Arja-Leena Kariniemi

Diagnostic Application of Monoclonal Antibodies to Intermediate Filamentsa

Most of the histopathologic practice in pathology laboratories is based on histochemical staining techniques that reveal the major components common to most tissues or cells, or on more specific staining reactions, that reveal structural components or products typical of certain tissues or cells. The assortment of the utilizable specific stains is, however, narrow and therefore the correct identification of the specific nature of a given lesion is critically dependent on the personal experience of the pathologist. In order to improve the accuracy and reliability of the histopathological diagnostics, much effort has been devoted to develop new and more specific techniques that could be used to reveal specific compositional features of cells and tissues. The most successful approach has been the use of antibodies-either conventional polyclonal or monoclonal antibodies raised against specific tissue components that, combined with immunohistochemical techniques, can be used to retrieve more specific information on the compositional features of a lesion, e.g. tumor, which may allow its identification. Such new reagents include, for instance, antibodies that enable us to identify specific cell types in tissue sections, such as endothelial cells, histiomonocytic cells, different types of lymphatic cells, epithelial cells, neuronal cells, and muscle-type of cells.’32

Demodex mites in acne rosacea

The hair follicle mites Demodex folliculorum and Demodex brevis and their role in the pathogenesis of rosacea have been the subject of much debate in the past. We studied the prevalence of Demodex mites in facial skin biopsies obtained from 80 patients with rosacea, 40 with facial eczematous eruption and 40 with lupus erythematosus discoides. The mite prevalence in the rosacea group (51%) was significantly higher than in the rest of the study population (eczema 28% and lupus discoides 31%). Demodex mites were found on all facial sites. The most infested areas in the whole study group were the forehead (49%) and the cheeks (44%). Males were more frequently infested (59%) than females (30%). We did not find any significant difference in mite counts of infested follicles between rosacea and the control group. A lympho‐histiocytic cell infiltration was seen around the infested hair follicles. Our results suggest that Demodex mites may play a role in the inflammatory reaction in acne rosacea.

Fluorochrome-coupled lectins reveal distinct cellular domains in human epidermis.

The distribution of saccharide moieties in human interfollicular epidermis was studied with fluorochrome-coupled lectins. In frozen sections Concanavalin A (Con A), Lens culinaris agglutinin (LCA), Ricinus communis agglutinin I (RCAI), and wheat germ agglutinin (WGA) stained intensively both dermis and viable epidermal cell layers, whereas peanut agglutinin (PNA) bound only to living epidermal cell layers. Ulex europaeus agglutinin I (UEAI) bound to dermal endothelial cells and upper cell layers of the epidermis but left the basal cell layer unstained. Dolichos biflorus agglutinin (DBA) bound only to basal epidermal cells, whereas both soybean agglutinin (SBA) and Helix pomatia agglutinin (HPA) showed strong binding to the spinous and granular cell layers. On routinely processed paraffin sections, a distinctly different staining pattern was seen with many lectins, and to reveal the binding of some lectins a pretreatment with protease was required. All keratin-positive cells in human epidermal cell suspensions, obtained with the suction blister method, bound PNA, whereas only a fraction of the keratinocytes bound either DBA or UEAI. Such a difference in lectin binding pattern was also seen in epidermal cell cultures both immediately after attachment and in organized cell colonies. This suggests that in addition to basal cells, more differentiated epidermal cells from the spinous cell layer are also able to adhere and spread in culture conditions. Gel electrophoretic analysis of the lectin-binding glycoproteins in detergent extracts of metabolically labeled primary keratinocyte cultures revealed that the lectins recognized both distinct and shared glycoproteins. A much different lectin binding pattern was seen in embryonic human skin: fetal epidermis did not show any binding of DBA, whereas UEAI showed diffuse binding to all cell layers but gave a bright staining of dermal endothelial cells. This was in contrast to staining results obtained with a monoclonal cytokeratin antibody, which showed the presence of a distinct basal cell layer in fetal epidermis also. The results indicate that expression of saccharide moieties in human epidermal keratinocytes is related to the stage of cellular differentiation, different cell layers expressing different terminal saccharide moieties. The results also suggest that the emergence of a mature cell surface glycoconjugate pattern in human epidermis is preceded by the acquisition of cell layer-specific, differential keratin expression.

