I. Olsen

Immunologic aspects of dermal and oral lichen planus : A review

There have been many investigations, both experimental and epidemiologic, of the forms of LP affecting the skin and oral mucosae. These studies have provided a varied range of hypotheses to explain not only the factors determining susceptibility to and onset of this disease, but also the immunologic mechanisms leading to the pathosis with which LP is associated. Much progress has been made, especially through in vitro studies, regarding detailed aspects of the immunology of LP. However, data is often conflicting or incomplete. In this review we attempt to bring together the currently available data regarding the immunologic basis of LP.

Polymerase chain reaction analysis of oestrogen and androgen receptor expression in human gingival and periodontal tissue

Oestrogen and androgen receptors mediate the effects of their respective hormones by acting as ligand-activated transcription factors that control a wide range of biological processes. In order to determine whether periodontal and gingival tissues could respond to oestrogen and androgen, the specific and highly sensitive reverse-transcribed polymerase chain reaction (PT-PCR) was used to examine the presence of the mRNAs corresponding to these receptors. Expression of the androgen receptor was readily detected in periodontal and gingival tissue and in fibroblasts derived from these tissues, but transcripts for the oestrogen receptor were not detected in these samples. Moreover, treatment of the cultured fibroblasts with the oestrogen diethylstilbesterol (DES) or the androgen dihydrotestosterone (DHT) did not induce the expression of the oestrogen receptor mRNA; nor did the hormones modify the activity of the androgen receptor gene. These results suggest that, while periodontal and gingival tissues are not able to respond directly to oestrogen, they may nevertheless be highly sensitive to the anabolic effects of androgens.

Flow Cytometry Analysis of Gingival and Periodontal Ligament Cells

Gingival and periodontal ligament (PDL) fibroblasts are the major cellular components of periodontal soft connective tissues, but the precise differences between these cells are not yet known. In the present study, we have therefore examined the phenotypic and functional features of the cells obtained from gingival and PDL biopsy samples. Spindle-shaped cells characteristic of fibroblasts were the main cell type observed in vitro, although epithelial cells were also present in primary gingival cell cultures. Flow cytometry was used to measure the size and granularity of the cultured cells, and showed that the gingival fibroblasts were smaller and less granular compared with the PDL cells. The expression of certain key extracellular matrix (ECM) proteins, fibronectin, collagen type I, and tenascin was measured by flow cytometry. Analysis of the fluorescence profiles of these cultures showed that the majority of cells expressed fibronectin and that the average fluorescence intensity of this antigen in the PDL cells was higher than that in the gingival fibroblasts. Moreover, the fibronectin-positive PDL cells apparently comprised two subpopulations which expressed fibronectin at different levels, suggesting that the cells in the PDL cultures were functionally heterogeneous. The level of collagen type I was also found to be up-regulated in the PDL compared with the gingival cells and, as with fibronectin, was expressed at two different levels by subsets of the PDL cells. In contrast, tenascin was expressed at very similar levels by both the gingival fibroblasts and PDL cells. In addition, measurement of alkaline phosphatase, a marker enzyme for mineralized tissue-forming cells, showed that the PDL cells had higher activity than the gingival fibroblasts and that the alkaline phosphatase activity in the PDL cells was far more markedly up-regulated by dexamethasone. Our findings demonstrate that, despite their similar spindle-shaped appearance, fibroblasts derived from gingival and PDL tissues appear to display distinct functional activities which are likely to play a vital part in the maintenance of tissue integrity and regenerative processes.

Excess matrix accumulation in scleroderma is caused partly by differential regulation of stromelysin and TIMP-1 synthesis

Scleroderma (systemic sclerosis: SSc) is an autoimmune disorder in which excessive extracellular matrix is deposited in skin and internal organs. Because of the importance of metalloproteinases in the turnover of connective tissue, in this study we have developed a novel procedure which utilises flow cytometry (FACS) to measure the production of stromelysin (MMP-3), gelatinase A (MMP-2), and the proteinase inhibitor TIMP-1, by SSc skin fibroblasts. In the presence of monensin, which prevents the secretion of these matrix proteins, there was a similar intracellular accumulation of gelatinase A in normal and SSc cells. However, whereas stromelysin levels also increased in the normal cells, no net synthesis could be detected in the SSc fibroblasts. In marked contrast, the synthesis of TIMP-1 was 50% greater in the SSc cells than in the normal fibroblasts. Our results thus show unequivocally, for the first time, that cells from SSc patients simultaneously produce less stromelysin but substantially higher amounts of TIMP-1 than do normal dermal fibroblasts, suggesting that abnormalities in the regulation of the matrix enzymes and their inhibitors play an important part in the molecular pathology of SSc.

Simultaneous measurement of cell surface and intracellular antigens by multiple flow cytometry

We have employed a method for permeabilizing lymphocytes with the detergent saponin in order to detect an intracellular protein simultaneously with surface antigens by flow cytometry (FCM). Using monoclonal antibodies specific for the murine CD2 receptor and for the lysosomal enzyme, beta-glucuronidase (Gus), we found that the expression of both of these antigens increased markedly when T cells were activated. Two sensitive methods were used to show that FCM provided an accurate measure of the actual number of CD2 and Gus molecules present in the lymphocytes. Immunogold electron microscopy revealed the precise ultrastructural localization of these different components and corroborated the specificity of the multiple labelling procedure for the simultaneous detection of surface and intracellular antigens. We also developed a three-colour FCM technique which we used to examine the changes in Gus expression in the CD4 and CD8 T cell sub-sets during activation.

