Regulation of adhesion and migration in the germinal center microenvironment

T cell dependent humoral immune responses are initiated by the activation of naive B cells in the T cell areas of the secondary lymphoid tissues. This primary B cell activation leads to migration of germinal center (GC) cell precursors into B cell follicles where they engage follicular dendritic cells (FDC) and T cells, and differentiate into memory B cells or plasma cells. Both B cell homing to the GC and interaction with FDC critically depend on integrin-mediated adhesion. We have recently indentified the c-met-encoded receptor tyrosine kinase and its ligand, the growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF), as a novel paracrine signalling pathway regulating B cell adhesion (van der Voort et al., 1997, J. Exp. Med. 185, 2121-2131). The c-Met protein is expressed on B cells localized in the dark zone of the GC (centroblasts) and is induced by CD40 plus BCR ligation. Stimulation of c-Met with HGF/SF, which is produced at high levels by tonsillar stromal cells and FDC, leads to receptor phosphorylation and to enhanced integrin-mediated adhesion of B cells to both VCAM-1 and fibronectin. Interestingly, these responses to HGF/SF are promoted by heparan-sulfate proteoglycan forms of CD44 (CD44-HS). Like c-Met, CD44-HS is induced on B cells by CD40 ligation. It efficiently binds HGF/SF and strongly promotes signalling through c-Met. We conclude that integrin regulation during antigen specific B cell differentiation involves cross-talk between the HGF/SF-c-Met pathway and CD44-HS.     

Hepatocyte growth factor/scatter factor induces not only scattering but also cohort migration of human colorectal-adenocarcinoma cells

We presented earlier a 2-dimensional cell-motility assay using a highly metastatic variant (L-10) of human rectal-adenocarcinoma cell line RCM-1 as a motility model of tumor cells of epithelial origin. In this model, L-10 cells moved as coherent cell sheets when stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA), and we called this type of movement "cohort migration". Electron- and immunoelectron-microscope study of the migrating cell sheets demonstrated localized release from cell-cell adhesion only at the lower portion of the cells with loss of E-cadherin immunoreactivity, and this change was associated with increased tyrosine phosphorylation of the E-cadherin-catenin complex, including beta-catenin. In the present study, to obtain evidence to support the relevance of our model to carcinoma-cell movement in vivo, we sought a naturally occurring motogenic factor(s) able to induce this cohort migration. Among the factors examined, hepatocyte growth factor/scatter factor (HGF/SF) clearly induced cohort migration of L-10 cells. Additionally, not only L-10 but several other human colorectal-carcinoma cell lines showed this type of migration in response to HGF/SF, while yet others showed scattering-type motility. In this HGF/SF-induced migration, localized release from cell-cell adhesion was induced only at the lower portion of the cells, allowing them to extend leading lamellae, whereas close cell-cell contacts remained at the upper portion of the cells, as seen in TPA-induced cohort migration. Scattering-type cell lines tended to express more c-Met (receptor for HGF/SF) mRNA than the cell lines that showed cohort-type migration. LoVo, one of the scattering-type cell lines, expressed more c-Met protein and less E-cadherin than L-10, which showed cohort-type migration. HGF/SF treatment of LoVo reduced the amount of alpha-catenin complexed with E-cadherin more markedly than in L-10, but in both cell lines this reduction was not accompanied by increased tyrosine phosphorylation of beta-catenin, suggesting the presence of a mechanism other than phosphorylation for release from cell-cell adhesion during cell motility.     

Hepatocyte growth factor/scatter factor (HGF/SF) is produced by human bone marrow stromal cells and promotes proliferation, adhesion and survival of human hematopoietic progenitor cells (CD34+)

