EnVision+, a new dextran polymer-based signal enhancement technique for in situ hybridization (ISH).

Seventy paraffin-embedded cervical biopsy specimens and condylomata were tested for the presence of human papillomavirus (HPV) by conventional in situ hybridization (ISH) and ISH with subsequent signal amplification. Signal amplification was performed either by a commercial biotinyl-tyramide-based detection system [GenPoint (GP)] or by the novel two-layer dextran polymer visualization system EnVision+ (EV), in which both EV-horseradish peroxidase (EV-HRP) and EV-alkaline phosphatase (EV-AP) were applied. We could demonstrate for the first time, that EV in combination with preceding ISH results in a considerable increase in signal intensity and sensitivity without loss of specificity compared to conventional ISH. Compared to GP, EV revealed a somewhat lower sensitivity, as measured by determination of the integrated optical density (IOD) of the positively stained cells. However, EV is easier to perform, requires a shorter assay time, and does not raise the background problems that may be encountered with biotinyl-tyramide-based amplification systems. (J Histochem Cytochem 49:1067-1071, 2001)

Surface-expressed invariant chain (CD74) is required for internalization of human leucocyte antigen-DR molecules to early endosomal compartments.

Transport of major histocompatibility complex (MHC) class II molecules to the endocytic route is directed by the associated invariant chain (Ii). In the endocytic pathway, Ii is proteolytically cleaved and, upon removal of residual Ii fragments, class II alpha beta dimers are charged with antigenic peptide and recognized by CD4+ T cells. Although distinct peptide-loading compartments such as MIIC (MHC class II loading compartment) and CIIV (MHC class II vesicles) have been characterized in different cells, there is growing evidence of a multitude of subcellular compartments in which antigenic peptide loading takes place. We employed a physiological cellular system in which surface Ii (CD74) and surface human leucocyte antigen (HLA)-DR were induced either alone or in combination. This was achieved by transient exposure of HT-29 cells to recombinant interferon-gamma (rIFN-gamma). Using distinct cellular variants, we showed that: (i) the majority of Ii molecules physically associate on the cell membrane with class II dimers to form DR alpha beta:Ii complexes; (ii) the presence of surface Ii is a prerequisite for the rapid uptake of HLA-DR-specific monoclonal antibodies into early endosomes because only the surface DR+/Ii+ phenotype, and not the DR+/Ii- variant, efficiently internalizes; and (iii) the HLA-DR:Ii complexes are targeted to early endosomes, as indicated by co-localization with the GTPase, Rab5, and endocytosed bovine serum albumin. Internalization of HLA-DR:Ii complexes, accommodation of peptides by DR alphabeta heterodimers in early endosomes and recycling to the cell surface may be a mechanism used to increase the peptide repertoire that antigen-presenting cells display to MHC class II-restricted T cells.

Early detachment of colon carcinoma cells during CD95(APO-1/Fas)-mediated apoptosis. I. De-adhesion from hyaluronate by shedding of CD44.

Ligation of CD95 (APO-1/Fas) cell surface receptors induces death in apoptosis-sensitive cells. Induction of apoptosis in adherent gamma interferon-stimulated HT-29 and COLO 205 colon carcinoma cells by cross-linking CD95 with anti-APO-1 monoclonal antibody resulted in detachment of the cells from hyaluronate starting about 1 h after antibody exposure. Loss of adhesion was paralleled by a substantial reduction of the multifunctional cell surface adhesion molecule CD44. As evidenced by cycloheximide treatment, this effect was not caused by impaired protein synthesis. Depletion of surface CD44 was also not due to membrane blebbing, since cytochalasin B failed to inhibit ascension from hyaluronate. Instead, ELISA and time kinetics showed increasing amounts of soluble CD44 in the supernatant of CD95-triggered cells. SDS-PAGE revealed that soluble CD44 had an apparent molecular mass of about 20 kD less than CD44 immunoprecipitated from intact cells. Thus, CD95-triggering induced shedding of CD44. Shedding is a novel mechanism operative in early steps of CD95-mediated apoptosis. Shedding surface molecules like CD44 might contribute to the active disintegration of dying epithelial cells in vivo.

