Injection Of Thioglycollate Research Articles

Exploring the complex immunomodulatory effects and gut defense via oral administration of Astragali radix water extract to normal mice

BackgroundAstragali radix (AR) is one of the most widely used traditional Chinese herbal medicines. It exhibits diverse biological activities, including immunomodulatory and anti-inflammatory properties; however, some of its activities have only been demonstrated in vitro.ObjectiveTo examine the effects of orally administered AR extract on immune cells and the intestine under physiological conditions, which bridges the gap between previously observed in vitro outcomes and in vivo results.MethodsAR extract was prepared by hot water extraction. Three separate animal experiments were conducted to isolate macrophages, splenocytes, and the small intestine epithelium. For the macrophage preparation experiment, an intraperitoneal injection of sterile thioglycolate was administered. The mice received oral AR extract at doses of 0.1, 0.5, or 2.5 g/kg for ten days. At the end of each experiment, cells or tissues were isolated. A portion of macrophages and splenocytes were analyzed for the phenotypic changes. The remaining cells were cultured and stimulated with lipopolysaccharide (LPS) or mitogen ex vivo to assess activation status, proliferation, and cytokine production. Samples of the intestine were subjected to real-time RT-PCR.ResultsPeritoneal macrophages from AR-treated mice exhibited increased expression of scavenger receptors, including SRA and CD36. Stimulation of these macrophages ex vivo with LPS selectively modulated the inflammatory response, including reduced expression of the costimulatory molecules CD40 and CD86, which are important for T cell responses, without affecting TNF-α and IL-6 production. Splenocytes from AR-treated mice exhibited a dose-dependent increase in CD4 and CD8 T cells; however, stimulation with mitogen decreased T cell proliferation and reduced IFN-γ production, which is essential for macrophage activation. An analysis of the small intestinal epithelium revealed an attenuated antimicrobial response, including reduced IgA content in the lumen and decreased expression of mucin-2 and polymeric Ig receptor genes.ConclusionThe response of immune cells following oral treatment with AR extract did not replicate the previously documented in vitro findings. Immune cells and intestinal epithelium from mice administered oral AR extract exhibited a selective anti-inflammatory phenotype. The overall findings indicate that the systemic effects after oral administration of AR extract include reduced sensitivity to inflammatory insults.

PKC-α is involved in the signaling of phagocytosis induced by two snake venom secretory PLA2S in macrophages

Phagocytosis, an essential process for host defense, requires the coordination of a variety of signaling reactions. MT-II, an enzymatically inactive Lys49 phospholipase A2 (PLA2) homolog, and MT-III, a catalytically-active Asp49 PLA2, are known to activate phagocytosis in macrophages. In this study, the signaling pathways mediating phagocytosis, focusing on protein kinases, were investigated. Macrophages from male Swiss mice peritoneum were obtained 96 h after intraperitoneal thioglycolate injection. Phagocytosis was evaluated using non-opsonized zymosan particles in the presence or absence of specific inhibitors, as well as PKC and PKC-α localization by confocal microscopy. Moreover, protein kinase C (PKC) activity was assessed by γP32 ATP in macrophages stimulated by both PLA2s. Data showed that both sPLA2s increased phagocytosis. Cytochalasin D, staurosporine/H7, wortmannin, and herbimycin, inhibitors of actin polymerization, PKC, phosphoinositide 3-kinase (PI3K), and protein tyrosine kinase (PTK), respectively, significantly reduced phagocytosis induced by both PLA2s. PKC activity was increased in macrophages stimulated by both PLA2s. Actin polymerization and talin were evidenced by immunofluorescence and talin was recruited 5 min after both PLA2s stimulation. PKC and PKC-α localization within the cell were increased after 60 min of MT-II and MT-III stimulation. These data suggest that the effect of both PLA2s depends on actin cytoskeleton rearrangements and the activation of PKC, PI3K, and PTK signaling events required for phagocytosis.

C5aR2 Deficiency Lessens C5aR1 Distribution and Expression in Neutrophils and Macrophages.

