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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.

The proinflammatory cytokine tumor necrosis factor (TNF) orchestrates complex multicellular processes through a wide variety of changes that it induces in cell functions. At various stages of the study of TNF, attention has been drawn to one of three different modes of its action. The work that led to the discovery of this cytokine addressed situations in which it inflicts massive damage to tissues through a mode of action that appeared to be unrestricted. In the years that followed, attention was drawn to the existence of negative feedback mechanisms that do restrict TNF formation and function, and of reciprocal mechanisms for negatively regulating TNF-induced gene activation and of cell death. Most recently, the discovery of the critical role of TNF in chronic inflammatory diseases directed attention to the ability of TNF also to act with no apparent time restriction. Major gaps still remain in our knowledge of the cellular and molecular basis for these three modes of TNF action.

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.

Necroptosis - a form of programmed necrotic cell death - and its resulting release of damage-associated molecular patterns (DAMPs) are believed to participate in the triggering of inflammatory processes. To assess the relative contribution of this cell death mode to inflammation, we need to know what other cellular effects can be exerted by molecules shown to trigger necrotic death, and the extent to which those effects might themselves contribute to inflammation. Here, we describe the technical approaches that have been applied to assess the impact of the main signaling molecules known to mediate activation of necroptosis upon generation of inflammatory cytokines in LPS-treated mouse bone marrow-derived dendritic cells. The findings obtained by this assessment indicated that signaling molecules known to initiate necroptosis can also initiate activation of the NLRP3 inflammasome, thereby inducing inflammation independently of cell death by triggering the generation of proinflammatory cytokines such as IL-1β.

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.

Expression of enzymatically inactive caspase-8, or deletion of caspase-8 from basal epidermal keratinocytes, triggers chronic skin inflammation in mice. Unlike similar inflammation resulting from arrest of nuclear factor κB activation in the epidermal cells, the effect induced by caspase-8 deficiency did not depend on TNF, IL-1, dermal macrophage function, or expression of the toll-like receptor adapter proteins MyD88 or TRIF. Both interferon regulatory factor (IRF) 3 and TANK-binding kinase were constitutively phosphorylated in the caspase-8-deficient epidermis, and knockdown of IRF3 in the epidermis-derived cells from these mice abolished the expression of up-regulated genes. Temporal and spatial analyses of the alterations in gene expression that result from caspase-8 deficiency reveal that the changes are initiated before birth, around the time that cornification develops, and occur mainly in the suprabasal layer. Finally, we found that caspase-8-deficient keratinocytes display an enhanced response to gene activation by transfected DNA. Our findings suggest that an enhanced response to endogenous activators of IRF3 in the epidermis, presumably generated in association with keratinocyte differentiation, contributes to the skin inflammatory process triggered by caspase-8 deficiency.

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

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.

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).

Previous evidence suggested that NF-κB-inducing kinase (NIK) might regulate IL-2 synthesis. However, the molecular mechanism is not understood. In this study, we show that NIK is involved in CD3 plus CD28 activation of IL-2 transcription. Splenic T cells from aly/aly mice (that have a defective NIK protein) have a severe impairment in IL-2 and GM-CSF but not TNF secretion in response to CD3/CD28. This effect takes place at the transcriptional level as overexpression of alyNIK inhibits DL-2 promoter transcription. NIK activates the CD28 responsive element (CD28RE) of the IL-2 promoter and strongly synergizes with c-Rel in this activity. We found that NIK interacts with the N-terminal domain of c-Rel, mapping this interaction to aa 771-947 of NIK. Moreover, NIK phosphorylates the c-Rel C-terminal transactivation domain (TAD) and induces Gal4-c-Rel-transactivating activity. Anti-CD28 activated Gal4-c-Rel transactivation activity, and this effect was inhibited by a NIK-defective mutant. Deletion studies mapped the region of c-Rel responsive to NIK in aa 456-540. Mutation of several serines, including Ser⁴⁷¹, in the TAD of c-Rel abrogated the NIK-enhancing activity of its transactivating activity. Interestingly, a Jurkat mutant cell line that expresses one of the mutations of c-Rel (Ser⁴⁷¹ Asn) has a severe defect in IL-2 and CD28RE-dependent transcription in response to CD3/CD28 or to NIK. Our results support that NIK may be controlling CD28RE-dependent transcription and T cell activation by modulating c-Rel phosphorylation of the TAD. This leads to more efficient transactivation of genes which are dependent on CD28RE sites where c-Rel binds such as the IL-2 promoter.

