Influence of gangliosides or LPS-like gangliosides on the tumoricidal activity of adherent leukocytes

We previously showed that highly metastatic clones derived from the poorly metastatic human melanoma cell line M4Be are very radiosensitive provided that they are deficient in complex gangliosides. Here, we report that the highly metastatic clone 4 appears more sensitive to activated adherent leukocytes than M4Be via a transmembrane TNF-alpha-dependent mechanism. Adherent leukocytes (AL) were freshly isolated from different blood donors and were activated with Esherichia coli lipopolysaccharide (LPS). These AL contain 80% (73-93%) monocytes, 15% (6-20%) B lymphocytes and 5% (1-8%) T lymphocytes. The tumour cell survival following contact with AL was estimated with a clonogenic assay where isolated tumour cells were plated for 14 days with AL. We show on the one hand that either exogenous bovine brain GM1 gangliosides or Campylobacter jejuni LPS with GM1-like structure (LPS-like GM1) significantly decrease the hypersensitivity of clone 4 to AL. On the other hand, the cleaving with neuraminidase of more than 50% of the sialic residues bound to endogenous gangliosides in resistant M4Be cells significantly increases their sensitivity to AL. Thus, our highly metastatic cells appear both very sensitive to activated AL when they are deficient in complex gangliosides and resistant to AL when they are transiently exposed to exogenous gangliosides or LPS-like gangliosides. These in vitro data may reflect the paradoxidal behaviour of highly metastatic cells in vivo which appear both very sensitive to physiological stresses and able to survive to form secondary tumours.     

The generation of anti-tumoral cells using dentritic cells from the peripheral bloood of patients with malignant brain tumors

 Dendritic cells (DCs) can be the principal initiators of antigen-specific immune responses. We analyzed the in vitro-responses against brain tumor cells using DCs from the peripheral blood of patients with brain tumors. Peripheral blood mononuclear cells (PBMC) were obtained from 19 patients with malignant brain tumors: 12 metastatic brain tumors of lung adenocarcinoma, 7 high-grade astrocytomas. PBMC were cultured with 100 ng/ml of GM-CSF and 10 ng/ml of IL-4 for 5–7 days in order to produce mature DCs. The autologous tumor lysate (5 mg/ml, containing 1 × 10⁶ cells) was then added to the cultured DCs. Using the DCs generated by these treatments, we assessed the changes that occurred in their immune responses against brain tumor via ⁵¹Cr-release and lymphocyte proliferation assays. We found that the matured DCs displayed the typical surface phenotype of CD3⁺, CD45⁺, CD80⁺ and CD86⁺. After the pulsation treatment with tumor lysate, DCs were found to have strong cytotoxic T lymphocyte activity, showing 42.5 ± 12.7% killing of autologous tumor cells. We also found an enhancement of allogeneic T cell proliferation after pulsing the DC with tumor lysate. These data support the efficacy of DC-based immunotherapy for patients with malignant brain tumors. Received: 2 October 2000 / Accepted: 26 April 2001     

Activation of EGF receptor family members suppresses the cytotoxic effects of tumor necrosis factor-α

Tumor necrosis factor (TNF)-α has a broad range of biological activities, which depend heavily on cell type and physiological condition. In a panel of human tumor cell lines we analyzed expression of the receptor tyrosine kinases EGFR, ErbB2 and ErbB3, and the response to TNF-α. Among the cell lines tested those resistant to TNF-α were found to express high levels of either EGFR, or ErbB2 and ErbB3. In TNF-sensitive breast and cervical carcinoma cells activation of EGFR or ErbB2 by the exogenous growth factors EGF and heregulin β1 resulted in a significant increase in the number of cells surviving TNF-α treatment. In contrast, inhibition of EGFR activation in TNF-resistant breast carcinoma cells by the novel antagonistic anti-EGFR antibody 14E1 sensitized the cells to the cytotoxic effects of TNF-α. A bacterially expressed fusion protein consisting of a 14E1 single-chain (sc) Fv antibody fragment linked to human TNF-α retained TNF-α activity. This scFv(14E1)-TNF-α molecule localized specifically to EGFR on the surface of tumor cells and activated the NF-κB pathway in co-cultured T cells, as demonstrated by electrophoretic mobility shift assays. Received: 6 May 1998 / Accepted: 16 July 1998     

