Binding of integrin alpha6beta4 to plectin prevents plectin association with F-actin but does not interfere with intermediate filament binding.

Hemidesmosomes are stable adhesion complexes in basal epithelial cells that provide a link between the intermediate filament network and the extracellular matrix. We have investigated the recruitment of plectin into hemidesmosomes by the alpha6beta4 integrin and have shown that the cytoplasmic domain of the beta4 subunit associates with an NH(2)-terminal fragment of plectin that contains the actin-binding domain (ABD). When expressed in immortalized plectin-deficient keratinocytes from human patients with epidermol- ysis bullosa (EB) simplex with muscular dystrophy (MD-EBS), this fragment is colocalized with alpha6beta4 in basal hemidesmosome-like clusters or associated with F-actin in stress fibers or focal contacts. We used a yeast two-hybrid binding assay in combination with an in vitro dot blot overlay assay to demonstrate that beta4 interacts directly with plectin, and identified a major plectin-binding site on the second fibronectin type III repeat of the beta4 cytoplasmic domain. Mapping of the beta4 and actin-binding sites on plectin showed that the binding sites overlap and are both located in the plectin ABD. Using an in vitro competition assay, we could show that beta4 can compete out the plectin ABD fragment from its association with F-actin. The ability of beta4 to prevent binding of F-actin to plectin explains why F-actin has never been found in association with hemidesmosomes, and provides a molecular mechanism for a switch in plectin localization from actin filaments to basal intermediate filament-anchoring hemidesmosomes when beta4 is expressed. Finally, by mapping of the COOH-terminally located binding site for several different intermediate filament proteins on plectin using yeast two-hybrid assays and cell transfection experiments with MD-EBS keratinocytes, we confirm that plectin interacts with different cytoskeletal networks.

hCG beta expression by cervical squamous carcinoma--in vivo histological association with tumour invasion and apoptosis.

AIMS: To investigate the correlation of beta-subunit of human chorionic gonadotrophin (hCG beta) expression by cervical carcinomas with measures of tumour apoptosis. METHODS AND RESULTS: Eighty-nine cervical carcinoma patients' samples were subject to hCG beta immunohistochemistry and scored with respect to intensity of immunopositivity and percentage of positive cells. Apoptosis was evaluated by three independent parameters: morphological characteristics [haematoxylin and eosin (H&E)], terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) and poly (ADP-ribose) polymerase (PARP) immunopositivity. Of the 12 adenocarcinomas, only one (8%) was hCG beta+. However, 87% (61/70) of the squamous cell and 100% (7/7) of adenosquamous cell carcinomas were hCG beta+. hCG beta reactivity and intensity was predominantly confined to peripheral tumour cells at the stromal-epithelial interface. Correlation analysis showed that H&E and PARP apoptotic immunopositivity negatively correlated with hCG beta expression (P < 0.001 and P = 0.028 respectively), whereas TUNEL did not (P = 0.12). However, immunopositivity for apoptotic cells by TUNEL was significantly less in tumours where hCG beta expression was greater (scoring >or= 6) and vice versa. hCG beta immunopositivity was also observed in newly formed blood vessels, as well as tumour cells within lymphatic vessels. When tumour vascularization was taken into account, samples with noted vascularization positively correlated with hCG beta scoring. CONCLUSIONS: hCG beta expression correlates with reduced tumour cell apoptosis and may be involved in tumour vascularization and dissemination.

Basal cell glycoprotein in pig epidermis closely resembles the beta 1 subunit of the integrin family of cell adhesion molecules.

