Production of colony-stimulating factor by tumor cells and the factor-mediated induction of suppressor cells

Spleen mononuclear cells of C3H/HeN mice were cultivated with mitomycin C-treated tumor cells, X5563, MH134, MM48, MM46, and FM3A/R, all of which were of syngeneic origin, in a medium containing normal syngeneic mouse serum but not FCS. There was a proliferative response to X5563, MH134, and MM48, but not to the two other tumor cells, MM46 and FM3A/R. The responder spleen cells were found to be nonadherent cells with a phenotype of Thy-1-L3T4-Lyt2-Ig-Macl-, which were neither mature T and B cells nor mature macrophage/granulocytes. It was also found that the proliferation of these nonadherent no-marker cells was mediated by tumor cell-derived soluble factors but not by direct stimulation with tumor cells. The responsible factor was a molecule(s) with a Mr of 23 to 25 kDa, which had a CSF activity inducing granulocyte (G)-, macrophage (M)- and G + M-colonies in the bone marrow cells. Neutralization tests of this factor-induced proliferation of spleen cells revealed that a major part of the factor may be GM-CSF or a molecule closely related to it. Incubation of spleen mononuclear cells with these GM-CSF-like tumor cell factors resulted in induction of myeloblastic/promyelocytic cells with a phenotype of Mac-1+2+Ia+ Thy-1-L3T4-Lyt2-Ig- in the spleen cell cultures, which could suppress mitogenic responses of the spleen cells to T and B cell mitogens. GM-CSF-like activity could also be detected in the serum of mice bearing X5563, MH134, and MM48, but not in those bearing MM46 and FM3A/R. Subcutaneous inoculation of C3H/HeN mice with these X5563, MH134, and MM48 tumor cells generated massive metastasis in the lung and lymph nodes, whereas MM46 and FM3A/R produced no macroscopic tumor cell metastasis. These results strongly suggest the possibility that in some tumor cell-host systems, a GM-CSF-like factor(s) produced constitutively by the tumor cells may play an important role in the development of tumor metastasis, mediating through suppression of lymphoid tissues of the host.     

Additional evidence for the augmented induction of tumor-specific resistance in vaccinia virus-primed mice by immunization with vaccinia virus-modulated syngeneic tumor cells

The augmenting effect of vaccinia virus infection of tumor cells on induction of tumor-specific resistance was examined in mice. C3H/HeN mice were primed intraperitoneally (ip) with live vaccinia virus after whole-body irradiation with 250 rad of X-rays. Three weeks later the mice were immunized ip 3 times at weekly intervals with syngeneic murine hepatoma MH134 or spontaneous myeloma X5563 which had been infected in vitro with vaccinia virus and subsequently irradiated with 7000 rad of X-rays. One week after the third immunization, the mice were challenged with 1 X 10(5) viable cells of MH134 or X5563 ip or 1 X 10(6) tumor cells intradermally (id). On ip challenge with viable MH134 cells all mice that had not been pretreated died within 3 weeks due to ascites tumor out-growth, whereas all mice that had been vaccinia virus-primed and immunized with vaccinia virus-infected MH134 cells survived. On ip challenge with X5563 cells, the percentage survival of vaccinia virus-primed and vaccinia virus-modified tumor-immunized mice was 80%. On id challenge with MH134 and X5563 tumor cells, in un-treated mice tumors grew to more than 5 mm in diameter within 3 weeks, whereas 90% and 60%, respectively, of the mice that had been vaccinia virus-primed and immunized with vaccinia virus-infected tumor cells showed no tumor out-growth. Pretreatment by only immunization with vaccinia virus-infected cells or vaccinia virus-priming and immunization with virus non-infected tumor cells were not effective for preventing induction of tumor-resistance to either ip or id challenge with MH134 or X5563 tumor cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific accumulation of gamma- and delta-tocotrienols in tumor and their antitumor effect in vivo

In contrast to extensive studies on tocopherols, very little is understood about tocotrienols (T3). We evaluated the antitumor activities of gamma-T3 and delta-T3 in murine hepatoma MH134 cells in vitro and in vivo. We found that delta-T3 inhibited the growth of MH134 cells more strongly than gamma-T3 by inducing apoptosis. In C3H/HeN mice implanted with MH134, it was found that gamma-T3 and delta-T3 feeding significantly delayed tumor growth. On the other hand, both T3 had no significant effect on body weight, normal-tissue weight and immunoglobulin levels. Intriguingly, we found that T3 was detected in tumor, but not in normal tissues. These results, to our knowledge, are the first demonstration of specific accumulation of gamma-T3 and delta-T3 in tumors and suggest that T3 accumulation is critical for the antitumor activities of T3.     