Surface expression of the invariant chain (CD74) is independent of concomitant expression of major histocompatibility complex class II antigens.

Whether or not intracellular transport and surface expression of the invariant chain (Ii; CD74) occurs independent of the presence of major histocompatibility complex (MHC) class II molecules was examined by comparing the class II-negative mutant lymphoblastoid cell line 174 x CEM.T2 (T2) and its class II-positive parental cell line 174 x CEM.T1 (T1). We found a similar proportion of Ii being transported to the Golgi complex in T1 and T2, as monitored by the degree of sialic acid addition to glycan side chains of Ii. In agreement with this result, T1 and T2 expressed comparable amounts of Ii at the cell surface, as measured by flow cytometry. This indicates that, although not associated with class II molecules, a proportion of Ii is transported to the plasma membrane. Both in T1 and T2, surface Ii (sIi) was rapidly internalized with a half-life of 3-4 min, suggesting that some Ii enters the endocytic route via the cell surface after being internalized. Our data demonstrate transport of Ii on a route alternative to the endocytic pathway. This alternative route could also account for delivery of newly synthesized class II-Ii complexes to processing compartments in antigen-presenting cells.

Expression of APO-1 (CD95), a member of the NGF/TNF receptor superfamily, in normal and neoplastic colon epithelium.

APO-1 is a 48-kDa cell-membrane protein identical to the Fas antigen now designated CD95. It is a member of the NGF/TNF receptor superfamily. Anti-APO-1 monoclonal antibody induces apoptosis in a variety of cell types expressing this antigen. We immunohistochemically investigated APO-1 expression in normal colon mucosa, 20 adenomas, 258 colon carcinomas and 10 liver metastases and carried out in vitro studies using a panel of colon-carcinoma cell lines. Immunohistochemically, APO-1 was regularly expressed at the basolateral membrane of normal colon epithelia. In a minor fraction of colon adenomas and in 39.1% of colon carcinomas APO-1 expression was diminished and in 48.1% of carcinomas, predominantly of the non-mucinous type, APO-1 expression was completely abrogated. The normal level of APO-1 in carcinomas was correlated with the mucinous type. Reduced/lost APO-1 expression was more frequent in rectal carcinomas. Complete loss of APO-1 was more frequent in tumors that had already metastasized. APO-1 expression in liver metastases essentially corresponded to that of the primary tumors. Comparative analysis with data from previous studies revealed that the mode of APO-1 expression is correlated with that of HLA-A,B,C./beta 2m, HLA-DR, HLA-D-associated invariant chain and of the secretory component. Surface expression of APO-1 was heterogeneous in colon-carcinoma cell lines; SW480 expressed considerable amounts of APO-1 on all cells, while HT-29 constitutively did less so and only in a minority of cells. Surface density of APO-1 and the fraction of positive cells in HT-29 was enhanced by interferon-gamma (IFN-gamma) and, additively, by tumor necrosis factor-alpha (TNF-alpha), whereas in SW480 APO-1 expression was not modulated by these cytokines. We conclude that neoplastic transformation of colon epithelium often leads to a loss of the physiologic, high level of surface APO-1 by giving rise either to a stable lack of APO-1 or to an IFN-gamma/TNF-alpha-sensitive phenotype of inducible APO-1 expression.

Comparative evaluation of integrin alpha- and beta-chain expression in colorectal carcinoma cell lines and in their tumours of origin.

