsibility of which is the defence of mucous surfaces. The distribution of these antibodies is recorded in Fig. 2 (Halliwell, 1971). I t will be immediately noted that the peak PK activity does not coincide with the peak concentration of any of the known immuno- globulin classes. This has led to the proposal of the name IgE to this antibody (Schwartzman et al., 1971). In all parameters so far investigated it has proved to be directly analogous to human IgE which has recently been shown as the carrier of atopic disease in man (Ishizaka & Ishizaka, 1968).

434 R. E. W. H A L L I W E L L

The amount of IgE in the serum is minute. I n man the normal mean level is around 0.2 pg (Johannsson, 1967) which is some 1/60,00Oth of the level of IgG. It is frequently elevated in allergic individuals, but the level seldom exceeds 1 pg/ml. We are not yet in a position to quantitate the levels in dogs, but they are likely to he of the same order.

How is it that this minute amount of antibody can be so pathogenic? For some time it has been known that IgE has strong tissue fixing properties, and the

C 0 I gA

._

.- ik? 8,": - L .I

- y l u l w I I ' , . 0 0 )

1 o a I

FIG. 2. Fractionation of canine pollen (ragweed) allergic serum by 3 in-series 4 x 60 cm columns of Sephadex G200. Localization of the PK reactivity and of the major im-

munoglobulin classes and albumin are demonstrated.

assumption was that the main target cells were tissue mast cells and blood baso- phils. More direct evidence for this contention is now emerging from studies in man (Hubscher, Watson & Goodfriend, 1970; Ishizaka, Tomioka & Ishizaka, 1971), and the dog (Halliwell, 1971).

Briefly, the skin of normal dogs was passively sensitized by injection of pollen (ragweed) allergic serum. By means of fluorescent antibody techniques it was shown that the ragweed binding antibodies injected attached to one cell type only in the skin (Fig. 3) . On counterstaining with toluidene blue these cells were shown to be mast cells. Mast cells are distributed throughout the dermis, but they are seen in greater numbers in the superficial layers (Fig. 4). Similarly immuno- fluorescent studies using antisera directed against the different immunoglobulin classes demonstrated that the only immunoglobulin detectable on the surface of mast cells was IgE (Fig. 5). Thus it can now be stated with some assurance that the target cell for IgE is the tissue mast cell. Further details of these findings will be published in due course. They do not, of course, preclude the possibility

T H E I M M U N O L O G Y O F A L L E R G I C S K I N D I S E A S E 43 5

FIG. 3. Biopsy of normal dog skin passively sensitized by pollen (ragweed) allergic serum. Immunofluorescent technique demonstrating the localization of ragweed binding antibodies. Three fluorescing cells are visible in the centre of the photograph. They are

tissue mast cells. x 1050.

that other immunoglobulin classes may, under certain circumstances, attach to mast cells, as will be discussed later.

When allergen reaches the mast cell coated with specific IgE antibody, de- granulation occurs with release of pharmacologically active compounds, in- cluding histamine, slow reactive substance, bradykinin, and proteolytic enzymes.

FIG. 4. Biopsy of normal dog skin demonstrating the localization of tissue mast cells within the dermis. Toluidene blue. x 280.

436 R . E . W. H A L L I W E L L

In some skin conditions, e.g. urticaria, the prompt relief obtained with anti- histamines implicates histamine as being of prime pathogenic importance. In other diseases which are clearly IgE mediated, little relief is obtained with antihistamines and i t may well be that the release of proteolytic enzymes, the pruritogenic properties of which are well known (Arthur & Shelley, 1958) are, at any rate in part, responsible for the symptoms.

IgE mediated skin conditions in the dog include atopic disease (i.e. the pollen allergies) and probably some food allergies, drug allergies, and possibly in part, hypersensitivity to external parasites. In the recently proposed classification by Gel1 & Coombs (1968) this is a Type I reaction. The pathway is outlined dia- gramatically in Fig. 6.

FIG. 5 . Same biopsy as in Fig. 3. Immunofluorescent technique to demonstrate the localization of IgE on tissue mast cells. Antisera to IgG, IgA, and IgM gave negative

results. x 1050.

The finding in man that immediate type hypersensitivities are not invariably IgE mediated, has led to a resurgence of interest in other possible mechanisms. There are two other well established immunological pathways which could be relevant, but it must be emphasized that the relevance of these pathways to skin allergies has not been conclusively demonstrated in man, let alone in animals. They both necessitate the formation of complement fixing antibodies, and the ratio of antigen to antibody in the initiation of the pathological process may well be crucial (Goldstein & Heiner, 1971; Cochrane, 1968). The possible mechan- isms are:

1. Circulating antibody may, on combination with antigen, lead to the for- mation of microcomplexes which lodge in small blood vessels, attract complement, and thus initiate tissue damage.