The fate of hematopoietic progenitor cells (HPCs) in the bone marrow (BM) microenvironment is determined by two different interactions: 1) they adhere (via integrins) to both extracellular matrix molecules and BM stromal cells; and 2) stromal cells produce cytokines that influence their survival, proliferation, differentiation, and mobilization. The ligands for the protein tyrosine kinase receptors c-KIT and FLT3/FLK2, stem cell factor (SCF), and FL are produced by BM stromal cells and are known to affect several facets of hematopoiesis. We studied another protein tyrosine kinase receptor, c-MET, and its ligand hepatocyte growth factor (HGF), also known as scatter factor (SF), which play a similar role in hematopoiesis. c-MET mRNA is expressed in immature human BM HPCs (CD34+CD33- or CD34+CD38-), but not in more mature HPCs (CD34+CD33+ or CD34+CD38+). The ligand HGF/SF is predominantly produced by BM stromal cells at both the mRNA and protein levels. We confirmed functionally that HGF/SF alone has no effect on proliferation of HPCs, but that when combined with granulocyte/macrophage colony-stimulating factor (GM-CSF) or interleukin-3 it acts as a synergistic proliferative factor, although not as potently as kit-ligand or FLT-3/FLK-2 ligand. Furthermore, HGF/SF promotes adhesion of HPCs to immobilized fibronectin. HGF/SF-induced adhesion to fibronectin is probably caused by activation of the integrins alpha4beta1 and alpha5beta1, insofar as we were able to block this interaction by using monoclonal blocking antibodies directed against these integrin subunits. Addition of the tyrosine-phosphorylation inhibitor genistein inhibited HGF/SF-induced adhesion, supporting the idea that HGF/SF-induced effects are the result of signaling via the receptor c-MET after ligand binding. The enhanced adhesion of HGF/SF to fibronectin proved to be beneficial for the maintenance of the colony-forming potential of HPCs. HGF/SF alone and especially in combination with fibronectin prolongs survival of GM colony-forming cells in liquid culture. Our data indicate that HGF/SF is a polyfunctional cytokine in the BM microenvironment. It is produced by human BM stromal cells and directly or indirectly promotes proliferation, adhesion, and survival of human HPCs.     

Cellular and molecular factors that regulate the differentiation and apoptosis of germinal center B cells. Anti-Ig down-regulates Fas expression of CD40 ligand-stimulated germinal center B cells and inhibits Fas-mediated apoptosis

To investigate the molecular and cellular mechanisms underlying the selection, differentiation, and apoptosis of germinal center (GC) B cells, we have established a culture system containing a follicular dendritic cell (FDC) line, HK. The mAb, 3C8, which is specific to HK cells and recognizes dendritic network in the GC, was developed and provided additional evidence that HK cells are related to FDC by sharing a unique surface Ag. The roles for CD40 ligand (CD40L) and T cell-derived cytokines in the differentiation of GC B cells were investigated in our culture system. We show that there are two distinct stages of GC B cell differentiation. In the early stage, GC B cells undergo spontaneous apoptosis unless they are stimulated by CD40L. In the secondary stage, IL-10 directs GC B cell differentiation toward the generation of plasma cells, while the absence of IL-10 stimulation leads to the generation of memory B cells. The major function of CD40L was found in the enhancement of cell recovery and the augmentation of memory B cell generation. Although GC B cells are Fas+, GC B cells are at first resistant to, but then become sensitive to, anti-Fas killing after 24 h in culture with CD40L, which coincides with the gradual increase in Fas expression on GC B cells. Furthermore, anti-Ig down-regulated Fas expression on CD40L-stimulated GC B cells, suggesting that Ag receptor engagement down-regulates Fas expression and prevents Fas-mediated apoptosis of GC B cells. Our data imply that GC T cells have an important role in the differentiation and apoptosis of GC B cells. GC T cells expressing both CD40L and Fas ligand have a dual function on GC B cells, helper or killer, depending on the status of target B cells. In the early stage, GC T cells stimulate the extensive proliferation of GC B cells, ensuring a large repertoire of B cells for selection. In the later stage, GC T cells kill B cells via Fas-Fas ligand interactions unless GC B cells are positively selected by Ags present on FDC.     

