A universal influenza A vaccine based on the extracellular domain of the M2 protein.

The antigenic variation of influenza virus represents a major health problem. However, the extracellular domain of the minor, virus-coded M2 protein is nearly invariant in all influenza A strains. We genetically fused this M2 domain to the hepatitis B virus core (HBc) protein to create fusion gene coding for M2HBc; this gene was efficiently expressed in Escherichia coli. Intraperitoneal or intranasal administration of purified M2HBc particles to mice provided 90-100% protection against a lethal virus challenge. The protection was mediated by antibodies, as it was transferable by serum. The enhanced immunogenicity of the M2 extracellular domain exposed on HBc particles allows broad-spectrum, long-lasting protection against influenza A infections.

Protection by alpha 1-acid glycoprotein against tumor necrosis factor-induced lethality.

We here report that alpha 1-acid glycoprotein, a typical acute phase protein, protects mice from lethal shock induced by tumor necrosis factor (TNF) or endotoxin. The protection is observed both in normal and in galactosamine-sensitized mice. Optimal desensitization requires at least 3 mg alpha 1-acid glycoprotein administered 2 h before the lethal challenge. Under these conditions, complete inhibition of all TNF-induced metabolic changes was observed: fall in body temperature, release of liver transaminases, enhanced clotting time, and mortality. The known platelet aggregation-inhibitory activity of alpha 1-acid glycoprotein provides a possible explanation for this protective capacity.

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.

The antitumor function of tumor necrosis factor (TNF), I. Therapeutic action of TNF against an established murine sarcoma is indirect, immunologically dependent, and limited by severe toxicity.

The ability of murine recombinant tumor necrosis factor (rTNF) and natural TNF in tumor-necrotizing serum (TNS) to cause regression of the SA1 sarcoma was investigated. We found that to cause regression of a 9-d SA1 sarcoma, near lethal quantities of rTNF and TNS had to be given to the host. However, even at these highly toxic doses, rTNF was not reliable at causing complete tumor regression. On the other hand, both types of TNF were reliable at causing a tumor hemorrhagic reaction that resulted in the destruction of greater than 75% of the tumor's center in 24 h. The TNF-induced hemorrhagic reaction involved the development of numerous petechial hemorrhages in the tumor's vascular bed, which apparently resulted from destruction of the tumor's blood vessels. It was possible to follow the development of the hemorrhagic reaction against time after giving rTNF or TNS by measuring the intratumor extravasation of 51Cr-labeled syngeneic red cells. According to this method, TNF-induced intratumor hemorrhaging was in progress within 1 h of giving TNF and continued for about a 6-h period. However, the hemorrhagic reaction was greatly reduced and complete regression of the rim of the living tumor tissue that survived hemorrhagic necrosis failed to occur, if SA1 sarcoma was growing in T cell-deficient (TXB) mice. This indicates that the TNF-induced hemorrhagic reaction is partly dependent, and the tumor regression that follows is completely dependent on host immunocompetence. This suggests in turn, that rTNF does not directly destroy SA1 tumor cells in vivo, even though it was shown that it can destroy SA1 tumor cells in vitro. This interpretation is supported by the additional findings that rTNF was no more therapeutic against a 3-d (3-mm) SA1 than against a 9-d (8-mm) SA1, and was no more therapeutic when injected directly into the tumor than when injected intravenously. Lastly it was possible to completely inhibit the ability of rTNF and TNS to cause tumor hemorrhagic necrosis and regression by infusing the host with a monospecific, polyvalent anti-rTNF antibody that neutralized the cytotoxic action of rTNF in vitro.

Inflammatory properties of recombinant tumor necrosis factor in rabbit skin in vivo.

