The molecular action of tumor necrosis factor-alpha.

Tumor necrosis factor-alpha (TNF-alpha) is a polypeptide hormone newly synthesized by different cell types upon stimulation with endotoxin, inflammatory mediators (C5a anaphylatoxin), or cytokines such as interleukin-1 and, in an autocrine manner, TNF itself. The net biological effect of TNF-alpha may vary depending on relative concentration, duration of cell exposure and presence of other mediators which may act in synergism with this cytokine. TNF-alpha may be relevant either in pathological events occurring in cachexia and endotoxic shock and inflammation or in beneficial processes such as host defense, immunity and tissue homeostasis. The biological effects of TNF-alpha are triggered by the binding to specific cell surface receptors. The formation of TNF-alpha-receptor complex activates a variety of biochemical pathways that include the transduction of the signal at least in part controlled by guanine-nucleotide-binding regulatory proteins (G proteins), its amplification through activation of adenyl cyclase, phospholipases and protein kinases with the generation of second messenger pathways. The transduction of selected genes in different cell types determines the characteristics of the cell response to TNF-alpha. The full understanding of the molecular mechanisms of TNF-alpha will provide the basis for a pharmacological approach intended to inhibit or potentiate selected biological actions of this cytokine.     

Oncostatin M: signal transduction and biological activity

Oncostatin M (OSM) is a multifunctional cytokine produced by activated T lymphocytes and monocytes that is structurally and functionally related to the subfamily of cytokines known as the IL-6-type cytokine family. OSM shares properties with all members of this family of cytokines, but is most closely related structurally and functionally to LIE OSM acts on a wide variety of cells and elicits diversified biological responses in vivo and in vitro which suggest potential roles in the regulation of gene activation, cell survival, proliferation and differentiation. OSM and LIF can bind to the same functional receptor complex (LIF-receptor beta and gp130 heteromultidimers) and thus mediate overlapping spectra of biological activities. There is a second specific beta receptor that binds OSM with high affinity and also involves the subunit gp130. The two receptors for OSM can be functionally different and be coupled to different signal transduction pathways. OSM-specific receptors are expressed in a wide variety of cell types and do not possess an intrinsic tyrosine kinase domain, but the JAK/STAT tyrosine kinase pathway mediates signal transduction.     

Measurement of cytokines by bioassays: theory and application

A large number of cytokines have been characterized, of which several have proved successful in the clinic as biotherapeutic agents for malignant, infectious or autoimmune diseases. As biologically active proteins, they cannot be fully characterized by physicochemical methods alone. Thus, biological assays (bioassays) have become increasingly important for their biological characterization and potency determinations. Since cytokines exert various biological activities in vitro, cultured cell line-based bioassay methods have mainly been developed to quantify potency. Such bioassays, like all biological systems, are inherently variable. Thus, measurement of potency of a particular cytokine must be made relative to a common, stable, reference preparation of the same cytokine to permit valid inter-assay and inter-laboratory comparisons. The development and establishment of appropriate primary reference preparations as World Health Organization (WHO) International standards (IS) and reference reagents (RR) is essential for the standardization of bioassays. This review addresses the practical and statistical considerations for the development of valid bioassays, the preparation and use of WHO IS and RR and, in brief, the types of bioassay methods applicable to potency measurements of individual cytokines. More extensive details for the potency determinations of tumor necrosis factor-alpha (TNF-alpha), related cytokines, and biotherapeutic anti-TNF-alpha products are provided.     

Modulation of innate and acquired immune responses by Escherichia coli heat-labile toxin: distinct pro- and anti-inflammatory effects of the nontoxic AB complex and the enzyme activity