Influence of lipopolysaccharides and lipids A from some marine bacteria on spontaneous and Escherichia coli LPS-induced TNF-α release from peripheral human blood cells

Some endotoxic properties of lipopolysaccharides (LPS) and lipids A (LA) from the marine bacteria Marinomonas communis ATCC 27118^T, Marinomonas mediterranea ATCC 700492^T, and Chryseobacterium indoltheticum CIP 103168^T were studied. The preparations tested were shown to have high 50% lethal doses (4 μg per mouse for LPS from M. mediterranea and more than 12 μg per mouse for two other LPS and LA from C. indoltheticum) and were moderate (371 ± 37 pg/ml at 10 μg/ml of C. indoltheticum LPS), weak (148 ± 5 pg/ml at 1 μg/ml of M. mediterranea LPS), and zero (LA and LPS from M. communis and LA from C. indoltheticum) inducers of tumor necrosis factor α (TNF-α) release from peripheral human blood cells. The capacity of the LA and LPS samples from marine bacteria to inhibit TNF-α release induced by LPS from Escherichia coli O55: B5 (10 ng/ml) was also studied. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 7, pp. 936–944.     

Interleukin-15 effectively potentiates the in vitro tumor-specific activity and proliferation of peripheral blood γδT cells isolated from glioblastoma patients

γδT cells play a regulatory role in both primary and metastatic tumor growth in humans. The mechanisms responsible for the activation and proliferation of circulating γδT cells should be fully understood prior to their adoptive transfer to cancer patients. We have examined in vitro functional effects of interleukin-15 (IL-15) on highly purified γδT cells isolated from glioblastoma patients. γδT cells constitutively express the heterotrimeric IL-2 receptor (IL-2R) αβγ, but the levels of IL-2Rβ or γ expression were not increased by incubation with saturating amounts of IL-15. IL-15 was shown to induce a maximal γδT cell proliferation, although at much higher concentrations (at least 2000 U/ml) than IL-2 (100 U/ml). Submaximal concentrations of IL-15 plus low concentrations of IL-2 produced an additive proliferative response. In contrast to the IL-2-induced response, this activity was completely or partially abrogated by anti-IL-2Rβ, or anti-IL-2Rγ antibodies, but not by anti-IL-2Rα antibodies. Incubation of γδT cells in the presence of IL-15 resulted not only in the appearance of NK and LAK activity, but also in specific autologous tumor cell killing activity, an additive effect being seen with IL-15 and IL-2. This IL-15-induced tumor-specific activity could be significantly blocked by anti-IL-2Rγ and anti-IL-2R-β mAb, but not by anti-IL-2Rα mAb. Thus, in contrast to IL-2, IL-15 activates tumor-specific γδT cells through the components of IL-2Rβ and IL-2Rγ, but not IL-2Rα. These enhanced in vitro tumor-specific and proliferative responses of γδT cells seen with IL-15 suggest a rational adjuvant imunotherapeutic use of γδT cells in cancer patients. Received: 23 January 1998 / Accepted: 20 May 1998     

Synergy between interleukin-2 and prothymosin α for the increased generation of cytotoxic T lymphocytes against autologous human carcinomas

 Peripheral blood mononuclear cells (PBMC) from cancer patients were cultured in vitro with irradiated autologous tumor cells isolated from malignant effusions (mixed lymphocyte tumor cultures, MLTC) and low-dose (50 IU/ml) recombinant interleukin-2 (IL-2). The combination of IL-2 and prothymosin α (ProTα) resulted in a greater PBMC-induced response to the autologous tumor than that brought about by IL-2 alone. In particular, ProTα specifically enhanced the CD4⁺ T-cell-mediated proliferation against the autologous tumor. CD4⁺ T cells seemed to recognize tumor antigens presented by HLA-DR molecules expressed on the autologous monocytes, since preincubation of the latter with an anti-HLA-DR monoclonal antibody (mAb) abrogated the response. In addition, MLTC set up with IL-2 and ProTα also generated more MHC-class-I-restricted cytotoxic T lymphocytes (CTL) against the autologous tumor than did MLTC set up with IL-2 alone. The MLTC-induced CTL contained high levels of cytoplasmic perforin and their development was strictly dependent on the presence of both autologous CD4⁺ T cells and monocytes. In the absence of either population there was a strong impairment of both proliferative and cytotoxic responses which was not restored by the presence of ProTα. In contrast, when both cell populations were present, ProTα exerted optimal enhancement of CD4⁺ T cell proliferation, which was associated with potentiated CTL responses. Our data emphasize the role of ProTα for the enhancement of IL-2-induced CTL responses against autologous tumor cells. Such responses require collaborative interactions between CD4⁺, CD8⁺ T cells and monocytes as antigen-presenting cells. Our data are relevant for adoptive immunotherapeutic settings utilizing IL-2 and ProTα-induced autologous-tumor-specific CTL. Received: 2 March 2000 / Accepted: 1 June 2000     

