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PubMed1996

Cell-to-cell communication in the anterior pituitary: evidence for gap junction-mediated exchanges between endocrine cells and folliculostellate cells.

The ability of rat anterior pituitary cells to communicate through gap junctions (GJ) was studied using a fluorescent molecule, Lucifer Yellow (LY), which freely passes through GJ channels. The probe was introduced into the cell cytoplasm by using either the cut-end loading method on intact tissue, or cell microinjection on cultured cells. The identification of communicating cells was performed by immunofluorescence labeling of specific hormones in endocrine cells and of S100 protein in folliculostellate (FS) cells. Rat anterior pituitary cells in their physiological organization, i.e. in the intact tissue, exhibited a high level of coupling through GJ. LY-labeled cells were found up to 300-microns apart from its site of introduction. The communicating cells were primarily PRL cells, GH cells, and FS cells. Only a few LH, TSH, and ACTH cells were labeled with LY. Anterior pituitary cells, isolated from the rat tissue by mild protease treatment and cultured for 3 days, reestablished functional GJ as demonstrated by microinjection of LY into individual cells. By immunolabeling of specific hormones and/or S100 protein, we found a GJ coupling between FS cells, and between FS cells and endocrine cells, including PRL cells. The communication between FS cells was by far the most frequent. In conclusion, we demonstrate the presence of functional GJ between anterior pituitary cells of the same type and between anterior pituitary cells having distinct differentiated functions.

Animals↗
PubMed2005

Airway smooth muscle cells enhance C3a-induced mast cell degranulation following cell-cell contact.

Growing evidence suggests that anaphylatoxins, C3a and C5a, play important roles in innate immunity and may also participate in the pathogenesis of asthma. Previous studies with animal models and immunohistochemistry analysis of lung tissue indicated that anaphylatoxins may regulate airway hyperresponsiveness (AHR) in asthma via the activation of their cell surface G protein-coupled receptors (C3aR and C5aR) in airway smooth muscle (ASM) cells. Using RT-PCR, flow cytometry, and confocal microscopy, we made the surprising observation that while C3aR and C5aR were expressed in human mast cells, they were not present in cultured primary human or murine ASM cells. Furthermore, we could not detect C3aR in smooth muscle-positive cells of human trachea or bronchus. Interestingly, incubation of human mast cells with ASM cells, but not its culture supernatant, caused a significant enhancement of C3a-induced mast cell degranulation. Although stem cell factor (SCF) and its receptor c-kit are constitutively expressed on ASM cells and mast cells, respectively, neutralizing antibodies to SCF and c-kit failed to inhibit ASM cell-mediated enhancement of mast cell degranulation. However, dexamethasone-treated ASM cells were normal for cell surface SCF expression but were significantly less effective in enhancing C3a-induced mast cell degranulation when compared with untreated cells. These findings suggest that cell-cell interaction between ASM cells and mast cells, via a SCF-c-kit-independent but dexamethasone-sensitive mechanism, enhances C3a-induced mast cell degranulation, which likely regulates ASM function, thus contributing to the pathogenesis of asthma.

Animals↗
PubMed1999

CD40-activated B-cell chronic lymphocytic leukemia cells for tumor immunotherapy: stimulation of allogeneic versus autologous T cells generates different types of effector cells.

Although spontaneous remissions may rarely occur in B-cell chronic lymphocytic leukemia (B-CLL), T cells do generally not develop a clinically significant response against B-CLL cells. Because this T-cell anergy against B-CLL cells may be caused by the inability of B-CLL cells to present tumor-antigens efficiently, we examined the possibility of upregulating critical costimulatory (B7-1 and B7-2) and adhesion molecules (ICAM-1 and LFA-3) on B-CLL cells to improve antigen presentation. The stimulation of B-CLL cells via CD40 by culture on CD40L expressing feeder cells induced a strong upregulation of costimulatory and adhesion molecules and turned the B-CLL cells into efficient antigen-presenting cells (APCs). CD40-activated B-CLL (CD40-CLL) cells stimulated the proliferation of both CD4(+) and CD8(+) T cells. Interestingly, stimulation of allogeneic versus autologous T cells resulted in the expansion of different effector populations. Allogeneic CD40-CLL cells allowed for the expansion of specific CD8(+) cytolytic T cells (CTL). In marked contrast, autologous CD40-CLL cells did not induce a relevant CTL response, but rather stimulated a CD4(+), Th1-like T-cell population that expressed high levels of CD40L and released interferon-gamma in response to stimulation by CD40-CLL cells. Together, these results support the view that CD40 activation of B-CLL cells might reverse T-cell anergy against the neoplastic cell clone, although the character of the immune response depends on the major histocompatibility complex (MHC) background on which the CLL or tumor antigens are presented. These findings may have important implications for the design of cellular immunotherapies for B-CLL.

