Thomas P. Shanley

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.

Suppression of macrophage inflammatory protein (MIP)-1α production and collagen-induced arthritis by adenosine receptor agonists

1 Ligands of the various adenosine receptor subtypes modulate the production of pro‐and anti‐inflammatory cytokines. Here we evaluated the effect of adenosine and various ligands of the adenosine receptor subtypes (A1, A2, A3) on the chemokine macrophage inflammatory protein (MIP) 1α production in immunostimulated RAW macrophages in vitro. Furthermore, we studied whether a selected A3 adenosine receptor agonist inhibits MIP‐1α production and affects the course of inflammation in collagen‐induced arthritis. 2 In the cultured macrophages, the A3 receptor agonist N6‐(3‐iodobenzyl)‐adenosine‐5′‐N‐methyluronamide (IB‐MECA), and, less potently, the A2 receptor agonist 2‐p‐(2‐carboxyethyl) phenethylamino‐5′‐N‐ethyl‐carboxamidoadenosine (CGS; 1–200 μM) dose‐dependently suppressed the production of MIP‐1α. The selective A1 receptor agonist 2‐chloro‐N6‐cyclopentyladenosine (CCPA, 1–200 μM) was ineffective, and adenosine was a weak inhibitor. The inhibition of MIP‐1α production by the A3 and A2 agonist was associated with suppression of its steady‐state mRNA levels. 3 Based on the in vitro data, we concluded that activation of A3, and to a lesser extent A2 adenosine receptors suppresses MIP‐1α expression. Since IB‐MECA was the most potent inhibitor of MIP‐1α expression, we next investigated whether it affects the production of other pro‐inflammatory mediators. We observed that IB‐MECA (1–300 μM) inhibited, in a dose‐dependent manner, the production of IL‐12, IL‐6, and, to a lesser extent, nitric oxide in the immunostimulated cultured macrophages. 4 Since MIP‐α is a chemokine which enhances neutrophil recruitment into inflammatory sites, we investigated whether the A3 agonist IB‐MECA affects the course of inflammation, MIP‐α production and the degree of neutrophil recruitment in arthritis. In a model of collagen‐induced arthritis in mice, IB‐MECA (0.5 mg/kg/day) reduced the severity of joint inflammation. IB‐MECA inhibited the formation of MIP‐1α, IL‐12 and nitrotyrosine (an indicator of reactive nitrogen species) in the paws, and suppressed neutrophil infiltration. 5 We conclude that adenosine receptor agonists, most notably the A3 agonist IB‐MECA suppress the production of MIP‐α, and exert anti‐inflammatory effects. Therefore, stimulation of adenosine receptor subtypes A3 and A2 may be a strategy worthy of further evaluation for the abrogation of acute or chronic inflammatory disorders.

Tristetraprolin (TTP)-14-3-3 Complex Formation Protects TTP from Dephosphorylation by Protein Phosphatase 2a and Stabilizes Tumor Necrosis Factor-α mRNA

Tumor necrosis factor (TNF)-α is a major cytokine produced by alveolar macrophages in response to pathogen-associated molecular patterns such as lipopolysaccharide. TNF-α secretion is regulated at both transcriptional and post-transcriptional levels. Post-transcriptional regulation occurs by modulation of TNF-α mRNA stability via the binding of tristetraprolin (TTP) to the adenosine/uridine-rich elements found in the 3′-untranslated region of the TNF-α transcript. Phosphorylation plays important roles in modulating mRNA stability, because activation of p38 MAPK by lipopolysaccharide stabilizes TNF-α mRNA. We hypothesized that the protein phosphatase 2A (PP2A) regulates this signaling pathway. Our results show that inhibition of PP2A by okadaic acid or small interference RNA significantly enhanced the stability of TNF-α mRNA. This result was associated with increased phosphorylation of p38 MAPK and MAPK-activated kinase 2 (MK-2). PP2A inhibition increased TTP phosphorylation and enhanced complex formation with chaperone protein 14-3-3. TTP physically interacted with PP2A in transfected mammalian cells. A functional consequence of TTP-14-3-3 complex formation appeared to be protection of TTP from dephosphorylation by inhibition of the binding of PP2A to phosphorylated TTP. Mutation of the MK-2 phosphorylation sites of TTP did not influence TNF-α adenosine/uridine-rich element binding and did not alter the increased TNF-α 3′-untranslated region-dependent luciferase activity induced by PP2A-small interference RNA silencing. Our data indicate that, although phosphorylation stabilizes TNF-α mRNA, PP2A regulates the mRNA stability by modulating the phosphorylation state of members of the p38/MK-2/TTP pathway.

