Effect of MiR-10b on Cervical Cancer Rats Through mTOR/P70S6K Signaling Pathway.

The purpose of this experiment was to observe the biological effect and mechanism of miR-10b on cervical cancer (CC) rats. For this purpose, the rat model of CC was established and divided into three groups (Inhibitors/ Mimics/Control). The miR-10b transfection efficiency was analyzed via RT-PCR in cervical tissues in each group. The content of CD3+, CD4+, and CD8+ was detected. The levels of IL-8, TNF-ß, IL-6, (CAT, SOD, and MDA were determined via ELISA, and the apoptosis of cervical tissues was detected usingTUNEL assay. The expressions of Caspase-3, Bcl-2, and the mTOR/P70S6K pathway genes and proteins were detected by qRT-PCR and Western blotting. Results showed that miR-10b was significantly increased in the Mimics group and decreased in the Inhibitors group. The content of IL-8, TNF-ß, IL-6, CAT and MDA was raised, while that of SOD notably declined in the Inhibitors group. There were remarkably more apoptotic cells in the Mimics group, dominated by gliocytes, and fewer apoptotic cells in the Inhibitors group, with increased content of CD3+, CD4+ and CD8+. The Bcl-2, mTOR, and P70S6K mRNA expressions in the Inhibitors group were up-regulated than those in the other two groups, and the Caspase-3 gene in the Mimics group was increased and close to that in the control group. In the Mimics group, the mTOR and P70S6K protein were remarkably lower than those in the Inhibitors group. In conclusion, miR-10b can inhibit the occurrence and development of CC in rats by suppressing mTOR/P70S6K signaling, reducing the level of inflammation and oxidative stress, and increasing the level of immune factors.

Calebin A Potentiates the Effect of 5-FU and TNF-ß (Lymphotoxin α) against Human Colorectal Cancer Cells: Potential Role of NF-κB.

OBJECTIVE: The majority of chemotherapeutic agents stimulate NF-κB signaling that mediates cell survival, proliferation and metastasis. The natural turmeric non-curcuminoid derivate Calebin A has been shown to suppress cell growth, invasion and colony formation in colorectal cancer cells (CRC) by suppression of NF-κB signaling. Therefore, we hypothesized here that Calebin A might chemosensitize the TNF-ß-treated tumor cells and potentiates the effect of 5-Fluorouracil (5-FU) in advanced CRC. MATERIALS AND METHODS: CRC cells (HCT116) and their clonogenic 5-FU chemoresistant counterparts (HCT116R) were cultured in monolayer or alginate-based 3D tumor environment culture and were treated with/without Calebin A, TNF-ß, 5-FU, BMS-345541 and DTT (dithiothreitol). RESULTS: The results showed that TNF-ß increased proliferation, invasion and resistance to apoptosis in chemoresistant CRC cells. Pretreatment with Calebin A significantly chemosensitized HCT116R to 5-FU and inhibited the TNF-ß-induced enhanced efforts for survival, invasion and anti-apoptotic effects. We found further that Calebin A significantly suppressed TNF-ß-induced phosphorylation and nuclear translocation of p65-NF-κB, similar to BMS-345541 (specific IKK inhibitor) and NF-κB-induced tumor-promoting biomarkers (NF-κB, ß1-Integrin, MMP-9, CXCR4, Ki67). This was associated with increased apoptosis in HCT116 and HCT116R cells. Furthermore, blocking of p65-NF-κB stimulation by Calebin A was imparted through the downmodulation of p65-NF-κB binding to the DNA and this suppression was turned by DTT. CONCLUSION: Our findings indicate, for the first time, that Calebin A chemosensitizes human CRC cells to chemotherapy by targeting of the p65-NF-κB signaling pathway.

Resveratrol Suppresses Cross-Talk between Colorectal Cancer Cells and Stromal Cells in Multicellular Tumor Microenvironment: A Bridge between In Vitro and In Vivo Tumor Microenvironment Study.