Aged↗
PubMed1989

Requirement for delivery of signals by physical interaction and soluble factors from accessory cells in the induction of receptor-mediated T cell proliferation. Effectiveness of IFN-gamma modulation of accessory cells for physical interaction with T cells.

We analyzed the mechanism by which accessory cells support the induction of the proliferation of human peripheral blood T cells by a monoclonal anti-CD3 antibody, OKT3. Cross-linking of T cell receptor/CD3 complex by anti-CD3 coupled to latex beads and the addition of IL-1 are not enough to induce the IL-2 production and proliferation of T cells extensively depleted of accessory cells, while the addition of both the culture supernatant of macrophages or a monoblastic cell line, U937 cells, and the paraformaldehyde-fixed macrophages or U937 cells which had been precultured with interferon-gamma before fixation into the culture of the T cells with anti-CD3-latex did induce the T cell proliferation. Lack of the addition of either one of these did not induce the response. These results indicate that the signal(s) delivered by soluble factors released from the accessory cells and that delivered by the physical interaction between accessory cells and T cells are both required for the induction of IL 2 production and proliferation of T cells by anti-CD3-latex. Importantly, the macrophages or U937 cells had to be cultured with Con A-stimulated lymphocyte culture supernatant or IFN-gamma prior to fixation with paraformaldehyde, suggesting that a molecule(s) inducible on accessory cells surface by IFN-gamma or other lymphokine is necessary for the effective accessory cell-T cell interaction to induce the T cell response. It was further revealed that the activity of the culture supernatant of accessory cells may be mediated synergistically by IL 1 and a certain other factor(s) and was actually shown to be replaced by the combined addition of rIL-1 and rIL-6 but not by rIL-1 alone. The experimental system described here will be very useful for dissecting the accessory functions for T cell activation.

Adjuvants, Immunologic↗
PubMed1995

The accumulation of B220+ CD4- CD8- (DN) T cells in C3H-lpr/lpr mice is not accelerated by the stimulation of CD8+ T cells or B220+ DN T cells with staphylococcal enterotoxin B and occurs independently of V beta 8+ T cells.

Mice homozygous for lpr or gld develop lymphoproliferative disease characterized by the progressive accumulation of functionally impaired B220+ double-negative (DN) T cells and primed CD4+ and CD8+ T cells. The mechanisms leading to the accumulation of these T cells subsets are poorly understood but are clearly dependent on lack of expression of Fas in lpr mice and expression of defective FasL in gld mice. A role for V beta 8+ T cells also has been reported. Recently, a variety of experimental approaches revealed that the majority of B220+ DN T cells are derived from MHC class I-selected CD8+ precursors. Here we used the potent mitogen, staphylococcal enterotoxin B (SEB): (i) to examine the effects of defective Fas-FasL expression on the deletion of peripheral V beta 8+ T cells in 6- to 8- and 20-week old C3H-lpr and -gld mice, (ii) to determine the immunocompetence of B220+ DN T cells in vivo, and (iii) to determine if activated V beta 8+ CD8+ T cells can differentiate into B220+ DN T cells. The role of V beta 8+ T cells in the accumulation of B220+ DN T cells also was reinvestigated. These studies showed that deletion pathways independent of Fas-FasL expression function in young lpr and gld mice and delete CD4+ T cells more efficiently than CD8+ T cells. As the mice age, these alternative pathways become less effective and this may explain the progressive accumulation of memory T cells. No abnormalities in tolerance induction were observed in young or diseased mice. Stimulation of +/+, lpr and gld V beta 8+ CD8+ T cells induced the expression of B220. B220 levels were maximal 2 days after SEB and were undetectable 5 days later, suggesting that B220 is a transiently expressed activation marker on CD8+ T cells. Neither the B220+ V beta 8+ CD8+ T cells nor other V beta 8+ T cell populations converted with detectable frequency into B220+ DN T cells after single or multiple doses of SEB. B220+ DN T cells, which are functionally anergic in vitro, did not proliferate or undergo deletion after SEB stimulation indicating that these cells also are functionally impaired in vivo. In contrast to previous reports, chronic elimination of V beta 8+ T cells had no effect on the accumulation of B220+ DN T cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Soluble antigen-primed inducer T cells activate antigen-specific suppressor T cells in the absence of antigen-pulsed accessory cells: phenotypic definition of suppressor-inducer and suppressor-effector cells.

