Instructing Human Macrophage Polarization by Stiffness and Glycosaminoglycan Functionalization in 3D Collagen Networks.

Dynamic alterations of composition and mechanics of the extracellular matrix are suggested to modulate cellular behavior including plasticity of macrophages (MPhs) during wound healing. In this study, engineered 3D fibrillar matrices based on naturally occurring biopolymers (collagen I, glycosaminoglycans (GAGs)) are used to mimic matrix stiffening as well as modification by sulfated and nonsulfated GAGs at different stages of wound healing. Human MPhs are found to sensitively respond to these microenvironmental cues in terms of polarization toward proinflammatory or wound healing phenotypes over 6 days in vitro. MPhs exhibit a wound healing phenotype in stiffer matrices as determined by protein and gene expression of relevant cytokines (IL10, IL12, and TNFα). Presence of sulfated and nonsulfated GAGs inhibits this polarization effect. Furthermore, control experiments on 2D matrices stress the relevance of using stiffness-controlled 3D matrices, as MPhs show a reciprocal polarization behavior depending on GAG presence. Hence, the results indicate a strong influence of dimensionality, stiffness, and GAG presence of the biomaterial scaffold on MPh polarization and emphasize the need for matrices closely mimicking the 3D in vivo context with a variable stiffness and GAG composition in in vitro studies.

A Potential Mechanism for the Anti-Apoptotic Property of Koumine Involving Mitochondrial Pathway in LPS-Mediated RAW 264.7 Macrophages.

Koumine is a kind of alkaloid extracted from Gelsemium elegans (G. elegans). Benth, which has shown promise as an anti-tumor, anxiolytic, and analgesic agent. In our present study, the effect of koumine on lipopolysaccharide (LPS)-mediated RAW 264.7 cell apoptosis was evaluated. MTT assays showed that koumine obviously increased cell viability in LPS-mediated RAW 264.7 macrophages. Preincubation with koumine ameliorated LPS-medicated apoptosis by decreasing reactive oxygen species (ROS) production, which resulted in a significant decrease in the levels of nitric oxide (NO) and inducible nitric oxide synthase (iNOS). In addition, koumine-pretreated RAW 264.7 macrophages exhibited reduction of LPS-induced levels of TNF-α, IL-1ß, and IL-6 mRNA. Furthermore, pretreatment with koumine suppressed LPS-mediated p53 activation, loss of mitochondrial membrane potential, caspase-3 activation, decrease of Bcl-2 expression, and elevation of Bax and caspase-3 expressions, suggesting that koumine might act directly on RAW 264.7 cells to inhibit LPS-induced apoptosis. It seems as though the mechanism that koumine possesses is the anti-apoptotic effect mediated by suppressing production of ROS, activation of p53, and mitochondrial apoptotic pathways in RAW 264 cells. Koumine could potentially serve as a protective effect against LPS-induced apoptosis.

Anti-Inflammatory Effects and Mechanisms of Action of Coussaric and Betulinic Acids Isolated from Diospyros kaki in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages.

Diospyros kaki Thunb. is widely distributed in East Asian countries, its leaves being mainly used for making tea. In this study, coussaric acid (CA) and betulinic acid (BA), both triterpenoid compounds, were obtained from D. kaki leaf extracts through bioassay-guided isolation. CA and BA showed anti-inflammatory effects via inhibition of the nuclear factor-κB (NF-κB) pathway, providing important information on their anti-inflammatory mechanism. Furthermore, they markedly inhibited nitric oxide (NO) and prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages, and suppressed tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß) levels. Furthermore, they decreased protein expression of inducible nitric oxide synthase and cyclooxygenase-2. Pre-treatment with CA and BA inhibited LPS-induced NF-κB. We further examined the effects of CA and BA on heme oxygenase (HO)-1 expression in RAW 264.7 macrophages: BA induced HO-1 protein expression in a dose-dependent manner, while CA had no effect. We also investigated whether BA treatment induced nuclear translocation of Nrf2. BA inhibited LPS-induced NF-κB-binding activity, as well as pro-inflammatory mediator and cytokine production (e.g., NO, PGE2, TNF-α, IL-1ß, IL-6), by partial reversal of this effect by SnPP, an inhibitor of HO-1. These findings further elucidate the anti-inflammatory mechanism of CA and BA isolated from D. kaki.