T H E I M M U N O L O G Y O F A L L E R G I C S K I N D I S E A S E 437

2. Circulating antibody may combine with antigen, fix complement, and lead to the formation of anaphylotoxins which are fragments of complement molecules. These substances have the property of initiating mast cell degranulation and release of the same substances that follow IgE mediated degranulation.

In these circumstances, as IgE is not involved, the PK test is negative. But it is still possible to transfer the hypersensitivity by means of intravenous administra- tion of serum. In the classification of Gel1 & Coombs (1968) these are Type I11

Predisposition to Access of form I g E antibodies allergen

(?hereditary) a Formotion of IgE antibodies

mast cel ls

Access of more

- 1 allergen

Degranulotion and releose o f pharmacologically active substances

\ Oedema and pruritus

Pruritus and inf iarnrna tion

8 Aliergen c] Antibody

np Estoblished site of action -no Possible site of action of indicated therapy of indicated therapy

FIG. 6. IgE mediated allergic disorders. Probable pathway of initiation and stages at which therapeutic measures are effective.

reactions. The relevance of these phenomena to veterinary dermatology is still unclear, but it seems probable that some of the food allergies may turn out to be mediated by these pathways. They are illustrated in Fig. 7.

The prerequisite for the development of immediate type hypersensitivities is thus the ability to form either IgE antibodies, or, alternatively, antibodies of other immunoglobulin classes which have the property of fixing complement in response to what is usually a very minor antigenic challenge. There is considerable

B

438 R . E . W . HALLIWELL

evidence that the tendency to form IgE antibodies may be hereditary (Schwartz- man et al., 1971). The factors predisposing to the formation of complement fixing antibodies are obscure.

Predisposition to Access of form complement fixing antibydies

allergen

I I Formation of complement fixing

arrtibody 1-1 I

Access of more allergen

/ Formation of micro comdexes

Fixotion’of comDlement

in small blood

t Production of anophylotoxin

Act iva t ion of complement

I8Adrener Ics Tissue

inf lammation Oedem’a and

rc- u pruritus [Antihistamine)

0 Allergen Antibody

Complement *:.:* Anaphylotoxin

*Established site o f action o f indicated therapy therapy

Possible site of action of indicated

FIG. 7. Complement fixing antibody mediated allergic disorders. Probable pathway of initiation and stages at which therapeutic measures are effective.

DELAY ED H Y P E R S E N S I T I V I T Y

The commonest clinical manifestation of this phenomenon is allergic contact dermatitis. Not a11 contact dermatitis is allergic in origin. Some substances may act as primary irritants, and no immune mechanism is then involved.

Substances implicated in causing allergic contact dermatitis in dogs have included constituents of plant foliage, dyestuffs, and polishes (Walton 1965). The

THE I M M U N O L O G Y O F A L L E R G I C S K I N D I S E A S E 439

agent responsible is often a simple chemical, and thus acts as a hapten, becoming allergenic following union with the sulphur rich epidermal proteins. Small quan- tities of the protein/hapten conjugate are then carried to the local lymph nodes where the immune response is initiated. Antigen specific mononuclear cells, particularly small lymphocytes, are produced and migrate chemotactically towards the skin, where interaction with allergen occurs. Mediators are then released which include macrophage migration inhibition factor (MIF) . Despite much recent work on the subject, little is known about this factor; but it has been shown to consist of a t least two substances with some five properties between them.

FIG. 8. Biopsy of skin of a dog in which delayed hypersensitivity was induced to 2 : 4 dinitrophenol. Twenty-four hour lesion. The cellular response is primarily mononuclear

with small lymphocytes prominent. H & E x 280.

Exactly how the inflammatory response that follows is caused, is still unclear. The initial response in the skin is thus essentially lyrnphocytic (Fig. 8) but in time other cells are attracted, notably macrophages, polymorphs, and plasma cells. At this stage the dermal reaction is no longer histologically specific for delayed hyper- sensitivity (Fig. 9). The pathogenesis is indicated diagrammatically in Fig. 10.

Although delayed hypersensitivity occurs most frequently following contact with allergen, it may occasionally result from antigenic challenge via other routes. An example is flea-bite hypersensitivity, which has both immediate, and delayed components (Benjamini, Keingold & Kartman, 1961). In the classification of Gel1 & Coombs (1968) this is a Type IV reaction.

