08_117_126 04.11.2005 15:39 Uhr Seite 117 Chapter 8 Ultrastructure of Irritant 8 and Allergic Contact Dermatitis Carolyn M. Willis Contents In the sections which follow, ultrastructural 8.1 Introduction . 117 changes seen in skin exposed to irritants and aller- gens are described.With the exception of the last sec- 8.2 Ultrastructural Changes in the Epidermis . 117 tion, which deals specifically with a recent study of 8.2.1 Stratum Corneum . 117 chronic chromate hand dermatitis, the data refer to 8.2.2 Viable Keratinocytes . 118 the effects of acute exposure. 8.2.2.1 Irritant Contact Dermatitis . 118 8.2.2.2 Allergic Contact Dermatitis . 121 8.2.3 Langerhans Cells . 121 8.2 Ultrastructural Changes 8.2.3.1 Allergic Contact Dermatitis . 122 in the Epidermis 8.2.3.2 Irritant Contact Dermatitis . 122 8.3 Ultrastructural Changes in the Dermis . 124 The stratified nature of the epidermis, and the pres- 8.4 Ultrastructural Changes ence of Langerhans cells and melanocytes in addi- in Chronic Contact Dermatitis . 124 tion to keratinocytes, presents a wide variety of bio- 8.5 Summary . 125 chemical and immunological targets for topically ap- References . 125 plied irritants and allergens. Primary contact occurs at the outermost stratum corneum,which,depending on the chemical characteristics of the substance, may show ultrastructural evidence of damage. Diffusion into and penetration of the viable epidermal regions then take place. Again depending upon the chemical 8.1 Introduction nature of the agent, as well as the severity of response and time of examination post-exposure, morpholog- Electron microscopy has provided us with a valuable ical indications of metabolic interruption may be tool to investigate the cellular and subcellular effects seen. of topical exposure to irritants and allergens, com- plementing histological examination at the light mi- croscope level. Most reported data are based on the 8.2.1 Stratum Corneum use of conventional preparative techniques, but de- velopments such as post-fixation in ruthenium te- The outermost diffusion barrier of the skin, the stra- troxide to visualize intercellular lipids and the par- tum corneum, is a 20- to 30-cell-thick layer of flat, allel examination of semi-thin and ultra-thin resin- hexagonal, protein-rich corneocytes surrounded by embedded samples have enhanced our understand- intercellular lipids.Generally speaking,chemical irri- ing of the cellular changes that take place. It is impor- tants rather than allergens produce marked changes tant to remember, however, that electron microscopy to its structure and behavior, as evidenced, biophysi- gives us only a snapshot of a minute fraction of a skin cally, by increased transepidermal water loss. Recent biopsy. Therefore, studies employing small sample ultrastructural studies utilizing ruthenium tetroxide numbers, with limited scrutiny of each specimen, as a post-fixative have greatly increased our under- should be viewed with a degree of caution. This is standing of the manner in which some irritant chem- particularly true for irritant contact dermatitis in- icals interact with this region of the epidermis and vestigations, where considerable inter-individual contribute to the development of irritant contact der- variation in the intensity of the response to chemicals matitis (ICD). The application of low concentrations occurs, and where the cellular damage inflicted is of the anionic surfactant sodium lauryl sulfate (SLS) rarely uniform across the application site. to normal human skin was found by Fartasch to re- 08_117_126 04.11.2005 15:39 Uhr Seite 118 118 Carolyn M.Willis While both induce varying degrees of spongiosis, sult not so much in an alteration of the existing lipid clearly visible by both light and electron microscopy, structure, but rather an alteration in the synthesis of chemical irritants also give rise to a heterogeneity of new lipids [1]. Hence, disturbance of lamellar body forms of intracellular damage that are time, dose lipid extrusion and transformation into lipid bilayers and, in some cases, irritant dependent. occurred, in the absence of any disruption to the intercellular lipid layers of the upper stratum corne- um. By way of contrast, acetone produced a different 8.2.2.1 Irritant Contact Dermatitis pattern of change.Epidermal lipid lamellae displayed disruption and loss of cohesion throughout the stra- Two early studies provided some of the first evidence tum corneum – the transformed, more nonpolar, la- that irritants can damage the skin by different mech- mellar lipids showing greater disruption than the anisms. A comparison between the effects of an acid more polar lamellar body sheets [1]. A similar dis- and an alkali on human epidermis found that sodium ruption of stratum corneum