Lipopolysaccharide-induced CXC Chemokine Research Articles

Rodent Model of Cardiopulmonary Bypass Demonstrates Systemic Inflammation and Neuromarker Changes

IntroductionThe physiologic derangements imposed by cardiopulmonary bypass (CPB) can result in complications such as postoperative delirium. We aim to validate a rodent survival model of CPB demonstrating a systemic inflammatory response and hypothesize that this contributes to post-CPB delirium. MethodsAdult Sprague–Dawley rats were randomized to three groups: 1) Sham peripheral surgical cannulation, 2) CPB followed by acute phase harvest, or 3) CPB followed by 24-h survival. CPB was carried out for 60 min before decannulation and weaning from mechanical ventilation. Physiological and biochemical endpoints were compared between groups. Gene expression analysis of hippocampal tissue was performed using quantitative RT-PCR panels and protein expression levels were confirmed with Western blot. ResultsSixteen animals underwent cannulation and were successfully decannulated without transfusion requirement or inotrope use with one procedure-related mortality. Serum acute phase proinflammatory chemokines cytokine-induced neutrophil chemoattractant 1, cytokine-induced neutrophil chemoattractant 3, fractalkine, and lipopolysaccharide-induced CXC chemokine as well as interleukin (IL)-10 were increased 1 h following CPB compared to sham (P < 0.05). Significant changes in hippocampal expression of biomarkers apolipoprotein 1, vascular epithelial growth factor A, and synapsin 1 were demonstrated following CPB. ConclusionsThis study validated a model of CPB that captures the resultant systemic inflammatory response, and identified differentially expressed proteins that may be associated with brain injury. Modulation of the CPB-induced inflammatory response may be a promising therapeutic target to attenuate post-CPB delirium, and this survival rat model of CPB with low surgical attrition will allow for more comprehensive evaluations of the short- and long-term effects of both CPB and potential therapeutic interventions.

Soft Tissue Manipulation Alters RANTES/CCL5 and IL-4 Cytokine Levels in a Rat Model of Chronic Low Back Pain.

Low back pain (LBP) is a common musculoskeletal complaint that can impede physical function and mobility. Current management often involves pain medication, but there is a need for non-pharmacological and non-invasive interventions. Soft tissue manipulation (STM), such as massage, has been shown to be effective in human subjects, but the molecular mechanisms underlying these findings are not well understood. In this paper, we evaluated potential changes in the soft tissue levels of more than thirty pro- or anti-inflammatory cytokines following instrument-assisted STM (IASTM) in rats with chronic, induced LBP using Complete Freund's Adjuvant. Our results indicate that IASTM is associated with reduced soft tissue levels of Regulated on Activation, Normal T cell Expressed and Secreted (RANTES)/Chemokine (C-C motif) ligand 5 (CCL5) and increased soft tissue levels of Interleukin (IL)-4, which are pro-inflammatory and anti-inflammatory factors, respectively, by 120 min post-treatment. IASTM was not associated with tissue-level changes in C-X-C Motif Chemokine Ligand (CXCL)-5/Lipopolysaccharide-Induced CXC Chemokine (LIX)-which is the murine homologue of IL-8, CXCL-7, Granulocyte-Macrophage-Colony Simulating Factor (GM-CSF), Intercellular Adhesion Molecule (ICAM)-1, IL1-Receptor Antagonist (IL-1ra), IL-6, Interferon-Inducible Protein (IP)-10/CXCL-10, L-selectin, Tumor Necrosis Factor (TNF)-α, or Vascular Endothelial Growth Factor (VEGF) at either 30 or 120 min post-treatment. Combined, our findings raise the possibility that IASTM may exert tissue-level effects associated with improved clinical outcomes and potentially beneficial changes in pro-/anti-inflammatory cytokines in circulation and at the tissue level.

The Mechanism of Cannabichromene and Cannabidiol Alone Versus in Combination in the Alleviation of Arthritis-Related Inflammation.