We have investigated the ability of recombinant TNF (mouse and human) to produce acute inflammatory lesions in an established experimental model of inflammation. Upon intradermal injection in rabbit skin, TNF, in amounts as low as 3 x 10(-14) mol/site, was found to be very potent at inducing local neutrophil accumulation and neutrophil-dependent oedema formation, thereby fulfilling two important criteria to be considered as an inflammatory mediator. Our findings further indicate that the pro-inflammatory properties of TNF are probably more related to its immediate stimulatory effects on neutrophils rather than to its slow (protein biosynthesis-dependent effects on endothelial cells. Our data thus show that very low amounts of mouse and human recombinant TNF can initiate an acute inflammatory reaction in vivo in rabbit skin and that TNF is able to evoke two of the four cardinal signs of inflammation.

Circulating interleukin 6 during a continuous infusion of tumor necrosis factor and interferon gamma.

Plasma samples obtained from patients receiving a 24-h continuous infusion of human recombinant (hr)TNF or a combination of two overlapping, 24-h continuous infusions of hrIFN-gamma and hrTNF were analyzed for IL-6 in a sensitive bioassay. A transient appearance of circulating IL-6 was observed with peak levels between 3 and 6 h after the start of the hrTNF infusion. These peak levels correlated quite well with the dose of hrTNF administered (r = 0.86; p less than 0.001). The maximal value observed was 27.5 ng/ml IL-6 in a sample of a patient receiving 545 micrograms/m2 hrTNF. The combination of hrIFN-gamma (200 micrograms/m2) and hrTNF in the infusions resulted in higher IL-6 levels than a comparable dose of hrTNF alone. A maximal value of 23.5 ng/ml IL-6 was observed in a patient receiving 205 micrograms/m2 hrTNF. No IL-6 was found in the plasma of patients during the 12-h infusion with hrIFN-gamma alone, except for two borderline samples.

Characterization of soluble factors that induce the cytolytic activity and the expression of T cell growth factor receptors of a T cell hybrid.

A rat X mouse T cell hybrid (PC60) proliferates in the absence of T cell growth factor (TCGF) and its cytolytic activity can be induced by culture in mixed leukocyte culture supernatants or concanavalin A-activated rat spleen cell supernatant (CS) to lyse 51Cr-labeled tumor target cells. To characterize the factor(s) responsible for this reversible induction, serum-free CS was fractionated by reverse phase high performance liquid chromatography and by phenyl-Sepharose chromatography. A cytotoxicity-inducing activity (CIA) was separated from TCGF and macrophage-activating factor/interferon-gamma. CIA was found to be a macromolecule with an apparent molecular weight of 12,000-18,000 and a pI of 5.0 and 6.2. Its activity on PC60 cells depended on the addition of TCGF. Thus TCGF may have other effects on T cells than the induction of entry into cell cycle. The number of TCGF surface receptors on PC60 cells was measured using purified 3H-TCGF. TCGF receptors were undetectable on noninduced cells but appeared during induction. The expression of TCGF receptors was not induced either by TCGF or by CIA-containing supernatants or fractions alone, only by a combination of both. These results show that TCGF plays a role in the regulation of the expression of its own receptors.

Interleukin 10 reduces the release of tumor necrosis factor and prevents lethality in experimental endotoxemia.

Because of its ability to efficiently inhibit in vitro cytokine production by activated macrophages, we hypothesized that interleukin (IL) 10 might be of particular interest in preventing endotoxin-induced toxicity. We therefore examined the effects of IL-10 administration before lipopolysaccharide (LPS) challenge in mice. A marked reduction in the amounts of LPS-induced tumor necrosis factor (TNF) release in the circulation was observed after IL-10 pretreatment at doses at low as 10 U. IL-10 also efficiently prevented the hypothermia generated by the injection of 100 micrograms LPS. Finally, pretreatment with a single injection of 1,000 U IL-10 completely prevented the mortality consecutive to the challenge with 500 micrograms LPS, a dose that was lethal in 50% of the control mice. We conclude that IL-10 inhibits in vivo TNF secretion and protects against the lethality of endotoxin in a murine model of septic shock.