As another receptor for complement activation product C5a, C5aR2 has been paid much attention these years. Although controversial and complex, its specific signals or roles in modulating the classic receptor C5aR1 have been investigated and gradually revealed. The hypothesis of the heterodimer of C5aR1 and C5aR2 has also been suggested and observed under extremely high C5a concentrations. In this article, we tried to investigate whether C5aR2 would affect C5aR1 expression under normal or inflammatory conditions in WT and C5ar2 -/- mice of C57BL/6 background. We focused on the innate immune cells-neutrophils and macrophages. The mRNA levels of C5ar1 in normal kidney, liver, and the mRNA or protein levels of naïve-bone marrow and peripheral blood leukocytes and peritoneal Mφs were comparable between WT and C5ar2 -/- mice, indicating the technique of C5aR2 knockout did not affect the transcription of its neighboring gene C5aR1. However, the mean fluorescence intensity of surface C5aR1 on naïve circulating C5ar2 -/- neutrophils detected by FACS was reduced, which might be due to the reduced internalization of C5aR1 on C5ar2 -/- neutrophils. In the peritonitis model induced by i.p. injection of thioglycollate, more neutrophils were raised after 10 hr in C5ar2 -/- peritoneal cavity, indicating the antagonism of C5aR2 on C5aR1 signal in neutrophil chemotaxis. After 3 days of thioglycollate injection, the mainly infiltrating macrophages were comparable between WT and C5ar2 -/- mice, but the C5ar1 mRNA and surface or total C5aR1 protein expression were both reduced in C5ar2 -/- macrophages, combined with our previous study of reduced chemokines and cytokines expression in C5ar2 -/- peritoneal macrophages, indicating that C5aR2 in macrophages may cooperate with C5aR1 inflammatory signals. Our article found C5aR2 deficiency lessened C5aR1 distribution and expression in neutrophils and macrophages with different functions, indicating C5aR2 might function differently in different cells.

ATP Consumption Is Coupled with Endocytosis in Exudated Neutrophils.

Neutrophil energy metabolism during phagocytosis has been previously reported, and adenosine triphosphate (ATP) plays a crucial role in endocytosis. Neutrophils are prepared by intraperitoneal injection of thioglycolate for 4 h. We previously reported a system established for measuring particulate matter endocytosis by neutrophils using flow cytometry. In this study, we utilized this system to investigate the relationship between endocytosis and energy consumption in neutrophils. A dynamin inhibitor suppressed ATP consumption triggered by neutrophil endocytosis. In the presence of exogenous ATP, neutrophils behave differently during endocytosis depending on ATP concentration. The inhibition of ATP synthase and nicotinamide adenine dinucleotide phosphate oxidase but not phosphatidylinositol-3 kinase suppresses neutrophil endocytosis. The nuclear factor kappa B was activated during endocytosis and inhibited by I kappa B kinase (IKK) inhibitors. Notably, IKK inhibitors restored endocytosis-triggered ATP consumption. Furthermore, data from the NLR family pyrin domain containing three knockout mice suggest that inflammasome activation is not involved in neutrophil endocytosis or concomitant ATP consumption. To summarize, these molecular events occur via endocytosis, which is closely related to ATP-centered energy metabolism.

Recombinant Interleukin-19 Suppresses the Formation and Progression of Experimental Abdominal Aortic Aneurysms.

