Wortmannin, an inhibitor of phosphoinositide 3-kinase, inhibits transcytosis in polarized epithelial cells.

Wortmannin, an inhibitor of phosphoinositide 3-kinase, inhibits both basolateral to apical and apical to basolateral transcytosis of ricin in Fisher rat thyroid (FRT) cells by 50% at 100 nM in a continuous transcytosis assay. In MDCK cells, a similar effect of wortmannin on basolateral to apical transcytosis of ricin was found, whereas apical to basolateral transcytosis was inhibited to a lesser degree. Transcytosis of dimeric IgA in MDCK cells expressing the polymeric immunoglobulin receptor was also reduced to 50% of controls, suggesting that wortmannin inhibits membrane translocation rather than sorting of specific proteins in the transcytotic pathway. This effect of wortmannin is selective, however, in that endocytosis at the basolateral domain and recycling at both the basolateral and apical membrane domains are unaffected, and apical endocytosis and apical secretion are only moderately reduced. We have shown previously that cAMP stimulates a late stage in basolateral to apical transcytosis in MDCK cells through activation of protein kinase A (Hansen, S. H., and Casanova, J.E. (1994) J. Cell Biol. 126, 677-687). Elevation of cellular cAMP still induced a 100% increase in transcytosis in wortmannin-treated cells, but transcytosis was no longer increased when compared to cells which received no drugs. In contrast, in experiments using a 17 degrees C block to accumulate ricin internalized from the basolateral surface in the apical compartment of MDCK cells, wortmannin had little effect on the stimulation of transcytosis by activators of protein kinase A observed under these conditions. The data thus suggest the existence of a wortmannin-sensitive step in the transcytotic pathway, positioned after endocytosis but prior to translocation into the protein kinase A-sensitive apical compartment, implying a role for phosphoinositide 3-kinase in an intermediate step in transcytosis in polarized epithelial cells.

Interleukin-1 alpha- and beta-, interleukin-6- and tumour necrosis factor-alpha-like immunoreactivities in chronic granulomatous skin conditions.

Paraformaldehyde-fixed tissue of chronic granulomatous skin conditions, such as cutaneous leishmaniasis, granuloma annulare, leprosy and hidroadenitis, was investigated for the presence of interleukin-1 alpha-, interleukin-1 beta-, interleukin-6- and tumour necrosis factor-alpha-like immunoreactivities among the cellular infiltrates. There was a weak to strong cytoplasmic labelling of plasma cells for interleukin-6 and tumour necrosis factor-alpha at the periphery of the granulomatous mass and around the skin appendages. The interleukin-6-like immunoreactivity seemed to be correlated with the coarseness of the chromatin material of the cells, being more intense with coarse chromatin. The cytoplasmic labelling for interleukin-1 alpha and interleukin-1 beta in the plasma cells was less intense. Epitheloid, Langhans' giant cells and small round cells exhibited a weak to moderate cytoplasmic labelling for interleukin-1 alpha and interleukin-1 beta, whereas the staining intensity for interleukin-6 and tumour necrosis factor-alpha was weak to strong. In addition, there was staining of the stroma in the centre of granuloma with antisera against interleukin-1 alpha, interleukin-1 beta, interleukin-6 and tumour necrosis factor-alpha. This area contained few cells, suggesting that the granuloma was in a resolution process. A contribution of interleukin-6 and tumour necrosis factor-alpha to the granulomatous reaction, at least during the maintenance period, is suggested by the occurrence of these cytokines in the skin conditions studied. The findings are also consistent with a suggested role of B cells in the late stages of the granulomatous reaction. In addition, they are in line with the reported declining role of interleukin-1 in the maintenance of granuloma.

Plasminogen activator regulation by transforming growth factor-beta in normal and neoplastic human urothelium.

Plasminogen activators (PA) are thought to participate in the invasive and metastatic processes of malignancies and are known to be modulated by certain growth factors (Danø, K; Andreasen, P.A.; Grondahl-Hansen, J.; Kristensen, P.; Nielsen, L.S.; Skriver, L. Adv. Cancer Res. 44:139-266; 1985 and Laiho, M.; Keski-Oja, J. Cancer Res. 49:2533-2553; 1989). This report describes the effect of TGF-beta on the regulation of secreted PA activity produced by human fetal urothelium and neoplastic urothelial cell lines. Epidermal growth factor was previously shown to induce substantially different effects on PA production by normal versus neoplastic urothelial (Dubeau, L.; Jones, P.A.; Rideout, W.M.; Laug, W.E. Cancer Res. 48:5552-5556; 1988). This report demonstrates that log phase normal urothelium, but not transformed cells, responded to TGF-beta (1-10 ng/mL) by diminishing the total secreted PA activity. Northern and western analyses showed that the reduction in protease activity resulted from an increased level of plasminogen activator inhibitor-1 (PAI-1) mRNA and protein. Additionally, northern analysis of total mRNA levels at varying cell densities demonstrated modulation of tPA, PAI-1, and TGF-beta transcripts in normal urothelial cells as a function of growth in vitro, suggesting the presence of an intact regulatory pathway to control extracellular proteolysis.