Effects of the New Generation Synthetic Reconstituted Surfactant CHF5633 on Pro- and Anti-Inflammatory Cytokine Expression in Native and LPS-Stimulated Adult CD14+ Monocytes.

BACKGROUND: Surfactant replacement therapy is the standard of care for the prevention and treatment of neonatal respiratory distress syndrome. New generation synthetic surfactants represent a promising alternative to animal-derived surfactants. CHF5633, a new generation reconstituted synthetic surfactant containing SP-B and SP-C analogs and two synthetic phospholipids has demonstrated biophysical effectiveness in vitro and in vivo. While several surfactant preparations have previously been ascribed immunomodulatory capacities, in vitro data on immunomodulation by CHF5633 are limited, so far. Our study aimed to investigate pro- and anti-inflammatory effects of CHF5633 on native and LPS-stimulated human adult monocytes. METHODS: Highly purified adult CD14+ cells, either native or simultaneously stimulated with LPS, were exposed to CHF5633, its components, or poractant alfa (Curosurf®). Subsequent expression of TNF-α, IL-1ß, IL-8 and IL-10 mRNA was quantified by real-time quantitative PCR, corresponding intracellular cytokine synthesis was analyzed by flow cytometry. Potential effects on TLR2 and TLR4 mRNA and protein expression were monitored by qPCR and flow cytometry. RESULTS: Neither CHF5633 nor any of its components induced inflammation or apoptosis in native adult CD14+ monocytes. Moreover, LPS-induced pro-inflammatory responses were not aggravated by simultaneous exposure of monocytes to CHF5633 or its components. In LPS-stimulated monocytes, exposure to CHF5633 led to a significant decrease in TNF-α mRNA (0.57 ± 0.23-fold, p = 0.043 at 4h; 0.56 ± 0.27-fold, p = 0.042 at 14h). Reduction of LPS-induced IL-1ß mRNA expression was not significant (0.73 ± 0.16, p = 0.17 at 4h). LPS-induced IL-8 and IL-10 mRNA and protein expression were unaffected by CHF5633. For all cytokines, the observed CHF5633 effects paralleled a Curosurf®-induced modulation of cytokine response. TLR2 and TLR4 mRNA and protein expression were not affected by CHF5633 and Curosurf®, neither in native nor in LPS-stimulated adult monocytes. CONCLUSION: The new generation reconstituted synthetic surfactant CHF5633 was tested for potential immunomodulation on native and LPS-activated adult human monocytes. Our data confirm that CHF5633 does not exert unintended pro-inflammatory effects in both settings. On the contrary, CHF5633 significantly suppressed TNF-α mRNA expression in LPS-stimulated adult monocytes, indicating potential anti-inflammatory effects.

Human neonates display altered ex vivo monokine production related to healthy adults.

The inflammatory response plays an important role during the induction of several neonatal diseases. Previous studies have shown that during newborn infections, the natural imbalance between pro- and anti-inflammatory responses shifts toward the production of pro-inflammatory cytokines. In this study, we employed an array system to detect 9 pro- and anti-inflammatory cytokines, and performed ELISA for 6 other cytokines. We then compared the immune response profiling in umbilical cord blood (UV) plasma samples with circulating levels in otherwise healthy donors (HD). Concentrations of ex vivo monokine levels, such as interleukins (IL)-18, IL-23 and IL-27, were profoundly reduced in the UV in relation to the HD group (p-values of 0.003, 0.009 and <0.0001, respectively). Conversely, UV-plasmatic TGF-ß1 levels displayed marked enhancement (p-value=0.005) in relation to HD. Several factors may be implicated in these neonatal alterations, and additional characterization of a broader cytokine panel is warranted to reveal other possible candidates.

Pro-inflammatory Macrophages Sustain Pyruvate Oxidation through Pyruvate Dehydrogenase for the Synthesis of Itaconate and to Enable Cytokine Expression.

