Monocyte Release Research Articles

Comparison of the effects of hypolipidemic treatment on monocyte proinflammatory cytokine release in men and women with type 2 diabetes and atherogenic dyslipidemia.

Statins and fibrates reduce monocyte release of proinflammatory cytokines, but it remains unknown whether this effect is sex dependent. We retrospectively analysed age-, weight-, and lipid-matched populations of type 2 diabetic patients of both sexes, who, because of atherogenic dyslipidaemia, were treated with simvastatin (40 mg daily), fenofibrate (200 mg daily), or simvastatin plus fenofibrate. Monocyte release of tumour necrosis factor α (TNF-α), inteleukin-1β, interleukin-6, and monocyte chemoattractant protein-1 (MCP-1), as well as circulating levels of high-sensitivity C-reactive protein (hsCRP) and free fatty acids (FFA) were assessed separately for men and women before and after 12 weeks of treatment. Baseline monocyte release of TNF-α, interleukin-1β, interleukin-6, and MCP-1, as well as plasma hsCRP and FFA levels were comparable in both sexes. Simvastatin, fenofibrate, and simvastatin/fenofibrate combination therapy reduced monocyte release of TNF-α, inteleukin-1β, interleukin-6, and MCP-1, with no difference between the treatment groups. The impact of simvastatin and fenofibrate administered alone on monocyte cytokine release and systemic inflammation did not differ between the men and women. The effect of simvastatin/fenofibrate combination therapy on monocyte release of interleukin-6 and MCP-1 was more pronounced in the male population. The impact of simvastatin administered together with fenofibrate on TNF-α, interleukin-1β, and hsCRP was also stronger in the men than in the women, but the difference did not reach the level of significance. The obtained results suggest that sex differences determine the strength of the monocyte-suppressing effect of simvastatin/ /fenofibrate combination therapy in type 2 diabetic patients with atherogenic dyslipidaemia.

Electronegative LDL (LDL(-)) induces IL-1b release in monocytes and macrophages through caspase-1 activation

Electronegative LDL (LDL(-)) induces IL-1b release in monocytes and macrophages through caspase-1 activation

P11 Induction of immunomodulatory cell death by synthetic RNA mimetics

Background Immune cells detect viral and bacterial DNA/RNA via specific so-called pattern recognition receptors (PRRs). Synthetic RNA and DNA mimetics stimulate defined immune cell subsets and are currently tested as novel immunotherapeutic agents against cancer with, however, varying clinical efficacy. Recent data showed the expression of RNA-recognizing receptors also on tumor cells. In this study we exposed human head and neck squamous cell carcinoma (HNSCC) cells to poly(I:C) and imiquimod, two commercially available immunotherapeutic RNA mimetics, and investigated immunological events associated with the induction of tumor cell death by these compounds. Methods A HNSCC cell line was exposed to poly(I:C) and imiquimod, which were delivered via culture medium or into the cytosol via electroporation. Cell death and cell biological consequences thereof were analyzed in vitro . For in vivo analyses a human HNSCC xenograft model was used. Results Poly(I:C) induced cell death only if delivered into the cytosol. Interestingly, injection of poly(I:C) into the tumor reduced tumor growth and induced tumor cell apoptosis in tumor bearing mice, whereas imiquimod did not affect tumor size. Cell death induced by poly(I:C) resulted in cytokine release (interleukin-6, type I interferon and CXCL-10) and activation of monocytes in vitro . Monocytes activated by the supernatant of cancer cells previously exposed to poly(I:C) recruited significantly more Th1 cells than monocytes exposed to control supernatants. If delivered exogenously, Imiquimod also induced tumor cell death and release of interleukin-6, but cell death was not associated with release of Th1 cytokines, interferons, monocyte activation and Th1 recruitment. Conclusion Our data demonstrate the induction of immunostimulatory HNSCC cell death by intracellular delivery of poly(I:C). Interestingly, imiquimod also induced cancer cell death, but failed to promote additional immunostimulatory mechanisms. These findings have implications for the use of RNA mimetics in cancer immunotherapy and suggest the local delivery of RNA compounds. In addition to the well-known activation of immune cells, local delivery of RNA mimetics to the malignant tissue will induce additional tumor cell death associated with compound-specific promotion of local cellular immunity.

Interleukin 23 levels are increased in carotid atherosclerosis: possible role for the interleukin 23/interleukin 17 axis.

