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Introduction Patients with frailty represent an increasing patient group in the intensive care medicine. A connection between frailty and inflammation has been suggested. An increased mortality rate in patients with high grade aortic valve stenosis (AS) and frailty, who underwent Transcatheter Aortic Valve Implantation (TAVI) has been observed. A systemic inflammatory reaction in the intensive care unit in the first days after TAVI is a positive predictive factor for an unfavorable outcome. Exact mechanisms are still not fully explained. Monocyte subpopulations are associated with both cardiovascular diseases and a high APACHE II score in critically ill patients. Purpose This study investigates the correlation between frailty and cellular and systemic inflammatory mechanisms and mortality after TAVI. Methods We examined 120 patients with symptomatic AS who underwent TAVI. Before the implantation, frailty status has been assessed. In all patients a flow cytometry analysis has been performed. Monocyte subpopulations were defined as follows: Mon1 (CD14++CD16–), Mon2 (CD14++CD16+) and Mon3 (CD14+CD16++). Expression of CD11b has been measured as a marker for monocyte activation. Pro-inflammatory cytokines such as interleukin IL-8, as well as CRP have been measured with Cytometric Bead Array or standard laboratory methods. Results After 3 months 15 of 120 patients died, primarily without relevant dysfunction of the implanted aortic valve. In 8 of 15 (53%) of the deceased patients and 20 of 100 (19%) of the surviving patients, frailty could be diagnozed before TAVI (p=0.003). Patients with frailty showed prior to TAVI signs of chronic inflammation: elevated CRP (3.7 vs. 5.9 mg/l, p=0.001) and elevated levels of considered as pro-inflammatory Mon2 monocytes (37 vs. 53, p=0.001). Expression of CD11b and IL-8 showed an increasing trend in patients with frailty. Frailty, the monocyte markers, IL-8 and CRP prior to TAVI correlated with increased early mortality after TAVI. Conclusion A considerable number of elderly patients with high grade aortic valve stenosis can be described as frail. This syndrome is associated with increased mortality and with signs of chronic systemic inflammation and pro-inflammatory monocytes. Funding Acknowledgement Type of funding source: Private hospital(s). Main funding source(s): Heart Center Dresden

Background Inhibition of neprilysin by the angiotensin receptor-neprilysin inhibitor has shown remarkable success in the treatment of heart failure with reduced ejection fraction (HFrEF). However, the exact mechanism of action is still a matter of debate. Neprilysin (NEP) is a ubiquitous transmembrane endopeptidase markedly expressed on the cell surface of neutrophils, in this context also known as CD10. Originally, CD10 has been identified as a cell surface marker to discriminate mature from immature neutrophils. Other inflammatory neutrophil surface markers such as CD11b, CD64 and CD66b are commonly used to determine the activation status of neutrophils. In our previous explorative study, NEP expression on neutrophils correlated inversely with HF severity and mortality. This study aimed to explore the relationship between NEP expression and typical neutrophil activation markers and to confirm its relation to HF severity reflected by NT-proBNP and NYHA class in a bigger cohort of HFrEF patients. Methods We prospectively enrolled 208 consecutive patients with stable HFrEF. Mean fluorescence intensity (MFI) of CD10 expression on peripheral blood neutrophils was determined by flow cytometry of whole blood samples. In addition, for a subset of 117 patients the expression of CD11b, CD64 and CD66b was measured. EDTA anticoagulated blood (100 μl) was stained using a combination of six antibodies with fluorescence minus one sample as control [CD10 (#332777), CD45 (#560178), CD16 (#335035), CD11b (#555388), CD64 (#561191), CD66b (#562254); BD Biosciences, San Jose, CA, USA]. Results Median age was 64 years (IQR 54–72), 57 (27%) patients were female. Median NT-proBNP values were 1819 pg/ml (IQR 740–4264). Approximately half of the study population with 115 (55.3%) patients had a non-ischemic etiology of HF. Median MFI of NEP (CD10) on neutrophils was 5542 (IQR 4168–6903). Neutrophil NEP expression decreased with HF severity reflected by NYHA stage (p=0.006) and tertiles of NT-proBNP (p=0.003). Interestingly, non-ischemic HFrEF was characterized by higher neutrophil NEP expression compared to ischemic HFrEF [5703 (IQR 4548–7235) vs 4994 (IQR 3844–6718), p=0.018]. Neutrophil NEP expression correlated highly significant with CD11b expression (r=0.61, p Conclusion These data support the evidence for an inverse correlation of neutrophil NEP expression with HF severity in stable HFrEF patients and for NEP upregulation on neutrophils in patients with non-ischemic cardiomyopathy. There was a strong relationship between neutrophil NEP regulation and the activation marker CD11b, proposing a link between the neutrophil NEP compartment and systemic inflammatory response. Figure 1 Funding Acknowledgement Type of funding source: None

BackgroundFormyl-peptide receptors (FPRs) are important pattern recognition receptors that sense specific bacterial peptides. Formyl-peptide receptors are highly expressed on neutrophils and monocytes, and their activation promotes the migration of phagocytes to sites of infection. It is currently unknown whether FPRs may also influence subsequent processes such as bacterial phagocytosis and killing. Staphylococcus aureus, especially highly pathogenic community-acquired methicillin-resistant S aureus strains, release high amounts of FPR2 ligands, the phenol-soluble modulins.MethodsWe demonstrate that FPR activation leads to upregulation of complement receptors 1 and 3 as well as FCγ receptor I on neutrophils and, consequently, increased opsonic phagocytosis of S aureus and other pathogens.ResultsIncreased phagocytosis promotes killing of S aureus and interleukin-8 release by neutrophils.ConclusionsWe show here for the first time that FPRs govern opsonic phagocytosis. Manipulation of FPR2 activation could open new therapeutic opportunities against bacterial pathogens.