This study was undertaken to characterize interactions among human T cell subpopulations involved in the generation of suppressor T cells specific for a soluble antigen. Purified PPD-primed Leu-3+ cells, when co-cultured for 7 days with fresh autologous Leu-2+ cells, induced differentiation of Leu-2+ but not Leu-3+ cells into specific suppressor T cells, which subsequently inhibited the proliferative response of fresh Leu-3+ cells to PPD but not to tetanus toxoid or allogeneic non-T cells. The PPD-specific suppressor effect of activated Leu-2+ cells was not due to altered kinetics of the PPD response and also extended to the secondary response of PPD-primed Leu-3+ cells. Furthermore, only those Leu-2+ cells that lacked the 9.3 marker, an antigen present on the majority of T cells including the precursors of cytotoxic T cells, differentiated into suppressor T cells. To analyze the inducer population, fresh Leu-3+ cells were separated into Leu-3+,8- and Leu-3+,8+ subpopulations with anti-Leu-8 monoclonal antibody, activated with PPD, and then were examined for inducer function. Although both Leu-3+,8- and Leu-3+,8+ cells proliferated in response to PPD and upon activation expressed comparable amounts of HLA-DR (Ia) antigens, the Leu-3+,8+ subpopulation alone induced Leu-2+ cells to become suppressor-effectors in the absence of PPD-pulsed autologous non-T cells. Once activated, however, Leu-2+ suppressor cells inhibited the PPD response of both Leu-3+,8- and Leu-3+,8+ cells. These results indicate that antigen-primed Leu-3+,8+ inducer cells can directly activate Leu-2+, 9.3- precursors of antigen-specific suppressor T cells in the absence of antigen-pulsed autologous non-T cells.

Antigens, Bacterial↗
PubMed1995

Relationships between intermediate TCR cells and NK1.1+ T cells in various immune organs. NK1.1+ T cells are present within a population of intermediate TCR cells.

Experiments to date have revealed a population of T cells that carry intermediate (int) levels of TCR (or CD3) and express IL-2R beta-chain (IL-2R beta) in mouse liver. Such int TCR cells also reside in other immune organs, although in low numbers. On the other hand, NK1.1+ T cells with int TCR do reside in the thymus and other peripheral organs. To determine the relationship of two types of cells, we characterized int CD3 cells and NK1.1+ T cells throughout the organs in terms of the phenotype, V beta repertoire, and morphology. Although both IL-2R beta+ T cells and NK1.1+ T cells are classified as int CD3 cells, NK1.1+ T cells are present within int CD3 cells. The majority of int CD3 cells in the liver and thymus were NK1.1+, whereas the minority of such cells in the spleen, lymph nodes, and bone marrow were NK1.1+. Among int CD3 cells, double-negative (DN) CD4-8- cells and/or CD4+ were abundant in NK1.1+ subset, whereas CD8+ cells were generally abundant in NK1.1- subset. Self-reactive V beta+ clones estimated by the M1s system were distributed to both NK1.1+ and NK1.1- subsets. High CD3 cells in the thymus and other organs contained neither DN cells nor forbidden clones. Int CD3 cells had the morphology of granular or agranular lymphocytes carrying perforin. Among int CD3 cells, NK1.1+ subset had a higher level of perforin-positive cells than NK1.1- subset. These results clearly demonstrate the relationship between int TCR cells and NK1.1+ T cells in various organs.

Animals↗
PubMed1996

CD4+ T cell mediated destruction of xenografts within cell-impermeable membranes in the absence of CD8+ T cells and B cells.