Interleukin (IL)-23 is a cytokine in the IL-12 family, mainly produced by antigen-presenting cells with a central role in inflammation. We hypothesize that IL-23 is also important in atherogenesis and investigate this in a population with carotid atherosclerosis. Plasma levels of IL-23 were measured in patients with carotid artery stenosis and in healthy controls. The mRNA levels of IL-23 and its receptor, IL-23R, were measured in atherosclerotic plaques, nonatherosclerotic vessels, peripheral blood mononuclear cells, and plasmacytoid dendritic cells. Our findings were as follows: (1) patients with carotid atherosclerosis (n=177) had significantly raised plasma levels of IL-23 when compared with healthy controls (n=24) with particularly high levels in those with the most recent symptoms. (2) mRNA levels of IL-23 and IL-23R were markedly increased in carotid plaques (n=68) when compared with nonatherosclerotic vessels (n=8-10). Immunostaining showed colocalization to plaque macrophages. (3) Patients with carotid atherosclerosis had increased mRNA levels of both IL-23 and IL-23R in plasmacytoid dendritic cells, but not in peripheral blood mononuclear cells. (4) IL-23 increased IL-17 release in monocytes and particularly in peripheral blood mononuclear cells from patients with carotid atherosclerosis, but not in cells from healthy controls. (5) IL-23 gave a prominent tumor necrosis factor release in monocytes from patients with carotid atherosclerosis but not in cells from healthy controls. (6) High plasma levels of IL-23 were associated with increased mortality during follow-up. We have shown an association between IL-23 and disease progression in patients with carotid atherosclerosis, potentially involving IL-17-related mechanisms.

Cessation of CCL2 inhibition accelerates breast cancer metastasis by promoting angiogenesis.

Secretion of C-C chemokine ligand 2 (CCL2) by mammary tumours recruits CCR2-expressing inflammatory monocytes to primary tumours and metastatic sites, and CCL2 neutralization in mice inhibits metastasis by retaining monocytes in the bone marrow. Here we report a paradoxical effect of CCL2 in four syngeneic mouse models of metastatic breast cancer. Surprisingly, interruption of CCL2 inhibition leads to an overshoot of metastases and accelerates death. This is the result of monocyte release from the bone marrow and enhancement of cancer cell mobilization from the primary tumour, as well as blood vessel formation and increased proliferation of metastatic cells in the lungs in an interleukin (IL)-6- and vascular endothelial growth factor (VEGF)-A-dependent manner. Notably, inhibition of CCL2 and IL-6 markedly reduced metastases and increased survival of the animals. CCL2 has been implicated in various neoplasias and adopted as a therapeutic target. However, our results call for caution when considering anti-CCL2 agents as monotherapy in metastatic disease and highlight the tumour microenvironment as a critical determinant of successful anti-metastatic therapy.

ADIPONECTIN LIMITS MONOCYTIC MICROPARTICLE-INDUCED INFLAMMASOME ACTIVATION AND ENDOTHELIAL DYSFUNCTION

Monocytic microparticles (mono-MPs) are small vesicles (100-1000 nm) that are shed from monocytes following cellular activation or apoptosis. Levels of mono-MPs are heightened in the inflammatory state and mono-MPs have been reported to play a fundamental role at all stages of the atherothrombotic process. Alterations in monocyte-endothelial cell (EC) interaction mediate immune activation and propagate inflammation within the developing atheroma. Adiponectin is an anti-inflammatory adipokine that primes monocyte differentiation into anti-inflammatory M2 macrophages vs. pro-inflammatory M1 macrophages and inhibits EC activation thereby retarding plaque development. The goal of this work was to uncover the mechanistic root underlying the pathophysiological and clinical benefits of adiponectin. Specifically, we hypothesized that mono-MPs trigger NLRP3 inflammasome-dependent EC activation and adiponectin inhibits this phenomenon, in part, through limiting mono-MP production and activation. Mono-MPs were characterized using electron microscopy, flow cytometry and NanoSight microscopy. Exosomes were excluded based on size and absence of the MP-specific marker phosphatidylyserine. Studies were conducted with THP-1 monocytes and peritoneal monocytes isolated from adiponectin knockout (Adipoq-/-) mice. We evaluated the effects of lipopolysaccharide (LPS; 5 μg/ml, 24 h) and recombinant adiponectin (10 μg/ml) on mono-MP production, expression of the NLRP3 inflammasome and its components, and key downstream inflammatory factors. We also studied how MPs alter the expression of critical adhesion molecules and inflammatory transcription factors in human umbilical vein endothelial cell (HUVEC) cultures. Inflammasome activation was examined in mono-MPs derived from THP-1 cells. Imaging analysis confirmed that the mono-MPs isolated were sized 100-1000 nm and expressed phosphatidylyserine. Absence of exosomes in the final mono-MP samples was further confirmed by a lack of CD63 expression. LPS concentration-dependently increased MP generation by THP-1 monocytes. Adiponectin suppressed MP release in untreated and LPS-stimulated monocytes. LPS-treated mono-MPs exhibited a marked upregulation in the NLRP3 inflammasome and its components - ASC, caspase-1, pro-IL1β and IL1β. Adiponectin attenuated these changes. Mono-MP-HUVEC co-cultures revealed significant increases in markers of endothelial activation (ICAM-1, VCAM-1) and inflammatory transcription factors (p-IkB, p-NFkB and p-ERK-1). Adiponectin inhibited these outcomes. Compared to monocytes from Adipoq+/+ mice, those from Adipoq-/- littermates showed a ∼6-fold greater production of MPs (Cd11b+/Ly6Chi/AnnexinV+) following LPS stimulation. We report a novel mechanism through which mono-MPs sustain NLRP3 inflammasome activation and endothelial dysfunction. These data reveal a previously unknown atheroprotective mechanism for adiponectin that may account for the inverse association observed between circulating adiponectin levels and the risk of diabetes and obesity.