The interaction between tumor cells and the tumor microenvironment (TME) is an important process for the development of tumor malignancy. Modulation of paracrine cross-talk could be a promising strategy for tumor control within the TME. The exact mechanisms of multi-targeted compound resveratrol are not yet fully understood. Whether resveratrol can modulate paracrine signal transduction-induced malignancy in the multicellular-TME of colorectal cancer cells (CRC) was investigated. An in vitro model with 3D-alginate HCT116 cells in multicellular-TME cultures (fibroblast cells, T-lymphocytes) was used to elucidate the role of TNF-ß, Sirt1-ASO and/or resveratrol in the proliferation, invasion and cancer stem cells (CSC) of CRC cells. We found that multicellular-TME, similar to TNF-ß-TME, promoted proliferation, colony formation, invasion of CRC cells and enabled activation of CSCs. However, after co-treatment with resveratrol, the malignancy of multicellular-TME reversed to HCT116. In addition, resveratrol reduced the secretion of T-lymphocyte/fibroblast (TNF-ß, TGF-ß3) proteins, antagonized the T-lymphocyte/fibroblast-promoting NF-κB activation, NF-κB nuclear translocation and thus the expression of NF-κB-promoting biomarkers, associated with proliferation, invasion and survival of CSCs in 3D-alginate cultures of HCT116 cells induced by TNF-ß- or multicellular-TME, but not by Sirt1-ASO, indicating the central role of this enzyme in the anti-tumor function of resveratrol. Our results suggest that in vitro multicellular-TME promotes crosstalk between CRC and stromal cells to increase survival, migration of HCT116 and the resveratrol/Sirt1 axis suppresses this loop by modulating paracrine agent secretion and NF-κB signaling. Fibroblasts and T-lymphocytes are promising targets for resveratrol in the prevention of CRC metastasis.

Different combinations of growth factors for the tenogenic differentiation of bone marrow mesenchymal stem cells in monolayer culture and in fibrin-based three-dimensional constructs.

Tendon injuries are severe burdens in clinics. The poor tendon healing is related to an ineffective response of resident cells and inadequate vascularization. Thanks to the high proliferation and multi-lineage differentiation capability, bone marrow-derived mesenchymal stem cells (BMSCs) are a promising cell source to support the tendon repair. To date, the association of various growth factors to induce the in vitro tenogenic differentiation of multipotent progenitor cells is poorly investigated. This study aimed to investigate the tenogenic differentiation of rabbit BMSCs by testing the combination of bone morphogenetic proteins (BMP-12 and 14) with transforming growth factor beta (TGF-ß) and vascular endothelial growth factor (VEGF) both in 2D and 3D cultures within fibrin-based constructs. After 7 and 14 days, the tenogenic differentiation was assessed by analyzing cell metabolism and collagen content, the gene expression of tenogenic markers and the histological cell distribution and collagen deposition within 3D constructs. Our results demonstrated that the association of BMP-14 with TGF-ß3 and VEGF enhanced the BMSC tenogenic differentiation both in 2D and 3D cultures. This study supports the use of fibrin as hydrogel-based matrix to generate spheroids loaded with tenogenic differentiated BMSCs that could be used to treat tendon lesions in the future.

Dendritic cell regulation of NK-cell responses involves lymphotoxin-α, IL-12, and TGF-ß.

Dendritic cell (DC) vaccines induce T-cell responses in cancer patients. However, there is a paucity of data regarding the role of DC vaccines in shaping natural killer (NK) cell responses. Here, we observe that NK cells are less activated following DC vaccination. In vitro, DC-mediated inhibition of NK cells did not require cell-to-cell contact, but required increased Signal transducer and activator of transcription 3 (STAT3) phosphorylation (pSTAT3) in DCs. When phosphorylation of STAT3 was inhibited in DCs, we found that DCs did not suppress NK cells, and observed an increase in the production of lymphotoxin-alpha (LTα) and interleukin-12 (IL-12) as well as reduced release of transforming growth factor beta (TGF-ß). The addition of recombinant LTα or IL-12 to the DC-NK-cell cocultures restored NK-cell activity, and neutralization of TGF-ß resulted in elevated production of LTα and IL-12 from DCs. Compared with LPS, DCs matured with a cocktail of R848, poly I:C, and IFN-γ showed reduced levels of pSTAT3 and higher levels of LTα and IL-12 and did not inhibit NK-cell activity. These results show that LTα, IL-12, and TGF-ß are involved in the cross-talk between NK cells and DCs. Our findings have important implications for the development of DC-based vaccination strategies to potentiate NK-cell responses in patients with cancer.

Identification of Tyrosine-Phosphorylated Proteins Upregulated during Epithelial-Mesenchymal Transition Induced with TGF-ß.