A 135-kD conA-binding glycoprotein isolated from pig epidermis was previously localized to the surface of basal cells in stratified epithelia using affinity-purified antibodies. Preembedding immunoperoxidase electron microscopy has now shown that this glycoprotein is concentrated on the lateral surfaces of basal cells but is not detectable on those surfaces adjacent to the basement membrane indicating a role in cell-cell rather than cell-substrate interactions. The basal cell glycoprotein was shown to resemble the beta 1 subunit of the integrin family following the generation of a specific monoclonal antibody (M5.25). The epidermal glycoprotein recognized by M5.25 and by antibodies against the beta 1 fibronectin receptor from human placenta co-migrated on SDS gels under both reducing and non-reducing conditions. Its response to disulphide reducing agents was characteristic of beta 1 integrin subunits. In addition, the basal cell glycoprotein was shown to bind to the 120-kD cell-binding fragment of fibronectin in a RGD-dependent manner. It was readily detected by immunoblotting whole cell lysates of cultured pig keratinocytes suggesting increased expression in cultured cells compared to fresh epithelial tissue. The results suggest that beta 1 integrin subunits may be involved in cell-cell interactions between basal keratinocytes in pig epidermis and that these receptors are lost from the cell surface during terminal differentiation. Thus modulation of beta 1 integrin subunit expression may play an important role in regulating differentiation in pig epidermis.

Keratin 19: predicted amino acid sequence and broad tissue distribution suggest it evolved from keratinocyte keratins.

The type I keratin 19 is unusual in its tissue distribution in that under normal circumstances it does not seem to be restricted, as the other keratins are, to expression in either stratified or simple epithelia. In addition to the previously reported distribution of keratin 19 in human tissues, we have observed keratin 19 in epidermal basal cells, in a defined region of the hair follicle, and in nipple epidermis. We noticed that expression of keratin 19 appears to be especially characteristic of regions of labile or variable cellular differentiation as indicated by the presence of multiple keratin phenotypes in close proximity to each other. Using a monoclonal antibody recognizing keratin 19 (LP2K) to screen a human placenta cDNA expression library, we have isolated, cloned, and sequenced cDNA coding for full-length human keratin 19, as confirmed by its reactivity with several other known anti-keratin 19 monoclonal antibodies and by the near identity of its sequence with that of the bovine keratin 19 homologue. This similarity extends to both proteins being truncated at the C-terminal end to only 13 amino acids beyond the rod domain. Although the amino acid homology over the N-terminal and helical rod domains is particularly high, the human and bovine proteins diverge substantially over the short C-terminal domain, which suggests that this region has no conserved function. Comparison with other type I keratins indicates that the closest evolutionary neighbors of keratin 19 are keratinocyte keratins, probably 13 and 14, and not the simple epithelial keratin 18. Assessing the histochemistry and sequence data together, we propose that the cell may use this apparently deficient keratin as a "neutral" keratin. While unimpaired in its ability to polymerize (keeping the cell integrated into the epithelial sheet via filament-desmosome networks), keratin 19 expression does not irrevocably commit a cell to any one of the local differentiation options. Such predicted differentiational flexibility may also imply vulnerability to transformation.

Expression of distinct desmocollin isoforms in human epidermis.

Previous evidence suggested the presence of two distinct desmocollin isoforms in human epidermis. These isoforms have now been distinguished at the protein level using monoclonal and polyclonal antibodies against N-terminal fragments of desmosomal glycoprotein (DG) IV/V isolated from plantar callus and antibodies against a fusion protein containing the extracellular domain of DGII/III. Immune blotting of glycoprotein fractions from whole epidermis, plantar callus, psoriatic scales and cultured keratinocytes showed that intact DGIV/V and its proteolytic fragments consistently migrated faster than DGII/III during SDS-PAGE. The apparent Mr difference between the two isoforms was in the range 2-5 kD. DGIV/V was the predominant species in epidermal tissue but was much less prominent in cultured cells by immune-blotting and immune precipitation. This is consistent with the differentiation-related expression of desmocollins revealed by immunofluorescence. DGIV/V was strongly expressed in the upper spinous/granular layer of the epidermis whereas DGII/III was more prominent in the basal layers of the tissue. The DGIV/V monoclonal (LH50) recognized an N-terminal, Ca(++)-sensitive epitope, because its staining of unfixed epidermal tissue was markedly influenced by Ca++ levels. Ca++ inhibition was observed at concentrations as low as 50 microM, suggesting its possible physiologic significance. Ca++ inhibition of LH50 binding was also observed in an enzyme-linked immunosorbent assay system using denatured glycoproteins although higher concentrations were required. It remains to be seen whether direct effects of Ca++ on desmocollin conformation are involved in the regulation of keratinization by extracellular Ca++.