Glioblastoma (GBM) still remains incurable. In search for new treatment modalities immunotherapy might be attractive but highly depends on a sufficient infiltration and function of effector T cells in an immunosuppressive microenvironment. In this study, we analyzed if blocking or reprogramming M2polarized glioma-associated microglia/macrophages (GAMs) could improve spontaneous effector T cell responses and thus enhance the effectiveness of immunotherapy. This was tested by sorting patient-derived CD11b+ cells from GBM tissues and treating these GAMs with small molecule inhibitors (SMI) targeting the colony stimulating factor1 receptor (CSF1R). Especially CD11b+ cells treated with the SMI GW2580 presented with a reduced expression of the M2 marker CD163 (p < 0.01) and an increased expression of HLA-DR (p < 0.05). Conditioned media of SMI-treated GAMs also contained significantly higher levels of nitrite (p < 0.001) and a reduced concentration of the immunosuppressive cytokine IL-6 (p < 0.05). Moreover, gene expression profiles of GW2580-treated GAMs showed a shift from an immunosuppressive towards a pro-inflammatory phenotype. Most importantly, the addition of conditioned media of GW2580-treated GAMs to a co-culture of autologous tumor and T cells significantly reduced the number of live tumor cells as compared to the use of conditioned media of untreated GAMs (p < 0.05). Interestingly, in some cases the ability of T cells to transmigrate through a dense barrier of autologous tumor-derived endothelial cells could also be increased. In summary, we showed that CSF-1R blockade with the SMI GW2580 can reprogram GAM phenotype and thereby improve T cell activation. This strongly suggests further studies on the use of GW2580 in combination with immunotherapeutic approaches for the treatment of GBM.

Background Alterations in the mesentery of Crohn’s disease (CD) patients have long been described, although the functional importance of this tissue is less clear. Some studies hypothesise an anti-inflammatory role for enhanced mesentery, whereas others consider a more pathologic function, given its close proximity to the inflamed intestine. Better phenotypic and functional evaluation of the cells present in this tissue is warranted to improve our understanding of its role in disease. We have previously shown a disproportionate presence of macrophages in CD mesorectum. In this study, we aimed to better characterise the phenotype and function of the macrophages in the mesentery in CD. Methods We collected mesenteric specimens from Crohn’s disease, ulcerative colitis (UC) and non-inflammatory bowel disease (IBD) patients, undergoing surgical bowel resections. In IBD patients, mesentery was collected contiguous to the inflamed region of the intestine, in non-IBD patients, mesentery adjacent to the resection margin. Macrophages were sorted by flow cytometry as CD45+ CD66b-CD14+CD11b+ cells. Sorted cells were analysed by expression profiling and functional analysis. Results Macrophages were present in all analysed tissues. Within the population, a discriminating expression pattern for CD11b was present, dividing CD14+ macrophages in a CD11bhigh and a CD11bdim subset. Transcriptional profiling and gene set enrichment showed the CD11bhigh population to be consistent with IFN-induced pro-inflammatory macrophages, while the CD11bdim population was most consistent with IL4-induced regulatory macrophages. Among the top upregulated genes in CD11bhigh macrophages were S100A8 and S100A9, the two heterodimeric subunits for calprotectin. Flow cytometry confirmed higher expression of calprotectin in the CD11bhigh population also on the protein level. Functionally, CD11bdim macrophages showed higher phagocytic capacity, again consistent a role in tissue repair. Strikingly, the CD11bdim subset was diminished significantly in the mesentery of CD patients, with a near absence in some patients, while the profile in UC was comparable to non-IBD patients. Conclusion Mesenteric macrophages contain two populations, CD11bhigh with a pro-inflammatory profile and CD11bdim with a regulatory profile. In the mesentery of CD patients, the CD11bdim population was strongly decreased, consistent with a pro-inflammatory role for this tissue.

Objectives Echinodorus macrophyllus (Kunth) Micheli (Alismataceae) is popularly used as an infusion to treat inflammatory diseases. This work fractionated the aqueous extract of E. macrophyllus (AEEm) to improve its anti-inflammatory effects. Methods Aqueous extract of E. macrophyllus was fractionated by Sephadex LH-20 and analysed by HPLC-DAD. Anti-inflammatory action was evaluated, in vivo, by air pouch model (total leucocyte, protein and leukotriene B4 (LTB4)), and, in vitro, by neutrophil migration (transwell assay) and its Mac1 expression (flow cytometry), and RAW 264.7 nitric oxide (NO) production (Griess reaction). Key findings Fr20 reduced total leucocyte at 2.5 mg/kg (29.7%) while ethanolic extract of E. macrophyllus (EAEm) increased it (94.0%). Fr20 showed higher (P < 0.05) inhibition (89.8%) of LTB4 in exudate than EAEm (75.0%). Fr20 and EAEm decreased exudate protein and inflammatory infiltrate in pouch tissues, in-vitro neutrophil migration, and NO production. Otherwise, Fr40 did not reduce leucocytes and exudate protein (until 50 mg/kg) nor tissue inflammation, and increased in-vitro NO production. The inhibition of neutrophil migration by EAEm, but not Fr20, was dependent on reduced Mac-1 expression. Conclusions The fractionation of AEEm provided a more potent anti-inflammatory fraction containing flavonoids (Fr20) that reduces the migration of neutrophils and LTB4 release, probably contributing to its mechanism of action.

