The antigen-presenting cell function of Reed-Sternberg cells

Reed-Sternberg cells, the neoplastic cells of Hodgkin's disease, express all membrane molecules required to function as antigen-presenting cells (APCs), such as major histocompatibility complex (MHC) class II antigens and the recently characterized B7 proteins, which are of critical importance for APC to adequately stimulate CD4+ T cells. As APC do, Reed-Sternberg cells also express the adhesion molecules ICAM-1 (CD54) and LFA-3 (CD58), via which T cells are able to adhere to the cell. MHC antigens, B7 proteins as well as the adhesion molecules are expressed by Reed-Sternberg cells in virtually all cases of Hodgkin's disease, irrespective of the subtype. In vitro studies have shown that Hodgkin's disease-derived cell lines are potent stimulators of mixed lymphocyte cultures and that the MHC antigens, B7 proteins and the adhesion molecules, expressed by Hodgkin's disease-derived cell lines, are essential for such a function. Taken together, these data strongly suggest that Reed-Sternberg cells function as APC in vivo, and that the APC function of the cell is a major common denominator of Hodgkin's disease. The APC function of Reed-Sternberg cells does not support the hypothesis that they derive from dendritic cells, since activated B and T cells may also exert an APC function. Analysis of the antigens that are potentially expressed by Reed-Sternberg cells may greatly advance our knowledge on the pathogenesis of Hodgkin's disease and may allow the development of immunotherapy as an alternative treatment method.     

Bystander apoptosis triggers dendritic cell maturation and antigen-presenting function

Physiologic cell death via apoptosis occurs without inflammation or autoimmunity. Here, we investigated the outcome of the interaction of apoptotic cells with dendritic cells (DCs), which are potent professional APCs. DCs internalized apoptotic cells and processed them for presentation to both MHC class I- and class II-restricted T cells with an efficiency that was dependent upon the number of apoptotic cells. The latter event was accompanied by the autocrine/paracrine secretion of IL-1beta and TNF-alpha, with eventual DC maturation. High numbers of apoptotic cells, mimicking a failure of their in vivo clearance, are therefore sufficient to trigger DC maturation and the presentation of intracellular Ags from apoptotic cells, even in the absence of exogenous "danger" signals.     

The dendritic cell differentiation and antigen-presenting function of the erythroleukemia cells induced by GM-CSF

OBJECTIVE: To investigate the generation of dendritic cells and the change of antigen-presenting function during the differentiation of FBL-3 erythroleukemia cells induced by GM-CSF. METHODS: The effects of GM-CSF on the phenotype, ultrastructure and antigen-presenting function of FBL-3 erythroleukemia cells were observed by FACS, electromicroscopy and 51Cr-release assay. RESULTS: After treatment with 100 ng/ml GM-CSF for 3 days, the expressions of 33 D1 and NLDC 145 which are the specific markers on dendritic cells were increased significantly; MHC-II B7-1, B7-2, ICAM-1, VCAM-1 and CD40 were also upregulated. The membrane of FBL-3 cells was changed into villous surface with dendritic projections. There were plenty of mitochondria in cytoplasm, the nucleus became lobulated. The GM-CSF-treated FBL-3 cells could apparently stimulate the proliferation of allogeneic T lymphocytes, induce the production of IL-2 and improve the specific cytotoxic activity of CTL on FBL-3 cells. CONCLUSION: Erythroleukemia cells were induced to differentiate into the dendritic cells by GM-CSF and obtained the antigen-presenting function.     

Localization of DNA damage and its role in altered antigen-presenting cell function in ultraviolet-irradiated mice

