Prostaglandins have wide-ranging effects in the body and are thought to be important mediators of inflammation. Cyclooxygenase (COX) plays a key regulatory role in prostaglandin synthesis, and occurs in both constitutive (COX-1) and inducible (COX-2) isoforms. COX-1 is thought to provide cytoprotective effects, whereas COX-2 is both inducible and the major isoform of inflammatory cells. Reduction of prostaglandin production by inhibition of cyclooxygenases appears to be the main mechanism of action of most non-steroidal anti-inflammatory drugs (NSAIDS). Here we present an animal model of COX-2 deficiency that was generated by gene targeting. Defects in null mice correlating with reduced viability included renal alterations, characteristic of renal dysplasia (100% penetrance), and cardiac fibrosis (50% penetrance). Female Cox-2-/- mice were infertile. COX-2 deficiency failed to alter inflammatory responses in several standard models, but striking mitigation of endotoxin-induced hepatocellular cytotoxicity was observed.

The ubiquitous transcription factor NF-B is an essential component in signal transduction pathways, in inflammation, and in the immune response. NF-B is maintained in an inactive state in the cytoplasm by protein-protein interaction with IB␣. Upon stimulation, rapid degradation of IB␣ allows nuclear translocation of NF-B. To study the importance of IB␣ in signal transduction, IB␣-deficient mice were derived by gene targeting. Cultured fibroblasts derived from IB␣-deficient embryos exhibit levels of NF-B1, NF-B2, RelA, c-Rel, and IB␤ similar to those of wild-type fibroblasts. A failure to increase nuclear levels of NF-B indicates that cytoplasmic retention of NF-B may be compensated for by other IB proteins. Treatment of wild-type cells with tumor necrosis factor alpha (TNF-␣) resulted in rapid, transient nuclear localization of NF-B. IB␣-deficient fibroblasts are also TNF-␣ responsive, but nuclear localization of NF-B is prolonged, thus demonstrating that a major irreplaceable function of IB␣ is termination of the NF-B response. Consistent with these observations, and with IB␣ and NF-B's role in regulating inflammatory and immune responses, is the normal development of IB␣-deficient mice. However, growth ceases 3 days after birth and death usually occurs at 7 to 10 days of age. An increased percentage of monocytes/macrophages was detected in spleen cells taken from 5-, 7-, and 9-day-old pups. Death is accompanied by severe widespread dermatitis and increased levels of TNF-␣ mRNA in the skin.Members of the Rel transcription factor family regulate the expression of numerous genes, including many of those involved in immune and inflammatory responses (for reviews, see references 4, 22, and 54). The known mammalian Rel proteins-NF-B1 (p50/p105), NF-B2 (p52/p100), RelA (p65), c-Rel, and RelB-contain a conserved 300-amino-acid Rel-homology domain, which is involved in protein-protein interaction, DNA binding, and nuclear localization. Nearly all possible homodimers and heterodimers have been observed (classical NF-B is a heterodimer of p50 and p65). These dimers are capable of binding to the B DNA binding site-a 10-or 11 bp sequence that conforms to a loose consensus. Rel proteins are expressed in most (perhaps all) cells, although their individual levels may vary with the cell type. With few exceptions, the various dimers are localized in the cytoplasm of unstimulated cells. A wide variety of agents (e.g., phorbol ester, lipopolysaccharide [LPS], and cytokines) induce the nuclear translocation of Rel proteins, initiating expression of numerous genes.Rel dimers are held in the cytoplasm by members of the IB family of inhibitor proteins, all of which contain five to seven copies of the ''ankyrin repeat''-a 33-amino-acid motif that is particularly abundant in the protein ankyrin. The inhibitor IB␣ is capable of binding to most (perhaps all) dimers, thereby blocking their nuclear localization signals (6,20,34). Upon stimulation of the cell, IB␣ undergoes phosphorylation and rapid degradation (5,9,16,26,41,57), resulting ...

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SummaryA new neutrophil-activating peptide, termed ENA-78, was identified in the conditioned media of stimulated human type II epithelial cell line A549. In response to stimulation with either interleukin 13 (II-13) or tumor necrosis factor c~ (TNF-o0, ENA-78 was produced and secreted concomitantly with II.-8, GtkOcz, and GRO3,. ENA-78 consists of 78 amino acids (AGPAAA-VLILELRCVCLQTTQGVHPKMISNLQVFAIGPQCSKVEVVASLKNGKEICLDPEAPFLKK-VIQKILDGGNKEN) and has a molecular weight of 8,357. It has four cysteines positioned identically to those of Ib8 and analogues, and thus belongs to the CXC family of peptides. ENA-78 is related to neutrophil-activating peptide 2 (NAP-2) and GtkOo~ (sequence identity, 53% and 52%, respectively) and II,8 (22% identity). Like NAP-2 and GILO~ ENA-78 stimulates neutrophils, inducing chemotaxis, a rise in intracellular free calcium and exocytosis. Crossdesensitization experiments indicate that ENA-78 acts through the same type of receptors as Ib8, NAP-2, and GtLOo~.