IFN-α regulates IL 10 production by CML cells in vitro

High levels of spontaneous in vitro IL 10 secretion by a subset of untreated chronic phase CML patients' cells are shown to be decreased in the presence of IFN-α. However, the lower level of spontaneous IL 10 secretion by healthy control cells are was not depressed by IFN-α. In contrast to its effects on IL 10 production, IFN-α increased the low spontaneous secretion of IL 1α by patients' cells, bud did not further increase the higher levels of spontaneous IL 1β secretion by normal cells. It had no effect on secretion of TNF-α by patients or normals. Spontaneous secretion of IL-1α (or IFN-γ) by patients' cells was not observed whether or not IFN-α was present. Therefore, one mechanism of action of IFN-α in vivo may involve decreasing endogenous IL 10 secretion (thereby reducing suppressive effects on T cell reactivity) and increasing IL 1β secretion (thereby enhancing antigen presentation). Received: November 1998 / Accepted: 1 March 1999     

Observed localization of the long-term cultured rat adherent natural killer cells in mammary tumor tissues

 Adherent natural killer (A-NK) cells were isolated from splenic lymphocytes and treated with long-term culture in the presence of recombinant interleukin-2 (rIL-2). Immunocytochemical and flow-cytometric analysis revealed that most of the A-NK cells strongly expressed lymphocyte-function-associated antigen 1 (LFA-1α, and LFA-1β) throughout the incubation. All A-NK cells from 8- to 150-day cultures, particularly those cultured for 8 days, showed significant cytolytic activity against all targets. Analysis of the tissue distribution of the injected [³H] uridine-labeled A-NK cells demonstrated that, in the first 3 h, most (over 60%) cells localized in the lungs, and that most cells remained temporally within the cavities of blood capillaries of the lungs and moved gradually into lymphoid and other tissues. Peritumoral injection of various kinds of adjuvant, particularly Freund's complete adjuvant (FCA) plus bacillus Calmetee-Guérin (BCG), resulted in a marked accumulation of [³H]A-NK cells in mammary tumor tissues 24 h after injection, and simultaneously in the formation of vessels resembling high-endothelium venules (HEV), infiltration of LFA-1⁺ lymphocytes and expression of the ICAM-1 molecule on the tumor cells in the sites of tumor tissues. When 30 × 10⁶ A-NK cells were intravenously administered, significant retardation of tumor growth and prolongation of survival of tumor-bearing rats were observed in the groups that received the prior injection of adjuvants, especially FCA + BCG and Freund's incomplete adjuvant (FIA) + BCG. The suppressive effect of combination therapy on tumor growth was blocked effectively by the injection of anti-ICAM-1 mAb. These results indicate that the prior injection of proper adjuvant into the peritumoral region is effective for the selective accumulation or infiltration of A-NK cells into the sites of tumor tissues, and results in the marked retardation of tumor growth. Received: 18 May 1999 / Accepted: 4 October 1999     

In vitro analysis of the melanoma/endothelium interaction increasing the release of soluble intercellular adhesion molecule 1 by endothelial cells

