Inhibition of colony formation by a human tumor degenerating factor (TDF); enhancement of this effect by interferons

Human tumor-degenerating factor (TDF), which is produced in the culture of human fibroblasts, causes the degenerative changes of the tumor cells and forms a cell-free area on the surface of culture dish. Its degenerating activity is enhanced by the treatment with interferons. In the present study, it was recognized that TDF preparation inhibited the colony formation of KB cells according to the TDF activity. When recombinant human leukocyte interferon (rHuIFN-alpha A) was added to the KB cell culture, the colony formation was inhibited. The combination of TDF preparation with rHuIFN-alpha A inhibited more strongly the colony formation than TDF alone or rHuIFN-alpha A alone. It was found that the combination of TDF preparation with rHuIFN-gamma inhibited more remarkably the colony formation than the combination of TDF preparation with rHuIFN-alpha A.     

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.     

Induction of antibody responses to influenza virus in human lymphocyte cultures. I. Role of interleukin 2

The in vitro T cell-dependent antibody response of human lymphocytes to influenza virus X31 was used to study the role of T cell-derived lymphokines in antigen-specific responses. Supernatant from cultures of phytohaemagglutinin-stimulated, pooled human tonsil cells (PHA-MLR) was capable of replacing T cells and inducing T-depleted tonsil cells to secrete influenza-specific antibody. The T cell-replacing activity of PHA-MLR supernatant co-purified with interleukin 2 (IL 2) on Ultrogel AcA54 gel filtration and reversed phase-high performance liquid chromatography. PHA-MLR supernatant and IL 2 also enhanced B cell proliferation induced by anti-mu or Staphylococcal aureus strain Cowan I (SAC). A murine monoclonal antibody directed against the human IL 2 receptor (Mab 2A3) was used to completely block the enhancement of influenza-specific antibody production mediated by PHA-MLR supernatant, purified IL 2, and recombinant human IL 2. Mab 2A3 did not affect the T-independent B cell proliferation induced by anti-mu or SAC, but abrogated the enhancing effect of the PHA-MLR supernatant and IL 2 in this culture system. Immunofluorescence studies failed to demonstrate binding of Mab 2A3 to B cells activated by the X31 influenza virus and IL 2, or by SAC. By using Mab 2A3 to mask out IL 2 effects in the influenza-specific culture system, no other B cell differentiating activities were revealed in supernatants from lymphocytic cultures stimulated with a variety of mitogens. Thus, our results indicate that the production of influenza-specific antibodies by T-depleted human lymphocyte cultures is absolutely dependent on the presence of both antigen and IL 2.     

Effect of interferons on the activity of human tumor degeneration factor (TDF)

KB cells were cultivated in the well with tumor-degenerating factor (TDF) and the natural and recombinant interferons. TDF alone induced the degenerative changes of the KB cells and formed the cell-free area, but the interferons alone did not induce the changes and did not form the cell-free area. When KB cells were cultivated with TDF and natural interferons (HuIFN-alpha, -beta and -gamma), the cell-free area was enlarged. Particularly, HuIFN-gamma enhanced the TDF activity most strongly. The recombinant interferons (HuIFN-alpha, -beta and -gamma) also augmented in the same ways as the natural interferons.     

Mitogen response and cell cycle kinetics of Swiss 3T3 cells in defined medium: differences from human fibroblasts and effects of cell density

The mitogen requirement and proliferative response of Swiss 3T3 cells in serum-free, chemically defined culture medium were compared with those of early-passage human diploid fibroblasts. The effects of platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin, transferrin, and dexamethasone on cell-cycle parameters were measured using 5'-bromo-deoxyuridine-Hoechst flow cytometry. Swiss 3T3 cells differ from human fibroblasts in several ways: (1) Swiss 3T3 cells showed a much higher dependence on PDGF than human fibroblasts; the growth of the latter, but not of the former, could be stimulated by the combination of EGF, insulin, and dexamethasone to the full extent of that when PDGF was present; (2) in the absence of PDGF, insulin was an absolute requirement for Swiss 3T3 cells to initiate DNA synthesis, while a substantial proportion of human fibroblasts could enter DNA synthesis without exogenous insulin or IGF-I; and (3) in the absence of PDGF, increasing insulin concentration increased the cycling fraction of Swiss 3T3 cells without an appreciable effect on the rate of cell exit from G0/G1, while under similar culture conditions, insulin showed its major effect on regulation of the G1 exit rate of human fibroblasts, without much effect on the cycling fraction. In addition, the proliferative response of high-density versus low-density, arrested Swiss 3T3 cells showed that the interaction of mitogens varied with cell density. At high cell density, the PDGF requirement was consistent with the "competence/progression" cell-cycle model. This growth response was not seen, however, when cells were plated at low density.     

