Individual Keratinocytes Research Articles

Lichen amyloidosus: A consequence of scratching

Background: Lichen amyloidosus (LA) is generally said to be a pruritic type of amyloidosis of unknown cause. Histopathologically, it is characterized by epidermal changes of lichen simplex chronicus and by deposits of amyloid in the papillary dermis that are derived from keratin peptides of necrotic keratinocytes. Chronic scratching is responsible for the development of lichen simplex chronicus and may lead to necrosis of individual keratinocytes. Objective: Our purpose was to evaluate whether chronic scratching may also be responsible for the formation of amyloid in LA. Methods: We studied patients with LA in regard to histopathologic findings, onset of pruritus, associated diseases, and response to treatment. Results: In most cases, pruritus had preceded the skin lesions. Eight of nine patients suffered from diseases other than LA that may be associated with pruritus. Histopathologically, amyloid was confined to areas that also showed signs of lichen simplex chronicus. Systemic treatment with sedating antihistamines and intense local treatment with corticosteroids were found to be effective. Conclusion: LA is considered to be a variant of lichen simplex chronicus in which scratching leads to necrosis of keratinocytes and eventually to the formation of amyloid in the papillary dermis. Because chronic scratching seems to be the cause and not the result of the deposits of amyloid, treatment should be directed at the amelioration of pruritus. (J Am Acad Dermatol 1997;37:923-8.)

Apoptosis is the mode of keratinocyte death in cutaneous graft-versus-host disease

Background: Satellite cell necrosis is a histopathologic hallmark of cutaneous graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation. Although this interaction of lymphocytes with keratinocytes has features of immune cytolytic destruction, the details of the process are not known. Objective: Our purpose was to investigate whether apoptosis is involved in the process of GVHD. Methods: We used TdT-mediated dUTP-biotin nick end labeling (TUNEL) technique with biotinylated nucleotides. Formalin-fixed, paraffin-embedded sections from skin biopsy specimens of patients with GVHD and normal skin were studied. Results: Labeling of scattered individual keratinocytes in the epidermis and adnexal epithelium undergoing cytolytic degeneration and a proportion of the adjacent lymphocytes was noted in all specimens of GVHD. Conclusion: The positive staining of keratinocytes by the TUNEL method indicates that cell destruction in cutaneous GVHD involves apoptosis.

Differential Cell Surface Distribution of Adhesion Molecules Demonstrated by Immuno‐Scanning Electron Microscopy

Scanning electron microscopy with immuno-gold labeling revealed that epidermal keratinocytes expressed ICAM-1 and HLA-DR molecules on their surfaces in patterns that differed in mycosis fungoides (MF) and lichenoid reaction (LR). ICAM-1 molecules, visualized as deposits of gold particles, were present as clusters adjacent to the junctions interconnecting the keratinocytes of MF lesions. LFA-1 molecules were seen as granules on the surfaces of all infiltrates, most of which also expressed ICAM-1. HLA-DR molecules were seen continuously along the borders of the individual keratinocytes. In LR, ICAM-1 and HLA-DR were expressed only sparsely on the undersurface of the epidermis, whereas the infiltrates expressed LFA-1 molecules on their surfaces. These findings may explain the differing histological features of MF and LR: ICAM-1 molecules present on the intercellular junctions of MF epidermis lead the LFA-1-bearing cells to migrate into the interspaces of the keratinocytes, thus producing "epidermotropism". These cells aggregate by means of co-expressed ICAM-1 to thus produce the appearance of a "microabscess". In LR, on the other hand, the minimal expression of ICAM-1 on the epidermal undersurface leaves most infiltrates within the dermis, thus producing a "band-like infiltrate" beneath the epidermis.

Calcium-independent increases in pericellular plasminogen activator activity in pemphigus vulgaris.