The integrin family consists of broadly expressed cell surface adhesion receptors, each member of which is composed of a non-covalently linked alpha/beta heterodimer. Integrin receptors are involved in the interaction with matrix proteins and may contribute to invasion and metastasis of carcinomas. To examine the biological role integrins play in colorectal carcinoma we compared the expression of integrin alpha- and beta-subunits in situ and in vitro. Eight newly established cell lines derived from immunohistochemically characterized colorectal carcinomas together with two sublines obtained after nude mouse passage and the commonly used colon carcinoma lines HT-29, SW480, SW620, and COLO 205 were investigated by immunocytochemistry and flow cytometry. The carcinomas in situ expressed alpha 1-, alpha 2-, alpha 3-, alpha 6-, alpha v- and beta 1-subunits in variable amounts while being devoid of alpha 4, alpha 5, and beta 3. The individual integrin profile of the tumour in tissue was essentially maintained in vitro. However, a neo expression of the alpha 5 chain was found, together with an induction or increase in alpha 1, alpha 2, alpha 3, alpha v and beta 1 levels. No decrease in integrin subunit expression was observed. Standard-serum and serum-free medium revealed no striking differences in alpha- and beta-chain expression in the cell lines HT-29 and COLO 205. In serum-free medium, SW480 showed a slight increase of alpha 1 and alpha 5 and a decrease of alpha 3 and alpha v while SW620 expressed more alpha 1. We conclude that the great variability of adhesion receptor expression of the integrin family in colorectal carcinomas in situ is essentially maintained in vitro, although culture conditions which are only marginally influenced by serum factors unpredictably lead to some increase in expression or even induction of several integrin subunits.

Synoviocytes in chronic synovitis in situ and cytokine stimulated synovial cells in vitro neo-express alpha 1, alpha 3 and alpha 5 chains of beta 1 integrins.

The expression of the beta 1 integrins was examined immunohistochemically in synoviocytes from normal synovitis membrane and from chronic synovitis of different aetiology and intensity. Normal synoviocytes were alpha 6 beta 1-positive but lacked alpha 1 through alpha 5. In mild inflammation type A synoviocytes neo-expressed alpha 1, alpha 3, and alpha 5 chains. In severe inflammation both type A and B synoviocytes expressed alpha 3, alpha 4, alpha 5, and alpha 6 chains. The effects of inflammatory cytokines, as single agents or in combination, on the beta 1 integrin expression in cultured normal synoviocytes was determined by immunocytochemistry and flow cytometry. The alpha 1 chain, while absent in unstimulated synoviocytes, was induced by interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma). This effect was enhanced by combining IL-1 beta and TNF-alpha. Expression of the alpha 3 chain was up-regulated by IL-1 beta and, more intensely, by IFN-gamma. Transforming growth factor beta (TGF-beta) inhibited the up-regulating effect of IL-1 beta and antagonized the effect of IFN-gamma on alpha 3 chain expression. Expression of the alpha 5 chain was up-regulated significantly by co-stimulation through IL-1 beta together with TGF-beta or TNF-alpha. Thus, the beta 1 integrin profile of cytokine activated synoviocytes in vitro resembled that of synoviocytes in synovitis in situ. These data suggest that IL-1 beta, TNF-alpha, IFN-gamma, and TGF-beta are likely to be among the effectors regulating beta 1 integrin expression in synoviocytes in vivo.

PMA-activation of peripheral blood and tonsillar B lymphocytes induces large adhesive cells reminiscent of large extrafollicular (monocytoid) B cells.

Extrafollicular (EF) B lymphocytes differ in size and morphology depending on the lymphatic organ involved and the kind of inflammatory reaction. On re-evaluating EF B cells in various sites and conditions we discriminated three forms: a small (lymphoid) and intermediate (centrocytoid), and a large (monocytoid) variant. Immunohistochemically, these variants could be discriminated by their differential expression of adhesion molecules CD62L (L-selectin) and CD11c: small EF B cells were strongly L-selectin+ and CD11c-; intermediate cells were moderately CD62L+ and CD11c-; large cells were faintly CD62L+ or - but expressed CD11c. In 72 h cultures of normal peripheral and tonsillar B cells, cross-linking surface immunoglobulin in the presence of interleukin-2 or interleukin-4 led to formation of clusters in vitro together with an increase in cell size and a slight up-regulation of CD11c, as determined by flow cytometry. Stimulation with phorbol 12-myristate 13-acetate (PMA), however, gave rise to large, plastic adherent cells which also showed strong homotypic adhesion, expressed CD62L at minimal levels and CD11c at comparably highest levels and altogether mimicked the large cell variant of EF B cells. We conclude that EF B cells are subjected to cytokine-induced metamorphosis and that differences in cell size and morphology reflect their state of activation and activation-associated adhesion properties. Our data suggest that EF B cells in all anatomical sites are functionally closely related cells which--possibly mediated by CD11c/CD18--may become sessile and proliferate locally once activated by appropriate signals.

