In vivo suppression of NF-kappa B and preservation of I kappa B alpha by interleukin-10 and interleukin-13.

IL-10 and IL-13 have powerful antiinflammatory activities in vitro and in vivo. In the IgG immune complex model of lung injury in rats, exogenously administered IL-10 or IL-13 have recently been shown to suppress neutrophil recruitment and ensuing lung injury by greatly depressing pulmonary production of TNF alpha. Transcriptional control of the TNF alpha gene is regulated by the nuclear factor kappa B (NF-kappa B). Activation of NF-kappa B involves the degradation of its cytoplasmic inhibitor I kappa B alpha, allowing the nuclear translocation of NF-kappa B, with ensuing transcriptional activation. In this study, we sought to determine whether the protective effects of IL-10 and IL-13 in IgG immune complex-induced lung injury were mediated by inhibition of NF-kappa B activation. Electrophoretic mobility shift analysis of nuclear extracts from alveolar macrophages and whole lung tissues demonstrated that both IL-10 and IL-13 suppressed nuclear localization of NF-kappa B after in vivo deposition of IgG immune complexes. Western blot analysis indicated that these effects were due to preserved protein expression of I kappa B alpha in both alveolar macrophages and whole lungs. Northern blot analysis of lung mRNA showed that, in the presence of IgG immune complexes, IL-10 and IL-13 augmented I kappa B alpha mRNA expression. These findings suggest that in vivo, IL-10 and IL-13 may operate by suppressing NF-kappa B activation through preservation of I kappa B alpha.

STAT4 and STAT6 regulate systemic inflammation and protect against lethal endotoxemia.

Members of the signal transducer and activator of transcription (STAT) family are transcription factors that mediate many of the effects of pro- and anti-inflammatory cytokines. The progressive systemic inflammatory response induced by endotoxin is mediated by overzealous cytokine production. Here we identify STAT4 and STAT6 as critical regulators of the systemic inflammatory response to endotoxin. Mice deficient for STAT4 or STAT6 were highly susceptible to lethal endotoxemia. In STAT4(-/-) mice, antibody blockade of IL-12 prevented mortality, suggesting that STAT4 confers protection, while another signaling pathway mediates the detrimental effects of IL-12. In STAT6(-/-) mice we observed dysregulated activation of the transcription factor NF-kappaB, resulting in augmented production of proinflammatory cytokines and chemokines. Furthermore, STAT6(-/-) mice displayed increased organ accumulation of leukocytes and significant hepatocellular injury. These findings demonstrate that STAT4 and STAT6 confer protection against endotoxin-induced death and that for STAT6 these protective effects occur through the regulation of NF-kappaB activation and subsequent production of proinflammatory cytokines and chemokines.

The role of cytokines, adhesion molecules, and chemokines in interleukin-2-induced lymphocytic infiltration in C57BL/6 mice.

IL-2 mediates the regression of certain malignancies, but clinical use is limited because of associated toxicities, including parenchymal lymphocytic infiltration with multiple organ failure. Secondarily induced cytokines are important mediators of IL-2 toxicity and IL-2-induced lymphocyte-endothelial adherence and trafficking. The recently discovered C-C chemokines, RANTES (regulated on activation, normal T expressed and secreted) and macrophage inflammatory protein-1alpha, have also been implicated in lymphocytic migration. We hypothesized that IL-2 alters cytokine, C-C chemokine, and adhesion molecule expression in association with parenchymal lymphocytic infiltration. C57BL/6 mice were injected with 3x10(5) IU of IL-2 or 0.1 ml of 5% dextrose intraperitoneally every 8 h for 6 d, then killed. IL-2 induced massive lymphocytic infiltration in the liver and lung and moderate infiltration in the kidney in association with organ edema and dysfunction. Immunostaining showed increased intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression in association with this organ-specific lymphocytic infiltration. Flow cytometry showed increased expression of the corresponding ligands (lymphocyte function-associated antigen-1 and very late antigen-4) on splenocytes. IL-2 increased TNF-alpha mRNA and protein expression in the liver. Organs infiltrated by lymphocytes had increased TNF-alpha mRNA, whereas RANTES mRNA was increased in all organs, regardless of lymphocytic infiltration. IL-2 toxicity involves organ-specific TNF-alpha and RANTES production with increased ICAM-1 and VCAM-1 expression as potential mechanisms facilitating lymphocytic infiltration and organ dysfunction.

