Contents
7.1 Introduction: General Considerations . . . . . 107 7.2 Histopathological Features
of Positive Allergic Patch Test Reactions . . . . 108 7.2.1 Epidermal Changes . . . . 108 7.2.2 Dermal Changes . . . . 108 7.3 Histopathological Features
of Positive Irritant Patch Test Reactions . . . . 110 7.3.1 Epidermal Changes . . . . 110 7.3.2 Dermal Changes . . . . 112 7.4 Histopathological Criteria
for Distinguishing Between Allergic
and Irritant Patch Test Reactions in Humans . 112 7.5 Comparative Immunohistochemical
and Immunocytochemical Characteristics of Allergic and Irritant Patch Test Reactions in Humans . . . . 114 7.5.1 Epidermal Langerhans Cells in Irritant
and Allergic Positive Patch Test Reactions . . . 114 7.5.2 Cells of the Infiltrate in Irritant
and Allergic Positive Patch Test Reactions:
Immunophenotypic Studies . . . . 114 7.6 Conclusions . . . . 115 References . . . . 115
7.1 Introduction: General Considerations Histopathological features of allergic and/or irritant contact dermatitis are not described in full detail in most textbooks of dermatology [1–3]. This is because they are not usually involved in the diagnostic proce- dures of both conditions. In most cases, contact der- matitis is suspected from anamnestic data and clini- cal signs [4]. Diagnosis is confirmed by patch testing and/or other tests, with additional information about the responsible agent(s). Nevertheless, in daily prac- tice, contact dermatitis may be superimposed onto an underlying skin disease, the diagnosis of which is sometimes difficult.
In those circumstances, skin biopsy is highly rec- ommended and considered an important tool of dif- ferential diagnosis.
Among such examples, the following can be quoted:
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Nummular dermatitis (eczema) versus para- psoriasis en plaques (benign type), psoriasis or tinea incognito.
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Seborrheic dermatitis versus lupus erythema- tosus or rosacea.
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Pompholyx versus pustular psoriasis, palmo- plantar pustulosis or bullous pemphigoid.
When an eczematous reaction is involved, the histo- pathological clue in diagnosis is the presence of a spongiotic (spongiform) dermatitis, notwithstand- ing its origin: irritant, allergic or endogenous.
In each individual case, the histopathological pic- ture is dependent on various parameters, which can play a confounding role, such as: (1) unknown dura- tion of the disease; (2) lesions related to scratching;
(3) infections; and (4) lichenification. Clinicians are sometimes advised to perform two biopsies instead of one, in order to focus on different stages of the dis- ease.
A full description of histopathological signs of al- lergic and/or irritant contact dermatitis is better achieved by a careful study of positive allergic and/or irritant patch test reactions.
This approach has two advantages: (1) the histo- pathological signs reflect a practical situation, en- countered daily at the patch test clinic; (2) a positive patch test reaction is a clear-cut, unmodified reaction – the direct consequence of the application of a sub- stance on previously intact skin.
The only possible drawback to using patch test re- actions is the role played by occlusion. This might be especially true for allergic reactions, and it is the rea- son for this description also being based upon open (unoccluded) reactions, the use of which is becoming commoner in many clinics.
This description will be a “freeze-frame photo- graph” of the situation at 48, 72 or 96 h; it does not take into account the chronology of events, starting at time 0 (with the application of the substance) and
Histopathological and Immuno-
histopathological Features of Irritant and Allergic Contact Dermatitis
Jean-Marie Lachapelle, Liliane Marot
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continuing for instance every 6 h – until 48 or 72 h.
This dynamic view has been achieved in previous re- search studies [5].
It has to be emphasized that this description is useful when expressed in scientific (more than prac- tical) terms, to improve our knowledge at the micro- scopic level. In this respect, patch testing has been used recently as a tool for evaluating the efficacy of topical drugs, such as pimecrolimus [6] or tacroli- mus, [7] versus corticosteroids regarding the out- come of allergic positive patch test reactions to nick- el sulfate in volunteers. The evaluation of results has been based on visual scoring and biometrical meas- urements using noninvasive technology, but not on the evaluation of histopathological parameters. In- deed, biopsy is considered an invasive procedure, re- jected nowadays by most ethical committees.
A lot of information delivered in the next para- graphs comes from our own material, used in former studies, at a time when legal procedures were not as strictly codified as they are today.