Patients suffering from arthritis have limited treatment options for nonoperative management. In search of pain relief, patients have been taking over-the-counter cannabinoids. Cannabidiol (CBD) and cannabichromene (CBC) are minor cannabinoids with reported analgesic and anti-inflammatory properties and have been implicated as potential therapeutics for arthritis-related pain. To this end, we utilized a murine model to investigate the effectiveness of and mechanism by which CBC alone, CBD alone, or CBD and CBC in combination may provide a reduction in arthritis-associated inflammation. Forty-eight mice were included in the study, which were separated into 4 groups: control group (n = 12), treatment with CBD alone (n = 12), treatment with CBC alone (n = 12), and treatment with CBD + CBC (n = 12). We induced inflammation in each mouse utilizing the collagen-induced arthritis model. At scheduled timepoints, mice were clinically assessed for weight gain, swelling, and arthritis severity. In addition, inflammation-associated serum cytokine levels were analyzed for each animal. Thirty-five of 48 mice survived the duration of the study resulting in the following group numbers: control group (n = 8), treatment with CBD alone (n = 9), treatment with CBC alone (n = 9), and treatment with CBD + CBC (n = 9). Animals treated with CBC and CBD + CBC showed significant weight gain between 3 and 5 weeks. Irrespective of treatment, regression analysis comparing all cytokine measurement and physical outcomes found a significant positive correlation between levels of 5 individual cytokines and both arthritis scores and swelling. Animals treated with CBD + CBC showed a significant decrease in swelling between 3 and 5 weeks compared with the control group. Cannabinoid treatment selectively affected the gene expression of eotaxin and lipopolysaccharide-induced CXC chemokine with combined treatment of CBC + CBD. Treatment with cannabinoids resulted in decreased clinical markers of inflammation. Further, the anti-inflammatory effect of CBC and CBD in conjunction was associated with a greater anti-inflammatory effect than either minor cannabinoid alone. Future work will elucidate the possibility of synergistic or entourage effects of minor cannabinoids used in combination for the treatment of arthritis-related pain and inflammation.

Baicalin Modulates Inflammatory Response of Macrophages Activated by LPS via Calcium-CHOP Pathway.

Studies on natural products that can alleviate the inflammatory response of macrophages caused by endotoxin (lipopolysaccharide) continue. This study investigated the anti-inflammatory activity of baicalin related to macrophage activation caused by lipopolysaccharide (LPS). Baicalin is a flavone glycoside found in plants such as Scutellaria baicalensis and Scutellaria lateriflora belonging to the genus Scutellaria. The multiplex cytokine assay (MCA), Griess reagent assay, fluo-4 calcium assay, dihydrorhodamine 123 (DHR123) assay, quantitative RT-PCR, and flow cytometry were performed using RAW 264.7 mouse macrophages. The MCA revealed that baicalin significantly decreased the production of interleukin (IL)-6, granulocyte colony-stimulating factor (G-CSF), vascular endothelial growth factor (VEGF), macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-2, and RANTES in LPS-stimulated RAW 264.7 macrophages at concentrations of 10, 25, and 50 μM. The DHR123 assay showed that baicalin significantly inhibited reactive oxygen species generation in LPS-stimulated RAW 264.7 macrophages. Flow cytometry revealed that baicalin significantly reduced the levels of phosphorylated p38 MAPK and Fas in LPS-stimulated RAW 264.7 macrophages. Baicalin also inhibited the mRNA expression levels of inflammatory genes such as Chop, Fas, Nos2, Ptgs2, Stat1, c-Jun, c-Fos, and At1a. The IC50 values of baicalin for IL-6, TNF-α, G-CSF, VEGF, interferon gamma-induced protein 10 (IP-10), leukemia inhibitory factor (LIF), lipopolysaccharide-induced CXC chemokine (LIX), MIP-1α, MIP-1β, MIP-2, RANTES, nitric oxide, intracellular calcium, and hydrogen peroxide were 591.3, 450, 1719, 27.68, 369.4, 256.6, 230.7, 856.9, 1326, 1524, 378.1, 26.76, 345.1, and 32.95 μM, respectively. Baicalin modulated the inflammatory response of macrophages activated by LPS via the calcium-CHOP pathway.

A diet rich in docosahexaenoic acid enhances reactive astrogliosis and ramified microglia morphology in apolipoprotein E epsilon 4-targeted replacement mice.

Docosahexaenoic acid (DHA) consumption reduces spatial memory impairment in mice carrying the human apolipoprotein E ε4 (APOE4) allele. The current study evaluated whether astrocyte and microglia morphology contribute to the mechanism of this result. APOE3 and APOE4 mice were fed either a DHA-enriched diet or a control diet from 4 to 12 months of age. Coronal brain sections were immunostained for GFAP, Iba1, and NeuN. Astrocytes from APOE4 mice exhibited signs of reactive astrogliosis compared to APOE3 mice. Consumption of DHA exacerbated reactive astrocyte morphology in APOE4 carriers. Microglia from APOE4-control mice exhibited characteristics of amoeboid morphology and other characteristics of ramified morphology (more processes, greater process complexity, and greater distance between neighboring microglia). DHA enhanced ramified microglia morphology in APOE4 mice. In addition, APOE4 mice fed the DHA diet had lower hippocampal concentrations of interleukin-7, lipopolysaccharide-induced CXC chemokine and monocyte chemoattractant protein 1, and higher concentration of interferon-gamma compared to APOE4-control mice. Our results indicate that a diet rich in DHA enhances reactive astrogliosis and ramified microglia morphology in APOE4 mice.