Antitumor activity of eosinophils activated by IL-5 and eotaxin against hepatocellular carcinoma

We examined the antitumor effects of eosinophils to explore the potential of eosinophils as effector cells in tumor cytotoxicity. We expressed eotaxin in hepatocellular carcinoma cells, MH134, and injected them into either normal or IL-5 TG mice intradermally and monitored cell growth. In normal mice, growth of MH134 cells containing the expression plasmid pCXN2-eotaxin was similar to that of vector-transfected MH134 cells for a period of 2 weeks, suggesting that expression of eotaxin does not change the growth rate of tumor cells. In IL-5 TG mice, however, the growth of eotaxin expressing MH134 cells was significantly suppressed. LPS induced eosinophils to produce TNF-alpha to kill MH134 cells in vitro. Intratumor injection of LPS is effective to kill MH134-pCXN2 and MH134-pCXN2-eotaxin only in normal mice. Administration of anti-CD4 or anti-CD8 antibodies suppressed growth of MH134-pCXN2-eotaxin cells compared with control antibodies, suggesting that T cells may interfere with immunity against MH134. Administration of anti-IL-5Ralpha and anti-asialo GM1 antibodies enhanced growth of MH134-pCXN2-eotaxin cells, suggesting involvement of eosinophils and NK cells in suppression of tumor cell growth. Although we cannot exclude the possibility that NK cells participate in tumor cell killing in vivo, the presence of NK markers such as DX5, asialo GM1, Ly49, and CD94, and NKG2D on large numbers of eosinophils activated by eotaxin suggests that eosinophils function in such suppression of tumor cell growth. Furthermore, we showed that anti-NKG2D antibodies could significantly inhibit the LPS-induced cytotoxicity against MH134 by highly enriched fraction of eosinophils.     

Significant antitumor effect of a synthetic lipid A analogue,DT-5461, on murine syngeneic tumor models

Summary The antitumor effect of a synthetic lipid A analogue, DT-5461, was investigated using syngeneic tumor models in mice. Intravenous injection of DT-5461 into mice transplanted with solid tumors of MethA fibrosarcoma, MH134 hepatoma, MM46 mammary carcinoma, Lewis lung carcinoma (3LL), and colon adenocarcinomas 26 and 38 resulted in significant reductions in the weight of all tumors except Colon 26, with marked hemorrhagic necrosis of tumor tissues. Efficacy was almost equal to that of anEscherichia coli-type synthetic lipid A (compound 506), and also to those of some chemotherapeutics including Adriamycin, mitomycin C, fluorouracil and cisplatin. Furthermore, DT-5461 was more effective than other immunotherapeutics, including picibanil (OK-432) and lentinan. However, its antitumor effects were inferior to those of Adriamycin or OK-432 against the malignant ascites caused by intraperitoneal inoculation with MethA or with MH134 cells; life span was not prolonged by either intraperitoneal or intravenous administration. In addition, although DT-5461 showed direct inhibitory effects on the in vitro growth of MethA or MH134, these were much weaker than those of Adriamycin. These findings clearly indicated that DT-5461 with systemic administration is a highly effective antitumor agent on solid tumors, and suggest that the antitumor effect of DT-5461 with potent necrotizing activity might derive from indirect mechanisms related to the activation of host immune systems and not to the weak direct cytotoxicity.     