189: A novel player for the regulation of IL-1α/β release in human monocytes

Interleukin (IL)-1α and IL-1β are pro-inflammatory cytokines mainly released by monocytes and macrophage in response to infection. Upon release, IL-1α/β bind to IL-1 receptor type 1 (IL-1R1) initiating a pro-inflammatory signaling pathway, which leads to the release of several inflammatory mediators. Dysregulation of IL-1α/β production is associated to the pathogenesis of several disorders, including sepsis, cancer and autoinflammatory diseases. The release of cytokines of the IL-1 family requires inflammasome-mediated caspase-1 activation. The inflammasome is a multi-protein complex that senses danger and microbial signals promoting the production of IL-1β, IL-18 and partially of IL-1α. The activation of the NLRP3 inflammasome is generally regulated by a two-step pathway: first, the cells have to be primed through the activation of NF-κB, inducing the expression of pro-IL-1β and NLRP3; second, an additional stimulus is required to induce the inflammasome complex formation, which in turn leads to the activation of caspase-1 and the subsequent cleavage of pro-IL-1β and pro-IL-18. Human monocytes do not follow this dual mode of activation inasmuch they release IL-1α/IL-1β when stimulated with lipopolysaccharide (LPS) alone in absence of any proper inflammasome activator. Recently, an alternative mechanism of NLRP3 inflammasome activation driven by the murine inflammatory caspase-11 by LPS has been described. We investigated at molecular level the role of the inflammatory caspase-5 in monocyte in response to LPS stimulation. We identified caspase-5 as key immune effector of NLRP3 inflammasome activation in response to LPS in human monocytes. We also elucidated the pathway leading to caspase-5 activation and the subsequent cytokine release. In this new scenario, we proposed caspase-5 as a new candidate target for future therapeutic strategies aimed at limiting excessive inflammatory response.

Can your hunch get this punch?

A 34 year-old Hispanic female with a history of nephrolithiasis presented with right flank pain and fever. A non-contrast computer tomography (CT) showed a 4.1 × 3.9 cm cystic lesion in the right kidney lower pole and a 6 mm non-obstructive renal calculus. She was treated with cephalexin for Escherichia coli (E. coli) urinary tract infection. One year later she returned to the emergency room with recurrent flank pain, fever, positive right renal punch and a bulge on her right flank (Figure 1 white arrow). A new contrast CT of the abdomen showed several fluid collections (Figure 2 black arrow) with septations located in the right kidney forming a ‘bear paw sign’ (Figure 2 white arrow inset). The largest collection (6.7 × 8.6 cm) had a sinus tract to the posterior lateral abdominal wall. The collections were drained by interventional radiology and grew Lactobacillus species. She subsequently underwent a right total nephrectomy. Pathology of the kidney showed a small tan-yellow, well circumscribed, pseudo-encapsulated, nodular mass located near the renal hilum extending from the medulla to part of the renal cortex (Figure 3, black arrows). This gross appearance is seen in xanthogranulomatous pyelonephritis, renal oncocytoma or chromophobe-type renal cell carcinoma. Histology of the mass revealed two hallmarks: (i) clusters of large lipid-laden macrophages with foamy cytoplasm (xanthoma cell seen in Figure 4, white arrow and black arrowheads) and (ii) multinucleated giant cells (Figure 4 inset black arrows). Fig. 1. Patient's back showing the right bulge noted on physical examination. Fig. 2. Coronal Section showing the right kidney with septations illustrated by black arrow and transverse section inset showing a ‘bear-paw’ appearance illustrated by the white arrow. Fig. 3. Small tan-yellow, well circumscribed, pseudo-encapsulated, nodular mass located near the renal hilum extending from the medulla to part of the renal cortex illustrated by black arrows. Fig. 4. White arrows and black arrowheads illustrate clusters of large lipid-laden macrophages with foamy cytoplasm (xanthoma cell) and black arrows illustrate multinucleated giant cells. Xanthogranulomatous pyelonephritis has a female preponderance compared with men with a ratio of 7:3. It occurs as a result of chronic destructive granulomatous inflammation of the renal parenchyma as a consequence of chronic infection [1]. The common organisms isolated include E. Coli, Proteus species, Staphylococcus aureus, group B Streptococcus, Candida, Klebsiella, and Bacteroides [1]. Nephrolithiasis is the primary etiology in up to 83% of cases of described, with occasional staghorn calculus formation. On CT scan, the classic ‘bear paw sign’ can be seen with the triad of unilateral renal enlargement, non-functional kidney, and pelvic or ureteric obstructing calculus [2]. A differential diagnosis would include transitional-cell carcinoma of the renal pelvis, lymphoma, tuberculosis (unlikely without pulmonary involvement), primary retroperitoneal tumors and primary or secondary retroperitoneal fibrosis [3]. The pathogenesis is thought to occur as a consequence of chronic obstructive nephropathy with bacterial infection leading to systemic chemokine release and stimulation of monocytes and macrophages with subsequent adipose tissue proliferation. Subsequent defective macrophage bacterial removal produces the foam cells [4]. The gross pathological features mimic benign to malignant diseases such as renal oncocytoma or renal cell carcinoma, respectively [5]. Both of these tumors are usually confined to the kidney and can be found in a rare autosomal dominant syndrome called Birt–Hogg–Dube syndrome, although our patient lacked the cutaneous and pulmonary features of this syndrome. Treatment is nephrectomy with an excellent post-operative prognosis provided good residual contralateral renal function exists. In conclusion, the present case re-emphasizes the importance of appropriate follow-up of patients with recurrent urinary tract infections and nephrolithiasis. Chronic infections can lead to granulomatous changes within the kidney and rarely complicate into xanthogranulomatous pyelonephritis with subsequent renal loss.