Melanoma cells constitutively release intercellular adhesion molecule 1 (ICAM-1) as soluble ICAM-1 (sICAM-1), and its levels are elevated in melanoma patients and correlate with disease progression. However, this correlation is not absolute, suggesting that specific characteristics of neoplastic cells and/or ICAM-1-positive non-neoplastic cells may influence the amounts of circulating sICAM-1. In this study, we found a weak correlation (r = 0.55; r ² = 0.3) between sICAM-1 release by 40 metastatic melanomas (36 primary cultures and 4 cell lines), and ICAM-1 expression on neoplastic cells. In addition, melanoma-secreted interleukin-1α (IL-1α) (1/40) but not vascular endothelial growth factor (VEGF) (29/40), significantly (P < 0.05) up-regulated the shedding of sICAM-1 by human umbilical vein endothelial cells (HUVEC). This was completely abolished by IL-1α/β neutralizing antibodies both at the protein and mRNA level. Altogether, our results suggest that (i) the extent of sICAM-1 release is distinctive for individual melanomas and can be independent of ICAM-1 expression; (ii) tumor endothelia may sustain levels of sICAM-1 in selected melanomas; (iii) melanoma-released VEGF does not affect ICAM-1 expression and sICAM-1 release by HUVEC. Melanoma-derived sICAM-1 inhibits cell-mediated cytotoxicity of melanoma cells; therefore, constitutive levels of sICAM-1 release and IL-1α secretion by individual melanomas can differentially influence tumor progression and the clinical effectiveness of cytotoxic-cell-based vaccines. Received: 15 October 1998 / Accepted: 17 February 1999     

Tyrosine kinase activation in LPS stimulated rat kupffer cells

Kupffer cells, a majority of the body's fixed macrophages, are a major site of bacterial lipopolysaccharide (LPS) metabolism and are mediators in the body's response to sepsis. Uptake of LPS is different in Kupffer cells than other macrophages. Signal transduction in other macrophages in response to LPS involves phosphorylation of proteins in the 50–60 kDa range. We hypothesized that Kupffer cells may have unique signal transduction pathways in response to LPS. Rat Kupffer cells were exposed to LPS (1 μg/mL) for varying times ranging from 15 to 90 min. Cell lysates were Western blotted using an anti-phosphotyrosine antibody. The blots showed an increase in the amount of tyrosine phosphorylation on two proteins of 119 kDa and 83 kDa. The effects of varying LPS concentration (1 ng/mL-1 μg/mL) showed an increasing amount of phosphorylation with increasing LPS concentration. To associate the importance of tyrosine phosphorylation in the response of Kupffer cells to LPS, the tyrosine kinase inhibitors, tyrphostin, lavendustin, and genisten were used to study the effects of inhibiting phosphorylation on TNF-α production. Kupffer cells were preincubated in the presence of the inhibitor and exposed to LPS (1 μg/mL). TNF-α was measured in the conditioned media by ELISA. A 70% or greater decrease in TNF-α production was observed. When phagocytosis of latex beads by rat Kupffer cells was measured in vivo using intravital video microscopy, LPS treatment significantly increased uptake. This increase in phagocytosis was inhibited by tyrphostin. These results show what may be unique phosphorylation events in Kupffer cells that are related to LPS induced production of TNF-α. Presented in part at the American Association for the Study of Liver Diseases Annual Meeting, Chicago, IL (USA), November 3–7, 1995.     

Phenotypic and functional alterations of Vγ2Vδ2 T cell subsets in patients with active nasopharyngeal carcinoma

