Function and regulation of SRBC-induced contrasuppressor T cells which modulate suppression of MOPC-315 cell secretory differentiation in vivo and in vitro

Supported by NIH Research Grants CA-28708 (J.W.R.), CA-35654 (J.W.R.), and CA-37252 (J.D.K.).     

Indomethacin stimulation of macrophage cytostasis against MOPC-315 tumor cells is enhanced by endogenous metabolites of lipoxygenase and counteracted by prostaglandin E2

Conclusions We have shown that indomethacin stimulation of macrophage cytostasis against MOPC-315 tumor cells can be counteracted by PGE₂ and by the lipoxygenase inhibitor NDGA. The results are congruent with the earlier finding, which showed that LTD₄ reinforces the effect of indomethacin on macrophage cytostasis, which thus is modulated by the balance between cyclooxygenase and lipoxygenase metabolites.     

Leukotriene C4 decreases the activity of respiratory cilia in vitro

In the present study we assessed the effect of leukotriene C4 on ciliary beat frequency in vitro. Chicken tracheas were sliced into thin rings and ciliary activity was viewed microscopically. Ciliary beat frequency was measured through an optical fibre by a fast Fourier transformer analyser and recorded on an oscilloscope. Ciliary beat frequency was measured at one fixed point during 30 min at room temperature in RPMI-HEPES medium alone and in medium containing leukotriene C4 at a range of concentrations (10(-6)-10(-9) M). Our results demonstrate that leukotriene C4 (10(-7) and 10(-8) M) caused a significant decrease in ciliary beat frequency in all tracheal rings tested (mean decrease of 24%). The specificity and Ca2(+)-dependency of leukotriene C4 activity was elucidated by the ability of FPL 55712 (SRS-A specific antagonist), Ca2(+)-free medium, calcium channel blockers (nifedipine and verapamil) and the calmodulin inhibitor trifluroperazine to abrogate its effect on ciliary beating.     

Leukotriene C4 and D4 in neonates with hypoxemia and pulmonary hypertension

Persistent pulmonary hypertension of the newborn is a syndrome consisting of severe hypoxemia and pulmonary hypertension that appears within hours of birth. Since certain leukotrienes (C4, D4, and E4) are known to produce some of the features of persistent pulmonary hypertension of the newborn, including pulmonary vasoconstriction, bronchoconstriction, decreased lung compliance, and pulmonary edema, we studied five newborns with the syndrome to determine whether these leukotrienes were present in their airways. We found leukotriene C4 and leukotriene D4 in the lung lavage fluids of all five newborns who had the clinical diagnosis of persistent pulmonary hypertension and who required ventilatory assistance. In contrast, leukotrienes were not demonstrated in a control group of 14 infants requiring ventilatory assistance who did not have the clinical diagnosis of persistent pulmonary hypertension. We conclude that leukotrienes may have a role in persistent pulmonary hypertension of the newborn.     

Leukotriene D4 (LTD4) activates charybdotoxin-sensitive and -insensitive K+ channels in Ehrlich ascites tumor cells

The putative role for ATP, UTP, bradykinin and leukotriene D₄ (LTD₄) in the activation of the charybdotoxin-insensitive, volume-activated K⁺ leak pathway has been assessed in Ehrlich cells. K⁺ channel activity is evaluated from bumetanide-insensitive ⁸⁶Rb⁺ efflux using Rb⁺ as a tracer for K⁺. Addition of the Ca²⁺-mobilizing agonists bradykinin, ATP, UTP or LTD₄ accelerates the regulatory volume decrease (RVD) response and activates a fast bumetanide-insensitive, charybdotoxin-sensitive efflux of K⁺. In addition LTD₄ activates a charybdotoxin-insensitive K⁺ efflux, whereas bradykinin, ATP and UTP do not. The charybdotoxin-insensitive K⁺ efflux dominates after addition of LTD₄ at concentrations too low to elicit an increase in [Ca²⁺]_i but still high enough to be effective in accelerating the RVD response. The EC₅₀ values for LTD₄-induced K⁺ effluxes are estimated at 2 nM and 15 nM for the charybdotoxin-insensitive and charybdotoxin-sensitive components, respectively. The LTD₄ (cysLT1) receptor antagonist L660,711(MK-571) blocks the activation of the charybdotoxin-sensitive but not the charybdotoxin-insensitive K⁺ efflux. Thus, LTD₄ activates two different K⁺ leak pathways in Ehrlich cells, one pathway activated by an increase in [Ca²⁺]_i and the other via an alternative signalling pathway. LTD₄ is thus a potential candidate for an autocrine messenger activating the Ca²⁺-independent, charybdotoxin-insensitive K⁺ channel during the RVD response in Ehrlich cells. Received: 5 January 1999 / Received after revision: 24 March 1999 / Accepted: 20 April 1999     