The epithelial-to-mesenchymal transition (EMT) is a unique process for the phenotypic changes of tumor cells characterized by a transition from polarized rigid epithelial cells to migrant mesenchymal cells, thus conferring the ability of tumor invasion and metastasis. A major challenge in the treatment of lung adenocarcinoma is to identify early stage patients at a high risk of recurrence or metastasis, thereby permitting the best therapeutic strategy and prognosis. In this study, we used a transforming growth factor-ß (TGF-ß)-induced EMT model to quantitatively identify protein tyrosine phosphorylation during the course of EMT in relation to malignant characteristics of lung adenocarcinoma cells. We performed relative quantitation analysis of tyrosine-phosphorylated peptides in TGF-ß-treated and -untreated lung adenocarcinoma cells and identified tyrosine-phosphorylated proteins that were upregulated in TGF-ß-treated cells. These include tensin-1 (TNS1) phosphorylated on Y1404, hepatocyte growth factor receptor (c-Met) phosphorylated on Y1234, and NT-3 growth factor receptor (TrkC) phosphorylated on Y516. We also found that these protein phosphorylation profiles were specifically observed in tissue samples of patients with poor prognostic lung adenocarcinoma. Tyrosine phosphorylations of these proteins represent possible candidates of prognostic prediction markers for lung adenocarcinoma.

Uterine wound healing after caesarean section: A systematic review.

The rate of caesarean section (CS) is increasing worldwide. Defects in uterine healing have a major gynaecological and obstetric impact (uterine rupture, caesarean scar defect, caesarean scar pregnancy, placenta accreta spectrum). The complex process of cellular uterine healing after surgery, and specifically after CS, remains poorly understood in contrast to skin wound healing. This literature review on uterine wound healing was mainly based on histological observations, particularly after CS. The primary objective of the review was to examine the effects of CS on uterine tissue at the cellular level, based on histological observations. The secondary objectives were to describe the biomechanical characteristics and the therapies used to improve scar tissue after CS. This review was performed using PRISMA criteria, and PubMed was the data source. The study included all clinical and animal model studies with CS and histological analysis of the uterine scar area (macroscopic, microscopic, immunohistochemical and biomechanical). Twenty studies were included: 10 human and 10 animal models. In total, 533 female humans and 511 female animals were included. Review articles, meeting abstracts, case series, case reports, and abstracts without access to full-text were excluded. The search was limited to studies published in English. No correlation was found between cutaneous and uterine healing. The histology of uterine scars is characterized by disorganized smooth muscle, fibrosis with collagen fibres and fewer endometrial glands. As for skin healing, the initial inflammation phase and mediation of some growth factors (particularly connective tissue growth factor, vascular endothelial growth factor, platelet-derived growth factor, tumour necrosis factor α and tumour necrosis factor ß) seem to be essential. This initial phase has an impact on the subsequent phases of proliferation and maturation. Collagen appears to play a key role in the initial granulation tissue to replace the loss of substance. Subsequent maturation of the scar tissue is essential, with a decrease in collagen and smooth muscle restoration. Unlike skin, the glandular structure of uterine tissue could be responsible for the relatively high incidence of healing defects. Uterine scar defects after CS are characterized by an atrophic disorganized endometrium with atypia and a fibroblastic highly collagenic stromal reaction. Concerning immunohistochemistry, one study found a decrease in tumour necrosis factor ß in uterine scar defects. No correlation was found between biomechanical characteristics (particularly uterine strength) and the presence of a collagenous scar after CS. Based on the findings of this review, an illustration of current understanding about uterine healing is provided. There is currently no validated prevention of caesarean scar defects. Various treatments to improve uterine healing after CS have been tested, and appeared to have good efficacy in animal studies: alpha lipoic acid, growth factors, collagen scaffolds and mesenchymal stem cells. Further prospective studies are needed.

Progranulin does not inhibit TNF and lymphotoxin-α signalling through TNF receptor 1.

Progranulin (proepithelin, granulin precursor) has been recently suggested to exhibit anti-inflammatory properties by directly binding to tumour necrosis factor (TNF) receptors and thereby inhibiting TNF signalling by Tang et al. This finding was challenged by Chen et al. and no interaction between progranulin and TNF receptor (TNFR) 1 or 2 was observed. We tested the ability of recombinant progranulin from different commercial sources to inhibit TNF- or lymphotoxin-α-induced signalling through TNFR1. We observed that progranulin does not affect signalling and cell death induction downstream of TNF or lymphotoxin-α. Our results suggest that the anti-inflammatory role of progranulin is not mediated through direct inhibition of TNFR1.