PURPOSE Radiotherapy (RT) is an essential component in the adjuvant treatment of glioblastoma (GBM); however, despite an initial benefit in tumor control, recurrence is nearly universal. The inflammasome is a multimeric cytosolic complex that has been shown to be activated in immune cells following RT. We investigated the relationship of inflammasome activation and disease progression after RT in GBM. METHODS We performed survival analysis for patients with recurrent GBM using the TCGA dataset, stratifying according to expression of inflammasome pathway molecules Caspase 1 (CASP1), IL-1β, and NLRP3. IL-1β expression and colocalization with CD11b+ myeloid cells were analyzed at different time points following RT in a murine glioma GL261 model. We detected the secreted IL-1β, CASP1 activity, and NLRP3 expression in both tumor and infiltrating immune cells, and the downstream effects of their interplay, including cellular apoptosis, proliferation, and viability. RESULTS High expression of CASP1, IL-1β, and NLRP3 are associated with poor survival in recurrent GBM. In a mouse model, recurrent tumors after treatment with RT display higher levels of the inflammasome effector molecule IL-1β, which colocalizes with infiltrating CD11b+ myeloid cells. Using a co-culture system of tumor cell and M2 macrophages, we found that RT enhances inflammasome activation in both cell types, and this interaction restores the proliferative capacity of surviving tumor cells following treatment. RT induces the inflammasome pathway activation in the setting of an associated immune microenvironment rich in tumor-associated macrophages and myeloid-derived suppressor cells (TAM/MDSCs) and not in tumor cells alone. CONCLUSIONS Inhibition of the oncoprogressive crosstalk between GBM tumor cells and M2 macrophages, possibly through disruption of the inflammasome pathway, might be a potential therapeutic strategy with RT.

International audience; The aim of our study was to analyze polymorphonuclear neutrophil (PMN) functions in elderly individuals compared with those in healthy young participants, directly in whole blood to avoid issues with data interpretation related to cell isolation procedures. Despite the presence of increased circulating levels of proinflammatory cytokines, resting PMNs from the elderly individuals were not activated as shown by normal CD62L and CD11b expression at the PMN surface and normal constitutive reactive oxygen species (ROS) production. However, suboptimal stimulation induced modulations of CD62L and CD11b expression, which positively correlated with the interleukin-6 circulating level, suggesting a possible in vivo preactivation of old PMNs by this cytokine. In addition, PMN phagocytosis of opsonized Escherichia Coli was decreased in elderly individuals. Furthermore, upon preincubation of elderly whole-blood samples with tumor necrosis factor-a or Toll Receptor agonists, we observed a reduced PMN oxidative burst in response to formyl peptides. Elderly participants also exhibited an increased percentage of the immunosuppressive CD16(bright)/CD62L(dim) PMN subpopulation, which was characterized by a lower phagocytic index and a reduced ROS production compared with the CD16(bright)/CD62L(bright) subset. Thus, the reduced phagocytosis and ROS production associated with an expansion of immunosuppressive CD16(bright)/CD62L(dim) PMN subpopulation might be involved in the increased susceptibility to bacterial and fungal infections with old age.

Although neutrophils are typically the first immune cells attracted to an infection site, little is known about how neutrophils dynamically interact with invading pathogens in vivo. Here, with the use of intravital microscopy, we demonstrate that neutrophils migrate to the arrested Cryptococcus neoformans, a leading agent to cause meningoencephalitis, in the brain microvasculature. Following interactions with C. neoformans, neutrophils were seen to internalize the organism and then circulate back into the bloodstream, resulting in a direct removal of the organism from the endothelial surface before its transmigration into the brain parenchyma. C. neoformans infection led to enhanced expression of adhesion molecules macrophage 1 antigen on neutrophils and ICAM-1 on brain endothelial cells. Depletion of neutrophils enhanced the brain fungal burden. Complement C3 was critically involved in the recognition of C. neoformans by neutrophils and subsequent clearance of the organism from the brain. Together, our finding of the direct removal of C. neoformans by neutrophils from its arrested site may represent a novel mechanism of host defense in the brain, in addition to the known, direct killing of microorganisms at the infection sites. These data are the first to characterize directly the dynamic interactions of leukocytes with a microbe in the brain of a living animal.

Introduction CHRFAM7A is a uniquely human gene that is a dominant negative inhibitor of the alpha-7 nicotinic acetylcholine receptor (a7nAchR). The relative balance of CHRFAM7A and a7nAchR expression may play an important role in mediating the human immune response to injury. We have previously shown that CHRFAM7A expression increased the hematopoietic stem cell reservoir in bone marrow and altered emergency myelopoiesis after injury. Here, we hypothesized that loss of the a7nAchR would produce a phenotype similar to CHRFAM7A overexpression, with an increased bone marrow reservoir of stem cells that would modulate myeloid cell mobilization after burn injury. Methods a7nAchR knockout (KO) mice (CHRNA7-/-) underwent 30% TBSA steam burn and were compared to age- and sex-matched wild-type mice. Bone marrow and blood were harvested 24 hours following burn injury for flow cytometry analysis. To measure bone marrow hematopoietic stem cells (HSC) we used flow cytometry to measure Lin−Sca1+c-Kit+ (LSK) cell expression. Bone marrow short term (ST) and long term (LT) HSCs were identified with CD48 and CD150. Immune cells were identified with myeloid markers including CD45, CD11b, Gr1, Ly6C, and Ly6G. Results a7nAchR KO mice have an increased bone marrow reservoir of LSK HSCs compared to WT. While bone marrow expression of ST-HSC and LT-HSC decreased after severe burn injury in WT mice, a7nAchR KO prevented the burn-induced depletion of ST-HSC and LT-HSC leaving a bone marrow HSC reservoir similar to sham. a7nAchR KO attenuated burn-induced mobilization of CD11b+ monocytes and CD11b+Ly6CHi inflammatory monocytes into the systemic circulation compared to WT. Conclusions Loss of the a7nAchR increases myeloid cell mobilization into the circulation after severe burn injury. There is significant variability in CHRFAM7A expression between individuals, suggesting that the relative balance of CHRFAM7A and a7nAchR expression may alter the host inflammatory response and influence human resilience after injury. Applicability of Research to Practice Understanding the factors that mediate the human response to burn injury could give important insights into variability in the inflammatory response between individuals and lead to therapeutics targeting the uniquely human response to burn injury.

