Publications

Cell-culture studies are our main source of knowledge of the various forms of programmed cell death. Yet genetic perturbations of death-protein function in animal models are almost the only source of our knowledge of the physiological roles of these programs. Shortcomings in the state of knowledge acquired by these two experimental approaches are exemplified in this Perspective by reference to research on the contribution of apoptosis to lymphocyte development, a subject on which there is already much knowledge, and on the role of necroptosis in inflammation, about which information is just beginning to emerge. To address these shortcomings, there is need to find ways to verify the notions obtained through the current experimental approaches by directly monitoring death programs within specific cells in vivo. Cell-culture studies are our main source of knowledge of the various forms of programmed cell death. Yet genetic perturbations of death-protein function in animal models are almost the only source of our knowledge of the physiological roles of these programs. Shortcomings in the state of knowledge acquired by these two approaches are exemplified in this Perspective by reference to research on the contribution of apoptosis to lymphocyte development, a subject on which there is already much knowledge, and on the role of necroptosis in inflammation, about which information is just beginning to emerge. To address these shortcomings, there is need to find ways to verify the notions obtained through the current experimental approaches by directly monitoring death programs within specific cells in vivo.

Until recently, programmed cell death was conceived of as a single set of molecular pathways. We now know of several distinct sets of death-inducing mechanisms that lead to differing cell-death processes. In one of them - apoptosis - the dying cell affects others minimally. In contrast, programmed necrotic cell death causes release of immunostimulatory intracellular components after cell-membrane rupture. Defining the in vivo relevance of necrotic death is hampered because the molecules initiating it [such as receptor-interacting protein kinase-1 (RIPK1), RIPK3, or caspase-1] also serve other functions. Proteins that participate in late events in two forms of programmed necrosis [mixed lineage kinase domain-like protein (MLKL) in necroptosis and gasdermin-D in pyroptosis] were recently discovered, bringing us closer to identifying molecules that strictly serve in death mediation, thereby providing probes for better assessing its role in inflammation.

A signaling pathway that induces programmed necrotic cell death (necroptosis) was reported to be activated in cells by several cytokines and various pathogen components. The major proteins participating in that pathway are the protein kinases RIPK1 and RIPK3 and the pseudokinase mixed lineage kinase domain-like protein (MLKL). Recent studies have suggested that MLKL, once activated, mediates necroptosis by binding to cellular membranes, thereby triggering ion fluxes. However, our knowledge of both the sequence of molecular events leading to MLKL activation and the subcellular sites of these events is fragmentary. Here we report that the association of MLKL with the cell membrane in necroptotic death is preceded by the translocation of phosphorylated MLKL, along with RIPK1 and RIPK3, to the nucleus.

Emerging evidence indicates that necrotic cell death can be regulated by a specific set of signaling molecules. Studies showing that the same signaling molecules also trigger inflammation, and that when cells die necrotically some of the molecules they release facilitate inflammation, raised the possibility that the death induced by these signaling molecules ("necroptosis") serves to trigger inflammation. Here we briefly discuss the work done on the anti-inflammatory function of caspase-8 and its relation to the inhibitory effect of this enzyme on the induction of necroptosis. The studies imply that caspase-8 and the other proximal signaling proteins known to participate in the induction and regulation of necroptosis are too pleiotropic to serve as reliable molecular probes for determining the relative contribution of this death mode to in vivo processes.

STING, a protein mediating signaling activation by cytosolic DNA and known to act downstream of DNA sensor proteins, is shown also to bind DNA directly, raising the need to clarify the relative contributions of these distinct binding modes to DNA sensing.

Keywords: DEATH DOMAIN; PROTEIN; RECEPTOR; MICE; APOPTOSIS; INTERACTS; FAS

Members of the TNF superfamily control numerous aspects of immune defense as well as various processes of homeostasis and embryonic development. Recent advances in our knowledge of both the beneficial and the deleterious activities of these cytokines were thoroughly discussed at this conference. Participants presented new information about signaling mechanisms that these cytokines activate, with special attention to cell-death regulation, ubiquitination of signaling-proteins as a means of regulating their function, and complex systems of gene and signaling regulation. Sessions were devoted specifically to aberrations in functions of the TNF-family that contribute to the pathology of infectious, autoimmune and neurodegenerative diseases and to cancer, and to the application of our knowledge to therapy.

Caspase-8, the proximal enzyme in the death-induction pathway of the TNF/nerve growth factor receptor family, is activated upon juxtaposition of its molecules within the receptor complexes and is then self-processed. Caspase-8 also contributes to the regulation of cell survival and growth, but little is known about the similarities or the differences between the mechanisms of these nonapoptotic functions and of the enzyme's apoptotic activity. In this study, we report that in bacterial artificial chromosome-transgenic mice, in which the aspartate residue upstream of the initial self-processing site in caspase-8 (D387) was replaced by alanine, induction of cell death by Fas is compromised. However, in contrast to caspase-8-deficient mice, which die in utero at mid-gestation, the mice mutated at D387 were born alive and seemed to develop normally. Moreover, mice with the D387A mutation showed normal in vitro growth responses of T lymphocytes to stimulation of their Ag receptor as well as of B lymphocytes to stimulation by LPS, normal differentiation of bone marrow macrophage precursors in response to M-CSF, and normal generation of myeloid colonies by the bone marrow hematopoietic progenitors, all of which are compromised in cells deficient in caspase-8. These finding indicated that self-processing of activated caspase-8 is differentially involved in the different functions of this enzyme: it is needed for the induction of cell death through the extrinsic cell death pathway but not for nonapoptotic functions of caspase-8.

Caspase-8 is frequently deficient in several kinds of human tumors, suggesting that certain effects of this enzyme restrict tumor development. To examine the nature of the cellular function whose regulation by caspase-8 contributes to its antitumor effect, we assessed the impact of caspase-8 deficiency on cell transformation in vitro. Caspase-8-deficient mouse embryonic fibroblasts immortalized with the SV40 T antigen did not survive when cultured in soft agar, and were nontumorogenic in nude mice. However, the rate of transformation of these cells during their continuous growth in culture, as reflected in the observed emergence of cells that do grow in soft agar and are able to form tumors in nude mice, was far higher than that of cells expressing caspase-8. These findings indicate that caspase-8 deficiency can contribute to cancer development in a way that does not depend on the enzyme's participation in killing of the tumor cells by host immune cytotoxic mechanisms, or on its involvement in the cell-death process triggered upon detachment of the cells from their substrate, but rather concerns cell-autonomous mechanisms that affect the rate of cell transformation.

Keywords: Biochemistry & Molecular Biology; Cell Biology; Immunology

Recruitment of the NF-κB-activating IKK signaling complex to the TNF receptor is shown to be driven by induced binding of NEMO, a regulatory component of this complex, to K63-linked polyubiquitin chains attached to RIP1, a receptor-associated adaptor protein (Ea et al., 2006 [in a recent issue of Molecular Cell]; Li et al., 2006; Wu et al., 2006a).

