Dermo-epidermal separation is associated with induced tenascin expression in human skin

Tenascin, a large glycoprotein of the extracellular matrix, shows a site-restricted distribution during embryogenesis. and can be found in adults in a variety of pathological conditions. In normal skin, tenascin is expressed at low levels, but it is upregulated in skin tumours, in a number of skin diseases with epidermal hyperproliferatkm and during wound healing. Several tenascin variants have been described, and these arise by alternative splicing. Using a monoclonal antibody recognizing all tenascin variants, and polyclonal antibodies specific for the large tenascin variants, we have investigated tenascin expression in bullous diseases such as epidermolysis bullosa, pemphigus, bullous pemphigoid and pemphigoid gestationis. By immunohistochemistry, we have found increased tenascin staining in ail patient skin samples, with a more pronounced tenascin expression in samples of autoimmune bullous diseases. The large tenascin variants seem to be major forms of tenascin occurring in healthy skin. In patients with blistering diseases, however, these large variants appear to represent a subpopulation of the induced tenascin accumulation. These findings suggest different functions for the tenascin variants in normal and diseased skin.     

Tenascin expression in hyperproliferative skin diseases

The expression of tenascin, a recently discovered extracellular matrix glycoprotein, was studied by immunohistochemistry in normal human skin and in a number of skin diseases with epidermal hyperproliferation such as psoriasis, basal cell carcinoma, Bowen's disease and solar keratosis. Tenascin expression in the upper dermis of normal skin was found to vary from almost absent to patchy along the basal membrane. Staining was continuous and intense around blood vessels, hair follicles and eccrine sweat ducts. In basal cell carcinoma a marked expression of tenascin was found in the tumour stroma, especially adjacent to the basal membrane surrounding the tumour cell nests. In Bowen's disease and solar keratosis, tenascin expression was found in the dermis next to the keratinocytes. In psoriasis the dermal papillae of clinically involved skin were intensely stained and a continuous band of tenascin was present in the upper dermis along the basal membrane. The distribution of tenascin differed from other known extracellular matrix components.     

Identification and partial characterization of pemphigoid antigen extracted from normal human skin

Antibodies in the sera of patients with the disease bullous pemphigoid define a normal component of the basement membrane of stratified squamous epithelia. Pemphigoid antigen has been shown to be synthesized by mouse and human epidermal cells in culture as an approximately 220 kd protein when reduced. The purpose of this study was to characterize pemphigoid antigen extracted directly from normal human skin and to determine its relationship to the high molecular weight protein found in culture. Suction blister-derived epidermis was extracted with 2% sodium dodecyl sulfate (SDS) and the solubilized proteins were separated, after reduction, by SDS-polyacrylamide gel electrophoresis (PAGE). The separated proteins were electrophoretically transferred to nitrocellulose sheets. Pemphigoid antigen was then specifically identified by immunoperoxidase staining using pemphigoid sera. IgG from 5 different bullous pemphigoid patients bound a band of apparent molecular weight 225 kd. Antibodies from 6 normal sera and 4 pemphigus sera did not bind this molecule. On a lower percentage (4%) polyacrylamide gel the pemphigoid antigen could be resolved as a doublet (two closely spaced bands) in the range of 220-240 kd. When unreduced, the pemphigoid antigen extracted from skin was also detected as a doublet in the 220-240 kd range. This suggests that the two chains are not necessarily disulfide-linked to each other in skin. Newly synthesized pemphigoid antigen immunoprecipitated from extracts of cultured human epidermal cells could also be identified on SDS-PAGE, when reduced, as a doublet in the 220-240 kd range. Taken together these data demonstrate that the pemphigoid antigen can be extracted directly from normal human skin and is a molecule similar in molecular weight to the antigen synthesized in human epidermal cell culture.     