The role of tumor-specific Lyt-1+2− T cells in eradicating tumor cells in vivo

Summary The present study investigates some of mechanisms for tumor-specific Lyt-1⁺2^– T cell-mediated tumor cell eradication in vivo through analyses of tumor specificity in the afferent tumor recognition and efferent rejection phases. When C3H/He mice which had acquired immunity against syngeneic MH134 hepatoma were challenged with other syngeneic X5563 plasmacytoma cells, these mice failed to exhibit any inhibitory effect on the growth of X5563 tumor cells. However, the inoculation of X5563 tumor cells into the MH134-immune C3H/He mice together with the MH134 tumor cells resulted in appreciable growth inhibition of antigenically distinct (bystander) X5563 tumor cells. Although the growth of X5563 cells was inhibited in an antigen-nonspecific way in mice immunized to antigenically unrelated tumor cells (bystander effect), the activation of Lyt-1⁺2^– T cells leading to this effect was strictly antigen-specific. Such a bystander growth inhibition also required the admixed inoculation of the bystander (X5563) and specific target (MH134) tumor cells into a single site in mice immunized against the relevant MH134 tumor cells. Furthermore, the results demonstrated that Lyt-1⁺2^– T cells specific to MH134 tumor cells were responsible for mediating the growth inhibition of antigenically irrelevant (bystander) and relevant tumor cells. These results are discussed in the context of cellular and molecular mechanisms involved in the Lyt-1⁺2^– T cell-initiated bystander phenomenon.This work was supported by Special Project Research-Cancer Bioscience from the Ministry of Education, Science and Culture     

Modification of tumor cells with Fas (CD95) antigen gene and Fas ligand (CD95L) gene transfection by electroporation for immunotherapy of cancer

Electroporation is a method for introducing DNA into cells by using a high-voltage electric field. This method is very simple and easily manipulated. We describe here a method for the modification of tumor cells with the Fas/Apo-1 (CD95) antigen-gene and Fas ligand (FasL)-gene transfection through the use of electroporation, and suggest that the Fas-FasL system is a good target for the induction of apoptosis-mediated antitumor activity. The Fas receptor/ligand system induces apoptosis and plays an important role in regulation of the immune system. In the method described, hepatoma MH134 (Fas⁻ and FasL⁻) is transfected with murine Fas and FasL cDNA. A single administration of monoclonal anti-Fas antibody efficiently suppresses the growth of F6b (MH134+Neo+Fas) tumors but not that of N1d (MH134+Neo) tumors in gld/gld lpr/lpr mice. MH134+Neo+FasL tumor cells were rejected after the induction of inflammation with infiltration of neutrophils in mice. These results suggest that electroporation and Fas-mediated apoptosis are a good method for inducing of antitumor activity.     

Intratumoral tumor necrosis factor induction in tumor-bearing mice by exogenous/endogenous tumor necrosis factor therapy as compared with systemic administration of various biologic response modifiers.

The relationship between the induction of tumor necrosis factor (TNF) (as an indicator of inflammatory reaction) in tumor tissues and its antitumor effect was investigated in tumor-bearing mice by using nine biologic response modifiers (BRMs) and by exogenous/endogenous TNF therapy following a previously reported protocol. Close correlation between the induction of TNF-rich inflammation in tumor tissues and the antitumor effect of BRM were observed. The results of this study suggest that the conditions necessary for exerting antitumor effects of biologic response modifiers may be the induction of TNF (50 to 200 U/g) at the tumor lesions at an early stage after BRM administration and maintenance of the detectable amount of TNF (approximately 10 U/g) for more than 6 hours. Tumor necrosis factor should also be induced in the liver and spleen so that its activity can be maintained in the tumor lesions.     

Feasibility of UV-inactivated vaccinia virus in the modification of tumor cells for augmentation of their immunogenicity