Glioblastoma (GBM) is the most common, highly aggressive and lethal primary brain tumor in adults, and has a median overall survival ranging from 12 to 15 months. Several human cancers including glioma are infiltrated with numerous immune cell types which play a critical role in tumor growth, invasion and resistance to treatment. Previous studies, including our group, have shown that resistance to anti-VEGF therapy is associated with myeloid cell infiltration and mesenchymal transition in GBM. Notably, most glioma patients have shown increase in CD68+ cells due to overproduction of colony stimulating factor 1 (CSF-1) by tumor cells, a growth factor for macrophages. Therefore, we hypothesized that CSF-1 inhibition may reduce macrophage and/or myeloid cell infiltration in glioma, thereby increasing animal survival as monotherapy or in combination with VEGF inhibitors in xenograft GBM mouse models. We tested two CSF-1R inhibitors (AZD 7507 and JNJ-28312141) alone and in combination with VEGF inhibition to prevent macrophage infiltration in xenograft GBM mouse models. CSF-1R and VEGF inhibitors reduced macrophage infiltration (F4/80 staining), tumor volume, and mesenchymal transition (YKL-40 staining), and there was a marginal survival benefit in this model. Interestingly, despite significant reduction in tumor macrophages, we observed a significant increase in neutrophil infiltration and hypoxia (HIF1α staining), particularly in the combinatorial treated. Considering these observations, we further evaluated tumor-associated neutrophil (TAN) infiltration in GBM patient tumors by fluorescence-activated cell sorting (FACS). FACS-isolated TANs were identified as CD11b+/CD15+/CD66b+ triple positive. Our results shown that the infiltrating TAN population vary from 0.5 to 5% in GBM patient tumors. Detailed characterization of TAN population and polarization in patient tumors are ongoing. Our findings revealed that CSF-1 and VEGF inhibition reduced macrophage infiltration and tumor growth, but significantly increased TAN infiltration which will likely hamper the potential therapeutic benefit of anti-CSF1-directed inhibitors.

Immunosuppressive tumor microenvironment (TME) in glioblastoma (GBM) attributes to poor efficacy of chemotherapy and/or immunotherapy. We recently reported that SLC9A1 gene expression (Na/H exchanger 1, NHE1) in GBM tumor correlates with increased macrophage tumor infiltration and worsened patient survival. Temozolomide (TMZ) monotherapy, the standard care therapy for GBM, stimulates NHE1 protein expression in mouse syngeneic glioma models. We speculate that in response to TMZ therapy, NHE1 promotes immunosuppressive TME development via deregulation of glucose metabolism and anti-tumor function of immune cells. CD57BL/6 mice bearing GL26 glioma tumor were treated with NHE1 inhibitor HOE-642 (H, 0.3 mg/kg/day), TMZ (T, 2.5 mg/kg/day), and/or anti-PD-1 (10 mg/kg/day) monotherapy or in combination. The T+H+anti-PD-1 combination therapy increased glucose uptake (2-NBDG) and mitochondrial mass (mitotracker fluorescent intensity) of tumor infiltrating CD8+ T-cells by ~2-fold compared to the monotherapies. This suggests that NHE1 protein is involved in metabolic transformation of T-cells and affects their responses to chemo and/or immunotherapy. Furthermore, we detected that specific deletion of Nhe1 in Cx3Cr1CreErT2r+/-;Nhe1f/f mice (Nhe1 KO) alters activation of glioma-associated microglia/macrophages (GAM) and T-cells in response to TMZ therapy. TMZ monotherapy stimulated macrophage (CD11b+CD45hi) infiltration by ~2.5-fold in Cx3Cr1CreErT2-/-;Nhe1f/f (Nhe1 Con) mice bearing SB28-GFP glioma tumor with abundant proinflammatory CD16/32+ GAMs. In contrast, TMZ treated Nhe1 KO mice displayed ~3-fold reduction in tumor infiltrating GAMs with nearly abolished Ym-1+ tolerogenic macrophages, ~3-fold reduction of tumor-promoting CD4+CD25+FoxP3+ regulatory T-cells (Treg) and increased CD8+/Treg ratio by ~2-fold than the Nhe1 Con mice. Importantly, TMZ followed by anti-PD-1 therapy prolonged survival of Nhe1 KO mice but not Nhe1 Con mice. Taken together, our study suggests that NHE1 is involved in transformation of GAMs and PD-1 check point activation of T-cells. Blocking NHE1 function in combination with TMZ and anti-PD-1 may restore glucose metabolism of immune cells necessary for their anti-tumor function.

