Lectin binding studies on C-28/I2 and T/C-28a2 chondrocytes provide a basis for new tissue engineering and drug delivery perspectives in cartilage research.

The present study was performed to evaluate the applicability of plant lectins as mediators of bioadhesion in cartilage research using human chondrocyte cell lines C-28/I2 and T/C-28a2. The bioadhesive properties of fluorescein-labelled lectins with different carbohydrate specificities were investigated by flow cytometry. Specificity of the lectin-cell interactions was ascertained by competitive inhibition using complementary carbohydrates. As compared to that of other lectins, the interaction between wheat germ agglutinin (WGA) and chondrocytic cells was characterised by remarkable cytoadhesion, adequate binding strength and a high degree of specificity for N-acetyl-glucosamine as contained in hyaluronan chains. We therefore suggest WGA to be a promising candidate for mediating bioadhesion to low-adhesive scaffolds in cartilage tissue engineering. Moreover, the WGA-association rate of C-28/I2 and T/C-28a2 cells was dependent on temperature indicating cellular uptake of membrane-bound WGA. Intracellular enrichment was confirmed by confocal microscopy. Equilibration of intracellular pH gradients with monensin resulted in the reversal of quenching effects indicating accumulation of WGA within acid compartments of chondrocytic cells. Thus, WGA might be internalised into chondrocytes together with hyaluronan via the CD44 receptor-mediated endocytosis pathway and accumulated within lysosomes. This physiological process could represent a feasible pathway to target WGA-functionalised drug delivery devices into chondrocytes.

Effects of phosphatidylinositol-3-kinase inhibitors on degranulation and gene induction in allergically triggered mouse mast cells.

BACKGROUND: Phosphatidylinositol-3-kinase (PI3-kinase) comprises an essential component in a number of signaling cascades, primarily of the growth factor type. Two specific inhibitors, wortmannin and demethoxyviridin (DMV), are widely used to block signaling via this molecule and link certain receptors to the PI3-kinase pathway. METHODS: We have studied the extent of involvement of PI3-kinase in signaling events by Fc epsilonRI in mast cells using a mouse mast cell line as a model system. This was done using beta-hexosaminidase release assays, a leukotriene ELISA, transient transfections with reporter gene constructs of TNF alpha and MARC, and in addition a TNF alpha ELISA. RESULTS: Consistent with previously published data in the rat basophilic cell line RBL-2H3, we find that wortmannin as well as DMV prevent the degranulation reaction in the mouse mast cell line CPII. DMV also inhibits the release of leukotrienes, leading to the conclusion that Fc epsilonRI activates PI3-kinase which then mediates these reactions. On the contrary, however, lymphokine and chemokine induction at the gene and protein level is not inhibited, suggesting that the activation of this gene set in mast cells is independent of PI3-kinase. CONCLUSION: PI3-kinase is activated in our mast cell model system via cross-linking of the Fc epsilonRI. This reaction is clearly necessary for the degranulation process and the release of leukotrienes. Activation of lymphokine and chemokine genes as well as secretion of their gene products are not triggered along the PI3-kinase signaling pathway. This is in agreement with our previous findings, showing that the MAP kinase pathway and Ca2+ influx are both involved in gene activation in this cell type.

Induction of the TNF-alpha promoter in the murine dendritic cell line 18 and the murine mast cell line CPII is differently regulated.

While it was recently shown that activation of dendritic cells (DC) results in the production of a number of cytokines, the signal pathways and transcription factors involved in this process have not been described. To address this issue we compared the events resulting in the activation of the human TNF-alpha promoter occurring in the fetal dendritic cell line 18 (DC18) with those in the well-characterized murine mast cell line CPII. As stimuli we employed the protein kinase C inducer, PMA, and the Ca2+ ionophore, ionomycin, both of which are known to activate a large variety of intracellular signaling pathways. In the DC18 cells, PMA alone induces the TNF-alpha promoter in a macrolide-insensitive manner. In contrast, in the mast cell line CPII, both stimuli (PMA plus ionomycin) are necessary for promoter activation which, in addition, is sensitive to immunosuppressive drugs. Mapping of the TNF-alpha promoter showed that in both cell types the so-called kappa factor binding site is the crucial promoter element for the induction. We show that in DC18 cells, this sequence is bound to and controlled by NF-kappaB proteins p50 (NF-kappaB1) and p65 (ReIA), whereas in CPII mast cells, NF-AT and AN factors are the predominant proteins that bind to and control the kappaB element of the TNF-alpha promoter. These and further experimental data indicate that in DC, NF-kappaB factors play a predominant role in the activation of the TNF-alpha promoter and, possibly, of other cytokine promoters.

