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The gram-negative anaerobe Porphyromonas gingivalis colonizes the gingival crevice and is etiologically associated with periodontal disease that can lead to alveolar bone damage and resorption, promoting tooth loss. Although susceptible to antibiotics, P. gingivalis can evade antibiotic killing by residing within gingival keratinocytes. This provides a reservoir of organisms that may recolonize the gingival crevice once antibiotic therapy is complete. Polymersomes are nanosized amphiphilic block copolymer vesicles that can encapsulate drugs. Cells internalize polymersomes by endocytosis into early endosomes, where they are disassembled by the low pH, causing intracellular release of their drug load. In this study, polymersomes were used as vehicles to deliver antibiotics in an attempt to kill intracellular P. gingivalis within monolayers of keratinocytes and organotypic oral mucosal models. Polymersome-encapsulated metronidazole or doxycycline, free metronidazole, or doxycycline, or polymersomes alone as controls, were used, and the number of surviving intracellular P. gingivalis was quantified after host cell lysis. Polymersome-encapsulated metronidazole or doxycycline significantly (P<0.05) reduced the number of intracellular P. gingivalis in both monolayer and organotypic cultures compared to free antibiotic or polymersome alone controls. Polymersomes are effective delivery vehicles for antibiotics that do not normally gain entry to host cells. This approach could be used to treat recurrent periodontitis or other diseases caused by intracellular-dwelling organisms.

Oral lichen planus (OLP) is a chronic inflammatory disease of the oral mucosa characterized by a band-like accumulation of lymphocytes in the connective tissue adjacent to the basement membrane as well as intraepithelially. Amalgam fillings can induce oral lichenoid reactions (OLR) that are similar to OLP. The adhesion molecule ICAM-1 and the chemokines interleukin-8 and RANTES all play central roles in leucocyte trafficking. The aim of this study was to investigate the possible role of these molecules in the migration of leucocytes into the oral mucosa in OLP and OLR. Standard immunoperoxidase techniques were used to visualize the expression of ICAM-1, RANTES and interleukin-8 in frozen biopsy sections. ICAM-1 was expressed by endothelial cells, but not by keratinocytes, in normal oral mucosa. ICAM-1 was expressed by keratinocytes in 11 of 12 biopsies of OLP and in six of seven biopsies of OLR. In all of these cases ICAM-1 was also expressed by endothelial cells and leucocytes. Although not present in normal oral mucosa, RANTES was expressed by keratinocytes in 21 of 24 biopsies of OLP and in seven of seven cases of OLR. Interleukin-8 was not detected in any of the samples. The expression of ICAM-1 and RANTES by epithelial keratinocytes in the oral mucosa in OLP and OLR could be a key inflammatory mechanism in these diseases.

Background: Despite the benefits of mercury-containing amalgam dental fillings there are growing concerns regarding the potential adverse health effects arising from exposure to mercury released from fillings. In some individuals this process may result in a local lichenoid reaction of the oral mucosa., Objectives: The aim of this study was to investigate the possibility that mercury salts released from amalgam fillings might act directly on oral keratinocytes to induce changes that could promote the development of such lesions., Methods: In vitro experiments were performed in which normal oral and cutaneous keratinocytes were cultured in the presence of mercuric chloride (HgCl2). ICAM-1 expression and the release of cytokines was determined by enzyme-linked immunosorbent assay techniques. T-cell binding to HgCl2-pretreated keratinocytes was assessed using a colorimetric method., Results: Subcytotoxic concentrations of HgCl2 induced a concentration-related increase in ICAM-1 expression and consequent T-cell binding on oral, but not cutaneous, keratinocytes. HgCl2 also stimulated the release of low levels of tumour necrosis factor-alpha and interleukin-8 (but not RANTES), and inhibited the release of interleukin-1alpha by oral keratinocytes., Conclusions: This study provides evidence that oral keratinocytes may play an integral part in initiating the pathogenesis of amalgam-induced lichenoid reactions.