Mutants of lymphotoxin-α with augmented cytotoxic activity via TNFR1 for use in cancer therapy.

The cytokine lymphotoxin-α (LTα) is a promising candidate for use in cancer therapy. However, the instability of LTαin vivo and the insufficient levels of tumor necrosis factor receptor 1 (TNFR1)-mediated bioactivity of LTα limit its therapeutic potential. Here, we created LTα mutants with increased TNFR1-mediated bioactivity by using a phage display technique. We constructed a phage library displaying lysine-deficient structural variants of LTα with randomized amino acid residues. After affinity panning, we screened three clones of lysine-deficient LTα mutant, and identified a LTα mutant with TNFR1-mediated bioactivity that was 32 times that of the wild-type LTα (wtLTα). When compared with wtLTα, the selected clone showed augmented affinity to TNFR1 due to slow dissociation rather than rapid association. In contrast, the mutant showed only 4 times the TNFR2-mediated activity of wtLTα. In addition, the LTα mutant strongly and rapidly activated caspases that induce TNFR1-mediated cell death, whereas the mutant and wtLTα activated nuclear factor-kappa B to a similar extent. Our data suggest that the kinetics of LTα binding to TNFR1 play an important role in signal transduction patterns, and a TNFR1-selective LTα mutant with augmented bioactivity would be a superior candidate for cancer therapy.

Transcriptional regulation of miR-224 upregulated in human HCCs by NFκB inflammatory pathways.

BACKGROUND & AIMS: miR-224 is up-regulated in human HCCs as compared to both paired peri-tumoral cirrhotic tissues and cirrhotic livers without HCC. Here, we have cloned the miR-224 regulatory region and characterized its transcriptional regulation by the NFκB-dependent inflammatory pathways. METHODS: Mature miRNA expression was evaluated by a 2 step stem-loop real-time RT-PCR. The recruitment of polymerase II and transcription factors on the pre-miR-224 promoter has been assessed by ChIPSeq and ChIP. RESULTS: We found miR-224 levels strongly up-regulated in both peri-tumoral cirrhotic livers and HCC samples as compared to normal livers. In silico analysis of the putative miR-224 promoter revealed multiple NFκB sites. We showed that LTα and TNFα activate transcription from the miR-224 promoter and of endogenous miR-224 expression in HCC cell lines, whereas the expression of miR-224 target API5 was reduced. Exogenously expressed p65/RelA activates the miR-224 promoter and a dominant negative form of IκBα (IκBSR) represses it. ChIP analysis showed that p65/NFκB is recruited on the miR-224 promoter and that its binding sharply increases after exposure to LPS, TNFα, and LTα. Altogether these findings link the inflammatory signals to NFκB-mediated activation of miR-224 expression. An antago-miR specific for miR-224 blocked LPS and LTα stimulated HCC cells migration and invasion. Conversely, the IKK inhibitor BMS-345541 blocks pre-miR-224-induced cellular migration and invasion. CONCLUSIONS: Our results identify p65/NFκB as a direct transcriptional regulator of miR-224 expression and link miR-224 up-regulation with the activation of the LPS, LTα, and TNFα inflammatory pathways and cell migration/invasion in HCC.

Microbial signals, MyD88, and lymphotoxin drive TNF-independent intestinal epithelial tissue damage.

Anti-TNF antibodies are effective for treating patients with inflammatory bowel disease (IBD), but many patients fail to respond to anti-TNF therapy, highlighting the importance of TNF-independent disease. We previously demonstrated that acute deletion of 2 IBD susceptibility genes, A20 (Tnfaip3) and Abin-1 (Tnip1), in intestinal epithelial cells (IECs) sensitized mice to both TNF-dependent and TNF-independent death. Here we show that TNF-independent IEC death after A20 and Abin-1 deletion was rescued by germ-free derivation or deletion of MyD88, while deletion of Trif provided only partial protection. Combined deletion of Ripk3 and Casp8, which inhibits both apoptotic and necroptotic death, completely protected against death after acute deletion of A20 and Abin-1 in IECs. A20- and Abin-1-deficient IECs were sensitized to TNF-independent, TNFR1-mediated death in response to lymphotoxin α (LTα) homotrimers. Blockade of LTα in vivo reduced weight loss and improved survival when combined with partial deletion of MyD88. Biopsies of inflamed colon mucosa from patients with IBD exhibited increased LTA and IL1B expression, including a subset of patients with active colitis on anti-TNF therapy. These data show that microbial signals, MyD88, and LTα all contribute to TNF-independent intestinal injury.