Summary We compared the immunity induced by tumor cells modified with UV-inactivated purified vaccinia virus (UV-VV) and with live purified vaccinia virus (L-VV). C3H/HeN mice were inoculated i.p. with UV-VV or L-VV after whole-body irradiation with 150 rads of X-rays (priming). After 3 weeks the mice were immunized i.p. 3 times at weekly intervals with syngeneic X5563 or MH134 cells that had been adsorbed in vitro with UV-VV or infected with L-VV and subsequently irradiated with 10⁴ rads of X-rays. Then 1 week after the last immunization, the mice were challenged s.c. with X5563 viable tumor cells or challenged i.p. with MH134 viable tumor cells. The 50% lethal dose (TLD₅₀) of X5563 in mice primed and immunized with UV-VV (UV-VV group) on s.c. challenge (10^6.06) was the same as for mice treated with L-VV (L-VV group), whereas the TLD₅₀ of unprimed or nonimmunized mice (control group) was 10^2.61. The TLD₅₀ of MH134 in the UV-VV treated group on i.p. challenge (10^6.48) was similar to that of the L-VV treated group (10^6.54), while the TLD₅₀ of the control group was 10^1.00. The difference between the TLD₅₀ values of X5563 on s.c. challenge of mice primed and immunized with UV-VV or L-VV and control mice was 10^3.4. The difference between the TLD₅₀ values of MH134 on i.p. challenge of primed and immunized mice and control mice was 10^5.5. These results indicate that the in vivo helper function of UV-VV is similar to that of L-VV and that the augmenting effect of this protocol depends on the kind of tumor.     

Cell-associated tumor necrosis factor (TNF) as a killing mechanism of activated cytotoxic macrophages

Different macrophage populations were investigated for their abilities to secrete tumor necrosis factor (TNF) and to lyse TNF-susceptible tumor cells. In this way we could demonstrate that TNF-secretion, although a feature of all activated macrophage populations, is no absolute requirement for the killing of the TNF-sensitive Wehi 164 target. Macrophage cytotoxicity against this cell but not against the TNF-resistant P815 mastocytoma, was completely inhibitable by a specific anti-TNF serum also in the absence of measurable secreted TNF. Moreover the TNF-dependent lysis of tumor cells could also be performed by activated macrophages that had been fixed with paraformaldehyde before the addition of the target cells. In the indirect radioimmunoassay, TNF could be demonstrated on the surface of fixed effector cells. Our results must be interpreted in terms of membrane-associated TNF as the lytic principle for TNF-susceptible tumor cells.     

Interferon-gamma enhances expression of cellular receptors for tumor necrosis factor

Incubation of several human tumor cell lines with human interferon-gamma (IFN-gamma) increased the specific binding of subsequently added 125I-labeled recombinant human tumor necrosis factor (TNF). A similar increase in TNF binding was seen in murine L929 cells after incubation with murine IFN-gamma, but not after incubation with human IFN-gamma. Increased TNF binding to cells incubated with IFN-gamma was due to an increase in the number of TNF receptors, with no demonstrable change in binding affinity. In one out of two human cell lines tested, IFN-alpha and IFN-beta also produced increased TNF binding, albeit with a lower efficacy than IFN-gamma. A maximal increase in TNF binding was seen after about 6 to 12 hr of incubation with IFN. Increased TNF binding due to enhanced TNF receptor expression may contribute to the enhancement of TNF cytotoxicity seen in some tumor cell lines after INF treatment. Modulation of TNF receptor expression by IFN may also influence other biological activities of TNF.     

Studies on macrophage-activating factor (MAF) in antitumor immune responses. I. Tumor-specific Lyt-1+2- T cells are required for producing MAF able to generate cytolytic as well as cytostatic macrophages

In the present study we investigated some of the cellular mechanisms for the generation of macrophage-activating factor(s) (MAF) in immune responses to tumor antigens. C3H/HeN mice were immunized to syngeneic MH134 hepatoma or MCH-1-A1 fibrosarcoma by intradermal inoculation of viable tumor cells, followed by the surgical resection of the tumor. Spleen and lymph node cells from these tumor-immune mice were stimulated in vitro with the corresponding tumor cells, and supernatant from such a culture was tested for an ability to activate macrophages to exert their cytostatic and cytolytic activities as detected on tumor cells unrelated to immunizing tumors. Peritoneal adherent cells as a macrophage source, which were preincubated with supernatant from co-culture of tumor-unimmunized normal spleen and lymph node cells plus tumor cells, failed to exhibit any significant antitumor effect on unrelated X5563 tumor cells, whereas the addition of supernatant from cultures containing immune lymphocytes to adherent cells resulted in appreciably potent cytostatic and cytolytic effects on X5563 tumor cells, indicating the generation of MAF in culture supernatant. The activation of tumor-immune spleen and lymph node cells for MAF generation was tumor-specific, because anti-MH134- and anti-MCH-1-A1-immune lymphocytes produced MAF by the stimulation with the respective but not with the other alternative tumor cells. Such MAF production was abolished by treatment of tumor-immune spleen and lymph node cells with anti-Thy-1.2 or anti-Lyt-1.1 but not with anti-Lyt-2.1 antibody plus complement before culturing. These results indicate that the tumor-specific Lyt-1+2- T cell subset has a crucial role in generating MAF by which an adherent cell population as a source of macrophages acquires the potential for inducing a cytolytic as well as a cytostatic effect on tumor cells.     