Expression of differentiation antigens in mammary and extramammary Paget's disease

Seven cases of mammary and twelve cases of extramammary Pagets disease were studied for the presence of carcinoembryonic antigen (CEA) and apocrine epithelial antigen (AEA) using the immunoperoxidase technique. CEA was found in the Paget cells in five out of seven mammary and in all twelve cases of extramammary Pagets disease, whereas the AEA reaction was positive in six mammary and all extramammary cases. The same antigens were also found in the cells of intraductal or ductal adenocarcinomas of the breast associated with mammary Pagets disease, suggesting a common origin for the cells. In the cases of extramammary Pagets disease studied no underlying malignant neoplasms could be detected. Our findings support the suggestion that Paget cells originate in sweat gland ducts and undergo an apocrine differentiation.

Metalloelastase (MMP-12) and 92-kDa gelatinase (MMP-9) as well as their inhibitors, TIMP-1 and -3, are expressed in psoriatic lesions

Abstract: In skin biology, matrix metalloproteinases (MMPs) have been implicated in inflammatory matrix remodeling, neovascularization, wound healing and malignant transformation. Psoriasis is histologically characterized by keratinocyte hyperproliferation, infiltration of inflammatory cells, neoangiogenesis and production of cytokines, such as TNF‐α, IL‐1β, TGF‐α, and IFN‐γ, also capable of regulating MMP transcription. To investigate the role of stromelysins‐1 and ‐2, matrilysin, metalloelastase, collagenases‐1 and ‐3 and 92‐kDa gelatinase as well as their inhibitors, TIMPs‐1 and ‐3, in psoriasis, we performed in situ hybridization using 35S‐labeled cRNA probes on 29 psoriatic lesions and 9 samples of normal looking skin from psoriatic patients. Metalloelastase mRNA was detected in 21/27 samples in macrophages that had migrated into the epidermis or in the inflammatory infiltrates of the superficial dermis. A quantity of 92‐kDa gelatinase was found in macrophages and neutrophils (25/27). Stromelysin‐1 mRNA was detected in basal keratinocytes in 4/21 lesions. Intracellular laminin‐5 immunosignal in basal keratinocytes of the same samples, suggested that stromelysin‐1 might participate in remodeling of the basement membrane zone. No signal for stromelysin‐2 or collagenase‐3 was found and only sweat glands were positive for matrilysin. TIMP‐1 was more abundantly expressed than TIMP‐3 in the inflammatory infiltrates and endothelial cells of dermal papillae (22/29). TIMP‐3 was expressed perivascularly in 9/16 samples. Our results suggest that overexpression of the investigated MMPs by keratinocytes is not associated with psoriasis. However, macrophages express MMPs in psoriatic skin. Also TIMPs, particularly TIMP‐1, were abundantly expressed, suggesting that mere MMP overexpression is unlikely to contribute to psoriatic tissue changes.

Integrins in human cells and tumors

We have studied the distribution of the alpha- and beta-subunits of integrins in developing and adult human kidney as well as in selected other tissues and cultured cells. In cultured cells some of the integrin subunits (beta 1, alpha 1, alpha 2 and alpha 5) colocalize with talin at focal adhesions when plated on an appropriate ligand. Similarly, in tissues the polarization of beta 1-integrins in colocalization with talin appears to indicate adhesive complexes, as demonstrated in adult glomeruli. In human kidney, the alpha subunits of integrins were seen to be segment-specifically expressed already in fetal tissues. In glomeruli the integrin alpha 1 subunit characterized mesangial cells while the alpha 2 and alpha 3 subunits showed immunoreactivity in endothelial cells and podocytes, respectively. In renal tubuli, the alpha 6 subunit, complexed with the beta 1 subunit, showed a typical polarized distribution coaligning with the tubular basement membrane while the alpha 3 and alpha 2 subunits were expressed in distal tubular cells. These results suggested that in kidney the alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 1 integrins can function as basement membrane receptors. The alpha 5 subunit was nearly lacking in the kidney and it appears to be mainly expressed in some smooth muscle cells. In other tissues distinct patterns in the expression of integrins were found. Thus, in many glandular epithelial cells the alpha 3 beta 1 integrin appeared to function as a basement membrane receptor while in various stratified epithelia and in the breast such a polarized localization could be found for the alpha 6 beta 4 integrin. Finally, although presenting a clearly polarized distribution for beta 1 integrins, none of the alpha subunits could be found in cardiac or skeletal muscle cells and none of the integrins could be revealed in neuronal cells of human developing and adult cerebrum or cerebellum, although neurons in peripheral tissues contained abundantly the alpha 6 beta 1 integrin complex. In human tumors, the tumor cells, including also metastastatic tumors, generally presented the same integrins as their tissues of origin. In some poorly differentiated tumors both a population heterogeneity and even a lack of expression or a disorganization of basement membrane receptor integrins was obvious.