Requiem protein links RelB/p52 and the Brm-type SWI/SNF complex in a noncanonical NF-kappaB pathway.

The SWI/SNF chromatin remodeling complex plays pivotal roles in mammalian transcriptional regulation. In this study, we identify the human requiem protein (REQ/DPF2) as an adaptor molecule that links the NF-kappaB and SWI/SNF chromatin remodeling factor. Through in vitro binding experiments, REQ was found to bind to several SWI/SNF complex subunits and also to the p52 NF-kappaB subunit through its nuclear localization signal containing the N-terminal region. REQ, together with Brm, a catalytic subunit of the SWI/SNF complex, enhances the NF-kappaB-dependent transcriptional activation that principally involves the RelB/p52 dimer. Both REQ and Brm were further found to be required for the induction of the endogenous BLC (CXCL13) gene in response to lymphotoxin stimulation, an inducer of the noncanonical NF-kappaB pathway. Upon lymphotoxin treatment, REQ and Brm form a larger complex with RelB/p52 and are recruited to the BLC promoter in a ligand-dependent manner. Moreover, a REQ knockdown efficiently suppresses anchorage-independent growth in several cell lines in which the noncanonical NF-kappaB pathway was constitutively activated. From these results, we conclude that REQ functions as an efficient adaptor protein between the SWI/SNF complex and RelB/p52 and plays important roles in noncanonical NF-kappaB transcriptional activation and its associated oncogenic activity.

Osteoclast differentiation independent of the TRANCE-RANK-TRAF6 axis.

Osteoclasts are derived from myeloid lineage cells, and their differentiation is supported by various osteotropic factors, including the tumor necrosis factor (TNF) family member TNF-related activation-induced cytokine (TRANCE). Genetic deletion of TRANCE or its receptor, receptor activator of nuclear factor kappaB (RANK), results in severely osteopetrotic mice with no osteoclasts in their bones. TNF receptor-associated factor (TRAF) 6 is a key signaling adaptor for RANK, and its deficiency leads to similar osteopetrosis. Hence, the current paradigm holds that TRANCE-RANK interaction and subsequent signaling via TRAF6 are essential for the generation of functional osteoclasts. Surprisingly, we show that hematopoietic precursors from TRANCE-, RANK-, or TRAF6-null mice can become osteoclasts in vitro when they are stimulated with TNF-alpha in the presence of cofactors such as TGF-beta. We provide direct evidence against the current paradigm that the TRANCE-RANK-TRAF6 pathway is essential for osteoclast differentiation and suggest the potential existence of alternative routes for osteoclast differentiation.

Fibromatosis stem cells rather than bone-marrow mesenchymal stem cells recapitulate a murine model of fibromatosis.

Palmar fibromatosis is a benign fibroproliferative tumor of unknown etiology, with a high rate of recurrence after excision. The offending cells of palmar fibromatosis are myofibroblasts and the cellular origin of other myofibroblasts has previously been reported to be the bone marrow. However, further clarification of the relationship between bone marrow precursors and palmar fibromatosis is required. Stem cells (SCs) are known to exist in various tissues, but whether SCs can be isolated from fibromatosis tissue is still unclear. The purpose of this study was to isolate and identify stem cells from human palmar fibromatosis, and to evaluate the differences in the differentiation and fibrogenic capacities of bone marrow stem cells (BMSCs) and fibromatosis-derived stem cells (FSCs). We found that FSCs had better fibrogenic differentiation potential than BMSCs, whereas BMSCs had better adipogenic and chondrogenic differentiation capacities. Treatment with transforming growth factor-ß1 increased the expression of α-smooth muscle actin, and types III and I collagen significantly more in FSCs than in BMSCs. An in vivo study further confirmed the results of fibrogenesis and suggested that FSCs can recapitulate the fibromatosis nodule. In summary, their myofibroblastic differentiation both in vivo and in vitro makes FSCs a potential cell source for future applications in murine models of fibromatosis or fibrogenesis.