Expression of an exogenous tumor necrosis factor (TNF) gene in TNF-sensitive cell lines confers resistance to TNF-mediated cell lysis.

TNF, a cytokine with cytotoxic activity on a variety of tumor cells, is mainly produced by macrophages; however, some tumor cell types of non-macrophage origin, apparently resistant to TNF-mediated cell lysis, can also produce TNF. It is not clear whether these cells were TNF-resistant a priori or whether protective mechanisms against toxicity of autocrine TNF may be induced in TNF-producing cells. Murine L929sA fibrosarcoma cells, which are highly sensitive to TNF cytotoxicity, were transfected with the neomycin resistance (neor) gene, alone or in combination with the human (h) or the murine (m) TNF gene. All exogenous genes were under control of the constitutive SV40 early promoter. After cotransfection, the number of neor colonies was 10 to 100% as compared with the number of colonies upon transfection with the neor gene alone. An appreciable fraction of these colonies (50-100%) constitutively produced biologically active TNF. mTNF-producing L929 cells were fully TNF resistant, whereas hTNF-producing cells showed partial TNF resistance. Specific TNF binding could not be detected on mTNF-producing L929sA transfectants, whereas hTNF-producing cells showed reduced TNF binding. Apparently, TNF gene expression, even in a priori TNF-sensitive cells, can induce mechanisms to prevent toxicity by both autocrine and exogenous TNF. No TNF resistance was induced by expression of a gene sequence encoding the 9-kDa membrane-bound presequence part of the 26-kDa mTNF proform. Expression of a mutant 26-kDa TNF gene coding for a quasi-inactive mature mTNF induced only weak TNF resistance as compared with the complete resistance obtained after transfection with the wild-type gene. These findings show that the membrane-bound TNF presequence as such is not sufficient for induction of TNF resistance and imply that the active site of mature TNF is involved in modulation of TNF responsiveness upon autocrine TNF production.     

Signalling pathway of tumor necrosis factor in normal and tumor cells

Summary Several aspects of the activity and effects of tumor necrosis factor (TNF) were investigated to gain further insight into its cytotoxic mechanism. The relation between number of TNF receptors and TNF susceptibility of both tumor cells and normal cells was studied, utilizing a specific binding assay. Among the tumor cells, a fairly close correlation (r=0.855) was observed between receptor number and sensitivity to TNF. No cytotoxic effect by TNF was observed on any of the normal cells tested, even though TNF receptors were shown to be present, and cell proliferation was apparently stimulated by TNF in some cases. TNF internalization and intracellular distribution were studied by pulse-labelling and Percoll density gradient centrifugation. In L-M (murine tumorigenic fibroblasts, highly sensitive to TNF cytotoxicity) cells and HEL (human embryonic lung cells, non-sensitive to TNF cytotoxicity) cells, receptor-bound ¹²⁵I-labelled recombinant human TNF was rapidly internalized and delivered to lysosomes within 15–30 min, and this was followed by degradation and release into the culture medium. The presence of either a cytoskeletal disrupting agent or a lysosomotropic agent was observed to inhibit the cytotoxic effect of TNF, thus also indicating that TNF internalization, followed by delivery to lysosomes, is essential in the cytolytic mechanism of TNF.As observed by [³H]uridine incorporation, TNF did not affect RNA synthesis in L-R cells (TNF-resistant cell lines derived from L-M cells) and HEL cells, but markedly stimulated (by 3.5 times) RNA synthesis in L-M cells.     