INTRODUCTION: Immuno-therapy directed at the PD-1/PD-L1 axis has produced significant treatment advances in various human cancers. Unfortunately, this progress has not extended to glioblastoma, with recent clinical trials failing to show efficacy of anti-PD-1 monotherapy in recurrent tumors. Commonly employed murine glioma models exhibit varied responsiveness to anti-PD-1 monotherapy, e.g. GL261 gliomas are sensitive while KR158 tumors are resistant. Previously, we reported that combining PD-1 blockade with either chemokine receptor CCR2 deficiency or a CCR2 antagonist improved survival over anti-PD-1 monotherapy in GL261 gliomas. This was associated with reduced numbers of MDSCs within tumors. The current study evaluated the combination of PD-1 blockade and a novel CCR2 antagonist in anti-PD-1 resistant gliomas. OBJECTIVE: Determine if combination anti-PD-1/CCR2 antagonist therapy is an effective treatment in anti-PD-1 insensitive gliomas. METHODS: Overall survival and immune cell characteristics were determined in KR158 and 005GSC gliomas established in either CCR2 deficient or wild type mice treated with CCR2 antagonist (CCX872) and/or PD-1 blockade. RESULTS: CCR2 deficiency unmasked an anti-PD-1 survival benefit in KR158 glioma-bearing mice. CCX872 increased median survival as a monotherapy in KR158 tumor-bearing animals, and significantly increased median and durable overall survival when combined with anti-PD-1. In 005GSC tumors, the combination of CCX872 and anti-PD-1 prolonged median survival time. Increases in overall CCR2(+) cells and CD11b(+)/Ly6C(hi) myeloid derived suppressor cells (MDSC, known to be CCR2(+)) were evident in the bone marrow of CCR2-deficient mice. Additionally, these mice exhibited decreased MDSCs within established gliomas. The data demonstrate CCX872/anti-PD-1 synergize to increase survival in clinically relevant glioma models via reduced MDSC infiltration, resulting in a tumor microenvironment favorable for anti-PD-1 efficacy. CONCLUSION: The combination of CCX872 and anti-PD-1 is effective in clinically relevant murine glioma models, providing a basis on which to progress this novel combinatorial treatment toward early phase human trials.

Streptococcus pyogenes of the M1 serotype can cause STSS, which is associated with significant morbidity and mortality. The purpose of the present study was to examine the role of NFAT signaling in M1 protein-induced lung injury. NFAT-luc mice were treated with the NFAT inhibitor A-285222 before administration of the M1 protein. Neutrophil infiltration, edema, and CXC chemokines were quantified in the lung, 4 h after challenge with the M1 protein. Flow cytometry was used to determine Mac-1 expression. Challenge with the M1 protein increased NFAT-dependent transcriptional activity in the lung, spleen, and liver in NFAT-luc mice. Administration of the NFAT inhibitor A-285222 abolished M1 protein-evoked NFAT activation in the lung, spleen, and liver. M1 protein challenge induced neutrophil recruitment, edema, and CXC chemokine production in the lung, as well as up-regulation of Mac-1 on circulating neutrophils. Inhibition of NFAT activity attenuated M1 protein-induced neutrophil infiltration by 77% and edema formation by 50% in the lung. Moreover, administration of A-285222 reduced M1 protein-evoked pulmonary formation of CXC chemokine >80%. In addition, NFAT inhibition decreased M1 protein-triggered Mac-1 up-regulation on neutrophils. These findings indicate that NFAT signaling controls pulmonary infiltration of neutrophils in response to streptococcal M1 protein via formation of CXC chemokines and neutrophil expression of Mac-1. Thus, the targeting of NFAT activity might be a useful way to ameliorate lung injury in streptococcal infections.

Neutrophils are mediators of innate immunity and motility is critical to their function. We used microcontact printing to investigate the relationship between density of adhesive ligands and the dynamics of neutrophil motility. We show that neutrophils adopt a well-spread morphology without a uropod on moderate densities of adhesion ligand. As density is increased, the morphology switches to a classic amoeboid shape. In addition to the morphological differences, the dynamics of motility were quantitatively distinct. Well-spread cells without uropods glide slowly with high persistence while amoeboid cells made frequent directional changes, migrating quickly with low persistence. Using an antibody panel against various integrin chains, we show that adhesion and motility on fibronectin were mediated by MAC-1 (αMβ2). The phenotypic switch could be generalized to other surface ligands, such as bovine serum albumin, to which the promiscuous MAC-1 also binds. These results suggest that neutrophils are capable of displaying multiple modes of motility as dictated by their adhesive environment.

The potential cardiovascular (CV) toxicity associated with combined antiretroviral therapy (cART) has been attributed mainly to the nucleoside reverse transcriptase inhibitors abacavir and didanosine. However, the other two components of cART--non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs)--may also be implicated, either directly or by influencing the action of the other drugs. This study evaluates the acute direct effects of the NNRTIs efavirenz and nevirapine and one of the most widely employed PIs, lopinavir, on leucocyte-endothelium interactions, a hallmark of CV disease.Drugs were analysed in vitro in human cells (interactions of peripheral blood polymorphonuclear or mononuclear cells with human umbilical vein endothelial cells) using a flow chamber system, and in vivo in rat mesenteric vessels by means of intravital microscopy. The expression of adhesion molecules in leucocytes and endothelial cells was studied by flow cytometry, and the role of these molecules in white cell recruitment was evaluated by pre-treating human cells or rats with blocking antibodies.Efavirenz and nevirapine, but not lopinavir, increased the rolling flux and adhesion of leucocytes in vitro and in vivo while inducing emigration in rat venules. Efavirenz, but not nevirapine, augmented the levels of CD11b, CD11c and CD18 in neutrophils and monocytes. The actions of efavirenz, but not of nevirapine, were reversed by antibodies against Mac-1 (CD11b/CD18), gp150,95 (CD11c/CD18) or ICAM-1 (CD54).NNRTIs, but not PIs, interfere with leucocyte-endothelial interactions. However, differences between efavirenz and nevirapine suggest a specific CV profile for each compound.

