Quantitative Image Analysis of Epithelial and Stromal Area in Histological Sections of Colorectal Cancer: An Emerging Diagnostic Tool.

In colorectal cancer (CRC), an increase in the stromal (S) area with the reduction of the epithelial (E) parts has been suggested as an indication of tumor progression. Therefore, an automated image method capable of discriminating E and S areas would allow an improved diagnosis. Immunofluorescence staining was performed on paraffin-embedded sections from colorectal tumors (16 samples from patients with liver metastasis and 18 without). Noncancerous tumor adjacent mucosa (n = 5) and normal mucosa (n = 4) were taken as controls. Epithelial cells were identified by an anti-keratin 8 (K8) antibody. Large tissue areas (5-63 mm(2)/slide) including tumor center, tumor front, and adjacent mucosa were scanned using an automated microscopy system (TissueFAXS). With our newly developed algorithms, we showed that there is more K8-immunoreactive E in the tumor center than in tumor adjacent and normal mucosa. Comparing patients with and without metastasis, the E/S ratio decreased by 20% in the tumor center and by 40% at tumor front in metastatic samples. The reduction of E might be due to a more aggressive phenotype in metastasis patients. The novel software allowed a detailed morphometric analysis of cancer tissue compartments as tools for objective quantitative measurements, reduced analysis time, and increased reproducibility of the data.

Functional expression of the human neonatal Fc-receptor, hFcRn, in isolated cultured human syncytiotrophoblasts.

Materno-fetal IgG transfer in the mature human placenta involves transport across the syncytiotrophoblast (STB) and fetal endothelial cell layer. The MHC class I-related Fc gamma-receptor (hFcRn) localized in STB as well as in endothelial cells is involved in overall IgG transfer from the maternal into the fetal circulation. Functional hFcRn is a heterodimer of a transmembrane alpha-chain and beta2-microglobulin. To establish the basis for future studies to unravel the mechanism of IgG transport in STB, we investigated hFcRn alpha-chain and beta2-microglobulin expression in cytotrophoblasts (CTB) isolated from human term placentae and cultured in vitro under conditions where differentiation into multinuclear STB takes place (>or=48 h). Northern blot analysis demonstrated up-regulation of alpha-chain mRNA after 48 h of in vitro cultivation. Likewise, hFcRn alpha-chain and beta2-microglobulin were at the limit of detection by immunofluorescence microscopy in CTB immediately after isolation, but their expression increased upon STB formation. hFcRn alpha-chain co-localized with beta2-microglobulin in multinuclear STB and formed a functional, i.e. low pH IgG binding, receptor as shown by affinity isolation. The in vitro differentiated STB exhibited specific, low pH-dependent IgG binding to the plasma membrane. In conclusion, these cultures can now be applied to study the role of hFcRn in IgG transport and trafficking in STB cultures in vitro.

Efficient apical IgG recycling and apical-to-basolateral transcytosis in polarized BeWo cells overexpressing hFcRn.

The human neonatal Fcgamma-receptor (hFcRn) involved in overall maternal-fetal IgG transmission is expressed in placental villous syncytiotrophoblast. However, the role of hFcRn in IgG transport and trafficking across this cell layer is poorly characterized. To gain insight into this mechanism we have overexpressed functional hFcRn in trophoblast-derived BeWo cells (BeWo/hFcRn cells) [Ellinger I, Reischer H, Lehner C, Leitner K, Hunziker W, Fuchs R. Placenta 2005;26:171-82] since parental BeWo cells endogenously express low levels of the receptor. We now demonstrate that hFcRn overexpression differentially affected apical-to-basolateral transcytosis and apical recycling: whereas IgG transcytosis was reduced by 35%, IgG recycling was stimulated by 100% as compared to parental cells, indicating that hFcRn plays a role in both processes. The endosomal compartments involved in hFcRn/IgG transport after apical IgG internalization were then analyzed by confocal immunofluorescence microscopy using compartment specific markers. hFcRn/IgG were found in apical early endosomes, in transferrin recycling compartments and in vesicles near the basolateral plasma membrane, presumably transcytotic vesicles. Neither hFcRn nor IgG was routed to the degradative pathway to lysosomes. These transport and localization data are in accordance with efficient hFcRn-mediated apical IgG recycling and basolateral directed IgG transcytosis in placental trophoblasts.

Overexpression of the human neonatal Fc-receptor alpha-chain in trophoblast-derived BeWo cells increases cellular retention of beta2-microglobulin.