Upon stimulation with Th1 cytokines or bacterial lipopolysaccharides, resting macrophages shift their phenotype toward a pro-inflammatory state as part of the innate immune response. LPS-activated macrophages undergo profound metabolic changes to adapt to these new physiological requirements. One key step to mediate this metabolic adaptation is the stabilization of HIF1α, which leads to increased glycolysis and lactate release, as well as decreased oxygen consumption. HIF1 abundance can result in the induction of the gene encoding pyruvate dehydrogenase kinase 1 (PDK1), which inhibits pyruvate dehydrogenase (PDH) via phosphorylation. Therefore, it has been speculated that pyruvate oxidation through PDH is decreased in pro-inflammatory macrophages. However, to answer this open question, an in-depth analysis of this metabolic branching point was so far lacking. In this work, we applied stable isotope-assisted metabolomics techniques and demonstrate that pyruvate oxidation is maintained in mature pro-inflammatory macrophages. Glucose-derived pyruvate is oxidized via PDH to generate citrate in the mitochondria. Citrate is used for the synthesis of the antimicrobial metabolite itaconate and for lipogenesis. An increased demand for these metabolites decreases citrate oxidation through the tricarboxylic acid cycle, whereas increased glutamine uptake serves to replenish the TCA cycle. Furthermore, we found that the PDH flux is maintained by unchanged PDK1 abundance, despite the presence of HIF1. By pharmacological intervention, we demonstrate that the PDH flux is an important node for M(LPS) macrophage activation. Therefore, PDH represents a metabolic intervention point that might become a research target for translational medicine to treat chronic inflammatory diseases.

Bioeffects of micron-size magnesium particles on inflammatory cells and bone turnover in vivo and in vitro.

Magnesium (Mg) is a promising biodegradable metal offering many potential advantages over current scaffold technologies. Many studies have reported on the corrosion characteristics the Mg and its bioeffects in vitro and in vivo, but there are few studies on the biological effects of the corrosive products of Mg - the micron-size Mg particles (MgMPs). In this study, the effects of size-selected commercial MgMPs on bone turnover and macrophages were investigated in vivo and in vitro. We found that MgMPs were susceptible to engulfment by macrophages, leading to cell lysis, likely resulting from H2 gas production. We also found that the inflammatory cytokines IL-1, IL-6, and TNF-α were induced more strongly by titanium particles (TiMPs) group than by either MgMPs or control. Examination of the expression of bone remodeling markers revealed that MgMPs are beneficial for bone regeneration. Micro-CT scanning indicated that, 30 days postimplantation, unlike TiMPs, MgMPs had no adverse effect on either bone quality or quantity. We have investigated the bioeffects of micron-size MgMPs in vivo and in vitro, and our results indicate that MgMPs may promote bone regeneration without inducing inflammation. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 923-931, 2016.

Dietary exposure to benzoxazinoids enhances bacteria-induced monokine responses by peripheral blood mononuclear cells.

SCOPE: To examine potentially immunomodulating effects of dietary benzoxazinoids (BXs), present in cereal grains. METHODS AND RESULTS: Nineteen healthy volunteers were randomly distributed into two groups, who received diets with high or low content of BXs for 3 wk. After a week's wash-out, the groups switched diets. Peripheral blood mononuclear cells (PBMCs) were stimulated with Porphyromonas gingivalis, Escherichia coli lipopolysaccharide (LPS), or tetanus toxoid (TT). PBMCs from a healthy donor received the same stimuli in presence of serum from each participant receiving BXs. The production of monokines, T-cell cytokines and T-helper cell proliferation were assessed. A 3-wk diet with high BX content enhanced IL-1ß responses against LPS and P. gingivalis, as well as TNF-α response against P. gingivalis, after 24 h of stimulation. Moreover, IL-6 was found to be increased after 7 days of stimulation with LPS. No effect was observed on T-cell cytokines or proliferation. BX levels in serum after a single meal did not modify cytokine responses. CONCLUSION: High dietary intake of BXs enhances bacteria-induced production of pro-inflammatory monokines by PBMCs, but not T-cell responses; presumably due to intrinsic changes within PBMCs, built up over 3 wk of BX-rich diet, rather than to an immediate effects of BXs contained in serum.

A Novel Role of Numb as A Regulator of Pro-inflammatory Cytokine Production in Macrophages in Response to Toll-like Receptor 4.