Mechanisms of interleukin-2-induced hepatic toxicity.

Interleukin 2 (IL-2) mediates the regression of metastatic cancer, but its clinical use is limited by associated toxicities including hepatic dysfunction. To determine the mechanism for IL-2-induced hepatic dysfunction, we hypothesized that IL-2 activation of Kupffer cells causes leukocyte-endothelial adhesion and decreases hepatic sinusoidal blood flow. C57BL/6 mice were given injections of latex particles and prepared for intravital hepatic microscopy 2 h after i.p. IL-2 administration. Liver tissue was also prepared to quantitate hepatic tumor necrosis factor (TNF) mRNA and processed for light and electron microscopy. Phagocytosing Kupffer cells and leukocytes adherent to the endothelium were counted, and surface sinusoidal blood flow was quantitated. Kupffer cell activity was quantitated as the ratio of phagocytosing Kupffer cells to sinusoidal blood flow. IL-2 significantly increased Kupffer cell activity (0.56 +/- 0.05 for controls versus 0.84 +/- 0.05 for IL-2), significantly caused leukocyte-endothelial adhesion (26.7 +/- 7.9 for controls versus 87.0 +/- 27.6 for IL-2, WBC/mm2 endothelial surface), and significantly decreased the number of sinusoids containing blood flow per microscopic field (6.66 +/- 0.15 for controls versus 5.79 +/- 0.13 for IL-2) without causing changes in systemic hemodynamic parameters. In IL-2 treated livers, light and electron microscopy showed the constriction of sinusoids associated with swollen or ruptured mitochondria, which was consistent with hypoxic deterioration near central venules. Adherent platelets, neutrophils, and lymphocytes within sinusoids and central venules were also observed. PCR revealed that IL-2 significantly induced TNF mRNA expression in the liver. These data suggest that IL-2 activates Kupffer cells in association with the release of monokines including TNF, which causes activation of circulating leukocytes as well as hepatic sinusoidal endothelial cells. The resultant leukocyte and platelet adhesion to the endothelium may then physically impede the sinusoidal microcirculation, resulting in microscopic areas of hepatic ischemia and explaining the mechanism of IL-2-induced hepatic dysfunction.

N omega-nitro-L-arginine methyl ester inhibits inflammatory liver injury induced by interleukin-2.

Administration of interleukin-2 (IL-2) for treatment of metastatic disease often results in inflammatory liver injury. Previous studies have implicated increased leukocyte and platelet adhesion and enhanced nitric oxide production as causative factors in the development of IL-2-induced hepatic injury. This study investigated the capacity of N omega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthesis inhibitor, to limit IL-2-induced hepatic edema and hepatocellular damage in mice. Using hepatic intravital microscopy, we also examined the effects of L-NAME on IL-2-induced increases in leukocyte and platelet adhesion. Administration of IL-2 increased leukocyte and platelet adhesion in post-sinusoidal venules and decreased hepatic perfusion. Cotreatment with L-NAME had no effect on leukocyte adhesion but increased platelet-endothelial adhesion and microvascular thrombosis. Chronic IL-2 treatment induced hepatic edema and hepatocellular injury. However, coadministration of L-NAME attenuated IL-2-induced edema and completely inhibited hepatocellular damage. These findings suggest that nitric oxide may play a central role in IL-2-induced inflammatory liver injury.

Interleukin-10 inhibits interleukin-2-induced tumor necrosis factor production but does not reduce toxicity in C3H/HeN mice.