Fibronectin and fibronectin-receptors of the integrin type in normal colon mucosa, adenoma and carcinoma.

Integrin alpha 3, alpha 4, alpha 5, and alpha v subunits heterodimerize with beta 1 and beta 3 chains to form functional fibronectin (FN) receptors. Two of them, alpha 4 beta 1 and alpha 5 beta 1, recognize FN as the only matrix molecule. Additional binding of laminin, collagen, and entactin is achieved by alpha 3 beta 1 while alpha v beta 1 and alpha v beta 3 also bind vitronectin. We investigated immunohistochemically the tissue distribution of FN and the expression of FN receptor alpha- and beta-chains in 20 normal colon tissues, 10 adenomas, 90 carcinomas, and 10 liver metastases derived thereof. In normal and adenomatous colon tissues FN was detected in association with reticular and fibrillar structures of the gut wall. The tumor stroma of carcinomas and liver metastases contained abnormally high amounts of FN which were detectable as chaotic deposits. Normal and adenomatous mucosa were alpha 3(+), alpha 4(-), alpha 5(-), alpha v(+/-), beta 1(+), beta 3(-) indicating that only promiscuous FN receptors were expressed. 21 of 90 carcinomas had a focal or complete loss of alpha 3 which was more often found in Dukes C/D tumors (p<0.005). The liver metastases more often had lower levels of alpha 3 expression compared to the primary tumor. The alpha v-expression was inconsistent and often weak. These losses of FN receptor constituents in carcinomas were not paralleled by any noticeable differences in stromal content or distribution pattern of FN, Enhanced expression/induction of FN receptors was found in only 2 of 90 carcinomas which focally neo-expressed the alpha 5-chain. This aberrant alpha 5 expression, however, was flow cytometrically found in 3/4 colon carcinoma cell lines which otherwise had receptor profiles corresponding to the in situ situation. FN-adherence of cell lines was variable. Anti-alpha 3 had no blocking effect on FN adherence of SW480, SW620, and COLO 205 but a minor effect on FN binding of HT-29. Anti-alpha 5 blocked FN adhesion whenever alpha 5 was expressed. Anti-av had no blocking effect while anti-beta 1 reduced FN adherence of all cell lines. The net biological effect(s) of aberrant expression of integrin type FN receptors in colon carcinomas is unpredictable as yet.

Expression of CD59, a complement regulator protein and a second ligand of the CD2 molecule, and CD46 in normal and neoplastic colorectal epithelium.

CD59 (protectin) and CD46 (membrane cofactor protein, MCP) are membrane-bound complement regulator proteins which inhibit complement-mediated cytolysis of autologous cells. CD59, a phosphatidyl-inositol-anchored glycoprotein, inhibits the formation of the terminal membrane attack complex (MAC) of complement and was found to be a second ligand for CD2 contributing to T-cell activation. In 20 colorectal normal mucosa samples, in ten adenomas, 71 carcinomas and in ten liver metastases derived thereof, CD59 was inconsistently expressed in the epithelial compartment. In carcinomas CD59 expression in the whole neoplastic compartment was more often found in well- and moderately differentiated tumours. By contrast, focal expression or even complete lack of CD59 was more often found in poorly differentiated tumours (P = 0.021). In addition, carcinomas without metastases at the time of operation (Dukes A/B) more often expressed CD59 in the entire neoplastic population compared to those carcinomas which had already metastasised (P = 0.018). There was no correlation between the mode of CD59 expression in colorectal carcinomas and the tumour type or location. CD46 has C3b/C4b binding and factor-I dependent cofactor activity and is broadly expressed in various cells and tissues. In the epithelial compartment of normal colorectal mucosa, of all adenomas, carcinomas and their liver metastases, CD46 was expressed throughout the epithelial compartment. Since CD46 was consistently expressed in colorectal carcinomas the low expression or even lack of CD59 in a subset of tumours might not lead to critical complement-mediated attack of CD59-negative tumour cells. Regarding CD59 as a natural T-cell ligand involved in cognate T-cell-target-cell interaction, however, loss of CD59 might well be a selection advantage, provided that tumour antigen-mediated T-cell toxicity in colorectal carcinoma exists.