Activation of macrophages triggers the release of pro-inflammatory cytokines leading to inflammation. Numb is a negative regulator of Notch signaling, but the role of Numb in macrophages is not fully understood. In this study, the role of Numb as a regulator of inflammatory responses in macrophages was investigated. Murine bone marrow-derived macrophages, in which expression of Numb was silenced, secreted significantly less TNFα, IL-6 and IL-12 and more IL-10 upon activation by lipopolysaccharide (LPS), a ligand for Toll-like receptor 4 (TLR4), despite increased Notch signaling. The Tnfα mRNA levels both in Numb-deficient and wild-type macrophages were not significantly different, unlike those of Il6 and Il12-p40. In Numb-deficient macrophages, the Tnfα mRNAs were degraded at faster rate, compared to those in control macrophages. Activation of p38 MAPK and NF-κΒ p65 were compromised in activated Numb deficient macrophages. Numb was found to interact with the E3 ubiquitin ligase, Itch, which reportedly regulates p38 MAPK. In addition, blocking the Notch signaling pathway in activated, Numb-deficient macrophages did not further reduce TNFα levels, suggesting a Notch-independent role for Numb. A proteomics approach revealed a novel function for Numb in regulating complex signaling cascades downstream of TLRs, partially involving Akt/NF-κB p65/p38 MAPK in macrophages.

Upregulations of glucocorticoid-induced leucine zipper by hypoxia and glucocorticoid inhibit proinflammatory cytokines under hypoxic conditions in macrophages.

Hypoxia and inflammation often develop concurrently in numerous diseases, and the influence of hypoxia on natural evolution of inflammatory responses is widely accepted. Glucocorticoid-induced leucine zipper (GILZ) is thought to be an important mediator of anti-inflammatory and immune-suppressive actions of glucocorticoid (GC). However, whether GILZ is involved in hypoxic response is still unclear. In this study, we investigated the effects of hypoxic exposure and/or the administration of dexamethasone (Dex), a synthetic GC on GILZ expression both in vitro and in vivo, and further explored the relationship between GILZ and proinflammatory cytokines IL-1ß, IL-6, and TNF-α under normoxic and hypoxic conditions. We found that hypoxia not only remarkably upregulated the expression of GILZ, but also significantly enhanced Dex-induced expression of GILZ in macrophages and the spleen of rats. ERK activity is found involved in the upregulation of GILZ induced by hypoxia. Inhibiting the expression of GILZ in RAW264.7 cells using specific GILZ small interfering RNA led to a significant increase in mRNA production and protein secretion of IL-1ß and IL-6 in hypoxia and abrogated the inhibitory effect of Dex on expression of IL-1ß and IL-6 in hypoxia. We also found that adrenal hormones played pivotal roles in upregulation of GILZ expression in vivo. Altogether, data presented in this study suggest that GILZ has an important role not only in adjusting adaptive responses to hypoxia by negatively regulating the activation of macrophages and the expression of proinflammatory cytokines, but also in mediating the anti-inflammatory action of GC under hypoxic conditions.

Monokine induced by interferon-gamma (MIG/CXCL9) is derived from both donor and recipient sources during rejection of class II major histocompatibility complex disparate skin allografts.

Chemokines, including monokine induced by interferon-gamma (Mig/CXCL9), are produced both in allografts and during the direct T-cell infiltration that mediates graft rejection. Neither the specific production nor contribution of allograft donor versus recipient Mig in allograft rejection is currently known. C57BL/6 mice with a targeted deletion in the Mig gene were used as both skin allograft donors and recipients in a class II major histocompatibility complex-mismatched graft model to test the requirement for donor- versus recipient-derived Mig for acute rejection. B6.Mig(-/-) allografts had a 10-day prolonged survival in B6.H-2(bm12) recipients when compared with wild-type C57BL/6 allograft donors, and B6.H-2(bm12) skin allografts had a 5-day prolonged survival in B6.Mig(-/-) versus wild-type recipients. Transplantation of B6.Mig(-/-) skin grafts onto B6.H-2(bm12).Mig(-/-) recipients resulted in further prolonged allograft survival with more than 30% of the grafts surviving longer than 60 days. Prolonged allograft survival was also associated with delayed cellular infiltration into grafts but not with altered T-cell proliferative responses to donor stimulators. Immunohistochemical staining of allograft sections indicated that Mig is produced by both donor- and recipient-derived sources, but Mig from each of these sources appeared in different areas of the allograft tissue. These results therefore demonstrate the synergy of donor- and recipient-derived Mig in promoting T-cell infiltration into allografts.

Ecstasy (3,4-methylenedioxymethamphetamine) limits murine gammaherpesvirus-68 induced monokine expression.

