Upregulation of adhesion complex proteins and fibronectin by human keratinocytes treated with an aqueous extract from the leaves of Chromolaena odorata (Eupolin)

ARTICLE

Eupolin ointment is a formulation of an extract from the leaves of Chromolaena odorata, manufactured and provided by the Vietnam National Institute of Burns, Hanoi. Traditionally, the aqueous extract or decoction is used in Vietnam and other tropical countries, for the treatment of soft tissue wounds, burns and infections [1, 2]. The leaf extract is a complex mixture of natural compounds including flavonoids [3], essential oils, saponin triterpenoids, tannins, organic acids, and numerous trace substances, and has previously been described [4]. A number of studies carried out by investigators in Vietnam, Nigeria and the Côte d'Ivoire on clinical and experimental wounds demonstrated that the extract of the leaves of Chromolaena odorata inhibited the growth of bacteria [5-8], and stimulated granulation tissue formation and re-epithelialisation [8].

The proteins of the basement membrane zone (BMZ) and extracellular matrix form the adhesion complex of the skin (Fig. 1). The importance of the adhesion complex in re-epithelialisation and wound healing, after injury or burns is well recognised [9]. More recently laminin 5, the major component of the anchoring filament, has been shown to be critical to the stability of epithelial cell attachment to their substrate, and to play an essential role in adhesion complex formation [9, 10].

We have previously shown that the Eupolin extract enhanced fibroblast and endothelial cell proliferation [11], and affected keratinocyte migration (unpublished). The extract stimulated keratinocyte growth in monolayer at low concentrations, but appeared to trigger cell differentiation at high concentrations. As a result of these studies we have investigated the effect of Eupolin on the expression of adhesion complex components and fibronectin.

As a preliminary technique, indirect immunofluorescence, although not quantitative, is rapid and easy to perform, and used in conjunction with specific monoclonal antibodies, can demonstrate expression of proteins under different culture conditions. We cultured human keratinocytes in a serum-free basal medium supplemented with increasing amounts of Eupolin and used indirect immunofluorescence to determine whether the clinical efficacy might be explained, in part, by an effect of the extract on the adhesion complex proteins of the skin.

Materials and methods

Plant extract material (Eupolin)

An extract for in vitro studies was prepared as previously described [11]. The Eupolin extract was stored as stock solutions of 10 or 12 mg/ml in the dark at 4° C. The different dilutions of the stock extract (0.1, 1.0, 10, 50 and 150 µg/ml of Eupolin solution diluted in keratinocyte serum-free basal medium) were prepared on the day of the experiment.

Cell culture

Human keratinocytes were isolated from normal breast skin removed during plastic surgery and maintained at 37° C in a humidified 5% CO₂/95% air atmosphere in serum-free medium. Keratinocyte Growth Medium (KGM) was prepared from Keratinocyte Basal Medium (KBM) supplemented with 5 µg/ml insulin, 0.5 µg/ml hydrocortisone, 0.1 ng/ml epidermal growth factor, 30 µg/ml BPE, and 5 µg/ml gentamycin (KBM and supplements were purchased from Tissue Culture Services Botolph Claydon, Buckingham, UK). All other chemicals were obtained from Sigma-Aldrich Co. Ltd., Poole, Dorset, Boehringer Mannheim, Lewes, East Sussex, and ICN BioMedicals, Thame, Oxon, UK.

For experiments, cells were thawed from frozen stock or used directly from subcultures. First or second passage keratinocytes were seeded onto 12-well slides at a density of 2 x 10³ cells/well in KGM. After 24 or 48 hours the medium was replaced by KBM or by KBM containing various doses of Eupolin solution (0.1, 1.0, 10, 50 and 150 µg/ml), with one set being maintained in KGM as a control. At 70-80% confluence (4-5 days), the slides were washed three times in PBS, air dried and stored at ­ 70°C until required for indirect immunofluorescence.

Indirect immunofluorescence and monoclonal antibodies

A standard indirect immunofluorescence technique was used to determine the expression of the adhesion complex components and fibronectin by cultured human keratinocytes maintained in KBM and treated with increasing doses of Eupolin extract (Fig. 1). A rhodamine counterstain (ICN Biomedicals, Thame, Oxon. UK), used at a 1:40 dilution was added to the fluorescent labelled anti-mouse secondary antibody (Dako Ltd, Eley, UK) to reduce the non specific green fluorescent background staining. Using this counterstain cells not expressing the antigen were a dull orange-brown colour, whilst cells expressing the protein of interest fluoresced green.