Expression of tenascin, biglycan and decorin in disorders of keratinization

Summary The distribution of three (recently discovered) extracellular matrix components (tenascin, biglycan and decorin) was studied in normal adult human skin and in a number of monogenic disorders of keratinization, using immunohistology. The expression of tenascin, which is sparsely distributed in normal human dermis, was found to be grossly increased in epidermolytic hyperkeratoses and in Darier's disease. Tenascin expression in three types of ichthyosis (X-linked recessive ichthyosis. autosomal dominant ichthyosis vulgaris. non-erythrodermic lamellar ichthyosis) was similar to that of normal skin. The presence of biglycan and decorin did not show a marked variation between the different disorders studied, suggesting that their expression is subject to regulatory mechanisms distinct from those of tenascin.
The increased expression of tenascin in two disorders of keratinization with a hyperproliferative phenotype, lends further support to the hypothesis that dermal tenascin expression is increased as a result of epidermal hyperproliferation.     

Caged fluorescent haptens reveal the generation of cryptic epitopes in allergic contact dermatitis

Allergic contact dermatitis (ACD) is the most prevalent form of human immunotoxicity. It is caused by skin exposure to haptens, i.e., protein-reactive, low-molecular-weight chemical compounds, which form hapten-protein complexes (HPCs) in the skin, triggering the immune system. These immunogenic HPCs are elusive. In this study a series of thiol-reactive caged fluorescent haptens, i.e., bromobimanes, were deployed in combination with two-photon fluorescence microscopy, immunohistochemistry, and proteomics to identify possible hapten targets in proteins in human skin. Key targets found were the basal keratinocytes and the keratins K5 and K14. Particularly, cysteine 54 of K5 was found to be haptenated by the bromobimanes. In addition, elevated levels of anti-keratin antibodies were found in the sera of mice exposed to bromobimanes in vivo. The results indicate a general mechanism in which thiol-reactive haptens generate cryptic epitopes normally concealed from the immune system. In addition, keratinocytes and keratin seem to have an important role in the mechanism behind ACD, which is a subject for further investigations.     

Organ-specific, phylogenetic, and ontogenetic distribution of the epidermolysis bullosa acquisita antigen

The organ-specific, phylogenetic, and ontogenetic distribution of the epidermolysis bullosa acquisita (EBA) antigen, a newly recognized basement membrane component, was determined using polyclonal sera from patients with EBA and a mouse monoclonal antibody, H3a. Both antibodies are distributed at the basement membrane zone of skin, oral, anal, and vaginal mucosae, and esophagus, but not in kidney, urinary bladder, lymph nodes, placenta, or blood vessels. Both polyclonal and monoclonal EBA antibodies react with a basement membrane antigen in primate and other mammalian skin, but not in avian, amphibian, or reptilian skin. The antigen is present initially in the 8-week-old human fetus, and increases in density until the adult linear pattern is reached at 15 weeks' gestational age.     

Urokinase- and tissue-type plasminogen activators in keratinocytes during wound reepithelialization in vivo

Urokinase- and tissue-type plasminogen activators (u-PA and t-PA) were identified immunohistochemically during reepithelialization of mouse and human skin wounds, by means of polyclonal and monoclonal antibodies. In incised mouse skin wounds u-PA immunoreactivity was found in keratinocytes at the edge of the wound after 12 h, and at days 2 to 10 after wounding it was found in virtually all keratinocytes of the epithelial outgrowth that gradually covered the wound. At day 14, the epidermis appeared normal and no u-PA immunoreactivity was detected. t-PA immunoreactivity was found from day 5 to day 10 in some keratinocytes located superficially in the epidermal outgrowths near the edge of the mouse wounds. In 3- and 5-day old human skin wounds, u-PA immunoreactivity was found in keratinocytes in the epithelial outgrowths, whereas no t-PA immunoreactivity was detected. No u-PA and no t-PA immunoreactivity was found in normal mouse and human epidermis. The specificity of the staining was supported by a variety of controls, including absorption of the polyclonal antibodies with highly purified u-PA and t-PA preparations and zymographic analysis of extracts of wound tissue. The function of the plasminogen activators during reepithelialization is discussed and it is suggested that the keratinocytes use plasmin activated by u-PA for dissecting their way through the provisional matrix in the upper part of the granulation tissue.     