Introduction  Human Vγ2Vδ2 T cells play important role in immunity to infection and cancer by monitoring self and foreign isoprenoid metabolites with their γδ T cell antigen receptors. Like CD4 and CD8 αβ T cells, adult peripheral Vγ2Vδ2 T cells represent a pool of heterogeneous cells with distinct functional capabilities. Purpose  The aim of this study was to characterize the phenotypes and functions of various Vγ2Vδ2 T cell subsets in patients with nasopharyngeal carcinoma (NPC). We sought to develop a better understanding of the role of these cells during the course of disease and to facilitate the development of immunotherapeutic strategies against NPC. Results  Although similar total percentages of peripheral blood Vγ2Vδ2 T cells were found in both NPC patients and normal donors, Vγ2Vδ2 T cells from NPC patients showed decreased cytotoxicity against tumor cells whereas Vγ2Vδ2 T cells from normal donors showed potent cytotoxicity. To investigate further, we compared the phenotypic characteristics of Vγ2Vδ2 T cells from 96 patients with NPC and 54 healthy controls. The fraction of late effector memory Vγ2Vδ2 T cells (T_{EM RA}) was significantly increased in NPC patients with corresponding decreases in the fraction of early memory Vγ2Vδ2 T cells (T_CM) compared with those in healthy controls. Moreover, T_{EM RA} and T_CM Vγ2Vδ2 cells from NPC patients produced significantly less IFN-γ and TNF-α, potentially contributing to their impaired cytotoxicity. Radiotherapy or concurrent chemo-radiotherapy further increased the T_{EM RA} Vγ2Vδ2 T cell population but did not correct the impaired production of IFN-γ and TNF-α observed for T_{EM RA} Vγ2Vδ2 T cells. Conclusion  We have identified distinct alterations in the Vγ2Vδ2 T cell subsets of patients with NPC. Moreover, the overall cellular effector function of γδ T cells is compromised in these patients. Our data suggest that the contribution of Vγ2Vδ2 T cells to control NPC may depend on the activation state and differentiation of these cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Role of adhesion molecules in recruitment of Vδ1 T cells from the peripheral blood to the tumor tissue of esophageal cancer patients

 The mechanism responsible for tissue specific localization of γδ T cell subsets is not well understood. In order to explain the sequestration of specific γδ T cell subsets in the peripheral blood and tumor tissue of patients with esophageal cancer, we examined the function and expression of adhesion molecules on these cells. A hierarchy in the expression of adhesion molecules was observed. In vitro activated γδ T cells showed dominant expression of LFA-1 (CD11a), VLA-α4 (CD49d), intermediate expression of VLA-α5 (CD49e) and L-selectin (CD62L), but low expression of CD44v6 and α_Eβ₇ (CD103). It was observed that the γδ T cells use LFA-1, L-selectin and CD44v6 to bind to squamous cell carcinoma (SCC) cells, whereas they adhere to fibroblast cells using LFA-1, VLA-α4 and VLA-α5. Vδ1 T cell subsets from the peripheral blood γδ T cells utilize a larger array of adhesion molecules, namely LFA-1, VLA-α4, VLA-α5, L-selectin and α_Eβ_7, to bind to SCC cells compared to the restricted usage of LFA-1, L-selectin and CD44v6 by the Vδ2 T cells. Flow cytometric analysis of tumor infiltrating lymphocytes from the esophageal tumors confirmed the selective accumulation of Vδ1⁺γδ T cells in the tumor compartment. It thus appears that adhesion molecules expressed on these lymphocytes play an important role in the recruitment and retention of Vδ1 T cells in the tumor milieu. Received: 27 November 2000 / Accepted: 1 March 2001     

l-NAME inhibits tumor cell progression and pulmonary metastasis of r/m HM-SFME-1 cells by decreasing NO from tumor cells and TNF-α from macrophages

Highly metastatic ras/myc-transformed serum-free mouse embryo (r/m HM-SFME-1) cells were injected subcutaneously to mice and the effects of Nω-nitro-l-arginine methyl ester (l-NAME) on the tumor progression and pulmonary metastasis were investigated. In addition, production of nitric oxide (NO), matrix metalloproteinases (MMPs) and tumor necrosis factor-alpha (TNF-α) in the tumor cells and in a mouse macrophage-like cell line, J774.1 cells, was analyzed. The increase in footpad thickness was significantly smaller in the mice which were fed the l-NAME containing water (4.24 ± 0.39 mg/day/mouse). The number of the tumor cells metastasized to the lungs was smaller in the l-NAME treated mice, although statistical significance was not found. Co-treatment of r/m HM-SFME-1 cells with interferon-gamma (IFN-γ; 100 U/ml) and lipopolysaccharide (LPS; 0.5 μg/ml) significantly enhanced NO production, and the presence of l-NAME at 1 mM significantly decreased this response. In r/m HM-SFME-1 cells, MMP-2 was undetectable and MMP-9 was also very little in the basal level, and both MMPs were unaffected by the IFN-γ and/or LPS treatments, not to mention by the l-NAME treatment. In J774.1 cells, any treatment including LPS appeared to enhance MMP-9 production, however, this upregulation was not inhibited by the additional presence of l-NAME. Production of TNF-α by J774.1 cells was markedly enhanced with LPS treatment, and this enhancement was significantly reduced in the presence of l-NAME. These results indicate that the inhibitory effects of l-NAME on the tumor cell progression and pulmonary metastasis could be due to suppression of NO from tumor cells and TNF-α from macrophages (Mol Cell Biochem, 2007). Hideaki Yamaguchi and Yumi Kidachi contributed equally to this work.     