Combined Steroid Treatment for Congenital Heart Surgery Improves Oxygen Delivery and Reduces Postbypass Inflammatory Mediator Expression

Background—Steroid administration during cardiopulmonary bypass is thought to improve cardiopulmonary function by modulating bypass-related inflammation. This study was designed to compare preoperative and intraoperative methylprednisolone (MP) to intraoperative MP alone with respect to postbypass inflammation and clinical outcome. Methods and Results—Twenty-nine pediatric patients undergoing bypass procedures were randomly assigned to receive preoperative and intraoperative MP (30 mg/kg 4 hours before bypass and in bypass prime, n=14) or intraoperative MP only (30 mg/kg, n=15). Myocardial inflammatory mediator mRNA expression was determined in paired atrial biopsies (before and after bypass) by ribonuclease protection. Before and after bypass, serum IL-6 and IL-10 were measured by ELISA. Postoperative outcome was assessed by intubation time, CICU length of stay, fluid balance, arterio-venous O2 difference (&Dgr;A−Vo2), and inotrope requirements. Compared with intraoperative MP alone, combined preoperative and intraoperative MP was associated with reduced myocardial mRNA expression for IL-6, MCP-1, and ICAM-1 both before and after bypass (P <0.05). Patients who received combined steroids had lower serum IL-6 and increased IL-10 at end-bypass (P <0.05), although differences were negligible by 24 hours. Combined MP treatment was associated with reduced fluid requirements, lower body temperature, and lower &Dgr;A−Vo2 for the first 24 hours after surgery (P <0.05), along with trends toward improvement in other clinical outcomes. Conclusions—Compared with intraoperative steroid treatment, combined preoperative and intraoperative steroid administration attenuates inflammatory mediator expression more effectively and is associated with improved indexes of O2 delivery in the first 24 hours after congenital heart surgery. These findings need to be confirmed in a larger multicenter trial.

Role of CC Chemokines (Macrophage Inflammatory Protein-1β, Monocyte Chemoattractant Protein-1, RANTES) in Acute Lung Injury in Rats

The role of the CC chemokines, macrophage inflammatory protein-1β (MIP-1β), monocyte chemotactic peptide-1 (MCP-1), and RANTES, in acute lung inflammatory injury induced by intrapulmonary deposition of IgG immune complexes injury in rats was determined. Rat MIP-1β, MCP-1, and RANTES were cloned, the proteins were expressed, and neutralizing Abs were developed. mRNA and protein expression for MIP-1β and MCP-1 were up-regulated during the inflammatory response, while mRNA and protein expression for RANTES were constitutive and unchanged during the inflammatory response. Treatment of rats with anti-MIP-1β Ab significantly decreased vascular permeability by 37% (p = 0.012), reduced neutrophil recruitment into lung by 65% (p = 0.047), and suppressed levels of TNF-α in bronchoalveolar lavage fluids by 61% (p = 0.008). Treatment of rats with anti-rat MCP-1 or anti-rat RANTES had no effect on the development of lung injury. In animals pretreated intratracheally with blocking Abs to MCP-1, RANTES, or MIP-1β, significant reductions in the bronchoalveolar lavage content of these chemokines occurred, suggesting that these Abs had reached their targets. Conversely, exogenously MIP-1β, but not RANTES or MCP-1, caused enhancement of the lung vascular leak. These data indicate that MIP-1β, but not MCP-1 or RANTES, plays an important role in intrapulmonary recruitment of neutrophils and development of lung injury in the model employed. The findings suggest that in chemokine-dependent inflammatory responses in lung CC chemokines do not necessarily demonstrate redundant function.