The following monoclonal antibodies (MoAbs) were used: anti-beta1 MoAb to the beta1 epitope (clone DF5 Hu) beta1 integrin (Chemicon, Harrow, UK), GoH3 to the alpha6 chain of the alpha6beta4 integrin (Dr A Sonnenberg, Amsterdam, The Netherlands), G71 (Dr J Aplin, Manchester, UK) and 3E1 (Chemicon) to the beta4 chain of the alpha6beta4 integrin, GB3 (Harlan Sera-Lab, Loughborough, UK), BM165, and K140 (Dr P Marinkovich, Stanford, California, USA) to laminin 5, anti-laminin MoAb to the A, B1 and B2 chains of laminin 1 (Sigma-Aldrich Co. Ltd, Poole, UK), anti collagen IV to the helical part of collagen type IV (Dako Ltd), LH7.2 (Professor IM Leigh, London, UK) to the NC 1 domain of collagen type VII, and anti-fibronectin to fibronectin (Harlan Sera-Lab, Loughborough, UK). The MoAbs were used at optimal dilutions for normal human skin (range undiluted to 1:2,000). The negative controls were MoAb anti-tenascin to tenascin (Professor IM Leigh), L15 tissue culture medium (undiluted) and non-immune ascites control fluid (1:100) (Nordic Immunologicals, Tilburg, The Netherlands). 8 µm cryostat sections of normal human skin served as the tissue control for the specificity of the monoclonal antibodies. The expression of antigens was assessed qualitatively, both for change in pattern and amount based on the area of fluorescence of the whole section, scored as +, ++, +++.

Results

The results in Table 1, and Figures 2-6 show that culture in Eupolin-supplemented serum-free basal medium increased the expression of several of the adhesion complex proteins and fibronectin. The effect of Eupolin on the expression of these proteins by cultured human keratinocytes was dependent on the dose of Eupolin in the culture medium.

Basal cell and hemidesmosome associated components

beta1 and alpha6beta4 integrins: The staining of the cells with G71, 3E1, and anti-beta1 demonstrated a focal pattern of staining which was dose-dependent. The expression of the beta4 and beta1 integrin chains was stimulated by low concentrations (0.1 and 1 µg/ml) of Eupolin, and decreased by higher concentrations (Fig. 2). The staining of the cells with GoH3 was weak and the expression of the alpha6 integrin chain was not altered by different concentrations (0.1 ­ 150 mg/ml) of Eupolin. The control skin showed expression of the beta1 integrin on the lateral walls of the basal cell whereas the antibodies to the alpha6beta4 integrin stained the hemidesmosome. There was staining of the blood vessel basement membrane by the antibodies to the beta1 and alpha6beta4 integrins.

Adhesion complex components

Anchoring Filament Components: Laminin 5 was identified by MoAbs: GB3, BM165 and K140, and there was copious extracellular secretion by keratinocytes dependent on the concentration of Eupolin in the culture medium. The expression and extracellular secretion was similar in KBM and 0.1 µg/ml Eupolin, was greatly enhanced with higher concentrations, and was most marked at the highest concentration (Fig. 3), and differed in pattern from that in KGM by the massive extracellular secretion. The antibodies bound the normal skin control tissue with bright linear fluorescence at the BMZ.

Lamina lucida: There was enhanced expression of laminin 1 at higher doses of the extract (50 to 150µg/ml), but no detection at low doses (0.1 µg, 1 µg or 10 µg/ml) or in KBM and KGM (Fig. 4). Antibodies to laminin 1 bound to the BMZ and blood vessels of the normal control tissue.

Lamina densa: Eupolin supplements had a very spectacular, dose-dependent effect on collagen IV synthesis and expression by human keratinocytes with extracellular secretion at the highest concentrations (Fig. 5). Collagen IV was weakly expressed by a few keratinocytes cultured in the basal (KBM) or growth (KGM) medium. There was strong staining of collagen IV in the BMZ and blood vessels of the control normal tissue.

Anchoring fibrils: Eupolin had little effect on collagen VII, the major structural protein of the anchoring fibrils, with expression varying from undetectable to very weak. There was normal strong linear staining of the BMZ in the control tissue.

Fibronectin

Fibronectin, a non-collagenous extracellular glycoprotein synthesised by both keratinocytes and fibroblasts in culture, was expressed by very few keratinocytes cultured in the KBM or KGM. The expression of fibronectin in Eupolin-supplemented KBM was significantly enhanced at low concentrations and strongly enhanced at 50 and 150 µg/ml of the extract (Fig. 6). The MoAb to fibronectin stained the papillary dermis and blood vessels in normal control skin.

Discussion

Our results show that the expression of adhesion complex proteins and fibronectin (visualised using a panel of MoAbs) was modified by supplements of Eupolin in the culture medium. These proteins are markers of cell attachment and migration, and could contribute to the mechanisms by which Eupolin influences wound healing.