Increased expression of tenascin C by keloids in vivo and in vitro

Tenascin C, undulin, collagen XIV and fibronectin are extracellular matrix glycoproteins with a partial DNA sequence homology. During embryogenesis, tenascin C is abundant in mesenchymal tissues but its distribution in human adult tissue is severely restricted. The levels of tenascin C expression are enhanced with skin inflammation, wound healing and hyperproliferative skin diseases and return to normal in normal scar tissue after wound contraction is completed. Undulin/collagen XIV is associated with collagen fibrils and fibronectin is present throughout the dermis in adult skin but it is produced by keloidal fibroblasts in an increased amount. In this study we investigated by immunohistochemistry the expression of the three extracellular matrix proteins in keloids and normal skin as well as in keloidal and normal fibroblasts in vitro. In keloids, increased tenascin C expression was observed especially in the reticular dermis associated with collagen fibrils sharply demarcating the limit of the lesion. In normal tissue, tenascin C was only expressed beneath the basal lamina and dermal-epidermal junction. Corresponding to the in vivo findings, tenascin C expression was increased in keloidal fibroblasts compared with normal fibroblasts in vitro (P < 0.003), whereas undulin/collagen XIV and fibronectin expression in keloids and keloidal fibroblasts was similar to that in normal tissue and normal fibroblasts, respectively. Therefore, tenascin C is a marker associated with keloids and we suggest that keloidal fibroblasts, once stimulated, continue to produce tenascin C independently from circulating factors.     

The TCR-delta repertoire in normal human skin is restricted and distinct from the TCR-delta repertoire in the peripheral blood

The skin and the intestinal mucosa form surfaces to external environments and share similarities in anatomic structure and immunologic defense. In healthy humans, intestinal gamma/delta T cells express a highly restricted gamma/delta T cell receptor repertoire whereas gamma/delta T cells of the skin were thought to express a polyclonal repertoire. Herein we report, using complementarity-determining region 3 size spectratyping and nucleotide sequencing of T cell receptor DV1 and DV2 rearrangements, that the human skin is also composed of clonally expanded gamma/delta T cells that are widely distributed. Identical complementarity-determining region 3 profiles and T cell receptor delta rearrangements were found in two separate skin samples that were obtained as far as 2-10 cm apart. Furthermore, analysis of peripheral blood mononuclear cells of these subjects clearly demonstrated that the skin harbors a unique population of gamma/delta T cells that is distinct from that in the peripheral blood. In addition comparable data were obtained irrespective of whether DNA or RNA was analyzed, indicating that the observed oligoclonality is not secondary to the expression of large amounts of mRNA from a few activated cells. Thus, gamma/delta T cells of the skin and the intestine both express an oligoclonal repertoire that enables them to respond to a variety of deleterious antigens without the need for diverse T cell receptors, possibly by recognition of stress-induced self-antigens or of conserved foreign antigens.     

Tenascin is overexpressed in vitiligo lesional skin and inhibits melanocyte adhesion

The aetiology of vitiligo remains obscure. In this study, the role of integrins in the observed inability of melanocytes to repopulate lesional skin was investigated. Antibodies directed to α₂, α₃, α₅, α_v, α₆, β₁ and β₃ integrin subunits were used. Immunohistology revealed no marked differences in the overall levels of expression of integrins between control, non-lesional, perilesional or lesional skin. Moreover, no differences were noted in the level of expression of integrins or the adhesive capacity between cultured control cells derived from three separate donors and vitiligo-derived melanocytes from two donors. Rather, it was clearly observed that towards the lesion, vitiligo skin contains increasing amounts of tenascin in the basal membrane and papillary dermis in five patients employing T2H5 antihuman tenascin antibody. The anti-adhesive effect observed in vitro for this extracellular matrix molecule using normal melanocytes may contribute to loss of pigment cells in vitiligo or to ineffective repopulation of the lesions.     

Radiation therapy induces tenascin expression and angiogenesis in human skin.