Major histocompatibility complex (MHC) class I and MHC class I-type molecules such as the neonatal Fcgamma-receptor, FcRn, are heterodimers consisting of a transmembrane alpha-chain non-covalently associated with beta2-microglobulin (beta2m). Human placental villous syncytiotrophoblast (STB) lacks MHC class I molecules, but express hFcRn that mediates materno-fetal transmission of immunoglobulin G (IgG). Trophoblast-derived BeWo cells that are used to study placental IgG transport likewise express beta2m and low levels of hFcRn alpha-chain. The contribution of FcRn alpha-chain in retention and subcellular distribution of beta2m in STB and BeWo cells is unclear. To investigate this issue, we increased expression of hFcRn alpha-chain in BeWo cells (BeWo/hFcRn) by cDNA transfection. Overexpressed hFcRn protein exhibited the characteristic pH-dependent IgG binding and association with beta2m. In comparison to parental BeWo cells, beta2m mRNA levels in BeWo/hFcRn cells were not significantly altered, but total cell-associated beta2m protein was increased by 120%. Treatment of BeWo and BeWo/hFcRn cells with brefeldin A, an inhibitor of the secretory pathway, abrogated this effect, demonstrating that hFcRn alpha-chain expression retained otherwise secreted beta2m. Flow cytometry revealed that beta2m plasma membrane expression was unaffected by alpha-chain overexpression whereas by fluorescence microscopy a preferential staining of beta2m in peripheral endosomes was observed.

Apical to basolateral transcytosis and apical recycling of immunoglobulin G in trophoblast-derived BeWo cells: effects of low temperature, nocodazole, and cytochalasin D.

The murine neonatal Fc receptor, FcRn, carries out two functions: materno-fetal IgG delivery and maintenance of serum IgG homeostasis. During human pregnancy maternal IgG is transferred across placental syncytiotrophoblasts presumably by the human homolog of FcRn, hFcRn. Trophoblast-derived BeWo cells express hFcRn endogenously and can be considered as a model system to investigate IgG transport in syncytiotrophoblasts. Using a pulse-chase protocol, we here demonstrate that polarized BeWo cells exhibit not only apical to basolateral transcytosis but also apical IgG recycling. Thus, for the first time we demonstrate that epithelial cells can be involved in both materno-fetal IgG transmission and regulation of serum IgG levels. Lowering the temperature from 37 to 16 degrees C reduced, but did not block, IgG recycling and transcytosis. Microtubule-disruption by nocodazole did not influence transcytosis or apical recycling. Disassembly of filamentous actin by cytochalasin D stimulated apical endocytosis and recycling, while transcytosis remained unaffected. In summary, in BeWo cells apically internalized IgG enters both a transcytotic and recycling pathway. While the transcytotic route is temperature-sensitive but independent from microtubules and actin filaments, the apical recycling pathway is temperature-influenced and stimulated by actin disassembly, suggestive for the involvement of distinct endosome subcompartments in transcytosis and recycling.

Placental alkaline phosphatase expression at the apical and basal plasma membrane in term villous trophoblasts.

Human placental alkaline phosphatase (PLAP) was localized at the apical and basal plasma membrane of syncytiotrophoblasts and at the surface of cytotrophoblasts in term chorionic villi using immunoelectron microscopy. Similarly, apical and basolateral PLAP expression was found in polarized trophoblast-derived BeWo cells. Trophoblasts isolated from term placentas exhibited mainly vesicular PLAP immunofluorescence staining immediately after isolation. After in vitro differentiation into syncytia, PLAP plasma membrane expression was upregulated and exceeded that observed in mononuclear trophoblasts. These data call for caution in using PLAP as a morphological marker to differentiate syncytiotrophoblasts from cytotrophoblasts or as a marker enzyme for placental brush-border membranes. (J Histochem Cytochem 49:1155-1164, 2001)

Intracellular traffic of the MHC class I-like IgG Fc receptor, FcRn, expressed in epithelial MDCK cells.

Transfer of passive immunity from mother to the fetus or newborn involves the transport of IgG across several epithelia. Depending on the species, IgG is transported prenatally across the placenta and yolk sac or is absorbed from colostrum and milk by the small intestine of the suckling newborn. In both cases apical to basolateral transepithelial transport of IgG is thought to be mediated by FcRn, an IgG Fc receptor with homology to MHC class I antigens. We have now expressed the human FcRn in polarized MDCK cells and analyzed the intracellular routing of the receptor. FcRn showed a predominant intracellular localization at steady state. Newly synthesized FcRn was delivered in a non-vectorial fashion to both the apical and basolateral surfaces of MDCK cell monolayers. Following internalization from the apical or basolateral domain, the receptor transcytosed to the opposite surface. These findings provide direct evidence for the transepithelial transport function of FcRn and indicate that the receptor undergoes multiple rounds of transcytosis.