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In clinical practice, when the diagnosis of allergic and/or irritant contact dermatitis is not clear-cut, skin biopsy is considered an important tool of differential diagnosis.
In contrast, biopsies of positive patch tests are not recommended, except for scientific purposes.
7.2 Histopathological Features of Positive Allergic Patch Test Reactions
The histopathological picture of a positive allergic patch test reaction (read at 48 h) is a typical example of a spongiotic dermatitis [3]. Features are very simi- lar in all cases.
7.2.1 Epidermal Changes
In the epidermis, spongiosis is an almost constant sign, resulting from the accumulation of fluid around individual keratinocytes (exoserosis) and the conse- quent stretching of intercellular desmosome com- plexes (or “prickles”).
Spongiosis is focally or evenly distributed along the length of the epidermis; it is either limited to the lower layers or extends from the basal to the granular
layer. In some but not all cases, it spares the cells of the sweat duct unit. Hair follicles are usually involved by the spongiotic process.
A more plentiful accumulation of fluid results in rupture of the intercellular prickles and the forma- tion of vesicles. Thus, in allergic contact dermatitis, spongiotic vesiculation can be defined as an intra- epidermal cavity with ragged walls and surrounding spongiosis. There is migration of inflammatory cells into the epidermis (exocytosis). These cells, mainly lymphocytes and occasionally polymorphonuclear neutrophils and eosinophils, accumulate in the spon- giotic vesicles.
Some vesicles are rounded and tense; they are lo- cated in the stratum spinosum, whereas others are flat and located in the stratum corneum. They finally rupture at the surface of the epidermis and vertical channels of fluid discharge are occasionally seen on serial sections. These channels are sometimes color- fully described as “Devergie’s eczematous wells.”
Intracellular edema of keratinocytes does occur, with accumulation of glycogen.
At the electron microscopic level, dissolution of interdesmosomal areas, or “microacantholysis,” can be demonstrated; remaining desmosomes show ten- sion and alignment of tonofilament bundles.
In photoallergic contact dermatitis, a biopsy of the photopatch test site, when positive, clearly shows transforming keratinocytes in sunburn cells.
7.2.2 Dermal Changes
Papillary blood capillaries are often congested and dilated; dilatation of lymphatic vessels is very con- spicuous in some but not all cases. Dermal edema is prominent with deposits of acid mucopolysaccha- rides. A dense mononuclear cell infiltrate is usually present around blood vessels of the lower dermis, and even in the subcutaneous tissue. The cells of the infiltrate migrate from the perivascular spaces to the epidermis and are found throughout the dermal tis- sue, either isolated or grouped in small clumps.
It is not uncommon to see a dermal infiltration of inflammatory cells around and within hair sheaths and sebaceous ducts, which show some degree of spongiosis and cellular degeneration. This picture could be partly due to direct penetration of the aller- gens through the pilosebaceous unit.
The infiltrate is of the lymphohistiocytic type, composed almost exclusively of mononuclear cells, varying in form and size. The occurrence of an inti- mate contact between the cell surfaces of lympho- cytes and the cell processes of macrophages was demonstrated many years ago at the ultrastructural
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Core Message
level. It was emphasized that, in delayed hypersensi- tivity, macrophages were thought to play an impor- tant role, together with lymphocytes. This view was later confirmed and broadened by the discovery of the role played by Langerhans cells.
Polymorphonuclear neutrophils are usually ab- sent. Some eosinophils can be found in the edema- tous tissue of the upper dermis, migrating towards the epidermis.
The histopathological picture is very similar when the biopsy is taken 72 h or 96 h after application of the allergen. The dermal infiltrate around blood ves-
sels is usually more pronounced. At this later stage, a few eosinophils can be observed very occasionally.
The role of the mast cell in allergic contact hyper- sensitivity remains controversial. Some studies showing histological evidence of mast cell degranu- lation suggest that early mast cell activation occurs [8].
In recent years, Hannuksela’s repeated open appli- cation test (see Sect. 22.10.2) has become popular for confirming the clinical relevance of positive allergic patch test reactions [9]. We have taken biopsies from positive allergic open test reactions on the volar as-
Fig. 1.