Rubi Fructus Water Extract Alleviates LPS-Stimulated Macrophage Activation via an ER Stress-Induced Calcium/CHOP Signaling Pathway.

Despite the availability of antibiotics and vaccines, many intractable infectious diseases still threaten human health across the globe. Uncontrolled infections can lead to systemic inflammatory response syndrome and the excessive production of inflammatory cytokines, known as a cytokine storm. As cytokines also play necessary and positive roles in fighting infections, it is important to identify nontoxic and anti-inflammatory natural products that can modulate cytokine production caused by infections. Rubi Fructus, the unripe fruits of Rubus coreanus Miquel, are known to possess antioxidative properties. In this study, the effect of the water extract of Rubi Fructus (RF) on the lipopolysaccharide (LPS)-induced inflammatory response in RAW 264.7 macrophages was investigated using biochemical and cell biology techniques. Our data indicated that RF inhibits p38 phosphorylation, intracellular calcium release, and the production of nitric oxide (NO), interleukin (IL)-6, monocyte chemotactic activating factor (MCP)-1, tumor necrosis factor (TNF)-α, leukemia inhibitory factor (LIF), lipopolysaccharide-induced CXC chemokine (LIX), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), macrophage colony-stimulating factor (M-CSF), macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-2, and regulated on activation, normal T cell expressed and secreted (RANTES) in LPS-treated macrophages. In addition, we observed decreasing mRNA expression of Chop, Camk2a, Stat1, Stat3, Jak2, Fas, c-Jun, c-Fos, Nos2, and Ptgs2 without cytotoxic effects. We concluded that RF demonstrated immunoregulatory activity on LPS-stimulated macrophages via an endoplasmic reticulum (ER) stress-induced calcium/CCAAT-enhancer-binding protein homologous protein (CHOP) pathway and the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway.

Neurotropin exerts neuroprotective effects after spinal cord injury by inhibiting apoptosis and modulating cytokines.

Background/objectiveSpinal cord injury (SCI) severely and irreversibly damages the central nervous system. Neurotropin (NTP), a nonprotein extract obtained from inflamed rabbit skin inoculated with vaccinia virus, is a drug that has been used for more than sixty years to alleviate neuropathic pain. It also reportedly exerts a neuroprotective role in peripheral nerves and in response to various central nervous system diseases, such as brain injury and Alzheimer disease. However, whether NTP promotes SCI recovery remains unknown. This study evaluated NTP's effects after SCI and explored its underlying mechanisms in a rat contusion model of SCI.MethodNTP was intraperitoneally administered to adult female Wistar rats subjected to contusion-induced SCI. Functional recovery was evaluated with behavioural scores and electrophysiological examinations. Tissue recovery was assessed with magnetic resonance imaging as well as histological staining with haematoxylin and eosin and Luxol Fast Blue. Neuronal survival and gliosis were observed after NeuN and glial fibrillary acidic protein immunofluorescence. Levels of apoptosis were demonstrated with TdT-mediated dUTP nick-end labeling (TUNEL) staining, Caspase-3 and B-cell lymphoma-2 (Bcl-2) Western blot, and Annexin V/propidium iodide flow cytometry. A protein antibody chip analysis was performed to evaluate the expression levels of 67 rat cytokines.ResultsNTP treatment improved the hindlimb locomotor recovery of the injured animals as well as their electrophysiological outcomes after SCI. A dosage of 50 NTP units/kg was found to optimize the efficacy of NTP. Magnetic resonance imaging revealed that lesion sizes decreased after NTP treatment. The haematoxylin and eosin and Luxol Fast Blue staining showed significant increases in the amount of spared tissue. The NeuN and glial fibrillary acidic protein immunofluorescence revealed that NTP treatment increased neuronal survival and reduced gliosis in tissue samples obtained from the lesion's epicentre. That NTP inhibited apoptosis was confirmed by the decreased number of TUNEL-positive cells, level of Caspase-3 expression, and number of Annexin V/propidium iodide–positive cells, as well as the increased level of Bcl-2 expression. The protein array analysis identified 28 differentially expressed proteins in the NTP group, and the gene ontology (GO) analysis showed that the enriched differentially expressed proteins implicate janus kinase-signal transducer and activator of transcription (JAK-STAT) signalling pathways. The expression levels of proinflammatory cytokines such as interleukin 6, thymus chemokine-1(TCK-1), and lipopolysaccharide-induced CXC chemokine (LIX) decreased after NTP treatment, whereas the levels of prorepair cytokine hepatocyte growth factor and adiponectin increased.ConclusionOur research provides evidence that NTP can improve functional outcomes and alleviate secondary injury after SCI by inhibiting apoptosis and modulating cytokines.The translational potential of this articleThe multicomponent NTP might have broad target spectra in SCI pathophysiology and halt the secondary injury cascade. As a safe drug that features sixty years of clinical use as an analgesic, translating this demonstrated efficacy of NTP to addressing SCI in human patients may potentially be accelerated.

