Calcium-induced changes in distribution and solubility of cadherins, integrins and their associated cytoplasmic proteins in human keratinocytes.

Studies with cultured human epidermal keratinocytes have shown that stratification, the movement of differentiating cells out of the basal layer, involves changes in cell-extracellular matrix and cell-cell adhesiveness mediated by receptors of the integrin and cadherin families, respectively. Keratinocytes normally lose their integrins when they initiate terminal differentiation. However, when stratification is inhibited by a low concentration of calcium ions in the medium (0.1 mM) or by addition of antibodies to P- and E-cadherin in standard medium (1.8 mM calcium ions), differentiating, involucrin-positive, cells continue to express functional integrins. In order to investigate the mechanism by which cadherins may regulate integrin expression, we have examined the distribution and detergent solubility of the receptors and associated cytoplasmic proteins in keratinocytes grown as a monolayer in low calcium medium or transferred to standard medium to induce stratification. Within 1 hour of raising the concentration of calcium ions, integrins, cadherins, alpha-catenin, beta-catenin, plakoglobin, vinculin and alpha-actinin appeared to accumulate at cell-cell borders, whereas the focal contact proteins, paxillin and talin, did not. The change in distribution was correlated with decreased solubility in 0.5% Triton X-100 of some of the proteins examined, but the integrins, alpha-actinin, paxillin and talin remained completely soluble. Addition of cytochalasin D inhibited both the redistribution of proteins and subsequent stratification of involucrin-positive cells. Cycloheximide treatment allowed protein redistribution and stratification, but involucrin-positive cells continued to express integrins. These results suggest that stratification requires the interactions of cadherins and integrins with the actin cytoskeleton and that the selective loss of integrins from differentiating cells requires de novo protein synthesis.

Integrin expression and function in HPV 16-immortalised human keratinocytes in the presence or absence of v-Ha-ras. Comparison with cervical intraepithelial neoplasia.

Keratinocytes express several receptors of the integrin family which regulate both adhesion and differentiation. We have investigated whether HPV immortalisation, which changes the growth and differentiation properties of keratinocytes, is associated with altered integrin expression or function. We compared two HPV 16-immortalised lines of human keratinocytes, up and vp, with the normal keratinocyte strains, u and v, from which they were derived and with upr, obtained by transfection of up with viral Harvey ras. Immunofluorescence, immunoprecipitation and flow cytometry demonstrated that up and vp had lower levels of integrins than u and v, the reduction in up being greater than in vp. Up and vp also had reduced levels of mRNA encoding the beta 1 integrin subunit. Reduced expression of the alpha 5 beta 1 and alpha 2 beta 1 integrins was correlated with reduced adhesion to fibronectin and collagen in up but not in vp and there were no significant differences between the normal and immortalised cells in adhesion to laminin. Reduced integrin expression was correlated with decreased motility, up showing a greater reduction in motility than vp. Introduction of activated ras into up had no effect on integrin levels, cell motility or tumorigenicity in nude mice; the only difference between up and upr was that upr showed increased adhesion to fibronectin. Examination of eight biopsies of cervical intraepithelial neoplasias with evidence of HPV infection revealed reduced or discontinuous integrin expression in the most severe lesions. We conclude that both in vivo and in culture keratinocytes the impaired differentiation that is associated with the presence of HPV is correlated with reduced integrin expression.

Cell adhesion. Fibronectin and integrin knockouts come unstuck.

Mice with homozygous null mutations in the genes encoding fibronectin or the alpha5 integrin subunit die as embryos. The types of embryonic defect confirm some ideas about fibronectin function but cast doubt on others.

Evidence that cadherins play a role in the downregulation of integrin expression that occurs during keratinocyte terminal differentiation.