Keratin 15 expression in stratified epithelia: downregulation in activated keratinocytes.

Keratin 15 (K15) is a type I keratin without a defined type II partner whose expression in epidermal diseases has not been investigated. In this study we have used LHK15, a monoclonal antibody raised against the last 17 amino acids of the K15 polypeptide, to show that K15 is expressed primarily in the basal keratinocytes of stratified tissues, including the fetal epidermis and fetal nail. Although K15 in normal hair follicles was virtually absent from hair bulbs, it was expressed by a subset of keratinocytes in the outer root sheath. By comparison, K14 expression was found throughout the outer root sheath of hair follicles; however, when both K14 alleles were naturally ablated, the expression of K15 was also observed throughout the outer root sheath of the follicles. Expression of K15 mRNA was assessed by in situ hybridization and corroborated the data from immunostaining. An increase in K15 mRNA and protein expression in hair follicles from the K14 ablated epidermis suggested an upregulation of the K15 gene in the absence of the K14 protein. In organotypical cultures where differentiating keratinocytes expressed markers of activated phenotype, i.e., K6 and K16, expression of K15 was undetectable. The expression of K15 mRNA and protein was also downregulated in two hyperproliferating situations, psoriasis and hypertrophic scars. Because keratinocytes in psoriasis and hypertrophic scars are activated, we conclude that K15 expression is not compatible with keratinocyte activation and the K15 gene is downregulated to maintain the activated phenotype.

Type VII collagen is a normal component of epidermal basement membrane, which shows altered expression in recessive dystrophic epidermolysis bullosa.

The murine monoclonal antibody LH 7:2, which reacts with the basement membrane of stratified squamous epithelia including epidermis, has been characterized biochemically and shown to bind to part of the type VII collagen molecule. Immunoblotting reveals that the antibody binding site lies in the non-helical carboxy terminal region of the type VII collagen dimer and immunoelectron microscopy shows that the epitope is within the lamina densa of the basement membrane. Loss of LH 7:2 binding in the hereditary blistering disease recessive dystrophic epidermolysis bullosa suggests that inadequate synthesis or excessive breakdown of type VII collagen may form the biologic basis for the disease.

AJ-p90: a novel protein of the perinuclear theca in human sperm subacrosome.

A perinuclear theca protein of the human sperm subacrosome was detected using LH43 monoclonal antibody which was originally raised against human keratinocytes. Using indirect immunofluorescence, the antibody stained the acrosomal zone (AZ) of dried ejaculated spermatozoa but did not react with viable cells, thus suggesting that the antigen was intracellular. This was confirmed by immunogold electron microscopy which also revealed the ultrastructural localisation of the antigen to the subacrosomal fibrils. Throughout spermatogenesis the antigen was only detected on the AZ of developed testicular spermatozoa and its expression was continued during their epididymal passage. Biochemically, the protein was insoluble in Triton, and dithiothreitol (DTT) was required for its solubilisation. In Western blotting of sperm and keratinocyte lysates, the antibody detected similar 90-kDa protein doublets (AJ-p90). These biochemical features exclude the identity of AJ-p90 with various cyto- and karyo-skeletal antigens, including the intermediate filaments and microfilaments. AJ-p90 therefore represents a novel product of the subacrosomal perinuclear theca. The significance of these data is discussed together with the importance of the antibody for probing the perinuclear theca in normal and abnormal germ cells, including multinucleated spermatids which also showed reactivity with the antibody.

Epidermal differentiation and dermal changes in healing following treatment of surgical wounds with sheets of cultured allogeneic keratinocytes.