Prior ultraviolet (UV) irradiation of the site of application of hapten on murine skin reduces contact sensitization, impairs the ability of dendritic cells in the draining lymph nodes (DLN) to present antigen, and leads to development of hapten-specific suppressor T lymphocytes. We tested the hypothesis that UV-induced DNA damage plays a role in the impaired antigen-presenting activity of DLN cells. First, we assessed the location and persistence of cells containing DNA damage. A monoclonal antibody specific for cyclobutyl pyrimidine dimers (CPD) was used to identify UV-damaged cells in the skin and DLN of C3H mice exposed to UV radiation. Cells containing CPD were present in the epidermis, dermis, and DLN and persisted, particularly in the dermis, for at least 4 d after UV irradiation. When fluorescein isothiocyanate (FITC) was applied to UV-exposed skin, the DLN contained cells that were Ia+, FITC+, and CPD+; such cells from mice sensitized 3 d after UV irradiation exhibited reduced antigen-presenting function in vivo. We then assessed the role of DNA damage in UV-induced modulation of antigen-presenting cell (APC) function by using a novel method of increasing DNA repair in mouse skin in vivo. Liposomes containing T4 endonuclease V (T4N5) were applied to the site of UV exposure immediately after irradiation. This treatment prevented the impairment in APC function and reduced the number of CPD+ cells in the DLN of UV-irradiated mice. Treatment of unirradiated skin with T4N5 in liposomes or treatment of UV-irradiated skin with liposomes containing heat-inactivated T4N5 did not restore immune function. These studies demonstrate that cutaneous immune cells sustain DNA damage in vivo that persists for several days, and that FITC sensitization causes the migration of these to the DLN, which exhibits impaired APC function. Further, they support the hypothesis that DNA damage is an essential initiator of one or more of the steps involved in impaired APC function after UV irradiation.     

Nuclear accumulation of Saccharomyces cerevisiae Mcm3 is dependent on its nuclear localization sequence

BACKGROUND: The proteins of the Mcm2-7 family are required for the initiation of DNA replication. In Saccharomyces cerevisiae the nuclear envelope does not break down during the mitotic phase of the cell cycle. Large nuclear proteins, such as the Mcm proteins, which accumulate in the nucleus during specific portions of the cell cycle, must have regulated mechanisms to direct their entry into the nucleus. RESULTS: We have identified a nuclear localization sequence (NLS) in Mcm3, and demonstrated that it is necessary for the translocation of Mcm3 into the nucleus and sufficient for directing Escherichia coli beta-galactosidase to the nucleus. Immediately adjacent to the nuclear localization sequence are four potential sites for phosphorylation by Cdc28. Mutagenesis of all four sites has no immediate phenotypic effect on cell growth or viability, nor does it affect nuclear accumulation of Mcm3, although two-dimensional protein gel analysis has shown that at least some of these sites are normally phosphorylated in vivo. Substitution of the Mcm3 NLS by the SV40 large T-antigen NLS also directs the nuclear accumulation of the Mcm3-T-antigen protein, although cell growth is compromised. Replication activity in cells bearing either the Mcm3-Cdc28 phosphorylation site mutations or the Mcm3 T-antigen NLS substitution, as measured by plasmid stability assays, is comparable to activity in wild-type cells. CONCLUSIONS: The Mcm3 protein is imported into the nucleus by a specific NLS. The cell cycle specific nuclear accumulation of Mcm3 appears to be a result of nuclear retention or nuclear targeting, rather than nuclear import regulated through the NLS.     

Costimulation through CD86 is involved in airway antigen-presenting cell and T cell responses to allergen in atopic asthmatics

Atopic allergic asthma is characterized by activation of Th2-type T cells in the bronchial mucosa. Previous reports have suggested an important role for costimulation through the CD28/CTLA4-CD80/CD86 pathway in allergen activation of T cells in animal models of inhaled allergen challenge. However, human allergen-specific lines and clones were reported to be costimulation independent. We therefore examined CD80 and CD86 dependence of allergen-induced T cell proliferation and cytokine production in peripheral blood and bronchoalveolar lavage from atopic asthmatic subjects and controls. Both allergen-induced proliferation and IL-5 production from PBMC were inhibited by CTLA4-Ig fusion protein and anti-CD86, but not anti-CD80 mAbs. When allergen-specific CD4+ T cell lines from peripheral blood were examined, proliferation and cytokine production were found to be independent of CD80 or CD86 costimulation. However, when cells obtained directly from the airways were examined, allergen-induced proliferation of bronchoalveolar lavage T cells from atopic asthmatic subjects was inhibited by anti-CD86 but not anti-CD80. In addition, bronchoalveolar lavage-adherent cells from asthmatic, but not control subjects showed APC activity to autologous T cells. This was also inhibited by anti-CD86 but not anti-CD80. Thus allergen-induced T cell activation and IL-5 production in the airway in asthmatic subjects is susceptible to blockade by agents interfering with costimulation via CD86, and this may hold therapeutic potential in asthma.     