In epidermis the onset of terminal differentiation normally coincides with inhibition of integrin function and expression, thereby ensuring that differentiating cells are selectively expelled from the basal layer. However, when stratification of cultured human epidermal keratinocytes is prevented by reducing the calcium concentration of the medium to 0.1 mM, keratinocytes initiate terminal differentiation while still attached to the culture substrate. We have examined the mechanism by which differentiating keratinocytes adhere to extracellular matrix proteins in low calcium medium and the consequences of inducing stratification by raising the calcium ion concentration to 1.8 mM (Standard Medium). In low calcium medium keratinocytes co-expressed integrins and terminal differentiation markers such as involucrin and peanut lectin-binding glycoproteins: differentiating cells contained integrin mRNA, synthesized integrin proteins de novo and expressed functional mature integrins. There were no differences in integrin synthesis, maturation or break down in low calcium or standard medium, although the level of beta 1 integrins on the surface of proliferating cells was higher in standard medium. Within 6 h of transfer from low calcium to standard medium integrin mRNA was no longer detectable in terminally differentiating cells, integrins were being lost from the cell surface, and selective migration out of the basal layer had begun. Antibodies to P- and E-cadherin, which block calcium-induced stratification, prevented the selective loss of integrin mRNA and protein from terminally differentiating cells. This suggests that cadherins may play a role in the down-regulation of integrin expression that is associated with terminal differentiation.

Detection of mature malaria infections in live mosquitoes.

A method has been developed which detects malaria parasites in the salivary glands of live Anopheles stephensi. The method exploits the sugar feeding behaviour of the mosquito and requires only routine Western blotting techniques on nitrocellulose membrane (NCM). Infectivity can be determined without any direct manipulation of individual mosquitoes. Female A. stephensi were infected with the rodent malaria parasite, Plasmodium berghei, and after 14-16 d were starved of fructose overnight (12-18 h), then resupplied with fructose presented through a small piece of NCM. Mosquitoes were allowed to probe the membrane for several hours; the NCM was then removed and subjected to a standard immunoblotting protocol using an anti-P. berghei circumsporozoite protein (CSP) monoclonal antibody as the primary reagent, and a horseradish peroxidase-coupled secondary antibody. NCMs taken from cages containing infected mosquitoes showed a variable number of small black dots where individual females had probed and deposited either CSP or sporozoites. Infectivity could be detected easily from 13-14 d after feeding, and in as few as 10 mosquitoes at 19 d after infection; in one instance, infection in a single mosquito was clearly determined. After blocking with goat serum, the NCMs could be stored for 3-4 months and still provided positive reactions, offering some potential for applicability to field research studies.

A liver-stage specific antigen of P. berghei identified by a monoclonal antibody.

Stage-specific immunity (to the sporozoite, the asexual blood-stages and the sexual stages of malaria) has been well documented and antigens from each stage are being tested for their potential as vaccine candidates. Recently it has become clear that the liver stage can also be the target of protective immune responses; however, only the circumsporozoite protein has been identified as a protective liver antigen. It is critical for vaccine evaluation and development to identify other liver antigens and assess their potential role in immunity. In this paper we describe a monoclonal antibody, which recognizes a liver-specific antigen of Plasmodium berghei (referred to as Pbl1). Passive immunization studies using this antibody suggest that it may influence the course of sporozoite-induced infections.

Contrasts in antigen expression in the erythrocytic and exoerythrocytic stages of rodent malaria.

The time and site of expression of five antigens, recognized by monoclonal antibodies raised against blood-stage parasites, were studied in the exoerythrocytic stage of Plasmodium berghei using indirect immunofluorescent antibody staining. Two monoclonal antibodies (W 3.5, I 2.6), which stain the cytoplasm of infected erythrocytes, did not stain the cytoplasm of the infected liver cell but stained the parasite itself suggesting a difference in the antigenic architecture of the erythrocytic and exoerythrocytic parasites. Another antibody (17.6.1) revealed a further difference in the antigenic composition of the blood and liver-stage parasites being expressed almost exclusively in the former. Two others (C139 and 17.3.9) showed broadly similar patterns of expression in these two stages of the malarial life-cycle.