We have examined the roles of enzyme activity and the nontoxic AB complex of heat-labile toxin (LT) from Escherichia coli on its adjuvant and immunomodulatory properties. LTK63, an LT mutant that is completely devoid of enzyme activity, enhanced Th1 responses to coinjected Ags at low adjuvant dose. In contrast, LTR72, a partially detoxified mutant, enhanced Th2 responses and when administered intranasally to mice before infection with Bordetella pertussis suppressed Th1 responses and delayed bacterial clearance from the lungs. LTR72 or wild-type LT inhibited Ag-induced IFN-gamma production by Th1 cells, and LT enhanced IL-5 production by Th2 cells in vitro. Each of the toxins enhanced B7-1 expression on macrophages, but enhancement of B7-2 expression was dependent on enzyme activity. We also observed distinct effects of the nontoxic AB complex and enzyme activity on inflammatory cytokine production. LT and LTR72 suppressed LPS and IFN-gamma induced TNF-alpha and IL-12 production, but enhanced IL-10 secretion by macrophages in vitro and suppressed IL-12 production in vivo in a murine model of LPS-induced shock. In contrast, LTK63 augmented the production of IL-12 and TNF-alpha. Furthermore, LTK63 enhanced NF-kappaB translocation, whereas low doses of LTR72 or LT failed to activate NF-kappaB, but stimulated cAMP production. Thus, E. coli LT appears to be capable of suppressing Th1 responses and enhancing Th2 responses through the modulatory effects of enzyme activity on NF-kappaB activation and IL-12 production. In contrast, the nontoxic AB complex can stimulate acquired immune responses by activating components of the innate immune system.     

Activation of nuclear factor kappaB and induction of apoptosis by tumor necrosis factor-alpha in the mouse uterine epithelial WEG-1 cell line

In order to better understand how tumor necrosis factor (TNF)-alpha may contribute to the local regulation of uterine cell death, cultures of mouse uterine epithelial WEG-1 cells were exposed to TNF-alpha and observed at different time intervals. Earliest decrease in cell viability was observed after 31 h of exposure to 50 ng/ml mouse TNF-alpha and was associated with the expression of several markers of apoptosis. Treatment with human TNF-alpha or addition of a neutralizing antibody against TNF-alpha receptor protein 80 to mouse TNF-alpha resulted in attenuated induction of apoptosis, suggesting that coengagement of the two TNF-alpha receptor types is required for maximal impact. Ceramide analogs failed to replicate the effect of TNF-alpha and the stress-activated protein kinase signaling pathway was not activated by the cytokine. Treatment with mouse TNF-alpha resulted in an increase in nuclear factor (NF)kappaB activity that receded after 24 h. The impact of human TNF-alpha on NFkappaB activation was more moderate. Addition of either one of three different inhibitors of NFkappaB (SN50, PDTC, and A771726) to mouse TNF-alpha sensitized WEG-1 cells to the toxicity of the cytokine. Our data suggest that WEG-1 cells initiate their response to TNF-alpha with an increase in NFkappaB activation that may have transiently biased these cells toward cell death resistance.     

Characterization of the receptor for tumor necrosis factor (TNF) and lymphotoxin (LT) on human T lymphocytes. TNF and LT differ in their receptor binding properties and the induction of MHC class I proteins on a human CD4+ T cell hybridoma

TNF-alpha and lymphotoxin (LT or TNF-beta) are structurally related cytokines that share several proinflammatory and immunomodulatory activities. The shared biologic activities of TNF and LT have been attributed to their binding to a common cell surface receptor(s). We observed that rTNF enhanced the expression of MHC class I proteins on the human T cell hybridoma, II-23.D7, however LT was largely unable to regulate MHC expression. To determine the molecular basis of this disparity between LT and TNF the receptor binding characteristics of rTNF and rLT were investigated by direct and competitive radioligand assays on the II-23.D7 T hybridoma, and for comparison, anti-CD3 activated human T lymphocytes. Specific 125I-rTNF binding to the II-23.D7 line revealed a single class of sites with a Kd = 175 pM and 3000 sites/cell; anti-CD3 activated T cells exhibited specific TNF binding with similar properties. The relationship of receptor occupancy to the induction of MHC class I Ag yielded a hyperbolic curve indicating a complex relationship between rTNF binding and biologic response. LT appeared to function like a partial agonist in that rLT was 10- to 20-fold less effective than rTNF in competitively inhibiting 125I-rTNF binding on the II-23.D7 line. Scatchard type analysis revealed a single class of low affinity binding sites for 125I-rLT. No differences in the competitive binding activity of rTNF and rLT were observed on the anti-CD3-activated T cells. Receptors for rTNF and rLT were immunoprecipitated from the II-23.D7 and activated T cells with anticytokine antibodies after cross-linking of radioiodinated rTNF or rLT to intact cells by using chemical cross-linking reagents. Analysis of the cross-linked adducts by SDS-PAGE and autoradiography indicated a major adduct of 92 kDa for rTNF and 104 kDa for rLT. Enzymatic digestion with neuraminidase or V8 protease revealed a unique structure to these adducts consistent with the cross-linking of a single chain of cytokine to a cell surface glycoprotein. rTNF inhibited the formation of the 104-kDa adduct formed with 125I-rLT on the II-23.D7 line, indicating these two cytokines bind to the same receptor of approximately 80 kDa. These results suggest that the disparate activities of LT and TNF to induce MHC class I proteins on the II-23.D7 cells are, in part, associated with a modified state of a common receptor.     