Genomic expression profiling across the pediatric systemic inflammatory response syndrome, sepsis, and septic shock spectrum

Objectives:To advance our biological understanding of pediatric septic shock, we measured the genome-level expression profiles of critically ill children representing the systemic inflammatory response syndrome (SIRS), sepsis, and septic shock spectrum. Design:Prospective observational study involving microarray-based bioinformatics. Setting:Multiple pediatric intensive care units in the United States. Patients:Children ≤10 years of age: 18 normal controls, 22 meeting criteria for SIRS, 32 meeting criteria for sepsis, and 67 meeting criteria for septic shock on day 1. The available day 3 samples included 20 patients still meeting sepsis criteria, 39 patients still meeting septic shock criteria, and 24 patients meeting the exclusive day 3 category, SIRS resolved. Interventions:None other than standard care. Measurements and Main Results:Longitudinal analyses were focused on gene expression relative to control samples and patients having paired day 1 and day 3 samples. The longitudinal analysis focused on up-regulated genes revealed common patterns of up-regulated gene expression, primarily corresponding to inflammation and innate immunity, across all patient groups on day 1. These patterns of up-regulated gene expression persisted on day 3 in patients with septic shock, but not to the same degree in the other patient classes. The longitudinal analysis focused on down-regulated genes demonstrated gene repression corresponding to adaptive immunity-specific signaling pathways and was most prominent in patients with septic shock on days 1 and 3. Gene network analyses based on direct comparisons across the SIRS, sepsis, and septic shock spectrum, and all available patients in the database, demonstrated unique repression of gene networks in patients with septic shock corresponding to major histocompatibility complex antigen presentation. Finally, analyses focused on repression of genes corresponding to zinc-related biology demonstrated that this pattern of gene repression is unique to patients with septic shock. Conclusions:Although some common patterns of gene expression exist across the pediatric SIRS, sepsis, and septic shock spectrum, septic shock is particularly characterized by repression of genes corresponding to adaptive immunity and zinc-related biology.

Glomerular inflammation induces resistance to tubular injury in the rat. A novel form of acquired, heme oxygenase-dependent resistance to renal injury.

Considerable attention is directed to a surprising biologic phenomenon wherein tissues exposed to one insult acquire resistance to another. We identify a novel example of acquired resistance to acute renal failure and a mechanism that contributes to such resistance. Nephrotoxic serum, administered to rats 24 h before the induction of glycerol-induced acute renal failure, reduces functional and structural injury that occurs in this model. Since heme oxygenase, the rate-limiting enzyme in heme degradation, protects against heme protein-induced renal injury, we questioned whether induction of heme oxygenase underlies the protection afforded by nephrotoxic serum. Kidney heme oxygenase (HO-1) mRNA was induced 6 h after nephrotoxic serum and renal tubules were identified as the site of expression of heme oxygenase protein. Induction of heme oxygenase was accompanied by increased renal content of ferritin but not by induction of other antioxidant enzymes. Inhibition of heme oxygenase prevented the protection afforded by nephrotoxic serum. Nephrotoxic serum did not protect against ischemic acute renal failure, a model in which heme oxygenase is not induced. Thus, nephrotoxic serum protects against glycerol-induced acute renal failure by inducing heme oxygenase in tubules. This study provides the first demonstration of resistance to tubular injury acquired from glomerular inflammation, uncovers a mechanism for such resistance, and exposes the dialogue that occurs between glomeruli and tubules.