Electronegative LDL induces il-1b release through the activation of cd14 and tlr4 in human monocytes

Objectives: Secretion of the proinflammatory cytokine IL-1β requires the presence of proIL-1b and its processing by caspase-1, which is activated by the NLRP3 inflammasome pathway. Since caspase-1 is constitutively active in monocytes, Toll-like receptor (TLR) ligands, such as lipopolysaccharide (LPS), are sufficient to trigger IL-1β secretion by inducing proIL-1β expression. Electronegative LDL (LDL(-)) is a minor LDL subfraction that promotes inflammatory cytokine release in monocytes through CD14-TLR4 activation.

Electronegative LDL-induced cytokine release in human monocytes is promoted through activation of p38-mitogen activated protein kinase and nuclear factor kappa-b

electronegative LDL-induced cytokine release in human monocytes is promoted through activation of p38-mitogen activated protein kinase and nuclear factor kappa-b

C1q/TNF-related protein-3 (CTRP-3) attenuates lipopolysaccharide (LPS)-induced systemic inflammation and adipose tissue Erk-1/-2 phosphorylation in mice in vivo

BackgroundThe C1q/TNF-related proteins comprise a growing family of adiponectin paralogous proteins. CTRP-3 represents a novel adipokine with strong expression in adipose tissue and was shown to inhibit chemokine and cytokine release in adipocytes and monocytes in vitro. The aim of the study was to gain the proof of principle that CTRP-3 is a potent anti-inflammatory adipokine in vivo. MethodsC57BL/6N mice were treated intraperitoneally (i.p.) with bacterial lipopolysaccharide (LPS) for 2h. The effects of a 30min pre-treatment with CTRP-3 i.p. or intravenously (i.v.) on systemic and on epididymal, perirenal and subcutaneous adipose tissue inflammation was analyzed via real-time RT-PCR, ELISA and Western blot analysis. ResultsLPS (1μg i.p.) significantly increased serum IL-6 and MIP-2 levels as well as epididymal adipose tissue expression of IL-6 and MIP-2 in mice, whereas CTRP-3 (10μg i.p.) alone or PBS (i.p.) had no effect. Pre-treatment of mice by CTRP-3 i.p. prior to LPS application significantly attenuated LPS-induced cytokine levels but had no effect on adipose tissue cytokine mRNA expression. In contrast to i.p. application of CTRP-3, systemic i.v. application was not sufficient to inhibit LPS-induced cytokine levels or mRNA tissue expression. CTRP-3 given i.p. significantly attenuated LPS-induced phosphorylation of Erk-1/-2 in inguinal adipose tissue. ConclusionThe present study shows the proof of principle that the novel adipokine CTRP-3 is a potent inhibitor of LPS-induced systemic inflammation and LPS-induced signaling in adipose tissue in vivo.