Xenogeneic cells encapsulated in cell-impermeable diffusion chambers die within 3 weeks when implanted into immunocompetent animals but not when implanted into immunodeficient animals. To determine which cells are necessary for this observation, we depleted normal mice in vivo of either CD4+ or CD8+ T cells using monoclonal antibodies. We also reconstituted the immune system of athymic CBA mice (T-lymphocyte deficient) and C.B17 SCID mice (T- and B-lymphocyte deficient) with different cell subsets from normal CBA and BALB/C mice, respectively. Depleted or reconstituted mice were implanted with a diffusion chamber containing COS (monkey kidney) cells. Membrane enclosed xenografts survived in CD4+ T cell depleted mice but not in CD8+ T cell depleted or nondepleted control mice. Encapsulated xenografts survived when implanted into either athymic or SCID mice but were destroyed in reconstituted athymic and SCID mice. Furthermore, encapsulated xenogeneic cells were destroyed in athymic or SCID mice reconstituted with CD4+ cell preparations depleted of CD8+ cells and/or B cells. In contrast, encapsulated xenogeneic cells were not destroyed in athymic or SCID mice reconstituted with CD8+ cell preparations depleted of CD4+ cells. These studies highlight the critical role of CD4+ T cells, in the absence of CD8+ cells and B cells, in the processes leading to the ultimate destruction of encapsulated xenografts. Because of the use of cell-impermeable membranes in these studies, the most likely involvement of CD4+ T cells is in the indirect antigen recognition by these cells and subsequent stimulation of inflammatory cells.

Animals↗
PubMed1989

Cognate interactions between helper T cells and B cells. III. Contact-dependent, lymphokine-independent induction of B cell cycle entry by activated helper T cells.

An Ag-specific, IL-2-dependent Th clone induced the growth of B cells in a class II-restricted, Ag-specific, IL-2-dependent manner. The formation of stable Th-3.1-B cell conjugates was restricted by Ag and class II MHC. After activation of Th-3.1 by insolubilized anti-T3 (Th-3.1T3), Th-3.1T3 induced the growth of B cells in a class II unrestricted, Ag nonspecific manner. The formation of stable conjugates between Th-3.1T3 and B cells was also class II unrestricted and Ag nonspecific. Although the interaction of Th-3.1T3 and B cells was class II unrestricted, the interaction was inhibited by the combination of anti-IA and anti-IE mAb. This suggested that monomorphic domains of class II MHC molecules were involved in Th-3.1T3-B cell interaction. Fixed Th-3.1T3 but not fixed resting Th-3.1 induced B cell cycle entry, as measured by an increase in B cell RNA synthesis. Trypsin-treatment of Th-3.1T3 before fixation reduced their ability to activate B cells, indicating that cell surface proteins on Th-3.1T3 were required for enhanced B cell RNA synthesis. Anti-IL-4, anti-IL-2R, or anti-IFN-gamma did not affect the ability of Th-3.1T3 to induce heightened B cell RNA synthesis. Progression into S phase by B cells activated with fixed Th-3.1T3 was supported by the addition of soluble factors. When stimulated with fixed Th-3.1T3, EL4 supernatant (SN) enhanced B cell DNA synthesis. Depletion of IL-4, but not IL-2, from EL4 SN ablated its supportive capabilities. IL-4 alone was completely ineffective in supporting entry into S phase. Therefore, IL-4 and another activity(ies) in EL4 SN were necessary for B cell cycle progression into S phase. Taken together, these data suggest that after Th activation, Th cell surface proteins are expressed that mediate the binding of Th to B cells via recognition of nonpolymorphic domains of class II MHC molecules. Contact of Th-3.1T3 with B cells, not lymphokines, results in the entry of B cells into the cell cycle and heightened B cell lymphokine responsiveness. The addition of exogenous lymphokines supports the progression of Th-3.1T3-activated B cells into S phase.

Animals↗
PubMed1993

Redistribution of epidermal growth factor receptor as a function of cell density, cell-cell adhesion and calcium in human (A-431) cells.