In analysing radiation-induced connective tissue changes, we studied tenascin expression, elastic fibres, angiogenesis and physio-mechanical properties in irradiated and contralateral healthy skin of radiotherapy-treated breast cancer patients. Skin biopsies were obtained from a radiotherapy-treated skin area and a corresponding non-treated skin area. Haematoxylin-eosin and Verhoeff stainings as well as immunohistochemical stainings for tenascin and factor VIII were performed. Epidermal and total skin thickness, together with the amount of elastic tissue calculated by computerized digital image analysis, were measured. Suction blisters were induced on both skin areas. Transepidermal water loss was analysed. Skin elasticity was also measured. Tenascin expression was found to be increased in irradiated human skin. In haematoxylin-eosin and factor VIlI-stained sections, an increase in the number of blood vessels was detected. Although skin stiffness measured by an elastometer was increased in irradiated skin, no marked difference in the elastic fibres could be found between treated and non-treated skin. The increased tenascin expression could be due to activation of cytokines as a result of irradiation. An increase in angiogenesis could be caused by an activation of angiogenetic factors by irradiation or due to direct radiation damage on blood vessel walls. Our findings suggest that the effects of irradiation tend to accumulate in the dermal parts of skin. The higher skin stiffness values measured by elastometer in irradiated skin could be due to an accumulation of dermal connective tissue as a result of irradiation.     

The skin immune system (SIS): distribution and immunophenotype of lymphocyte subpopulations in normal human skin

The complexity of immune response-associated cells present in normal human skin was recently redefined as the skin immune system (SIS). In the present study, the exact immunophenotypes of lymphocyte subpopulations with their localizations in normal human skin were determined quantitatively. B cells were not found to be present in normal human skin. Lymphocytes were always of T-cell type, and 90% of these T cells were clustered in 1-3 rows around postcapillary venules of the papillary vascular plexus or adjacent to cutaneous appendages. In such perivascular localizations, they were found to differ from their circulating counterparts in three ways. First, skin perivascular cells were found to be approximately evenly distributed over CD4+ inducer and CD8+ suppressor-cytotoxic T-cell subsets (mean CD4/CD8 ratio: papillary layer 0.96, reticular layer 0.99). Second, within the category of CD4+ inducer T cells, most were phenotyped as CD4+, 4B4+ helper inducer T lymphocytes, whereas CD4+, 2H4+ suppressor inducer T lymphocytes were found to be relatively rare (less than 5%). Third, the majority of skin perivascular T cells were activated as they expressed HLA-DR and interleukin 2 receptors. Intraepidermal, directly subepidermal, and other ("free") lymphocytes were mostly of the CD8+ suppressor-cytotoxic T-cell subset but accounted for less than 10% of the total number of lymphocytes. Intraepidermally localized T cells accounted for less than 2% of the total number of lymphocytes present in normal skin. Our results indicate that preferential perivascular localization of activated T lymphocytes is the characteristic of normal human skin. This might be a reflection of continuous antigen recognition upon endothelial cell presentation and/or continuous T cell-mediated endothelial cell activation thereby inducing enhanced antigen clearing by the skin's endothelium.     

Coordinate upregulation of tenascin C expression with degree of photodamage in human skin