Signals emanating from receptors of the tumor necrosis factor/nerve growth factor (TNF/NGF) family control practically all aspects of immune defense and, as such, constitute potential targets for therapeutic intervention through rational drug design. Indeed, arrest of these signals by blocking ligand-receptor interactions enables effective suppression of a variety of activities that are implicated in various pathologies, such as T and B lymphocyte activation and growth, inflammation, fibroblast proliferation, and cell death. To be therapeutically useful, however, inhibition of signaling should be restricted by determinants of specificity, at least to the same degree observed when blocking activation of individual receptors. In spite of their broad range of functions, receptors of the TNF/NGF family are known to activate just a few signaling pathways. Of these, the most extensively studied are the activation of the caspase protease cascade, which leads to cell death, and the activation of NF-kappaB (nuclear factor-kappaB) transcription factors through protein phosphorylation cascades. Until recently, most studies of the two pathways have solely focused on the core signaling complexes that are shared by the different receptors: death-inducing complexes containing the cysteine proteases caspase-8 and caspase-10, bound to the adapter protein MORT1/FADD (mediator of receptor-induced toxicity/Fas-associated DD protein), and the NF-kappaB-activating complex, composed of the protein kinases IKK1 (IkappaB kinase 1) and IKK2 (IkappaB kinase 2) and the regulatory subunit NEMO (NF-kappaB essential modulator; the 'IKK signalosome'). Knowledge has begun to emerge of additional molecules and mechanisms that affect these basic signaling complexes and impose specificity on their function.

Significant caspase-8 activity has been found in normal and certain tumor cells, suggesting that caspase-8 possesses an alternative, nonapoptotic function that may contribute to tumor progression. In this article, we report that caspase-8 promotes cell motility. In particular, caspase-8 is required for the optimal activation of calpains, Rac, and lamellipodial assembly. This represents a novel nonapoptotic function of caspase-8 acting at the intersection of the caspase-8 and calpain proteolytic pathways to coordinate cell death versus cell motility signaling.

Knockout of caspase-8, a cysteine protease that participates in the signaling for cell death by receptors of the TNF/nerve growth factor family, is lethal to mice in utero. To explore tissue-specific roles of this enzyme, we established its conditional knockout using the Cre/loxP recombination system. Consistent with its role in cell death induction, deletion of caspase-8 in hepatocytes protected them from Fas-induced caspase activation and death. However, application of the conditional knockout approach to investigate the cause of death of caspase-8 knockout embryos revealed that this enzyme also serves cellular functions that are nonapoptotic. Its deletion in endothelial cells resulted in degeneration of the yolk sac vasculature and embryonal death due to circulatory failure. Caspase-8 deletion in bone-marrow cells resulted in arrest of hemopoietic progenitor functioning, and in cells of the myelomonocytic lineage, its deletion led to arrest of differentiation into macrophages and to cell death. Thus, besides participating in cell death induction by receptors of the TNF/nerve growth factor family, caspase-8, apparently independently of these receptors, also mediates nonapoptotic and perhaps even antiapoptotic activities.

Derangement of the apoptotic program is considered an important cause of liver disease. It became clear that receptor-mediated apoptosis is of specific interest in this context, and CD95 and CD120a, both members of the tumor necrosis factor (TNF) receptor superfamily, are the most prominent cell death receptors involved. The death signal is induced upon ligand binding by recruitment of caspases via the adapter molecule MORT1/FADD to the receptor and their subsequent activation. To investigate the role of MORT1/FADD in hepatocyte apoptosis, we generated transgenic mice expressing liver-specific dominant negative mutant. Mice looked grossly normal; breeding and liver development were not different compared with wild-type littermates. Expression of the transgene completely protected animals from liver failure induced by the anti-Fas antibody Jo2, whereas control animals died as expected 3 to 6 hours after i.p. injection of 15 μg antibody from acute hemorrhagic liver failure. Histology demonstrated only moderate inflammatory changes in the transgenic animals, whereas severe hemorrhagic hepatitis was observed in controls. Similar results were obtained in a model of TNF-mediated liver failure, in which transgenic animals survived significantly better than wild-type animals. In conclusion, our experiments provide evidence that MORT1/FADD is indispensable for Fas and TNF-mediated hepatic injury. This is not only of great importance for targeting future therapies for liver disease but might also serve as an intriguing model to study other causes of liver injury.

David Wallach of the Weizmann Institute, Zhaodan Cao of Tularik Inc., Michael Karin of the University of California, San Diego, and Ebrahim Zandi of the University of Southern California, Norris Cancer Center, discuss how protein kinases regulate NF-kappa B activity.

FIP-3 (14.7K interacting protein) was discovered during a search for cell proteins that could interact with an adenovirus protein (Ad E3-14.7K) that had been shown to prevent tumor necrosis factor (TNF)-α-induced cytolysis. FIP-3, which contains leucine zippers and a zinc finger domain, inhibits both basal and induced transcriptional activity of NF-κB and causes a late-appearing apoptosis with unique morphologic manifestations. Ad E3- 14.7K can partially reverse apoptotic death induced by FIP-3. FIP-3 also was shown to bind to other cell proteins, RIP and NIK, which previously had been described as essential components of TNF-α-induced NF-κB activation. In addition, FIP-3 inhibited activation of NF-κB induced by TNF-α, the TNFR-1 receptor, RIP, NIK, and IKKβ, as well as basal levels of endogenous NF-κB in 293 cells. Because the activation of NF-κB has been shown to inhibit apoptosis, FIP-3 appears both to activate a cell-death pathway and to inhibit an NF-κB-dependent survival mechanism.

The Epstein-Barr virus oncoprotein latent infection membrane protein 1 (LMP1) is a constitutively aggregated pseudo-tumor necrosis factor receptor (TNFR) that activates transcription factor NF-κB through two sites in its C-terminal cytoplasmic domain. One site is similar to activated TNFRII in associating with TNFR-associated factors TRAF1 and TRAF2, and the second site is similar to TNFRI in associating with the TNFRI death domain interacting protein TRADD. TNFRI has been recently shown to activate NF-κB through association with TRADD, RIP, and TRAF2; activation of the NF-κB-inducing kinase (NIK); activation of the IκBα kinases (IKKα and IKKβ); and phosphorylation of IκBα. IκBα phosphorylation on Ser-32 and Ser-36 is followed by its degradation and NF-κB activation. In this report, we show that NF-κB activation by LMP1 or by each of its effector sites is mediated by a pathway that includes NIK, IKKα, and IKKβ. Dominant negative mutants of NIK, IKKα, or IKKβ substantially inhibited NF-κB activation by LMP1 or by each of its effector sites.

CD27 is a member of the tumor necrosis factor (TNF) receptor superfamily and is expressed on T, B, and NK cells. The signal via CD27 plays pivotal roles in T-T and T-B cell interactions. Here we demonstrate that overexpression of CD27 activates NF-κB and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK). Deletion analysis of the cytoplasmic domain of CD27 revealed that the C-terminal PIQEDYR motif was indispensable for both NF-κB and SAPK/JNK activation and was also required for the interaction with TNF receptor-associated factor (TRAF) 2 and TRAF5, both of which have been implicated in NF-κB activation by members of the TNF-R superfamily. Co-transfection of a dominant negative TRAF2 or TRAF5 blocked NF-κB and SAPK/JNK activation induced by CD27. Recently, a TRAF2-interacting kinase has been identified, termed NF-κB-inducing kinase (NIK). A kinase- inactive mutant NIK blocked CD27-, TRAF2-, and TRAF5-mediated NF-κB and SAPK/JNK activation. These results indicate that TRAF2 and TRAF5 are involved in NF-κB and SAPK/JNK activation by CD27, and NIK is a common downstream kinase of TRAF2 and TRAF5 for NF-κB and SAPK/JNK activation.