Inhibition of colony-stimulating factor-stimulated macrophage proliferation by tumor necrosis factor-alpha, IFN-gamma, and lipopolysaccharide is not due to a general loss of responsiveness to growth factor

The role of stimulatory factors, such as the CSF, in the regulation of hemopoiesis has been extensively documented. Less is known of the negative regulators of hemopoiesis. In this report, we show that the macrophage activating agents, TNF-alpha, IFN-gamma, and LPS, are all potent inhibitors of CSF-1-stimulated murine bone marrow-derived macrophage (BMM) DNA synthesis and increase in cell numbers. The inhibitory effects of TNF-alpha and IFN-gamma do not appear to be due to endotoxin contamination in the recombinant cytokine preparations. The inhibition of proliferation is reversible and is not due to a general loss of growth factor responsiveness, inasmuch as the three agents do not inhibit CSF-1-stimulated BMM survival, protein synthesis, or fluid phase pinocytosis. Because TNF-alpha and LPS are known to rapidly and potently down-modulate CSF-1 receptor levels in BMM, the results also suggest that low levels of receptor occupancy are sufficient for biological responses to CSF-1. The inhibitory effects of TNF-alpha, IFN-gamma, or LPS were also seen when granulocyte-macrophage-CSF or IL-3 was used to stimulate BMM DNA synthesis. The results suggest that TNF-alpha, IFN-gamma, and LPS appear to be inhibiting CSF-stimulated proliferation by acting at a post-receptor level, possibly by regulation of some critical event(s) in the mitogenic signaling pathway.     

Biological functions and therapeutic opportunities of soluble cytokine receptors

Cytokines control the immune system by regulating the proliferation, differentiation and function of immune cells. They activate their target cells through binding to specific receptors, which either are transmembrane proteins or attached to the cell-surface via a GPI-anchor. Different tissues and individual cell types have unique expression profiles of cytokine receptors, and consequently this expression pattern dictates to which cytokines a given cell can respond. Furthermore, soluble variants of several cytokine receptors exist, which are generated by different molecular mechanisms, namely differential mRNA splicing, proteolytic cleavage of the membrane-tethered precursors, and release on extracellular vesicles. These soluble receptors shape the function of cytokines in different ways: they can serve as antagonistic decoy receptors which compete with their membrane-bound counterparts for the ligand, or they can form functional receptor/cytokine complexes which act as agonists and can even activate cells that would usually not respond to the ligand alone. In this review, we focus on the IL-2 and IL-6 families of cytokines and the so-called Th2 cytokines. We summarize for each cytokine which soluble receptors exist, were they originate from, how they are generated, and what their biological functions are. Furthermore, we give an outlook on how these soluble receptors can be exploited for therapeutic purposes.     

