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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.

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.

Emerging evidence indicates that the molecular mechanisms of cell death have regulatory roles in inflammation and that the molecular changes that are associated with different forms of cell death affect the course of inflammation in different ways. In this Timeline article, we discuss how our understanding of the mechanisms and functional roles of tissue injury and cell death in inflammation has evolved on the basis of almost two centuries of study. We describe how such ideas have led to our current models of cell death and inflammation, and we highlight the remaining gaps in our knowledge of the subject.

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

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.

Early in the exploration of the chemical nature of life, it was widely believed that the molecules of living organisms, by their very nature, differ from those of inorganic material molecules and possess a vital force ('élan vital'). Similarly, early scientific thinking on the subject of cell death and its induction by cytotoxic cells of the immune system was pervaded by a sense that the molecules mediating these functions possess intrinsic deadly activity and are dedicated exclusively to death-related tasks. This impression was also reflected in the initial notions of the mode of action of intracellular proteins that signal for death. It is now gradually becoming clear, however, that proteins participating in death induction also have functions unrelated to death. Nevertheless, as exemplified by studies of the function of caspase-8 (an enzyme that signals both for activation of the extrinsic cell-death pathway and for non-death-related effects), analysis of the mechanistic basis for such heterogeneity might allow identification of distinct structural determinants in the proteins participating in death induction that do bear death specificity.

Keywords: Biochemistry & Molecular Biology; Cell Biology; Immunology

Hsp90 is a highly abundant chaperone whose clientele includes hundreds of cellular proteins, many of which are central players in key signal transduction pathways and the majority of which are protein kinases. In light of the variety of Hsp90 clientele, the mechanism of selectivity of the chaperone toward its client proteins is a major open question. Focusing on human kinases, we have demonstrated that the chaperone recognizes a common surface in the amino-terminal lobe of kinases from diverse families, including two newly identified clients, NFκB-inducing kinase and death-associated protein kinase, and the oncoprotein HER2/ErbB-2. Surface electrostatics determine the interaction with the Hsp90 chaperone complex such that introduction of a negative charge within this region disrupts recognition. Compiling information on the Hsp90 dependence of 105 protein kinases, including 16 kinases whose relationship to Hsp90 is first examined in this study, reveals that surface features, rather than a contiguous amino acid sequence, define the capacity of the Hsp90 chaperone machine to recognize client kinases. Analyzing Hsp90 regulation of two major signaling cascades, the mitogen-activated protein kinase and phosphatidylinositol 3-kinase, leads us to propose that the selectivity of the chaperone to specific kinases is functional, namely that Hsp90 controls kinases that function as hubs integrating multiple inputs. These lessons bear significance to pharmacological attempts to target the chaperone in human pathologies, such as cancer.

Recruitment of the NF-kappa B-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.

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.

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 (

We recently demonstrated that closed head injury (CHI) in the rat triggers the production of tumor necrosis factor alpha (TNFα) in the contused hemisphere. Other investigations have shown that this cytokine plays a role in the inflammatory response following trauma. The present study was designed to determine whether inhibition of TNFα production or activity affects the development of cerebral edema as well as neurological dysfunction and hippocampal cell loss after CHI. To this end, we used two pharmacological agents, each acting via a different mechanism: pentoxifylline (PTX), which attenuates the production of TNFα, and tumor necrosis factor binding protein (TBP), a physiological inhibitor of TNFα activity. Both agents significantly lessened peak edema formation at 24 h and facilitated the recovery of motor function for ≤14 days postinjury. In addition, TBP attenuated disruption of the blood-brain barrier and protected hippocampal cells. PTX significantly lowered the brain TNFα level (by ~80%), and TBP completely abolished the activity of recombinant human TNF when they were added at the same time in the in vitro bioassay. We suggest, therefore, that a decrease in TNFα level or the inhibition of its activity is accompanied by reduced brain damage.

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 long‐term presence of the cytokine. TNF's superior cytotoxicity, which requires only short‐term 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 200‐fold 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.

