Functional discrepancies between tumor necrosis factor and lymphotoxin α explained by trimer stability and distinct receptor interactions

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.     

Interleukin-12 activates human γδ T cells: synergistic effect of tumor necrosis factor-α

γδ T cell populations are known to expand in response to intracellular bacterial infectious agents regardless of previous priming. We have shown previously that soluble factor(s) produced by Mycobacterium-stimulated monocytes activate cord blood γδ T cells to proliferate. In this study, we investigated whether cytokines produced by monocytes are responsible for γδ T cell activation in vitro: interleukin (IL)-1β, IL-6, IL-8, IL-12, tumor necrosis factor (TNF)-α and granulocyte/macrophage colony-stimulating factor were examined. Recombinant human IL-12 stimulated γδ T cells, but not αβ T cells in peripheral blood mononuclear cells, to express CD25 on their surfaces, and to expand in number in vitro. IL-12-primed γδ T cell numbers increased to a greater extent in the culture to which exogenous IL-2 (5 U/ml) was added. Anti-TNF-α monoclonal antibody inhibited IL-12-induced up-regulation of CD25 on γδ T cells, suggesting that endogenous TNF-α may play a role in IL-12-induced activation of γδ T cells. Recombinant TNF-α synergistically augmented IL-12-induced activation of γδ T cells. Furthermore, IL-12 up-regulated TNF receptors on γδ T cells in vitro: TNF-α binding to its receptor induced CD25 expression on the γδ T cells in an autocrine or paracrine fashion, or perhaps both. It also became evident that both IL-12 and TNF-α were produced by mycobacterial lysate-stimulated monocytes. Taken together, these results suggest that upon confrontation with mycobacterial organisms, γδ T cells can be quickly and antigen-nonspecifically activated by soluble factors including IL-12 and TNF-α, both of which are produced by mononuclear phagocytes in response to mycobacterial organisms.     

Purified native and recombinant human alpha lymphotoxin [tumor necrosis factor (TNF)-beta] induces inflammatory reactions in normal skin

These studies report findings that demonstrate that human alpha lymphotoxin (LT) induces local, visible, and microscopic inflammatory reactions in normal skin. Skin sites in rabbits, when inoculated with a single injection of native or recombinant human alpha lymphotoxin, demonstrated erythema, swelling, and warmth within 5 hr. Erythema peaked between 24 and 48 hr and resolved by 72 hr. Histologic studies of skin sites injected with native LT revealed polymorphonuclear neutrophil (PMN) infiltration and edema beginning as early as 3 hr posttreatment. Individual skin sites that received three daily injections of native LT exhibited persistent erythema and swelling. Palpable induration was evident 24 hr after the second injection in the series. Histologic examination revealed the presence of many PMNs with associated focal dermal destruction, in the form of microabscesses, and scattered mononuclear cells. In contrast, control materials and recombinant human tumor necrosis factor (TNF-alpha) did not induce visible skin reactions in the rabbit. Several additional controls excluded endotoxin as being the agent responsible for the inflammatory skin reactions observed. The ability of LT to induce inflammation may have a role in its antitumor activity and it may be an important endogenous mediator in other immunologic reactions.     

Identification of unique murine tumor associated antigens by tumor infiltrating lymphocytes using tumor specific secretion of interferon-γ and tumor necrosis factor

Stimulation of multiple CD8⁺ murine tumor infiltrating lymphocyte (TIL) lines and one TIL clone with the tumor of origin of the TIL induced at least three-fold more secretion of TNF and/or INF-γ than was elicited by other syngeneic, methylcholanthrene (MCA) induced sarcomas. TIL which specifically secreted lymphokines were generated from three different sarcomas. Specific lymphokine secretion was a stable characteristic of the lines over time. IL-2 was necessary for maximal lymphokine secretion by TIL. These investigations demonstrate that lymphokine secretion by CD8⁺ lymphocytes derived from tumor bearing mice can be used to define unique tumor associated antigens on at least three different sarcomas and may be valuable in studies of the biologic nature of these antigens and of the adoptive immunotherapy of cancer.     