We previously demonstrated that p38 MAPK is a crucial mediator in the NF-κB-dependent gene activation induced by TNF. Here, we have studied the role of several TNF receptor-associated proteins and caspases in p38 MAPK activation by TNF. The latter appears to be dependent on TRAF2, but independent of FADD or caspases. Remarkably, p38 MAPK activation by TNF proceeds independently of the TRAF2-associated NF-κB-inducing kinase NIK, which is known to bind and activate two recently identified IλB kinases. These results demonstrate that two kinase pathways involved in NF-κB regulation, viz. NIK and p38 MAPK-mediated, diverge at the level of TRAF2.

Keywords: TUMOR-NECROSIS-FACTOR; INTERLEUKIN-1-BETA CONVERTING-ENZYME; FACTOR-INDUCED APOPTOSIS; SMALL NUCLEAR RIBONUCLEOPROTEIN; 70-KDA PROTEIN-COMPONENT; ZINC FINGER PROTEIN; FACTOR-ALPHA; C-ELEGANS; POLY(ADP-RIBOSE) POLYMERASE; CAENORHABDITIS-ELEGANS

Several members of the tumour-necrosis/nerve-growth factor (TNF/NGF) receptor family activate the transcription factor NF-κB through a common adaptor protein, Traf2 (refs 1-5), whereas the interleukin 1 type-I receptor activates NF-κB independently of Traf2 (ref. 4). We have now cloned a new protein kinase, NIK, which binds to Traf2 and stimulates NF-κB activity. This kinase shares sequence similarity with several MAPKK kinases. Expression in cells of kinase-deficient NIK mutants fails to stimulate NF-κB and blocks its induction by TNF, by either of the two TNF receptors or by the receptor CD95 (Fas/Apo-1), and by TRADD, RIP and MORT1/FADD, which are adaptor proteins that hind to these receptors. It also blocked NF-κB induction by interleukin-1. Our findings indicate that NIK participates in an NF-κB- inducing signalling cascade common to receptors of the TNF/NGF family and to the interleukin-1 type-I receptor.

That leukocytes can produce proteins with cell-killing activities has been known for almost 30 years (Granger GA, Kolb EP. J Immunol 1968, 101; Ruddle NH, Waksman BH. J Exp Med 1968, 128, 1267-1279; Carswell EA, Old LJ, Kassel S, Green S, Fiore N, Williamson B. Proc Natl Acad Sci USA 1975, 72, 3666-3670). However, it is only recently that the nature of this cell killing activity has become clear. What appeared initially to be merely a toxic effect of the leukocyte-produced proteins (hence their initial name, 'lymphotoxins') has turned out to represent a new kind of biological mechanism, whose understanding required a radical change of concepts concerning the ways in which the life and death of the cell are controlled. The leukocyte-produced 'toxic' proteins turned out to act, not through any toxic feature of the proteins themselves, but by activating destructive mechanisms that pre-exist within the target cell. Their action thus represents a way by which one cell can dictate suicide to another. Within the last few years more has been learnt about their mechanisms of action than was learnt throughout the two preceding decades. Nevertheless, many questions still remain unresolved. The purpose of this exposition is to spell out some of these open questions.

Fas/APO-1 and p55 tumor necrosis factor (TNF) receptor (p55-R) activate cellular mechanisms that result in cell death. Upon activation of these receptors, Fas/ APO-1 binds a protein called MORT1 (or FADD) and p55-R binds a protein called TRADD. MORT1 and TRADD can also bind to each other. We have cloned a novel protein, MACH, that binds to MORT1. This protein exists in multiple isoforms, some of which contain a region that has proteolytic activity and shows marked sequence homology to proteases of the ICE/ CED-3 family. Cellular expression of the proteolytic MACH isoforms results in cell death. Expression of MACH isoforms that contain an incomplete ICE/ CED-3 region provides effective protection against the cytotoxicity induced by Fas/ APO-1 or p55-R triggering. These findings suggest that MACH is the most upstream enzymatic component in the Fas/APO-1-and p55-R-induced cell death signaling cascades.

Purpose: To study the role of tumor necrosis factor (TNF) in the male reproductive system by examining the occurrence, source, and possible functional significance of soluble TNF receptors in seminal fluids of normal and infertile men. Materials and Methods: Concentrations of soluble TNF receptors (p55-sTNF-R and p75-sTNF-R) were measured by ELISA in human sera, seminal fluids, prostatic fluid and fluid obtained from an epididymal spermatocele. Results: The level of p55-sTNF-R in seminal fluids of normospermic men was ≃20-fold higher than in normal serum (13.9 ± 6.9 ng./ml. versus 0.7 ± 0.2 ng./ml.). In contrast, p75-sTNF-R, which occurs in serum at amounts higher than p55-sTNF-R, was almost indiscernible in the seminal fluids (

All references are of the Abstracts presented at the Sixth International Congress on TNF and Related Cytokines, published in Eur. Cyt. Net., Vol 7 No 2. Only the abstract and page numbers are cited.

High levels of circulating soluble tumor necrosis factor receptors (sTNF-R) are associated with HIV-1 infection and disease. To understand better this association, we have investigated p55 and p75 TNF-R expression on peripheral blood mononuclear cell (PBMC) subsets and in the promonocytic cell line U937, with or without HIV infection. Using flow cytometry and monoclonal antibodies both to sTNF-R and to PBMC subsets, TNF-R were found to be expressed mostly by monocytes and in decreasing amounts and intensity in the following order: CD14⁺ cells > CD8⁺ cells > CD4⁺ cells. Expression of TNF-R was higher on cells obtained from HIV-infected than from noninfected subjects, and expression of p75 sTNF-R was much higher than that of p55 sTNF-R. Studying the U937 cells revealed that over 80% of the cells expressed both sTNF-R, but with greater fluorescence intensity in the HIV-1 chronically infected cells (U-937-IIIB). Treatment of the cells with PMA caused an accelerated release into the medium of both sTNF-R, with a sharp decline in their cell surface expression. Basal levels of mRNA transcripts for p75 TNF-R were higher in the U-937-IIIB cells than in the uninfected cells, but p55 TNF-R mRNA was expressed only in the HIV-1-infected cells. These findings show that HIV-1 infection is accompanied by predominant elevation of p75 TNF-R surface expression on monocytes and CD8⁺ lymphocytes, and results in both increased message and expression of these receptors in monocytes. It is very likely that increased shedding of these receptors into the serum accounts for the increased serum levels of both sTNF-R found in HIV-infected people.

Tumor necrosis factor (TNF) and lymphotoxin α (LTα) are closely related cytokines which bind with nearly identical affinities to the same pair of cell surface receptors, p55 and p75TNFR. Therefore it is assumed that TNF and LTα are redundant cytokines. This study, however, demonstrates that TNF and LTα differ significantly with regard to their mitogenic and cytotoxic potentials. LTα's superior mitogenic effect could be explained by its formation of a more stable trimer. In contrast to the TNF trimer, which disintegrated under physiological conditions into biologically inactive monomers, the LTα trimer remained stable for several days. Accordingly, LTα more effectively induced fibroblast growth which demands longterm presence of the cytokine. TNF's superior cytotoxicity, which requires only shortterm impact of the cytokine, could be attributed to a distinct interaction with the human p55TNFR. This was demonstrated in NIH 3T3 cells transfected with the human p55TNFR, where cytotoxicity is mediated exclusively by the transfected receptor. Although the p55TNFR had virtually identical affinities for TNF and LTα, as defined by Scatchard analysis, it nevertheless discriminated between binding of each cytokine and showed a 200fold enhanced cytotoxicity mediated by TNF.