Rapid isolation of nucleoli from detergent purified nuclei of various tumor and tissue culture cells

A rapid isolation procedure of nucleoli from detergent purified nuclei of some tumor and tissue culture cells is described. The procedure makes use of a non-ionic detergent, Nonidet P40 and sodium deoxycholate to purify nuclei followed by the addition of Ca²⁺ or Mg²⁺ to strengthen the nucleoli against sonication. Enzymatically active (with respect to nucleolar RNA polymerase) nucleoli containing undegraded nucleolar RNAs may be isolated from a mouse hepatoma MH134, Ehrlich ascites tumor, HeLa cells, L cells and C3H2K cells with this procedure.     

Helper T cells against tumor-associated antigens (TAA): preferential induction of helper T cell activities involved in anti-TAA cytotoxic T lymphocyte and antibody responses

This study establishes assay systems for helper T cell activities assisting cytotoxic T lymphocyte (CTL) and antibody responses to tumor-associated antigens (TAA) and demonstrates the existence of TAA that induce preferentially anti-TAA CTL helper and B cell helper T cell activities in two syngeneic tumor models. C3H/HeN mice were immunized to the syngeneic X5563 plasmacytoma or MH134 hepatoma. Spleen cells from these mice were tested for anti-TAA helper T cell activity capable of augmenting anti-trinitrophenyl(TNP) CTL and anti-TNP antibody responses from anti-TNP CTL and B cell precursors (responding cells) by stimulation with TNP-modified X5563 or MH134 tumor cells. The results demonstrate that cultures of responding cells plus 85OR X-irradiated tumor-immunized spleen cells (helper cells) failed to enhance anti-TNP CTL or antibody responses when in vitro stimulation was provided by either unmodified tumor cells or TNP-modified syngeneic spleen cells (TNP-self). In contrast, these cultures resulted in appreciable augmentation of anti-TNP CTL or antibody response when stimulated by TNP-modified tumor cells. Such anti-TAA helper activities were revealed to be Lyt-1+2- T cell mediated and TAA specific. Most interestingly, immunization with X5563 tumor cells resulted in anti-TAA helper T cell activity involved in CTL, but not in antibody responses. Conversely, TAA of MH134 tumor cells induced selective generation of anti-TAA helper T cell activity responsible for antibody response. These results indicate that there exists the qualitative TAA-heterogeneity as evidenced by the preferential induction of anti-TAA CTL- and B cell-helper T cell activities. The results are discussed in the light of cellular mechanisms underlying the preferential anti-TAA immune responses, and the interrelationship between various types of cell functions including CTL- and B cell-help.     

In vivo activity of tumor necrosis factor (TNF) mutants. Secretory but not membrane-bound TNF mediates the regression of retrovirally transduced murine tumor.

We have previously demonstrated that murine tumor cells transduced with a retrovirus containing the cDNA encoding wild-type human TNF regress in vivo when injected into immunocompetent mice; this regression is T cell mediated. To determine whether membrane-associated or secreted TNF was responsible for tumor regression, we transduced a cloned murine fibrosarcoma 205 F4 with retroviruses encoding modified human TNF genes. The cloned tumor lines of one retroviral transduction expressed only membrane bound 26-kDa TNF. This TNF could not be cleaved or secreted, but was present on the cell surface. A second retrovirus caused the expression of only secretory 17-kDa TNF, as the transmembrane domain of the cDNA was deleted. The TNF produced by tumor cells transduced with either retroviral vector was functional in vitro as direct lysis of the TNF-sensitive target L929 by transduced tumor cells was demonstrated. The TNF present on 26-kDa expressing tumors was membrane bound as supernatants from cultured 17-kDa TNF expressing tumor cells but not 26-kDa TNF expressing tumors mediated the lysis of L929 cells. Both tumors were injected s.c. into syngeneic mice and tumor growth was measured serially. In repeated experiments, 26-kDa TNF expressing tumors grew progressively in all mice. In contrast, 17-kDa TNF expressing tumors grew for 10 days and then regressed with all animals free of tumor at 28 days. Tumor regression was abrogated by in vivo injection of an anti-TNF antibody. Similar results were obtained in a second tumor model, 203 E4. Thus regression of TNF transduced tumors in vivo requires secretion of TNF, as membrane-bound TNF is insufficient to elicit the host response.     