intercellular bilayers hydroxide dissolved the contents of horny cells and was also seen in human skin patch-tested with water disrupted tonofilament–desmosome complexes, alone [2], which would have the effect, as pointed out while hydrochloric acid did not [3]. Similarly, in a by the investigators, of enhancing skin permeability comparative study of two lipid solvents, the response and susceptibility to irritants. to acetone, which was characterized by intracellular 8 edema of keratinized cells and vacuolation of spi- Core Message nous cells, was conspicuously different to that to ker- osene, in which the formation of large lacunae and í cytolysis of spinous cells were seen [4]. In our own Chemical irritants generally have a greater study, designed to systematically compare the mor- impact than allergens on the ultrastructure phological effects of six structurally unrelated irri- of the stratum corneum. tants on normal human skin, electron microscopy al- so revealed significant differences in the nature of the cellular damage induced by different chemicals after 48 h of exposure [5]. Patch test reactions to SLS 8.2.2 Viable Keratinocytes were characterized by parakeratotic cells in the upper epidermis, containing dense osmiophilic cyto- The greatest diversity of ultrastructural effects on vi- plasm with numerous lipid droplets and vesicles, but able keratinocytes within the epidermis is undoubt- an absence of keratohyalin granules (Figs. 1, 2). In edly exerted by irritants, rather than by allergens. contrast, the cationic detergent benzalkonium chlo- Fig. 1. The interface between dark, osmiophilic, vesiculated, par- akeratotic cells in the upper epidermis and paler cells of the stratum spinosum in a 48-h patch test reaction to sodium lauryl sulfate (SLS) (4%) 08_117_126 04.11.2005 15:39 Uhr Seite 119 Ultrastructure of Irritant and Allergic Contact Dermatitis Chapter 8 119 ride produced distinct areas of necrosis (Fig. 3). Ap- plication of the 12-C-long chain fatty acid nonanoic acid resulted in the formation of tongues of dyskera- totic cells, largely composed of dense, wavy aggre- gates of osmiophilic keratin filaments associated with prominent intercellular desmosomes, and con- taining shrunken nuclei with condensed, marginated heterochromatin (Fig. 4). Exposure to dithranol pro- duced different changes again, namely markedly en- larged upper epidermal keratinocytes, containing finely dispersed filaments and ribosomes, and, in keeping with previous findings [6, 7], disrupted mi- tochondria (Fig. 5). The concept of ultrastructural changes being irri- tant-dependent was further supported by a recent study of the effects of a wide variety of irritant chem- icals on the skin of hairless guinea pigs [8].Although the skin changes described were not identical to those seen in human skin, partly perhaps as a result of concentration differences, it was clear, that again the nature of the epidermal damage elicited by SLS differed markedly from that of benzalkonium chlo- ride. Fig. 2. Basal keratinocytes in a 48-h SLS (4%) patch test reac- tion, illustrating lipid droplet accumulation and prominent intracytoplasmic vesiculation Table 1. Ultrastructural changes induced in the viable epider- mis by acute exposure to selected irritants.Changes depend on the irritant, its concentration, and time Irritant Ultrastructural changes Sodium lauryl Spongiosis, vesiculation, nuclear/intra- sulfate cytoplasmic/mitochondrial vacuolation, lipid droplet accumulation, hydropic swelling, decreased desmosomes with aggregation of tonofilaments Benzalkonium Nuclear/intracytoplasmic vacuolation, chloride nuclear pyknosis, mitochondrial swell- ing, organelle disruption, hydropic swelling, spongiosis Dithranol Hydropic swelling, mitochondrial mem- brane disruption, spongiosis, intracyto- plasmic vacuolation, dyskeratosis, apop- tosis, colloid bodies Croton oil Marked spongiosis, intracytoplasmic vacuolation, pyknotic/enlarged nuclei Nonanoic acid Dyskeratosis, nuclear/intracytoplasmic vacuolation, vesiculation, lipid droplet accumulation, pyknotic nuclei Acetone Acantholysis, spongiosis, nuclear/intra- cytoplasmic edema and vacuolation Sodium Disrupted tonofilament–desmosome Fig. 3. An area of necrosis induced in the mid epidermis by hydroxide complexes 48-h patch testing with benzalkonium chloride (0.5%). Kerati- nocytes show extensive vacuolation, pyknotic nuclei, and dis- Combined human and animal data [3–12] rupted organelles and membranes 08_117_126 04.11.2005 15:39 Uhr Seite 120 120 Carolyn M.Willis Fig. 4. Dyskeratotic upper epider- mal cells, containing dense, wavy aggregates of osmio- philic keratin filaments, pro- duced by 48-h patch testing with nonanoic acid (80%) 8 Fig. 5. Enlarged upper epidermal keratinocyte, with cytoplasm containing