Two monokines, interleukin 1 and tumor necrosis factor, render cultured vascular endothelial cells susceptible to lysis by antibodies circulating during Kawasaki syndrome.

Kawasaki syndrome (KS) is an acute febrile illness of early childhood characterized by diffuse vasculitis and marked immune activation. The present study was undertaken to determine whether the acute phase of KS is associated with circulating cytotoxic antibodies directed to target antigens induced on vascular endothelium by the monokines, IL-1, or tumor necrosis factor (TNF). Sera from 20 patients with acute KS, 11 patients in the convalescent phase of KS, and 17 age-matched controls were assessed for complement-dependent cytotoxic activity against 111In-labeled human endothelial cells (HEC), dermal fibroblasts, and vascular smooth muscle cells. Sera from patients with acute KS but not the other subject groups caused significant (p less than 0.01) complement-mediated killing of IL-1- or TNF-stimulated HEC. None of the sera tested had cytotoxicity against control HEC cultures or the other target cell types, with or without IL-1 or TNF pretreatment. Expression of the IL-1- or TNF-inducible target antigens on endothelial cells was rapid and transient, peaking at 4 h and disappearing after 24 h despite continued incubation with monokine. In contrast, we have previously shown that IFN-gamma requires 72 h to render HEC susceptible to lysis with acute KS sera. Serum adsorption studies demonstrated that IL-1- and TNF-inducible endothelial target antigens are distinct from IFN-gamma-inducible antigens. These observations suggest that mediator secretion by activated monocyte/macrophages could be a predisposing factor to the development of vascular injury in acute KS. Although our present observations have been restricted to KS, the development of cytotoxic antibodies directed to monokine-inducible endothelial cell antigens may also be found in other vasculitides accompanied by immune activation.

Functional characterization of the human tumor necrosis factor receptor p75 in a transfected rat/mouse T cell hybridoma.

We investigated the biological role of the human tumor necrosis factor p75 (hTNF-R75), making use of the species specificity of TNF responses in murine (m) T cell lines. Several TNF-mediated activities on mouse T cells, such as cytokine induction or proliferation, showed a 100-500-fold difference in specific biological activity between mTNF and hTNF. After transfection of hTNF-R75 cDNA in a rat/mouse T cell hybridoma (PC60), however, the 100-fold lower specific biological activity of hTNF was converted to the same specific biological activity as mTNF. The TNF-mediated induction of granulocyte/macrophage colony-stimulating factor was strongly synergized by the addition of interleukin 1. In the presence of the latter cytokine, ligand-competing monoclonal antibodies against hTNF-R75 (utr-1, utr-2, utr-3) were agonistic on transfected PC60 cells. This agonistic activity was further enhanced by crosslinking with sheep anti-murine immunoglobulin antibodies. These data provide direct evidence for a functional role of TNF-R75, without ligand-dependent TNF-R55 involvement, in the induction of cytokine secretion in T cells.

Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor.

Murine L929 fibrosarcoma cells treated with tumor necrosis factor (TNF) rapidly die in a necrotic way, due to excessive formation of reactive oxygen intermediates. We investigated the role of caspases in the necrotic cell death pathway. When the cytokine response modifier A (CrmA), a serpin-like caspase inhibitor of viral origin, was stably overexpressed in L929 cells, the latter became 1,000-fold more sensitive to TNF-mediated cell death. In addition, TNF sensitization was also observed when the cells were pretreated with Ac-YVAD-cmk or zDEVD-fmk, which inhibits caspase-1- and caspase-3-like proteases, respectively. zVAD-fmk and zD-fmk, two broad-spectrum inhibitors of caspases, also rendered the cells more sensitive, since the half-maximal dose for TNF-mediated necrosis decreased by a factor of 1,000. The presence of zVAD-fmk also resulted in a more rapid increase of TNF-mediated production of oxygen radicals. zVAD-fmk-dependent sensitization of TNF cytotoxicity could be completely inhibited by the oxygen radical scavenger butylated hydroxyanisole. These results indicate an involvement of caspases in protection against TNF-induced formation of oxygen radicals and necrosis.