BackgroundInterleukin‐19 is an immunosuppressive cytokine produced by immune and nonimmune cells, but its role in abdominal aortic aneurysm (AAA) pathogenesis is not known. This study aimed to investigate interleukin‐19 expression in, and influences on, the formation and progression of experimental AAAs.Methods and ResultsHuman specimens were obtained at aneurysm repair surgery or from transplant donors. Experimental AAAs were created in 10‐ to 12‐week‐old male mice via intra‐aortic elastase infusion. Influence and potential mechanisms of interleukin‐19 treatment on AAAs were assessed via ultrasonography, histopathology, flow cytometry, and gene expression profiling. Immunohistochemistry revealed augmented interleukin‐19 expression in both human and experimental AAAs. In mice, interleukin‐19 treatment before AAA initiation via elastase infusion suppressed aneurysm formation and progression, with attenuation of medial elastin degradation, smooth‐muscle depletion, leukocyte infiltration, neoangiogenesis, and matrix metalloproteinase 2 and 9 expression. Initiation of interleukin‐19 treatment after AAA creation limited further aneurysmal degeneration. In additional experiments, interleukin‐19 treatment inhibited murine macrophage recruitment following intraperitoneal thioglycolate injection. In classically or alternatively activated macrophages in vitro, interleukin‐19 downregulated mRNA expression of inducible nitric oxide synthase, chemokine C‐C motif ligand 2, and metalloproteinases 2 and 9 without apparent effect on cytokine‐expressing helper or cytotoxic T‐cell differentiation, nor regulatory T cellularity, in the aneurysmal aorta or spleen of interleukin‐19–treated mice. Interleukin‐19 also suppressed AAAs created via angiotensin II infusion in hyperlipidemic mice.ConclusionsBased on human evidence and experimental modeling observations, interleukin‐19 may influence the development and progression of AAAs.

CD137-CD137L Signaling Affects Angiogenesis by Mediating Phenotypic Conversion of Macrophages.

Angiogenesis in atherosclerotic plaque is an important factor causing plaque hemorrhage, vulnerability, and rupture, and different phenotypes of macrophages have different effects on angiogenesis. Our previous study has demonstrated CD137-CD137L signaling, a pair of inflammatory costimulatory molecules, can promote angiogenesis in atherosclerotic plaque. Therefore, we aimed to investigate whether this signaling could affect angiogenesis by regulating phenotypic transition of macrophages. Male mouse primary peritoneal macrophages were extracted by intraperitoneal injection of thioglycollate, and then flow cytometry was used to detect the expression of CD137. Flow cytometry, Western blotting, quantitative real-time PCR, and enzyme-linked immunosorbent assay (ELISA) were used to assess the phenotypic changes of macrophages after different treatment. Mouse brain microvascular endothelial cells (bEnd.3) were cocultured with macrophages, and tube formation was assessed to examine angiogenesis. We found that the number of junctions and branches of bEnd.3 were increased when CD137-CD137L signaling was activated, while such number was further increased when bEnd.3 were cocultured with macrophages. Flow cytometry showed that CD137 was expressed on almost all primary peritoneal macrophages. The expression of CD86 was decreased in the agonist CD137L group and increased in the agonist CD137L + inhibitory anti-CD137 antibody group after adding the CD137 inhibitor. The expression of CD206 in each group exhibited opposite trend compared with CD86. Moreover, the expression of inducible nitric oxide synthase at the mRNA, and protein levels were decreased after stimulating CD137-CD137L signaling, and such downward trend was reversed when CD137-CD137L signaling was inhibited. Furthermore, the expression of arginase-1 was opposite to that of inducible nitric oxide synthase. Enzyme-linked immunosorbent assay indicated that the content of interleukin-12 (IL-12) in the supernatant of macrophages in the agonist CD137L group was lower than that in the control group, and its content in the inhibited group was higher than that in the activated group. The change of interleukin-10 (IL-10) content in macrophage supernatant was opposite to that of IL-12. When AKT serine/threonine kinase 1 (Akt1) inhibitor was used to inhibit the phenotypic transformation of macrophages induced by CD137-CD137L, the number of junctions and branches formed by bEnd.3 was decreased compared with the coculture group. These results indicated that CD137-CD137L signaling could promote angiogenesis by regulating phenotypic transition of macrophages of male mice.

ACE overexpression in myeloid cells increases oxidative metabolism and cellular ATP

ACE overexpression in myeloid cells increases oxidative metabolism and cellular ATP

Angiotensin‐converting enzyme (ACE) up‐regulates metabolic function in myeloid‐derived cells