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.

Keywords: Gastroenterology & Hepatology

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.

Chronic inflammatory diseases are associated with increased soluble tumour necrosis factor (TNF) receptor concentrations in serum. To obtain such an increase, we implanted mice, with ethylene vinyl-acetate or poly (lactic-co-glycolic) acid copolymers containing human soluble p55 TNF receptor. Copolymers containing rather small amounts of the receptor (about 20 μg) maintained prolonged increases in serum receptor concentrations. Mice implanted with these copolymers were effectively protected against lethal wasting and from arthritis resulting from chronic exposure to TNF. These findings suggest that the increased production of soluble TNF receptors in chronic inflammatory diseases counteracts deleterious effects of TNF, and suggest a therapeutic application for the natural forms of the receptors in such diseases.

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.

Tumor necrosis factor (TNF) functions both as a soluble molecule and as a cell surface 26 kDa transmembrane protein, from which the soluble form is proteolytically derived. The 26 kDa TNF molecules isolated from ³²P labeled HeLa cells that had been transfected with the cDNA of a partially clearable TNF mutant were found labeled. Phosphorylated 26 kDa TNF molecules could also be isolated from human LPS stimulated monocytic Mono Mac 6. Phosphoaminoacid analysis revealed that the labeled phosphate is bound to serine residues. No label was found incorporated in soluble 17 kDa TNF, indicating that the phosphorylated residue(s) of membrane-associated TNF occur in the cytoplasmic portion of the molecule. Phosphorylation of the intracellular domain of the 26 kDa TNF molecules may play a role in the regulation of expression or proteolytic processing of TNF, modulate TNF bioactivity, or take part in intracellular signaling by cell-surface TNF.

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.

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.

Objective. To determine the value of measurement of serum soluble tumor necrosis factor receptor (sTNFR), compared with established parameters such as antidoublestranded DNA, in monitoring systemic lupus erythematosus (SLE) disease activity, and to determine whether serum sTNFR are bioactive and can effectively inhibit TNF bioactivity. Methods. Fiftythree consecutive ambulatory or hospitalized SLE patients and 140 consecutive healthy subjects were enrolled in a prospective cohort study. Serum levels of sTNFR were measured by a unique 2sided capture enzymelinked immunosorbent assay using mouse monoclonal antibodies and rabbit antisera against the sTNFR. Results. The mean ± SD concentrations of both the p55 (type I) and p75 (type II) soluble receptors were significantly higher in a group of 46 SLE patients than in controls: 1.89 ± 0.89 ng/ml versus 0.77 ± 0.19 ng/ml and 7.25 ± 3.89 ng/ml versus 3.02 ± 0.57 ng/ml, respectively (P

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.

The receptors for tumor necrosis factor (TNF) exist in cell-associated as well as soluble forms, both binding specifically to TNF. Since the soluble forms of TNF receptors (sTNF-Rs) can compete with the cell-associated TNF receptors for TNF, it was suggested that they function as inhibitors of TNF activity; at high concentrations, the sTNF-Rs indeed inhibit TNF effects. However, we report here that in the presence of low concentrations of the sTNF-Rs, effects of TNF whose induction depend on prolonged treatment with this cytokine are augmented, reflecting an attenuation by the sTNF-Rs of spontaneous TNF activity decay. Evidence that this stabilization of TNF activity by the sTNF-Rs follows from stabilization of TNF structure within the complexes that TNF forms with the sTNF-Rs is presented here, suggesting that the sTNF-Rs can affect TNF activity not only by interfering with its binding to cells but also by stabilizing its structure and preserving its activity, thus augmenting some of its effects.

Iodinated natural human urinary tumor necrosis factor binding protein I (¹²⁵I-uTBP) was iv injected into BALB/c mice, and its pharmacokinetics and tissue distribution were assessed during a short-term (0-1 hr) and for a long-term (0-24 hr) period. The blood ¹²⁵I-uTBP concentration displayed a biphasic pattern that was adequately described by a biexponential function with estimated half-lives of 0.1 and 3.8 hr. The apparent volume of distribution (V(c)) of the central compartment was 3 ml, which approximated the mouse blood volume. The clearance (CL) derived either from a model- dependent or a model-independent method of analysis was 2.5 and 2.9 ml/hr, respectively. One hr after the iv administration of ¹²⁵I-uTBP, the radioactivity accumulated in the major organs and tissues. The highest concentrations in terms of pg per organ were seen in the skin and in the liver. When expressed as pg ¹²⁵I-uTBP per mg organ, the distribution was the highest in the gallbladder, bladder, kidneys, and lungs. At 24 hr, the distribution of ¹²⁵I-uTBP represented about 10% of the amount measured at 1 hr. The rank order of accumulation of the radiolabeled uTBP in the major organs, expressed as pg per organ at 24 hr was skin > liver > kidneys > lungs > gut > spleen > gallbladder.