Topical analgesic, anti-inflammatory and antioxidant properties of Oxybaphus nyctagineus: Phytochemical characterization of active fractions

Ethnopharmacological relevanceOxybaphus nyctagineus (Michx.) Sweet has traditionally been used by several Native American tribes predominantly as a topical anti-inflammatory and analgesic agent. Aim of the studyTo evaluate the antioxidant, analgesic and anti-inflammatory activity of the extracts prepared from the aerial parts of Oxybaphus nyctagineus and to characterize the major chemical constituents of the bioactive extracts. Materials and methodsCrude polar and apolar extracts (PCE and ACE) of the herb of Oxybaphus nyctagineus were prepared and tested in the models of the CFA-induced hyperalgesia in rat knee and carrageenan-induced paw edema in rat. To identify the active compounds, subfractions were prepared by column chromatography and subjected in vitro assays, such as antioxidant assays (DPPH, peroxynitrite (ONOO−) scavenging), and the LPS-induced IL-1β release test in human monocytes. Preparative HPLC was employed for the isolation of active substances, while phytochemical analysis was performed by mean of LC–MS/MS and NMR. ResultsThe topically administered PCE and ACE of Oxybaphus nyctagineus demonstrated a significant analgesic and anti-inflammatory effect in the inflammation animal models. The subfraction A4 of ACE and the subfraction P5 of PCE considerably inhibited the LPS-induced IL-1β release in human monocytes, while the strongest activity was localized in the subfraction P5 in the antioxidant assays. The HPLC–MS/MS and NMR analysis revealed that 6-methoxyflavonol diglycosides, namely patuletin-3-O-robinobioside (1), 6-methoxykaempferol-3-O-robinobioside (2), spinacetin-3-O-robinobioside (3), and hydroxy-polyenoic fatty acids, namely corchorifatty acid B (4), 9-hydroxy-10E,12Z,15Z-octadecatrienoic acid (9-HOT acid) (5), and 9-hydroxy-10E,12Z-octadecadienoic acid (9-HOD acid) (6) were present in PCE, and in ACE as major compounds. ConclusionThe results of this study established a pharmacological evidence for the traditional use of Oxybaphus nyctagineus as an anti-inflammatory agent used topically, and provided data on its phytochemical composition for the first time.

Ceramide-enriched LDL induces cytokine release through TLR4 and CD14 in monocytes. Similarities with electronegative LDL

IntroductionIn vitro ceramide-enriched LDL (CER-LDL) reproduces most of the properties of electronegative LDL (LDL(−)), a heterogeneous subfraction of LDL found in plasma. LDL(−) comprises several modifications of LDL and has an increased content in ceramide (CER). It promotes cytokine release in monocytes through CD14 and TLR4. CER-LDL also induces cytokine release in these cells but the mechanism is unknown. AimTo evaluate TLR4 andCD14 as the putative receptors involved in cytokine release induced by CER-LDL. MethodsCER-LDL was obtained by incubating native LDL with CER-enriched liposomes. CER content in CER-LDL was assessed by thin layer chromatography of lipid extracts. CER-LDL and LDL(−) were incubated for 20h with human monocytes in the presence or absence of a TLR4 signaling inhibitor. Both LDLs were also incubated with two human monocytic cell lines, normal and THP1 overexpressing CD14 (THP1-CD14) cells. The release of IL-6, IL-10 and MCP-1 was evaluated by ELISA in culture medium. ResultsThe release of IL-6, IL-10 and MCP-1 induced by CER-LDL in monocytes was inhibited by VIPER (90% inhibition), a specific TLR4 inhibitor. The cytokine release induced by CER-LDL was negligible in THP1, cells presenting a very low CD14 expression. In contrast, the induction of cytokine release in THP1-CD14 was high and dependent on the CER content in LDL. ConclusionCER-LDL induces IL-6, IL-10 and MCP-1 release through the activation of CD14 and TLR4 in monocytes, reproducing the behavior of LDL(−). The increased content of CER in LDL(−) is then related to the inflammatory action of LDL(−).