Cell surface expression of the epidermal growth factor (EGF) receptor in several cell lines declines as a function of increased cell density and is associated with diminished responsiveness to EGF. However, the mechanism whereby this density-induced down regulation of receptors occurs has not been discerned. In the present study the distribution of the EGF receptor in A-431 cells as a function of cell density using (1) two polyclonal antibodies raised against peptide specific sequences of the EGF receptor that recognize either the cytoplasmic or extracellular domains of the receptor, respectively, and (2) biotinylated EGF, a specific probe for the cell surface receptor is now investigated. Immunolocalization of the receptor using the polyclonal antibodies or the biotin-EGF revealed that the receptor was homogeneously distributed on the cell surface of individual cells, or in cells plated at low density. In contrast, as cell density increased, prominent EGF immunoreactivity and biotin-EGF staining became limited to the periphery of the cells, at sites of cell-cell apposition, and was characterized by a honeycomb pattern, typical of a basolateral distribution. The effects of low Ca++ treatment, known to cause cells to round up and detach from one another, on EGF receptor distribution in cells at high cell density were then examined. Confocal microscopy of immunostained preparations revealed that incubation of high density cultures in Ca(++)-free media for as little as 10 min restored the homogeneous distribution of the EGF receptor and resulted in strong intracellular staining. Three-dimensional reconstruction of serial optical sections revealed that redistribution of the EGF receptor following low Ca++ treatment involved a heretofore undetected 'ruffling', an immunostaining pattern characterized by stripes of intense fluorescence signal interspersed with complete absence of fluorescence. Next, cell-cell adhesion was disrupted with antisera to the cell adhesion molecule E-cadherin. Although the antisera caused cells to detach from one another, eventually leading to cell rounding and redistribution of the EGF receptor, the receptor 'ruffling' immunostaining pattern rendered by the low Ca++ treatment was not detected. These results suggest that an association may exist between the plasma membrane EGF receptor distribution, density-induced EGF receptor down regulation, and the growth effects of low Ca++ observed in previous studies.

Amino Acid Sequence↗
PubMed2006

A novel cell division factor from tobacco 2B-13 cells that induced cell division in auxin-starved tobacco BY-2 cells.

Effects of auxin as plant hormones are widespread; in fact in almost all aspects of plant growth and development auxin plays a pivotal role. Although auxin is required for propagating cell division in plant cells, its effect upon cell division is least understood. If auxin is depleted from the culture medium, cultured cells cease to divide. It has been demonstrated in this context that the addition of auxin to auxin-starved nondividing tobacco BY-2 cells induced semisynchronous cell division. On the other hand, there are some cell lines, named habituated cells, that can grow without auxin. The cause and reason for the habituated cells have not been clarified. A habituated cell line named 2B-13 is derived from the tobacco BY-2 cell line, which has been most intensively studied among plant cell lines. When we tried to find the difference between two cell lines of BY-2 and 2B-13 cells, we found that the addition of culture filtrated from the auxin-habituated 2B-13 cells induced semisynchronous cell division in auxin-starved BY-2 cells. The cell division factor (CDF) that is responsible for inducing cell division in auxin-starved BY-2 cells was purified to near-homogeneity by sequential passage through a hydroxyapatite column, a ConA Sepharose column and a Sephadex gel filtration column. The resulting purified fraction appeared as a single band of high molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels by silver staining and was able to induce cell division in auxin-starved BY-2 cells. Identification of the protein by MALD-TOF-MS/MS revealed that it is structurally related to P-glycoprotein from Gossypioides kirkii, which belongs to ATP-binding cassette (ABC)-transporters. The significance of CDF as a possible ABC-transporter is discussed in relationship to auxin-autotrophic growth and auxin-signaling pathway.

Amino Acid Sequence↗
PubMed1991

Proliferating cells in psoriatic dermis are comprised primarily of T cells, endothelial cells, and factor XIIIa+ perivascular dendritic cells.