Tenascin C is a large extracellular matrix glycoprotein involved in morphogenesis and wound healing. The distribution and expression levels of tenascin were examined in photodamaged skin to investigate the hypothesis that photoaged skin displays characteristics of wound repair. In situ hybridization and semiquantitative immunohistochemistry were performed on paired skin biopsies from patients with varying levels of photodamage, using monoclonal antibodies and cRNA probes for tenascin and its large isoform. In sun-protected skin, tenascin protein was distributed adjacent to the dermoepidermal junction, usually sparsely and discontinuously; tenascin mRNA was detected in dermal fibroblasts and some keratinocytes. In photodamaged skin, tenascin protein was increased in proportion to the clinical level of photodamage (analysis of variance: P < 0.0001, n = 29). With increased photodamage, tenascin protein expression became continuous along the dermoepidermal junction, extending deeper into and sometimes throughout the papillary dermis; tenascin mRNA was detected throughout the epidermis. Large tenascin isoform protein and mRNA distribution mirrored that of pantenascin, suggesting that it may be the predominant species in photodamaged skin. There was no correlation between tenascin expression levels and age or sex, and no seasonal variation was noted. The results indicate that photodamaged skin demonstrates tenascin increases consistent with an early wound healing response. However, tenascin increases in photodamage appear to be permanent and may therefore interfere with effective repair of ultraviolet-induced damage. In conclusion, this study has shown that dermal tenascin expression increases in proportion to the degree of photodamage. In normal skin, the temporal and spatial patterns of tenascin expression during morphogenesis and tissue remodelling are crucial to their correct progression. In photoageing, the 'normal' control of tenascin expression seems to be abrogated.     

Dermcidin is constitutively produced by eccrine sweat glands and is not induced in epidermal cells under inflammatory skin conditions

BACKGROUND: Antimicrobial peptides (AMPs) are important effector molecules of innate immunity, protecting epithelial surfaces of multicellular organisms. In human skin two classes of AMPs-the beta-defensins and the cathelicidins-are produced by keratinocytes primarily under inflammatory conditions. In contrast, dermcidin (DCD), a recently discovered AMP with broad-spectrum activity, is expressed in eccrine sweat glands and transported via sweat to the epidermal surface. OBJECTIVES: To investigate whether DCD expression is induced under inflammatory conditions in epidermal keratinocytes. METHODS: Lesional skin of the inflammatory skin diseases atopic dermatitis, psoriasis and lichen planus was analysed by immunohistochemistry using a polyclonal anti-DCD antiserum. We also examined whether DCD RNA expression is induced in cultured human keratinocytes, fibroblasts, melanocytes and melanoma cells. RESULTS: Whereas DCD was constitutively expressed in eccrine sweat glands of all skin biopsies, we found that, independent of the type of the inflammatory skin lesion, DCD protein expression was not induced in human epidermal keratinocytes. In contrast, beta-defensin 2 was expressed in epidermal keratinocytes of inflammatory human skin, but not in keratinocytes of healthy human skin. Upon stimulation of the cultured cells with 12-O-tetradecanoyl-phorbol-13-acetate, tumour necrosis factor-alpha, lipopolysaccharide or H2O2, DCD mRNA expression was not detected in primary keratinocytes, fibroblasts and melanocytes, but was detected in MeWo and SKMEL28 melanoma cells. CONCLUSIONS: These results indicate that, unlike human cathelicidins and beta-defensins which are inducible peptides that primarily function in response to injury and inflammation, DCD is exclusively part of the constitutive innate defence of human skin. By modulating surface colonization, DCD may help to prevent local and systemic invasion of pathogens.     

Immune responses to soluble antigens of treponemes.

Sonicates from five cultivable treponemes were used as antigens in delayed hypersensitivity tests and macrophage inhibition assays. Immunodiffusion analysis showed that the sonicate comprised two major antigenic components which were not distinguishable in the skin tests. The sonicate antigens elicited significant cell-mediated immunity in guinea-pigs. Treponema refringens biotype Nichols proved to induce the strongest delayed response. Delayed skin hypersensitivity to the antigens of Treponema pallidum was found in eight rabbits without orchitis, but not in six rabbits with T. pallidum orchitis. In contrast, the rabbits with syphilitic orchitis gave the strongest reactions with the non-pathogenic spirochetes. In terms of the cell-mediated immunity responses, Treponema phagedenis Reiter was found to be related to T. phagedenis Kazan 4 and Treponema denticola. Treponema scoliodontum was related to T. phagedenis Kazan 5, and T. refringens biotypes Nichols and refringens. The antigens of T. pallidum had the closest relationship to T. refringens biotypes refringens and Nichols ,T. phagedenis biotype Reiter, and T. scoliodontum. It was also demonstrated that some three of 12 human syphilitic sera reacted with the antigens of T. pallidum but not with control sera.     