While most studies of ACE focus on the blood pressure effects of angiotensin II, it is now recognized that ACE also plays an important role in both the innate and adaptive immune responses1. To study the mechanism of this effect, we made two mouse strains: ACE 10/10, which over‐express ACE in macrophages, and NeuACE, which over‐express ACE in neutrophils. NeuACE and ACE 10/10 mice have a better immune response in several different disease models (e.g. bacteria, tumor, Alzheimer's disease)1,2. We hypothesized that ACE affects myeloid function by inducing changes in cellular energy metabolism. To examine this, metabolic intermediates were studied by mass spectrometry and chemical methods in ACE over‐expressing cells.MethodsNeutrophils were isolated from bone marrow; macrophages were collected from the peritoneum 4 days after thioglycolate injection. Cells were purified by Percoll gradient.ResultsMass spec. showed more than 32 differences, with virtually all affected metabolites increased in the ACE over expressing cells. In ACE 10/10 macrophages, cellular ATP, ADP and AMP was 3.4‐, 2.7‐, and 2.7‐fold the levels in WT cells. In NeuACE neutrophils, the pattern was the same but numerically less than in macrophages. Chemical analysis showed a 2.8‐fold and a 1.5‐fold increase of cellular ATP in ACE 10/10 macrophages and NeuACE neutrophils. In ACE 10/10 macrophages, the ATP/ADP ratio was 2.1 vs. 1.4 for WT cells. For NeuACE neutrophils, the ATP/ADP ratio was 3.0 vs. 1.9 in WT neutrophils. The increase of cellular ATP is due to the catalytic activity of ACE; when animals were treated with an ACE inhibitor, peritoneal macrophages or bone marrow derived neutrophils showed ATP levels that were no different from similarly treated WT mice. The effect of ACE is not due to increased angiotensin II.ConclusionsACE overexpression in macrophages or neutrophils of mice upregulates ATP concentrations in these immune cells. This is due to the catalytic activity of ACE but not the production of angiotensin II. Increased cellular ATP plays an important role in the enhanced innate and adaptive immune ability of myeloid cells over expressing ACE.Support or Funding InformationNIH P01 HL129941 and R01 AI143599 imageThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Macrophages from Mice Administered Rhus verniciflua Stokes Extract Show Selective Anti-Inflammatory Activity.

The bark of Rhus verniciflua Stokes (RVS) is used as a food additive and herbal medicine for various inflammatory disorders and cancer in Eastern Asia. RVS has been shown to exert anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated macrophages in vitro, but whether oral administration of RVS affects the inflammatory response of macrophage needs to be verified. RVS was given orally to mice for ten days. For isolation of macrophages, intraperitoneal injection of thioglycollate was performed. For determination of serum inflammatory response, intraperitoneal injection of LPS was applied. RVS stimulated monocyte differentiation in thioglycollate-induced peritonitis by increasing the population of cells expressing CD11b and class A scavenger receptors. These monocyte-derived macrophages showed an increased uptake of acetylated low-density lipoprotein. When peritoneal macrophages from the RVS group were stimulated with LPS, the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in the supernatant decreased, but the level of IL-12 increased. The surface expression of CD86 was reduced, but surface expression of class II major histocompatibility complex molecules was increased. RVS suppressed the serum levels of LPS-induced TNF-α and IL-6. Collectively, RVS promoted monocyte differentiation upon inflammatory insults and conferred selective anti-inflammatory activity without causing overall inhibitory effects on immune cells.

Regulator of G protein signaling 5 restricts neutrophil chemotaxis and trafficking

Neutrophils are white blood cells that are mobilized to damaged tissues and to sites of pathogen invasion, providing the first line of host defense. Chemokines displayed on the surface of blood vessels promote migration of neutrophils to these sites, and tissue- and pathogen-derived chemoattractant signals, including N-formylmethionylleucylphenylalanine (fMLP), elicit further migration to sites of infection. Although nearly all chemoattractant receptors use heterotrimeric G proteins to transmit signals, many of the mechanisms lying downstream of chemoattractant receptors that either promote or limit neutrophil motility are incompletely defined. Here, we show that regulator of G protein signaling 5 (RGS5), a protein that modulates G protein activity, is expressed in both human and murine neutrophils. We detected significantly more neutrophils in the airways of Rgs5-/- mice than WT counterparts following acute respiratory virus infection and in the peritoneum in response to injection of thioglycollate, a biochemical proinflammatory stimulus. RGS5-deficient neutrophils responded with increased chemotaxis elicited by the chemokines CXC motif chemokine ligand 1 (CXCL1), CXCL2, and CXCL12 but not fMLP. Moreover, adhesion of these cells was increased in the presence of both CXCL2 and fMLP. In summary, our results indicate that RGS5 deficiency increases chemotaxis and adhesion, leading to more efficient neutrophil mobilization to inflamed tissues in mice. These findings suggest that RGS5 expression and activity in neutrophils determine their migrational patterns in the complex microenvironments characteristic of inflamed tissues.