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.

Keywords: Biochemistry & Molecular Biology; Oncology; Immunology

Keywords: Biochemistry & Molecular Biology; Endocrinology & Metabolism; Genetics & Heredity

Affinity chromatography of crude human urinary proteins on either human rIL-6, human rIFN-γ, or anti-IFN-γ-R mAb yielded the two respective soluble receptors in significant quantities. A single sequence of 30 amino acid residues was obtained by NH₂-terminal microsequencing of the protein peak purified in tandem by affinity chromatography on an IL-6 column and reversed-phase HPLC. This sequence was identical to the predicted NH₂-terminal sequence of IL-6-R as previously reported. Analyis of the eluted proteins from both IFN-γ and anti-IFN-γ-R columns by inhibition of solid phase RIA, ELISA, SDS-PAGE, and Western blotting proved the existence of soluble IFN-γ-R in normal urine. Our finding, together with the already known presence of urinary TNF binding proteins and a soluble IL-2-R both in plasma and in urine, indicates that release of soluble cytokine receptors into body fluids is a general phenomenon that occurs under normal physiological conditions.

Unfractionated preparations of the proteins of human urine provided protection against the in vitro cytocidal effect of tumor necrosis factor (TNF). In certain cells, the proteins decreased expression of the receptors for TNF in a temperature-dependent way. In all cells examined, the proteins were found to interfere also with the binding of both TNF and interleukin-1 when applied directly into the binding assays. That effect could be observed in the cold, suggesting that it was independent of cellular metabolism. A protein which protects cells against the cytotoxicity of TNF was purified from human urine by chromatography on CM-Sepharose followed by high performance liquid chromatography on Mono Q and Mono S columns and reversed phase high performance liquid chromatography. This protein is a very minor constituent of normal urine, with an apparent molecular weight of about 27,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. Homogeneity of the purified protein was confirmed by microsequence analysis which revealed a single N-terminal sequence: Asp-Ser-Val-Cys-Pro-. The protein protected cells from TNF toxicity at concentrations of a few nanograms per ml and interfered with the binding of both TNF-alpha and TNF-beta to cells, when applied simultaneously with the cytokines. However, unlike crude preparations of the urinary proteins, the purified protein did not induce in cells a decrease in ability to bind TNF nor did it interfere with the binding of interleukin-1 to its receptor. Direct, specific binding to the protein of TNF-alpha and, to a lesser extent, also TNF-beta, but not of interleukin-1 nor interferon-gamma could be demonstrated. It is suggested that this protein blocks the function of TNF by competing for TNF with the TNF receptor and not by interacting with the target cell.

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.

The mechanisms underlying differences in vulnerability to the cytocidal effect of TNF, among various cell lines and strains, were explored by examining the response to TNF of heterokaryons formed by fusing TNF-resistant and -sensitive cells. Several combination pairs of human and murine cells, differing significantly in response to TNF toxicity, yet expressing a similar level of TNF receptors, were examined. In all combinations tested, the heterokaryons exhibited resistance to TNF toxicity, comparable, in extent, to that of the more resistant of the two parental cell lines. This dominance of resistance suggests that differences in vulnerability to TNF toxicity reflect activities which are expressed by resistant cells and are deficient in vulnerable ones, activities which perhaps protect the cell against the cytocidal effect of TNF.

Cells are sensitized to the cytolytic effect of tumor necrosis factor (TNF) by simultaneous application of inhibitors of RNA or protein synthesis. Treating cells, in the absence of such inhibitors, with cytokine preparations produced by stimulated mononuclear leukocytes may render them resistant to the cytolytic effect of TNFthe inhibitors. One of the cytokines which induces that resistance was identified as TNF itself (17). As shown in the present study, similar resistance against TNF-mediated killing can be effectively induced also with preparations of cytokines which are depleted of TNF. Fractionation of such TNF-free preparations revealed that their resistance-inducing activity is mediated by interleukin 1 (IL 1). In part of the cell lines in which IL 1 induced resistance to TNF killing, when applied without inhibitors of protein/RNA synthesis, it was found to exert cytolytic effect in the presence of such inhibitors, however, less effectively than TNF. Both TNF and IL 1 thus appear to activate in cells cytolytic mechanisms as well as antagonizing mechanisms which can protect cells from cytolysis.