A novel protein that binds specifically to the intracellular domain of the p55 tumor necrosis factor (TNF) receptor was cloned by two-hybrid screening of a HeLa cell cDNA library. Data bank searches revealed high sequence similarity of the protein (55.11) to yeast, nematode and plant proteins, whose functions are yet unknown. Significant similarity was also found between 55.11 and SEN3, the yeast equivalent of the p112 subunit of the 26S proteasome. Deletion analysis showed that the protein binds to the p55 receptor upstream to the region involved in induction of cell death.

Soluble tumour necrosis factor receptors (sTNF-R) are up-regulated at sites of chronic inflammation such as rheumatoid synovial joints. The p75 sTNF-R is the more abundant, suggesting an important role for this TNF inhibitor in regulating TNF bioactivity in vivo. As the precise cellular source of these soluble receptors is not known, we investigated the production and regulation of sTNF-R by T lymphocytes, an abundant cell type in inflammatory infiltrates, which upon activation express high levels of p75 surface receptors. Using panels of T-cell lines and clones expressing high levels of p75 TNF-R, we found that p75 sTNF-R production upon stimulation is a feature common to all subsets of T cells, including cells of the CD4^-CD8^- double negative phenotype expressing either αβ or γδ T-cell receptors (TCR). In contrast, levels of p55 sTNF-R were only detected when T cells were stimulated at higher densities and by potent mitogens such as phorbol 12-myristate 13-acetate (PMA). Detailed kinetic analyses revealed that the production of p75 sTNF-R was biphasic, the first phase was activation dependent, occurring in the absence of detectable TNF, while the second phase of p75 sTNF-R production was regulated by cytokines such as TNF. Unlike short-term exposure to TNF which enhances sTNF-R production in vitro and in vivo, prolonged exposure of T lymphocytes to TNF suppressed p75 sTNF-R (but not p55 sTNF-R) production in a dose- and time-dependent fashion. These results suggest that in patients with chronic inflammatory disease, which are exposed to augmented levels of bioactive TNF for prolonged periods, the production of p75 sTNF-R may be impaired.

Signaling by the p55 tumor necrosis factor (TNF) receptor and by the structurally related receptor Fas/ APO1 is initiated by receptor clustering. Data presented here and in other recent studies (Wallach, O., Boldin, M., Varfolomeev, E. E., Bigda, Y., Camonis, H. J. and Mett, I. (1994) Cytokine 6, 556; Song, H. Y., Dunbar, J. D., and Bonner, D. B. (1994) J. Biol. Chem. 269, 22492-22495) indicate that part of that region within the intracellular domains of the two receptors that is involved in signaling for cell death, as well as for some other effects (the "death domain"specifically self-associates. We demonstrate also the expected functional consequence of this association; a mere increase in p55 TNF receptor expression, or the expression just of its intracellular domain, is shown to trigger signaling for cytotoxicity as well as for interleukin 8 gene induction, while expression of the intracellular domain of Fas/APO1 potentiates the cytotoxicity of co-expressed p55 TNF receptor. These findings indicate that the p55 TNF and Fas/APO1 receptors play active roles in their own clustering and suggest the existence of cellular mechanisms that restrict the self-association of these receptors, thus preventing constitutive signaling.

Objective: To examine the effect of glycosylated recombinant human tumor necrosis factor binding protein-1 (r-hTNF binding protein-1), the extracellular domain of the tumor necrosis factor receptor p55 produced in mammalian cells, in a rabbit model of circulatory shock due to Escherichia coli. Design: Prospective, randomized, controlled trial. Setting:University hospital research laboratory.Subjects: Eighteen female, New Zealand white rabbits. Interventions: Anesthetized rabbits, infused with E. coli (10⁹ organisms/kg), were pretreated with either r-hTNF binding protein-1 or saline. Mean arterial pressure, central venous pressure, cardiac output, and heart rate were recorded every 20 mins for 1 hr before, and for 4 hrs after, the infusion of E. coli. Blood samples were obtained at 1-hr intervals for platelet count and white blood cell count, r-hTNF binding protein-1, and tumor necrosis factor (TNF) measurements. Measurements and Main Results: Administration of r-hTNF binding protein-1 resulted in improvement of mean arterial pressure, cardiac output, and systemic vascular resistance, as compared with the vehicle-treated group (p

Whereas there is ample evidence for involvement of the p55 tumor necrosis factor (TNF) receptor (p55-R) in the cytocidal effect of TNF, the role of the p75 TNF receptor (p75-R) in this effect is a matter of debate. In this study, we probed the function of p75-R in cells sensitive to the cytotoxicity of TNF using a wide panel of antibodies (Abs) against the receptor's extracellular domain. Two distinct Ab effects were observed. The Abs triggered signaling for cytotoxicity. This effect: (a) was correlated with the extent of p75-R expression by the cells; (b) was dependent on receptor cross-linking by the Abs; (c) occurred in HeLa cells, but not in A9 cells transfected with human p75-R or in HeLa cells expressing cytoplasmically truncated p75-R mutants, indicating that it involves cell-specific activities of the intracellular domain of the receptor; (d) was synergistic with the cytocidal effect of Abs against p55-R. Moreover, it seemed to reverse induced desensitization to the cytocidal effect of anti p55-R Abs, suggesting that it involves mechanisms different from those of the signaling by the p55 TNF-R. In addition, the Abs affected the response to TNF in a way that does not involve the signaling activity of p75-R. These effects: (a) could be observed also in cells in which only p55-R signaled for the cytocidal effect; (b) were not dependent on receptor cross-linking by the Abs; (c) varied according to the site at which the Abs bound to the receptor; and (d) were correlated inversely with the effects of the Abs on TNF binding to p75-R. That is, Abs binding to the membrane-distal part of the receptor's extracellular domain displaced TNF from the p75 receptor and enhanced cytocidal effect, whereas Abs that bind to the membrane-proximal part of the extracellular domain - a region at which a conformational change seems to take place upon TNF binding - decreased the dissociation of TNF from p75-R and inhibited its cytocidal effect. The above findings suggest that p75-R contributes to the cytocidal effect of TNF both by its own signaling and by regulating the access of TNF to p55-R.

Certain inflammatory cytokines and growth factors have been previously shown to interact with glycosaminoglycan moieties of the extracellular matrix (ECM). We have examined the association of the pleiotropic cytokine TNF-α with glycoprotein constituents of ECM. TNF-α interacted with fibronectin (FN) and laminin, and to a lesser degree with collagen. The major binding site for TNF-α on FN was localized to its 30-kDa N-terminal fragment (FN- N') with a K(i) in the sub-nM range. The binding of ¹²⁵I-labeled TNF-α to immobilized FN or FN-N' persisted for at least 24 h, and was specifically inhibited by antibodies to FN, mAb directed against the FN-N' domain, unlabeled TNF-α, and by the truncated forms of TNF-α receptors. Once bound to immobilized FN or FN-N', the cytokine could not be released by the soluble TNF-α-receptors, although it could be released by anti-TNF-α Ab. TNF-α was also found to interact with soluble FN, although with a lower affinity. Similar to the soluble cytokine, the FN-bound TNF-α appears to be functional; it augmented the β₁-integrin-mediated adhesiveness of activated CD4⁺ human T cells to the glycoprotein. Hence, binding of TNF-α to immobilized FN, which modifies its functional accessibility to soluble TNF- α receptors, does not abolish but rather may locally restrict its activity. This study suggests that a major ECM glycoprotein can present, in a restricted manner, a functional adhesion-modulating cytokine to immune cells, and that ECM glycoproteins may regulate their intrinsic cell-adhesive properties by associating with cytokines.