Direct enzyme transfer from lymphocytes corrects a lysosomal storage disease

Fibroblasts from patients with mannosidosis, the lysosomal storage disease resulting from an inherited deficiency of lysosomal alpha-D-mannosidase (EC 3.2.1.24), accumulate specific mannose-containing oligosaccharides which are characteristic of the disease (1,2). The present study shows that these substances were extensively degraded following transfer of the missing enzyme from normal lymphocytes to mannosidosis fibroblasts on direct contact in tissue culture. Moreover, prolonged correction of the metabolic abnormality of the recipient cells was sustained if contact with fresh donor lymphocytes was periodically renewed. These findings may be highly relevant to lymphocyte function in enzyme replacement therapy by transplantation procedures currently being attempted.

Lysosomal storage diseases: mechanisms of enzyme replacement therapy

SummaryLysosomal diseases result from deficiency of one of the many enzymes involved in the normal, step-wise breakdown of macromolecules. Studies in vitro have shown that cells from enzyme-deficient patients can be corrected by an exogenous supply of the missing enzyme. This occurs by receptor-mediated endocytosis of normal enzyme added to tissue culture medium and also by direct transfer from normal leukocytes during cell-to-cell contact. Immunohistochemical analysis has revealed that these processes have similar pathways of intracellular transport of the acquired enzymes, which ultimately reach mature lysosomes in the recipient cells. Moreover, recent studies suggest that both mechanisms are important in the therapy of lysosomal storage diseases by bone marrow transplantation. Advances in gene technology are likely to improve the successful treatment of these disorders, by facilitating the large scale production of clinically effective proteins and also by enabling the stable and safe introduction of normal lysosomal genes into cells of affected patients.

Expression of ectopeptidases in scleroderma.

OBJECTIVES--To examine the expression and concentrations of three ectopeptidases likely to be involved in regulating the functional levels of adhesion molecules and the turnover of connective tissue components, in patients with scleroderma (systemic sclerosis) (SSc) and in normal individuals. METHODS--Monoclonal antibodies against these antigens were used for immunoperoxidase staining of cryostat skin sections and for flow cytometric (fluorescence activated cell sorter) analysis of cultured dermal fibroblasts grown from SSc patients and normal controls. RESULTS--Although neutral endopeptidase-24.11 (NEP) (CD10) was not detected in either SSc or normal skin, aminopeptidase N (APN) (CD13) and dipeptidyl peptidase IV (DPPIV) (CD26) were both readily visualised. However, DPPIV appeared to be present in smaller concentrations in the SSc biopsy specimens. Moreover, while fibroblasts grown in vitro from both SSc and normal skin also had similar concentrations of APN, the expression of DPPIV in the cultured SSc cells was found to be very much less than that present in the normal fibroblasts. It is noteworthy that NEP, which was not detected in the tissue sections, was nevertheless readily detected in fibroblasts in culture. CONCLUSIONS--These results show that a number of cell surface proteases are expressed by dermal fibroblasts both in vivo and in vitro, and it is suggested that the marked downregulation of DPPIV in SSc could be at least partly responsible for the increased concentrations of adhesion molecules and matrix proteins associated with the molecular pathology of this disease.

Cell contact induces the synthesis of a lysosomal enzyme precursor in lymphocytes and its direct transfer to fibroblasts

The activity of a lysosomal enzyme, alpha-D-mannosidase (EC 3.2.1.24), increased markedly in normal lymphocytes when they were cultured together with fibroblasts from a patient with an inherited deficiency of this enzyme. Cell-to-cell contact was obligatory for this increase in activity, which also required new protein synthesis. The enzyme induced in the co-cultured lymphocytes was a high molecular weight form of alpha-D-mannosidase that was not detected in lymphocytes cultured alone, which had only the low molecular weight mature enzyme. It was this precursor form alone that was directly transferred to the mannosidosis fibroblasts, where it was present initially in organelles of low density. When the culture period was extended the lymphocyte precursor enzyme was transported to the heavy lysosomes in the recipient cells, and correctly processed to the functionally effective mature enzyme.

Control of the human cell cycle by a bacterial protein, gapstatin

The oral gram-negative bacterium Actinobacillus actinomycetemcomitans is a major pathogen in human periodontal disease. Saline extraction releases a range of surface-associated components from this bacterium, including one which exhibits potent anti-proliferative activity as assessed by its capacity to inhibit DNA synthesis by human and other mammalian cells. Cultures incubated with this bacterial fraction for a prolonged period comprise a high proportion of cells containing a 4n level of DNA. Studies using hydroxyurea-synchronized cultures showed that cells treated with the surface-associated fraction were arrested in the G2 phase of the cell cycle and did not enter mitosis. This G2/M blockade was observed only when the bacterial fraction was added to the cells during early S phase. Our data also suggest that the active bacterial component binds to surface receptors expressed by the human cells and may act by a novel mechanism which involves down-regulation of cyclin B1 expression. The anti-proliferative activity of the bacterial fraction, purified by a combination of ammonium sulphate precipitation, HPLC anion exchange and gel filtration, has been shown to be an 8 kDa protein, which we have called gapstatin. Purified gapstatin was shown to be responsible for the the inhibitory effects of the surface-associated fraction on mammalian cells.