New concepts of IL-10-induced lung fibrosis: fibrocyte recruitment and M2 activation in a CCL2/CCR2 axis

IL-10 is most commonly recognized as an anti-inflammatory cytokine possessing immunosuppressive effects necessary for regulated resolution of proinflammation. However, its role in the development of fibrosis during inflammatory resolution has not been clear. Few prior studies have linked IL-10 with the inhibition of fibrosis principally on the basis of regulating inflammation thought to be driving fibroproliferation. In contrast, in a model of long-term overexpression of IL-10, we observed marked induction of lung fibrosis in mice. The total cell number retrieved by bronchoalveolar lavage (BAL) increased 10-fold in the IL-10 overexpression (IL-10 OE) mice, with significant infiltration of T and B lymphocytes and collagen-producing cells. The presence of increased fibrocytes, isolated from collagenase-digested lungs, was identified by flow cytometry using dual staining of CD45 and collagen 1. Quantitative PCR analysis on an array of chemokine/chemokine receptor genes showed that receptor CCR2 and its ligand, CCL2, were highly upregulated in IL-10 OE mice, suggesting that IL-10-induced fibrocyte recruitment was CCL2/CCR2 specific. Given the prior association of alternatively activated (M(2)) macrophages with development of fibrosis in other disease states, we also examined the effect of IL-10 OE on the M(2) macrophage axis. We observed significantly increased numbers of M(2) macrophages in both BAL and whole lung tissue from the IL-10 OE mice. Administration of rabbit anti-CCL2 antiserum to IL-10 OE mice for three consecutive weeks significantly decreased fibrosis as evidenced by lung hydroxyproline content, compared with mice that received preimmune rabbit serum. These results indicate that overexpression of IL-10 induces fibrosis, in part, by fibrocyte recruitment and M(2) macrophage activation, and likely in a CCL2/CCR2 axis.

IL-17–Induced Pulmonary Pathogenesis during Respiratory Viral Infection and Exacerbation of Allergic Disease

Severe respiratory syncytial virus (RSV) infections are characterized by airway epithelial cell damage, mucus hypersecretion, and Th2 cytokine production. Less is known about the role of IL-17. We observed increased IL-6 and IL-17 levels in tracheal aspirate samples from severely ill infants with RSV infection. In a mouse model of RSV infection, time-dependent increases in pulmonary IL-6, IL-23, and IL-17 expression were observed. Neutralization of IL-17 during infection and observations from IL-17(-/-) knockout mice resulted in significant inhibition of mucus production during RSV infection. RSV-infected animals treated with anti-IL-17 had reduced inflammation and decreased viral load, compared with control antibody-treated mice. Blocking IL-17 during infection resulted in significantly increased RSV-specific CD8 T cells. Factors associated with CD8 cytotoxic T lymphocytes, T-bet, IFN-γ, eomesodermin, and granzyme B were significantly up-regulated after IL-17 blockade. Additionally, in vitro analyses suggest that IL-17 directly inhibits T-bet, eomesodermin, and IFN-γ in CD8 T cells. The role of IL-17 was also investigated in RSV-induced exacerbation of allergic airway responses, in which neutralization of IL-17 led to a significant decrease in the exacerbated disease, including reduced mucus production and Th2 cytokines, with decreased viral proteins. Taken together, our data demonstrate that IL-17 plays a pathogenic role during RSV infections.

The Serine/Threonine Phosphatase, PP2A: Endogenous Regulator of Inflammatory Cell Signaling

We have investigated the regulation of kinases and phosphatases in early gene activation in monocytes because these cells are implicated in the pathogenesis of acute inflammatory states, such as sepsis and acute lung injury. One early gene up-regulated by endotoxin is c-Jun, a member of the activating protein (AP) family. C-Jun is phosphorylated by c-Jun N-terminal kinase (JNK) and associates with c-Fos to form the AP-1 transcriptional activation complex that can drive cytokine expression. Inhibition of the serine/threonine phosphatase, PP2-A, with okadaic acid resulted in a significant increase in JNK activity. This finding was associated with increased phosphorylation of c-Jun, AP-1 transcriptional activity, and IL-1β expression. Activation of PP2A inhibited JNK activity and JNK coprecipitated with the regulatory subunit, PP2A-Aα, supporting the conclusion that PP2A is a key regulator of JNK in the context of an inflammatory stimulus.