Lesions of the common inflammatory skin disease psoriasis are characterized by epidermal hyperproliferation, leucocyte adhesion molecule expression and leucocyte infiltration. The local release of proinflammatory cytokines, such as TNF-α, may play an important role in the induction of these events. We have, therefore, analysed aqueous extracts of lesional and uninvolved (clinically normal) stratum corneum for the presence of TNF-α immunoreactivity and biological activity. TNF-α immunoreactivity and bioactivity were consistently higher in lesional compared with uninvolved samples. By using an anti-TNF-α neutralizing antibody it was demonstrated that the biological activity measured was due to the presence of TNF-α alone. Concentrations of soluble TNF receptors (p55 and p75) were also higher in lesional stratum corneum extracts, with the p55 form predominating. The plasma of psoriatic patients was also found to contain elevated concentrations of soluble p55 compared with normal controls. These results confirm the presence of immunoreactive TNF-α and, for the first time, conclusively demonstrate TNF-α biological activity and quantifiable concentrations of soluble TNF receptors (p55 and p75) in lesional psoriatic samples. TNF-α recovery from stratum corneum probably reflects synthesis in deeper, viable layers, where it is likely to exert its biological effects. Local and systemic release of soluble TNF receptors, in particular p55, may serve to regulate the effects of TNF-α in psoriasis.

Tumour necrosis factor (TNF) concentrations are increased in the plasma during a malarial illness, and are highest in patients with severe or fatal disease. We have studied the plasma concentrations of two soluble receptors (sTNF-R1 and sTNF-R2), which act as binding proteins for TNF, in children with falciparum malaria. In 52 Malawian children with malaria, plasma concentrations of both sTNF-R1 (mean (S.D.) 4759(2552) pg/ml) and sTNF-R2 (59077(37102) pg/ml) were greatly increased when compared with levels of convalescence (sTNF-R1 718(68), and sTNF-R2 8015(7021) pg/ml), and in controls without malaria (486(1353) and 4380(2168)). Concentrations of both receptors correlated with plasma levels of TNF measured by immunoradiometric assay, but not with those of another cytokine, IL-6. The mean plasma concentrations of both immunoreactive TNF and soluble TNF receptors were greater in patients with cerebral malaria than those with uncomplicated malaria. Despite high levels of immunoreactive TNF in the plasma of patients acutely ill with malaria, no bioactive TNF could be detected in these patients by the WEHI cell bioassay. Soluble TNF receptors are present in greatly increased concentrations in the plasma of patients with malaria and may play a role in mediating or modulating the pathogenetic effects of the cytokine.

The in vivo protective effect of urinary TNF-binding protein (uTBP) on acute TNF-induced lesions and lethality was assessed in BALB/c mice. Two animal models, the local Shwartzman reaction and galactosamine (GaLN) induced TNF sensitization, were used. In the former, local cutaneous haemorrhagic necrosis induced by 10 μg of recombinant human TNF alpha (r-hTNF) was prevented with iv doses of uTPB as low as 1 μg when administered concomitantly or 10 μg when injected intravenously 60 min before or 30 min after the lesion eliciting-dose of r-hTNF. In the latter model, injection of 1 μg or r-hTNF caused the death of all mice within 36 h. Either 100 or 250 μg of uTBP given intravenously simultaneously with r-hTNF/GaLN totally prevented this mortality. In contrast to anti-human TNF monoclonal antibodies, these very same doses of uTBP significantly protected mice even when injected after the lethal r-hTNF dose. These data confirm in relevant in vivo pathological models the TNF inhibiting capacity of the natural soluble TNF receptor I.

Tumor necrosis factor (TNF-α) acts as a growth stimulatory factor on leukemic B lymphocytes from many patients with chronic lymphocytic leukemia (CLL). Because TNF induces production of interleukin-6 (IL-6), which has been shown to be a growth factor for myeloma and other transformed B cells, we examined the possibility that IL-6 mediates the growth-stimulatory effect of TNF on B-CLL cells. In fact, we found that IL-6 is an inhibitor of B-CLL growth. The addition of recombinant human IL-6 markedly decreased the TNF-induced B-CLL growth, and this decrease was even greater when soluble IL-6 receptor, known to act as IL-6 agonist, was added with recombinant IL-6. Conversely, neutralizing monoclonal antibodies to IL-6 and to the IL-6 receptor potentiated the growth stimulation of TNF on B-CLL cells, in line with the possibility that IL-6 functions as a negative feedback regulator of an autocrine TNF action on these B-leukemic cells. Evidence is presented that production of IL-6 by monocytes and B cells of CLL patients is low, suggesting that administration of IL-6 may be beneficial in CLL to reduce the eventual growth stimulation by TNF and, possibly, also the deficiency in platelets and Ig production in this disease.

T lymphocytes and macrophages (Mφ) have been seen to accumulate at sites of lesions in blood vessel walls, suggesting that these cells may contribute to the pathogenesis of inflammatory reactions. Tumour necrosis factor-α (TNF-α), a cytokine produced by both MΦ and T lymphocytes, plays a major role in inflammatory reactions, blood vessel formation, thrombosis and atherosclerosis. We now report that secretion of TNF-α by cloned CD4⁺ rat T cells, and to a lesser degree by peripheral T cells, and MΦ can be induced in vitro in the absence of antigen, in a major histocompatibility complex (MHC) class II-independent manner by integrin-mediated recognition of immobilized components of extracellular matrix such as fibronectin and laminin; the secretion of TNF-α by the interacting resting cells on fibronectin was partially abrogated by the presence of the Arg-Gly-Asp (RGD)-containing amino acid sequence. This T cell-MΦ interaction involves CD2 and CD4 molecules and requires a signal transduced in the T cells by a protein tyrosine kinase. Thus, a multicellular interaction with extracellular matrix protein exposed as a consequence of vascular wall injury can serve to signal the secretion of TNF-α which induces the recruitment of additional immune cells to the developing lesion.

Expression of the two known receptors for TNF was studied in the promyelocytic leukemia cell line HL-60 before and after differentiation of the cells along the granulocyte lineage (induced by incubation with retinoic acid), or along the macrophage lineage (induced by incubation with the phorbol diester, PMA). The extent of inhibition of TNF binding by receptor-specific antisera, as well as the size of the complexes formed after cross-linking TNF to its receptors on intact cells, indicated that both receptor species were expressed on the surface of the undifferentiated HL60 cells. Differentiation into granulocyte-like cells resulted in some increase in TNF binding. The increase was apparently due to enhanced expression of the 75-kDa TNF-R, whereas the amounts of the 55-kDa TNF-R did not change significantly. In contrast, in HL-60 cells induced to differentiate into macrophage-like cells, expression of the 55-kDa TNF-R species was completely abolished. The pattern of TNF-R expression in the differentiated HL-60 cells was similar to that observed in leukocytes isolated from peripheral blood: on granulocytes, there were about equal amounts of both receptor species, whereas on monocytes the 75-kDa receptor was predominant. The loss of 55-kDa receptors during differentiation of HL-60 cells into macrophage-like cells was accompanied by a pronounced decrease in the level of the mRNA for that receptor, suggesting that at least part of the change in TNF-R expression is due to mechanisms that control the amounts of receptor mRNA. Although little is yet known regarding the functional differences between the two receptor species, marked changes in the pattern of their expression, as observed during HL-60 cell differentiation, are likely to alter the kind of response of the cells to TNF and may therefore play an important role in the coordination of TNF effects in the organism.