Novel Markers of Early Neutrophilic and Monocytic Engraftment after Hematopoietic Stem Cell Transplantation

BackgroundNumerous studies tried to find new markers that after hematopoietic stem cell transplantation predict engraftment earlier than the conventional marker, absolute neutrophil count (ANC >500/µL). Early engraftment prediction can be achieved by a marker that reflects the release of neutrophils and monocytes into the leukopenic peripheral blood.MethodsWe analyzed blood cell parameters, including cell population data such as volume, conductivity, and light scatter in 77 patients who underwent HSCT (allogeneic, n=63; autologous, n=11) to detect possible markers.ResultsWe identified 2 early engraftment markers of neutrophils (NEUTRO) and monocytes (MONO); a pair of mean-volume-neutrophils (MNV) and mean-conductivity-neutrophils (MNC) for NEUTRO; and a pair of mean-volume-monocytes (MMV) and mean-conductivity-monocytes (MMC) for MONO. The new markers showed distinct patterns for early engraftment wherein 1) on the engraftment day, MNV peaked as MNC notched simultaneously for every case, and 2) MMV peaked as MMC notched simultaneously in most cases. Engraftment was predicted 3.8±2.7 days earlier than by ANC in 74 successful engraftment cases by using NEUTRO and/or MONO: 1) 72 cases (97.3%), in which NEUTRO and/or MONO predicted earlier engraftment than ANC, 2) 1 case, in which the 3 markers predicted engraftment on the same day, and 3) 1 case, in which NEUTRO predicted engraftment on the same day as ANC and MONO failed to predict engraftment.ConclusionsBy analyzing the data from daily complete blood counts, engraftment can be predicted approximately 4 days earlier than ANC >500/µL using NEUTRO as a base marker and MONO as a supplementary marker.

In Vitro and In Vivo Effects of Human Monocytes and their Subsets on New Vessel Formation

Human monocytes can be divided into CD16(-) monocytes and CD16(+) monocytes. Studies in mice suggested differential effects of monocyte subsets during new vessel formation. The functional role of human monocyte subsets in neovascularization processes was investigated. For in vivo experiments, nude mice underwent unilateral hindlimb ischemia surgery before being injected with either total monocytes, CD16(-) monocytes or CD16(+) monocytes isolated from healthy individuals. In vitro, cytokine array analysis demonstrated that monocytes release numerous angiogenic cytokines, some of which were differentially expressed in monocyte subsets. Sprout length was enhanced in EC spheroids being cultured in conditioned medium obtained from total monocytes and, to a lesser extent, also in supernatants of CD16(-) monocytes. Laser Doppler perfusion imaging up to day 28 after surgery revealed a trend toward improved revascularization in mice treated with monocytes, but no significant differences between monocyte subsets. Histological analyses four weeks after surgery showed an increased arteriole size in mice having received CD16(+) monocytes, whereas the number of capillaries did not significantly differ between groups. Our findings suggest additive and differential effects of monocyte subsets during neovascularization processes, possibly due to an altered secretion of angiogenic factors and their paracrine capacity to stimulate new vessel formation.

Invited Commentary

Cardiopulmonary bypass, as developed over the last 50 years, is an extremely effective technique allowing correction of many cardiac lesions. Despite its wide use, many questions still surround its safety and potential risks. Although there have been many improvements in technique and equipment, the inflammatory process initiated with circulatory support remains surprisingly common and surprisingly incompletely understood. This inflammatory process can be system wide or most pronounced in one organ. Pulmonary dysfunction after cardiopulmonary bypass can be particularly problematic. Occasionally, pulmonary injury and inflammatory response can be severe and life threatening. Response of the lungs to cardiopulmonary bypass has been studied clinically and experimentally since the technique's introduction. From these studies, we have come to understand that the initiating insult is most often cardiogenic, infectious, or inflammatory in origin. The inflammatory response is theorized to arise from contact of blood with the artificial material of the bypass circuit, leading to complement activation, neutrophil and leukocyte activation, and activation of various enzyme systems. Molecular biologists have made a plethora of advances in understanding the inflammatory response. Thoracic surgeons have been slow to apply these advances to our own field in general and to pulmonary dysfunction in particular. In this issue of The Annals, Goto and colleagues [1Goto Y. Hiramatsu Y. Ageyama N. et al.Cardiopulmonary bypass induces recruitment of bone marrow-derived leukocytes to the lungs in monkeys.Ann Thorac Surg. 2014; 97: 617-623Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar] present an experimental study concerning the inflammatory response mounted in response to cardiopulmonary bypass. Using an elegant technique that they developed in a primate model, they document a rapid release of both polymorphonuclear leukocytes (PMN) and monocytes (MO) from the bone marrow into both the circulation and bronchoalveolar lavage fluid after cardiopulmonary bypass. A thymidine analog, 5'-bromo-2'-deoxyuridine (BrdU), was used to label dividing leucocytes in the bone marrow. Flow cytometry was used to document the increased accumulation of BrdU-positive polymorphonuclear neutrophils and monocytes in the peripheral blood and in the bronchoalveolar lavage fluid in both experimental and control animals. The PMNs and MOs first appear in the peripheral blood at 24 hours and peak at 120 hours after bypass. In contrast to the peripheral blood, bronchoalveolar fluid washings from experimental animals contain significantly more BrdU-labeled cells (PMNs and MOs) within 24 hours than do control animals. This type of elegant primate study has somewhat fallen out of favor owing to regulatory and procedural difficulties. Goto and colleagues [1Goto Y. Hiramatsu Y. Ageyama N. et al.Cardiopulmonary bypass induces recruitment of bone marrow-derived leukocytes to the lungs in monkeys.Ann Thorac Surg. 2014; 97: 617-623Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar] are to be commended for their important and challenging work using a classic animal experimental design that includes a control group. This type of study is extremely important in improving our understanding of cardiopulmonary bypass. This improved understanding of the inflammatory reaction after cardiopulmonary bypass opens the door to therapeutic interventions at the molecular level that might limit and ameliorate pulmonary dysfunction after cardiac surgery. Cardiopulmonary Bypass Induces Recruitment of Bone Marrow–Derived Leukocytes to the Lungs in MonkeysThe Annals of Thoracic SurgeryVol. 97Issue 2PreviewA bone marrow (BM) response induced by cardiopulmonary bypass (CPB) as a systemic inflammatory reaction has previously been postulated but not clarified. Newly released polymorphonuclear leukocytes (PMNs) and monocytes from the BM are known to be immature, indicating their greater potential to damage tissue. The present study aimed to examine the kinetics of BM–derived leukocytes associated with CPB in a nonhuman primate model. Full-Text PDF