Immunotherapy with interleukin-2 (IL-2) is limited by severe side effects thought to be mediated by the activation of immune effector cells and the induction of secondary cytokines including tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). In C3H/HeN mice the primary IL-2 toxicity is the production of pleural effusion with subsequent respiratory compromise. IL-10 is a cytokine that has been shown to inhibit the generation of secondary cytokines in vitro and in vivo. In this study, in C3H/HeN mice, we tested the ability of IL-10 to inhibit IL-2-induced mononuclear cell and alveolar macrophage activation and IL-2-induced increases in serum TNF-alpha and IFN-gamma, all of which may contribute to the generation of toxicity. IL-10 was ineffective at reducing IL-2-induced pleural effusion. However, IL-10 did inhibit IL-2-induced increases in serum TNF-alpha, which was accompanied by a decrease in Golgi apparatus and rough endoplasmic reticulum in alveolar macrophages. In addition, IL-10 combined with IL-2 increased mononuclear cell activation, which may limit the ability of IL-10 to inhibit IL-2-induced IFN-gamma production and pulmonary injury.

Regulatory effects of interleukin-11 during acute lung inflammatory injury.

The role of interleukin-11 (IL-11) was evaluated in the IgG immune complex model of acute lung injury in rats. IL-11 mRNA and protein were both up-regulated during the course of this inflammatory response. Exogenously administered IL-11 substantially reduced, in a dose-dependent manner, the intrapulmonary accumulation of neutrophils and the lung vascular leak of albumin. These in vivo anti-inflammatory effects of IL-11 were associated with reduced NF-kappaB activation in lung, reduced levels of tumor necrosis factor alpha (TNF-alpha) in bronchoalveolar lavage (BAL) fluids, and diminished up-regulation of lung vascular ICAM-1. It is interesting that IL-11 did not affect BAL fluid content of the CXC chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-inducible neutrophil chemoattractant (CINC); the presence of IL-11 did not affect these chemokines. However, BAL content of C5a was reduced by IL-11. These data indicate that IL-11 is a regulatory cytokine in the lung and that, like other members of this family, its anti-inflammatory properties appear to be linked to its suppression of NF-kappaB activation, diminished production of TNF-alpha, and reduced up-regulation of lung vascular ICAM-1.

Role of complement in in vitro and in vivo lung inflammatory reactions.

Complement is one of the integral buttresses of the inflammatory response. In addition to host defense activities, proinflammatory properties of several complement components are described. This overview elucidates the role of complement in inflammatory reactions in vitro and in vivo, focusing on the complement activation products, C5a, and the membrane attack complex, C5b-9. Using several approaches, the impact of these complement components in mechanisms relevant to neutrophil recruitment is emphasized. In addition, the participation of complement in endothelial superoxide generation and its essential requirement for full expression of lung injury is demonstrated, as are the involved intracellular signal transduction pathways. Understanding the mechanisms of complement-induced proinflammatory effects may provide a basis for future therapeutic blockade of complement and/or its activation products.

The acute inflammatory response and its regulation.

The acute inflammatory response is composed of an elaborate cascade of both proinflammatory and anti-inflammatory mediators. The balance between these mediators often determines the outcome after injury. In clinical scenarios, such as trauma or sepsis, there is often unregulated production of proinflammatory mediators that can cause multiple organ failure. Further understanding of the endogenous mechanisms that control the inflammatory response is needed to facilitate development of therapeutic options. In this review, we discuss the current knowledge of the mechanisms leading to development of acute inflammatory injury as well as the factors that regulate this response.

The NFkappaBb/IkappaB system in acute inflammation.

The transcription factor NFkappaB is a primary regulator of a wide variety of proinflammatory mediators. Under normal conditions, NFkappaB is retained in the cytoplasm bound to inhibitory proteins of the IkappaB family. Following cell activation, a number of signal transduction pathways lead to degradation of IkappaB proteins which results in nuclear translocation of NFkappaB and the ensuing transcriptional activation of proinflammatory genes. Several endogenous regulatory mediators, which function to prevent uncontrolled inflammation, exert their effects by blocking the activation of NFkappaB. Thus, NFkappaB appears to be at the heart of the acute inflammatory response. The present review discusses the role of NFkappaB in the induction and propagation of the acute inflammatory response as well as the regulation and resolution of this process.