Somatic and axonal LIGHT signaling elicit degenerative and regenerative responses in motoneurons,respectively

A receptor–ligand interaction can evoke a broad range of biological activities in different cell types depending on receptor identity and cell type‐specific post‐receptor signaling intermediates. Here, we show that the TNF family member LIGHT, known to act as a death‐triggering factor in motoneurons through LT‐βR, can also promote axon outgrowth and branching in motoneurons through the same receptor. LIGHT‐induced axonal elongation and branching require ERK and caspase‐9 pathways. This distinct response involves a compartment‐specific activation of LIGHT signals, with somatic activation‐inducing death, while axonal stimulation promotes axon elongation and branching in motoneurons. Following peripheral nerve damage, LIGHT increases at the lesion site through expression by invading B lymphocytes, and genetic deletion of Light significantly delays functional recovery. We propose that a central and peripheral activation of the LIGHT pathway elicits different functional responses in motoneurons.     

Suppression of dendritic cell activation by anthrax lethal toxin and edema toxin depends on multiple factors including cell source, stimulus used, and function tested

Bacillus anthracis produces lethal toxin (LT) and edema toxin (ET), and they suppress the function of LPS-stimulated dendritic cells (DCs). Because DCs respond differently to various microbial stimuli, we compared toxin effects in bone marrow DCs stimulated with either LPS or Legionella pneumophila (Lp). LT, not ET, was more toxic for cells from BALB/c than from C57BL/6 (B6) as measured by 7-AAD uptake; however, ET suppressed CD11c expression. LT suppressed IL-12, IL-6, and TNF-alpha in cells from BALB/c and B6 mice but increased IL-1beta in LPS-stimulated cultures. ET also suppressed IL-12 and TNF-alpha, but increased IL-6 and IL-1beta in Lp-stimulated cells from B6. Regarding maturation marker expression, LT increased MHCII and CD86 while suppressing CD40 and CD80; ET generally decreased marker expression across all groups. We conclude that the suppression of cytokine production by anthrax toxins is dependent on variables, including the source of the DCs, the type of stimulus and cytokine measured, and the individual toxin tested. However, LT and ET enhancement or suppression of maturation marker expression is more related to the marker studied than the stimuli or cell source. Anthrax toxins are not uniformly suppressive of DC function but instead can increase function under defined conditions.     

细胞药物的种类及生物学特性——细胞药物连载之一

细胞药物是以不同细胞为基础的用于疾病治疗的制剂、药物或产品的统称,是继放疗、化疗之后又一种临床有效的治疗手段,可实施个性化治疗。细胞药物的种类很多,按其生物学特性可分为传统体细胞、免疫细胞以及各种不同的干细胞等。经体外操作过的细胞群,如肝细胞、胰岛细胞、软骨细胞、树突状细胞、细胞因子诱导的杀伤细胞、淋巴因子激活的杀伤细胞、体外加工的骨髓或造血干细胞和体外处理的肿瘤细胞(瘤苗)等。细胞药物已在一些难治性疾病中得到应用,包括血液系统疾病、心血管系统疾病、消化系统疾病、神经系统疾病、免疫系统疾病和抗衰老等。细胞治疗涉及的细胞种类很多,且不同细胞或不同治疗方法各有特点。运用不同的细胞药物来修复病变细胞,以重建受损的功能细胞和组织,恢复其生物学功能,为细胞丢失或损伤性疾病的防治提供了崭新的思路。     

Lectin activities of cytokines: functions and putative carbohydrate-recognition domains

The discovery that some cytokines have carbohydrate-binding (lectin) properties opens new concepts in the understanding of their mechanism of action. The carbohydrate-recognition domain (CRD), which is localized at the opposite of the receptor-binding domain, makes these molecules bi-functional. The expression of the biological activity of the cytokine relies on its carbohydrate-binding activity, which allows the association of the cytokine receptor with molecular complexes comprising the specific kinase/phosphatase involved in receptor phosphorylation/dephosphorylation and in specific signal transduction. As a correlate, a cytokine can act only on cells possessing both the receptor and the ligand. Two cytokines using the same receptor can have different target cells and functions because of their different lectin activities. Based on a few examples, the CRD can be predicted based on the 3-D structures of the molecules.     