Tumor necrosis factor (TNF)-α has been shown to be increased in brain tissue of AIDS patients and may function as a mediator of cerebral damage. We initiated a study to determine the cellular localization and degree of protein and mRNA expression of the two specific TNF-α receptors (TNF-Rs), p55 and p75, in brain tissues from AIDS patients. Cerebral white matter obtained at autopsy from 13 AIDS patients, 10 unhealthy controls, and 4 healthy controls was evaluated. Double-label immunohistochemistry revealed prominent up-regulation of p55 and p75 TNF-Rs on activated macrophages and microglial cells in all AIDS patients; no increased staining was found on astrocytes. Staining was most prominent in patients with opportunistic infection of the brain and in microglial nodules of patients with HIV encephalitis. Brain tissues also showed increased expression of interleukin (IL)-1β, IL-6, and TNF-α, cytokines known to up-regulate the TNF-Rs. Increased staining for TNF-Rs was also found in patients with multiple sclerosis, chronic cerebral edema, and radiation necrosis but not in an asymptomatic HIV-positive patient without AIDS. Reverse transcriptase polymerase chain reaction performed on adjacent sections from five AIDS patients revealed up-regulation from normal for p55 in all patients and for p75 in three patients. The up-regulation of both TNF-Rs in AIDS suggests that macrophages and microglial cells may be important in amplifying the TNF-α response.

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

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 anti–double‐stranded DNA, in monitoring systemic lupus erythematosus (SLE) disease activity, and to determine whether serum sTNFR are bioactive and can effectively inhibit TNF bioactivity. Methods. Fifty‐three 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 2‐sided capture enzyme‐linked 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

In cells of the fibroblastoid line SV-80, rapid down-modulation of TNF binding in response to TNF itself, or to IL-1, was followed by a gradual recovery of binding, which occurred even in the continuous presence of the cytokines. Untreated cells carried mainly the 55-kDa receptor species. In cells treated with TNF or IL-1, the 55-kDa TNF-R, although increasing after initial down-modulation, remained lower than before treatment. Expression of the 75-kDa TNF-R species upon treatment with either cytokine was markedly increased. Both TNF and IL-1 also induced a strong increase of the mRNA for the 75-kDa receptors, whereas the amount of mRNA for the 55-kDa receptors decreased. The effects of TNF on cell surface expression of the TNF-R could not be blocked with antibodies to IL-1, nor could the effects of IL-1 be blocked with antibodies to TNF, indicating that each cytokine affects the cell surface expression of the receptors independently of the other. Applying both cytokines together resulted in much stronger increase in expression of the 75-kDa TNF-R than applying each alone. Similar changes in cell surface expression and mRNA levels of the two TNF-R as observed in SV-80 cells were also found in TNF and IL-1-treated human foreskin fibroblasts. It is suggested that these sustained changes in the pattern of receptor expression contribute to the adjustment of the cellular response to TNF when formation of TNF and IL-1 takes place over a prolonged period.

Two distinct types of soluble tumor necrosis factor α receptors (sTNFRs), which are felt to represent proteolytic cleavage products of the extracellular domains of membrane-bound TNFRs of molecular mass, 55 and 75 kDa, are found in the serum and urine of humans. We have measured the serum concentrations of these receptors in eight patients with metastatic renal cell carcinoma during treatment with interleukin-2 (IL-2)-based immunotherapy. The mean pretreatment concentration of sTNFR-55 kDa (p

The objective of this study was to determine sTNF-R, type I (p55) and type II (p75) in sera of HIV-infected male homosexuals and correlate them to T lymphocyte subpopulations and course of HIV infection. Serum samples were obtained from 39 HIV-1⁺ asymptomatic male homosexuals, 10 symptomatic (ARC and AIDS) male homosexuals and 44 HIV^- non-homosexual healthy controls. sTNF-R levels were determined by ELISA with specific MoAbs and polyclonal antibodies to the sTNF-R proteins. sTNF-RI and II levels were significantly elevated in 72% and 74% respectively of HIV⁺ asymptomatic male homosexuals and in all of the symptomatic male homosexuals. In sequential studies a highly significant positive correlation was found between sTNF-RI and sTNF-RII (r = 0.8, P + lymphocyte counts (r = 0.6 and 0.92, respectively, P + lymphocytes during the asymptomatic stage of HIV infection.

Soluble forms of the two receptors for tumor necrosis factor (TNF) are present in human sera at concentrations that increase greatly in various disease states as well as varying among healthy individuals. Measurements of the soluble TNF receptor (sTNF-R) concentrations in healthy individuals at time lapses of 3 months (17 individuals) or 1 year (51 individuals) showed a significant correlation between the first and the second measurements from each individual, implying that individual differences are stable. Since the sTNF-Rs are believed to function as physiological attenuators of TNF activity, these steady individual differences may contribute to differences in the severity of the harmful effects of TNF in disease states.

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.