In vivo recruitment of neutrophils: consistent requirements for L-arginine and variable requirements for complement and adhesion molecules.

The current studies examined the mechanisms of neutrophil recruitment into the rat peritoneal cavity following injection of glycogen and into rat lungs following alveolar deposition of IgA immune complexes or airway instillation of phorbol ester (PMA). Unexpectedly, in each model a requirement for L-arginine for neutrophil recruitment was demonstrated, since administration of the L-arginine analogue, NG-monomethyl L-arginine acetate (L-NMA), greatly reduced neutrophil accumulation as assessed by quantitation of neutrophils in peritoneal exudates and bronchoalveolar lavage fluids, and by lung myeloperoxidase content. In the case of IgA immune complex deposition, lung recruitment of neutrophils was also suppressed by soluble recombinant human complement receptor-1 (sCR1) and antibody to CD18 but not by antibody to E-selection. In contrast, neutrophil accumulation following airway instillation of PMA exhibited, surprisingly, no requirement for complement but requirements for both E-selection and CD18. These data demonstrate variable requirements for complement, E-selectin and CD18 but a consistent requirement for L-arginine for neutrophil recruitment. These findings provide evidence suggesting that L-arginine or its derivatives regulate neutrophil recruitment.

Cytokine and adhesion molecule requirements for lung injury induced by anti-glomerular basement membrane antibody.

Acute hemorrhagic lung injury occurs in humans with anti-GBM antibody (Goodpasture's syndrome), however, the mechanism of this injury is still largely unknown. To date, treatment has been confined to steroids and plasmaphoresis. Infusion of anti-GBM antibody into rats caused lung injury with intra-alveolar hemorrhage and intrapulmonary accumulation of neutrophils. Lung injury was dependent on the presence of neutrophils and complement and required both TNF alpha and IL-1. Experiments employing blocking antibodies to adhesion molecules demonstrated requirements for the beta 1 integrin VLA-4, beta 2 integrins LFA-1 and Mac-1, and L-selection. The endothelial cell adhesion molecules, E-selectin and ICAM-1, were also required for the full development of lung injury. Inhibition of TNF alpha or IL-1 or adhesion molecules reduced both lung injury and tissue neutrophil accumulation. Thus, this study underscores cytokine and adhesion molecule requirements for neutrophil mediated injury in lung and kidney caused by anti-GBM, suggesting potential targets for the treatment of Goodpasture's syndrome in humans.

CXCR1 deficiency does not alter liver regeneration after partial hepatectomy in mice.

Previous studies have shown that CXC chemokines containing Glu-Leu-Arg (ELR) in their amino-terminus stimulate hepatocyte proliferation and liver regeneration after partial hepatectomy. These ELR+CXC chemokines bind to two receptors, CXCR1 and CXCR2. Previous work has shown that CXCR2 is involved in the proliferative effects of CXC chemokines. However, the function of CXCR1 during the regenerative response has not been studied. The aim of the current study was to investigate the role of CXCR1 in liver regeneration after partial hepatectomy. C57BL/6 (wild type) or CXCR1-/- mice were subjected to 70% partial hepatectomy or sham surgery and sacrificed on day 2 and 4 after operation. There were no significant differences in liver-to-body weight ratio or hepatocyte proliferation. The data suggest that CXCR1 does not mediate the proliferative effects of ELR+ CXC chemokines during liver regeneration after partial hepatectomy.

The Ron receptor promotes prostate tumor growth in the TRAMP mouse model.