Lateral asymmetries of pupillary responses

Migration of mature B lymphocytes within secondary lymphoid organs and recirculation between these sites are thought to allow B cells to obtain T cell help, to undergo somatic hypermutation, to differentiate into effector cells, and to home to sites of antibody production. The mechanisms that direct migration of B lymphocytes are unknown, but there is evidence that G protein-coupled receptors, and possibly chemokine receptors, may be involved. Stromal cell- derived factor (SDF)-1alpha is a CXC chemokine previously characterized as an efficacious chemoattractant for T lymphocytes and monocytes in peripheral blood. Here we show with purified tonsillar B cells that SDF-1alpha also attracts naive and memory, but not germinal center (GC) B lymphocytes. Furthermore, GC B cells could be converted to respond to SDF-1alpha by in vitro differentiation into memory B lymphocytes. Conversely, the migratory response in naive and memory B cells was significantly reduced after B cell receptor engagement and CD40 signaling. The receptor for SDF-1, CXC chemokine receptor 4 (CXCR4), was found to be expressed on responsive as well as unresponsive B cell subsets, but was more rapidly downregulated on responsive cells by ligand. Finally, messenger RNA for SDF-1 was detected by in situ hybridization in a layer of cells surrounding the GC. These findings show that responsiveness to the chemoattractant SDF-1alpha is regulated during B lymphocyte activation, and correlates with positioning of B lymphocytes within a secondary lymphoid organ.     

Specialization of mucosal follicular dendritic cells revealed by mucosal addressin-cell adhesion molecule-1 display

Follicular dendritic cells (FDC) in the mucosal lymphoid organs, Peyer's patches (PP), display mucosal vascular addressin-cell adhesion molecule-1 (MAdCAM-1). MAdCAM-1 decorates interlacing dendritic cells throughout PP follicles and is accentuated on FDC of the germinal center (GC) light zone and on "junctional" dendritic cells overlapping the B/T border and subepithelial dome region, sites associated with microenvironmental homing decisions. In contrast, MAdCAM-1 is rarely displayed by coronal or junctional FDC in peripheral lymph node follicles and is largely confined to the GC after lymph node immunization. FDC-associated MAdCAM-1 plays a prominent role in lymphocyte adhesion to GC in PP frozen sections and participates significantly in binding to GC in chronically stimulated lymph nodes and spleen, but not to GC in lymph nodes after primary immunization (where binding is dominated by vascular cell adhesion molecule-1). Differential display of MAdCAM-1 by FDC could contribute to the specialization of mucosal vs nonmucosal immune responses.     

B lymphocytes induce the formation of follicular dendritic cell clusters in a lymphotoxin alpha-dependent fashion

Lymphotoxin (LT)alpha is expressed by activated T cells, especially CD4(+) T helper type 1 cells, and by activated B and natural killer cells, but the functions of this molecule in vivo are incompletely defined. We have previously shown that follicular dendritic cell (FDC) clusters and germinal centers (GCs) are absent from the peripheral lymphoid tissues of LTalpha-deficient (LTalpha-/-) mice. LTalpha-/- mice produce high levels of antigen-specific immunoglobulin (Ig)M, but very low levels of IgG after immunization with sheep red blood cells. We show here that LTalpha-expressing B cells are essential for the recovery of primary, secondary, and memory humoral immune responses in LTalpha-/- mice. It is not necessary for T cells to express LTalpha to support these immune functions. Importantly, LTalpha-expressing B cells alone are essential and sufficient for the formation of FDC clusters. Once these clusters are formed by LTalpha-expressing B cells, then LTalpha-deficient T cells can interact with B cells to generate GCs and productive class-switched antibody responses. Thus, B cells themselves provide an essential signal that induces and maintains the lymphoid microenvironment essential for GC formation and class-switched Ig responses.     

Hepatocyte growth factor (HGF/SF) is a muscle-derived survival factor for a subpopulation of embryonic motoneurons

Muscle-derived factors are known to be important for the survival of developing spinal motoneurons, but the molecules involved have not been characterized. Hepatocyte growth factor/scatter factor (HGF/SF) plays an important role in muscle development and motoneuron axon outgrowth. We show that HGF/SF has potent neurotrophic activity (EC50=2 pM) for a subpopulation (40%) of purified embryonic rat motoneurons. Moreover, HGF/SF is an essential component of muscle-derived support for motoneurons, since blocking antibodies to HGF/SF specifically inhibited 65% of the trophic activity of media conditioned by C2/C7 skeletal myotubes, but did not inhibit the trophic activity secreted by Schwann cell lines. High levels of expression of the HGF/SF receptor c-Met in the spinal cord are restricted to subsets of motoneurons, mainly in limb-innervating segments. Consistent with this distribution, cultured motoneurons from limb-innervating brachial and lumbar segments showed a more potent response to HGF/SF than did thoracic motoneurons. By the end of the period of motoneuron cell death, levels of c-Met mRNA in motoneurons were markedly reduced, suggesting that the effects of HGF/SF may be limited to the period of motoneuron cell death. HGF/SF may play an important role during motoneuron development as a muscle-derived survival factor for a subpopulation of limb-innervating motoneurons.     