Interleukin-10 controls interferon-γ and tumor necrosis factor production during experimental endotoxemia

Interleukin-10 (IL-10) is a potent inhibitor of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) production and has been shown to protect mice from endotoxin shock. As IFN-γ is another important mediator of LPS toxicity, we studied the effects of IL-10 on LPS-induced IFN-γ synthesis in vitro and in vivo. First, we found that the addition of recombinant human IL-10 (rhIL-10) (10 U/ml) to human whole blood markedly suppressed LPS-induced IFN-γ release while neutralization of endogenously synthesized IL-10 resulted in increased IFN-γ levels. The ability of rIL-10 to inhibit LPS-induced IFN-γ synthesis was also observed in vivo in mice. Indeed, administration of 1000 U recombinant mouse IL-10 (rmIL-10) 30 min before and 3 h after challenge of BALB/c mice with 100 μg LPS resulted in a threefold decrease in peak IFN-γ serum levels. We then examined the production and the role of IL-10 during murine endotoxemia. We found that LPS injection causes the rapid release of IL-10, peak IL-10 serum levels being observed 90 min after LPS challenge. Neutralization of endogenously produced IL-10 by administration of 2 mg JES5-2A5 anti-IL-10 monoclonal antibody (mAb) 2h before LPS challenge resulted in a marked increase in both TNF and IFN-γ serum levels while irrelevant isotype-matched mAb had no effect. The enhanced production of inflammatory cytokines in anti-IL-10 mAb-treated mice was associated with a 60% lethality after injection of 500 μg LPS, while all mice pretreated with control mAb survived. We conclude that the rapid release of IL-10 during endotoxemia is a natural antiinflammatory response controlling cytokine production and LPS toxicity.     

Monoclonal antibodies to rabbit α-2-macroglobulin and their use in a sensitive ELISA assay

alpha-2-macroglobulin (alpha-2-M), a plasma proteinase inhibitor, plays an important role in the pathogenesis of lung inflammation. The purpose of this study was to develop a sensitive ELISA assay for rabbit alpha-2-M to allow us to define the role of this protein in a rabbit model of lung inflammation/injury. Therefore, we developed hybridomas which secrete monoclonal antibodies (mAbs) against rabbit alpha-2-M. From the antibodies produced, two (5B6 and 3C5) were selected. Both of them were of the IgG1 subclass. 5B6 reacted with native alpha-2-M as well as with the "fast" form of alpha-2-M (native alpha-2-M or the "slow" form is converted to the "fast" form by reaction with a proteinase). On the other hand, 3C5 reacted only with "fast" form of alpha-2-M. With these antibodies, we developed two ELISA assays which were used to determine the concentration of alpha-2-M in the lung fluids from rabbits with lung injury.     

Distinct roles for lymphotoxin-α and tumor necrosis factor in organogenesis and spatial organization of lymphoid tissue

Specialized roles for the pro-inflammatory cytokines tumor necrosis factor (TNF) and lymphotoxin (LT) were characterized in TNF/LTα^?/? and TNF^?/? mice established by direct gene targeting of C57BL/6 ES cells. The requirement for LT early in lymphoid tissue organogenesis is shown to be distinct from the more subtle and varied role of TNF in promoting correct microarchitectural organization of leukocytes in LN and spleen. Development of normal Peyer's patch (PP) structure, in contrast, is substantially dependent on TNF. Only mice lacking LT exhibit retarded B cell maturation in vivo and serum immunoglobulin deficiencies. A temporal hierarchy in lymphoid tissue development can now be defined, with LT being an essential participant in general lymphoid tissue organogenesis, developmentally preceeding TNF that has a more varied and subtle role in promotion of correct spatial organization of leukocytes in LN and spleen. PP development in TNF^?/? mice is unusual, indicating that TNF is a more critical participant for this structure than it is for other lymphoid tissues.     

Highly sensitive enzyme immunoassays for antibodies to human tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta).