While Ecstasy (3,4-methylenedioxymethamphetamine, MDMA) has been shown to modulate immune responses, no studies have addressed drug-induced alterations to viral infection. In this study, bone marrow-derived macrophages were exposed to MDMA, then infected with murine gammaherpesvirus-68, and the expression of monokines assessed. MDMA-induced reductions in virus-stimulated monokine mRNA expression were observed in a dose-dependent manner. In particular, IL-6 mRNA expression and secretion was significantly decreased in gammaherpesvirus-infected macrophages exposed to MDMA. Concentrations of MDMA capable of reducing monokine production did not induce significant cell death and allowed normal viral gene expression. These studies represent the first to demonstrate the ability of this drug of abuse to alter a viral-induced macrophage response.

Sulfated glycosphingolipid as mediator of phagocytosis: SM4s enhances apoptotic cell clearance and modulates macrophage activity.

Sulfoglycolipids are present on the surface of a variety of cells. The sulfatide SM4s is increased in lung, renal, and colon cancer and is associated with an adverse prognosis, possibly due to a low immunoreactivity of the tumor. As macrophages significantly contribute to the inflammatory infiltrate in malignancies, we postulated that SM4s may modulate macrophage function. We have investigated the effect of SM4s on the uptake of apoptotic tumor cells, macrophage cytokine profile, and receptor expression. Using flow cytometry and microscopic analyses, we found that coating apoptotic murine carcinoma cells from the colon and kidney with SM4s promoted their phagocytosis by murine macrophages up to 3-fold ex vivo and in vivo. This increased capacity was specifically inhibited by preincubation of macrophages with oxidized or acetylated low density lipoprotein and maleylated albumin, indicating involvement of scavenger receptors in this interaction. The uptake of SM4s-coated apoptotic cells significantly enhanced macrophage production of TGF-beta1, expression of P-selectin, and secretion of IL-6. These data suggest that SM4s within tumors may promote apoptotic cell removal and alter the phenotype of tumor-associated macrophages.

Mycolactone-mediated inhibition of tumor necrosis factor production by macrophages infected with Mycobacterium ulcerans has implications for the control of infection.

The pathogenicity of Mycobacterium ulcerans, the agent of Buruli ulcer, depends on the cytotoxic exotoxin mycolactone. Little is known about the immune response to this pathogen. Following the demonstration of an intracellular growth phase in the life cycle of M. ulcerans, we investigated the production of tumor necrosis factor (TNF) induced by intramacrophage bacilli of diverse toxigenesis/virulence, as well as the biological relevance of TNF during M. ulcerans experimental infections. Our data show that murine bone marrow-derived macrophages infected with mycolactone-negative strains of M. ulcerans (nonvirulent) produce high amounts of TNF, while macrophages infected with mycolactone-positive strains of intermediate or high virulence produce intermediate or low amounts of TNF, respectively. These results are in accordance with the finding that TNF receptor P55-deficient (TNF-P55 KO) mice are not more susceptible than wild-type mice to infection by the highly virulent strains but are more susceptible to nonvirulent and intermediately virulent strains, demonstrating that TNF is required to control the proliferation of these strains in animals experimentally infected by M. ulcerans. We also show that mycolactone produced by intramacrophage M. ulcerans bacilli inhibits, in a dose-dependent manner, but does not abrogate, the production of macrophage inflammatory protein 2, which is consistent with the persistent inflammatory responses observed in experimentally infected mice.

Monocyte activation in response to polyethylene glycol hydrogels grafted with RGD and PHSRN separated by interpositional spacers of various lengths.

Polyethylene glycol (PEG) is often cited as a "stealth" polymer, capable of resisting both protein adsorption and cell adhesion. By extension, PEG would then be expected to limit the host response. Monocyte-derived macrophages play an integral role in inflammation, and thus their response to a material can potentially dictate the overall host response to a biomaterial. In the present study, monocyte responses following interaction with a photopolymerized PEG hydrogel were compared with those from standard tissue culture polystyrene (TCPS). Additionally, the effect of the spacing between RGD and PHSRN, the corresponding synergy sequence on fibronectin (FN), was evaluated using peptides with differing spacer lengths grafted to the PEG hydrogel. Monocyte adherent density on the PEG-only hydrogel was comparable with that of TCPS; however, the secretion of the proinflammatory molecules interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and granulocyte-macrophage colony stimulating factor (GM-CSF) increased dramatically following monocyte interaction with PEG-only hydrogels as compared with TCPS. The matrix metalloproteinase-2 (MMP-2) concentration was similar for all surfaces, while both the matrix metalloproteinase-9 (MMP-9) and FN concentrations were above the range of the assay for all substrates. Cell density was higher on the PHSRNG(13)RGD grafted substrate as compared with PHSRNG(6)RGD, but neither sequence increased cell density versus RGD alone. Although protein concentration did sometimes vary with different peptides, this variation was minimal in comparison with the surface effects between TCPS and the PEG-only hydrogel. This study explores the roles of PEG and FN-derived peptides on monocyte activation.