Coexpression of hepatocyte growth factor and c-Met proto-oncogene product in synovial sarcoma

Hepatocyte growth factor (HGF) is a heterodimeric polypeptide growth factor that has pleiotropic roles, including those of mitogen, motogen and morphogen. The HGF receptor is characterized as a c-Met proto-oncogene product (c-Met), which is a heterodimeric tyrosine kinase receptor. Hepatocyte growth factor acts as a mediator between the mesenchymal and epithelial tissues because HGF is produced by mesenchymal cells and c-Met is mainly expressed on various epithelial cells. Furthermore, the HGF/c-Met system plays an important role in embryogenesis and the regeneration of various organs. Synovial sarcoma (SS) are unique sarcoma that show epithelial differentiation, but little is known about their histogenesis. The expression of HGF and c-Met was examined by immunohistochemistry in SS specimens from 12 patients (six each of biphasic and monophasic fibrous types). Immunohistochemical coexpression of HGF and c-Met was demonstrated in the epithelial component of five biphasic SS, while only c-Met was expressed in the epithelioid nests of three monophasic fibrous SS. The spindle cell component was negative for HGF and c-Met. In SS, positivity for epithelial markers, such as cytokeratins and epithelial membrane antigen, was diffusely observed in the epithelial component and was focally observed in spindle cells, while vimentin was positive predominantly in the spindle cell component. The areas expressing HGF and c-Met corresponded to distinct epithelial structures; however, HGF and c-Met expression were not found in any other tumor cells expressing epithelial markers in the spindle cell component of SS. Considering the morphogenic effect of HGF, which has been known to be one of its most important roles, the unique immunohistochemical localization of HGF and c-Met in SS suggests that the HGF/c-Met system may be closely related to the formation of epithelial (glandular) structures in biphasic SS.     

Recombinant tumor necrosis factor enhances the locomotion of memory and naive B lymphocytes from human tonsils through the selective engagement of the type II receptor

Recent studies performed in mice knocked out for the tumor necrosis factor (TNF ), the lymphotoxin-alpha, or the type I TNF receptor (R), genes have shown that these animals display gross defects in germinal center (GC) formation, suggesting that members of the TNF and TNFR superfamilies are involved in the control of B-cell migration. Based on these premises, we have here investigated the effects of human recombinant (r) TNF on the polarization and locomotion of tonsillar B cells. rTNF increased the spontaneous polarization and locomotion of unfractionated tonsillar B lymphocytes in a dose-dependent manner by inducing a true chemotactic response. Memory (IgD-, CD38(-)) and naive (IgD+, CD38(-)), but not GC (IgD-, CD38(+)) B cells purified from total tonsillar B lymphocytes, showed a significantly higher locomotion in the presence than in the absence of rTNF. Accordingly, type I and II TNF receptors (TNFRs) were detected by flow cytometry on the surface of memory and naive, but not GC, B lymphocytes. Blocking experiments with monoclonal antibodies to type I or II TNFR showed that rTNF enhanced the spontaneous chemotaxis of memory and naive B cells through the selective engagement of type II TNFR. Finally, the TNF gene was found to be expressed in memory, naive and GC B lymphocytes; the cytokine was released in culture supernatants from the three B-cell subsets after stimulation. These data may support the hypothesis that human TNF is involved in the paracrine and perhaps autocrine control of B-cell migration in secondary lymphoid tissues.     