Constitutive and induced expression of APO-1, a new member of the nerve growth factor/tumor necrosis factor receptor superfamily, in normal and neoplastic cells.

BACKGROUND: APO-1 is a 48 kilodalton transmembrane, cysteine-rich glycoprotein identical with the Fas antigen which belongs to the nerve growth factor/tumor necrosis factor receptor superfamily. Cross-linking of APO-1 induces apoptotic cell death in sensitive cells. EXPERIMENTAL DESIGN: As suggested by our preliminary results, APO-1 expression is not restricted to cells of the hematopoietic lineage. We therefore investigated APO-1 expression in normal human tissues and in various epithelial and nonepithelial tumors. RESULTS: We show by immunohistochemistry that APO-1 is a non-lineage antigen constitutively expressed in a variety of epithelial cells. This includes the basal layers of various squamous epithelia, transitional epithelium and columnar epithelium of the biliary tract and intestine. Among the epithelial cell types of the reproductive system of both genders, APO-1 expression is complex. Except the satellite cells of autonomic ganglia, all cells of the nervous tissue are APO-1-negative. Among mesenchymal cells, constitutive APO-1 expression is rare but detectable in various kinds of activated cells, e.g. fibroblasts, osteoblasts, and subpopulations of endothelial cells. Within the immune system, APO-1 is broadly distributed among histiocytic cells but restricted to minor subpopulations of peripheral T and B cells. Immature T cells, i.e., thymocytes, do not express detectable APO-1-antigen. Expression of APO-1 was induced in phytohemagglutinin activated T cells and in a mammary carcinoma cell line by interferon-gamma alone and in combination with tumor necrosis factor alpha. Consistently, there was an in situ induction of APO-1 in several types of glandular epithelium in microtopographic association with lymphohistiocytic infiltrates. This inflammation-associated APO-1 induction went along with increased expression of this molecule within the lymphocytic compartment of the lesion. In tumors. APO-1 expression was heterogeneous. In comparison to their normal counterparts, some tumors showed abnormal hypo-expression or loss of APO-1. However, abnormal neo-expression was also found. CONCLUSIONS: Tissue distribution, in vitro expression, and reaction upon cytokine-induced activation suggest that APO-1 might not only transmit apoptotic signals but might play a more general role in growth control.

Coregulation of the APO-1 antigen with intercellular adhesion molecule-1 (CD54) in tonsillar B cells and coordinate expression in follicular center B cells and in follicle center and mediastinal B-cell lymphomas.