Two 'inverse sandwich' enzyme immunoassays (ELISAs) were developed for the detection and quantification of antibodies to human tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta), respectively. In these one-step assays, antibodies present in the sample linked antigen which had been covalently coupled to horseradish peroxidase to antigen bound to a solid phase (microtiter plates). The limits of detection of the assays were lower than those of neutralization bioassays; antibodies to TNF-alpha and TNF-beta being detected at concentrations as low as 2 ng/ml and 0.5 ng/ml, respectively, and no cross-reactivity was observed. The advantages of these ELISAs over other assay methods currently in use for the detection of antibodies include: (i) the convenience of the microtiter plate format and its suitability for testing large numbers of samples; (ii) the absence of radioactive tracers and precipitation steps; (iii) the high stability of the reagents; (iv) the avoidance of second antibodies and, thus, the possibility of testing samples from various species without modification of the assay and (v) the ability to detect low-affinity antibodies due to the absence of competitive reactions. The assays may be used without modification for the detection of antibodies in serum samples from both man and laboratory animals as well as in other samples such as hybridoma supernatants.     

β-Amyloid precursor protein (βAPP) as a marker for axonal injury after head injury

It has been demonstrated recently that β-amyloid protein (βAP), generally associated with the plaques of Alzheimer's disease, can also be found in the brains of survivors of head injury. In this study the distribution of the βAP precursor protein (βAPP) was examined immunohistochemically to determine if it is colocalized with βAP in such cases. βAPP immunoreactivity was observed in neuronal perikarya in the neocortex and in dystrophic neurites surrounding βAP immunoreactive plaques i.e. in a distribution similar to that seen in Alzheimer's disease. In addition, βAPP immunoreactivity was noted within white matter tracts where it marked damaged axons. However, no colocalisation of βAPP with βAP was observed in any white matter region. These results indicate that processing of βAPP to produce βAP occurs in the synaptic terminal field of axons and illustrate the utility of βAPP immunoreactivity as a general marker for axonal injury.     

Modulation of the transcripts for tumor necrosis factor-α and its receptors in vivo

We investigated the effects of a single bacterial lipopolysaccharide (LPS) injection in vivo on the gene expression of tumor necrosis factor-α (TNF) and its receptors: TNF receptor type I (TNF-R 55 kDa or TNF-R1) and TNF receptor type II (TNF-R 75 kDa or TNF-R2) in various tissues and white blood cells. While TNF mRNA rapidly accumulated in most tissues, TNF-R1 and TNF-R2 mRNA levels were found to be differentially regulated in lung, spleen, lymph nodes and white blood cells. In most cases, TNF-R mRNA levels did not parallel TNF mRNA levels. These observations indicate that TNF-R of both types are capable of modulating the host response to LPS, not only by shedding of their extracellular domains, but also by strict regulation of their gene expression.     

Mice heterozygous for a deletion of the tumor necrosis factor-α and lymphotoxin-α genes: biological importance of a nonlinear response of tumor necrosis factor-α to gene dosage

The tumor necrosis factors (TNF-α and lymphotoxin, or LT-α) are important mediators of the immune and inflammatory responses, and it has been proposed that a positive feedback loop could boost the expression of the TNF to sufficiently high levels to fend off infections. To investigate this phenomenon and its biological consequences, we have generated LT-α/TNF-α knockout mice and compared mice having one or two functional LT-α/TNF-α alleles. In response to lipopolysaccharide (LPS) stimulation, TNF-α levels in the circulation or in the supernatant of macrophage cultures were 20- to 100-fold lower in heterozygous samples than in their wild-type counterparts. This differential increased with the intensity of stimulation and throughout the response, supporting the involvement of a positive feedback loop. Moreover, the heterozygous mice had an increased bacterial load following Listeria monocytogenes infection and exhibited a bimodal response to the association of D -galactosamine and LPS which was similar to that of wild-type mice at low doses of LPS and more like that of homozygous mutants at high doses. These results therefore establish the biological importance of the nonlinear response of TNF-α levels to gene dosage, and these mice provide a unique tool to study how the propensity to produce TNF can determine the immunological fitness of individuals.     

Specific and sensitive quantitation of transforming growth factor β3 by sandwich enzyme-linked immunosorbent assay

Transforming growth factors beta (TGF-β) consist of a highly homologous family of 25 kDa dimers involved in a diverse array of biological functions. Progress in understanding the biology of the third isoform (TGF-β3) of this family has been limited by the absence of a quantitative assay for TGF-β3. Here we report the development of a sensitive and specific sandwich enzyme-linked immunosorbent assay (SELISA), which is based on an anti-TGF-β mouse monoclonal IgG as the capture antibody and chicken anti-recombinant-hTGF-β3 IgY as the secondary antibody. This assay can quantitate TGF-β3 in complex biological fluids, with a detection limit of 2 pg and no cross-reactivity or inteference with as high as 1000-fold molar excesses of either TGF-βs 1, 2 or 1.2. This TGF-β3 SELISA is the first reported assay for the direct and sensitive quantitation of TGF-β3 in complex biological fluids.     