Cooperative interaction between alpha- and beta-chains of hepatocyte growth factor on c-Met receptor confers ligand-induced receptor tyrosine phosphorylation and multiple biological responses

Hepatocyte growth factor (HGF) is a heterodimeric molecule composed of the alpha-chain containing the N-terminal hairpin domain, four kringle domains, and the serine protease-like beta-chain. We prepared HGF/NK4 and HGF/beta from the entire HGF after single-cut digestion with elastase. HGF/NK4 contains the N-terminal hairpin and four kringle domains, while HGF/beta is composed of the C-terminal 16 amino acids of the alpha-chain and the entire beta-chain, linked by a disulfide bridge. HGF/NK4 competitively inhibited the binding of 125I-HGF to the receptor, and affinity cross-linking analysis indicated that HGF/NK4 alone can bind to the c-Met receptor. In contrast, HGF/beta alone did not competitively inhibit the binding of 125I-HGF to the receptor and did not bind to the c-Met/HGF receptor. Scatchard analysis and affinity cross-linking experiments indicated that HGF/beta specifically binds to c-Met in the presence of HGF/NK4 but not HGF/NK2. Neither HGF/NK4 nor HGF/beta alone induced mitogenic, motogenic (cell scattering), and morphogenic (induction of branching tubulogenesis) responses; however, HGF/beta did induce these biological responses in the presence of HGF/NK4. Consistent with these results, although neither HGF/NK4 alone nor HGF/beta alone induced tyrosine phosphorylation of the c-Met/HGF receptor, HGF/beta induced tyrosine phosphorylation of the receptor when c-Met/HGF receptor was occupied by HGF/NK4. These results indicate that HGF/beta binds to the c-Met/HGF receptor that is occupied by HGF/NK4 and induces receptor tyrosine phosphorylation and the subsequent biological activities of HGF. We propose that there exists a unique cooperative interaction between alpha- and beta-chains, this interaction leading to beta-chain-dependent receptor tyrosine phosphorylation and subsequent biological responses.     

T cell receptor- and beta 1 integrin-mediated signals synergize to induce tyrosine phosphorylation of focal adhesion kinase (pp125FAK) in human T cells

The beta 1 subfamily of integrins is thought to play an important role in both the adhesion/migration and proliferation/differentiation of T cells. beta 1 integrins can provide T cell costimulation through interaction of very late antigen (VLA) 4 (VLA-4) (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) with the extracellular matrix protein fibronectin (FN), or by VLA-4 binding to its cell surface ligand, vascular cell adhesion molecule (VCAM) 1. The mechanism by which beta 1 integrin members transduce T cell-costimulatory signals is poorly understood. Studies in non-T cells have demonstrated regulation of the tyrosine focal adhesion kinase pp125FAK by beta 1 integrin engagement and, most recently, indicate a role for pp125FAK in linking integrin-mediated signal transduction to the Ras pathway (Schaller, M. D., and J. T. Parsons, 1994, Curr. Opin. Cell. Biol. 6: 705-710; Schlaepfer, D. D., S. K. Hanks, T. Hunter, and P. Van der Geer. 1994. Nature (Lond.), 372:786-790). Although pp125FAK kinase occurs in T cells, there are no reports on its regulation in this cell type. The studies described in this article characterize novel regulation of pp125FAK by the T cell receptor (TCR)-CD3 antigen complex and beta 1 integrins, and provide the first account, in any cell type, of integrin alpha 4 beta 1-mediated pp125FAK tyrosine phosphorylation. We demonstrate a rapid and sustained synergistic increase in tyrosine phosphorylation of human pp125FAK in Jurkat T cells after simultaneous (a) triggering of the TCR-CD3 complex, and (b) alpha 4 beta 1 and alpha 5 beta 1 integrin-mediated binding of these cells to immobilized FN or alpha 4 beta 1 integrin-mediated binding to immobilized VCAM-1. Studies with normal peripheral blood-derived CD4+ human T blasts confirm the synergistic action of a TCR-CD3 complex-mediated costimulus with a FN- or VCAM-1-dependent signal in the induction of T cell pp125FAK tyrosine phosphorylation. In vitro kinase assays performed on pp125FAK immunoprecipitates isolated from Jurkat cells and normal CD4+ T cells identified a coprecipitating 57-kD tyrosine-phosphorylated protein (pp57), distinct from pp59fyn or pp56lck. These results indicate, for the first time, the involvement of a specific kinase, pp125FAK, in alpha 4 beta 1- and alpha 5 beta 1-mediated T cell-costimulatory signaling pathways. In addition, the data demonstrate novel regulation of pp125FAK tyrosine phosphorylation by the TCR-CD3 complex.     