IgG transport across trophoblast-derived BeWo cells: a model system to study IgG transport in the placenta.

In primates, prenatal transfer of IgG from mother to offspring occurs predominantly across the placenta. Although a number of Fcgamma-receptors and IgG binding proteins have been detected in human placental tissue, an involvement of any of these receptors in IgG transport across the syncytiotrophoblast remains to be demonstrated. Therefore, we investigated the mechanism of IgG transcytosis in trophoblast-derived BeWo cells. BeWo cells were not only found to express the MHC class I-related IgG Fc receptor, human FcRn, but also specifically bound fluorescein isothiocyanate (FITC)-labeled human IgG (FITC-hIgG) at the apical surface at mildly acidic pH. The cells preferentially transcytosed FITC-hIgG from the apical to the basolateral side when compared to the fluid-phase marker FITC-dextran and to FITC-hIgG transcytosis in the opposite direction. However, endocytosis of FITC-hIgG at the apical plasma membrane at physiological pH required the continuous presence of FITC-hIgG at concentrations similar to those present in the maternal circulation. These results suggest a mechanism by which IgG is internalized by BeWo cells via fluid-phase endocytosis. Tight binding of IgG to hFcRn may then occur in acidic endosomes, followed by selective sorting into the transcytotic pathway. Thus, the main function of this receptor is to prevent entry of IgG into the degradative pathway in lysosomes.

In vitro fusion of tissue-derived endosomes and lysosomes.

We investigated the in vitro fusion of different endocytic compartments derived from perfused rat liver, where the cells are assumed to be in their physiological state. Specifically labelled early, late and transcytotic endosomes, as well as lysosomes were tested for their fusion properties. In addition to the expected ATP-dependent fusion between early endosomes, we observed fusion between early and late endosomes with similar efficiency, kinetics and cytosol dependence. Fusion between early endosomes and transcytotic vesicles could not be detected. Prolonged incubation of complementary labelled early endosomes under fusion-supporting conditions followed by Percoll gradient centrifugation revealed the occurrence of fusion product at a dense position, indicating fusion events between light and dense compartments. Incubation of membrane preparations containing avidin-labelled endosomes and biotin-dextran-loaded lysosomes resulted in the formation of avidin-biotin complexes, indicating that fusion between early and late endosomes is followed by fusion with lysosomes. This was verified by colocalization of fluorescently labelled endosomes and lysosomes, as assessed by laser scanning microscopy. Endosome fusion, as well as content mixing between endosomes and lysosomes, were dependent on temperature and ATP, and could be inhibited by N-ethylmaleimide (NEM). The NEM-sensitivity was localised on endosomes and in the cytosol, but not on lysosomes. These observations indicate that early and late endosomes of rat liver exhibit a high fusion competence in vitro, promoting not only homotypic, but also heterotypic fusion.

Fluid-phase marker transport in rat liver: free-flow electrophoresis separates distinct endosome subpopulations.

Free-flow electrophoresis (FFE) was used to investigate the intracellular compartments involved in fluid-phase marker, fluoresceine isothiocyanate (FITC)-dextran, transport in the isolated perfused rat liver. One to 2 min after uptake at 37 degrees C, FITC-dextran was found in endosomes with the same electrophoretic mobility as early sorting endosomes labeled either by the hepatocyte-specific marker asialoorosomucoid (ASOR) or by transferrin that enters all liver cells. Labeling at low temperature (16 degrees C) blocked transport of ASOR and dextran in early endosomes. With increasing internalization time (3-13 min) at 37 degrees C, FITC-dextran-labeled compartments co-localized with late, ASOR-containing endosomes. Since localization of FITC-dextran in late transcytotic compartments was not observed upon FFE separation, it is concluded that the majority of internalized markers is directed to lysosomes. The FITC-label did not account for the predominant lysosomal targeting of the dextran, since [3H]dextran-labeled endosomes exhibited an identical FFE pattern. Taken together, these data indicate that the fluid-phase marker dextran is transported through intracellular compartments with identical characteristics as endosome subcompartments of the receptor-mediated lysosomal route.