Sphingosine 1-phosphate receptors regulate TLR4-induced CXCL5 release from astrocytes and microglia.

Sphingosine 1-phosphate receptors (S1PR) are G protein-coupled and compose a family with five subtypes, S1P1R-S1P5R. The drug Gilenya® (Novartis, Basel, Switzerland) (Fingolimod; FTY720) targets S1PRs and was the first oral therapy for patients with relapsing-remitting multiple sclerosis (MS). The phosphorylated form of FTY720 (pFTY720) binds S1PRs causing initial agonism, then subsequent receptor internalization and functional antagonism. Internalization of S1P1R attenuates sphingosine 1-phosphate (S1P)-mediated egress of lymphocytes from lymph nodes, limiting aberrant immune function in MS. pFTY720 also exerts direct actions on neurons and glial cells which express S1PRs. In this study, we investigated the regulation of pro-inflammatory chemokine release by S1PRs in enriched astrocytes and microglial cultures. Astrocytes and microglia were stimulated with lipopolysaccharide (LPS) and increases in C-X-C motif chemokine 5 (CXCL5), also known as LIX (lipopolysaccharide-induced CXC chemokine) expression were quantified. Results showed that pFTY720 attenuated LPS-induced CXCL5 (LIX) protein release from astrocytes, as did the S1P1R selective agonist, SEW2871. In addition, pFTY720 blocked messenger ribonucleic acid (mRNA) transcription of the chemokines, (i) CXCL5/LIX, (ii) C-X-C motif chemokine 10 (CXCL10) also known as interferon gamma-induced protein 10 (IP10) and (iii) chemokine (C-C motif) ligand 2 (CCL2) also known as monocyte chemoattractant protein 1 (MCP1). Interestingly, inhibition of sphingosine kinase attenuated LPS-induced increases in mRNA levels of all three chemokines, suggesting that LPS-TLR4 (Toll-like receptor 4) signalling may enhance chemokine expression via S1P-S1PR transactivation. Lastly, these observations were not limited to astrocytes since we also found that pFTY720 attenuated LPS-induced release of CXCL5 from microglia. These data highlight a role for S1PR signalling in regulating the levels of chemokines in glial cells and support the notion that pFTY720 efficacy in multiple sclerosis may involve the direct modulation of astrocytes and microglia.

Anti-inflammatory effects of oroxylin A on RAW 264.7 mouse macrophages induced with polyinosinic-polycytidylic acid.

Oroxylin A (5,7-dihydroxy-6-methoxy-2-phenylchromen-4-one; Baicalein 6-methyl ether) is an active flavonoid compound originally isolated from Scutellaria radix, which has been used to treat pulmonary infection in Korea, China, and Japan. Oroxylin A is known to possess dopamine reuptake inhibitor activity. However, the effects of oroxylin A on virus-induced macrophages has not been fully elucidated. In the present study, the anti-inflammatory effects of oroxylin A on double-stranded RNA-induced macrophages were examined. Production of nitric oxide (NO), various cytokines, as well as calcium release and the mRNA expression of signal transducer and activator of transcription 1 (STAT1) in dsRNA polyinosinic-polycytidylic acid (PIC)-induced RAW 264.7 mouse macrophages were evaluated. Oroxylin A restored the cell viability in PIC-induced RAW 264.7 mouse macrophages at concentrations of up to 50 µM. Additionally, oroxylin A significantly inhibited the production of nitric oxide (NO), interleukin (IL)-1α, IL-1β, IL-6, IL-10, interferon gamma-induced protein 10, granulocyte-colony stimulating factor (CSF), granulocyte macrophage-CSF, leukemia inhibitory factor (IL-6 class cytokine), lipopolysaccharide-induced CXC chemokine (LIX), monocyte chemoattractant protein 1, macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-2, Regulated on Activation, Normal T Expressed and Secreted, tumor necrosis factor-α, and vascular endothelial growth factor as well as calcium release and the mRNA expression of STAT1 in PIC-induced RAW 264.7 cells (P<0.05). Thus, the present results suggest that oroxylin A has anti-inflammatory properties, associated with its inhibition of NO, cytokines, chemokines and growth factors in PIC-induced macrophages via the calcium-STAT pathway.