Abstract 493: Neutrophil and Macrophage Cell Surface Colony-Stimulating Factor-1 is Shed by a Disintegrin and Metalloprotease 17 and Contributes to Stimulating Macrophage Proliferation in Inflammation

Recent studies highlight a role for macrophage proliferation post monocyte recruitment in inflammatory conditions, such as atherosclerosis. However, the mechanisms regulating macrophage proliferation are not well understood. Using an acute peritonitis model, we identified a 40 ± 8% reduction (mean ± SEM; n=5; p<0.01) in macrophages in the S phase of the cell cycle 40 hrs post thioglycollate injection in mice lacking the transmembrane protease ADAM17 in hematopoietic cells. ADAM17 is a member of a disintegrin and metalloprotease family that cleaves many cell surface proteins involved in inflammation. The macrophage proliferation defect in ADAM17 null chimeras was associated with a 60 ± 10% (n=5; p<0.001) decrease in soluble macrophage colony-stimulating factor (CSF)-1 in the peritoneum, and was rescued by intraperitoneal injection of CSF-1. We demonstrate that neutrophils, the first innate immune cell that migrates into inflammatory sites, and macrophages are both major sources of cell surface (cs)CSF-1 in this acute inflammation model, adding to the current knowledge that stromal, epithelial and endothelial cells are sources of CSF-1. We show that ADAM17 is critical in mediating csCSF-1 release in the inflamed peritoneum, and that this release following neutrophil extravasation is associated with elevated expression of iRhom2, a member of the rhomboid-like superfamily, which promotes ADAM17 maturation and trafficking to the cell surface. Accordingly, deletion of hematopoietic iRhom2 is sufficient to prevent csCSF-1 release from neutrophils and macrophages, thus recapitulating the defect in macrophage proliferation seen in ADAM17 null cells. Our data indicate that csCSF-1 release and macrophage proliferation is self-limiting in states of acute inflammation due to the transient nature of leukocyte recruitment and temporally restricted csCSF-1 expression and release. In chronic inflammation such as atherosclerosis, we show that macrophage proliferation is decreased by 30 ± 7% (n=6; p<0.01) in atherosclerotic lesions of ADAM17 hematopoietic null LDL receptor-deficient mice fed a high fat diet for 16 weeks. Together, these results demonstrate a novel mechanism governing macrophage proliferation in both acute and chronic inflammation.

Signaling pathways involved in zymosan phagocytosis induced by two secreted phospholipases A2 isolated from Bothrops asper snake venom in macrophages

Phagocytosis, a process involved in host defense, requires coordination of a variety of signaling reactions. MT-II, a catalytically-inactive Lys49-PLA2¸ and MT-III, an active Asp49-PLA2 isolated from Bothrops asper snake venom, activate phagocytosis in macrophages. In this study the signal pathways mediating zymosan phagocytosis, focusing in lipidic second messengers, were investigated. Macrophages collected from male Swiss mouse peritoneum were obtained 96h after i.p. injection of thioglycollate. Phagocytosis was evaluated with non-opsonized zymosan in the presence or absence of specific inhibitors. Data showed that both venom PLA2s increased phagocytosis. Zileuton, Etoricoxib, PACOCF3 (5-LO, COX-2 and iPLA2 inhibitors, respectively), as well as WEB2170 (PAF receptor antagonist) significantly reduced phagocytosis induced by both venom PLA2s. However, Indomethacin (COX-1/COX-2 inhibitor) and Montelukast (CysL receptor antagonist) did not affect the toxins-induced phagocytosis. Moreover, while PACOCF3 (iPLA2 inhibitor), reduced the phagocytosis induced by MT-II and MT-III, AACOCF3 (cPLA2 inhibitor) significantly reduced the MT-II, but not MT-III-induced phagocytosis. These data suggest the effect of both sPLA2s depends on iPLA2 and that the effect of MT-II depends on activation of cPLA2. COX-2 and 5-LO-derived metabolites as well as PAF are involved in the signaling events required for phagocytosis induced by both venom sPLA2s.