Expression and purification of bioactive high-purity mouse monokine induced by IFN-gamma in Escherichia coli.

MIG (monokine induced by IFN-gamma) is a CXC-chemokine (CXCL9). It plays important roles in regulation of immune activities, and knowledge of the protein in areas of allograft transplants, autoimmune diseases, and cancer therapy is evolving quickly. The non-tagged recombinant murine MIG (rMuMIG) is therefore required to facilitate the functional studies of this important chemokine. Here we present the use of a bacteria expression system to produce non-tagged rMuMIG. The coding sequence for MIG was cloned into the pET28a (+) vector that was transformed into Escherichia coli BL21 (DE3). Expression of rMuMIG was induced by IPTG. Bacteria inclusion bodies containing the protein were isolated and washed to remove contaminated bacteria proteins, and resolved in Urea buffer. Renaturation of the denatured protein was carried out in the defined protein refolding buffer, and the refolded protein was purified using S-Sepharose cation exchange chromatography. The final preparation of the rMuMIG was more than 99% pure as measured by capillary electrophoresis and SDS-PAGE analysis. The biological activity of rMuMIG was demonstrated in a murine spleen cell chemotaxis assay with ED50 30 ng/ml. Further experiments showed that rMuMIG could inhibit proliferation of mouse bone marrow cells in vivo.

Interleukin-1 mediates thermal injury-induced lung damage through C-Jun NH2-terminal kinase signaling.

OBJECTIVE: The molecular mechanisms of lung damage following thermal injury are not clear. The purpose of this study was to determine whether interleukin (IL)-1 mediates burn-induced inducible nitric oxide synthase (iNOS) expression, peroxynitrite production, and lung damage through c-Jun NH2-terminal kinase (JNK) signaling. DESIGN: Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Thermal injury models in the mice. INTERVENTIONS: IL-1 receptor type 1 (IL-1R1) mice, Tnfrsf1a mice, and wild-type (WT) mice were subjected to 30% total body surface area third-degree burn. The JNK inhibitor, SP600125, was given to mice to study the involvement of the JNK pathway in thermal injury-induced lung damage. WT --> WT, WT --> IL-1R1, and IL-1R1 --> WT chimeric mice were generated to determine the role of hematopoietic cells in IL-1-mediated lung damage. Neutrophils were harvested and treated in vitro with N-formyl-methionyl-leucyl-phenylalanine (fMLP). MEASUREMENTS AND MAIN RESULTS: IL-1R1 mice rather than Tnfrsf1a mice showed less thermal injury-induced lung damage. IL-1R1 mice displayed less lung JNK activity; intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), chemokine receptor 2 (CXCR2), and macrophage inflammatory protein-2 (MIP2), messenger RNA expression; myeloperoxidase activity; and neutrophil p38 mitogen-activated protein kinase (MAPK) phosphorylation after thermal injury. SP600125 significantly reduced thermal injury-induced blood dihydrorhodamine (DHR) 123 oxidation, iNOS expression, and lung permeability in WT mice but not in IL-1R1 mice. IL-1R1 --> WT chimeric mice rather than WT --> IL-1R1 chimeric mice showed less thermal injury-induced lung damage. fMLP increased reactive oxygen species (ROS) production of neutrophils in WT mice but not in IL-1R1 mice. SP600125 decreased ROS production of neutrophils in WT mice but not in IL-1R1 mice. CONCLUSIONS: Thermal injury-induced lung JNK activation; lung ICAM, VCAM, CXCR2, and MIP2 expression; and DHR 123 oxidation are IL-1 dependent. JNK inhibition decreases IL-1-mediated thermal injury-induced lung damage. Given that the IL-1 receptor is critical in thermal injury-induced p38 MAPK phosphorylation and ROS production of neutrophils, we conclude that IL-1 mediates thermal injury-induced iNOS expression and lung damage through the JNK signaling pathway.