Lipopolysaccharide Upregulates the Expression of CINC-3 and LIX in Primary NG2 Cells.

Numerous NG2 cells, also called oligodendrocyte progenitor cells (OPCs), exist ubiquitously in the gray and white matter in the adult central nervous system (CNS). Although NG2 cells could become active by upregulation of NG2 expression and hypertrophy or extension of their processes under various neuropathological conditions, their actual role in the brain remains to be illustrated. In view of the fact that the synergy of cytokine and chemokine networks plays an important role in CNS inflammation and immunity, we have assumed that the NG2 cells might take part in brain inflammation and immunity by making a contribution to the pool of cytokines or chemokines. In the current study, NG2-expressing OPCs were prepared from cerebral hemispheres of postnatal day 0 or 1 Sprague-Dawley rats. Our results showed that NG2-expressing OPCs, verified by immunohistological staining of anti-NG2 antibody and anti-platelet-derived growth factor receptor alpha (PDGFRα) antibody, presented binding affinity to lipopolysaccharide (LPS), a commonly used stimulator in a neuroinflammatory model. Using cytokine antibody array, QPCR and ELISA, we have further shown that LPS could upregulate the expression of cytokine induced neutrophil chemoattractant-3 (CINC-3) and LPS induced CXC chemokine (LIX) in primary NG2-expressing OPCs, without the alteration in cell number of NG2-expressing OPCs. In addition, the cells bearing the receptor for these two cytokines included microglia and OPCs. Taken together, our results suggest that NG2-expressing OPCs could response to LPS and may take part in neuroinflammatory process, through secreting cytokines and chemokines to exert an effect on target cells (OPCs and microglia).

Primed Mycobacterial Uveitis (PMU): Histologic and Cytokine Characterization of a Model of Uveitis in Rats.

The purpose of this study was to compare the histologic features and cytokine profiles of experimental autoimmune uveitis (EAU) and a primed mycobacterial uveitis (PMU) model in rats. In Lewis rats, EAU was induced by immunization with interphotoreceptor binding protein peptide, and PMU was induced by immunization with a killed mycobacterial extract followed by intravitreal injection of the same extract. Clinical course, histology, and the cytokine profiles of the aqueous and vitreous were compared using multiplex bead fluorescence immunoassays. Primed mycobacterial uveitis generates inflammation 2 days after intravitreal injection and resolves spontaneously 14 days later. CD68+ lymphocytes are the predominant infiltrating cells and are found in the anterior chamber, surrounding the ciliary body and in the vitreous. In contrast to EAU, no choroidal infiltration or retinal destruction is noted. At the day of peak inflammation, C-X-C motif ligand 10 (CXCL10), IL-1β, IL-18, and leptin were induced in the aqueous of both models. Interleukin-6 was induced 2-fold in the aqueous of PMU but not EAU. Cytokines elevated in the aqueous of EAU exclusively include regulated on activation, normal T cell expressed and secreted (RANTES), lipopolysaccharide-induced CXC chemokine (LIX), growth-related oncogene/keratinocyte chemokine (GRO/KC), VEGF, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), and IL-17A. In the vitreous, CXCL10, GRO/KC, RANTES, and MIP-1α were elevated in both models. Interleukin-17A and IL-18 were elevated exclusively in EAU. Primed mycobacterial uveitis generates an acute anterior and intermediate uveitis without retinal involvement. Primed mycobacterial uveitis has a distinct proinflammatory cytokine profile compared with EAU, suggesting PMU is a good complementary model for study of immune-mediated uveitis. CXCL10, a proinflammatory cytokine, was increased in the aqueous and vitreous of both models and may be a viable therapeutic target.

Stimulation of nuclear receptor peroxisome proliferator-activated receptor-γ limits NF-κB-dependent inflammation in mouse cystic fibrosis biliary epithelium.