Immune responses to soluble antigens of treponemes.

Sonicates from five cultivable treponemes were used as antigens in delayed hypersensitivity tests and macrophage inhibition assays. Immunodiffusion analysis showed that the sonicate comprised two major antigenic components which were not distinguishable in the skin tests. The sonicate antigens elicited significant cell-mediated immunity in guinea-pigs. Treponema refringens biotype Nichols proved to induce the strongest delayed response. Delayed skin hypersensitivity to the antigens of Treponema pallidum was found in eight rabbits without orchitis, but not in six rabbits with T. pallidum orchitis. In contrast, the rabbits with syphilitic orchitis gave the strongest reactions with the non-pathogenic spirochetes. In terms of the cell-mediated immunity responses, Treponema phagedenis Reiter was found to be related to T. phagedenis Kazan 4 and Treponema denticola. Treponema scoliodontum was related to T. phagedenis Kazan 5, and T. refringens biotypes Nichols and refringens. The antigens of T. pallidum had the closest relationship to T. refringens biotypes refringens and Nichols ,T. phagedenis biotype Reiter, and T. scoliodontum. It was also demonstrated that some three of 12 human syphilitic sera reacted with the antigens of T. pallidum but not with control sera.     

The regional distribution of neuropeptides in human skin as assessed by radioimmunoassay and high-performance liquid chromatography

In this study radioimmunoassay was used to determine neuropeptide levels in extracts from 17 differing anatomical regions of human skin. Marked regional variations of neuropeptide content for human skin were found and these variations are likely to reflect true physiological functions for the neuropeptides studied. In general the tachykinins, substance P (SP), neurokinin A (NKLA) and calcitonin gene-related peptide (CGRP) were found in highest concentrations in regions of skin with the greatest tactile sensation. By contrast, highest concentrations of vasoactive intestinal peptide (VIP) and peptide histidine methionine (PHM) were found in axillary skin, where they probably play a part in axillary eccrine sweat production. Neurotensin was not found in any of the skin areas sampled, suggesting that it is relatively unimportant in human physiological skin control. Reverse-phase high-performance liquid chromatography (rpHPLC) was used to verify the results of radioimmunoassay. Both SP and NKA occurred in several regions in both their reduced and oxidized forms, as well as displaying molecular heterogeneity. CGRP occurred as one molecular species, this being α-CGRP, suggesting that this is the predominant molecular form in human skin. Likewise, both VIP and PHM displayed molecular homogeneity in the regions investigated by rpHPLC.     

Calcium enhances the sensitivity of immunofluorescence for pemphigus antibodies

Indirect immunofluorescence (IF) to detect pemphigus and pemphigoid autoantibodies is commonly performed with monkey esophagus (ME) as substrate and phosphate-buffered saline (PBS) as a diluent. The purpose of this study was to evaluate comparative IF titers using human skin (HS) as substrate with variations in the buffers employed. Substrates (ME or HS) were incubated in PBS, Tris-acetate-buffered saline (TAS), TAS with 5 mM CaCl+2 (TAS-Ca+2), and PBS or TAS with 1 mM EDTA, prior to incubation with pemphigus or pemphigoid sera for indirect IF. We examined sera from 11 patients with pemphigus vulgaris (PV), 10 patients with Brazilian pemphigus foliaceus (BPF), and 4 patients with bullous pemphigoid. In 20 of 21 pemphigus sera, endpoint indirect IF titers were highest on normal skin with TAS-Ca+2. Six sera (2 PV and 4 BPF) had endpoints that were 5 double dilutions higher than the endpoints obtained with ME and PBS. Six sera (3 PV and 3 BPF) were 4 double dilutions higher, 7 sera (3 PV and 4 BPF) were 2-3 double dilutions higher, and 2 PV sera were equivalent with both substrate/buffers. Preincubation of either tissue with EDTA prior to indirect IF abolished PV and BPF antibody binding completely. Exposure to EDTA after the tissue was incubated with PV or BPF sera did not affect indirect IF titers. In the presence of Ca+2, the antigen was resistant to trypsin in concentrations of 0.001%; however, in the absence of added Ca+2 it was destroyed by 0.0001% trypsin. These differences were not observed with bullous pemphigoid sera; all 4 sera had similar endpoint indirect IF titers. This study shows a significant increase in the sensitivity of indirect IF assays for pemphigus autoantibodies by the use of Ca+2-supplemented buffers on human skin. This finding may also have implications for procedures designed to purify and/or detect pemphigus antigens.     