Mutual inhibitory activities of tumor-degenerating factor (TDF) and fibronectin

It was reported in our previous studies that the "spongy degeneration-like" changes of human tumor cells were detected by coculture with human embryonic fibroblasts in vitro. It was discovered that the degenerative changes were mediated by a factor secreted from human embryonic fibroblasts. This factor was named the tumor-degenerating factor (TDF). The present study found that fibronectin inhibited TDF activity while TDF inhibited cell attachment mediated by fibronectin. It was possible that these mutual inhibitions were due to the direct binding of the TDF molecule to the fibronectin molecule. Since it is well known that fibronectin is composed of multiple domains which differ in their biological activities, this study also attempted to clarify which domain(s) inhibit TDF activity, through the use of trypsin, thermolysin and 2-nitro-5-thiocyanobenzoic acid (NTCB). It is concluded that multiple domains of fibronectin are required for the inhibition of TDF activity.     

Crosstalk between Cancer Cells and Fibroblasts for the Production of Monocyte Chemoattractant Protein-1 in the Murine 4T1 Breast Cancer

The chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2) is shown to promote the progression of breast cancer. We previously identified cancer cell-derived granulocyte-macrophage colony-stimulating factor (GM-CSF) as a potential regulator of MCP-1 production in the murine 4T1 breast cancer, but it played a minimum role in overall MCP-1 production. Here, we evaluated the crosstalk between 4T1 cells and fibroblasts. When fibroblasts were co-cultured with 4T1 cells or stimulated with the culture supernatants of 4T1 cells (4T1-sup), MCP-1 production by fibroblasts markedly increased. 4T1 cells expressed mRNA for platelet-derived growth factor (PDGF)-a, b and c, and the PDGF receptor inhibitor crenolanib almost completely inhibited 4T1-sup-induced MCP-1 production by fibroblasts. However, PDGF receptor antagonists failed to reduce MCP-1 production in tumor-bearing mice. Histologically, 4T1 tumors contained a small number of αSMA-positive fibroblasts, and Mcp-1 mRNA was mainly associated with macrophages, especially those surrounding necrotic lesions on day 14, by in situ hybridization. Thus, although cancer cells have the capacity to crosstalk with fibroblasts via PDGFs, this crosstalk does not play a major role in MCP-1 production or cancer progression in this model. Unraveling complex crosstalk between cancer cells and stromal cells will help us identify new targets to help treat breast cancer patients.     

Cryoprotective effects of D-allose on mammalian cells

D-allose, an aldo-hexose, is a rare sugar whose biological functions remain largely unclear. Recently, we demonstrated a novel inhibitory effect of D-allose on production of reactive oxygen species (ROS). Here, we focused on investigating cryoprotective effects of D-allose on cell viability. Mammalian cell lines including OVCAR-3 (human ovarian cancer), HeLa (human cervical cancer), HaCaT (human skin keratinocytes), HDF (human dermal fibroblasts) and NIH3T3 (murine fibroblasts) cells were frozen at -80 degrees C in culture media with various D-allose concentrations. Cells were allowed to recover for 24 h, 1 week or 1 month prior to survival assessment using the trypan blue dye exclusion test, when cell proliferation was evaluated by MTT assay. A beneficial protective role of D-allose on cell survival was found, similar to that of trehalose (disaccharide of glucose), a recognized cryoprotectant. The results suggest that D-allose as a sole additive may provide effective protection for mammalian cells during freezing. Practical studies now need to be performed with D-allose, for example to determine optimal freezing protocols and explore potential for preservation of tissues or organs at non-freezing temperatures.     