Lymphotoxin α stimulates proliferation and pro-inflammatory cytokine secretion of rheumatoid arthritis synovial fibroblasts.

OBJECTIVE: TNFα plays a crucial role in rheumatoid arthritis (RA) by stimulating fibroblast-like synoviocytes (FLS). Lymphotoxin α (LTα) is a pro-inflammatory cytokine with significant homology to TNFα. We compared the effects of both cytokines on cultured RA FLS. METHODS: Receptor expression on RA FLS was analyzed by FACS. Cells were stimulated with LTα or TNFα and proliferation was measured by [3H]thymidine incorporation and secretion of inflammatory cytokines and metalloproteinase 3 by ELISA. Activation of MAP kinases and Akt was analyzed by Western blotting. Nuclear translocation of NFκB was visualized by immunofluorescence. RESULTS: 60-80% and 30-50% of the RA FLS tested expressed TNF receptors I and II, respectively, and 70-75% expressed HVEM. LTα induced RA FLS proliferation at the same level of TNFα, which was blocked by etanercept. Both LTα and TNFα induced activation of MAP kinases ERK1/2 and p38 as well as Akt. 95-98% of FLS showed nuclear translocation of NFκB after stimulation with either cytokines. LTα and TNFα were potent to induce secretion of IL-6, IL-8 and metalloproteinase 3 in FLS. CONCLUSION: LTα is as effective as TNFα in stimulating RA FLS. Blocking both cytokines might allow a better control of inflammation and synovial proliferation in RA.

A cytokine receptor-masked IL2 prodrug selectively activates tumor-infiltrating lymphocytes for potent antitumor therapy.

As a potent lymphocyte activator, interleukin-2 (IL-2) is an FDA-approved treatment for multiple metastatic cancers. However, its clinical use is limited by short half-life, low potency, and severe in vivo toxicity. Current IL-2 engineering strategies exhibit evidence of peripheral cytotoxicity. Here, we address these issues by engineering an IL-2 prodrug (ProIL2). We mask the activity of a CD8 T cell-preferential IL-2 mutein/Fc fusion protein with IL2 receptor beta linked to a tumor-associated protease substrate. ProIL2 restores activity after cleavage by tumor-associated enzymes, and preferentially activates inside tumors, where it expands antigen-specific CD8 T cells. This significantly reduces IL-2 toxicity and mortality without compromising antitumor efficacy. ProIL2 also overcomes resistance of cancers to immune checkpoint blockade. Lastly, neoadjuvant ProIL2 treatment can eliminate metastatic cancer through an abscopal effect. Taken together, our approach presents an effective tumor targeting therapy with reduced toxicity.

Interferon gamma and lymphotoxin or tumor necrosis factor act synergistically to induce macrophage killing of tumor cells and schistosomula of Schistosoma mansoni.

Macrophages play a crucial role in the defense against tumors and parasites. Activation of tumoricidal and microbicidal effector mechanisms requires stimulation of macrophages with macrophage-activating factors (MAF). One such MAF is interferon gamma (IFN-gamma). In some assays, substantial activity of IFN-gamma on murine macrophages, however, is only observed in synergy with lipopolysaccharide (LPS) or other cytokines (1). In addition, certain cytokines have been shown to induce monocyte or macrophage activation in the absence of IFN-gamma (2-5). We previously described lymphokines in the supernatant of a murine T cell clone that synergized with IFN-gamma in the induction of tumoricidal and schistosomulicidal murine macrophages (1). We called this lymphokine(s) macrophage cytotoxicityinducing factor 2 (MCIF2)(1). A candidate for MCIF2 was lymphotoxin (LT), because the T cell clone supernatant contained high amounts of LT. LT is functionally homologous and structurally related to the macrophage product tumor necrosis factor (TNF). Therefore, we tested whether recombinant (r) LT or rTNF can function as MAF. We report here that rLT or rTNF synergize with rIFN-gamma in the induction of tumoricidal and schistosomulicidal murine macrophages.

Characterization of binding and biological effects of monoclonal antibodies against a human tumor necrosis factor receptor.