INTRODUCTION: Nonfunctional pituitary adenomas (NFPAs), while typically benign, display variable invasiveness, and can present with devastating symptoms of intercranial mass effect. Tumor-associated macrophages (TAMs) have long been accepted as active participants in tumor microenvironments, particularly their M1 and M2 subtypes, correlating with anti- and pro-tumoral effects respectively. We investigated TAMs in NFPA biology and clinical behavior. METHODS: Patient NFPAs were FACS sorted for total macrophages and M1 and M2 subtypes. Functional assays utilized monocyte cell line THP1. RESULTS: Macrophage isolation from fourteen NFPAs revealed TAMs average 8.5% of dissociated cells (range=0.5%-29.1%), displaying bimodal distribution with most cases clustering below 8% CD11b fraction. Two cases exhibited 18–29% CD11b fractions and were more expansile (size>3.5 cm or MIB1>3%). M2/M1 gene expression ratios assessed by qPCR were above one in NFPAs with cavernous sinus invasion, including the TAM-rich expansile tumors, while M2/M1 ratios were below one in NFPAs without cavernous sinus invasion. Increasing TAM percentage correlated with decreased M2/M1 ratio determined by qPCR of subtype-specific TAM markers. THP1 cells were polarized and used to created media conditioned with M1 and M2 TAMs. Primary cultured NFPA patient cell lines showed that M2 conditioned media induced more invasion (P < 0.01), migration (P < 0.05) and proliferation (P < 10(–3)). qPCR screening of patient cell lines identified S100A9 and EZH2 as mediators of M2-induced invasion and proliferation, respectively. CONCLUSION: Our work identified two subtypes of NFPAs based on TAM status: (1) Larger, more proliferative NFPAs exhibited increased TAMs and decreased M2/M1 ratio but sufficient M2-induced EZH2 can drive proliferative expansion with sheer TAM numbers; and (2) NFPAs invading the cavernous sinuses exhibited lower TAMs and higher M2/M1 ratios, with M2-induced S100A9 driving invasion. These results suggest that recruitment of TAMs to the pituitary microenvironment influences tumor development and M2 TAMs promote a more pro-tumoral state.

Regulation of nutrient availability is a critical way by which tumors exert their immunosuppressive effects on the host. The glioblastoma microenvironment, in particular, is enriched in expression of indoleamine 2,3- dioxygenase 1 (IDO1), which depletes tryptophan from the microenvironment. Upon depletion of tryptophan, the amino acid stress kinase, general control non-derepressible 2 (GCN2), inhibits general protein synthesis, and upregulates a number of genes to promote the cellular survival. In this study, we explored the effects of a deficiency of the GCN2 pathway on infiltrating immune cells in the context of the glioma. Interestingly after GL-261 (Glioma mouse model cell line) injections in GCN2 knock out (KO), and wild type (WT) mice, GCN2 KO mice survived significantly (p(value):0.0066) longer compared to WT mice. In-vivo flow cytometric analyses of these mice, showed significant reduction in CD45(hi)CD11b(+) (myeloid cell marker) and Ly6C (monocytic MDSC marker) population in GCN2 KO mice. To study the mechanisms responsible for this, we generated in-vitro bone-marrow-derived MDSCs from both WT and GCN2KO mice. Surprisingly, the in-vitro T-cell suppressive effects of GCN2 KO MDSCs were significantly attenuated, with a decrease in arginase-1 (Arg-1) expression as a potential mechanism for this observation. To validate this phenomena in-vivo, we injected GCN2 KO and WT mice with GL-261 tumor cells, and after 18 days post tumor injection, we isolated Gr-1 (pan-MDSC marker) positive cells from tumor bearing brains, and checked for Arg-1 level by RNA and protein. Our data, both by protein and RNA demonstrated significant reduction in Arg-1 levels. Importantly, ex-vivo isolated Gr-1 myeloid cells from tumor bearing brain of GCN2 KO and WT mice shows dramatically reduced suppressive capabilities. In conclusion, our data demonstrate the importance of GCN2 pathway on MDSC functionality, and provide insight on how amino acid sensing enzymes may regulate immunity in glioblastoma.

Leukocyte infiltration and acinar cell necrosis are hallmarks of severe AP, but the signaling pathways regulating inflammation and organ injury in the pancreas remain elusive. In the present study, we investigated the role of geranylgeranyltransferase in AP. Male C57BL/6 mice were treated with a geranylgeranyltransferase inhibitor GGTI-2133 (20 mg/kg) prior to induction of pancreatitis by infusion of taurocholate into the pancreatic duct. Pretreatment with GGTI-2133 reduced plasma amylase levels, pancreatic neutrophil recruitment, hemorrhage, and edema formation in taurocholate-evoked pancreatitis. Moreover, administration of GGTI-2133 decreased the taurocholate-induced increase of MPO activity in the pancreas and lung. Treatment with GGTI-2133 markedly reduced levels of CXCL2 in the pancreas and IL-6 in the plasma in response to taurocholate challenge. Notably, geranylgeranyltransferase inhibition abolished neutrophil expression of Mac-1 in mice with pancreatitis. Finally, inhibition of geranylgeranyltransferase had no direct effect on secretagogue-induced activation of trypsinogen in pancreatic acinar cells in vitro. A significant role of geranylgeranyltransferase was confirmed in an alternate model of AP induced by L-arginine challenge. Our findings show that geranylgeranyltransferase regulates neutrophil accumulation and tissue damage via expression of Mac-1 on neutrophils and CXCL2 formation in AP. Thus, these results reveal new signaling mechanisms in pancreatitis and indicate that targeting geranylgeranyltransferase might be an effective way to ameliorate severe AP.