The integrins are cell-surface receptor molecules expressed in the basal cells of the epidermis, by keratinocytes in culture, and during wound healing. Expression of the beta1 integrin and beta4 chain of the alpha6beta4 integrin was enhanced at low concentrations of Eupolin, but was decreased at higher concentrations (10-150 µg/ml). The beta1 family of integrins is the dominant class of integrin extracellular matrix receptors and controls cell-cell contact adhesion, spreading and migration of epidermal keratinocytes on extracellular matrix regulating the onset of terminal differentiation within the epidermis [12]. We showed a decrease in the expression of the beta1 at higher concentrations (10-150 µg) of Eupolin in our experiments, and suggest this is due to the switch from lateral migration to cell differentiation as demonstrated in Figure 2C [13]. Keratinocytes use beta1 integrins to adhere to fibronectin, laminin 1, collagens I and IV (14). The alpha6beta4 integrin, the cellular receptor for laminin 5, is involved in hemidesmosome formation and the establishment of adhesion complexes during wound healing [15, 16]. Human mucosal wound healing shows increased expression of the beta1 integrin and of the alpha6beta4 integrin during migration of the keratinocytes to cover the wound bed [17]. The increase in beta1 and beta4 expression at low concentrations of Eupolin may increase adhesion and stability of the epithelium on the underlying matrix.

We have shown that the expression and secretion of laminin 5 are strongly enhanced in cultured keratinocytes by increasing amounts of Eupolin. Laminin 5 is crucial to the stability of epithelial attachment, and has been called a "biological glue" as it increases adherence of epithelial sheets to wound surfaces and the rate of assembly of a new basement membrane zone [9]. It is the first extracellular matrix component to be expressed and deposited by migrating keratinocytes during wound healing and is essential for the regulation of keratinocyte migration and motility [17-20]. Laminin 5 induces the assembly of hemidesmosomes by cultured epithelial cells and regulates cell adhesion by the alpha3beta1 integrin and the alpha6beta4 integrin, its extracellular ligand [21]. These processes are all essential to wound healing. We believe that the enhanced secretion of laminin 5 by Eupolin is a major finding that partly explains the clinical effectiveness of Eupolin as a traditional herbal remedy in the healing of soft tissue wounds, burns and infections [1, 2].

The expression of laminin 1 by human keratinocytes was enhanced by increasing amounts of Eupolin in the culture medium. Laminin 1 plays a major role in maintaining the stability and integrity of the skin, and may also promote cell attachment, spreading, proliferation and differentiation [22]. Laminin 1 interacts with the beta1 integrin [14]. Any treatment which enhances the production of laminin 1 would therefore be beneficial to wound repair and healing

Expression of collagen IV was enhanced with increasing doses of Eupolin. This could be significant as collagen IV may stimulate the proliferation of human keratinocytes, and is believed to be secreted on contact with granulation tissue and perhaps act to stabilise the new epidermis on this granulation tissue [23].

Fibronectin is an extracellular protein found in the papillary dermis and surrounding blood vessels. We found enhanced expression of fibronectin in keratinocytes cultured with increasing amounts of Eupolin in the medium. In wound healing fibronectin acts as a potent stimulus for in vitro cell attachment, cell migration and wound re-epithelialisation [24], and it is a major component of the provisional matrix in granulation tissue [25]. Human keratinocytes synthesise, secrete and deposit fibronectin in the extracellular matrix [24, 26-29], and upregulation by Eupolin may contribute to the effectiveness of Eupolin in wound healing.

We have demonstrated upregulation of laminin 5, collagen type IV, fibronectin and to a lesser extent laminin 1 in human keratinocytes cultured in KBM supplemented with varying doses of Eupolin. The most striking upregulation was of those members of the adhesion complex which span or abut the lamina lucida, previously described as the locis minoris restitentiae, and thus crucial to re-epithelialisation and wound healing.

Eupolin ointment has been shown to enhance tissue repair and regeneration in the healing of burns, soft tissue wounds and skin infections in Vietnam and other developing countries. Our results in an in vitro cell culture model suggest that the increased expression of adhesion complex proteins and fibronectin by Eupolin may contribute to its clinical effectiveness. Further studies using quantitative and molecular biology techniques are necessary to determine the specific mechanisms involved. The traditional Eupolin treatment provides an effective herbal remedy which is readily available to a majority of people in tropical countries, regardless of income and status, and which has the potential to greatly improve the quality of life of patients.

Article accepted on17/7/00

CONCLUSION

Acknowledgements

We would like to thank Professor I.M. Leigh, Professor D. Garrod, Dr M.P. Marinkovich, Dr J. Aplin, and Dr A. Sonnenberg for their kind gift of monoclonal antibodies. We would also like to thank Professor Le The Trung and Professor Pham Manh Hung, the National Institute of Burns, Hanoi for providing the Eupolin. We would like to thank the organisation, Medical and Scientific Aid for Vietnam, Laos and Cambodia, and The Dunhill Medical Trust for their financial support of this project.

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