The mechanistic relationship between the signalling for the TNF effects by the human p55 TNF receptor (hu-p55-TNF-R) and the formation of a soluble form of the receptor, which is inhibitory to these effects, was explored by examining the function of C-terminally truncated mutants of the receptor, expressed in rodent cells. The 'wild-type' receptor signalled for a cytocidal effect when cross-linked with specific antibodies and exhibited spontaneous shedding. Shedding of the receptor was not affected by TNF but was markedly enhanced by 4β-phorbol-12-myristate-13-acetate (PMA). Receptor mutants with 53%, 83% and 96% C-terminal deletions could not signal for the cytocidal effect. Furthermore, they were found to associate with the endogenous rodent receptors, interfering with their signalling. Yet even the deletion of 96% of the intracellular domain did not abolish shedding of the receptor in response to PMA. These findings suggest that signalling and shedding of the p55 TNF-R are mechanistically distinct.

Serum levels of the soluble form of tumour necrosis factor receptor type II (p75) (sTNF-R) were determined in HIV-infected individuals and risk groups and were then correlated with the course of infection and prognosis. sTNF-R levels were determined by an ELISA with MoAbs and polyclonal antibodies to urine-derived sTNF-R proteins. The mean ± s.e. levels of sTNF-R in the sera of 49 HIV⁺ male homosexuals, 34 HIV^- male homosexuals and 44 matched controls were 6.1 ± 0.3 ng/ml, 4.4 ± 0.3 ng/ml and 3.4 ± 0.2 ng/ml, respectively. All these values were significantly different between each of the groups (P + male homosexuals and from healthy controls. This was independent of stage of HIV infection, serum sTNF-R level and type of ELISA kit used. These findings suggest that TNF-α/TNF-R system is turned on before and during HIV infection and raise the possibility that sTNF-R, the natural inhibitor of TNF, may be of importance in determining the course and probably prognosis of the disease.

The nature of soluble factors that regulate fibroblast proliferation have not been finally characterized. Our aim was to study the role of tumour necrosis factor α (TNF-α) and interleukin-1 (IL-1) in the suppressive activity of alveolar macrophages on autologous lung fibroblasts proliferation in sarcoidosis. We found that supernatants recovered from alveolar macrophages suppressed the proliferation of alveolar fibroblast in sarcoidosis by 35.5 ± 1.13% compared to 3 ± 16% in controls (p

Soluble forms of the two molecular species of the cell surface receptors for tumor necrosis factor (TNF) have been detected in normal urine. Using enzyme-linked immunosorbent assays for these soluble receptors, we determined their levels in the sera of 40 healthy subjects and 59 patients with solid tumors. The mean ± SD concentrations of both the soluble type I (p55) and type II (p75) receptors were significantly higher in the cancer patients than in the healthy controls: 1.96 ± 1.19 versus 0.79 ± 0.19 ng/ml (P

Modulation of cellular responsiveness to tumor necrosis factor (TNF) was studied in the human SV80 cells. A marked cytocidal effect is exhibited by these cells at about 4 to 8 h after application of TNF together with protein synthesis inhibitors. Sensitivity of the cells to TNF toxicity was shown to be markedly decreased following their pretreatment with TNF itself or with interleukin (IL) 1 in the absence of protein synthesis inhibitors. The SV80 cells respond to TNF also with enhanced phosphorylation of the small heatshock protein, HSP27. This TNF effect is much more rapid than the cytocidal effect; it is observed within minutes of TNF application. The response to this effect, just like the response to the cytocidal effect, is markedly decreased following preexposure of the cells to either TNF or IL 1. Responsiveness to both effects of TNF is regained at the same time, about 15 to 20 h following removal of TNF or IL 1. The decrease in responsiveness after pretreatment with TNF or IL 1 does not reflect an inability of the pretreated cells to bind TNF. Although there is an initial decrease in TNF binding after such pretreatment, it is fully reversed already about 5 h following removal of the cytokines. The rate of uptake of TNF by the pretreated cells is also normal. In view of the rapidity of the effect of TNF on the phosphorylation of HSP27, it seems likely that the observed hyporesponsiveness reflects impairment of an early step in a signaling pathway, perhaps common to both the stimulation of phosphorylation and the induction of cell death by TNF. By restricting the duration of the effects of TNF this desensitization mechanism may safeguard against harmful consequences of these effects.

Keywords: TNF-ALPHA

Affinity chromatography of crude human urinary proteins on either human recombinant interleukin-6 (rIL-6) or human recombinant interferon-γ (rIFN-γ) or anti IFN-γ receptor (IFN-γ-R) monoclonal antibodies (McAb) yielded the two respective soluble receptors in significant amounts. A single sequence of 30 amino acid residues was obtained by N-terminal microsequencing of the protein peak purified in tandem by affinity chromatography on an IL-6 column and reveresed-phase high-performance liquid chromatography. This sequence was identical with the predicted N-terminal sequence of IL-6-R as previously reported. The purified IL-6-R retained its biological activity. It was used for the preparation of specific anti IL-6-R monoclonal antibodies. Analysis of the eluted proteins from both IFN-γ and anti IFN-γ-R columns by inhibition of solid-phase radioimmunoassay, enzyme-linked immunosorbent assay, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting proved the existence of soluble IFN-γ-R in normal urine. This finding together with the already known presence of soluble TNF receptors and a soluble IL-2 receptors found both in plasma and in urine indicates that release of soluble cytokine receptors into body fluids is a general phenomenon which occurs under normal physiological conditions.

Two proteins which specifically bind tumor necrosis factor (TNF) have recently been isolated from human urine in our laboratory. The two proteins crossreact immunologically with two species of cell surface TNF receptors (TNFR). Antibodies against one of the two TNF binding proteins (TBPI) were found to have effects characteristic of TNF, including stimulating phosphorylation of specific cellular proteins. Oligonucleotide probes designed on the basis of the NH2terminal amino acid sequence of TBPI were used to clone the cDNA for the structurally related cell surface type 1 TNFR. It is notable that although this receptor can signal the phosphorylation of cellular proteins, it appears from its amino acid sequence to be devoid of intrinsic protein kinase activity. The extracellular domain of the receptor is composed of four internal cysteinerich repeats, homologous to structures repeated four times in the extracellular domains of the nerve growth factor receptor and the B lymphocytes surface antigen CDw40. The amino acid composition and size of the extracellular domain of the type I TNFR closely resemble those of TBPI. The COOHterminal amino acid sequence of the four cysteine rich repeats within the extracellular domain of the type I TNFR matches the COOHterminal sequence of TBPI. Amino acid sequences in the extracellular domain also fully match other sequences found in TBPI. On the other hand, amino acid sequences in the soluble form of the type II TNFR (TBPII), while indicating a marked homology of structure, did not suggest any identity between this protein and the extracellular domain of the type I TNFR. CHO cells transfected with type I TNFR cDNA produced both cell surface and soluble forms of the receptor. The receptor produced by CHO cells was recognized by several monoclonal antibodies against TBPI, reacting with several distinct epitopes in this molecule. These data suggest that the soluble forms of the TNFRs are structurally identical to the extracellular cytokine binding domains of these receptors and are consistent with the notion that the soluble forms are, at least partly, derived from the same transcripts that encode the cell surface receptors.