人胎肝基质细胞培养上清液中造血生长因子的分析

采用人胎肝造血基质细胞的体外液体培养技术,结合造血干细胞和祖细胞的体外测试方法,研究了造血基质细胞所释放的造血生长因子与造血干细胞和祖细胞之间的相互作用。结果表明,在适宜的条件下,人胎肝造血基质细胞可在体外传代培养达100d之久。培养过程中,对不同时间收集的培养上清液进行测试的结果表明,这些贴壁细胞可以不断地释放多种造血活性物质。在100d培养过程中,上清液中始终都可以检出CFU-S增殖刺激物活性。培养第24天的上清液中还可检出BPF和GM-CSF活性。这些造血活性物质对CFU-S的生理状态和祖细胞的增殖与分化有着深刻的影响。但是在培养上清液中未检出IL-3样活性物质。     

In vitro production of anti-histone antibodies by spleen cells from young autoantibody negative NZB/NZW mice

Anti-histone antibodies (AHA) are spontaneously produced in NZB/NZW mice as part of their autoimmune disease. IgM AHA are usually not detected until after 4 mo of age, and older female mice switch to the production of IgG AHA. We studied the in vitro production of AHA by spleen cells from young (less than or equal to 12-wk-old) NZB/NZW mice. Despite the absence of elevated serum AHA activity, spleen cells from these mice demonstrated marked spontaneous autoantibody production in culture. In kinetic studies, little in vitro production was detectable after 1 day of culture, and maximal accumulation occurred on day 5. Elevated AHA production was apparent by cells from 2-wk-old NZB/NZW mice, and an age-dependent increase in autoantibody production was also noted. Only AHA of the IgM class were detected in cultures of young spleen cells. The in vitro production of IgM AHA in culture was T cell dependent, depletion of T cells resulting in a 70 to 90% reduction in production, which was corrected by the readdition of T cells. In cultures where both IgM AHA and total IgM secretion were measured, a much greater T cell dependence for AHA production was apparent. The requirement for T cells could also be partially replaced by factors present in concanavalin A supernatant. AHA secretion was induced by lipopolysaccharide by using cells from both NZB/NZW and non-autoimmune mice. Although production was greater with NZB/NZW cells, the difference was much less than that for spontaneous production. Thus, AHA-secreting cells that are dependent on in vitro T cell help are present in young NZB/NZW mice. These studies may help define the mechanisms responsible for selective autoantibody secretion in lupus-like disease.     

Stimulation of sugar uptake in cultured fibroblasts by epidermal growth factor (EGF) and EGF-binding arginine esterase.

Mouse epidermal growth factor causes a rapid increase in 2-deoxyglucose uptake in stationary phase mouse (3T3) cells or human fibroblasts. Maximum effect is approximately two fold over control levels for 3T3 cells and about 50% over controls for human fibroblasts. Maximum effect on 3T3 cells is seen about two hours after addition of 10 ng/ml EGF to the culture medium. Stimulation is easily measureable within the first fifteen minutes after addition of the hormone and may be detected at hormone concentrations as low as 0.1 ng/ml. The EGF-binding arginine esterase found associated with EGF in the mouse submaxillary gland causes an enhancement of the EGF effect. In serum-free medium, the EGF effect is still readily observed, but no enhancement by the esterase is seen. SV40 virus-transformed 3T3 cells show no effect on deoxyglucose uptake after addition of 10 ng/ml EGF to the culture medium, but a response may be demonstrated after these cells are incubated for 12 hours or more in serumless medium. EFG stimulates transport of 3-O-methylglucose in stationary phase 3T3 and human fibroblasts but no EGF stimulation of alpha-amino-isobutyrate uptake in 3T3 cells is seen under conditions is reproted to inhibit intracellular degradation of human EGF by human fibroblasts, does not diminish the EGF effect on deoxyglucose uptake in human fibroblasts.     

IgE class-specific suppressor T cells and factors in humans

An in vitro experimental system for the induction of IgE production has been established with human peripheral blood lymphocytes (PBL). The stimulation of human PBL with PWM plus Cowan I induced IgM-, IgG-, and IgE- producing cells when assessed by reverse plaque assay. T cells, which had been isolated from patients with pulmonary tuberculosis and incubated with PPD plus IgE for 5 days, showed a suppressive effect on the polyclonal IgE response induced by PWM plus Cowan I. The direct addition of activated T cells, as well as the culture supernatant from activated T cells, abrogated the IgE response; the IgG response was not affected. The IgE-specific suppressive activity in the supernatant was specifically absorbed by an IgE column and could be eluted with acid buffer. The results demonstrated the presence of a human IgE binding factor(s), which showed its suppressive effect selectively in the IgE antibody response.     