Monocyte chemoattractant protein-1 (MCP-1) is a member of the CC subfamily of chemokines. It is chemoattractant for monocytes, activated memory T cells and dendritic cells. We studied MCP-1 mRNA and protein production in cultured human oral keratinocytes (OK) and skin keratinocytes (SK). MCP-1 production was undetectable in resting keratinocytes. However, stimulation with tumour necrosis factors (TNF-alpha) or interferon-gamma (IFN-gamma) induced MCP-1 mRNA and protein synthesis in both cell types. Together, TNF-alpha and IFN-gamma acted synergistically to increase MCP-1 production. In each case MCP-1 production was greater for SK than OK. The kinetics of IFN-gamma-induced MCP-1 production were similar for both cell types, peaking around 72 h. In contrast, TNF-alpha induced a more rapid increase in MCP-1 production by SK than OK, peak production occurring after 24 and 72 h, respectively. IL-1alpha and IL-4 did not induce MCP-1 production. However, in combination with IFN-gamma, they induced a small extra increase in MCP-1 production in SK but not OK.

Growth regulated peptide (GRO-alpha) is chemotactic for neutrophils. It also stimulates keratinocyte proliferation and migration, and angiogenesis in cutaneous wound healing. We compared GRO-alpha production by normal human skin and oral keratinocytes, and the effects of cytokine stimulation. Resting keratinocytes produced little, if any, GRO-alpha. TNF-alpha induced a large increase in GRO-alpha mRNA and protein production in both cell types (P<0.001). However, the response of oral keratinocytes was significantly higher (P<0.01). Oral, but not skin, keratinocytes also produced significant amounts of GRO-alpha in response to IL-1 alpha (P<0.005) and IL-4 (P<0.01) stimulation. Indeed, there was an additive effect on GRO-alpha production when oral keratinocytes were stimulated with combinations of TNF-alpha and IL-1 alpha or TNF-alpha and IL-4. Neither cell type responded to interferon gamma. Keratinocyte GRO-alpha production may help selectively recruit neutrophils in mucocutaneous inflammatory diseases, and differences in production by skin and oral keratinocyte could explain the different presentation of these diseases at the two sites. The increased GRO-alpha responsiveness of oral keratinocytes may also help explain the excellent wound healing properties of oral mucosa., (Copyright 2000 Academic Press.)

Intraepithelial lymphocytes (IEL) utilize the integrin alphaebeta7 on their surface to bind to E-cadherin on epithelial cells in the gut and breast. In oral mucosa and skin IEL express alphaebeta7 and the cutaneous lymphocyte-associated antigen (CLA) but the mechanisms of adhesion of these subsets to keratinocytes are unknown. Levels of alphaebeta7 and CLA were up-regulated on peripheral blood lymphocytes (PBL) by transforming growth factor-beta (TGF-beta) and interleukin-12 (IL-12), respectively, and both groups of lymphocytes adhered onto oral and skin keratinocytes. Adhesion of IL-12-activated PBL was totally abolished by anti-lymphocyte-associated function antigen type 1 (anti-LFA-1) antibodies but was unaffected by anti-alphaebeta7 antibodies indicating that adhesion of the CLA-positive subset is mediated via LFA-1 interaction with intercellular adhesion molecule-1 (ICAM-1). Adhesion of TGF-beta-activated PBL to E-cadherin-positive oral and skin keratinocytes was partially inhibited by anti-alphaebeta7 antibodies but was unaffected by the blocking antibody E4.6 against E-cadherin which detects the binding site for alphaebeta7-positive lymphocytes in breast and gut epithelium. TGF-beta-activated PBL also bound to an E-cadherin-negative oral keratinocyte cell line and adhesion was inhibited by anti-alphaebeta7 antibodies. These results strongly suggest that in oral epithelium and epidermis alphaebeta7-positive lymphocytes do not bind to E-cadherin and there may be a novel second ligand for the alphaebeta7 integrin.

Adhesion molecules are known to play a crucial role in the recruitment of inflammatory cells to sites of inflammation. In this study endothelial cell and keratinocyte adhesion molecule expression in recurrent oral ulcers (ROU) (n = 13) was compared with that found in normal oral mucosa (NOM) (n = 11) and experimentally induced ulcers (EIU) (n = 5) by using immunohistochemistry. Significantly greater expression of both vascular cell adhesion molecule- (VCAM-1) and E-selectin was demonstrated on vasculature in ROU compared with that found in both NOM and EIU. Induction of keratinocyte intercellular adhesion molecule-1 (ICAM-1) was also a prominent feature of ROU. The expression of VCAM-1 and E-selectin on blood vessels in ROU is likely to be important in the accumulation of lymphocytes that characterise early aphthous lesions. The induction of keratinocyte ICAM-1 may facilitate lymphocyte invasion of the epithelium in ROU, which may ultimately result in ulcer formation.

