For a long time, superoxide generation by an NADPH oxidase was considered as an oddity only found in professional phagocytes. Over the last years, six homologs of the cytochrome subunit of the phag...

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The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology

Mammalian cells proteolytically release (shed) the extracellular domains of many cell-surface proteins. Modification of the cell surface in this way can alter the cell's responsiveness to its environment and release potent soluble regulatory factors. The release of soluble tumour-necrosis factor-alpha (TNF-alpha) from its membrane-bound precursor is one of the most intensively studied shedding events because this inflammatory cytokine is so physiologically important. The inhibition of TNF-alpha release (and many other shedding phenomena) by hydroxamic acid-based inhibitors indicates that one or more metalloproteinases is involved. We have now purified and cloned a metalloproteinase that specifically cleaves precursor TNF-alpha. Inactivation of the gene in mouse cells caused a marked decrease in soluble TNF-alpha production. This enzyme (called the TNF-alpha-converting enzyme, or TACE) is a new member of the family of mammalian adamalysins (or ADAMs), for which no physiological catalytic function has previously been identified. Our results should facilitate the development of therapeutically useful inhibitors of TNF-alpha release, and they indicate that an important function of adamalysins may be to shed cell-surface proteins.

A metalloproteinase disintegrin that releases tumour-necrosis factor-α from cells

Inflammation, the most ancient aspect of the host immune response, is surely of great value to the host, insofar as agents that suppress inflam­ mation in a nonspecific fashion predispose to infection. Yet, the inflam­ matory response to invasive organisms may also, if sufficiently intense or inappropriately prolonged, cause injury or death. A delicate balance has thus been achieved, one which clinicians strive to maintain through judicious application of anti-inflammatory medications (e.g. glucocorti­ coids, nonsteroidal anti-inflammatory agents, and cytotoxic drugs). Only recently have we come to understand that many aspects of this primitive response to host invasion are governed by polypeptide hormones, in turn produced by immune effector cells. Moreover, we have come to appreciate the pleiotropic properties of these so-called "cytokines." It would appear that a ' limited number of cytokines are capable of orches­ trating disease states that scarcely resemble one another; among them, endotoxic shock, graft-vs-host disease, cerebral malaria, and cancer cachexia. Cells of monocyte/macrophage lineage play a central role in cytokine ("mono kine") production and so act to modulate many aspects of the inflammatory response. While devoid of specificity and immunologic mem-

The biology of cachectin/TNF--a primary mediator of the host response

Tumour-necrosis factor-alpha (TNF-alpha) is a cytokine that contributes to a variety of inflammatory disease states. The protein exists as a membrane-bound precursor of relative molecular mass 26K which can be processed by a TNF-alpha-converting enzyme (TACE), to generate secreted 17K mature TNF-alpha. We have purified TACE and cloned its complementary DNA. TACE is a membrane-bound disintegrin metalloproteinase. Structural comparisons with other disintegrin-containing enzymes indicate that TACE is unique, with noteable sequence identity to MADM, an enzyme implicated in myelin degradation, and to KUZ, a Drosophila homologue of MADM important for neuronal development. The expression of recombinant TACE (rTACE) results in the production of functional enzyme that correctly processes precursor TNF-alpha to the mature form. The rTACE provides a readily available source of enzyme to help in the search for new anti-inflammatory agents that target the final processing stage of TNF-alpha production.

Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha.

A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity

Different macrophage populations were investigated for their abilities to secrete tumor necrosis factor (TNF) and to lyse TNF-susceptible tumor cells. In this way we could demonstrate that TNF-secretion, although a feature of all activated macrophage populations, is no absolute requirement for the killing of the TNF-sensitive Wehi 164 target. Macrophage cytotoxicity against this cell but not against the TNF-resistant P815 mastocytoma, was completely inhibitable by a specific anti-TNF serum also in the absence of measurable secreted TNF. Moreover the TNF-dependent lysis of tumor cells could also be performed by activated macrophages that had been fixed with paraformaldehyde before the addition of the target cells. In the indirect radioimmunoassay, TNF could be demonstrated on the surface of fixed effector cells. Our results must be interpreted in terms of membrane-associated TNF as the lytic principle for TNF-susceptible tumor cells.

Cell-associated tumor necrosis factor (TNF) as a killing mechanism of activated cytotoxic macrophages.

A fast and easy method to determine the production of reactive oxygen intermediates by human and murine phagocytes using dihydrorhodamine 123.

In this study, acidic arabinogalactan, a highly purified polysaccharide from plant cell cultures of Echinacea purpurea, with a molecular weight of 75,000, was effective in activating macrophages to cytotoxicity against tumor cells and micro-organisms (Leishmania enriettii). Furthermore, this polysaccharide induced macrophages to produce tumor necrosis factor (TNF-alpha), interleukin-1 (IL-1), and interferon-beta 2. Arabinogalactan did not activate B cells and did not induce T cells to produce interleukin-2, interferon-beta 2, or interferon-gamma, but it did induce a slight increase in T-cell proliferation. When injected ip, this agent stimulated macrophages, a finding that may have therapeutic implications in the defense against tumors and infectious diseases.

Macrophage Activation by the Polysaccharide Arabinogalactan Isolated From Plant Cell Cultures of Echinacea purpurea

Plasma membranes were isolated from thioglycolate-induced peritoneal mouse macrophages and tested directly in a 51Cr-release assay against WEHI 164 tumor cells. These membranes showed anti-TNF antibody inhibitable killing of the TNF-sensitive tumor cell line, indicating that membrane-associated TNF is present on mouse macrophages. In order to elucidate whether membrane TNF is an integral protein or a molecule attached to a receptor, cells and plasma membranes were treated with low pH buffer. A partial reduction in TNF activity was observed which could be restored by incubation with exogenous TNF. In a Western blot analysis the integral membrane TNF could be identified as the 26-kDa molecule on activated mouse macrophages. These results indicate that both forms of membrane-associated TNF exist on macrophages and are responsible for cell-mediated cytotoxicity against TNF-alpha-sensitive targets.

M L Lohmann-Matthes

Papers

A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity

Cell-associated tumor necrosis factor (TNF) as a killing mechanism of activated cytotoxic macrophages.

A fast and easy method to determine the production of reactive oxygen intermediates by human and murine phagocytes using dihydrorhodamine 123.

Macrophage Activation by the Polysaccharide Arabinogalactan Isolated From Plant Cell Cultures of Echinacea purpurea

Evidence for the existence of two forms of membrane tumor necrosis factor: an integral protein and a molecule attached to its receptor.