T R E A T M E N T

A closer understanding of the mechanisms involved in allergic disorders is of major interest to Dractitioners because it facilitates diagnosis and treatment of

440 R. E . W. H A L L I W E L L

FIG. 9. Skin biopsy of same dog as Fig. 8. Severe reaction at 96 hr. The cellular infiltrate is more intense, and now includes polymorphs. H & E x 280.

the condition concerned. It is of value, therefore, to re-examine the mechanisms that have been described, and discuss how current therapeutic techniques can modify the outcome.

Immediate hypersensitivity-IgE mediated (Fig. 6 ) (a) Cortisone. Cortisone suppresses the immune response, and so reduces the rate

of formation of IgE antibodies. There is evidence that it reduces mast cell popu- lation in some species (Bloom, 1960). Although this effect has not been directly

Contact Union with Contact with - epidermal with more haDten protein allergen

Pruritus ond inflammation

.;:A. 4 -- -%F--- mediators Release of -- ' a. - --8-

Tm7TT77T- v - I 0

Q a: I Allergen

carried to local lymph Release of node allergen

specific

:: Hapten 1 lymphocytes

e Union of hapten with epidermal protein + Estoblished site of action of indicated therapy

-a+ ortisone

FIG. 10. Delayed hypersensitivity. Probable pathway of initiation of allergic contact der- matitis and stage at which therapeutic measures are effective.

T H E I M M U N O L O G Y O F A L L E R G I C S K I N D I S E A S E 44 1

demonstrated in the dog, the fact that mast cell tumours frequently respond, albeit temporarily, to corticosteroid therapy (Bloom, 1952), suggests that it could well be significant. The exact mechanism by which IgE antibody and allergen initiate mast cell degranulation is still speculative (Levine, 1965; Ishizaka & Campbell, 1959) but it could involve enzyme activation. The membrane stabilizing and anti-enzymic activities of cortisone may thus be inhibitory to degranulation. Both the inflammatory response, and the oedema which may follow release of phar- macological mediators from mast cells are inhibited by cortisone.

Cortisone thus acts a t many stages in the pathway, and its remarkable efficiency in alleviating the effects of this type of hypersensitivity can be readily understood.

(b) Antihistamines. The pathogenesis of histamine in this type of condition has been questioned (Halliwell & Schwartzman, 1971), but where urticaria, or angioneurotic oedema occurs, it is clearly implicated, and prompt relief will follow antihistamine therapy. These drugs do not inhibit the release of histamine-they merely antagonize its pharmacological actions. They relieve both the oedema and the accompanying pruritus.

(c) Hyposensitization. This is the only type of hypersensitivity that may be effectively controlled by this procedure. The rationale is that when a series of injections of the offending allergen are given, so-called blocking antibodies are produced. These are probably of the IgG class, and have the effect of binding any allergen in the circulation before it has a chance to reach the site of fixation of IgE. I t has been suggested that these blocking antibodies may sometimes attach to mast cells and are able to bind allergen without initiating mast cell disruption. I n this way they may be competing with IgE for sites on the target cell (Hubscher et al., 1970). One thus builds up a picture of protective antibodies (IgG) and sensitizing antibodies (IgE). The aim of hyposensitization is to encourage for- mation of the former.

(d) Disodium cromoglycate. Current research is turning more towards attempting to block the release of mediators that follows the interaction of sensitized mast cell and allergen. A major breakthrough in this respect was the development of disodium cromoglycate (Intal-Fisons) (Cox, 1967). Unfortunately the toxicity of this product in dogs prevents its application to this species. But it is highly likely that other similar drugs will be produced in due course.

(e) P-adrenergic stimulation. The effects of adrenergic stimulation, and of drugs with adrenergic activity, have been classified as a or p. Some drugs have pri- marily u-activity and some p, and some a mixture of both. The same applies to adrenergic blocking agents, some compounds specifically antagonize a-activity, and some antagonize /I-activity. I t has been known for some time that p- adrenergic stimulation or a-adrenergic blockade (and hence a relative increase in P-activity) could diminish release of pharmacologically active substances from sensitized mast cells. It is believed that these substances act by increasing the intracellular levels of cyclic 3’,5’,adenosine monophosphate (cyclic AMP) (Szentivanyi, 1968; Koopman, Orange & Austen, 1971). The main problem here

442 R . E . W . H A L L I W E L L

is to find an effective agent without undesirable side effects. I t is, however, a most promising line of research.

Immediate hypersensitiuip-complement$xing antibody mediated (Fig. 7 ) (a) Cortisone. The effects of cortisone on this type of hypersensitivity are es-

sentially the same as in the previous type. It is also effective in preventing the tissue damage and subsequent inflammation induced by microcomplexes. I t is thus a highly effective therapeutic measure.

(b) Antihistamines. As described in IgE mediated disorders, when the symptoms include urticaria or angioneurotic oedema, antihistamines are usually effective.