Fusarium solani Activates Dectin-1 in Experimentally Induced Keratomycosis.

In this study, the response of Dectin-1 on macrophages to Fusarium solani (F. solani) and the expression patterns of Dectin-1 in experimentally F. solani-induced keratomycosis were investigated. Peritoneal macrophages isolated after intraperitoneal injection of sodium thioglycollate were co-cultured with laminarin and spores of F. solani for 12 h. The expression levels of Dectin-1 and CARD9 were detected by immunofluorescence and real-time quantitative polymerase chain reaction. A mouse model of fungal keratitis was established by substromal inoculation with spores of F. solani. Corneal lesions and inflammatory responses were observed by slit-lamp and histopathology at 1, 2, 3, 5, and 7 day(s) after infection. Dectin-1 expression was significantly upregulated on macrophages stimulated by spores of F. solani. Dectin-1 was not detected in normal corneas of C57BL/6 mice, but detected in infected corneas from the first day after inoculation, with high mRNA levels observed on days 2 and 3. CARD9, a key transducer of Dectin-1 signaling, was also upregulated in infected corneas. In conclusion, Dectin-1 is an important recognition receptor in F. solani-induced keratitis, but the molecular mechanisms warrant further investigation.

Paradoxical Upregulation of Macrophage CXCR7 by Myeloid‐specific Gene Editing in Mice

ObjectiveWe previously reported that the chemokine receptor CXCR7 is induced during monocyte‐to‐macrophage differentiation in vitro, leading to a switch of SDF‐1 signaling profile and increased macrophage migration/phagocytosis. However, the role of monocyte/macrophage CXCR7 in vivo remains unknown.Methods and ResultsThe CXCR7‐floxed mice (CXCR7‐floxed+/+) were crossed with the LyzM‐Cre mice (LyzM‐Cre+/+) to obtain myeloid‐specific deletion of CXCR7 in vivo. Unlike the systemic knockouts, this new line was viable, fertile, and did not show obvious gross physical or behavioral abnormalities. However, we found significantly higher number of periphery blood monocytes in CXCR7‐floxed+/+/LyzM‐Cre+/+ mice compared with CXCR7‐floxed+/+/LyzM‐Cre−/− mice, indicating a role of CXCR7 in monocytosis. Further study indicated that this phenotype cannot be solely attributed to the disruption of LyzM gene by Cre insertion or the inclusion of floxed sequence to CXCR7 gene. Surprisingly, CXCR7 mRNA and protein expression were dramatically upregulated in CXCR7‐floxed+/+/LyzM‐Cre+/+ mice compared with littermate CXCR7‐floxed+/+/LyzM‐Cre−/− mice, both in bone marrow‐derived macrophages and in peritoneal macrophages, suggesting a potential compensatory mechanism. This is supported by our finding that PCR mapping indicated partial deletion of myeloid CXCR7 gene via the Cre‐LoxP approach and that adenovirus delivery of the Cre gene into CXCR7‐floxed macrophages in vitro mimicked CXCR7 induction in vivo. In addition, in the mouse peritonitis model, injection of thioglycolate led to more macrophage infiltration into the peritoneum in CXCR7‐floxed+/+/LyzM‐Cre+/+ mice.ConclusionsMyeloid‐specific manipulation of CXCR7 gene by using Cre‐LoxP method leads to compensatory upregulation of CXCR7 in monocytes/macrophages, which is associated with increased monocytosis and cell migration in vivo.Support or Funding InformationAHA national SDG award

Prolyl-glycyl-proline (PGP) Peptide Prevents an Increase in Vascular Permeability in Inflammation