Hyperoxia increases ventilator-induced lung injury via mitogen-activated protein kinases: a prospective, controlled animal experiment.

INTRODUCTION: Large-tidal volume (VT) mechanical ventilation and hyperoxia used in patients with acute respiratory distress syndrome can damage pulmonary epithelial cells through lung inflammation and apoptotic cell death. Hyperoxia has been shown to increase ventilator-induced lung injury, but the mechanisms regulating interaction between large VT and hyperoxia are unclear. We hypothesized that the addition of hyperoxia to large-VT ventilation would increase neutrophil infiltration by upregulation of the cytokine macrophage inflammatory protein-2 (MIP-2) and would increase apoptosis via the mitogen-activated protein kinase pathways. METHODS: C57BL/6 mice were exposed to high-VT (30 ml/kg) mechanical ventilation with room air or hyperoxia for one to five hours. RESULTS: The addition of hyperoxia to high-VT ventilation augmented lung injury, as demonstrated by increased apoptotic cell death, neutrophil migration into the lung, MIP-2 production, MIP-2 mRNA expression, increased DNA binding activity of activator protein-1, increased microvascular permeability, and c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 activation. Hyperoxia-induced augmentation of high-VT-induced lung injury was attenuated in JNK-deficient mice and in mice with pharmacologic inhibition of ERK activity by PD98059. However, only JNK-deficient mice, and not mice with ERK activity inhibition by PD98059, were protected from high-VT-induced lung injury without hyperoxia. CONCLUSION: We conclude that hyperoxia increased high-VT-induced cytokine production, neutrophil influx, and apoptotic cell death through activation of the JNK and ERK1/2 pathways.

CXC chemokine ligand 9/monokine induced by IFN-gamma production by tumor cells is critical for T cell-mediated suppression of cutaneous tumors.

The role of tumor-produced chemokines in the growth of malignancies remains poorly understood. We retrieved an in vivo growing MCA205 fibrosarcoma and isolated tumor cell clones that produce both CXCL9/monokine induced by IFN-gamma (Mig) and CXCL10/IFN-gamma-inducible protein 10 following stimulation with IFN-gamma and clones that produce IFN-gamma-inducible protein 10 but not Mig. The Mig-deficient variants grew more aggressively as cutaneous tumors in wild-type mice than the Mig-producing tumor cells. The growth of Mig-expressing, but not Mig-deficient, tumor cells was suppressed by NK and T cell activity. Transduction of Mig-negative variants to generate constitutive tumor cell production of Mig resulted in T cell-dependent rejection of the tumors and in induction of protective tumor-specific CD8(+) T cell responses to Mig-deficient tumors. The results indicate a critical role for tumor-derived Mig in T cell-mediated responses to cutaneous fibrosarcomas and suggest the loss of Mig expression as a mechanism used by tumor cells to evade these responses.

Alcohol intoxication inhibits pulmonary S100A8 and S100A9 expression in rats challenged with intratracheal lipopolysaccharide.

BACKGROUND: Alcohol is known to inhibit the recruitment of polymorphonuclear leukocytes (PMNs) into tissue sites including the lung. During infection and inflammation, recruited neutrophils (PMNs) release S100 proteins that function to promote the recruitment of additional phagocytes. METHODS AND RESULTS: This study investigated the effects of alcohol intoxication on S100 protein production in the lung in response to lipopolysaccharide (LPS). Animals were administered alcohol (5.5 g/kg) or saline 30 minutes before intratracheal challenge with LPS (100 microg/rat). Alcohol suppressed PMN recruitment into the lung following intratracheal LPS, which was associated with an inhibition of increase in S100A8 levels in both the bronchoalveolar lavage (BAL) fluid and lysates of cells recovered by BAL at 90 minutes and 4 hours post-LPS challenge. S100A8 and S100A9 mRNA expression in cells recovered by BAL was significantly up-regulated at both 90 minutes and 4 hours after the LPS challenge, and alcohol also suppressed this response. In addition, intratracheal LPS caused a transient increase in S100A8 mRNA expression in circulating leukocytes at 90 minutes after the challenge. Similarly, this LPS-induced up-regulation of S100A8 mRNA expression was inhibited in rats intoxicated with alcohol. CONCLUSION: These data show that alcohol inhibits the S100 protein response in the lung, which may serve as a mechanism underlying alcohol-induced suppression of PMN recruitment into the terminal airways during pulmonary infection.