The Ron receptor tyrosine kinase (TK) is overexpressed in many cancers, including prostate cancer. To examine the significance of Ron in prostate cancer in vivo, we utilized a genetically engineered mouse model, referred to as TRAMP mice, that is predisposed to develop prostate tumors. In this model, we show that prostate tumors from 30-week-old TRAMP mice have increased Ron expression compared with age-matched wild-type prostates. Based on the upregulation of Ron in human prostate cancers and in this murine model of prostate tumorigenesis, we hypothesized that this receptor has a functional role in the development of prostate tumors. To test this hypothesis, we crossed TRAMP mice with mice that are deficient in Ron signaling (TK-/-). Interestingly, TK-/- TRAMP+ mice show a significant decrease in prostate tumor mass relative to TRAMP mice containing functional Ron. Moreover, TK-/- TRAMP+ prostate tumors exhibited decreased tumor vascularization relative to TK+/+ TRAMP+ prostate tumors, which correlated with reduced levels of the angiogenic molecules vascular endothelial growth factor and CXCL2. Although Ron loss did not alter tumor cell proliferation, a significant decrease in cell survival was observed. Similarly, murine prostate cancer cell lines containing a Ron deficiency exhibited decreased levels of active nuclear factor-κB, suggesting that Ron may be important in regulating prostate cell survival at least partly through this pathway. In total, our data show for the first time that Ron promotes prostate tumor growth, prostate tumor angiogenesis and prostate cancer cell survival in vivo.

Potential role for IL-23 in hepatic ischemia/reperfusion injury.

IL-12 and IL-23 are related cytokines that share a p40 subunit. Our previous studies identified IL-12 as a primary initiator of the cytokine cascade induced after hepatic ischemia/reperfusion. Because those studies were conducted prior to the discovery of IL-23, it is not clear whether IL-12 or IL-23 is the relevant cytokine in this response. The current studies show that the antibodies used in our original study cross-react with IL-23. We also found that both IL-12 p35 and IL-23 p19 mRNA are expressed rapidly in the liver after ischemia/reperfusion. Finally, isolated Kupffer cells produced TNFalpha in response to IL-23, but not IL-12, suggesting that IL-23 may be the relevant initiator of the hepatic inflammatory response to ischemia/reperfusion.

Regulation of inflammatory vascular damage.

The acute inflammatory response is comprised of an elaborate cascade of mediators that control an ordered sequence of events resulting in the recruitment of neutrophils to the site of infection or injury. Microvascular injury occurring during acute inflammation often results in increased vascular permeability and microvascular haemorrhage. Damage to vascular endothelial cells, basement membrane, and matrix components results from both neutrophil-dependent and neutrophil-independent mechanisms and is also dependent on the organ/tissue source of the endothelial cells. Neutrophil-mediated injury of endothelial cells involves a complex cascade in which products from both cell types affect the cytotoxic outcome. It is also clear that the acute inflammatory response is carefully regulated by the endogenous gene expression of both pro-inflammatory and anti-inflammatory mediators. Control of acute inflammation seems to relate to activation of the transcription factor NFkappaB. To appreciate the interrelationship between multiple contributing factors of inflammatory vascular injury, one must first have an understanding of the inflammatory mediator cascades which bring about the recruitment of neutrophils to the site of inflammation. In this review it is discussed how inflammatory mediators, as well as the products of activated neutrophils, affect the outcome of the acute inflammatory response.

Activation and regulation of NFkappaB during acute inflammation.

During the acute inflammatory response, there is induction of a mediator cascade which functions to activate residential macrophages and recruit blood leukocytes to the site of the inflammatory insult. Dysregulation of this process can cause an exaggerated inflammatory response and lead to tissue injury. Recent studies have focused on the transcription factor NFkappaB, which controls the gene expression of many pro-inflammatory mediators. Under normal conditions, NFkappaB is retained in the cytosol by inhibitory proteins of the IkappaB family. In response to an inflammatory insult, IkappaB proteins are degraded and the free NFkappaB complex translocates to the nucleus where it initiates gene transcription. An understanding of the in vivo mechanisms leading to the activation of NFkappaB, and the regulatory mechanisms that exist to limit this activation, may lead to the development of novel new therapeutic options for inflammatory injury. In this review we will discuss the current knowledge of the role of NFkappaB in the development of acute inflammation, as well as the regulatory mechanisms that exist to prevent the activation of NFkappaB and resolve inflammatory tissue injury.