Degradation of IkappaBalpha in activated RAW264.7 cells is blocked by the phosphatidylinositol 3-kinase inhibitor LY294002

The mechanism by which lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA) induces production of proinflammatory cytokines in murine macrophages, and the role of phosphatidylinositol 3-kinase (PI3-kinase) have not been well investigated. Activation of nuclear factor κB (NF-κB) is initiated by the phosphorylation of the inhibitory subunit, IκB, which targets IκB for degradation and leads to the release of active NF-κB. In this study we demonstrate that 2- (4-morpholinyl)-8-phenylchromone (LY294002), which inhibits PI3-kinase, specifically inhibited degradation of IκBα in RAW264.7 cells stimulated with interferon-γ (IFN-γ) plus LPS or IFN-γ plus PMA. To elucidate the importance of this activity in RAW264.7 cells, we examined tumor necrosis factor-α (TNF-α) and interleukin IL)-6 production in the activated cells. Pretreatment of the cells with LY294002 resulted in the inhibition of TNF-α and IL-6 production in RAW264.7 cells stimulated with IFN-γ plus LPS or IFN-γ plus PMA. Furthermore, LY294002 inhibited the production of nitric oxide NO) in RAW264.7 cells stimulated with IFN-γ plus LPS or IFN-γ plus PMA. LY294002 also inhibited inducible NO synthase (iNOS) mRNA expression in the activated RAW264.7 cells. In conclusion, the present results suggest that PI3-kinase is involved in the signal transduction pathway responsible for LPS- or PMA-mediated TNF-α and IL-6 production, and that LY294002 inhibits NO generation through blocking the degradation of IκBα in activated RAW264.7 cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

The Expression of Tumor Necrosis Factor-α (TNF-α) by the Intrathecal Injection of Lipopolysaccharide in the Rat Spinal Cord

The proinflammatory and lipopolysaccharide (LPS)-inducible cytokine tumor necrosis factor α (TNF-α) has been shown to enhance primary sensory nociceptive signaling. However, the precise cellular site of TNF-α synthesis is still a matter of controversy. Therefore, we focused our study on TNF-α protein synthesis and expression patterns in spinal dorsal horn of naives and rats under intrathecal challenge with LPS. The enzyme-linked immunosorbent (ELISA) assay showed that the protein level of TNF-α reached peak at 8 h. Double immunofluorescence revealed that LPS-induced expression of TNF-α exclusively located in a subpopulation of microglia, which increased at 8 h in the rat spinal dorsal horn (the injected side). Positive staining of TNF receptor 1 (TNFR1) were also found in microglia. These observations have demonstrated the production of this proinflammatory cytokine by central nerve glia especially microglia. Synthesized TNF-α might directly act on microglia via TNFR1, but the inherent mechanisms remain unknown. Further studies are needed to confirm the pathogenic role of tumor necrosis factor in the early stage of inflammation. Aiguo Shen and Dan Zhou contributed equally to this work.     

Induction of TNF-α by LPS in Schwann Cell is Regulated by MAPK Activation Signals