Intracellular free calcium ([Ca2+]i), an important second messenger, plays a crucial role in a variety of biochemical reactions leading to cell activation and protein secretion. This study examines the potential role of [Ca2+]i in mediating increases in pericellular plasminogen activator activity of canine keratinocytes observed upon binding of human pemphigus vulgaris IgG (hPV IgG). Using the calcium-sensitive fluorescent probe fura-2 and digital video fluorescence imaging microscopy, [Ca2+]i levels were determined in individual keratinocytes for up to 29 minutes after addition of 0.1-5 mg/ml hPV IgG to monolayers of subconfluent and confluent cultures. Extracellular ATP (a known [Ca2+]i-agonist in canine keratinocytes) and normal human IgG (nh IgG) served as positive and negative controls, respectively. HPV IgG and nh IgG failed to induce significant increases in [Ca2+]i, whereas 500 microM ATP induced a rapid, 3- to 12-fold transient increase above resting levels. Binding of hPV IgG to these keratinocyte cultures was demonstrated by immunofluorescence at the end of selected experiments. ATP stimulation of cultures previously treated with hPV IgG showed normal responsiveness and more than 90% of the cells were still viable at the end of [Ca2+]i imaging, thus demonstrating that failure to respond to hPV IgG was not due to an experimental artifact. Plasminogen activator activity in supernatants of confluent cultures incubated with 0.1-1 mg/ml hPV IgG or nh IgG and harvested at various time intervals was dependent on the IgG dose used and increased steadily over time. Increases in activity were 47-92% higher in cultures treated with hPV IgG than those incubated with the same dose of nh IgG.(ABSTRACT TRUNCATED AT 250 WORDS)

Histologic, pharmacologic, and immunocytochemical effects of injection of bleomycin into viral warts

The plasma concentration of bleomycin after injection of bleomycin into warts is unknown, as is the long-term stability of bleomycin solution. Our purpose was to measure plasma bleomycin concentration after injection of bleomycin into warts, to relate histologic and immunocytochemical changes in warts to possible mechanisms of action of bleomycin, and to asses the long-term stability of stored frozen bleomycin solution. One milligram of bleomycin was injected into warts on the hands of seven men. Blood samples were taken 15 to 120 minutes after injection, and plasma bleomycin was measured by radioimmunoassay. Warts were removed 2 hours and 48 hours after treatment and studied histologically by light microscopy and for the presence of bleomycin by immunocytochemistry. The bleomycin concentration in 8 aliquots of solution stored at -20 degrees C for varying periods was measured by radioimmunoassay. Peak levels of bleomycin of 7 to 113 ng/ml were reached by 45 minutes after injection. Plasma bleomycin exposure ranged from 515 to 5137 ng/ml/min between 15 and 120 minutes after injection. The most pronounced histologic changes at 48 hours were individual keratinocyte apoptosis throughout the epidermis merging into areas of complete epidermal necrosis, diffuse neutrophil accumulation, and microabscess formation at the granular layer. Immunocytochemistry demonstrated tissue-fixed bleomycin in all levels of the epidermis except the basal layer and most prominently in the granular layer. Bleomycin in solution stored for up to 27 months at -20 degrees C in glass showed no significant loss of immunoreactivity. The use of bleomycin for the treatment of warts results in significant systemic drug exposure; thus it would be prudent to exclude pregnancy before treating women of child-bearing age. Bleomycin probably has a direct toxic effect on keratinocytes. Dilute bleomycin solution stored at -20 degrees C in glass is stable.

Differential localization of ICAM-1 and HLA-DR expression on epidermal basal surface in mycosis fungoides and lichenoid reaction.

Scanning electron microscopy with immunogold labeling revealed that epidermal keratinocytes expressed ICAM-1 (intercellular adhesion molecule-1) and HLA-DR molecules on their surfaces in patterns that differed in mycosis fungoides (MF) and lichenoid reaction (LR). ICAM-1 molecules, visualized as deposits of gold particle, were visualized as clusters adjacent to the junctions interconnecting the keratinocytes of MF lesions. LFA-1 (leukocyte function-associated antigen-1) molecules were seen as granules on the surfaces of all infiltrates, most of which also expressed ICAM-1. HLA-DR molecules were seen continuously along the borders of the individual keratinocytes, thus producing a cobblestone appearance on the epidermal undersurface. In contrast, ICAM-1 and HLA-DR were found only sparsely on the undersurface of the epidermis from LR. These findings may help to explain the differing histological features of MF and LR: ICAM-1 molecules present on the intercellular junctions of MF epidermis lead the LFA-1-bearing cells to migrate into the interspaces, thus producing epidermotropism. These cells aggregate by means of co-expressed ICAM-1 to thus produce Pautrier's microabscess. In LR, the minimal expression of ICAm-1 on the epidermal undersurface leaves most infiltrates within the dermis, thus producing a band-like infiltrate.