Cardiopulmonary Bypass Induces Recruitment of Bone Marrow–Derived Leukocytes to the Lungs in Monkeys

A bone marrow (BM) response induced by cardiopulmonary bypass (CPB) as a systemic inflammatory reaction has previously been postulated but not clarified. Newly released polymorphonuclear leukocytes (PMNs) and monocytes from the BM are known to be immature, indicating their greater potential to damage tissue. The present study aimed to examine the kinetics of BM-derived leukocytes associated with CPB in a nonhuman primate model. Normothermic CPB was performed in cynomolgus monkeys for 2 hours through a median sternotomy. Leukocyte precursors were labeled in the BM of the monkeys in vivo by an intravenous injection of 5-bromo-2'-deoxyuridine (BrdU), and their release into the circulation and recruitment to the lungs after operation with or without CPB (control group) were monitored over time by flow cytometry. In normal-state monkeys, the calculated transit time of BrdU-labeled PMNs (PMNBrdU) through the BM was 143.6±4.5 hours and that of monocytes was 100.9±7.6 hours. CPB caused a rapid release of PMNs and monocytes from the BM, shortened their transit through the BM to 92.0±4.1 and 60.3±2.9 hours, respectively, and further induced their increased appearance in the alveolar spaces, with a significant increase in both interleukin (IL)-6 and IL-8 levels in the bronchoalveolar lavage fluid (BALF) 24 hours after CPB. CPB accelerated the release of PMNs and monocytes from the BM and their recruitment to the lungs in our monkey model, indicating that this model is relevant for monitoring the kinetics of BM-derived leukocytes in humans.

Ex Vivo Cytokine Release and Pattern Recognition Receptor Expression of Subjects Exposed to Dampness: Pilot Study to Assess the Outcome of Mould Exposure to the Innate Immune System

In rooms with moisture damage, the indoor air can be enriched with microorganisms causing a variety of symptoms. Due to the highly diverse composition of bioaerosols and the multiple effects on humans, an assessment of the health risk is not sufficiently possible. The aim of this study was to characterize the features of innate immunity using blood from subjects exposed to moisture damage compared to control subjects living in houses without visible moisture damage. We investigated the expression of TLR-2, TLR-4 and dectin-1 on the surface of monocytes from both fresh blood and after in vitro stimulation with the model substances E. coli endotoxin, zymosan A, Pam3Cys and Aspergillus versicolor in 25 exposed subjects and 25 control subjects. In vitro stimulation of whole blood with the same components was performed for 20 h and the release of inflammatory mediators IL-8 and IL-1β were quantified. In addition to an enhanced number of blood leucocytes, the expression of the receptors TLR-2, TLR-4 and dectin-1 on blood monocytes was significantly enhanced in exposed subjects. In contrast, no different alteration in expression was detected between exposed and control group after in vitro stimulation with the model substances. The release of IL-8 and IL-1β after stimulation of whole blood with A. versicolor was increased in subjects exposed to moisture damage. Furthermore, in the exposed subjects the IL-1β release was significantly enhanced after in vitro stimulation with E. coli endotoxin (1000 pg/mL). In conclusion, features of the innate immune system (receptor expression and mediator release of monocytes) are altered in subjects exposed to moisture damage which may be a potential explanation for the increased incidence of respiratory health diseases observed in these populations.