Mitogen-activated protein kinases (MAPKs) are important mediators of cytokine expression and are critically involved in the immune response. The lipopolysaccharide (LPS) of gram-negative bacteria induces the expression of cytokines and proinflammatory genes via the toll-like receptor 4 (TLR4) signaling pathway in diverse cell types. In vivo, Schwann cells (SCs) at the site of injury may also produce tumor necrosis factor-- α (TNF-α). However, the precise mechanisms of TNF-α synthesis are still not clear. The purpose of the present study was to elucidate the underlying molecular mechanisms in the cultured SCs for its ability to activate the MAPKs and TNF-α gene, in response to LPS. Using enzyme-linked immunosorbent assay (ELISA), it was confirmed that treatment with LPS stimulated the synthesis of TNF-α in a concentration- and time-dependent manner. Intracellular location of TNF-α was detected under confocal microscope. Moreover, LPS activated extracellular signal-regulated kinase (ERK1/2), P38 and stress activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and induced their phosphorylation. LPS-elicited SCs TNF-α production was also drastically suppressed by PD98059 (ERK inhibitor), SB202190 (P38 inhibitor), or SP600125 (SAPK/JNK inhibitor). Additionally, the expression of CD14 and TLR4 was examined by RT–PCR. It was demonstrated that the expression of CD14, TLR4 was crucial for the SCs responses to LPS. In conclusion, the results provide novel mechanisms for the response of SCs to LPS stimulation, through MAPKs signaling pathways. Chun Cheng and Yongwei Qin contributed equally to this work.     

The regulation of TACE catalytic function by its prodomain

Aim To study the function of the prodomain of ADAM17 (TACE) and to develop an approach for interfering with inflammation processes. Method The expression plasmids of the TACE ectodomain (T1300), prodomain (T591), signal peptide and prodomain (T648), full length (T2472), and the turncated TACE without prodomain (T57-T1824) were constructed and designated as pET-28a-T300, pET-28a-T591, pIRES2-EGFP-648, pEGFP-N1-T648, pIRES2-EGFP-T2472, and pIRES2-EGFP-T57-T1824, respectively. After Ni²⁺-NTA resin-affinity chromatography, the recombinant T591 and T1300 proteins were obtained and assayed by western blotting and circular dichroism. The experiment was carried out on THP1 cell lines stimulated by LPS in vitro. The inhibition of recombinant protein T591 to TACE activity was detected by ELISA and immunohistochemical detection. The expression plasmids (pIRES2-EGFP-T648, pIRES2-EGFP-T2472, and pIRES2-EGFP-T57-T1824) were used to transfect the U937 cells. HeLa cells were also transfected with pEGFP-N1-T648. The transfected U937 cells were then stimulated by LPS and the effect of expression plasmids on TNF-α secretion was detected by ELISA and flow cytometry (FCM). Results The recombinant prodomain protein inhibited 57% of the TNF-α secretion and mediated an accumulation of TNF-α on the surface of THP1 cells. An intense green fluorescence was seen in the membranes of HeLa cells transfected with pEGFP-N1-T648. The plasmid pIRES2-EGFP-T648 inhibited TNF-α secretion by 61.09% and mediated an accumulation of mTNF-α on the surface of the U937 cells. The secretion of sTNF-α and the level of the mTNF-α in the pIRES2-EGFP-T57-T1824 transfected cells gave no difference when compared with the pIRES2-EGFP transfected cells. Also the secretion of sTNF-α from the cells transfected by the plasmid pIRES2-EGFP-T2472 increased, while the level of mTNF-α decreased, compared with the pIRES2-EGFP-transfected cells. Conclusion The prodomain has dual effects and might be useful in the molecular design of an anti-inflammatory drug.     

The Role of TNF-<Emphasis Type="Italic">α</Emphasis> and its Receptors in the Production of <Emphasis Type="Italic">β</Emphasis>-1,4-galactosyltransferase I mRNA by Rat Primary Type-2 Astrocytes