Background The activation of polymorphonuclear leucocytes (PMNLs) causes inflammation and as a result cardiovascular disease, which is a main risk factor for increased morbidity and mortality in patients with chronic kidney disease. Toxins accumulating in uraemic patients play a major role in modulating essential PMNL functions and apoptosis, the latter being crucial for a coordinated resolution of inflammation. One uraemic toxin is phenylacetic acid (PAA). We therefore investigated whether PAA contributes to the deranged immune response in uraemia by modulating PMNL activities. Methods PMNL oxidative burst, phagocytosis and surface expression of the activation markers CD11b and CD18 were measured by flow cytometry in whole blood from healthy subjects in the presence and absence of PAA. Spontaneous apoptosis of isolated PMNLs was assessed by evaluating morphological features under the fluorescence microscope and by measuring the DNA content by flow cytometry. PMNL chemotaxis was tested by the under-agarose method. Results PAA significantly enhanced the stimulation of PMNL oxidative burst by Escherichia coli, phagocytosis of E. coli by PMNLs and the expression of CD11b and CD18 at the PMNL surface. PAA significantly decreased PMNL apoptosis resulting in an increased percentage of viable cells. PAA affected neither the oxidative burst stimulated by phorbol-12-myristate-13-acetate nor PMNL chemotaxis. Conclusions PAA increases the activation of various PMNL functions and the expression of surface activation markers, while it attenuates PMNL apoptotic cell death. Therefore, PAA may contribute to the inflammatory state and consequently to increased cardiovascular risk in uraemic patients.

Summary Eosinophils contribute to the pathogenesis of bullous pemphigoid (BP) by secretion of proinflammatory cytokines and proteases. Trafficking of eosinophils into tissue in animal models and asthma depends on interleukin-5 and a family of chemokines named eotaxins, comprising CCL11, CCL24 and CCL26. Up-regulation of CCL11 has been described in BP, but the expression of the other two members of the eotaxin-family, CCL24 and CCL26, has not been investigated. In addition to these chemokines, expression of adhesion molecules associated with eosinophil migration to the skin should be analysed. We demonstrate that similar to CCL11, the concentration of CCL26 was up-regulated in serum and blister fluid of BP patients. In contrast, the concentration of CCL24 was not elevated in sera and blister fluid of the same BP patients. In lesional skin, CCL11 and CCL26 were detected in epidermis and dermis by immunohistochemistry. In contrast to CCL11, CCL26 was expressed strongly by endothelial cells. In line with these findings, eosinophils represented the dominating cell population in BP lesional skin outnumbering other leucocytes. The percentage of eosinophils expressing very late antigen (VLA): VLA-4 (CD49d) and CD11c correlated with their quantity in tissue. Macrophage antigen (MAC)-1 (CD11b/CD18) was expressed constitutively by tissue eosinophils. In conclusion, these data link the up-regulation of the eosinophil chemotactic factor CCL26 in BP to the lesional accumulation of activated eosinophils in the skin. Thereby they broaden the understanding of BP pathogenesis and might indicate new options for therapeutic intervention.

Staphylococcus epidermidis is a frequent cause of nosocomial infections. The central virulence factor of S. epidermidis is biofilm formation. Polysaccharide intercellular adhesin (PIA) constitutes the major biofilm matrix-component. PIA and biofilm have been implicated in S. epidermidis evasion of host immune defence. We examined the effects of S. epidermidis PIA on the inflammatory response with focus on complement activation. We used a human whole-blood ex vivo model of infection and compared the effects of a PIA-positive S. epidermidis strain (SE1457) and its PIA-negative isogenic mutant (M10). The independent effect of purified PIA on complement activation was investigated. In glucose-rich media, the mutant formed a proteinacious DNA-rich biofilm, whereas SE1457 formed a thick PIA-biofilm. In biofilm growth, SE1457 induced a stronger activation of the complement system compared with M10. We verified that purified PIA was independently responsible for a strong activation of the complement system. In contrast, M10 induced higher granulocyte activation by expression of CD11b and higher secretion of cytokines. We conclude that PIA has potent pro-inflammatory properties by activating the complement system. However, in a complex balance of the immune response, the decreased activation of granulocytes and cytokines by a PIA biofilm may limit host eradication of S. epidermidis.

Zymosan, a mimic of fungal pathogens, and its opsonized form (ZOP) are potent stimulators of monocyte NADPH oxidase, resulting in the production of O2.–, which is critical for host defense against fungal and bacterial pathogens and efficient immune responses; however, uncontrolled O2.– production may contribute to chronic inflammation and tissue injury. Our laboratory has focused on characterizing the signal transduction pathways that regulate NADPH oxidase activity in primary human monocytes. In this study, we examined the involvement of various pattern recognition receptors and found that Dectin-1 is the primary receptor for zymosan stimulation of O2.– via NADPH oxidase in human monocytes, whereas Dectin-1 and CR3 mediate the activation by ZOP. Further studies identified Syk and Src as important signaling components downstream of Dectin-1 and additionally identified PKCδ as a novel downstream signaling component for zymosan-induced O2.– as well as phagocytosis. Our results show that Syk and Src association with Dectin-1 is dependent on PKCδ activity and expression and demonstrate direct binding between Dectin-1 and PKCδ. Finally, our data show that PKCδ and Syk but not Src are required for Dectin-1-mediated phagocytosis. Taken together, our data identify Dectin-1 as the major PRR for zymosan in primary human monocytes and identify PKCδ as a novel downstream signaling kinase for Dectin-1-mediated regulation of monocyte NADPH oxidase and zymosan phagocytosis.