PReS-FINAL-2324: PAPA syndrome clinical spectrum and IL-1Β release

Results Monocytes isolated from PAPA patients tent to secrete higher levels of IL-1b but this was not consistent in all the patients (p = 0.144). Variability in IL-1b release occurred even in the presence of the same PSTPIP1 variant and it did not parallel disease activity when the whole cohort was taken in consideration. However, IL-1b levels varied according to the clinical picture (p = 0.0197), and it was significantly higher (P < 0.05) in patients displaying articular manifestations (N = 3) compared to those affected by cutaneous (N =6 ) or combined (N = 2) lesions. In the former, IL-1b secretion was increased in the presence of acute phase reactants elevation and active joint lesions, and treatment with Anakinra resulted in acute phase reactans normalization and symptom-free period. Silencing of of NLRP3 activity consistently inhibited LPS-induced IL-1b release in both PAPA and HD monocytes. Conclusion IL-1b secretion is higher in PAPA patients displaying prevalent articular manifestations. In these patients, IL-1b release correlates with disease activity and it is mediated by NLRP3.

Pivotal Role Of Heat Shock Proteins In Monocyte and Endothelial Cell Pathology In Sickle Cell Disease

BackgroundIt is well established that sickle cell disease (SCD) manifests global perturbations of hemostasis. Vaso-occlusion, inflammation and coagulopathy all likely contribute to the protean complications of SCD. Central to both inflammation and coagulation is the monocyte. These cells can be profoundly pro-inflammatory and can express tissue factor on their surface and thus influence both inflammation and coagulation. Monocytosis is common in SCD, as is steady state monocyte activation. Exaggerated monocytic response to stimulus may also contribute to the severity of acute events. Thus, agents that regulate monocyte function are potentially of significant relevance in SCD.To this end, we have discovered that the heat shock proteins (HSPs) are potential master regulators of these cells. We previously demonstrated that inhibition of one such HSP, HSP90, could completely ablate the profound and hyper-responsive monocytic inflammatory release upon lipopolysacchiride (LPS) challenge. Inhibition of HSP90 also blocked LPS induced NFk-B translocation to the nucleus. Thus, these results suggested a potent role for HSP90 in LPS-induced monocyte based inflammation. However, the role of HSP90 is cytokine-induced monocyte activation was speculative. The role of HSP90 in monocyte tissue factor expression, or reactive oxygen species (ROS) generation remained unknown. ObjectivesWe sought to determine the role of HSP90 in regulating the pro-inflammatory, pro-coagulant, and ROS generating potential in monocytes. We then wanted to establish a potential role for HSP90 inhibition. MethodsPeripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll density separation. Flow cytometry was employed to measure the LPS- or cytokine-induced monocyte tissue factor expression and ROS generation. Monocyte ROS generation was detected using L012 based chemiluminescence or visualized in individual cells using flow cytometry with CELLROX. Inflammatory cytokines and tissue factor gene expression was evaluated using quantitative real-time polymerase chain reaction (qRT-PCR). NFkB translocation to the nucleus was detected via cell fractionation followed by western blotting or indirect immunofluorescence. PBMCs or THP-1 cells were pre-treated with the HSP90 inhibitors 17-DMAG or AUY922 prior to assay. Monocyte- induced endothelial cell permeability was measured using endothelial cell-substrate impedance sensing (ECIS). Unless otherwise stated p<0.05. ResultsAt baseline, PBMCs from patients with SCD demonstrated elevated monocyte ROS generation and tissue factor expression than those from healthy controls. Inhibition of significantly reduced these measures of steady state monocyte activation. HSP90 inhibition also inhibited both LPS and cytokine induced tissue mRNA accumulation and subsequent cell surface expression of tissue factor in monocytes. Cytokine- induced ROS generation was significantly interrupted in monocytes upon inhibition of HSP90. A panel of monocyte pro-inflammatory genes could be inhibited with AUY922, whereas the anti-inflammatory cytokine, IL-10, was induced upon HSP90 inhibition with AUY922. Mechanistically, we also noted a profound translocation of NFkB to the monocyte nucleus upon cytokine stimulation. Consistent with the effects on tissue factor expression, pro-inflammatory potential, and ROS generation, this translocation could be completely ablated with HSP90 inhibition. Importantly, HSP90 inhibitors significantly attenuated the LPS-activated monocyte -induced lung microvascular endothelial permeability. ConclusionOur data suggest that Hsp90 inhibitors significantly reduced the both pro-inflammatory and pro-coagulatory potential of PBMCs from patients with SCD. These results thus position HSP90 as a potential master regulator of hemostasis, endothelial cell permeability, and thus suggest that HSP90 is an attractive therapeutic target in patients with SCD. Disclosures:Kutlar:Celgene Corporation: Research Funding. Meiler:Celgene Corporation: Research Funding.