In vitro anti-inflammatory activity of the Artemisia montana leaf ethanol extract in macrophage RAW 264.7 cells

Artemisia is one of the largest genera of the family Asteraceae or Compositae, consisting of 500 species. Some Artemisia species, such as Artemisia afra, A. sacrorum, and A. annua, have been widely used as traditional medicine to treat inflammatory and malarial diseases. However, the biological activity of A. montana has not been broadly studied. Therefore, in this study, we investigated the anti-inflammatory activity of A. montana leaf extract (ALE) and its molecular mechanisms in lipopolysaccharide-activated RAW 264.7 cells. Non-cytotoxic concentrations of ALE significantly reduced the expression of inducible nitric oxide (NO) synthase and cyclooxygenase-2, resulting in the decrease in NO and prostaglandin E₂. Moreover, ALE inhibited the production of tumour necrosis factor-α and interleukin-6. We also observed that ALE treatment repressed mitogen-activated kinase pathways by inhibiting the phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38, suggesting that ALE is a therapeutic candidate to treat inflammatory diseases.     

吲哚美辛对乳腺癌细胞体外迁移能力的影响研究

目的 观察吲哚美辛对乳腺癌细胞系MCF-7体外迁移能力的影响,探讨可能存在的机制.方法 采用Transwell实验方法检测吲哚美辛刺激后MCF-7细胞的迁移能力,运用流式细胞术、Real-timePCR及ELISA方法分别检测吲哚美辛刺激后MCF-7细胞中趋化因子受体(CXCR4)、环氧合酶(COX-2)、表皮生长因子受体( EGFR)和血管内皮生长因子(VEGF)的表达情况.结果 吲哚美辛刺激组MCF-7细胞的迁移能力较未刺激组明显降低;吲哚美辛可呈时间-剂量依赖性地抑制MCF-7细胞表面CXCR4的表达,并显著下调细胞中CXCR4、COX-2和EGFR mRNA水平的表达,但对VEGF的产生并无明显的影响.结论MCF-7细胞中CXCR4、COX-2和EGFR表达的显著下调,可能是吲哚美辛抑制MCF-7细胞体外迁移能力的重要机制.     

Long-lived fusions of human haematological tumour cells and B-lymphoblastoid cells induce tumour antigen-specific cytotoxic T-cell responses in vitro

Tumour-specific cytotoxic T-cells (CTL) are important anti-cancer immune effectors. Most tumour cells, however, do not stimulate effective anti-tumour immune responses, in vivo or in vitro. To enhance tumour cell immunogenicity, we fused human tumour cells from haematological malignancies with the B-lymphoblastoid cell line (LCL), HMy2, to generate a panel of long-lived, self replicating LCL/tumour hybrid cell lines. The LCL/tumour hybrid cell lines expressed HLA class I and class II molecules, CD80 and CD86, and a range of known tumour associated antigens (TAAs). In vitro stimulation of PBLs from healthy, HLA-A2+ individuals by hybrid cell lines induced tumour antigen-specific CTLs to TAAs, including survivin, MAGE-A1, NY-ESO-1 and WT-1. Individual hybrid cell lines simultaneously induced CTL to multiple TAAs. In vitro stimulation of PBL from 2 patients with acute myeloid leukaemia by autologous LCL/tumour hybrid cell lines induced CTL capable of killing the patient's own tumour cells. Our data show, for the first time, that hybrid cell lines formed by fusion of HMy2 cells and haematological tumour cells induce tumour- and tumour antigen-specific cytotoxic T-cell responses in vitro. Hybrid cell lines such as those described may represent novel reagents for use in the immunotherapy of haematological malignancies.     