Cystic fibrosis-associated liver disease is a chronic cholangiopathy that negatively affects the quality of life of cystic fibrosis patients. In addition to reducing biliary chloride and bicarbonate secretion, up-regulation of toll-like receptor 4/nuclear factor kappa light-chain-enhancer of activated B cells (NF-κB)-dependent immune mechanisms plays a major role in the pathogenesis of cystic fibrosis-associated liver disease and may represent a therapeutic target. Nuclear receptors are transcription factors that regulate several intracellular functions. Some nuclear receptors, including peroxisome proliferator-activated receptor-γ (PPAR-γ), may counterregulate inflammation in a tissue-specific manner. In this study, we explored the anti-inflammatory effect of PPAR-γ stimulation in vivo in cystic fibrosis transmembrane conductance regulator (Cftr) knockout mice exposed to dextran sodium sulfate and in vitro in primary cholangiocytes isolated from wild-type and from Cftr-knockout mice exposed to lipopolysaccharide. We found that in CFTR-defective biliary epithelium expression of PPAR-γ is increased but that this does not result in increased receptor activity because the availability of bioactive ligands is reduced. Exogenous administration of synthetic agonists of PPAR-γ (pioglitazone and rosiglitazone) up-regulates PPAR-γ-dependent genes, while inhibiting the activation of NF-κB and the secretion of proinflammatory cytokines (lipopolysaccharide-induced CXC chemokine, monocyte chemotactic protein-1, macrophage inflammatory protein-2, granulocyte colony-stimulating factor, keratinocyte chemoattractant) in response to lipopolysaccharide. PPAR-γ agonists modulate NF-κB-dependent inflammation by up-regulating nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha, a negative regulator of NF-κB. Stimulation of PPAR-γ in vivo (rosiglitazone) significantly attenuates biliary damage and inflammation in Cftr-knockout mice exposed to a dextran sodium sulfate-induced portal endotoxemia. These studies unravel a novel function of PPAR-γ in controlling biliary epithelium inflammation and suggest that impaired activation of PPAR-γ contributes to the chronic inflammatory state of CFTR-defective cholangiocytes.

Lung Angiogenesis Requires CD4(+) Forkhead Homeobox Protein-3(+) Regulatory T Cells.

Angiogenesis in ischemic organs is modulated by immune cells. Systemic neovascularization of the ischemic lung requires macrophages, with chemokines playing a central role in new vessel growth. Because regulatory T (Treg) cells modulate tumor-induced neovascularization, we questioned whether this CD4(+) lymphocyte subset impacts blood vessel growth during ischemia. In a model of left lung ischemia, an increase in CD4(+) CD25(+) forkhead homeobox protein-3 (Foxp3)(+) cells was observed 3-5 days after the onset of ischemia in wild-type C57Bl/6 mice. Using transgenic mice where Foxp3(+) Treg cells can be depleted with diphtheria toxin (DT; Foxp3(DTR)), we unexpectedly found that Foxp3(+) Treg depletion led to markedly reduced lung angiogenesis (90% reduction from Foxp3(gfp) controls). Adoptive transfer studies using CD4(+) CD25(+) splenocytes from congenic CD45.1 mice into Foxp3(+) Treg-depleted mice showed an almost complete recovery of the angiogenic phenotype (80% of Foxp3(gfp) controls). A survey of lung gene expression of angiogenic (lipopolysaccharide-induced CXC chemokine [LIX], IL-6, IL-17) and angiostatic (IFN-γ, transforming growth factor-β, IL-10) cytokines showed Treg-dependent differences only in LIX (CXCL5) and IL-6. Protein confirmation demonstrated a significant reduction in LIX in Treg-deficient mice compared with controls 5 days after the onset of ischemia. Phenotyping other inflammatory cells in the lung by multicolor flow cytometry demonstrated a significantly reduced number of macrophages (major histocombatibility complex class II [MHCII](int), CD11C(+)) in Treg-deficient lungs compared with Treg-sufficient lungs. Treg cells are essential for maximal systemic angiogenesis after pulmonary ischemia. One likely mechanism responsible for the decrease in angiogenesis in Treg-depleted mice was the decline in the essential CXC chemokine, LIX.

Inflammatory response mechanisms exacerbating hypoxemia in coexistent pulmonary fibrosis and sleep apnea.

Mediators of inflammation, oxidative stress, and chemoattractants drive the hypoxemic mechanisms that accompany pulmonary fibrosis. Patients with idiopathic pulmonary fibrosis commonly have obstructive sleep apnea, which potentiates the hypoxic stimuli for oxidative stress, culminating in systemic inflammation and generalized vascular endothelial damage. Comorbidities like pulmonary hypertension, obesity, gastroesophageal reflux disease, and hypoxic pulmonary vasoconstriction contribute to chronic hypoxemia leading to the release of proinflammatory cytokines that may propagate clinical deterioration and alter the pulmonary fibrotic pathway. Tissue inhibitor of metalloproteinase (TIMP-1), interleukin- (IL-) 1α, cytokine-induced neutrophil chemoattractant (CINC-1, CINC-2α/β), lipopolysaccharide induced CXC chemokine (LIX), monokine induced by gamma interferon (MIG-1), macrophage inflammatory protein- (MIP-) 1α, MIP-3α, and nuclear factor- (NF-) κB appear to mediate disease progression. Adipocytes may induce hypoxia inducible factor (HIF) 1α production; GERD is associated with increased levels of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and tumor necrosis factor alpha (TNF-α); pulmonary artery myocytes often exhibit increased cytosolic free Ca2+. Protein kinase C (PKC) mediated upregulation of TNF-α and IL-1β also occurs in the pulmonary arteries. Increased understanding of the inflammatory mechanisms driving hypoxemia in pulmonary fibrosis and obstructive sleep apnea may potentiate the identification of appropriate therapeutic targets for developing effective therapies.