Allergic positive patch test reaction to balsam of Peru ( Myroxylon pereirae) at 2 - days: spongiotic vesiculation in epidermis with exocytosis of mononuclear cells and dermal edema. Hematoxy- lin–eosin–saffron stain ( ×150)
Fig. 2.
Allergic positive patch test reaction to wool wax alco- hols (lanolin alcohol) at 2 - days: dense perivascular in- filtrate of mononuclear cells.
Hematoxylin–eosin–saffron
stain ( ×250)
pect of the forearm, or the cubital fossa, 48, 72, or 96 h after application of the allergen. In all cases, the his- topathological picture was quite similar to that ob- served in positive allergic patch test reactions.
Two additional features can be observed occasion- ally:
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In some positive allergic patch test reactions, particularly to azo dyes, purpuric lesions are clinically present [10]: in those cases, there is an important extravasation of erythrocytes, mainly located around blood capillaries, but extending also to interstitial dermal tissue and invading epidermis (exocytosis).
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In some other positive allergic patch test reac- tions, e.g., to gold (more often at 96 h than 48 h), the infiltrate may be lymphomatoid and mimics pseudolymphoma [11]. It is dense, with a few mitotic figures, and subtle nuclear atypia. Rarely, the lymphoid cells may be very bizarre.
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Histopathological features of positive allergic patch test reactions are typical of a spongiotic dermatitis, similar to that observed in different eczematous (exogenous or endogenous) reactions.
7.3 Histopathological Features of Positive Irritant Patch Test Reactions
The histopathological picture of positive allergic patch test reactions has been shown to be very simi- lar (“monotonous and uniform”) in most cases (see above). When irritants are applied – under occlusion – on the skin, a wide range of different lesions can be seen. This kaleidoscope of lesions concerns mainly epidermal alterations.
Various factors play a role in the formation of le- sions: (1) the nature of the irritant agent, and conse- quently its mode of deleterious action on the cells, (2) the concentration of the irritant applied on the skin, (3) the ways of penetration of the skin, and (4) the in- dividual reactivity of the skin to a well-defined irri- tant.
It is therefore possible that the same irritant chemical can produce different types of lesions in
different patients, even when it is applied for the same duration and under the same conditions. There is no general rule in this respect.
7.3.1 Epidermal Changes
Various alterations of epidermal cells can be ob- served. In some cases, these alterations are limited to the superficial layers of the epidermis, the stratum granulosum and the upper part of the stratum spino- sum; in others they extend to the dermo-epidermal junction, invading all layers of the epidermis. At first cells become karyopyknotic and lose their cytoplas- mic staining properties on hematoxylin and eosin sections. These changes are known as “Bandmann’s achromasia” [12]. When the irritation process be- comes more severe, complete necrosis (or cytolysis) of epidermal cells occurs, leading to the formation of intra- or subepidermal vesicles and bullae.“Chemical acantholysis” of epidermal cells can be seen, mainly, but not exclusively, with certain irritants, such as cantharidin and trichloroethylene [13]. Polymorpho- nuclear neutrophils accumulate in the damaged epi- dermis, leading to the formation of subcorneal or intra-epidermal pustules.
In some cases, the formation of pustules is prefe- rentially limited to the hair follicles. Follicular pus- tules are preferentially provoked by some irritants, such as croton oil (“croton oil effect”), or metal salts such as chromates, and those of mercury and nickel.
Pustules due to metals are observed mainly, but not exclusively, in atopics. As already noted many years ago, some irritant reactions do not show any of the
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Core Message
Table 1. Epidermal lesions observed in relation to certain com- mon irritants
Irritants Epidermal lesions
Nonchlorinated organic Achromasia; superficial solvents (i.e., alkanes, necrosis; karyopyknosis;
such as n-hexane; toluene; very occasional acantholy- xylene; white spirit; sis; subepidermal vesicles turpentine, etc.) and/or bullae
Chlorinated organic Acantholysis ++; karyopyk- solvents (i.e., trichloro- nosis; complete necrosis of ethane; trichloroethylene; epidermal cells; intraepi- carbon tetrachloride; etc.) dermal vesicles and/or
bullae
Acids, alkalis, surfactants, Achromasia; superficial or detergents, aldehydes complete necrosis of epi-
dermal cells; subepidermal
vesicles and/or bullae; no
acantholysis
aforementioned histopathological signs; they are ex- clusively spongiotic (with or without vesicles).