Vitamin D modulation of the activity of estrogenic compounds in bone cells in vitro and in vivo

Vitamin D analogs modulate different organs, including modulation of energy metabolism, through the induction of creatine kinase (CK) activity. Skeletal organs from vitamin D-depleted rats showed lower constituent CK than those from vitamin D-replete rats. Moreover, estradiol-17beta (E2) or dihydrotestosterone (DHT), which increased CK in organs from intact female or male rats, respectively, stimulated much less CK in vitamin D-depleted rats. Treatment of intact female rats with noncalcemic vitamin D analogs significantly upregulated E2- and DHT-induced CKresponse. These analogs upregulated the CK response to selective estrogen receptor modulators (SERMs) in organs from intact or ovariectomized (Ovx) female rats but abolished SERMs' inhibitory effect on E2-induced CK. These analogs significantly increased estradiol receptor alpha (ERalpha) protein in skeletal organs as well as histomorphological and biochemical changes due to this treatment followed by E2 or DHT. The analogs alone markedly altered the growth plate and the trabeculae and increased trabecular bone volume (%TB V) and trabecular width. The addition of E2 or DHT to this treatment restored all parameters as well as increased %TBV and cell proliferation. Treatment of Ovx female rats with JK 1624 F2-2 (JKF) decreased growth-plate width and increased %TB V, whereas QW1624 F2-2 (QW) restored growth-plate width and %TB V. Treatment of E2 with JKF restored %TBV and growth-plate width, whereas E2 with QW restored all parameters, including cortical width. There was also upregulation of the response of CK to E2 in both combined treatments. Our human-derived osteoblast (hObs)-like cell cultures respond to estrogenic compounds, and pretreating them with JKF upregulated the CK response to E2, raloxifene (Ral), and some phytoestrogens. ERalpha and ERbeta proteins, as well as mRNA, were modulated by CB 1093 (CB) and JKF. JKF increased specific nuclear E2 binding in female hObs but inhibited specific membranal E2 binding. hObs express 25 hydroxyvitamin D3-1alpha hydroxylase (1-OHase)-mRNA and its biological activity, which are both modulated by parathyroid hormone (PTH) and estrogenic compounds. Our results demonstrate mutual interaction between vitamin D and estrogenic compounds. We therefore conclude that combined treatment with less-calcemic analogs of vitamin D and estrogenic compounds might be superior for treatment of bone damage caused by ovariectomy in female rats, with possible application for postmenopausal osteoporosis.     

Vitamin D analogs decrease in vitro secretion of RANTES and enhance the effect of budesonide

PurposeEosinophils appear to be central inflammatory cells in the pathogenesis of rhinosinusitis with nasal polyps (NP). One of the most predominantly recognized eosinophil chemoattractants is RANTES. The aim of this study was to assess the influence of vitamin D (VD) derivates on RANTES expression in the culture of nasal polyp fibroblasts.Material and methodsNP fibroblast cell cultures derived from 16 patients with NP were first stimulated with bacterial LPS and than incubated in increasing concentrations (from 10^?7M to 10^?4M) of calcitriol, tacalcitol or budesonide and in combination with one of VD derivate with budesonide in 1:1, 1:3 and 3:1 ratios. Quantitative analysis of RANTES level was conducted in culture supernatants using an ELISA method.ResultsThe highest calcitriol concentration (10^?4M) as well as tacalcitol at 10^?5M and 10^?4M reduced RANTES production significantly compared to the control (201.1pg/ml, 338.7pg/ml, 211.3pg/ml v 571.78pg/ml; p<0.05). Budesonide and calcitriol administered in 1:3 ratio and budesonide and tacalcitol in 1:1 and 1:3 reduced RANTES concentration significantly better than each of the drug used in monotherapy (p<0.05). Budesonide and tacalcitol in 1:1 and 1:3 ratios suppressed RANTES production to the lowest level (171.8±97.6pg/ml and 178.7±105.22pg/ml, respectively).ConclusionActive VD compounds via downregulation of RANTES production exert a potential role as a complementary element in the therapy of chronic rhinosinusitis with NP. Compounds consisting of budesonide and VD derivate have an advantage over both drugs used in monotherapy.     