Novel mechanisms of sildenafil in pulmonary hypertension involving cytokines/chemokines, MAP kinases and Akt.

Pulmonary arterial hypertension (PH) is associated with high mortality due to right ventricular failure and hypoxia, therefore to understand the mechanism by which pulmonary vascular remodeling initiates these processes is very important. We used a well-characterized monocrotaline (MCT)-induced rat PH model, and analyzed lung morphology, expression of cytokines, mitogen-activated protein kinase (MAPK) phosphorylation, and phosphatidylinositol 3-kinase-Akt (PI-3k-Akt) pathway and nuclear factor (NF)-κB activation in order to elucidate the mechanisms by which sildenafil's protective effect in PH is exerted. Besides its protective effect on lung morphology, sildenafil suppressed multiple cytokines involved in neutrophil and mononuclear cells recruitment including cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-2α/β, tissue inhibitor of metalloproteinase (TIMP)-1, interleukin (IL)-1α, lipopolysaccharide induced CXC chemokine (LIX), monokine induced by gamma interferon (MIG), macrophage inflammatory protein (MIP)-1α, and MIP-3α. NF-κB activation and phosphorylation were also attenuated by sildenafil. Furthermore, sildenafil reduced extracellular signal-regulated kinase (ERK)1/2 and p38 MAPK activation while enhanced activation of the cytoprotective Akt pathway in PH. These data suggest a beneficial effect of sildenafil on inflammatory and kinase signaling mechanisms that substantially contribute to its protective effects, and may have potential implications in designing future therapeutic strategies in the treatment of pulmonary hypertension.

Immunomodulatory Effects of Lycii Radicis Cortex Water Extract on Lipopolysaccharide-Activated RAW 264.7 Mouse Macrophages

The root bark of Lycium chinense Miller (Solanaceae), also known as Lycii radicis cortex, is one of famous plant-originated drugs in Korean medicine owing to its anti-tussive, anti-asthmatic, and antipyretic properties. In this study, the effect of Lycii radicis cortex water extract (LRC; 10 - 200μg/mL) on inflammatory mediators from lipopolysaccharide (LPS)-activated RAW 264.7 mouse macrophages was inspected. After 24 h incubation with LRC, cell viability, nitric oxide (NO), and various cytokines from RAW 264.7 were measured. LRC increased the cell viability of RAW 264.7 at concentrations of up to 200 μg/mL. LRC significantly inhibited the production of NO, granulocyte colony-stimulating factor (G-CSF), tumor necrosis factor-α (TNF-α), platelet derived growth factor-BB (PDGF-BB), interleukin (IL)-2, and IL-10 in LPS-activated RAW 264.7 (P &lt; 0.05). As well, LRC diminished the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) and lipopolysaccharide-induced CXC chemokine (LIX). The current results suggest that LRC has immunomodulatory property to alleviate excessive immune reactions during the activation of macrophages by LPS.

Oral Caffeine During Voluntary Exercise Markedly Inhibits Skin Carcinogenesis and Decreases Inflammatory Cytokines in UVB-Treated Mice

Ultraviolet B (UVB)-pretreated SKH-1 mice were treated with water, caffeine (0.1 mg/ml), voluntary running wheel exercise (RW) or caffeine together with RW for 14 wk. Treatment of the mice with caffeine, RW, or caffeine plus RW decreased skin tumors per mouse by 27%, 35%, and 62%, respectively, and the tumor volume per mouse was decreased by 61%, 70%, and 85%, respectively. In mechanistic studies, mice were treated with water, caffeine, RW, or caffeine plus RW for 2 wk prior to a single irradiation with UVB. Caffeine plus RW increased RW activity by 22% when compared with RW alone. Caffeine ingestion was not significantly different between groups. Treatment of mice with caffeine plus RW for 2 wk decreased the weight of the parametrial fat pads and stimulated the formation of UVB-induced apoptosis to a greater extent than treatment with caffeine or RW alone. An antibody array revealed that caffeine plus RW administered to mice fed a high-fat diet and irradiated with UVB decreased the epidermal levels of lipopolysaccharide-induced CXC chemokine, soluble TNF alpha receptor-1, and macrophage inflammatory protein-1γ. Overall, caffeine during RW exerts a stronger effect than either treatment alone for decreasing tissue fat, increasing UVB-induced apoptosis, lowering the levels of cytokines associated with inflammation and for inhibiting UVB-induced carcinogenesis.