Such observations can be made: (1) with weak irri- tants, (2) with strong irritants, applied on the skin at a low concentration, and (3) in the “excited” (or irri- table) skin syndrome.
Examples of epidermal lesions classically ob- served with certain categories of irritants are given in Table 1.
Many years ago, ultrastructural studies threw some light on the mode of action of certain irritants, including croton oil, sodium hydroxide, and hydro-
chloric acid. More recently, Willis et al. [14, 15] com- pleted an extensive study comparing the action of several categories of irritants, using semi-thin sec- tion technology. They noted in particular that vari- ous kinds of detergents damaged epidermal cells in different ways when applied at a low concentration.
For instance, the major response to the anionic deter- gent sodium lauryl sulfate was parakeratosis, indicat- ing increased epidermal cell turnover, whilst benzal- konium chloride, a cationic detergent, caused a dif- ferent type of reaction – spongiosis and exocytosis with focal necrotic damage [16].
Fig. 3.
Irritant positive patch test reaction to croton oil at 2 - days: spongiotic vesiculation in epidermis with exocytosis of mononuclear cells. This picture is indistinguishable from an allergic reaction.
Masson’s trichrome blue stain ( ×150)
Fig. 4.
Irritant positive patch test
reaction to trichloroethylene
at 2 days: epidermal necrosis
with acantholytic keratino-
cytes, exocytosis of inflam-
matory cells. Masson’s tri-
chrome blue stain ( ×150)
Phototoxic reactions are characterized by the presence of eosinophilic necrotic keratinocytes (“sunburn cells”).
7.3.2 Dermal Changes
Dermal changes are also related to the mechanisms involved in the mode of action of each individual ir- ritant. Dermal edema is absent or slight. Blood capil- laries and lymphatics are discretely dilated, but usu- ally to a lesser extent than in positive allergic patch test reactions.
In some cases, there is an important inflammatory response distributed around the blood vessels of the upper and mid-dermis. It is either homogeneously mononuclear or mixed (polymorphonuclear neu- trophils and lymphocytes/macrophages). Eosino- phils are absent. In cases of severe irritation, it is usu- al to find pyknotic remnants of neutrophils in the upper part of the dermis.
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Histopathological features of positive irritant patch test reactions are varied, according to the nature and/or concen- tration of irritant chemicals and to the individual reactivity of the skin. This
“kaleidoscope” of lesions concerns mainly epidermal alterations.
7.4 Histopathological Criteria
for Distinguishing Between Allergic and Irritant Patch Test Reactions in Humans
In the preceding paragraphs, the various histopatho- logical signs encountered in allergic and irritant patch test reactions have been reviewed in detail [12, 17–20]. We must remember that this description re-
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Core Message
Fig. 5. Irritant positive patch test reaction to croton oil at 2 - days: a follicular pustule is filled with neutrophils and lym- phoid cells. There is a perivascular infiltrate of mononuclear cells. Masson’s trichrome blue stain ( ×75)
Fig.6. Irritant positive patch test reaction to sodium lauryl sul-
fate at 2 days: the epidermis is partly necrotic with infiltration
of mononuclear cells. There is a dermal perivascular infiltrate
of mononuclear cells. Hematoxylin–eosin stain ( ×150)
fers to “typical” cases: irritant (without allergic com- ponent) or allergic (without irritant component).
Comparative signs are presented in Table 2. These distinctive criteria are of limited value in practice for many reasons: (1) most criteria are present in irritant as well as in allergic positive patch test reactions; (2) other criteria are predominant either in irritant or in allergic reactions, but they lack specificity; (3) most allergens also have irritant properties. Even when al- lergens are patch tested at a concentration below the level of clinical irritancy to avoid “mixed” pictures, it is just possible that subclinically, at the microscopic level, they might show a mixed picture of irritation and allergy.
In practice, when a positive patch test reaction is clinically doubtful (irritant versus allergic), the help from a biopsy is minimal, due to the differential bias explained above.