Nuclear localization signal receptor importin alpha associates with the cytoskeleton

Importin alpha is the nuclear localization signal (NLS) receptor that is involved in the nuclear import of proteins containing basic NLSs. Using importin alpha as a tool, we were interested in determining whether the cytoskeleton could function in the transport of NLS-containing proteins from the cytoplasm to the nucleus. Double-labeling immunofluorescence studies showed that most of the cytoplasmic importin alpha coaligned with microtubules and microfilaments in tobacco protoplasts. Treatment of tobacco protoplasts with microtubule- or microfilament-depolymerizing agents disrupted the strands of importin alpha in the cytoplasm, whereas a microtubule-stabilizing agent had no effect. Biochemical analysis showed that importin alpha associated with microtubules and microfilaments in vitro in an NLS-dependent manner. The interaction of importin alpha with the cytoskeleton could be an essential element of protein transport from the cytoplasm to the nucleus in vivo.     

Murine thymic lymphoma is associated with a species-specific hematopoietic progenitor cell subpopulation

Many strains of laboratory mouse are uniquely susceptible to the development of T cell lymphoma/leukemia, either spontaneously or as a result of chemical or radiation exposure. In contrast, T cell leukemias or lymphomas which are relatively uncommon in human populations, are not easily induced by radiation, and are not generally associated with chemotherapy or chemical exposure. Evidence is presented to suggest that differences in the susceptibility to the development of these malignancies is related to subtle but important variations in the regulation of hematopoietic stem cell differentiation between these two species.     

Epidermal growth factor receptor expression is associated with rapid tumor cell proliferation in renal cell carcinoma

Epidermal growth factor receptor (EGF-r) expression and tumor cell proliferation rate have been proposed as potential prognostic parameters in renal cell carcinoma (RCC). In this study, immunohistochemical stains using antibodies to EGF-r and the cell proliferation marker Ki-67 (MIB-1) were used to study the relationship between EGF-r expression, tumor cell proliferation, and prognosis in 50 non-papillary RCC extending beyond the renal capsule (pT3). A high Ki-67 labeling index (LI) was associated with poor patient prognosis (P < .05). Thirty-eight cases (76%) expressed strong cell membrane immunoreactivity for EGF-r. There was a tendency toward a shortened survival for EGF-r-positive tumors (P = .08). Tumor growth fraction (Ki-67 LI) was significantly higher in EGF-r-positive tumors than in EGF-r-negative tumors (P < .05), suggesting that rapid tumor proliferation might be responsible for the poor prognosis associated with EGF-r-positive RCC.     

Nuclear localization of soluble CTP:phosphocholine cytidylyltransferase

The soluble form of CTP:phosphocholine cytidylyltransferase, which has previously been assumed to be cytosolic, has been localized to the nucleus of several cell types. Indirect immunofluorescence microscopy indicated a nuclear location in HepG2, NIH-3T3, and L-cells. A comparison of the fluorescence pattern of wild-type CHO cells with a cytidylyltransferase-deficient mutant provided genetic evidence that cytidylyltransferase is nuclear in CHO cells. The enzyme is also predominantly nuclear in rat liver, as revealed by staining frozen sections of that tissue. When L-cells were fractionated by enucleation, over 95% of cytidylyltransferase activity was found in the nuclear fraction, providing biochemical evidence for a nuclear location in these cells. In light of the demonstration that the membrane-bound cytidylyltransferase in CHO cells is associated with the nuclear envelope (Watkins, J. D., and Kent, C. (1992) J. Biol. Chem. 267, 5686-5692), these results suggest that this enzyme is predominantly an intranuclear enzyme.     

Defective cell migration in an ovarian cancer cell line is associated with impaired urokinase-induced tyrosine phosphorylation