Expression of Intercellular Adhesion Molecule-1 (ICAM-1) in the Skin of Patients with Systemic Scleroderma

The expression and tissue distribution of intercellular adhesion molecule-1 (ICAM-1) in skin biopsies from 12 patients with systemic (SSc) and localized (LS) scleroderma was studied and compared to the biopsies from patients with lupus erythematosus (LE) and normal individuals. In normal human skin ICAM-1 expression was restricted to the vascular endothelium, infiltrating mononuclear cells (MNC), and to few individual keratinocytes. In the inflammatory stage of SSc, however, the expression of ICAM-1 was dramatically increased at the site of MNC infiltrates and could also be detected on fibroblast-like cells lying well apart from these infiltrates in the deep dermis. In contrast, in LS ICAM-1 was expressed mainly at the sites of MNC infiltrates. In LE ICAM-1 expression was confined to the keratinocytes, endothelial cells, and mononuclear cells in the upper parts of the dermis. Analysis of serial tissue sections from patients with SSc demonstrated also colocalization of staining of ICAM-1 around blood vessels with LFA-1-positive lymphocytes. Increased expression of ICAM-1 in the dermis of patients with SSc may represent an important mechanism by which MNC become localized and retained at a site of connective tissue inflammation, leading to the activation of fibroblasts.

Acute Graft-vs-Host Disease in an Immunodeficient Newborn Possibly due to Cytomegalovirus Infection

Acute graft-vs-host disease occurring during the early weeks of life has been previously reported as a rare disease entity. We report a case of acute graft-vs-host disease in a female infant with an immunodeficiency that was thought to be secondary to intrauterine or neonatal cytomegalovirus infection or, less likely, to a severe combined immunodeficiency. The patient presented with a triad of failure to thrive, diarrhea, and maculopapular and petechial rash. The first clue to diagnosis was the skin biopsy finding of an epidermal lymphocytic infiltrate in association with individual necrotic keratinocytes. The diagnosis was confirmed at autopsy. In the absence of an obvious graft, the disease is believed to have been the result of maternofetal T-cell transfer in utero or at delivery.

Calcium-induced changes in cytoskeleton and motility of cultured human keratinocytes

In normal epidermis keratinocytes migrate upward from the basal layer as they undergo terminal differentiation, yet they also have the capacity for lateral movement during wound healing. The purpose of our experiments was to investigate these two types of movement by manipulating the calcium ion concentration of the medium so that keratinocytes formed monolayers (0.1 m M calcium) or stratified sheets (2.0 m M calcium). Time-lapse video recording indicated that keratinocytes in low-calcium medium were laterally more motile than keratinocytes in normal medium. This was consistent with the ultrastructural appearance of the cells and the lack of desmosomal junctions, determined by scanning and transmission electron microscopy. During calcium-induced stratification keratinocytes moved upward from the basal layer by gliding over their neighbors and forming contacts with other suprabasal cells. Keratinocytes in low-calcium medium migrated into wounds made in the cultures, a process which was inhibited by monensin; however, stratified keratinocytes in normal medium did not enter wounds. Cytochalasin D caused rapid cell rounding and disruption of actin filaments in keratinocytes grown in low-calcium but not in normal medium, indicating more rapid treadmilling of actin and consistent with the greater motility of keratinocytes in low-calcium medium. Our results suggest that desmosome formation may place constraints on the movement of individual keratinocytes and that the actomyosin cytoskeleton is involved in lateral migration.

The Use of Human Pemphigoid Autoantibodies to Study the Fate of Epidermal Basal Cell Hemidesmosomes After Trypsin Dissociation

It is known that during trypsinization of the skin, the epidermis is first separated from the dermis and individual keratinocytes are dissociated by disruption of the epidermal intercellular spaces. The desmosomal unit is separated at the level of the intercellular space and the split desmosomes are internalized in plasma membrane-limited vesicles; however the fate of the hemidesmosome under such conditions has not been studied. We have recently shown (Mutasim et al: J Invest Dermatol 84:47-53, 1985) that autoantibodies from the sera of patients with bullous pemphigoid bind in vitro to hemidesmosomes but not to desmosomes providing a highly specific marker for these organelles. Utilizing these autoantibodies, we studied the fate of the hemidesmosome during trypsin dissociation of epidermal basal cells derived from the skin of neonatal BALB/c mice. During trypsinization, portions of the dermal face of the plasma membrane which include hemidesmosomes formed pits which pinched off to produce vesicles that moved toward the nucleus. This was accompanied by retraction of the tonofilaments away from the cell periphery. The mechanism of this internalization process is not yet known, but may involve contractile elements of the cytoskeleton. The highly specific binding of bullous pemphigoid autoantibodies to the hemidesmosome may prove helpful in future biochemical and immunocytochemical studies of this organelle.

Electron microscopic demonstration of virus particles in hand, foot and mouth disease.