Regulation of experimental lung inflammation.

Acute lung inflammation is an important component of a number of pulmonary diseases, including acute respiratory distress syndrome (ARDS). Much has been learned about the manner in which various insults to lung, such as infection or trauma, bring about recruitment of neutrophils into alveoli and small airways, resulting in parenchymal damage and organ dysfunction. In this brief review, we discuss the endogenous mechanisms in which the lung regulates the acute inflammatory response in rats to intrapulmonary deposition of IgG immune complexes. Emphasis is given to the participation of the transcription factor, NF-kappaB, in the development of lung injury and the endogenous mediators which attempt to control the extent of lung inflammation by modulating the activation of NF-kappaB.

Involvement of the neuropeptide substance P in lung inflammation induced by hepatic ischemia/reperfusion.

OBJECTIVE AND DESIGN: The purpose of this study was to examine the potential role of substance P in accumulation of neutrophils in the lung following hepatic ischemia/reperfusion. MATERIALS AND METHODS: Male C57BL/6 mice (8-10 weeks of age) were subjected to either sham surgery, partial hepatic ischemia with or without reperfusion, or intratracheal administration of saline or 1 ng substance P. Lung neutrophil accumulation was assessed by tissue content of myeloperoxidase. Activation of the transcription factor, NF-kappaB, was determined by electrophoretic mobility shift assay. Levels of substance P and macrophage inflammatory protein-2 (MIP-2) in bronchoalveolar lavage (BAL) fluid was measured using enzyme-linked immunosorbent assays. RESULTS: Significant pulmonary neutrophil accumulation was observed just prior to hepatic reperfusion in association with increased BAL levels of substance P. Intratracheal administration of substance P resulted in a similar pattern of neutrophil accumulation which was associated with activation of NF-kappaB and increased BAL levels of the chemokine, MIP-2. CONCLUSIONS: The data suggest that hepatic ischemia causes substance P release in the lung which activates NF-kappaB leading to the production of MIP-2 and accumulation of neutrophils.

Interleukin-2 induces increased platelet-endothelium interactions: a potential mechanism of toxicity.

Cancer immunotherapy with interleukin-2 (IL-2) is limited by side effects that may cause organ dysfunction. The role of platelets in the generation of IL-2-induced organ dysfunction has not been studied, although various studies have shown that IL-2 therapy activates both platelets and the vascular endothelium. We hypothesized that IL-2 therapy may enhance the thrombogenic response to inflammatory stimuli through increased platelet-endothelial interactions and that these effects could lead to the development of organ dysfunction. C57BI/6 mice were treated with IL-2 intraperitoneally for 2 hours (short term) or 2 to 5 days (long term) and prepared for in vivo microscopy of the ear microcirculation. Mice were injected intra-arterially with fluorescein isothiocyanate conjugated to bovine serum albumin (FITC-BSA). Blue light activation of the FITC-BSA in ear arterioles induced thrombus formation. The time to initial thrombus formation was measured as an index of thrombogenicity. Platelet function was analyzed by aggregometry and platelet expression of IL-2 receptors, and the adhesion molecule lymphocyte function-associated antigen-1 (LFA-1) was analyzed by flow cytometry. Organ dysfunction was evaluated by serum markers. The administration of both short-term and long-term IL-2 reduced the time to initial thrombus formation as compared with controls. In vitro platelet aggregometry revealed no acute alterations in platelet function; however, long-term IL-2 treatment resulted in decreased disaggregation rates. There were no platelet IL-2 receptors present, and the expression of the adhesion molecule LFA-1 was not altered by IL-2. Increased thrombogenicity occurred before the generation of organ dysfunction. These data suggest that increased platelet adherence induced by IL-2 is caused by effects on the endothelium that could result in microvascular thrombus formation and contribute to organ dysfunction.