The urokinase receptor (u-PAR), a protein anchored to cell membrane by a glycosyl phosphatidylinositol, plays a central role in cancer cell invasion and metastasis by binding urokinase plasminogen activator (u-PA), thereby facilitating plasminogen activation. Plasmin can promote cell migration either directly or by activating metalloproteinases that degrade some of the components of the extra cellular matrix. However, the IGR-OV1-Adria cell line contains the u-PAR but does not migrate even in the presence of exogenous u-PA, although the parental IGR-OV1 cell line migrates normally in the presence of u-PA. We therefore investigated the role of cell signalling for u-PA induced cell locomotion. We show that cell migration induced by u-PA-u-PAR complex is always associated with tyrosine kinase activation for the following reasons: (1) the blockade of the u-PAR by a chimeric molecule (albumin-ATF) inhibits not only the u-PA-induced cell migration, but also the signalling in IGR-OV1 line; (2) the binding of u-PA to u-PAR on non-migrating IGR-OV1-Adria cells was not associated with tyrosine kinase activation; (3) the inhibition of tyrosine kinase also blocked cell migration of IGR-OV1. Therefore tyrosine kinase activation seems to be essential for the u-PA-induced cell locomotion possibly by the formation of a complex u-PAR-u-PA with a protein whose transmembrane domain can ensure cell signalling. Thus, IGR-OV1 and IGR-OV1-Adria cell lines represent a good model for the analysis of the mechanism of u-PA-u-PAR-induced cell locomotion.     

Stem cell factor induction is associated with mast cell accumulation after canine myocardial ischemia and reperfusion

BACKGROUND: Myocardial infarction is associated with an intense inflammatory reaction leading to healing and scar formation. Because mast cells are a significant source of fibrogenic factors, we investigated mast cell accumulation and regulation of stem cell factor (SCF), a potent growth and tactic factor for mast cells, in the healing myocardium. METHODS AND RESULTS: Using a canine model of myocardial ischemia and reperfusion, we demonstrated a striking increase of mast cell numbers during the healing phase of a myocardial infarction. Mast cell numbers started increasing after 72 hours of reperfusion, showing maximum accumulation in areas of collagen deposition (12.0+/-2.6-fold increase; P<0.01) and proliferating cell nuclear antigen (PCNA) expression. The majority of proliferating cells were identified as alpha-smooth muscle actin-positive myofibroblasts or factor VIII-positive endothelial cells. Mast cells did not appear to proliferate. Using a nuclease protection assay, we demonstrated induction of SCF mRNA within 72 hours of reperfusion. Immunohistochemical studies demonstrated that a subset of macrophages was the source of SCF immunoreactivity in the infarcted myocardium. SCF protein was not found in endothelial cells and myofibroblasts. Intravascular tryptase-positive, FITC-avidin-positive, CD11b-negative mast cell precursors were noted in the area of healing and in the cardiac lymph after 48 to 72 hours of reperfusion. CONCLUSIONS: Mast cells increase in number in areas of collagen deposition and PCNA expression after myocardial ischemia. The data provide evidence of mast cell precursor infiltration into the areas of cellular injury. SCF is induced in a subset of macrophages infiltrating the healing myocardium. We suggest an important role for SCF in promoting chemotaxis and growth of mast cell precursors in the healing heart.     

Notch1-induced delay of human hematopoietic progenitor cell differentiation is associated with altered cell cycle kinetics

Hematopoiesis is a balance between proliferation and differentiation that may be modulated by environmental signals. Notch receptors and their ligands are highly conserved during evolution and have been shown to regulate cell fate decisions in multiple developmental systems. To assess whether Notch1 signaling may regulate human hematopoiesis to maintain cells in an immature state, we transduced a vesicular stomatitis virus G-protein (VSV-G) pseudo-typed bicistronic murine stem cell virus (MSCV)-based retroviral vector expressing a constitutively active form of Notch1 (ICN) and green fluorescence protein into the differentiation competent HL-60 cell line and primary cord blood-derived CD34(+) cells. In addition, we observed endogenous Notch1 expression on the surface of both HL-60 cells and primary CD34(+) cells, and therefore exposed cells to Notch ligand Jagged2, expressed on NIH3T3 cells. Both ligand-independent and ligand-dependent activation of Notch resulted in delayed acquisition of differentiation markers by HL-60 cells and cord blood CD34(+) cells. In addition, primary CD34(+) cells retained their ability to form immature colonies, colony-forming unit-mix (CFU-mix), whereas control cells lost this capacity. Activation of Notch1 correlated with a decrease in the fraction of HL-60 cells that were in G0/G1 phase before acquisition of a mature cell phenotype. This enhanced progression through G1 was noted despite preservation of the proliferative rate of the cells and the overall length of the cell cycle. These findings show that Notch1 activation delays human hematopoietic differentiation and suggest a link of Notch differentiation effects with altered cell cycle kinetics.     