TGFbeta1 regulation and collagen-release-independent connective tissue re-modelling by the ruthenium complex NAMI-A in solid tumours

PURPOSE: The objective of this study is to evaluate the fibrotic process induced in vivo by NAMI-A in mice with solid tumours. In addition, the in vitro effects of NAMI-A on collagen fibres and the expression of TGFbeta1 in TS/A adenocarcinoma cells, NIH/3T3 fibroblasts and co-culture of fibroblasts and tumour cells have also been studied. METHODS: Collagen fibres release was assayed in supernatant of culture cells treated with 0.1 and 0.01 mM NAMI-A. TGFbeta1 was detected by RT-PCR and immunoblot on cellular lysates. RESULTS: NAMI-A, given to mice bearing MCa mammary carcinoma at advanced stages of growth, increased the thickness of connective tissue and induced recruitment of leukocytes, particularly in the peritumour capsule. In vitro NAMI-A stimulated collagen production by NIH/3T3 fibroblasts and decreased collagen release by TS/A tumour cells after prolonged exposure, either after single cell treatment or in co-cultures. In co-cultures, NAMI-A, in a dose-dependent manner, down-regulated the expression of TGFbeta1 mRNA and protein in tumour cells and up-regulated it in fibroblasts. The isoform of this cytokine is involved in fibrosis, invasion and metastatic processes. CONCLUSIONS: These data emphasize the ability of NAMI-A to evoke beneficial effects from healthy cells against tumour growth and metastases. The contribution of fibroblasts to the fibrosis arising in tumour masses is due to TGFbeta1, and its down-regulation in tumour cells might explain the documented reduction of gelatinase release.     

Neutrophil elastase treated dendritic cells promote the generation of CD4(+)FOXP3(+) regulatory T cells in vitro

We have previously shown that neutrophilic elastase converts human immature dendritic cells (DCs) into TGF-β secreting cells and reduces its allostimulatory ability. Since TGF-β has been involved in regulatory T cells (Tregs) induction we analyzed whether elastase or neutrophil-derived culture supernatant treated DCs induce CD4⁺FOXP3⁺ Tregs in a mixed lymphocyte reaction (MLR). We found that elastase or neutrophil-derived culture supernatant treated DCs increased TGF-β and decreased IL-6 production. Together with this pattern of cytokines, we observed a higher number of CD4⁺FOXP3⁺ cells in the MLR cultures induced by elastase or neutrophil-derived culture supernatant treated DCs but not with untreated DCs. The higher number of CD4⁺FOXP3⁺ T cell population was not observed when the enzymatic activity of elastase was inhibited with an elastase specific inhibitor and also when a TGF-β1 blocking antibody was added during the MLR culture. The increased number of CD4⁺ that express FOXP3 was also seen when CD4⁺CD25^- purified T cells were cocultured with the TGF-β producing DCs. Furthermore, these FOXP3⁺ T cells showed suppressive activity in vitro.These results identify a novel mechanism by which the tolerogenic DCs generated by elastase exposure contribute to the immune regulation and may be relevant in the pathogenesis of several lung diseases where the inflammatory infiltrate contains high numbers of neutrophils and high elastase concentrations.     

Characterization of an antigen-specific suppressive factor derived from a cloned suppressor effector T cell line

A cloned effector-type suppressor T cell line, 3D10, which is known to suppress the antibody response against dinitrophenylated keyhole limpet hemocyanin (KLH), produced a soluble KLH-specific factor (TsF) that can replace the function of parental T cell clones. High activity of TsF was released spontaneously into the culture supernatant when cultured in interleukin 2 (IL 2)-containing medium, requiring no antigenic stimulation. The culture supernatant of 3D10 was also capable of inhibiting the KLH-induced proliferative response of primed T cells in an antigen-specific manner. The direct target of TsF was found to be Lyt-1+2- T cells undergoing an early stage of antigen-specific proliferation. TsF was antigen binding but lacked any other serologic markers such as I-J and immunoglobulin heavy chain-linked allotypic determinants on T cells. No genetic restriction was found in its action on allogeneic T cells. The production of IL 2 in proliferative T cells by antigenic stimulation was not inhibited by TsF. These results indicate that the TsF described here is the legitimate mediator produced by the effector-type suppressor T cell that suppresses the antigen-specific responses of Lyt-1+2- T cells. The m.w. of TsF was approximately 75,000.     