This study was aimed at investigating the effect of prolyl-glycyl-proline (PGP) tripeptide on vascular permeability in rats with an inflammation. It was found that the peptide reduces the rat paw edema induced by a subcutaneous administration of histamine to the same extent as the conventional anti-inflammatory agent diclofenac. However, an assessment of the relative expression level of the cox-2 gene at the inflammation focus using real-time PCR showed that, in contrast to diclofenac, PGP does not affect the cox-2 gene expression. This is indicative of the fact that they have different mechanisms of action. We used the model of acute peritonitis induced by an intraperitoneal injection of thioglycolate to demonstrate that the inflammatory response of an organism is accompanied by increased vascular permeability in the tissues of the stomach and small intestine. Pre-administration (30 minutes before the induction of the inflammation) of PGP prevented this increase, whereby the level of vascular permeability, exudate volume in the peritoneal cavity, and the amount of the Evans Blue dye in this exudate remained at the control level. Therefore, these results suggest that the anti-inflammatory action of PGP is based on its ability to prevent an increase in vascular permeability.

Strain-dependent response to stimulation in middle-aged rat macrophages: A quest after a useful indicator of healthy aging

Rats of Albino Oxford (AO) strain in our animal facility exhibit a longer average healthy life span than rats of Dark Agouit (DA) strain. Since chronic activation of macrophages contributes to chronic low level inflammation common in older age, elucidation of the changes in middle-aged rats could be useful in prevention of unbalanced inflammatory response in advanced age. We have analysed the phenotype of unelicited and thioglycollate-elicited peritoneal macrophages from young and middle-aged DA and AO rats and tested functions of these cells following stimulation with lipopolysaccharide (LPS) in vitro. Unelicited cells from middle-aged DA rats produced higher amounts of proinflammatory mediators interleukin-6 (IL-6) and nitric oxide (NO), but have a diminished response to LPS stimulation then cells from young rats, in spite of increased frequency of TLR4- and CD14-expressing mature macrophages. Injection of thioglycollate robustly increased overall cytokine production in young rats' macrophages, while diminishing their response to LPS stimulation. In middle-aged DA rats injection of thioglycollate diminished IL-6 production, but increased it in response to LPS stimulation. Quite the contrary to DA rats, the macrophages from middle-aged AO rats have released diminished levels of TNF-α and NO, whereas urea production was strongly increased, when compared to the macrophages from young rats. Although the thioglycollate injection has increased the proportion of CD86+MHCII+ mature macrophages in young rats, and percentages of activated TLR4+ macrophages in both age groups of AO rats, it has not affected the cytokine production in young rats' macrophages, and the TNF-α production in middle-aged rats' macrophages. Moreover, the injection of thioglycollate has robustly increased the production of urea in macrophages derived from both age groups of AO rats. Although middle-aged rats of both strains were healthy during experiment, differences between the inflammatory responses of peritoneal macrophages of middle-aged rats of these strains might be one of the contributing factors defining their health in their advanced age. Development of strategies for the prevention of undesirable inflammatory changes in the elderly would benefit from the prospective study of the middle-aged.

Thymic E4bp4 gene transcription is up-regulated in the chicken during experimental peritonitis modified by the season-related lighting conditions

Immunity, like other vertebrate processes, undergoes the diurnal and seasonal rhythmicity generated and synchronized by the endogenous clock. In the present study a transcription of the selected clock genes was evaluated in the chicken thymus to verify its supposed role as a peripheral clock and to check its relation with the seasonality of immune function. Chickens kept from hatch in the season-related lighting conditions (LD 16:8 in summer vs 8:16 in winter) and in a controlled temperature were exposed to the experimental peritonitis elicited by i.p. thioglycollate injection. Previously described seasonality of the inflammatory response has been confirmed and the diurnal rhythms of a core clock gene Per3 and its repressor E4bp4 in the thymus has been evidenced. E4bp4 transcription was up-regulated in inflamed chickens while that of Per3 appeared independent of the locally induced inflammation. Our results suggest an interconnecting role of E4BP4 between molecular clock and immunity in the chicken.

11β-Hydroxysteroid Dehydrogenase Type 1 Is Expressed in Neutrophils and Restrains an Inflammatory Response in Male Mice.