Paget cells express cytokeratins typical of glandular epithelia

The expression of cytokeratins in Paget cells in mammary and extramammary Pagets disease was studied using different keratin antibodies and immunofluorescence microscopy. Antibodies to epidermal keratin did not react with the Paget cells but stained the surrounding epidermis. Two monoclonal cytokeratin antibodies (PKKi and RGE 53), which reacted typically with simple glandular epithelia in normal tissues, brightly stained the Paget cells and left the surrounding epidermal cells unstained. The results indicate that Paget cells are derived from mammary or sweat duct epithelium rather than from epidermal cells.

Matrix Metalloproteinase-19 is Expressed by Keratinocytes in Psoriasis

Keratinocyte hyperproliferation, inflammatory infiltrates, neoangiogenesis and alterations in cytokine levels are hallmarks of psoriatic skin. Matrix metalloproteinases (MMPs) have been associated with the remodeling of the extracellular matrix during inflammation, neovascularization, and malignant transformation. We have previously shown that particularly MMP-12 is abundantly expressed by macrophages and MMP-9 in macrophages and neutrophils of psoriatic lesions. In this work the expression of two novel metalloproteinases, MMP-19 and MMP-28, was investigated in psoriatic lesional and non-lesional skin. MMP-19 protein was detected by immunohistochemistry in 28/29 samples in keratinocytes in the same regions as Ki67 (marker of proliferating keratinocytes) and p63 (marker of keratinocyte stem cells). Immunosignaling was also seen in endothelial cells and fibroblasts. Furthermore, MMP-19 mRNA was upregulated in psoriatic keratinocytes and skin as assessed by quantitative real-time polymerase chain reaction. In lichen planus and lichenoid chronic dermatitis, MMP-19 staining was found in keratinocytes in areas where the basement membrane was abnormal. MMP-28 was not detected in psoriatic or non-lesional skin. Our results suggest that keratinocytes as well as the previously reported cell types (smooth muscle, endothelial and macrophages) can express MMP-19 in psoriasis and lichen planus. Upregulation of MMP-19 in keratinocytes may be influenced by changes in the architecture of the basement membrane zone.

Cellular differentiation of basal cell carcinoma studied with fluorescent lectins and cytokeratin antibodies

The expression of cellular glycoconjugates and cytokeratin polypeptides in 8 basal cell carcinomas (BCC) was studied using fluorochrome‐coupled lectins and different keratin‐antibodies. Peanut agglutinin and Wistaria floribunda agglutinin, binding to all layers of normal human epidermis, also stained all cells in the basal cell carcinomas. Dolichos biflorus agglutinin, which in normal epidermis binds only to the basal cells, gave a mottled staining pattern in most of the tumors. Instead, Ulex europaeus I agglutinin and soybean agglutinin, which in normal epidermis only bind to the spinous and granular cell layers, did not stain tumor cells in basal cell carcinomas. Rabbit antibodies to human 43–50kD epidermal keratin polypeptides and 2 monoclonal cytokeratin antibodies, PKK1 reacting only with follicular epithelium, and PKK2 reacting also with the basal epidermal cells, brightly stained all cells of the basal cell carcinomas studied, whereas antibodies to human 60–67kD epidermal keratin polypeptides did not bind to the carcinoma cells. The results suggest that the cells in basal cell carcinomas resemble epidermal basal cells both by their glycoconjugate pattern and keratin expression. However, the tumor cells also express cytokeratins, which can be found only in the follicular epithelium, but not in normal interfollicular epidermis.

Altered binding of Ulex europaeus I lectin to psoriatic epidermis

We have used Ulex europaeus I (UEA I) lectin, specific for α‐l‐fucose‐containing glycoconjugates, in fluorescence microscopy to stain cryostat sections of human skin from normal persons and patients with psoriasis and lichen simplex. In normal skin the upper layers of the stratum spinosum and the stratum granulosum were strongly reactive with UEA I, whereas the lower layers of the epidermis did not react. The staining intensity of the upper epidermis was similar to that of the endothelium of dermal blood vessels. Biopsies of the lesional skin of lichen simplex showed an intense UEA I‐specific staining throughout the whole epidermis, similar in intensity to that seen in the upper epidermis of normal skin.