Effect of IFN-α on KC and LIX expression: Role of STAT1 and its effect on neutrophil recruitment to the spleen after lipopolysaccharide stimulation

The spleen is a crucial lymphoid organ. It is involved in the recruitment of various immunocytes to their correct locations using specific chemokines, but little is known concerning the role of type-I interferon (IFN) in the regulation of chemokines. In this study, we first used protein microarrays to assess the expression of keratinocyte-derived chemokine (KC) and lipopolysaccharide-induced CXC chemokine (LIX) in murine spleens. Both expressions were smoothly enhanced by IFN-α pretreatment after LPS injection. Then, we focused on the IFN-α regulation of KC, LIX, and their target cells, neutrophils, using an IFN-α neutralizing antibody and fludarabine (specific signal transducers and activators of transcription 1 – STAT1 inhibitor). Next, LPS was found to attenuate the production of KC and LIX in spleen. Even the elevated production of chemokines caused by exogenous IFN-α was found to be attenuated by fludarabine pretreatment. We later determined that the marginal zone and red pulp are the main sites of KC and LIX production. Last, we determined that the number of neutrophils was slightly increased by IFN-α treatment and diminished by IFN-α neutralization or fludarabine treatment. Further, the elevated neutrophils due to exogenous IFN-α were partially reversed by fludarabine pretreatment. In this way, these results indicate that IFN-α facilitates KC and LIX expression in mouse spleens after an LPS challenge. This effect was found to be mainly dependent upon the activation of STAT1, it may be involved in the recruitment of neutrophils to the spleen for the clearance of pathogens.

Characterization of three newly established rat sarcoma cell clones

Establishment of new animal models using selected cell lines with different behaviour is very important for cancer investigations. In this study, we describe three morphologically distinct rat sarcoma clones-C4, C7 and D6-isolated from the R5-28 cell line. Cells of all clones expressed vimentin, fibronectin, laminin, collagen IV and matrix metalloproteinases 2 and 9. However, desmin, cytokeratins 8 and 18, ZO-1 and desmoplakins I and II were not detected. Significant proliferative capacity was documented by proliferating cell nuclear antigen expression and BrdU positivity. Karyotype of the C4, C7 and D6 cells greatly differed from diploid chromosome number of normal rat somatic cells. High expression of three cytokines-monocyte chemoattractant protein 1, tissue inhibitor of metalloproteinases 1 and vascular endothelial growth factor-was observed in all three clones. However, they varied in concentration of chemokines associated with neutrophil migration and activation-cytokine induced neutrophil chemoattractant 2 and lipopolysaccharide induced CXC chemokine. The C4 clone showed spontaneous tumour regression in vivo that was associated with significant changes in lymphocyte subpopulations.

Study of gastric fluid induced cytokine and chemokine expression in airway smooth muscle cells and airway remodeling

Asthma is a chronic airway inflammatory disease. Chronic aspiration by gastric fluid in gastroesophageal reflux disease (GERD) is considered a primary inflammatory factor exacerbating or predisposing patients to asthma. Airway smooth muscle cells (SMCs) are considered an important component in airway remodeling. To investigate the role of gastric fluid in airway SMC inflammation and airway remodeling, we examined gastric fluid-induced cytokine and chemokine profiles, airway SMC migration and matrix metalloproteinase expression in rat primary rat airway SMCs. The T helper cell type 2 (Th2) cytokines interleukin 4, interleukin 6 and tumor necrosis factor 2 (TNF-α) and the chemokines, lipopolysaccharide-induced CXC chemokine (LIX/CXCL5), cytokine-induced neutrophil chemoattractant 2 (CINC-2), CINC-3, fractalkine, ciliary neurotrophic factor (CNTF), and vascular endothelial growth factor were induced by gastric fluid in primary cultured rat airway SMCs. Migration of rat airway SMCs was enhanced by gastric fluid and conditioned medium. The migration of rat airway SMCs enhanced by gastric fluid was associated with actin polymerization and activation of focal adhesion kinase. Matrix metalloproteinase 2 expressions in airway SMCs was enhanced by gastric fluid and conditioned medium. The results suggest potential mechanisms by which gastric fluid aspiration might influence SMC-mediated airway remodeling.