To localize the protease(s) involved in shedding of tumor necrosis factor receptors (TNF-R) from activated neutrophils (PMN) (Porteu, F., and C. Nathan (1990) J. Exp. Med. 172, 599-607), we tested subcellular fractions from PMN for their ability to cause loss of TNF-R from intact cells. Exposure of PMN to sonicated azurophil granules at 37°C resulted in inhibition of ¹²⁵I-TNF binding; 50% inhibition ensued when PMN were treated for ∼1 min with azurophil granules equivalent to 2-3 PMN per indicator cell. The TNF-R-degrading activity in azurophil granules was identified as elastase by its sensitivity to diisopropyl fluorophosphate (DFP), α₁-antitrypsin and N-methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone (MSAAPV-CK), and by the ability of purified elastase to reproduce the effect of azurophil granules. Elastase preferentially acted on the 75-kDa TNF-R, reducing by 85-96% the binding of ¹²⁵I-TNF to mononuclear cells expressing predominantly this receptor, while having no effect on endothelial cells expressing almost exclusively the 55-kDa TNF-R. Elastase-treated PMN released a 32-kDa soluble fragment of p75 TNF-R that bound TNF and reacted with anti-TNF-R monoclonal antibodies. In contrast, fMet-Leu-Phe-activated PMN shed a 42-kDa fragment from p75 TNF-R, along with similar amounts of a 28-kDa fragment from p55 TNF-R. Shedding of both TNF-Rs by intact activated PMN was more extensive than shedding caused by elastase and was completely resistant to DFP and MSAAPV-CK. Thus, the TNF-R-releasing activity of azurophil granules is distinct from that operative in intact stimulated PMN and could provide an additional mechanism for the control of cellular responses to TNF at sites of inflammation.

Modulation of cellular responsiveness to tumor necrosis factor (TNF) was studied in the human SV‐80 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 SV‐80 cells respond to TNF also with enhanced phosphorylation of the small heat‐shock 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: Immunology; Medicine, Research & Experimental; Pathology; Physiology

Two proteins which specifically bind tumor necrosis factor (TNF) have recently been isolated from human urine in our laboratory. The two proteins cross‐react immunologically with two species of cell surface TNF receptors (TNF‐R). 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 NH2‐terminal amino acid sequence of TBPI were used to clone the cDNA for the structurally related cell surface type 1 TNF‐R. 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 cysteine‐rich 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 TNF‐R closely resemble those of TBPI. The COOH‐terminal amino acid sequence of the four cysteine rich repeats within the extracellular domain of the type I TNF‐R matches the COOH‐terminal 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 TNF‐R (TBPII), while indicating a marked homology of structure, did not suggest any identity between this protein and the extracellular domain of the type I TNF‐R. CHO cells transfected with type I TNF‐R 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 TNF‐Rs 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.

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)‐alpha‐C. 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 anti‐IFN antibodies could be detected in their sera. in vitro studies of colony formation from the peripheral blood of all responding patients showed that rIFN‐alpha‐C 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, rIFN‐alpha‐C 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 IFN‐alpha‐C 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.

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.

Keywords: Pediatrics

Binding of radiolabeled tumor necrosis factor (TNF) to cell surface receptors was markedly reduced in human foreskin fibroblasts and cells from SV-80 and HeLa cell lines subsequent to treatment with interleukin 1 (IL-1) or 4β-phorbol-12-myristate-13-acetate (PMA). The decrease in TNF binding was initiated within minutes of application of IL-1 or PMA and could not be blocked by cycloheximide, suggesting that it is independent of protein synthesis. Scatchard plot analysis of TNF binding to the SV-80 cells indicated that its decrease in response to IL-1 and PMA reflects a reduced amount of TNF receptors, with no change in their affinity. IL-1 and PMA together had an additive effect on TNF binding. Treatment with TNF did not result in decreased binding of IL-1 to its receptors nor did TNF and IL-1 compete directly for their respective receptors. Human U937 cells on which receptors for IL-1 were below detectable levels exhibited no decrease in TNF binding when treated with IL-1, but did so in response to PMA. In addition to a decrease in TNF receptors, cells treated with IL-1 or PMA exhibited a lesser vulnerability to the cytolytic effect of TNF. The two kinds of changes were not completely correlated. A particularly notable dissimilarity was evident when comparing the rate of their reversal: the TNF receptor level was fully recovered within a few hours of removal of IL-1 or of the water-soluble analogue of PMA, 4β-phorbol-12,13-dibutyrate, from pretreated SV-80 cells; yet at that time resistance to the cytotoxicity of TNF was still prominent. These findings indicate that IL-1 as well as tumor-promoting phorbol diesters can down regulate cellular response to TNF by inducing a decrease in the number of receptors for TNF, and apparently through some other effect(s) as well.

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 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.

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).

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 (10–20% of control value). The decreased activity could usually be correlated to predominance of blast cells in the peripheral blood.

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 “anti-NK” 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.

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.