Ia expression by vascular endothelium is inducible by activated T cells and by human gamma interferon.

We have used monoclonal antibody binding, measured by radioimmunoassay, fluorescence flow cytometry, and ultrastructural immunocytochemistry, to measure expression of Ia antigens on cultured human umbilical vein endothelial (HUVE) cells. Under standard culture conditions, HUVE cells do not express Ia antigens. However, treatment of primary HUVE cultures with phytohemagglutinin induces the expression of Ia antigens. Every endothelial cell in the culture becomes Ia-positive and endothelial cells appear to synthesize Ia. HLA-A,B is concomitantly increased. The expression of Ia appears to be mediated by T cells because (a) pretreatment of primary HUVE cultures with OKT3 plus complement blocks the action of the lectins but not of medium conditioned by lectin-activated peripheral blood mononuclear cells; (b) co-culture of endothelial cells with allogeneic T cells, in the absence of lectin, also induces endothelial Ia; and (c) human immune (gamma) interferon, produced by Chinese hamster ovary cells transfected with the human gamma interferon gene, directly induces endothelial Ia. During co-culture with lymphocytes, about one-third of the endothelial cells are Ia-positive after 24 h and all of the endothelial cells are Ia-positive by 72 h. Proliferation of allogeneic T cells starts by 96 h and peaks at 144 h. Thus, endothelial Ia appears sufficiently early to be a determinant for the proliferation of allogeneic T cells. Inducible expression of Ia by endothelium may be important both for allograft rejection and for recruitment of circulating T cells into the site of an immune response.

Dual signaling of the Fas receptor: initiation of both apoptotic and necrotic cell death pathways.

Murine L929 fibrosarcoma cells were transfected with the human Fas (APO-1/CD95) receptor, and the role of various caspases in Fas-mediated cell death was assessed. Proteolytic activation of procaspase-3 and -7 was shown by Western analysis. Acetyl-Tyr-Val-Ala-Asp-chloromethylketone and benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone++ +, tetrapeptide inhibitors of caspase-1- and caspase-3-like proteases, respectively, failed to block Fas-induced apoptosis. Unexpectedly, the broad-spectrum caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone and benzyloxycarbonyl-Asp(OMe)-fluoromethylketone rendered the cells even more sensitive to Fas-mediated cell death, as measured after 18 h incubation. However, when the process was followed microscopically, it became clear that anti-Fas-induced apoptosis of Fas-transfected L929 cells was blocked during the first 3 h, and subsequently the cells died by necrosis. As in tumor necrosis factor (TNF)-induced necrosis, Fas treatment led to accumulation of reactive oxygen radicals, and Fas-mediated necrosis was inhibited by the oxygen radical scavenger butylated hydroxyanisole. However, in contrast to TNF, anti-Fas did not activate the nuclear factor kappaB under these necrotic conditions. These results demonstrate the existence of two different pathways originating from the Fas receptor, one rapidly leading to apoptosis, and, if this apoptotic pathway is blocked by caspase inhibitors, a second directing the cells to necrosis and involving oxygen radical production.

Characterization of interleukin 5 receptors on eosinophilic sublines from human promyelocytic leukemia (HL-60) cells.