Avnstorp et al. [21] conducted a semi-quantitative histopathological study of individual morphological parameters in allergic and irritant patch test reac- tions. Their conclusions were as follows: statistical analysis by correlation of 17 selected variables gives a diagnostic specificity of 87% and a sensitivity of 81%
for allergic reactions. For irritant reactions, the spec- ificity is 100% and the sensitivity 46%. By multiple regressive analysis, an index was calculated for the differentiation of allergic and irritant reactions. If this index were to be used in cases of allergic patch test reactions, all would also be reported as allergic reactions while half of the irritant reactions would be reported as allergic. Although this study has shed some light on the problem of the histopathological differentiation between allergic and irritant contact
dermatitis, many difficulties remain in making such a differentiation [21].
When considering all these potential criteria of differential diagnosis, it is worth saying that spongio- sis is in bulk a more consistent feature in allergic than in irritant reactions. Vestergaard et al. [22] have recently conducted a human study comparing aller- gic and irritant reactions. Biopsy samples were taken at a very early stage (6–8 h) after applying (1) an irri- tant (benzalkonium chloride) and (2) an allergen (that is colophony or quaternium-15) to individuals with known allergy to one of these allergens, selected because they rarely give rise to unspecific or irritant reactions. The significant finding was that focal spongiosis was present only in allergic reactions.
It is likely that the aggregation of monocytes/mac- rophages and proliferating T-cells, along with their chemical mediators, is responsible for the epidermal spongiosis in allergic contact dermatitis [23].
In conclusion, though conventional histopatholo- gy of positive patch test reactions can provide some useful information, it is of little help in separating al- lergic from irritant or mixed reactions. Drawing such a conclusion at the end of this section might appear to be negative, since a different view has prevailed for decades in so many European contact dermatitis clinics. Nevertheless, it is based on a careful review of the literature and a reappraisal of our own material.
It coincides with the views of the basic scientists and must be considered by practicing dermatologists as reflecting reality.
Table 2. Distinctive histopathological criteria between allergic and irritant patch test reactions in humans (modified from [8]) Allergic reactions Irritant reactions
Epidermis
Spongiosis + to +++ + or –
Exocytosis + to +++ +++
Vesicles + (spongiotic) + (rarely spongiotic)
Formation of bullae Facultative (spongiotic) Facultative (rarely spongiotic)
Pustules – + or –
Necrosis of epidermal cells – + to +++
Acantholysis of epidermal cells – + or –
Distribution of the infiltrate in epidermis Focal [21] Diffuse [21]
Dermis
Perivascular infiltrate Mononuclear Mononuclear or mixed
(mononuclear + neutrophils)
Eosinophilic leucocytes + or – –
Dilatation of lymphatic vessels + or – –
Dilatation of blood capillaries + or – + or –
Edema + or – Very unusual
that allergic and irritant patch test reactions cannot be differentiated reliably by counting LC, in spite of the small differences observed [27]. Moreover, lym- phocyte/LC apposition is observed in both types of reaction [28, 29]. The presence of human leukocyte antigen (HLA) DR antigens on keratinocytes in aller- gic reactions may reflect an immunological response [30].
7.5.2 Cells of the Infiltrate in Irritant and Allergic Positive Patch Test Reactions:
Immunophenotypic Studies
Early human studies showed little evidence of diffe- rential cytokine release between allergic and irritant contact dermatitis.
This strongly suggests that, although initiating events vary considerably, the cascade mechanisms responsible for the induction and release of regulat- ing mediators are similar [31, 32]. Clearly, most, if not all, pro-inflammatory phenomena can be caused both by irritants and allergens. Therefore, they do not unambiguously discriminate between irritants and contact allergens [33].
In the various studies conducted so far, the com- position of the infiltrates is similar in allergic and ir- ritant reactions, and consists of T lymphocytes of helper/inducer types in association with T-cell acces- sory cells, that is, LC and HLA-DR-positive macro- phages.
Probably, true differences between these types of compounds depend on whether or not allergen-spe- cific T-cells become involved [33]. Thus, only after specific T-cell triggering might distinctive features be observed, e.g., local release of certain chemokines, such as CXCL10 (IP-10) and CXCL11 (I-TAC/IP9) [34].
The latter chemokines are produced by interferon- γ-activated keratinocytes and T lymphocytes [35].
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New immunocytopathological techniques are of no real help in distinguishing between irritant and allergic patch test reactions, since there is little evidence of differential cytokine release.
Clearly, most, if not all, of pro-inflamma- tory phenomena can be caused by both irritants and allergens. Therefore, they do not discriminate between irritants and contact allergens.
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