Increased expression of tenascin in the dermis in scleroderma

The expression of tenascin, a recently discovered extracellular matrix protein, was studied by immunohistochemical techniques in scleroderma skin and compared with its distribution in normal skin. In progressive systemic sclerosis, a marked increase in tenascin content was observed in the superficial reticular dermis. In localized scleroderma, the deposition of tenascin was increased both in the superficial and deep dermis of involved skin, whereas in clinically uninvolved skin the distribution of tenascin was the same as in normal control skin, i.e. the papillary dermis and peri-appendiceal zone. The distribution of tenascin did not strictly parallel that of fibronectin. These findings and the current knowledge of tenascin biology suggest that the overproduction of tenascin in scleroderma dermis could be secondary to stimulation of fibroblasts by immune cell-derived cytokines, or could be due to abnormal fibroblasts, or a subpopulation of fibroblasts, producing high levels of this extracellular matrix protein.     

Apoptotic keratin bodies as autoantigen causing the production of IgM-anti-keratin intermediate filament autoantibodies

The presence of numerous keratin bodies in the upper dermis is a characteristic finding in skin lesions of patients with various dermatoses such as cutaneous graft-versus-host disease, lichen planus, or chronic discoid lupus erythematosus. These keratin bodies are generated by apoptotic keratinocyte death, consist largely of keratin intermediate filaments (KIF), and are constantly covered with immunoglobulins, mainly IgM. Apoptosis is also thought to occur under physiologic conditions in the skin as it does in other organs, but keratin bodies are not frequently reported as being found in nonlesional skin. In order to assess the frequency of keratin bodies in normal skin, we examined serial sections of 10 normal human skin specimens and 5 dermal sheets prepared from normal human skin for the presence of keratin bodies. They were visualized by direct immunofluorescence using a fluorescein isothiocyanate (FITC) rabbit antihuman IgM conjugate. In addition the KIF origin of keratin bodies was demonstrated by a double-staining immunofluorescence procedure using a FITC-conjugated rabbit antihuman IgM followed by a mouse monoclonal antibody against keratin and a sheep antimouse immunoglobulin conjugated with Texas Red. One specimen was also examined for keratin bodies at the ultrastructural level. In serial sections, all 10 normal human skin specimens had numerous keratin bodies as assessed by visualization of globular IgM deposits. Evaluated on dermal sheets, the number of keratin bodies ranged from 39-262 per mm2. Nearly all keratin bodies also stained with the antikeratin antibodies. Ultrastructurally the remarkable number of keratin bodies, which consist of filaments measuring approximately 10 nm in diameter or of more granular material, in normal human skin was confirmed. In order to investigate the capacity of KIF material in keratin bodies to function as autoantigen, we examined the sera of the 10 skin donors and, in addition, of 30 normal healthy individuals and 10 patients with rheumatoid arthritis for the occurrence and specificity of IgM-anti-KIF autoantibodies by an enzyme-linked immunosorbent assay and by immunoblot. IgM-anti-KIF autoantibodies were found in all 50 test sera. In the majority of the sera the specificity of these autoantibodies included the 51 kD and the 58 kD KIF protein, which are constituents of KIF in keratin bodies and basal keratinocytes. Quantitatively, the antibody activity of the IgM-anti-KIF autoantibodies varied from serum to serum, being highest in the sera of patients with rheumatoid arthritis.(ABSTRACT TRUNCATED AT 400 WORDS)