The T cell product interleukin 5 (IL-5) has been shown to be a key factor in the development and the maturation of the eosinophilic cell lineage. We report here on the detection of human IL-5 receptors on eosinophilic sublines of the promyelocytic leukemia HL-60. Sodium butyrate, which initiates differentiation to mature eosinophils, also induces the appearance of high affinity (Kd 1-5 X 10(-11) M) IL-5 binding sites on these cells. The receptors are specific for IL-5, since binding of radiolabeled ligand can only be inhibited with homologous or murine IL-5 and not by other cytokines. We further show that the receptors are functional, since IL-5 can stimulate the proliferation of these cells. Affinity crosslinking of surface-bound 125I human IL-5 or 35S mouse IL-5 identified two membrane polypeptides of approximately 60 and approximately 130 kD to which IL-5 is closely associated. The presence of granulocyte/macrophage-colony-stimulating factor or tumor necrosis factor during butyrate induction decreased the expression of IL-5 binding sites compared with control cultures. The identification and characterization of human IL-5 receptors on HL-60 sublines should provide new insight into the role of this cytokine in eosinophil differentiation.

Two tumour necrosis factor receptors: structure and function.

Tumour necrosis factor (TNF) exerts two main effects: a beneficial one as an anti-infection, anti-tumour cytokine, and a detrimental one in the systemic inflammatory response syndrome (SIRS). Two receptors (TNF-R) mediate these effects, but their precise role in different cell types is far from solved. TNF induces receptor oligomerization, an event that is believed to connect the receptors to downstream signalling pathways. Recent research suggests that several TNF-R-associated proteins, including kinases, may initiate cytoplasmic signal transduction.

Induced expression of trimerized intracellular domains of the human tumor necrosis factor (TNF) p55 receptor elicits TNF effects.

The various biological activities of tumor necrosis factor (TNF) are mediated by two receptors, one of 55 kD (TNF-R55) and one of 75 kD (TNF-R75). Although the phenotypic and molecular responses elicited by TNF in different cell types are fairly well characterized, the signaling pathways leading to them are so far only partly understood. To further unravel these processes, we focused on TNF-R55, which is responsible for mediating most of the known TNF effects. Since several studies have demonstrated the importance of receptor clustering and consequently of close association of the intracellular domains for signaling, we addressed the question of whether clustering of the intracellular domains of TNF-R55 (TNF-R55i) needs to occur in structural association with the inner side of the cell membrane, where many signaling mediators are known to reside. Therefore, we investigated whether induced intracellular clustering of only TNF-R55i would be sufficient to initiate and generate a full TNF response, without the need for a full-length receptor molecule or a transmembrane region. Our results provide clear evidence that inducible forced trimerization of either TNF-R55i or only the death domain elicits an efficient TNF response, comprising activation of the nuclear factor kappaB, induction of interleukin-6, and cell killing.

Autocrine tumor necrosis factor (TNF) and lymphotoxin (LT) alpha differentially modulate cellular sensitivity to TNF/LT-alpha cytotoxicity in L929 cells.

Tumor necrosis factor (TNF) and lymphotoxin (LT) alpha are structurally and functionally related cytokines. We expressed the TNF and LT-alpha genes in murine fibrosarcoma L929r2 cells, which can be sensitized to TNF/LT-alpha-dependent necrosis by inhibitors of transcription or translation. Autocrine production of murine TNF in L929r2 cells completely downmodulated the expression of the 55- and 75-kD TNF receptors, resulting in resistance to TNF/LT-alpha cytotoxicity. Partial downmodulation of the 55-kD receptor was observed in human TNF-producing L929r2 cells. In contrast, an unaltered TNF receptor expression was found on LT-alpha L929r2 transfectants. Hence, although similar cytotoxic effects are induced by extracellularly administered TNF and LT-alpha, endogenous expression of these cytokines fundamentally differs in the way they modulate TNF receptor expression. Unlike LT-alpha, secreted by the classical pathway, TNF is first formed as a membrane-bound protein, which is responsible for receptor downmodulation. To explore whether the different pathways for secretion of TNF and LT-alpha explain this difference, we examined the effect of membrane-bound LT-alpha expression. This was obtained by exchange of the classical signal sequence of LT-alpha for the membrane anchor of chicken hepatic lectin. Membrane retention of LT-alpha resulted indeed in receptor downmodulation and TNF/LT-alpha resistance. We conclude that membrane retention of newly synthesized TNF or LT-alpha is absolutely required for receptor downmodulation and TNF/LT-alpha resistance.