Three different antibodies against a human TNF receptor (htr-1, htr-5, and htr-9) have been examined for their binding pattern to U937 cells and ability to mimic TNF-alpha activity in U937 cells, Fs4 fibroblasts, and human endothelial cells. Flow cytometric analysis revealed that htr-5 and htr-9 bound specifically to a TNF receptor on U937 cells that could be blocked by pretreatment with rTNF-alpha. Pretreatment of U937 cells with rTNF-beta blocked the binding of htr-9, but to a lesser extent htr-5 binding. Pretreatment with htr-5 inhibited the binding of htr-9 to U937 cells while pretreatment with htr-9 did not inhibit htr-5 binding. These results indicate that htr-5 and htr-9 recognize distinct but overlapping epitopes of a human TNF receptor on U937 cells and that htr-5 may be close to a TNF-alpha-specific domain of the binding site. Pretreatment with htr-5 or htr-9 only minimally reduced binding of BrTNF-alpha to U937 cells; however, these antibodies were much more effective in inhibiting BrTNF-alpha binding to HL-60 cells. Furthermore, it was found that htr-1 and htr-9, but not htr-5, had TNF-alpha activity on U937 cells, Fs4 fibroblasts, and endothelial cells and that the TNF-alpha activity induced by htr-9 was completely inhibited by htr-5. However, the cytotoxic activity of TNF-alpha was only partially inhibited by htr-5 on U937 cells while htr-5 had no effect on TNF-alpha activity on Fs4 cells. The data suggest that a common epitope is involved in inducing TNF-alpha activity in three different cell systems.

Three distinct classes of regulatory cytokines control endothelial cell MHC antigen expression. Interactions with immune gamma interferon differentiate the effects of tumor necrosis factor and lymphotoxin from those of leukocyte alpha and fibroblast beta interferons.

Recombinant preparations of TNF and lymphotoxin (LT) increase the expression of class I MHC antigens on cultured human endothelial cells (EC) without inducing expression of class II antigens. These actions are similar to those of rIFN-alpha or rIFN-beta. However, TNF and LT differ from IFN-alpha/beta in that the former synergize with IFN-gamma for class I regulation whereas the latter do not. Furthermore, LT or TNF do not affect IFN-gamma-mediated class II induction at optimal class I inducing concentrations (100 U/ml), whereas IFN-alpha and IFN-beta (at their optimal concentrations of 1,000 U/ml) are strikingly inhibitory. LT and TNF also can further increase expression of class I antigens on cells already maximally stimulated by IFN-alpha or IFN-beta. A recombinant preparation of IL-6 (formerly called 26-kD protein, IFN-beta 2, or B cell stimulating factor 2) was without effect on class I expression in EC. These data make it seem unlikely that the actions of LT or TNF on EC expression of MHC antigens are mediated through autocrine or paracrine production of IFN-alpha, IFN-beta or IL-6. More importantly, they suggest that LT or TNF are more likely to be immunostimulatory, whereas IFN-alpha or IFN-beta are more likely to be immunoinhibitory in vivo, a consideration of potential relevance for cytokine administration to various patient populations.

Tumor necrosis factor and lymphotoxin induce differentiation of human myeloid cell lines in synergy with immune interferon.

We show that the cytotoxins tumor necrosis factor (TNF) or lymphotoxin (LT), at concentrations of approximately 10(-11) M induce monocytic differentiation of human myeloid cell lines. After 5 d of culture in the presence of rTNF and LT, a significant proportion of the myeloid cell lines express monocyte differentiation antigens and ANAE activity, and become able to reduce nitroblue tetrazolium (NBT) and mediate low levels of ADCC against tumor target cells. These markers of differentiation, however, are maximally induced when rIFN-gamma, at concentrations as low as 4 U/ml, is present simultaneously with the cytotoxins, and the two classes of cytokines act synergistically to induce terminal differentiation. The appearance of monocytic antigens is accompanied by acquisition of morphology and other functional properties of mature monocytic cells, such as chemiluminescence and phagocytosis, and by expression of FcR for monomeric IgG. A decrease in cell proliferation accompanies induced differentiation, and is not due to the cytotoxic properties of TNF or LT, as indicated in simultaneous analysis of surface phenotype and cell cycle. The lack of cytotoxicity of TNF on the HL-60 cell line is also demonstrated by the enhancing effect of TNF on HL-60 cell growth and nucleoside uptake in the first 2 d of culture. These data show that the cytotoxins TNF and LT mediate complex effects on cells of the myelomonocytic lineage and, in synergy with IFN-gamma, can fully induce immature myeloid cells to differentiate into cells with phenotypic, functional, and proliferative characteristics of terminally differentiated myelomonocytic cells.