Micro-heterogenous expression of peanut agglutinin-binding sites in the extracellular matrix of cultured cells

Using double-label techniques with fluorochrome-conjugated peanut agglutinin (PNA) and indirect immunofluorescence with rabbit species-specific anti-fibronectin antibodies and a mouse monoclonal anti-fibronectin, the extracellular matrix (ECM) of cultured human and mouse fibroblasts (Hell7 and 3T3K) and human bladder epithelial cells (T24) was studied. The antibodies and PNA co-localized extensively. However, a small but consistent degree of micro-heterogeneity was revealed insofar as both PNA-positive fibronectin-negative fibrils as well as PNA-negative fibronectin-positive fibrils were observed. Fibronectin production by T24 cells (but not fibroblasts) was influenced by the growth medium, but this did not affect the heterogeneity. Trypsin removed most cell-surface fibronectin and all PNA-binding sites, but did not account for the observed phenomenon. Intracellular fibronectin, whether present naturally or induced to accumulate by culture in presence of Monensin, was PNA-negative. These data suggest that PNA-binding sites appear on fibronectin as a consequence of incorporation into the extracellular matrix, and that the resultant heterogeneity of spatial expression of beta-galactose-like residues may offer a mechanism whereby mesenchymal cells could modulate the behaviour of overlying cell-types.     

In vitro stimulation of natural killer (NK) cells by soluble factor(s) generated in antigen-stimulated rhesus monkey peripheral blood lymphocyte cultures.

Peripheral blood lymphocytes from rhesus monkeys (Macaca mulatta) sensitized to keyhole limpet hemocyanin (KLH), when stimulated in vitro with KLH, developed natural killer (NK) cell activity that was assayed with Rous Sarcoma virus-transformed marmoset fibroblasts as targets in a 4-hr 51Cr-release assay. The supernatant fluids from 24- to 25-hr KLH-activated cultures were capable of stimulating NK development in nonsensitive lymphocyte cultures. The effector cells were neither macrophages nor B cells (plastic and nylon-wool nonadherent) and did not form E-rosettes with neuraminidase-treated sheep red blood cells. Cultures depleted of EA-rosetting cells, i.e., Fc-receptor-bearing lymphocytes, were incapable of generating NK activity when stimulated in vitro. Kinetic studies showed that peak DNA synthesis, as measured by 3H-T incorporation, preceded maximum cytotoxicity. Elimination of dividing cells by 5-bromo-2'deoxyuridine (BrdU) and light treatment during the interval from day 1 to day 4 inhibited the development of cytotoxicity on day 7. Cell replication was required for the induction of NK cells with KLH as well as with antigen-activated culture supernatant fluids. When cultures were left unstimulated for 4 days, NK activity could not be developed subsequently either by adding antigen, mitogen (PHA), or supernatant fluids from activated cultures.     

Ligand-activated T cell growth factor-induced proliferation: absorption of T cell growth factor by activated T cells.

The fate in culture of the T cell growth factor (TCGF), which is required for continued growth of human cultured T cells (CTC) in vitro, was studied. TCGF activity was stable for 7 days at 37 degrees C. However, it was no longer detectable after incubation with actively growing CTC at 37 degrees C for 3 days. This loss of TCGF activity also occurred quite rapidly and was detectable within 1 hr of incubation of 0.3 ml supernatant with 2 to 5 x 10(7) CTC at 23 degrees C. 2 x 10(8) mononuclear peripheral blood leukocytes were not effective in removing TCGF activity, and incubation with similar numbers of cells from B and T cell lines had no effect. Three-day-old concanavalin A and phytohemagglutinin blasts were very reactive with TCGF, so that 10(7) or 2 x 10(7) cells consistently removed TCGF activity. These experiments suggested specific absorption of TCGF by activated T cells, and led us to develop a model of ligand-activated TCGF-induced proliferation of T cells: Ligands induce production of TCGF by T-producer cells and deliver a first signal to the T-responder cells. This causes a receptor for TCGF to appear on T-responder cells. Only then does TCGF deliver the obligatory second signal that is needed to drive the T-responder cells into proliferation.