Determination of the cell types proliferating in the dermis of patients with psoriasis should identify those cells experiencing activation or responding to growth factors in the psoriatic dermal milieu. Toward that end, sections of formalin-fixed biopsies obtained from 3H-deoxyuridine (3H-dU)-injected skin of eight psoriatic patients were immunostained, followed by autoradiography. Proliferating dermal cells exhibit silver grains from tritium emissions. The identity of the proliferating cells could then be determined by simultaneous visualization with antibodies specific for various cell types. UCHL1+ (CD45RO+) T cells (recall antigen-reactive helper T-cell subset) constituted 36.6 +/- 3.1% (mean +/- SEM, n = 6) of the proliferating dermal cells in involved skin, whereas Leu 18+ (CD45RA+) T cells (recall antigen naive T-cell subsets) comprised only 8.7 +/- 1.5% (n = 6). The Factor XIIIa+ dermal perivascular dendritic cell subset (24.9 +/- 1.5% of proliferating dermal cells, n = 6) and Factor VIII+ endothelial cells (23.0 +/- 2.3%, n = 6) represented the two other major proliferating populations in lesional psoriatic dermis. Differentiated tissue macrophages, identified by phase microscopy as melanophages or by immunostaining with antibodies to Leu M1 (CD15) or myeloid histiocyte antigen, comprised less than 5% of the proliferating population in either skin type. In addition to calculating the relative proportions of these cells to each other as percent, we also determined the density of cells, in cells/mm2 of tissue. The density of proliferating cells within these populations was increased in involved versus uninvolved skin: UCHL1+, 9.0 +/- 1.7 cells/mm2 versus 1.8 +/- 0.6 cells/mm2, p less than 0.01; Factor XIIIa+, 6.0 +/- 0.7 cells/mm2 versus 1.5 +/- 0.5 cells/mm2, p less than 0.01; Factor VIII+, 5.5 +/- 1.4 cells/mm2 versus 0.0 cells/mm2, p less than 0.05. The presence of preferential active proliferation of a T-cell subset in lesional dermis suggests that activating signals specific for this subset are contained within the psoriatic dermis in vivo. The activation of recall antigen-reactive T cells may be a driving force behind the dendritic cell and endothelial cell proliferation. Alternatively, the selective proliferation and expansion of these two constitutive cell types (Factor XIIIa+ and Factor VIII+) may result in signals that promote activation of UCHL1+ (CD45RO+) T cells.

Cell Division↗
PubMed2006

Induction of autologous CD4- and CD8-mediated T-cell responses against acute lymphocytic leukemia cell line using apoptotic tumor cell-loaded dendritic cells.

OBJECTIVE: Several studies have demonstrated that dendritic cells (DCs) pulsed with tumor lysate or apoptotic tumor cells can elicit effective T-cell responses. This technique does not require the identification of the tumor antigen or HLA haplotype of the patient. We applied this approach to induce HLA class I- and class II-restricted T-cell responses directed against autologous acute lymphocytic leukemia (B-ALL) cell line NH-1. METHODS: Autologous T cells were stimulated by apoptotic tumor cell-loaded DCs generated from a patient with ALL. The stimulated and expanded T cells were isolated into CD8(+) T-cell line and CD4(+) T-cell line, and each of them was examined as to their functions. RESULTS: Both CD8(+) and CD4(+) T-cell lines demonstrated cytotoxicity against NH-1 in an major histocompatibility complex-dependent manner. Finally, we established two independent CD4(+) T-cell clones restricted to HLA-DR. The CD4(+) T-cell line responded strongly to autologous Epstein-Barr virus-transformed lymphoblastoid cell lines (EBV-LCL) but not to autologous normal cells. Furthermore, the T-cell clones also responded to allogeneic EBV-LCLs and B-ALL cell lines in the context of the HLA-DRB1( *)04051 molecule. Interestingly, 293T and COS-7 cells, which had been transfected with the HLA-DRB1( *)04051, were also recognized by T-cell clones. CONCLUSION: These findings indicate that B-ALL has shared and strong immunogenic epitopes expressed on HLA class II molecules, the expression of which is limited to immortalized cells. These data suggest that vaccinations using DCs loaded with apoptotic tumor cells might be a potent strategy in the treatment of B-ALL.

Apoptosis↗
PubMed2001

Generation in vitro of B-cell chronic lymphocytic leukaemia-proliferative and specific HLA class-II-restricted cytotoxic T-cell responses using autologous dendritic cells pulsed with tumour cell lysate.