The potential for interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) to induce neutrophil and mononuclear phagocyte accumulation in the lungs of patients with pulmonary sarcoidosis and idiopathic pulmonary fibrosis (IPF) was investigated. Bronchoalveolar lavage (BAL) fluids from 12 patients with IPF and 15 with sarcoidosis were concentrated by reversed-phase chromatography, and their IL-8 and MCP-1 concentrations assessed by enzyme-linked immunosorbent assay (ELISA), chemotaxis, and enzyme-releasing assays with monocytes and neutrophils. ELISA revealed significantly elevated concentrations of MCP-1 (20.1 ng/mg albumin) in the BAL fluids of patients with pulmonary sarcoidosis and those with IPF (41.8 ng/mg) in comparison to 11 normal individuals (4.24 ng/mg) and 15 patients with chronic bronchitis (CB) (5.16 ng/mg). Similarly, the chemotactic activity for monocytes (MCP-1 equivalent) was strongly increased in patients with sarcoidosis (86.03 ng/mg) as well as in those with IPF (54.47 ng/mg). The chemoattractant activity of normal individuals and CB patients was 7- or 3-fold lower, respectively. Patients with IPF and sarcoidosis also had elevated IL-8 levels (15.5 and 26.0 ng/mg, respectively; normals: 2.14 ng/mg; and CB patients: 4.23 ng/mg) and greater neutrophil chemotaxis (60.25 and 49.68 ng/mg, respectively; normals: 0.35 ng/mg; and CB patients: 11.06 ng/mg). These data suggest that increased levels of both MCP-1 and IL-8 may be characteristic for sarcoidosis or IPF. It appears likely that both of these chemoattractants contribute to the influx of monocytes and neutrophils into the pulmonary alveolus and interstitium in these diseases.

To investigate the roles of tumor necrosis factor (TNF) and lymphotoxin (LT)-alpha in the development and function of the immune system, the Tnf and Ltalpha genes were simultaneously inactivated in mice by homologous recombination. These mutant mice are highly susceptible to Listeria monocytogenes infection and resistant to endotoxic shock induced by the combined administration of D-galactosamine (D-GaIN) and lipopolysaccharide (LPS). Their splenic microarchitecture is disorganized, characterized by the loss of the clearly defined marginal zone, ill defined T and B cell areas, and absence of MAdCAM-1 and reduced ICAM-1, VCAM-1 and Mac-1 expression. They are devoid of peripheral lymph nodes and Peyer's patches, and show a strong reduction of IgA+ plasma cells in the intestinal lamina propria. The alymphoplasia is accompanied by a marked B lymphocytosis and reduced basal lg levels. Ig depositions in the renal glomerulus and a strong up-regulation of MHC class I antigen expression on endothelial cells of different tissues are observed. The primary humoral immune response towards sheep red blood cells reveals a defective IgG isotype switch, while that against vesicular stomatitis virus is normal. The cytotoxic T cell responses are attenuated, although still effective, against vaccinia, lymphocytic choriomeningitis virus (LCMV-ARM) and LCMV-WE. In conclusion, the combined inactivation of Tnf and Ltalpha confirms their essential role in the normal development and function of the immune system.

SeminaryAntibody neutralization studies have established interferon qr (IFN-3') as a critical mediator of endotoxic shock. The advent of IFN-3, receptor negative (IFN3,R-/-) mutant mice has enabled a more direct assessment of the role of IFN-'y in endotoxin (lipopolysaccharide [LPS]-induced shock. We report that IFN3'R-/-mice have an increased resistance to LPS-induced toxicity, this resistance manifesting well before the synthesis and release of LPS-induced IFN-% LPSinduced lymphopenia, thrombocytopenia, and weight loss seen in wild-type mice were attenuated in IFN3,R-/-mice. IFN3,R-/-mice tolerated 100-1,000 times more LPS than the minimum lethal dose for wild-type mice in a D-galactosamine (D-GalN)/LPS model. Serum tumor necrosis factor (TNF) levels were 10-fold reduced in mutant mice given LPS or LPS/D-GalN. Bone marrow and splenic macrophages from IFN'yR-/-mice had a four-to sixfold decreased LPS-binding capacity which correlated with similar reduction in CD14. Serum from mutant mice reduced macrophage LPS binding by a further 50%, although LPS binding protein was only 10% reduced. The expression of TNF receptor I (p55) and II (p75) was identical between wild-type and mutant mice. Thus, depressed TNF synthesis, diminished expression of CD14, and low plasma LPSbinding capacity, in addition to blocked IFN-3' signaling in the mutant mice, likely to combine to manifest in the resistant phenotype of IFN'yR-/-mice to endotoxin.

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