The zinc finger protein A20 inhibits TNF-induced NF-kappaB-dependent gene expression by interfering with an RIP- or TRAF2-mediated transactivation signal and directly binds to a novel NF-kappaB-inhibiting protein ABIN.

The zinc finger protein A20 is a tumor necrosis factor (TNF)- and interleukin 1 (IL-1)-inducible protein that negatively regulates nuclear factor-kappa B (NF-kappaB)-dependent gene expression. However, the molecular mechanism by which A20 exerts this effect is still unclear. We show that A20 does not inhibit TNF- induced nuclear translocation and DNA binding of NF-kappaB, although it completely prevents the TNF- induced activation of an NF-kappaB-dependent reporter gene, as well as TNF-induced IL-6 and granulocyte macrophage-colony stimulating factor gene expression. Moreover, NF-kappaB activation induced by overexpression of the TNF receptor-associated proteins TNF receptor-associated death domain protein (TRADD), receptor interacting protein (RIP), and TNF recep- tor-associated factor 2 (TRAF2) was also inhibited by expression of A20, whereas NF-kappaB activation induced by overexpression of NF-kappaB-inducing kinase (NIK) or the human T cell leukemia virus type 1 (HTLV-1) Tax was unaffected. These results demonstrate that A20 inhibits NF-kappaB-dependent gene expression by interfering with a novel TNF-induced and RIP- or TRAF2-mediated pathway that is different from the NIK-IkappaB kinase pathway and that is specifically involved in the transactivation of NF-kappaB. Via yeast two-hybrid screening, we found that A20 binds to a novel protein, ABIN, which mimics the NF-kappaB inhibiting effects of A20 upon overexpression, suggesting that the effect of A20 is mediated by its interaction with this NF-kappaB inhibiting protein, ABIN.

Treatment of murine colitis by Lactococcus lactis secreting interleukin-10.

The cytokine interleukin-10 (IL-10) has shown promise in clinical trials for treatment of inflammatory bowel disease (IBD). Using two mouse models, we show that the therapeutic dose of IL-10 can be reduced by localized delivery of a bacterium genetically engineered to secrete the cytokine. Intragastric administration of IL-10-secreting Lactococcus lactis caused a 50% reduction in colitis in mice treated with dextran sulfate sodium and prevented the onset of colitis in IL-10(-/-) mice. This approach may lead to better methods for cost-effective and long-term management of IBD in humans.

Cloning and expression of human interferon-beta: from bc to ac.

In 1963 I started the Laboratory of Molecular Biology (LMB) at the University of Ghent. Molecular Biology was then a new scientific discipline. Nucleic acids (NA) could be sequenced, manipulated, and recombined to form genetic information which never before had existed. Cloning of DNA-segments allowed multiplying a single molecule a billion-fold. By 1975 recombinant DNA-technology had sufficiently progressed that one could start the pursuit of a medically important goal. Our choice was to go for Interferon, a mysterious substance which could protect against viral infection, and might possibly be used as an anti-cancer agent if available in unlimited quantities. Fortunately, Piet De Somer, then Rector of the KUL, was one of the pioneers in interferon research. He encouraged his young colleague Erik De Clercq to collaborate with us. Erik brought extensive interferon expertise and reagents to the collaboration. But molecular biologists work with NA and interferon is a protein. There was a missing link which was provided by Jean Content of the Brussels Pasteur Institute. Jean had developed a system to convert interferon mRNA into protein, which was send to Leuven for quantification. This close collaboration between 3 laboratories led in January 1980 to the cloning of the human fibroblast, now interferon-beta, gene, and to the primary structure of the protein. However, 2 months earlier, Tadatsugu Taniguchi had succeeded already to obtain such a clone. But we were the first to express the clone in E. coli, and this was the definite proof that the cloned gene coded for human interferon-beta.