APO-1 is a 48-Kd transmembrane glycoprotein identical to the Fas antigen and belongs to the nerve growth factor (NGF)/tumor necrosis factor (TNF) receptor family of surface molecules. Cross-linking of APO-1 induces apoptotic cell death in sensitive cells. We show here that APO-1 is an activation molecule on B cells. It was induced/enhanced on dense and buoyant tonsillar B cells, respectively, through surface Ig cross-linking in combination with interleukin-2 or by interferon-gamma together with tumor necrosis factor-alpha. These conditions also increased the amount of intercellular adhesion molecule-1 (CD54) on these cells. Epstein-Barr virus transformants of peripheral B cells coexpressed APO-1 and CD54 at very high levels. Immunohistologically, Apo-1 was detectable at low levels in a subpopulation of follicle center B blasts and, at higher levels, in sinusoidal B cells. APO-1 was undetectable in follicular mantle B cells and plasma cells. In isolated tonsillar B cells, APO-1 was expressed in CD10+ follicle center cells. In acute B lymphoblastic leukemia, chronic B lymphocytic leukemia, and Burkitt's lymphomas, APO-1 and CD54 molecules were immunohistochemically undetectable. Coordinate expression of these antigens was found in mediastinal B-cell lymphomas. The mode of APO-1 and CD54 expression was correlated in follicle center cell lymphomas (P < .0019), but less stringently in hairy cell leukemia. No association was found in plasmacytomas. This was in line with the differential expression of these molecules found in reactive plasma cells. Expression of APO-1 in B cells of different stages of differentiation and, correspondingly, in certain B-cell neoplasias might suggest a role of this molecule in the induction of B-cell apoptosis. This function might be influenced by CD54 and CD54-mediated signals.

Decay-accelerating factor (DAF, CD55) in normal colorectal mucosa, adenomas and carcinomas.

Decay-accelerating-factor (DAF, CD55), a phosphatidyl-inositol anchored glycoprotein, is a member of the cell membrane bound complement regulatory proteins that inhibit autologous complement cascade activation. DAF was found expressed on cells that are in close contact with serum complement proteins, but also on cells outside the vascular space and on tumour cells. Using CD55(BRIC110) and CD55(143-30) we show here that DAF(CD55) is only sporadically expressed on the luminal surface of normal colonic epithelium. However, 5/20 adenomas expressed DAF(CD55) on the cell surface of all tumour cells, 5/20 adenomas were completely negative, 10/20 adenomas expressed DAF(CD55) in various amounts. DAF(CD55) was expressed in various intensities on almost all tumour cells of the colon carcinoma cell line HT29. In 5/88 colorectal carcinomas DAF(CD55) was localised on the apical cell surface of all tumour cells, 31/88 were completely negative, 52/88 expressed DAF(CD55) in parts of their neoplastic populations. There was no correlation between the tumour grading, staging and location and the mode of DAF(CD55) expression, but DAF(CD55) was found more often in mucinous carcinomas (P = 0.007). Although the mode of DAF(CD55) expression is not correlated with tumour prognostic parameters, the upregulation of DAF(CD55) in a subset of adenomas and carcinomas needs further investigation concerning protection of tumour cells against complement cytotoxicity.

[Expression of APO-1, a cell surface molecule mediating apoptosis, during normal B cell ontogeny and in B cell tumors. Co-expression and coregulation of APO-1 and ICAM-1 (CD54) in germinal central cells]. / Expression von APO-1, eines Apoptose-vermittelnden Zelloberflächenmoleküls, während der normalen B-Zell-Ontogenese und in B-Zell-Tumoren. Koexpression und Koregulation von APO-1 und ICAM-1 (CD54) in Keimzentrumszellen.

APO-1 is a 48kDa transmembrane glycoprotein and belongs to the NGF/TNF receptor family of surface molecules. Cross-linking of APO-1 induces apoptotic cell death in sensitive cells. Here we show that APO-1 is an activation molecule on B cells. It could be induced/enhanced on dense and buoyant tonsillar B cells, respectively, through surface immunoglobulin cross-linking in combination with interleukin-2 or by interferon-gamma together with tumor necrosis factor-alpha. These conditions also increased the amount of intercellular adhesion molecule-1 (ICAM-1; CD54) on these cells. Epstein-Barr virus transformants of peripheral B cells co-expressed APO-1 and CD54 at very high levels. Immunohistologically, APO-1 was detectable at low levels in a subpopulation of follicular center B blasts and, at higher levels, in sinusoidal B cells. APO-1 was undetectable in follicular mantle B cells and plasma cells. Neoplastic B cell essentially mimicked their reactive counterpart with regard to APO-1 and CD54 expression.