The murine homolog of TB2/DP1, a gene of the familial adenomatous polyposis (FAP) locus.

The cDNA of the murine counterpart of the human TB2/DP1 (deleted in polyposis) gene, one of the six genes deleted in severe cases of familial adenomatous polyposis (FAP) disease, was isolated and analyzed. The murine transcript is 734-bp long and thereby considerably shorter than the 3100-bp human counterpart. This is due to a completely different 3' untranslated region in mouse which starts immediately after the translational stop codon, thereby deleting a RFLP (restriction-fragment length polymorphism) marker for this disease. The amino acid sequence, however, is 92% conserved between mouse and man. Triggering of murine mast cells by IgE plus antigen results in a decrease of TB2/DP1 mRNA up to 60% after 2 h implying a possible role of this gene in regulation of the allergic effector cell. Reverse transcription-polymerase chain reaction (RT-PCR) analysis shows an ubiquitous expression pattern in a number of mouse cell lines and tissues.

p21ras links Fc epsilon RI to NF-AT family member in mast cells. The AP3-like factor in this cell type is an NF-AT family member.

Very recently, an AP3-like transcription factor regulating the chemokine gene MARC and an NF-AT family member regulating IL-5 were the first components of the transcription factor repertoire to be described as activated in mast cells after an allergic triggering. In this study, we show that with respect to cross-competition in a gel shift analysis using an NF-AT consensus oligonucleotide binding site, the antigenicity to a recently generated serum against T cell NF-AT, and the sensitivity to macrolide immunosuppressants, the AP3-like activity on the MARC promoter is indistinguishable from that described for NF-AT in T cells. Additionally, we show that this factor functions on the MARC chemokine promoter without the AP1 cofactor, a situation reminiscent of the function of NF-AT in Th2-type T cells. In all of these aspects, and strengthened further by a gel shift competition analysis, the AP3-like transcription factor is identical to the NF-AT family member recently described by an analogous set of experiments as regulating IL-5 in mast cells. Our finding that p21ras, but probably not protein kinase C, is necessary to activate this factor after Fc epsilon RI triggering indicates a situation in which a common transcription factor denominator in mast cells induces chemokine (MARC) and lymphokine (IL-5) gene expression in a manner closely similar to Th2-type T cells, which are induced along the ras/raf signal pathway.

A nuclear factor of activated T cell-like transcription factor in mast cells is involved in IL-5 gene regulation after IgE plus antigen stimulation.

IL-5, which is produced mainly by activated T cells and allergically triggered mast cells, is a major survival and differentiation factor for eosinophils, and therefore, is of relevance to diseases associated with this type of cell infiltration, most importantly asthma. In this study, we have examined the transcriptional regulation of human IL-5 in a mouse mast cell line, CPII, stimulated with IgE and Ag. We report that an inducible activity in the region between -177 and -80, and a constitutive activity between -80 and -70, in the promoter of the human gene, are both necessary for the allergically triggered activation. A computer-assisted search for transcription factor binding motifs revealed a nuclear factor of activated T cell (NF-AT) and a GATA consensus site in the two regions. Corresponding binding activities were detected to be present in nuclear extracts from the mouse mast cell line by defined NF-AT and GATA binding sites as probes for a gel shift analysis. Competition analysis, in combination with probes from the human IL-5 promoter, confirmed that these factors indeed bind to the consensus sequences identified by computer analysis. An oligonucleotide spanning the IL-5 NF-AT consensus site is shown to confer allergic stimulation to a basal IL-5 promoter only in conjunction with the GATA site downstream, indicating that an inducible NF-AT-like factor cooperates with a constitutive member of the GATA transcription factor family in mediating the allergic stimulation of the human IL-5 gene.