T cells which do not express membrane tumor necrosis factor-α activate macrophage effector function by cell contact-dependent signaling of macrophage tumor necrosis factor-α production

Previous studies have suggested that T cell contact-dependent signaling of macrophages (MΦ) is mediated by membrane tumor necrosis factor-α (memTNF-α), based on the observation that anti-TNF-α could inhibit T cell-mediated MΦ activation. The current report confirms that anti-TNF-α does inhibit activation of interferon-γ (IFN-γ)-primed MΦ by paraformaldehyde-fixed activated T cells. However, the involvement of membrane molecules other than memTNF-α in the contact-dependent signaling is suggested by two lines of evidence. First, the T_H2 clone, AK8, displayed neither secreted TNF-α/β nor memTNF-α/β detectable by bioassay or immunofluorescence. Nonetheless, AK8 cells were equally effective, on a per cell basis, in contact-dependent signaling of MΦ activation as T_H2 and T_H1 cells which do express memTNF-α. Second, the expression of memTNF-α by the T_H clone, D10.G4, is maximal 24 h after activation, whereas the ability of this clone to activate MΦ is maximal at 6–8 h of activation and declines thereafter. Since TNF-α is known to play a critical role in activation of MΦ effector function, it was hypothesized that T cell membrane components other than memTNF-α might signal MΦ production of TNF-α, thus allowing autocrine TNF-α stimulation of MΦ effector function. In support of this, it is demonstrated that paraformaldehyde-fixed activated T_H2 cells can induce de novo production and release of TNF-α by MΦ. This effect was not an artifactual result of paraformaldehyde fixation since paraformaldehyde-fixed resting T cells did not induce TNF-α gene expression. Previous studies have demonstrated a role for autocrine TNF-α stimulation in LPS induction of effector function in recombinant IFN-γ-primed MΦ. The current study confirms that TNF-α plays a critical role in T cell contact-dependent signaling of MΦ but indicates that memTNF on the T cells may not be a sine qua non factor for contact-dependent signaling. The data suggest that other T cell membrane molecules contribute to activation of MΦ effector function by stimulation of MΦ TNF-α production.     

Biphasic control of nuclear factor-χB activation by the T cell receptor complex: role of tumor necrosis factor α

The regulation of nuclear factor (NF)-χB activation by the T cell receptor (TcR)/CD3 complex in primary human T cells has been studied at various times after activation. Only p50 NF-χB protein bound the χB element of interleukin-2 receptor (IL-2R) α chain promoter on resting T cells. However, immediately after TcR/CD3 cross-linking (after approximately 1 h; immediate) binding of p50.p65 heterodimers was observed. p50.c-rel heterodimers were also detected bound to this sequence at early time points (7–16 h; early), and both remained active at later time points (40 h; late) after activation. This regulation takes place mainly at the level of nuclear translocation of p65 and c-rel, at immediate and early time points. Activation also induced c-rel and p105/p50 mRNA synthesis, but not p65 mRNA whose expression was constitutive. Interestingly, all those early and late events, but not the immediate ones, were inhibited by a neutralizing anti-tumor necrosis factor α (TNF-α) monoclonal antibody. Similarly, cycloheximide prevented the p65 and c-rel translocation and consequent formation of active binding heterodimers, at early and late times. Cyclosporin A impaired not only early and late, but also immediate events; however, addition of TNF-α prevented all inhibition. These results indicate that the regulation of NF-χB activation during T cell activation by TcR/CD3 signals is biphasic: TcR/CD3 triggers its immediate translocation, which is transient if no TNF-α is present. TNF-α, therefore, emerges as the main factor responsible for a second phase of NF-χB regulation, controlling both translocation of p65 and c-rel, and new mRNA synthesis for c-rel and p105/p50.