Endogenous glucocorticoid action within cells is enhanced by prereceptor metabolism by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which converts intrinsically inert cortisone and 11-dehydrocorticosterone into active cortisol and corticosterone, respectively. 11β-HSD1 is highly expressed in immune cells elicited to the mouse peritoneum during thioglycollate-induced peritonitis and is down-regulated as the inflammation resolves. During inflammation, 11β-HSD1-deficient mice show enhanced recruitment of inflammatory cells and delayed acquisition of macrophage phagocytic capacity. However, the key cells in which 11β-HSD1 exerts these effects remain unknown. Here we have identified neutrophils (CD11b+,Ly6G+,7/4+ cells) as the thioglycollate-recruited cells that most highly express 11β-HSD1 and show dynamic regulation of 11β-HSD1 in these cells during an inflammatory response. Flow cytometry showed high expression of 11β-HSD1 in peritoneal neutrophils early during inflammation, declining at later states. In contrast, expression in blood neutrophils continued to increase during inflammation. Ablation of monocytes/macrophages by treatment of CD11b-diphtheria-toxin receptor transgenic mice with diphtheria toxin prior to thioglycollate injection had no significant effect on 11β-HSD1 activity in peritoneal cells, consistent with neutrophils being the predominant 11β-HSD1 expressing cell type at this time. Similar to genetic deficiency in 11β-HSD1, acute inhibition of 11β-HSD1 activity during thioglycollate-induced peritonitis augmented inflammatory cell recruitment to the peritoneum. These data suggest that neutrophil 11β-HSD1 increases during inflammation to contribute to the restraining effect of glucocorticoids upon neutrophil-mediated inflammation. In human neutrophils, lipopolysaccharide activation increased 11β-HSD1 expression, suggesting the antiinflammatory effects of 11β-HSD1 in neutrophils may be conserved in humans.

Purified and Recombinant Hemopexin: Protease Activity and Effect on Neutrophil Chemotaxis.

Infusion of the heme-binding protein hemopexin has been proposed as a novel approach to decrease heme-induced inflammation in settings of red blood cell breakdown, but questions have been raised as to possible side effects related to protease activity and inhibition of chemotaxis. We evaluated protease activity and effects on chemotaxis of purified plasma hemopexin obtained from multiple sources as well as a novel recombinant fusion protein Fc-hemopexin. Amidolytic assay was performed to measure the protease activity of several plasma-derived hemopexin and recombinant Fc-hemopexin. Hemopexin was added to the human monocyte culture in the presence of lipopolysaccharides (LPS), and also injected into mice intravenously (i.v.) 30 min before inducing neutrophil migration via intraperitoneal (i.p.) injection of thioglycolate. Control groups received the same amount of albumin. Protease activity varied widely between hemopexins. Recombinant Fc-hemopexin bound heme, inhibited the synergy of heme with LPS on tumor necrosis factor (TNF) production from monocytes, and had minor but detectable protease activity. There was no effect of any hemopexin preparation on chemotaxis, and purified hemopexin did not alter the migration of neutrophils into the peritoneal cavity of mice. Heme and LPS synergistically induced the release of LTB4 from human monocytes, and hemopexin blocked this release, as well as chemotaxis of neutrophils in response to activated monocyte supernatants. These results suggest that hemopexin does not directly affect chemotaxis through protease activity, but may decrease heme-driven chemotaxis and secondary inflammation by attenuating the induction of chemoattractants from monocytes. This property could be beneficial in some settings to control potentially damaging inflammation induced by heme.

The dual effect of paradoxical sleep deprivation on murine immune functions

We aimed to evaluate the effect of paradoxical sleep deprivation on the cellular migration during inflammation, the peritoneal macrophage phenotype and the infectious stimulus outcomes. A/J mice were inoculated with thioglycollate and exposed to paradoxical sleep deprivation. Sleep-deprived animals presented decreased cell migration compared to controls. Nitric oxide production was reduced in macrophages from sleep-deprived mice compared to controls. Cell surface analysis showed that sleep deprivation reduced F4/80+/CD80low peritoneal cell population induced by thioglycollate injection. Sleep-deprived mice were not more susceptible to infection than control mice. Our findings challenge the general perception that sleep loss always increases infection susceptibility.