Early skin and oral mucous membrane lesions of hand, foot and mouth disease (HFMD) were biopsied and investigated by light and electron microscopy. Histology showed spongiosis, intraepidermal splits indicating beginning vesicle formation, exocytosis of lymphoid cells and macrophages, and individual necrotic keratinocytes. Electron microscopy revealed naked viruses with a mean diameter of 24 nm which were arranged in sheets in the cytoplasm. Many keratinocyte nuclei contained tonofibrils. Cellular injury ranged from slight cytoplasmic edema to keratinocyte necrosis. Some nuclei showed dissolution of the nuclear membrane and dislocation of cytoplasm into the nucleus.

Long-term organ culture of human skin: an ultrastructural and immunochemical study.

Human skin grown in tissue culture medium for periods of up to 18 wk undergoes characteristic morphological changes. After an initial period of degeneration, new foci of epidermal cells arise at the dermo-epidermal junction and by rapid proliferation these cells spread out to form a complete second epidermal layer beneath the degenerating original strata. Stratification occurs in this new epidermis with incomplete keratinisation. The individual keratinocytes show ultrastructural similarities with fetal cells. There is a loss of complexity of the cytoplasm typified by a marked reduction in the numbers of keratin filaments and a decrease in the numbers of desmosomal contact areas. In addition, the formation of cilia and accumulation of glycogen in the cell cytoplasm are characteristic. The cytoplasm of the basal cells contains numerous polyribosomes and there is evidence of synthetic activity as illustrated by the proliferation of rough-surfaced endoplasmic reticulum and Golgi complex. Fluorescent and EM immunocytochemical staining with an anti-fetal antiserum demonstrates the development of fetal substances on the surface of cells during the regeneration stage occurring in the cultured skins. The significance of these observations concerning transplantability of cultured tissues is discussed.

Foreign cell acantholysis

1. “Foreign cell acantholysis” is defined as a special type of microacantholysis that occurs in the epidermis in the course of exocytosis. Characteristic is the loss of single desmosomes or the loss of desmosomal contacts in limited areas of the surface of individual keratinocytes, and the concomitant rearrangement of the tonofilament system. 2. Exocytosis in the widest sense is the immigration of all “foreign cells” into the epidermis; these foreign cells may be inflammatory (e.g., lymphocytes, macrophages, granulocytes) or “neoplastic” (e.g., in Paget's, Dubreuilh's diseases, histiocytosis X). 3. With the approach of foreign cells to the dermo-epidermal junction, desintegration of the basement membrane takes place first, followed by the opening of a gap between neighbouring basal cells. The gap is closed after the penetration of single or a group of foreign cells by the projection of tongue-like processes of the basal cells from both sides. 4. The desmosomes are opening during this process without any signs of mechanical rupture, and at the same time the desmosome-tonofilament system changes continuously in the functionally optimal way. Foreign cells may get into the upper epidermal layers either by active migration or by passive transport (transepidermal elimination). 5. The foreign cell acantholysis is independent of spongiosis (microacantholysis caused by intercellular edema) which in many cases exists at the same time. Under special conditions “symbiosis” between epidermal and foreign cells may develop. 6. Morphological signs of epidermal cells damage may appear depending on the type and density of exocytosis and eventual other noxes. 7. The phenomenon of foreign cell acantholysis gives further support to the widely accepted hypothesis that desmosomes and tonofilaments are no static structures but a system undergoing continuous functional changes.

The epidermal component of pityriasis lichenoides.

Summary.— The scaling papules of pityriasis lichenoides (PL) can at times simulate psoriasis clinically and histologically. In this study particular attention has been paid to the epidermal changes in PL, and 126 biopsy specimens from 94 patients were examined. Parakeratosis was found in 72%, invasion of the epidermis by mononuclear cells in 69%, and focal cell death in 57%. Skin surface biopsies from 8 patients and cryostat sections from 12 patients were examined histochemically. The parakeratotic scale and scattered individual keratinocytes within the epidermis demonstrated strong hydrolytic enzyme activity, and there was a diminution in the reactivity for mitochondrial enzymes within the PL papules. The labelling index was determined for 7 PL lesions after injection of tritiated thymidine and found to be 15·2%. In addition, slices of tissue from 8 PL papules were incubated with tritiated proline, and this manoeuvre also demonstrated the existence of dead and dying cells within the Malpighian layer. The suggestion is made that parakeratosis in PL is caused more by damage to the epidermis than by increased epidermopoiesis.