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.

A new type of human interferon (IFN), IFN-α-C, produced in Escherichia coli and purified on monoclonal antibodies, was given at a dose of 3 x 10⁶ u (3 μg)/day to a terminally ill unsplenectomized patient with hairy cell leukemia, who had had severe recurrent infections and pancytopenia. There was marked reduction in the size of the spleen after 2 weeks, and platelet counts returned to normal after 1 month of treatment. The IFN treatment also raised the granulocyte counts and hemoglobin levels, improved the normal repopulation of the bone marrow, and restored resistance to infections. IFN-α-C was well tolerated, without serious side effects, and treatment has been continued for 10 months.

Interferons are produced in response to viral infections. Among the biochemical changes they cause in cells is the induction of the enzyme (2-5)-oligo-isodenylate synthetase. The activity of this enzyme can be measured and this can indicate exposure and response of cells to interferon. The efficacy of such an assay of peripheral blood of pregnant women may aid in establishing screening guidelines for potentially teratogenic viral infections. The blood of 44 primigravidas with complaints of fever, myalgia, cough, vaginal discharge and/or costovertebral angle tenderness was assayed for activity of the enzyme (2-5)-oligo-isoadenylate synthetase and compared to assays of the enzyme activity in a group of 37 healthy primigravidas which served as a control group. It was found that the group with viral infections had an increase in enzyme activity from twice to 15 times the normal value, with characteristic rises of enzyme activity in several viruses known or suspected to cause human defects. Several general guidelines are proposed to assist the obstetrician in determining a viral etiology of acute illness in pregnancy, It is suggested that the assay of enzyme activity of (2-5)-oligo-isoadenylate synthetase may provide a simple tool for rapid diagnosis of viral infections in pregnancy.

The interferon (IFN)-induced enzyme 2-5A synthetase was found in human peripheral blood polymorphonuclear cells (PMNL). The average enzyme activity in a group of 15 patients with various viral infections was significantly higher (25-fold) than in healthy individuals. Eight patients with multiple sclerosis and six patients with bacterial infections were found to have normal 2-5A synthetase levels in the PMNL. Relationship of PMNL 2-5A synthetase levels to IFN was confirmed by finding enzyme increases in PMNL incubated in vitro with IFN, as well as in patients undergoing IFN therapy. These findings suggest that in PMNL, as in other cells, the level of 2-5A synthetase can be regulated by IFN and can be increased as a result of IFN information in diseases.

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.

Using a simplified technique for the determination of oligoisoadenylate synthetase activity, we have compared the cellular level of this interferon (IFN)-induced enzyme in multiple samples of peripheral blood leukocytes. In mononuclear cells (PBMC) of healthy donors the enzyme level was remarkably constant, but in the cells of about 85% of patients with viral infections enzyme activity was significantly elevated. In contrast, the incidence of elevated activity in bacterial infections was low. Synthetase activity could be also detected in granulocytes, although normally its level in these cells was considerably lower than in PBMC. A sharp increase in the enzyme level in granulocytes was found in cells exposed in vitro to IFN, as well as in cells from patients undergoing IFN therapy. Increased synthetase activity was also detected in the granulocytes of patients with viral infections. We have also determined the level of the enzyme in patients with various types of leukemias. In a large proportion of the patients with acute lymphoblastic leukemia (ALL) we found severely decreased enzyme levels (1020% of control value). The decreased activity could usually be correlated to predominance of blast cells in the peripheral blood.

A procedure for rapid isolation of the products of the interferon-induced enzyme, oligoisoadenylate synthetase, is described. After incubation of [α-³²P]ATP with the poly(rI): (rC)-adsorbed fraction of cellular proteins, the products are treated with phosphatase. Aliquots of the nucleotides are then applied on small columns which contain 300 μl of alumina powder and eluted with 3 ml of 1 m glycine-HCl buffer, pH 2.0. The labeled free phosphate, released by the phosphatase treatment, is efficiently adsorbed by the alumina, while the phosphatase-resistant cores of the oligo-isoadenylates, up to the length of pentamer, are eluted. Larger oligomers are only partially recovered. We successfully applied this method for determination of the level of the enzyme in multiple samples of cell homogenates.

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.