Immunotherapy using dendritic cells has shown encouraging results in both haematological and non-haematological malignancies. In this study, monocyte-derived dendritic cells from patients with B-CLL were cultured for 6 days in the presence of IL-4 and GM-CSF. Autologous B-CLL T-cells were cultured alone or with B-CLL lysate-pulsed and unpulsed autologous dendritic cells. IFN-gamma secretion was assessed using ELISA. Cytotoxicity was assessed, after 21 days in culture and re-stimulation, using flow cytometry with and without blockade by anti-HLA class I, anti-HLA class II, anti-CD4, anti-CD8 and anti-TCRalphabeta monoclonal antibodies. B-CLL T cells stimulated with B-CLL lysate-pulsed autologous dendritic cells showed a significant (P = 0.0004) increase in IFN-gamma secretion and a significant (P = 0.0008) increase in specific cytotoxicity to autologous B-cell targets, but none to autologous T cell or B cell targets from healthy individuals. B-CLL T cells cultured with (non-B-CLL) B-cell lysate-pulsed B-CLL dendritic cells showed no significant response. Pulsing dendritic cells from healthy volunteers with an autologous (non-B-CLL) B-cell lysate did not stimulate proliferation, cytokine production or cytotoxicity by autologous T cells. Pulsing B-CLL dendritic cells with allogeneic B-CLL lysates and culturing with autologous T-cells elicited cytotoxicity against autologous B-CLL targets in some cases, but not in others. Cytotoxicity was significantly reduced by blocking with anti-HLA class II (P = 0.001), anti-TCRalphabeta (P = 0.03) and anti-CD4 (P = 0.046) antibodies. Phenotyping of the responding T-cell population demonstrated the majority to be CD4 positive. Our data demonstrate that HLA class II-restricted proliferative and cytotoxic T-cell responses to B-CLL can be generated using autologous dendritic cells pulsed with tumour cell lysate.

Antibodies, Monoclonal↗
PubMed2001

Cell death mediated by alloreactive cytotoxic T cells via the granule exocytosis or the Fas pathway is independent of p34cdc2 kinase: Fas dependent killing of cells arrested in the cell cycle.

Inappropriate activation of p34cdc2 kinase has been shown to occur during apoptosis induced by cytotoxic T-cell derived perforin and fragmentin. We analysed the effect of two inhibitors of p34cdc2 kinase on alloreactive Tc-cell-mediated lysis and DNA fragmentation of P815 and L1210 target cells. Olomoucine, a specific inhibitor of cyclin dependent kinases, did not affect DNA fragmentation in the target cells. Lysis of olomoucine-treated target cells as assessed by 51Cr release over a typical 8-h period was also unaffected. We also examined the effects of thapsigargin on target cell death. This toxin causes increased intracellular calcium rises that then result in irreversible inhibition of cyclin dependent kinases, including p34cdc2 kinase. The same extent of specific cell lysis was induced by cytotoxic T cells from perforin(-/-), granzyme B(-/-), granzyme A(-/-), perforin(-/-) X granzymeB(-/-) X granzymeA(-/-) KO mice or normal mice in untreated target cells or target cells treated with either olomoucine or thapsigargin. Similarly DNA fragmentation measured by release of tritiated DNA was also unaffected. Thus inhibition of p34cdc2 kinase affects neither the Fas nor the perforin/granzyme pathways of alloreactive cytotoxic T-cell killing as measured by DNA fragmentation or chromium release. P815 cells treated with olomoucine were arrested in the cell cycle after 12-16 h exposure to the toxin. After cell cycle arrest, target cells now showed enhanced 51Cr release induced by effector cytotoxic T cells (CTL) derived from perforin(-/-) mice compared to untreated cells. This lysis was accompanied by an increase in cell surface Fas expression. Olomoucine induced cell cycle arrest and expression of Fas was reversible and when cells re-entered the cell cycle, surface expression of Fas was lost.

Animals↗
PubMed1998

Isolation of human conjunctival mast cells and epithelial cells: tumor necrosis factor-alpha from mast cells affects intercellular adhesion molecule 1 expression on epithelial cells.

PURPOSE: To isolate and purify mast cells and epithelial cells from human cadaveric donor conjunctival tissue and to characterize interactions between these cell types in vitro. METHODS: Monodispersed cell suspensions obtained by enzymatic digestion of conjunctival tissue were applied to a single-density Percoll gradient. Epithelial cells obtained from the top layer of the gradient were cultured to confluence. Mast cells obtained from the pellet were equilibrated in culture medium and further purified using a two-step Percoll gradient. Using reverse transcription-polymerase chain reaction (RT-PCR), RNA from the purified mast cell preparation was probed for tumor necrosis factor-alpha (TNF alpha) message. Fluorescence activated cell sorting (FACS) analysis of intracellular immunostained mast cells was used to detect the TNF alpha protein. An examination for intercellular adhesion molecule 1 (ICAM-1) on epithelial cells was performed after 24-hour incubations with either recombinant TNF alpha supernatants from calcium ionophore A23187 (CaI)-stimulated mast cells or appropriate controls using FACS analysis. RESULTS: Highly purified human conjunctival mast cells and epithelial cells (each > 95%) were obtained from human cadaveric donor tissue. RT-PCR analysis of purified mast cell RNA revealed the expression of TNF alpha mRNA. An evaluation of mast cells for intracellular protein demonstrated positive staining for tryptase and TNF alpha. ICAM-1 was found on purified epithelial cells, and incubation of epithelial cell monolayers with supernatants from Cal-stimulated mast cells resulted in upregulation of this receptor. This upregulation was blocked by incubation with TNF alpha-neutralizing antibody. CONCLUSIONS: This work provides the methods for isolating and purifying mast cells and epithelial cells from human donor tissue and the opportunity for studying mechanisms of conjunctival inflammation by evaluating the interactions between these cells.