K+ currents activated by leukotriene D4 or osmotic swelling in Ehrlich ascites tumour cells

K+ and Cl- currents activated by hypoosmotic cell swelling (IK,vol and Icl,vol) or after addition of leukotriene D4 (LTD4) to cells in isotonic medium were studied in Ehrlich ascites tumour cells. IK,vol and Icl,vol were not affected by strong buffering of intracellular Ca2+ or by additional removal of extracellular Ca2+. In isotonic media, 5 nmol/l LTD4 activated large K+ but not Cl- currents. The LTD4-activated IK was, as has been shown previously for IK,vol, insensitive to charybdotoxin (ChTX) but was blocked by the antiarrhythmic drug clofilium. The current/voltage (I/V) relation for the LTD4-activated IK was, as recently demonstrated for IK,vol, well fitted by the Goldman-Hodgkin-Katz current equation between -130 mV and 30 mV in both physiological and K+-rich extracellular solutions. LTD4 had no additional effect on the magnitude of IK in Ehrlich cells already activated by the hypoosmotic stimulus. Nevertheless, the onset time for IK after hypoosmotic cell swelling was significantly less in the presence of LTD4. The similar I/V relation, pharmacological sensitivity and lack of additivity suggest that hypoosmotic swelling and addition of LTD4 activate the same K+ channels in Ehrlich cells. The influence of [Ca2+]i appears, however, to differ between IK,vol and the IK activated by LTD4 in that the latter was reduced significantly by strong buffering of [Ca2+]i. This might reflect the involvement of some additional factor in the hypoosmotic activation of K+ channels besides the stimulation mediated by LTD4.     

Transfer of myelin-specific cells deviated in vitro towards IL-4 production ameliorates ongoing experimental allergic neuritis

A causal role of IL-4 (Th2) production for recovery in experimental allergic neuritis (EAN) was indicated by experiments where Th1-like autoreactive cell populations, taken from the induction phase of the disease, were deviated to extensive secretion of IL-4 in a selective fashion, by ex vivo stimulation with autoantigen in the presence of IL-4. The deviated cells were adoptively transferred to EAN rats at a time just prior to the onset of clinical signs. This treatment ameliorated EAN compared with sham treatment. This therapeutic approach, with generation of autoreactive IL-4-secreting cells ex vivo followed by subsequent adoptive transfer, may become a new selective treatment of organ-specific autoimmune diseases since, in contrast to previous attempts, it is done in a physiological and technically easy way.     

Detection of an inhibitor of cell division in cultures of tumour cells with immunosuppressive activity in vitro.

A number of previous reports have described the presence of factors which inhibit the response of lymphoid cells to phytohaemagglutinin. The present study describes an inhibitor of cell division synthesized and released directly from cultured tumour cells which appears to have similar immunosuppressive effects in vitro. This factor(s) was detected in a wide variety of cultured tumour cells and some cultures of normal foetal tissue. It inhibited the mitogenic response of lymphocytes to PHA and also the spontaneous cell division of a variety of cultured cells including the cells producing the factor. Pulse cytophotometry showed that cells were inhibited in the G1 phase. Production of the factor increased with time and was related to the number of cells in culture. Its synthesis was inhibited by cycloheximide. DNA and RNA synthesis was not required for its production. The factor appeared to have a high degree of biological activity in terms of the number of cultured cells required for production and in regard to the number of cells inhibited. The biological significance of this factor in vivo is unknown but in vitro it was shown to inhibit immunoglobulin and mitogen-induced responses which suggests it may play an important role in suppression of the immune response against tumour cells in the host.     

Combination of 3D tissue engineered scaffold and non-viral gene carrier enhance in vitro DNA expression of mesenchymal stem cells

The objective of this study is to enhance the expression of a plasmid DNA for mesenchymal stem cells (MSC) by combination of 3-dimensional (3D) tissue engineered scaffolds and non-viral gene carrier. As a carrier of plasmid DNA, dextran-spermine cationic polysaccharide was prepared by means of reductive-amination between oxidized dextran and the natural oligoamine, spermine. As the MSC scaffold, collagen sponges reinforced by incorporation of poly(glycolic acid) (PGA) fibers were used. A complex of the cationized dextran and plasmid DNA of BMP-2 was impregnated into the scaffolds. MCS were seeded into each scaffold and cultured by a 3D culture method. When MSC were cultured in the PGA-reinforced sponge, the level of BMP-2 expression was significantly enhanced by the cationized dextran-plasmid DNA complex impregnated into the scaffold than by the cationized dextran-plasmid DNA complex in 2-dimensional (2D) (tissue culture plate) culture method. The alkaline phosphatase activity and osteocalcin content of transfected MSC cultured in the PGA-reinforced sponge were significantly higher compared with 2D culture method. We conclude that combination of cationized dextran plasmid DNA complex and 3D tissue engineered scaffold was promising to promote the in vitro gene expression for MSC.     