AIMS: To establish the structural changes that occur in deep surgical wounds engrafted with allogeneic sheets, their time course and inter-relation. METHODS: Deep surgical wounds following shave excision of tattoos (down to deep dermis/subcutaneous fat) were treated with sheets of sex mismatched allogeneic keratinocytes in 19 patients and then biopsied weekly until wound healing was complete. More superficial surgical wounds--that is, 20 standard skin graft donor sites, were biopsied at seven to 10 days (all healed) following application of keratinocyte allografts. All biopsy specimens were examined with a large panel of monoclonal antibodies to keratins, envelope proteins, basement membrane components, and to extracellular matrix components. RESULTS: The hyperproliferative keratin pair K6/16 was expressed in all wounds, for up to six weeks in keratinocyte grafted deep wounds, and up to six months in split thickness skin grafted wounds. CONCLUSIONS: Keratins 6 and 16 have not been detected in normal skin, although the relevant mRNA has. This raises the possibility of regulation at a post-transcriptional level allowing a rapid response to injury with cytoskeletal changes that may aid cell migration. This keratin pair offers the most sensitive marker for altered epidermis following wounding.

Clinical practice and biological effects of keratinocyte grafting.

Skin grafts can be produced in the laboratory from simple sheets of cultured keratinocytes (keratinocyte grafts) or in combination with different mixtures of connective tissue components (composite culture grafts). Autologous keratinocyte grafts have been used most extensively in patients with major body surface burns and have proved life saving. Further attention to clinical factors has improved graft take to 50-60% in optimal circumstances although there is some short term instability of the graft. Keratinocyte autografts can also be used to treat other chronic wounds such as leg ulcers, and surgical excisions. Keratinocyte allografts do not survive transplantation but have effects on wound healing by the release of growth factors and matrix components. Composite grafts have been little used in clinical practice and there are inherent problems with the stability of the matrix components in the presence of high levels of wound collagenases, but banks of allogenic skin grafts may provide temporary cover in burns patients. The roles and clinical indications for keratinocyte grafting are now becoming clear following wider clinical experience.

Survival of cultured allogeneic keratinocytes transplanted to deep dermal bed assessed with probe specific for Y chromosome.

To determine the survival of cultured allogeneic keratinocytes transplanted to a deep dermal bed 24 tattoos that had been removed by deep shave excision in 19 patients were grafted with sheets of cultured allogeneic keratinocytes from donors of the opposite sex. Cells carrying the Y chromosome were identified in biopsy specimens taken from the graft site by in situ DNA hybridisation with a biotinylated Y probe (pHY 2.1) and visualised with a technique using immunoperoxidase. The cultured allograft sites were biopsied one, two, and three weeks after transplantation. No male cells were identified in any biopsy specimen from female patients who were given transplants of male cultured keratinocytes, and all biopsy specimens from male patients, who received female cultured keratinocytes, showed percentages of male cells within the normal range for male skin. The beneficial effects of cultivated allogeneic keratinocytes result from effects on wound healing other than forming a successful graft that "takes."

Cholera toxin and analogues of cyclic AMP stimulate the growth of cultured human mammary epithelial cells.

The growth of human epithelial cells is stimulated by cholera toxin and analogues of cyclic AMP, while the growth of breast derived fibroblasts is inhibited. These compounds have little effect on DNA synthesis in the absence of other mitogens but show a synergistic effect with serum and/or EGF. The results suggest that high intracellular levels of cyclic AMP in human mammary epithelial cells increase the growth response of the cell to mitogens.

Co-culture of synovial fibroblasts and differentiated U937 cells is sufficient for high interleukin-6 but not interleukin-1beta or tumour necrosis factor-alpha release.

Inflamed synovium is characterized by high concentrations of cytokines [interleukin (IL)-6, IL-1beta and tumour necrosis factor (TNF)-alpha] and the abundant presence of infiltrated monocytes, many of which are found adjacent to the resident fibroblast-like synoviocytes. We have used a co-culture of fibroblast-like synoviocytes and differentiated U937 cells to study IL-6, IL-1beta and TNF-alpha release. After a 3 day co-culture, 35% of the U937 cells had adhered and were fully differentiated towards monocytes, as determined by expression of p47phox, CD14, MSE-1, Mac-1, collagenase and NADPH oxidase activity. IL-6 release from fibroblast-like synoviocytes was induced 4-fold by co-culture with differentiated U937 cells. However, co-culture of differentiated U937 cells with fibroblast-like synoviocytes failed to release detectable levels of IL-1beta and TNF-alpha from the U937 cells. Addition of synovial fluid further increased IL-6 release, but again had no effect on IL-1beta or TNF-alpha, although U937 cells differentiated by phorbol ester were able to release these two cytokines and, in the case of the co-culture, mRNAs for both cytokines were highly expressed in the U937 cells. We postulate that the influx of monocytes into the synovium is instrumental in the elevation of IL-6 levels, but this is not sufficient to explain high levels of IL-1beta or TNF-alpha.