Production in Escherichia coli of human thymosin beta 4 as chimeric protein with human tumor necrosis factor

We have produced thymosin beta 4 protein in Escherichia coli as a chimeric protein with tumor necrosis factor (TNF). The protein was abundantly expressed, was immunoreactive against both anti-thymosin beta 4 and anti-TNF antibodies, and retained cytotoxicity in a TNF assay using mouse L929 fibroblasts. This latter characteristic enabled the easy and simple purification of thymosin beta 4 merely by following the TNF activity. The chimeric protein was designed to have an Asp-Pro bridge between thymosin beta 4 and TNF which could be specifically cleaved under suitable acidic conditions to release the thymosin beta 4 from the chimeric protein. These results indicate that the expression system in E.coli of a chimeric protein composed of thymosin beta 4 and TNF is appropriate for obtaining an abundant amount of the beta 4 peptide, especially since its purification from tissues is usually difficult because of limited yield and obscurity of its biological activity.     

Role of human beta-defensin-2 during tumor necrosis factor-alpha/NF-kappaB-mediated innate antiviral response against human respiratory syncytial virus

Human respiratory syncytial virus (RSV) constitutes a highly pathogenic virus that infects lung epithelial cells to cause a wide spectrum of respiratory diseases. Our recent studies have revealed the existence of an interferon-alpha/beta-independent, innate antiviral response against RSV that was dependent on activation of NF-kappaB. We demonstrated that NF-kappaB inducing pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF) confers potent antiviral function against RSV in an NF-kappaB-dependent fashion, independent of interferon-alpha/beta. During our efforts to study this pathway, we identified HBD2 (human beta-defensin-2), a soluble secreted cationic protein as an antiviral factor induced during NF-kappaB-dependent innate antiviral activity in human lung epithelial cells. Our results demonstrated that HBD2 is induced by TNF and RSV in an NF-kappaB-dependent manner. Induction of HBD2 in infected cells was mediated by the paracrine/autocrine action of TNF produced upon RSV infection. HBD2 plays a critical role during host defense, because purified HBD2 drastically inhibited RSV infection. We also show that the antiviral mechanism of HBD2 involves blocking of viral cellular entry possibly because of destabilization/disintegration of the viral envelope. The important role of HBD2 in the innate response was also evident from loss of antiviral activity of TNF upon HBD2 silencing by short interfering RNA. The in vivo physiological relevance of HBD2 in host defense was apparent from induction of murine beta-defensin-4 (murine counterpart of HBD2) in lung tissues of RSV-infected mice. Thus, HBD2 functions as an antiviral molecule during NF-kappaB-dependent innate antiviral immunity mediated by the autocrine/paracrine action of TNF.     

Possible role of prostaglandins as negative regulators in growth stimulation by tumor necrosis factor and epidermal growth factor in human fibroblasts

Recombinant tumor necrosis factor (TNF), epidermal growth factor (EGF), and transforming growth factor beta (TGF-beta) stimulated growth of confluent human diploid fibroblasts (FS-4 cells) in the presence of fetal calf serum. TGF-beta synergistically enhanced both the TNF- and EGF-stimulated cell growth, whereas synergism between the mitogenic action of EGF and that of TNF was not observed. When indomethacin or acetylsalicylic acid, an inhibitor of prostaglandin production, was added to FS-4 cells, cell growth stimulated by EGF or TNF was increased, suggesting that prostaglandins induced by these mitogens antagonize their growth stimulatory actions. In contrast, neither indomethacin nor acetylsalicylic acid had a significant effect on the TGF-beta-induced growth of FS-4 cells. Mitogenic responses of indomethacin-treated cells to EGF, TNF, and TGF-beta were similarly suppressed by the addition of exogenous prostaglandin D2 (PGD2). Other prostaglandins such as PGE2 and PGF2 produced less inhibition of the cell growth.