Keywords: Biochemistry & Molecular Biology; Oncology; Immunology

Human recombinant tumor necrosis factor-α (TNF-α) inhibited the growth of Chlamydia trachomatis (L₂/434/Bu) in HEp-2 cells. The effect was synergistic with that of gamma interferon (IFN-γ). TNF-induced resistance to chlamydiae could be blocked with cycloheximide, suggesting that it involves the function of some induced proteins. Tryptophan degradation was enhanced in the TNF-treated cells and was much further increased when the cells were treated with both TNF and IFN-γ at concentrations at which IFN-γ by itself had very little effect. Antibodies to IFN-β blocked the augmentation of tryptophan degradation by TNF and decreased but did not fully eliminate the antichlamydial effect of TNF. Increased concentration of tryptophan in the growth medium (>100 μg/ml) resulted in reversion of the antichlamydial effect of TNF. This study suggests that the inhibition of chlamydial growth by TNF is mediated partly through an autocrine function of IFN-β which, in synergism with TNF, enhances the activity of a tryptophan-degrading enzyme(s) and partly by some other activities of TNF which can be blocked by tryptophan.

Thirteen patients with advanced hairy cell leukemia were treated with a new subspecies of interferon (IFN): recombinant IFN (rIFN)alphaC. The timing of the peripheral hematologic remission of individual blood elements was similar to that reported for other interferons, and 60% of patients attained a complete peripheral hematologic remission at 9 months. Two patients relapsed despite a good initial response to IFN. No antiIFN antibodies could be detected in their sera. in vitro studies of colony formation from the peripheral blood of all responding patients showed that rIFNalphaC did not inhibit the growth of colonies but favorably affected the maturation of their elements towards monocytes, granulocytes, and erythroid elements. The relapsing patient examined initially experienced a similar beneficial in vitro response which paralleled his in vivo improvement. During relapse, rIFNalphaC inhibited both the colony formation and the myelomonocytic differentiation in the in vitro cultures. These findings may suggest that the acquired resistance to IFN in our patient could be due either to an acquired stem cell maturation arrest in response to IFN or to emergence of a new clone indifferent to IFNalphaC differentiation effect.

BALB/c mice were sensitized to lethal effects to human rTNF-α and of human rIL-1α by simultaneous treatment with sublethal doses of actinomycin D (Act D) or D-galactosamine (Ga1N). In contrast, treatment with sublethal doses of TNF or IL-1 themselves resulted in desensitization of the mice to the lethal effect of these cytokines: mice injected with TNF or IL-1 in the absence of Act D or Ga1N responded to a second injection of TNF or IL-1, this time together with Act D or Ga1N, by a significantly delayed death, or even survived. Desensitization developed rapidly (0.5-1.0 h) and abated 24 to 48 h postinjection. Each of the two cytokines induced hyporesponsiveness to its own lethal effect as well as to that of the other. Injection of TNF or IL-1 at sublethal doses resulted also in hyporesponsiveness to the lethal effect of LPS on mice primed with bacillus Calmette-Guerin, an effect which most likely is mediated by TNF and IL-1 produced in those mice in response to the LPS. TNF and IL-1 in combination had an additive effect both in lethality and in desensitization of the mice. These findings suggest that some of the deleterious effects of TNF and IL-1 are modualted by antagonistic mechanisms; mechanisms which can be suppressed by sensitizing agents, specifically by agents inhibiting the synthesis of RNA or protein; but which, in the absence of such agents, are found to be augmented in response to TNF and IL-1, thus resulting in desensitization.

Purified human recombinant tumor necrosis factor (rTNF) alpha inhibited the growth of Chlamydia trachomatis (L₂/434/Bu) in HEp-2 cell cultures. The inhibition of C. trachomatis yield could be achieved even when the rTNF alpha (200 ng/ml) was added up to 12 h after infection. The effect of rTNF alpha on chlamydial infection was synergistic with that of gamma interferon.

Keywords: Pediatrics

Bloksmu: I have some comments on the induction of haemorrhagic necrosis in Meth A tumours transplanted subcutaneously into the abdominal skin of syngeneic BALB/c mice. Endotoxin-induced tumour necrosis has been described in the literature as haemorrhage with necrosis, or haemorrhagic necrosis (Shear & Perrault 1943, Parr et a1 1973, Freudenberg et al 1984). However, morphological studies on the induction of necrosis in nine-day-old Meth A tumours (diameter about 7mm) by various agents have prompted us to propose that Meth A can show two distinct types of necrosis, as judged by pathogenesis and ultimate appearance. We have designated these as haemorrhagic necrosis (HN) and coagulation necrosis (CN), which is defined by cell decay with preservation of cell contours, nuclear condensation and nuclear disintegration. Both kinds of necrosis can be considered as different forms of coagulation necrosis. Evidence for the distinction has been compiled in Table 1. [first paragraph]

The cytotoxic effect of lymphotoxin (LT) and its modulation by interferon (IFN) was quantitatively assessed in uninfected and vesicular stomatitis virus (VSV)-infected cultured cells. Preparations of human LT, which were depleted of IFN, had a significant cytotoxic effect on VSV-infected HeLa, SV-80, WISH, and Vero cells. IFN, most notably IFN-γ, further potentiated destruction of the infected cells by these LT preparations, when applied on the cells at sub-antiviral IFN concentrations. In contrast, no cytotoxic effect could be observed in any of the examined cells, when applying LT, IFN, or their combination, in the absence of viral infection. Infected cells in which VSV replication was suppressed by treatment with antiviral concentrations of IFN also resisted destruction by LT. These findings indicate that LT cytotoxicity can be selectively directed against virus-infected cells and that IFN can augment this cell-killing mechanism when failing to exert an antiviral effect.

Crude preparations of cytotoxins (CTXs) produced by human peripheral blood mononuclear cells exert a marked cytotoxic effect when applied to cells in the presence of cycloheximide but in its absence can induce resistance to cytotoxicity. To examine the relationship between these cytotoxic and protective activities, we attempted to fully dissociate the CTX from the other proteins secreted by mononuclear cells. Mice injected with preparations of the cytokines secreted by peripheral blood mononuclear cells developed significant titers of serum antibodies to CTX(s). Splenocytes of such immunized mice were fused with NSO myeloma cells; a few among the resulting hybridoma cells secreted CTX-binding antibodies. Immunoadsorbents constructed with a monoclonal antibody produced by one of these hybridomas were used to purify to homogenecity a CTX (M(r) ~17,500) from crude preparations of cytokines, by a single adsorption and elution cycle. Purified CTX was cytotoxic in the presence of cycloheximide but in its absence induced resistance to cytotoxicity; this resistance was manifested by decreased vulnerability to CTX in a subsequent incubation in the presence of cycloheximide. We conclude that CTX itself can induce certain changes in cells, which are reflected in resistance to its own cytotoxic effect.