Anti-inflammatory activity of 4-arylcoumarins from endophytic Streptomyces aureofaciens CMUAc130 in murine macrophage RAW 264.7 cells

This research was undertaken to test the in vitro anti-inflammatory action of 5,7,4'-trimethoxy-4-phenylcoumarin and 5,7-dimethoxy-4-phenylcoumarin produced by Streptomyces aureofaciens CMUAc130. The effects of the two coumarins were investigated on the formation of NO, PGE2, and TNF-alpha and also on inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cells. The data obtained were consistent with the modulation of iNOS enzyme expression. A similar effect was also observed when LPS-induced PGE2 release and COX-2 expression were tested. The inhibitory effects were shown in concentration-dependent manners. The 5,7,4'-Trimethoxy-4-phenylcoumarin and 5,7-dimethoxy-4-phenylcoumarin also mildly but significantly reduced the formation of TNF-alpha.     

Cytotoxic activity against tumour cells mediated by intermediate TCR cells in the liver and spleen.

Morphological and phenotypic characterization in previous studies has indicated that intermediate (int) T-cell receptor (TCR) cells or T natural killer (TNK) cells may stand at an intermediate position between NK cells and high TCR cells of thymic origin in phylogenetic development. In this study, a functional study on cytotoxic activity against various tumour targets was performed in each purified subset. When a negative selection method entailing in vivo injection of anti-asialo GM, antibody or anti-interleukin (IL)-2R beta monoclonal antibody (mAb) was applied, IL-2R beta 1 CD3 NK cells were found to have the highest NK activity while IL-2R beta 1 int CD3 (or TCR) cells had a lower level of the NK activity. High CD3 cells (freshly isolated) did not have any such activity. Sorting experiments further revealed that the NK function mediated by int CD3 cells was augmented when they were exposed to anti-CD3 mAb. anti-TCR alpha beta, or anti-TCR-delta mAb. This phenomenon was not observed in NK cells and high CD3 cells. More importantly, when anti-CD3 mAb (or anti-TCR mAb) was added to the assay culture, int CD3 cells became cytotoxic against even NK-resistant tumour (Fc gamma R-. Fas+) targets. Liver mononuclear cells or int CD3 cells exposed to anti-CD3 mAb for 6 hr showed an elevated level of perforin in their cytoplasms. The present results suggest that int CD3 cells are usually non-cytotoxic against various tumours but become functional after being stimulated via the TCR CD3 complex.     

In vitro test to determine the effect of cytostatic drugs on co-cultured rat hepatocytes and hepatoma cells

Using uptake of the fluorescent bile acid derivative cholylglycylamido-fluorescein (FITC-GC) as a measurement of liver cell population size and functional, the antiproliferative and toxic effects of the well known cytostatic drug, cisplatin was evaluated on rapidly growing rat hepatoma McA-RH7777 cells and rat hepatocytes in primary culture under non-proliferating conditions. Co-culture set up to mimic the in vivo situation of tumour and extratumoural liver tissue exposed to cytostatic chemotherapy does not markedly affect the survival or the growth dynamics of both cell types. FITC-GC uptake as corrected for DNA and protein contents in the dish was significantly lower in hepatoma cells than in rat hepatocytes throughout the experimental period (96 h). Effect of 0.1–100 μ M cisplatin exposure from 24 to 96 h of culture on cell population size, as measured by protein and DNA contents in the culture dishes, were consistent with changes observed in total FITC-GC uptake. Cisplatin concentrations lower than 50 μ M did not affect FITC-GC uptake by rat hepatocytes. By contrast, a progressively increasing effect on hepatoma cells as from 2 μ M cisplatin was observed. Two phases in the decay of FITC-GC uptake versus cisplatin concentrations were found in co-cultures exposed to this drug. The first segment, between 2 μ M and 50 μ M , was characterized by a slow decay that matched the response of hepatoma cells to cisplatin exposure. This was considered to be due to the antiproliferative effect of cisplatin. The second segment, with a steeper decay, matched the effect of cisplatin on hepatocytes. This was interpreted as being due to non-specific toxicity. These results suggest that FITC-GC uptake by co-culture of hepatocytes and tumour cells provides a useful experimental model to explore the mechanism of action and the size of beneficial effect window for new drugs in vitro .