Autocrine hepatocyte growth factor/scatter factor-Met signaling induces transformation and the invasive/metastastic phenotype in C127 cells

Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic effector of cells expressing the Met tyrosine kinase receptor. C127 is a non-tumorigenic mouse cell line which expresses negligible levels of HGF/SF and Met proteins. In the present report we have generated C127 cells which overexpress HGF/SF and/or Met proteins, and have analysed the effect of HGF/SF-Met signaling in these cells. We show that this signaling pathway stimulates the growth and invasiveness of C127 cells in vitro and that cells overexpressing both HGF/SF and Met proteins (but neither alone) are phenotypically transformed and highly tumorigenic and metastatic in vivo. Our data unequivocally demonstrates the autocrine dependency of HGF/SF-Met-induced transformation and metastasis in this system and supports the theory that the inappropriate expression of HGF/SF and Met proteins could play a role in the development and spread of human tumors. In addition, this system may be useful for identifying metastasis-associated genes that are activated by HGF/SF-Met signaling.     

Pyk2 is differentially regulated by beta1 integrin- and CD28-mediated co-stimulation in human CD4+ T lymphocytes

Beta1 integrins can provide T cell co-stimulation, but little is known concerning their downstream signaling pathways. We found that Pyk2, a focal adhesion kinase-related tyrosine kinase, is regulated by beta1 integrin signaling in human T cells. Stimulation of Jurkat T cells with the alpha4beta1 integrin ligand VCAM-1 results in Pyk2 tyrosine phosphorylation, and combined stimulation with VCAM-1 and anti-CD3 mAb induces rapid and sustained synergistic Pyk2 phosphorylation. Studies with mAb suggest that in synergistic CD3- and alpha4beta1 integrin-mediated Pyk2 tyrosine phosphorylation, a major contribution of CD3-derived signals is independent of their effects on regulating integrin adhesion. Analysis of resting human CD4+ T cells confirmed the ability of CD3-derived signals to synergize with beta1 integrin-dependent signals in the induction of Pyk2 tyrosine phosphorylation. In addition, although CD28-mediated co-stimulatory signals were able to synergize with CD3-mediated signals in inducing ERK and JNK activation and secretion of IL-2 in the primary T cells, they did not contribute to the induction of Pyk2 phosphorylation. Taken together, these results indicate a potential role for Pyk2 in T cell co-stimulation mediated specifically by beta1 integrins.     

Overexpression and activation of hepatocyte growth factor/scatter factor in human non-small-cell lung carcinomas

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates the invasive growth of epithelial cells via the c-MET oncogene-encoded receptor. In normal lung, both the receptor and the ligand are detected, and the latter is known to be a mitogenic and a motogenic factor for both cultured bronchial epithelial cells and non-small-cell carcinoma lines. Here, ligand and receptor expression was examined in 42 samples of primary human non-small-cell lung carcinoma of different histotype. Each carcinoma sample was compared with adjacent normal lung tissue. The Met/HGF receptor was found to be 2 to 10-fold increased in 25% of carcinoma samples (P = 0.0113). The ligand, HGF/SF, was found to be 10 to 100-fold overexpressed in carcinoma samples (P < 0.0001). Notably, while HGF/SF was occasionally detectable and found exclusively as a single-chain inactive precursor in normal tissues, it was constantly in the biologically-active heterodimeric form in carcinomas. Immunohistochemical staining showed homogeneous expression of both the receptor and the ligand in carcinoma samples, whereas staining was barely detectable in their normal counterparts. These data show that HGF/SF is overexpressed and consistently activated in non-small-cell lung carcinomas and may contribute to the invasive growth of lung cancer.     