Apc gene mutation is associated with a dominant-negative effect upon intestinal cell migration

Apc-associated intestinal tumor formation appears to require functional loss of both Apc alleles. Apc has, therefore, been classified as a tumor suppressor gene. Loss of APC protein function results in increased intracellular beta-catenin, a molecule important to both cell-cell adhesion and regulation of cellular growth. In mice bearing a germ-line Apc mutation, we found that enterocyte beta-catenin expression was also increased in histologically normal intestinal mucosa. Enterocyte crypt-villus migration was decreased by 25%, and treatment of Min/+ animals with sulindac sulfide normalized both beta-catenin expression and enterocyte migration. Our data suggest that alterations in enterocyte migration occur in cells bearing a single mutant Apc allele, and that sulindac sulfide may normalize enterocyte growth in these cells.     

Protection against ascending infection of the genital tract by Chlamydia trachomatis is associated with recruitment of major histocompatibility complex class II antigen-presenting cells into uterine tissue

A mouse model of ascending infection following intravaginal inoculation with a strain of Chlamydia trachomatis isolated from humans has been used to identify immune mechanisms associated with protection against genital infection. BALB/c and C3H mice differed in their susceptibilities to infection and inflammatory disease. In both mouse strains, ascension of the organism and recruitment of bone marrow-derived mononuclear leukocytes were evident in uterine tissue 1 week postinfection. By 3 weeks the organism had been cleared and inflammation had been resolved in the BALB/c mice, but both persisted in the C3H animals. In athymic nude BALB/c mice both the organism and inflammation persisted, indicating the influence of the hosts' immune response on the outcome of infection. Both BALB/c and C3H mice had a Th1 response in draining lymph nodes, with predominant production of gamma interferon and tumor necrosis factor alpha, low levels of interleukin-10, and no detectable levels of interleukin-4. However, the composition of the early uterine infiltrate differed in these two mouse strains. Cell surface labeling and analysis of light scatter properties by flow cytometry identified a population of large, CD45(+) major histocompatibility complex class II mononuclear cells, which were a prominent feature of the infiltrates in BALB/c mice but were present in significantly lower numbers in C3H mice. These cells expressed the costimulatory molecules CD86 and CD40 and stimulated allogeneic T cells, suggesting that these mononuclear cells are a population of antigen-presenting cells and that they may play a role in clearing antigen and protecting against inflammatory disease in BALB/c mice. An additional level of immunological control may thus exist in genital chlamydial infection.     

Nuclear localization of the testis determining gene product SRY

We have studied the expression of the human SRY protein (termed p27SRY) in two different cell lines by using specific antibodies. Confocal microscopy enabled us to localize p27SRY precisely in the nucleus in a discrete punctuate pattern. Furthermore, through microinjection experiments, we have demonstrated that the localization of the p27SRY protein into the nucleus was an event involving the NH2-terminal part of the high mobility group (HMG) domain. With the help of several synthetic peptides and various p27SRY mutants, we have characterized a bipartite basic motif in this part of the protein corresponding to a nuclear localization signal. This nuclear localization signal appears to be highly conserved in SRY box- and HMB box-containing proteins, suggesting common properties of nuclear targeting within the HMG box protein family.     

Myosin I is associated with zymogen granule membranes in the rat pancreatic acinar cell

BACKGROUND & AIMS: The mechanisms whereby intracellular messengers mediate zymogen granule transport and exocytosis in the pancreatic acinar cell are not well defined. Electron microscopy has shown a periluminal network of actin in the acinar cell, suggesting a role for actin and myosin in the transport process. The possible involvement of two types of myosin in the secretory process was investigated, and their distribution in acinar cells was determined. METHODS: Antibodies specific to myosin I or to myosin II were used for immunocytochemistry and Western blot analysis. Ultrastructural studies were also performed. RESULTS: Western blot analysis showed that myosin I and myosin II were present in total pancreatic homogenate but that only myosin I was present on isolated zymogen granules and their membranes. By immunocytochemistry, myosin I was shown in the apical aspect of acinar cells colocalized with glycoprotein 2, a marker for zymogen granules, and actin. By immunocytochemistry, myosin I was also localized on isolated zymogen granules. CONCLUSIONS: The immunolocalization of myosin I to zymogen granule membranes and its close association with periluminal actin suggest that myosin I plays a direct role in the process of transport and exocytosis of zymogen granules in the pancreatic acinar cell.