β-1,4-galactosyltransferase I (β-1,4-GalT I) plays an important role in the synthesis of the backbone structure of adhesion molecules involved in leukocyte–endothelial cell interaction. The expression of β-1,4-GalT I mRNA increased in primary human endothelial cells after exposure to tumor necrosis factor-α (TNF-α). In the central nervous system (CNS), astrocytes play a pivotal role in immunity as immunocompetent cells by secreting cytokines and inflammatory mediators, there are two types of astrocytes. Type-1 astrocytes can secrete TNF-α when stimulated with Lipopolysaccharide (LPS), while the responses of type-2 astrocytes during inflammation are unknown. So we examined the expression change of β-1,4-GalT I mRNA in type-2 astrocytes after exposure to TNF-α and LPS. Real-time PCR showed that TNF-α or LPS affected β-1,4-GalT I mRNA expression in a time- and dose-dependent manner. RT-PCR analysis revealed that TNFR1 and TNFR2 were present in normal untreated type-2 astrocytes, and that TNF-α, TNFR1 and TNFR2 increased in type-2 astrocytes after exposure to TNF-α or LPS. Immunocytochemistry showed that TNFR1 was expressed in the cytoplasm, nucleus and processes of normal untreated type-2 astrocytes, and distributed mainly in the cytoplasm and processes after exposure to LPS. TNFR2 was mainly expressed in the nucleus of normal untreated type-2 astrocytes, and distributed mainly in the processes of type-2 astrocytes after exposure to LPS. Both anti-TNFR1 and anti-TNFR2 antibodies suppressed β-1,4-GalT I mRNA expression induced by TNF-α or LPS. From these results, we conclude that TNF-α signaling via both TNFR1 and TNFR2 translocated from nucleus to cytoplasm or processes is sufficient to induce β-1,4-GalT I mRNA. In addition, we observed that not only exogenous TNF-α but also TNF-α produced by type-2 astrocytes affected β-1,4-GalT I mRNA production in type-2 astrocytes. These results suggest that an autocrine loop involving TNF-α contributes to the production of β-1,4-GalT I mRNA in response to inflammation. Chunlin Xia is the co-first author.     

Importance of signaling via the IFN-α/β receptor on host cells for the realization of the therapeutic benefits of cyclophosphamide for mice bearing a large MOPC-315 tumor

Here we show that low-dose cyclophosphamide (CY), that depends for its therapeutic effectiveness on the immunopotentiating activity of the drug for T cell-mediated tumor-eradicating immunity, is curative for ~80% of wild-type (WT) mice bearing a large s.c. MOPC-315 tumor, but only for ~10% of IFN-α/βR^−/− mice bearing a large s.c. MOPC-315 tumor. Histopathological examination of the s.c. tumors of such mice on day 4 after the chemotherapy revealed that the low dose of CY led to accumulation of T lymphocytes in both the WT and the IFN-α/βR^−/− mice. However, in the CY treated tumor bearing WT mice the T lymphocytes were present throughout the tumor mass and in direct contact with tumor cells, but in the CY treated tumor bearing IFN-α/βR^−/− mice most of the T lymphocytes remained in blood vessels. In addition to being important for CY-induced transendothelial migration of T lymphocytes into the tumor mass, we show here that signaling via the IFN-α/βR is also important for CY-induced control of metastatic tumor progression in the spleen and liver of the tumor bearing mice. Finally, CY cured tumor bearing WT mice were resistant to a subsequent challenge with MOPC-315 tumor cells, but the few CY cured tumor bearing IFN-α/βR^−/− mice were not. Thus, signaling via the IFN-α/βR on host cells in MOPC-315 tumor bearers is important for CY-induced: (a) transendothelial migration of T lymphocytes into the tumor mass and the eradication of the primary tumor, (b) control of metastatic tumor progression, and (c) resistance to a subsequent tumor challenge. This work was supported by Research Grant 03-19 from the American Cancer Society-Illinois Division.     

Sensitization of resting T cells to autologous natural-killer-cell-mediated lysis by phytohemagglutinin

Natural killer (NK) cells are non-T, non-B cell lymphocytes that lyse a variety of tumor and virus-infected cells. In this study, we demonstrated that phytohemagglutinin (PHA) rendered resistant autologous T cells extremely sensitive to natural-killer(NK)-cell-mediated lysis. The sensitization was very rapid and concentration-dependent (0.01–1 μg/ml); 62% and 95% of autologous T cells were lysed by interleukin-2-activated NK cells 5 min and 18 h respectively after treatment with PHA (1 μg/ml). The maximal decrease in the level of MHC class I molecules observed on T cells was 22%. Induction of susceptibility to NK-mediated lysis was correlated with the expression of activation markers on T cells treated for relatively long intervals (more than 18 h) with high concentrations of PHA (more than 0.1 μg/ml). Sensitization of T cells required RNA and protein synthesis, although DNA synthesis was not essential. We propose that this unique system is suitable for studying the mechanisms involved in recognition and killing of target cells by NK cells. Received: 11 February 1999 / Accepted: 28 July 1999