Opposing actions of hepatocyte growth factor and basic fibroblast growth factor on cell contact, intracellular free calcium levels, and rat ovarian surface epithelial cell viability

Previous studies demonstrated that cell-to-cell contact stimulates a tyrosine phosphorylation signal transduction pathway that prevents rat ovarian surface epithelial (ROSE) cells from undergoing apoptosis. Hepatocyte growth factor (HGF), also know as scatter factor (SF), is expressed by ovarian stromal and thecal cells and has been shown to reduce cell contact in nonovarian tissues. The present studies were designed to determine whether HGF/SF promotes ROSE cells to dissociate and subsequently become apoptotic. Because an increase in intracellular free calcium ([Ca2+]i) is often an early event in the apoptotic cascade, the effects of HGF/SF on [Ca2+]i levels were also assessed. ROSE cells were cultured in serum-free medium with HGF/SF, basic fibroblast growth factor (bFGF), thapsigargin, Bay K, actinomycin D, cycloheximide, and/or BAPTA depending on the experimental design. Cell contact was assayed by time-lapse photography; [Ca2+]i levels were measured with Fluo-3, and apoptosis was assessed by in situ DNA staining. HGF/SF decreased cell contact within 1 h, increased [Ca2+]i levels by 3 h, and induced apoptosis by 6 h of culture. bFGF inhibited these HGF/SF-induced responses. The increase in [Ca2+]i appears to represent a point in the apoptotic cascade that commits ROSE cells to die. This concept is based on the observations that: 1) in the presence of the calcium chelator BAPTA, HGF/SF decreased cell contact but did not increase [Ca2+]i or apoptosis; 2) bFGF blocked HGF/SF-induced increase in [Ca2+]i; 3) bFGF did not attenuate HGF/SF's apoptotic action if exposed to cells after the increase in [Ca2+]i; and 4) RNA and protein synthesis were required for HGF/SF to increase [Ca2+]i, whereas the thapsigargin- and Bay K-induced increase in [Ca2+]i and apoptosis were independent of RNA/protein synthesis. These observations indicate that the components of the apoptotic cascade distal to the increase in [Ca2+]i are present within ROSE cells and are activated by a sustained elevation of [Ca2+]i. The present studies also show that when ROSE cells establish contact with 3T3 cells that express N-cadherin, [Ca2+]i levels are maintained at low basal levels. In contrast, cell contact with 3T3 cells that do not express N-cadherin results in elevated [Ca2+]i levels. Similarly, a synthetic N-cadherin peptide, which inhibits homophilic N-cadherin binding, increases [Ca2+]i levels. Taken together, these data indicate that homophilic N-cadherin binding between adhering cells plays an important role in maintaining calcium homeostasis. Further, these data support the concept that HGF/SF's ability to promote the dissociation of ROSE cells accounts in part for its ability to increase [Ca2+]i levels.     

Expression of several members of the TNF-ligand and receptor family on tonsillar lymphoid B cells

Although the expression patterns of the members of the tumour necrosis factor receptor and ligand families have extensively been studied by flow-cytometry on stimulated peripheral blood mononuclear cells (PBMNC), little or no flow-cytometric or immunohistological data exist about their expression in lymphoid tissue. According to the data obtained from stimulated PBMNC, several members of these molecule families (e.g. CD40 ligand [CD40L], CD30, CD27, hOX40) have been considered to be either T-cell restricted or strongly T-cell associated. The present study on samples from palatine tonsils revealed that most of these molecules are also expressed by tonsillar B cells. The additional analysis of the co-expression of these molecules also disclosed the existence of CD40+/CD40L+ and CD27+/CD70+ (CD27L+) lymphoid cells in tonsillar tissue.     

Follicular dendritic cells in non-Hodgkin's lymphomas

Follicular dendritic cells (FDC) are restricted to the B-cell regions of secondary lymphoid tissue and to non-Hodgkin's lymphomas derived from the follicular center or the mantle zone. With their cytoplasmic ramifications they form a dense network which contains the B-lymphocytes. In situ, FDC are only detectable at the ultrastructural level or when stained with anti FDC-reagents. On the surface of their dendritic extensions they express transferrin receptors (CD71), the B-cell epitope CD20, class II antigens, the myelomonocytic molecule CD14, the glycoprotein gp50 (CD40), and several receptors for components of the complement system (CD11b, CD21, CD35). Subsequent to an antigen challenge, FDC trap and retain immune-complexes for a long period of time. In vitro FDC and neoplastic lymphocytes spontaneously form small cellular aggregates. This adhesion is mediated by the LFA-1-alpha/beta = ICAM-1, the VLA-4 = VCAM-1, and the ICAM-1 = C3bi- receptor ligand pathways on B-cells and on FDC, respectively. The loss of LFA-1- alpha/beta and ICAM-1 molecules may enable neoplastic lymphocytes to detach from FDC. The monoclonal B-cells now invade new compartments. In vitro, FDC have the capacity to activate resting B-cells and to save them from dying by apoptosis. Signals involved in this activation include cell-surface immunoglobulin and CD40. Immunocytochemistry and autoradiography with single cell suspensions of neoplastic B cells suggest that FDC also provide signals leading to the continued stimulation of lymphoma lymphocytes. During the early stage of HIV infection lymph nodes show an immense follicular hyperplasia, with a massive increase of the dendritic network of FDC. In the later stage of the disease, the continuous involution of the germinal centers is associated with a progressive destruction of FDC.     