Calcimycin↗
PubMed1995

Differential expression of the T cell receptor/CD3 genes and their lymphoid-specific transcription factor genes in murine T cell x fibroblast and T cell x B cell hybrids.

We generated cell hybrids between mouse T cell lymphoma EL4 cells and mouse fibroblast B82 cells (BELIII and BELIV) to examine the expression of T cell receptor (TcR)/CD3 genes and their lymphoid-specific transcription factor genes, which are normally detected in EL4 cells. In BELIII and BELIV, expression of the TcR alpha, TcR beta and CD3 delta genes was extinguished, whereas expression of the CD3 epsilon gene was still detected. Expression of the (lymphoid enhancer binding factor 1) LEF-1 gene was extinguished and that of the GATA-3 gene was hardly detected in BELIII and BELIV. Ets-1 gene expression, observed not only in EL4 cells but also in B82 cells, was considerably reduced in BELIII and BELIV. A much higher level of PEBP2 alpha A gene expression was observed in B82 cells than in EL4 cells and was preserved in BELIII and BELIV. To examine whether reduced expression of these genes is also found in T cell x B cell hybrids, we generated an additional cell hybrid between EL4 cells and mouse plasmacytoma S194 cells (SELIII). Marked differences were observed in the expression of the TcR alpha, CD3 delta, LEF-1 and PEBP2 alpha A genes in BEL and SEL hybrids. Expression of the TcR alpha, CD3 delta and LEF-1 genes, which was extinguished in BELIII and BELIV, was detected in SELIII. PEBP2 alpha A gene expression, not detected in S194 cells, was considerably reduced in SELIII. Almost the sum of the chromosomes from the parental cells were retained by, and the presence of every gene was proven, in each cell hybrid. These results suggest that suppression of the expression of lymphoid-specific transcription factor genes may precede that of the TcR/CD3 genes in the cell hybrids, and that the presence of a different trans-acting negative regulatory mechanism(s) suppresses the expression of T cell specific genes in fibroblasts and B cells.

Animals↗
PubMed1998

Mouse mammary carcinoma cell line (BALB/c-MC) stimulates osteoclast formation from mouse bone marrow cells through cell-to-cell contact.

We recently reported that numerous osteoclasts (OC) were formed in cocultures of some mouse cancer cell lines and bone marrow cells. In this study, we examined mechanisms by which one of the cell lines, BALB/c-MC, induces OC. BALB/c-MC dose dependently stimulated OC formation in cocultures. In cocultures where direct cell-to-cell contact between BALB/c-MC and bone marrow cells was inhibited by membrane filters, OC formation was not stimulated. The stimulation of OC formation in the coculture was completely abolished by adding 10(-7)-10(-6) mol/L indomethacin. The concentration of prostaglandin E2 (PGE2) in the culture media of cocultures with cell-to-cell contact was higher than that of cocultures without cell-to-cell contact or marrow cultures alone, and it reached levels sufficient to induce OC (11.9 +/- 5.3 ng/mL [about 3.4 x 10(-8) mol/L]). When BALB/c-MC or bone marrow cells were fixed with formalin and then cocultured with bone marrow cells or BALB/c-MC, respectively, the concentration of PGE2 in the culture media of cocultures of fixed BALB/c-MC and bone marrow cells increased, whereas that of cocultures of BALB/c-MC and fixed bone marrow cells did not increase. These results indicate that BALB/c-MC stimulate OC formation through direct cell-to-cell contact with bone marrow cells, and PGE2 released from bone marrow cells through direct cell-to-cell contact are involved in OC formation by the cell line.

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