A technique is described for measurement of the antiviral activity of interferon by an immunoenzymatic assay for viral proteins. Cells treated by tested samples of interferon (IFN) are infected with vesicular stomatitis virus (VSV) and following the development of viral cytopathy are lysed by the addition of deoxycholate and then transferred into ELISA microplates. The viral proteins bind effectively to the microplates proportionally to their level in the culture and may be measured by incubating the plates sequentially with (1) rabbit antiserum against VSV, (2) a conjugate of alkaline phosphatase either to protein A or to an antibody against rabbit IgG and (3) p-nitrophenylphosphate. This procedure may be further simplified by using antibodies against VSV to which alkaline phosphatase has been directly conjugated. We found this immunoenzyme assay to be superior to the 'cytophatic effect inhibition' assay in precision and sensitivity and in being independent of the effectiveness of viral cytopathy.

A two-part study was done to assess the value of human fibroblast interferon (IFN-β) in the treatment of condylomata acuminata. The first part was an open study of different IFN-β preparations, which showed that intramuscular injection was the most suitable mode of administration of IFN-β. In the double-blind placebo section 22 patients were given injections of 2×10⁶ units IFN-β or placebo for 10 consecutive days and followed up for 3 months. In 9 of the 11 in the IFN-β group and 2 in the placebo group lesions disappeared from about 5 weeks after completion of the course of injections. After 3 months 8 of the non-responders were given a course of IFN-β and all responded to treatment. None of those who had responded has had a recurrence, the disease-free period now being 12 months. Changes in (2'-5') oligo A synthetase levels in white blood cells confirm that intramuscular injections of IFN-β produce a systemic response.

Intraperitoneal injection of vesicular stomatitis virus (VSV) into mice causes marked and rapid changes in leukocyte distribution. The virus induces an increase in peripheral blood (PB) granulocytes and an extensive decrease in the lymphocyte count which reaches a nadir of less than 10% of preinfection values, 12 hr after virus inoculation. In the lymph nodes and spleen extensive lymphocyte translocation and granulocyte infiltration are observed. Most changes abate 48 hr following virus inoculation. Injection of poly(rI):(rC) causes similar changes to those observed with VSV. The lymphocyte changes observed after injection of VSV or poly(rI):(rC) coincide with high levels of Interferon (IFN) in the serum. We have examined the effects of anti-IFN antibody on those changes and investigated whether they can be mimicked by injecting IFN. Our findings suggest that the IFN induced by VSV or poly(rI):(rC), rather than those agents themselves, causes the observed lymphopenia as well as some of the changes observed in the spleen. On the other hand, the effects of VSV on granulocyte localization do not appear to be mediated by IFN.

HeLa cells show a decrease of susceptibility to the killing by natural killer (NK) cells when treated with IFN-α, β, or γ. The concentrations at which preparations of IFN-α or β induce the resistance to killing are those which also induce resistance of HeLa cells to infection by vesicular stomatitis virus (VSV). Stimulation of the killing activity of NK cells is also induced at that same range of concentrations of IFN-α and β. In contrast with preparations of IFN-γ, induction of the resistance to killing occurs at IFN concentrations which have only marginal stimulatory effect on the activity of NK cells and have no antiviral effect against VSV. IFN-γ, produced with cloned IFN-γ cDNA, is as effective as lymphocyte-produced IFN in inducing the resistance to natural killing. The potent effect of IFN-γ on the target cells is, therefore, not due to the function of lymphokines which might contaminate lymphocyte-produced preparations of IFN-γ, but a genuine property of the IFN itself.

Interferon (IFN) is an effective activator of the natural killer (NK) cells but can also induce, in a variety of target cells, resistance to the NK induced cytolysis. We have compared the ability of the various types of human IFN to carry out this function, by quantitating the cytolysis induced by NK cells in cultured fibroblasts pretreated with each IFN and comparing this effect of the IFNs to their effectivity in suppressing viral replication. Relative to their antiviral potency, preparations of IFN-γ were significantly more effective than the alpha or beta interferons in inducing what we call the \u201canti-NK\u201d effect. Significant inhibition of the natural killing was observed with IFN-γ even at concentrations at which no antiviral effect was induced. Preparations of IFN-γ were also more effective than the other interferons in suppressing cell growth. While the induction of the enzyme oligo-isoadenylate synthetase correlated to the antiviral effectivity of the interferons, the quantitation of the HLA proteins in the cells had shown that their induction by the interferons correlates to the induction of the anti-NK effect, being stimulated much more effectively by IFN-γ than by IFN-α or IFN-β.⁽¹⁾ The anti-NK, HLA and antiviral-inducing activities co-purified in fractionation of IFN-γ preparations. These findings suggest the existence of several independent mechanisms for the regulation of cell functions by interferons. The gamma IFN is particularly effective in regulating functions involved in the interaction of cells with the immune system while the gamma virus-induced IFN-α and -β are more effective as inducers of resistance to viral growth.

Interferons produce a variety of biological effects on cells. They induce resistance to virus proliferation^1,2, inhibit cell growth ³, modify cell structure and differentiation^3-5, stimulate some immune functions and inhibit others⁶. However, the different interferon (IFN) species may vary in their mechanism of action and, hence, in their relative efficiency for inducing each of the effects. IFN-γ (type II) appears to show stronger immunoregulatory^7-9 and growth inhibitory effects^10-12 than antiviral effects¹³, but this conclusion has been challenged in other reports¹⁴. The aim of the present work is to compare the action of IFN-γ and other (type I) interferons on the induction of (2-5) oligo(A) synthetase which is probably part of the antiviral response^15,16 and the induction of the histocompatibility HLA-A,-B,-C antigens^17-19. We have shown previously that the induction of both proteins is regulated by interferons at the mRNA level^20,21, but show here that IFN-γ from stimulated human lymphocytes²² and from monkey cells transfected by cloned human IFN-γ cDNA²³ induced the HLA-A,-B,-C and β₂- microglobulin mRNAs or proteins at concentrations over 100 times lower than those needed to induce the (2-5)oligo(A) synthetase and the antiviral state. This difference was not found with IFN-α and -β (type I).

There are three enzymic activities elevated in cells that have been exposed to interferon: a double-stranded (ds) RNA-activated protein kinase (PK-i) phosphorylating initiation factor eIF-2's small subunit, the dsRNA-dependent (2'-5')-oligoadenylate synthetase (E), and a 2'- phosphodiesterase (2'-PDi), which splits the (2'-5')ppp(A2'p)_nA oligonucleotide formed by the synthetase. The chapter discusses several applications for these enzyme measurements with a detailed description of the assay procedures used. The experiments described are concerned with the (2'-5')- oligoadenylate synthetase, which shows the largest variations, and with the 2'-phosphodiesterase. The (2'-5')-oligoadenylate produced by this enzyme inhibits protein synthesis in cell-free systems by activating a specific ribonuclease, F. This property can be used to assay the synthetase. The incorporation of [a-³²P]ATP into pppA2'p5'A or longer chains, is measured. The enzyme in crude cell extracts is subjected to a one-step purification by adsorption to poly(rI)· poly(rC)-agarose beads.

Two mRNA species that produce biologically active interferon were isolated from human fibroblasts and studied by size fractionation and cloning in E. coli plasmid pBR322. The major fibroblast interferon (Hu IFN-β1) is coded for by the smaller of the two mRNAs, an 11S species, 900 nucleotides long, which in cell-free systems yields a 20,000 M(r) protein. The second interferon mRNA species (Hu IFN-β2) is 14S, abou 1300 nucleotides long, and codes for another protein of 23,000-26,000 M(r). The two interferon mRNAs do not cross-hybridize. Both are induced by poly(rI x rC), but IFN-β2 mRNA is induced to about 10% in cells by cycloheximide treatment alone, whereas under these conditions IFN-β1 is not induced.