(c) Intd and j3-adrenergics. It is still unclear as to whether these substances are effective antagonists to mast cell degranulation in this situation.

Delayed hypersensitivity (Fig. 10) Immunologically specific therapy in delayed hypersensitivity is currently

limited almost exclusively to corticosteroids. They are inhibitory to the initiation of the immune response by the local lymph node, and they prevent the subsequent inflammation induced by contact between allergen and antigen specific lymphocytes in the skin. Fortunately, as allergic contact dermatitis is the commonest clinical manifestation of this phenomenon, avoidance of the offending allergen, once it has been elucidated, is often relatively simple.

C O N C L U S I O N S

The immunological mechanisms that have been described are quite varied. The ultimate goal in research in this field is to be able to inhibit the different mechan- isms at various phases of their development. Even now a clearer understanding of the pathways involved helps to provide an indication not only of whether the condition under investigation is allergic in origin or not, but also as to what type of mechanism is likely to be operative, and the possible allergens involved.

If we try to see below the surface, and resist the temptation to call every itchy skin ‘allergic’ and think no further, then, and only then, can a rational therapeutic regimen evolve.

A C K N O W L E D G M E N T S

Thanks are due to Dr A. C. Palmer and Dr P. Whittlestone for valuable assistance with the photomicrographs. Some of the observations recorded by the author were made during the tenure of a Wellcome Research Fellowship, which is gratefully acknowledged.

R E F E R E N C E S

ARTHUR, R.F. & SHELLEY, W.B. (1958) Ann. intern. Med. 49, 900. BENJAMIN, E., KEINGOLD, B.F. & KARTMAN, L. (1961) Proc. SOC. exp. Biol. Med. 108, 700. BLOOM, F. (1952) Proc. SOC. exp. Biol. Med. 79, 65 1.

T H E I M M U N O L O G Y OF A L L E R G I C S K I N DISEASE 443

BLOOM, G. (1960) Thesis, University of Stockholm. BRUNNER, M. & WALZER, M. (1928) Arch. intern. Med. 42, 173. COCHRANE, C.G. (1968) 3. Allergy, 42, 1 13. Cox, J.S.G. (1967) Nature, Lond. 216, 1328. GELL, P.G.H. & COOMBS, R.R.A. (1968) In: Clinical Aspects of Immunology, p. 575. Blackwell

GOLDSTEIN, G.B. & HEINER, D.C. (197 1) 3. Allergy, 46, 270. HALLWELL, R.E.W. (197 1) Unpublished observations. HALLIWELL, R.E.W. & SCHWARTZMAN, R.M. (1971) Vet. Rec. In press. HUBSCHER, T., WATSON, J.I. & GOODFRIEND, L. (1970) J. Immun. 104, 1187. ISHIZAKA, K. & CAMPBELL, D.H. (1959) 3. Immun. 83, 318. ISHIZAKA, K. & ISHIZAKA, T. (1968) 3. Allergy, 42, 330. ISHIZAKA, K., TOMIOKA, H. & ISHIZAKA, T. (1971) 3. Immun. 105, 1459.

JOHANNSSON, S.G.O. (1967) Lancet, ii, 95 1. KOOPMAN, W. J. ORANGE, R.P. & AUSTEN, K.F. (1970) 3. Immun. 105, 1082. LEVINE, B.B. (1965) 3. Immun. 94, 12 1. RATNER, B. & GRUEHL, H.L. (1934) 3. Clin. Invest. 13, 51 7 . SCHWARTZMAN, R.M., ROCKEY, J.H. & HALLWELL, R.E.W. (1971) Clin. Ex$. Immunol. In press. SZENTIVANYI, A. (1968) 3. Allergy, 42,203. WALTON, G.S. (1964) In: Compurutiue Physiology and Pathology of the Skin (Ed. by A. J. Rook and

WITITCH, F.W. (1941) 3. Allergy, 12, 247.

Scientific Publications, Oxford.

G. S. Walton), p. 515. J. B. Lippincott, Philadelphia.

RCsumC. Les voies immunologiques principal= qui mtnent A l’apparition d’affections dermiques caustes par une allergie sont dtcrites. Les tendances Wrapeutiques actuelles et futures sont examintes en fonction du stade pathogtnique oh elles sont efficaces.

Zusammenfassung. Die immunologischen Hauptwege, die zur Entstehung von allergischen Hautstorungen fiihren, werden beschrieben. Die gegenwartigen und vorauszusehenden Ent- wicklungen der Therapie werden unter Beriicksichtigung des Stadiums in der Pathogenese besprochen, in dem sie wirksam werden.