TLR4 polymorphisms mediate impaired responses to respiratory syncytial virus and lipopolysaccharide

Severe bronchiolitis following respiratory syncytial virus (RSV) infection occurs in only a small subset of infected infants and the basis for variations in disease severity is not understood. Innate immune responses to RSV are mediated by TLR-4, and the (299)Gly and (399)Ile alleles of the TLR4 gene have been linked epidemiologically with increased severity of RSV disease in children. We hypothesized that cellular immune responses to RSV mediated by these variant forms of the receptor are defective relative to responses mediated via the common form of the receptor. Human bronchial epithelial cells were transfected with TLR4 constructs encoding the common TLR4 gene sequence ((299)Asp/(399)Thr), or the (299)Gly or (399)Ile alleles, and cytokine responses to in vitro RSV challenge were analyzed in the different transfected cells. Follow-up studies compared RSV-induced responses in PBMC from children expressing these same TLR4 genotypes. Human bronchial epithelial expressing (299)Gly or (399)Ile displayed normal levels of intracellular TLR4 but failed to efficiently translocate the receptor to the cell surface. This was associated with reduced NF-kappaB signaling post-TLR4 engagement, reduced production of IFNs, IL-8, IL-10, IL-12p35, IL-18, and CCL8, and the absence of acute-phase TNF-alpha. These findings were mirrored by blunted PBMC responses to RSV in children expressing the same TLR4 variants. Compromised first-line defense against RSV at the airway-epithelial surface of children expressing these TLR4 variants may thus confer increased susceptibility to severe infections with this virus.     

Distinct effector cytokine profiles of memory and naive human B cell subsets and implication in multiple sclerosis

Although recent animal studies have fuelled growing interest in Ab-independent functions of B cells, relatively little is known about how human B cells and their subsets may contribute to the regulation of immune responses in either health or disease. In this study, we first confirm that effector cytokine production by normal human B cells is context dependent and demonstrate that this involves the reciprocal regulation of proinflammatory and anti-inflammatory cytokines. We further report that this cytokine network is dysregulated in patients with the autoimmune disease multiple sclerosis, whose B cells exhibit a decreased average production of the down-regulatory cytokine IL-10. Treatment with the approved chemotherapeutic agent mitoxantrone reciprocally modulated B cell proinflammatory and anti-inflammatory cytokines, establishing that the B cell cytokine network can be targeted in vivo. Prospective studies of human B cells reconstituting following in vivo depletion suggested that different B cell subsets produced distinct effector cytokines. We confirmed in normal human B cell subsets that IL-10 is produced almost exclusively by naive B cells while the proinflammatory cytokines lymphotoxin and TNF-alpha are largely produced by memory B cells. These results point to an in vivo switch in the cytokine "program" of human B cells transitioning from the naive pool to the memory pool. We propose a model that ascribes distinct and proactive roles to memory and naive human B cell subsets in the regulation of memory immune responses and in autoimmunity. Our findings are of particular relevance at a time when B cell directed therapies are being applied to clinical trials of several autoimmune diseases.     

Aspartic acid 50 and tyrosine 108 are essential for receptor binding and cytotoxic activity of tumour necrosis factor beta (lymphotoxin).

Single amino acid substitutions were generated in predicted hydrophilic loop regions of the human tumour necrosis factor beta (TNF-beta) molecule, and the mutant proteins were expressed in Escherichia coli and purified. Mutants with single amino acid changes at either of two distinct loop regions, at positions aspartic acid 50 or tyrosine 108, were found to have greatly reduced receptor binding and cytotoxic activity. These two regions in TNF-beta correspond to known loop regions where mutations also result in loss of biological activity of TNF-alpha, a related cytokine which shares the same cellular receptors with TNF-beta. The two distinct loops at positions 31-34 and 84-89 in the known three-dimensional structure of TNF-alpha (equivalent to positions 46-50 and 105-110 respectively in TNF-beta), lie on opposite sides of the TNF-alpha monomer. When the TNF-alpha monomer forms a trimer, the two loops, each from a different subunit of the trimer, come together and lie in a cleft between adjacent subunits. Together, these findings suggest that a TNF receptor binds to a cleft between subunits via surface loops at amino acid residues 31-34 and 84-89 in TNF-alpha, and similarly via surface loops including amino acids aspartic acid 50 and tyrosine 108 in TNF-beta.