Dynamics of beta-catenin interactions with APC protein regulate epithelial tubulogenesis

Epithelial tubulogenesis involves complex cell rearrangements that require control of both cell adhesion and migration, but the molecular mechanisms regulating these processes during tubule development are not well understood. Interactions of the cytoplasmic protein, beta-catenin, with several molecular partners have been shown to be important for cell signaling and cell-cell adhesion. To examine if beta-catenin has a role in tubulogenesis, we tested the effect of expressing NH2-terminal deleted beta-catenins in an MDCK epithelial cell model for tubulogenesis. After one day of treatment, hepatocyte growth factor/scatter factor (HGF/ SF)-stimulated MDCK cysts initiated tubulogenesis by forming many long cell extensions. Expression of NH2-terminal deleted beta-catenins inhibited formation of these cell extensions. Both DeltaN90 beta-catenin, which binds to alpha-catenin, and DeltaN131 beta-catenin, which does not bind to alpha-catenin, inhibited formation of cell extensions and tubule development, indicating that a function of beta-catenin distinct from its role in cadherin-mediated cell-cell adhesion is important for tubulogenesis. In cell extensions from parental cysts, adenomatous polyposis coli (APC) protein was localized in linear arrays and in punctate clusters at the tips of extensions. Inhibition of cell extension formation correlated with the colocalization and accumulation of NH2-terminal deleted beta-catenin in APC protein clusters and the absence of linear arrays of APC protein. Continued HGF/ SF treatment of parental cell MDCK cysts resulted in cell proliferation and reorganization of cell extensions into multicellular tubules. Similar HGF/SF treatment of cysts derived from cells expressing NH2-terminal deleted beta-catenins resulted in cells that proliferated but formed cell aggregates (polyps) within the cyst rather than tubules. Our results demonstrate an unexpected role for beta-catenin in cell migration and indicate that dynamic beta-catenin-APC protein interactions are critical for regulating cell migration during epithelial tubulogenesis.     

Evidence for an important interaction between a complement-derived CD21 ligand on follicular dendritic cells and CD21 on B cells in the initiation of IgG responses

The addition of Ags to mononuclear leukocyte cultures typically elicits modest Ab responses, implying that cosignals beyond those provided by T cells and macrophages may be needed. Recently, we reported that Ab responses could be dramatically enhanced (10-1000-fold) by the addition of follicular dendritic cells (FDC), suggesting that FDC may provide an important costimulatory signal. This result prompted a study of molecules involved in FDC-mediated enhancement of Ab responses stimulated by specific Ag with memory T and B cells or nonspecifically by the addition of LPS. In this study, we report evidence supporting the concept that FDC bear a ligand that engages complement receptor II (CR2 or CD21) on B cells and provides a critical cosignal for both Ag-specific and polyclonal responses. A blockade of the CR2 ligand on FDC by the use of soluble CR2 or a blockade of CR2 on B cells by use of CR2 knockout mice (or B cells with CR2 blocked) reduced Ab responses from the microg/ml to the ng/ml range (10-1000-fold reductions). FDC from C3 knockout mice, which cannot generate the CR2-binding fragments (iC3b, C3d, and C3dg), were unable to provide costimulatory activity, suggesting the CR2 ligand on FDC consists of C3 fragments. FDC trap complement-activating Ag-Ab complexes, and it appears that FDC present B cells with both specific Ag to engage B cell receptors and a CR2 ligand to engage B cell-CR2. In short, optimal induction of specific Ab responses appears to require the combination of specific Ag and costimulatory molecules from both T cells and FDC.