The expression, and cytokine modulation, of major histocompatibility complex (MHC) class I and class II molecules on oral and skin keratinocytes were compared in cell culture. Both cell types expressed class I, but not class II, constitutively. However, stimulation with interferon-gamma, but not interleukin-1 alpha, and -1 beta, tumour necrosis factor-alpha or lymphotoxin, induced increased expression of class I and de-novo expression of HLA-DR on both cell types. Oral keratinocytes differed from skin keratinocytes in that they exhibited greater sensitivity to interferon-gamma stimulation and higher stimulated expression of both class I and HLA-DR. In addition, interferon-gamma stimulated oral, but not skin, keratinocytes to express HLA-DP and -DQ. These observations suggest that, like skin keratinocytes, under certain conditions, oral keratinocytes may be able to act as antigen-presenting cells. This may be important in the initiation and progression of some immune-mediated mucocutaneous diseases. Moreover, differences in MHC expression may help to explain differences in the presentation of these diseases on the skin and oral mucosa.

IL-1 alpha and IL-6 are important pro-inflammatory and immunomodulatory cytokines and their production by oral (OK) and skin keratinocytes (SK) was compared. OK and SK produced IL-1 alpha, but not IL-6, constitutively. TNF alpha stimulation increased IL-1 alpha production by both cell types and exhibited synergy with interferon gamma (IFN gamma), although the latter had no effect by itself. In contrast, both cell types produced IL-6 in response to TNF alpha, IFN gamma or IL-4, and IFN gamma and IL-4 exhibited synergy with TNF alpha. For all cytokines the levels of IL-6 production were greater for OK than SK and OK, but not SK, produced IL-6 in response to IL-1 alpha stimulation. In addition, the IL-6 response to IL-4 stimulation was more rapid for OK than SK. These observations may explain the similarities and differences in wound healing and immuno-inflammatory diseases affecting the skin and oral mucosa.

RANTES, interleukin-8 (IL-8), and macrophage inflammatory protein-1-alpha (MIP-1 alpha) exhibit different and highly selective chemotactic activity for leukocytes. Resting cultured normal oral and skin keratinocytes produced little if any of these chemokines. Stimulation with 250-1,000 U/ml of tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma) induced both cell types to produce RANTES. Protein levels peaked after 48 h and mRNA levels peaked after 8 h of stimulation. Used combination, TNF-alpha, and IFN-gamma synergistically increased mRNA and protein levels. Amounts of 100-1,000 U/ml of TNF-alpha also induced IL-8 production with peak mRNA levels after 4-24 h of stimulation and maximal protein production after 72 h or more. IL-8 production by oral keratinocytes was significantly greater than that by skin keratinocytes. Although IFN-gamma alone did not induce IL-8 production, it enhanced the effect of TNF-alpha on both cell types. Stimulation for 24 h with 100-1,000 U/ml of IL-alpha also induced IL-8 production by oral but not skin keratinocytes. No MIP-1 alpha production was detected under the conditions investigated. Keratinocyte production of RANTES and IL-8, under the influence of cytokines such as TNF-alpha or IFN-gamma, provides a mechanism for the selective accumulation of leukocytes into immunoinflammatory diseases of the skin and oral mucosa. Differences in their production may help to explain differences in the presentation of these diseases on the skin and oral mucosa.

Differences in expression of intercellular adhesion molecule-1 (ICAM-1) and ICAM-1 mRNA levels were studied in cultured skin and oral keratinocytes before and after stimulation with different pro-inflammatory cytokines. Basal expression of ICAM-1 was undetectable on skin keratinocytes but oral keratinocytes expressed ICAM-1 at high levels. Interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) increased ICAM-1 expression on both cell types, although TNF-alpha had a greater effect on oral than skin keratinocytes (P<0.05) and IFN-gamma was more effective than TNF-alpha on both cell types (P<0.01). In combination, TNF-alpha and IFN-gamma synergistically increased ICAM-1 expression on skin keratinocytes only, although ICAM-1 mRNA was synergistically increased in both cell types. IL-1alpha and IL-1beta induced a small increase in ICAM-1 expression on oral keratinocytes but had no effect on skin keratinocytes.