Effects of SV40 transformation on the cytoskeleton and behavioural properties of human keratinocytes.

A cloned cell line (SVK14) with apparently unlimited growth potential was isolated from simian virus 40 (SV40)-infected human foreskin keratinocytes which did not appear to pass through any obvious 'crisis' (Girardi et al., J. Cell. Comp. Physiol., 1965, 65, 69-84). Indirect immunofluorescence microscopy showed that the transformed cells have the SV40 large T antigen in their nuclei and stain positively with LE61, a monoclonal antibody that reacts with a tonofilament determinant normally only found in non-keratinizing simple epithelia. SVK14 cells can be grown in the absence of 3T3 feeders and show an impaired ability to differentiate into squames, and this impairment becomes more marked with passage. At later passages the cells acquire the ability to form colonies in agar and to produce a factor with mitogenic activity which stimulates DNA synthesis in quiescent 3T3 cells. Concomitantly, the SVK14 cells become less sensitive to the growth inhibitory effect of human alpha interferons.

Identification of an epidermal basement membrane defect in recessive forms of dystrophic epidermolysis bullosa by LH 7:2 monoclonal antibody: use in diagnosis.

LH 7:2 is a monoclonal antibody that was raised against an extract of human epidermal cells and identifies an epitope within the lamina densa of the basement membrane of stratified squamous epithelia. Using indirect immunofluorescence we found intense labelling with LH 7:2 at the epidermal basement membrane (EBM) of normal skin, and in skin samples from patients with simplex, junctional, dominantly inherited dystrophic and acquired forms of epidermolysis bullosa (EB), as well as bullous pemphigoid. Staining was absent or only very faint in generalized recessive dystrophic EB (RDEB), and patchily reduced in the localized form of RDEB. We conclude that LH 7:2 recognizes an EBM antigen which may be important in the pathogenesis of RDEB. Moreover, the antibody provides a useful probe for the rapid diagnosis of RDEB and is of special value in helping to discriminate between localized RDEB and typical dominant dystrophic EB--conditions which closely resemble each other clinically and which cannot be distinguished by means of transmission electron microscopy.

Treatment of chronic venous ulcers with sheets of cultured allogenic keratinocytes.

Cultured keratinocytes were used as allografts to treat 51 patients with chronic venous ulceration or rheumatoid ulcers unresponsive to all previous conventional treatments including split skin grafts. Although early epithelialization could be seen in the centre of some ulcers, a major effect appeared to be healing from the previously indolent edge. This treatment appears to provide some clinical benefit in healing of chronic ulceration.

Monospecific monoclonal antibodies to keratin 1 carboxy terminal (synthetic peptide) and to keratin 10 as markers of epidermal differentiation.

Monospecific antibodies to individual keratin polypeptides can be used to examine the tissue and cellular coexpression of members of keratin pairs. Monospecific monoclonal and polyclonal antibodies have been raised to keratins 1 and 10 using both crude cytoskeletal extracts and synthetic peptides. The tissue distribution of these keratins has been determined against a panel of freshly frozen normal tissues from humans, rodents and pigs. Epidermal expression has been examined in psoriatic plaques, and healing wounds, as examples of epidermal hyperproliferation. Cultured keratinocytes in monolayer (low calcium), stratified (high calcium), and complex cultures, transformed keratinocytes, and tumour cell lines, have been examined for the in vitro expression of these keratins. The sensitivity and precise localization of reactivity with these monospecific antibodies gives a highly accurate picture of individual cell expression. There is confirmation of coexpression of keratins 1 and 10 in epidermal and mucosal sites, and with keratin 16 in hyperproliferative states. These monospecific antibodies provide an important means of examining keratin expression in epidermal tumours and keratinizing disorders, and of seeking keratin mutations in cell lines and in skin diseases.