Resident peritoneal macrophages (PEMs) express SIGNR1 on the cell surface as a major mannose receptor. These cells also ingest oligomannose-coated liposomes (OMLs) in an oligomannose-dependent manner following intraperitoneal administration. Therefore, the current study was conducted to investigate the possible role of SIGNR1 in capture of OMLs. Transient expression of several SIGN-related lectins potentially expressed on PEMs in CHO cells revealed that only SIGNR1 contributed to capture of OMLs. When SIGNR1 was introduced into mouse macrophage-like RAW264.7 cells, SIGNR1-expressing RAW (RAW-SIGNR1) cells recognized OMLs under serum-free conditions. OML recognition by RAW-SIGNR1 cells as well as that by PEMs was partially inhibited by an anti-SIGNR1 antibody (ER-TR9) and by mannan, and completely inhibited by EDTA. Interestingly, OML recognition by RAW-SIGNR1 cells was accelerated in the presence of serum, partially inhibited by an anti-complement receptor 3 (CR3) antibody (M1/70), and almost completely inhibited by a combination of ER-TR9 and M1/70. Complete inhibition of OML ingestion by the combination of ER-TR9 and M1/70 was also observed under serum-free conditions, suggesting that SIGNR1 and CR3 cooperate in an additive way in capture of OMLs by macrophage-like RAW cells. Administration of ER-TR9 or M1/70 into the peritoneal cavity led to a significant decrease of OML uptake by PEMs. Therefore, SIGNR1 expressed on macrophages acts as a receptor for recognition of OMLs under physiological conditions.

Neutrophils play an important role in immune complex (IC)-mediated diseases, but the mechanisms underlying their recruitment to sites of IC deposition remain largely undefined. Fur- thermore, neutrophils encounter cytokines that prime their effector functions, yet the physiologi- cal relevance of priming to neutrophil functions is unclear. Using intravital microscopy, we demon- strate that TNF treatment of neutrophils ex vivo significantly increased their adhesion in a model of intravascular ICs deposited in the cremaster mus- cle. Notably, TNF priming had no effect on neu- trophil adhesion in the absence of ICs. Analyses of relevant knockout mice and neutrophil reconstitu- tion revealed a critical role for FcRs and the CD18 integrin Mac-1 in IC-mediated neutrophil adhesion. Furthermore, ICAM-1, a major Mac-1 ligand constitutively expressed on unactivated en- dothelium, significantly contributed to this pro- cess. These data suggest that TNF priming pro- motes FcR interaction with intravascular ICs, leading to the binding of Mac-1 to ICAM-1 and subsequent neutrophil arrest. J. Leukoc. Biol. 83: 1423-1430; 2008.

The protective immune response against the parasite, including the role of dendritic cells (DC) in the course of infection, plays a fundamental role. This study shows that wild-type (WT) Leishmania promastigotes and specifically the phosphoglycans family of virulence-associated antigens inhibit human monocyte-derived dendritic cells (MoDC) maturation and detachment to distinct surfaces. Immature phagocytosis of Leishmania donovani promastigotes by immature MoDC results in the increased expression of CD11b and CD51, and inhibition of cell detachment to distinct surfaces, which was dependent on the presence of phosphoglycans. These findings demonstrate that phosphoglycans of WT L. donovani might also inhibit human DC migration to lymphoid organs.

Candida albicans infections often occur during or shortly after antibacterial treatment. Phagocytosis by polymorphonuclear neutrophil granulocytes (PMN) is the most important primarily defence mechanism against C. albicans. Certain antibiotics such as some fluoroquinolones (FQ) are known to influence phagocyte functions. Thus, we investigated the influence of older and newer FQ on the phagocytosis and killing of C. albicans by human PMN paying special attention to CD11b expression of these cells as an indicator of the degree of their activation. In order to obtain comprehensive and comparable results we tested 13 FQ over a wide range of concentrations and in a time dependent manner in a standardized approach. When used at therapeutic concentrations, the FQ tested did not influence to a clinically significant degree the phagocytosis or the killing of C. albicans by human PMN and also not their activation. However, at high concentrations those FQ with cyclopropyl-moiety at position N1 showed increase in CD11b expression and diminished phagocytosis and oxidative burst.

In Crohn’s disease and ulcerative colitis patients, the numbers of neutrophils recovered from stool directly correlates with the severity of disease, implying that neutrophils in the lumen contribute to the tissue destruction; therefore, it is important to understand the mechanisms behind transintestinal epithelial migration. Neutrophil transintestinal epithelial migration to fMLP is appreciated to be CD11b/CD18 integrin (Mac-1)-dependent, while we recently reported that migration to C5a is Mac-1-independent. Here, we investigated whether phospholipase D (PLD), a signaling molecule linked to chemoattractant activation of neutrophils, is necessary for both Mac-1-dependent and Mac-1-independent migration. Both fMLP and C5a increased neutrophil expression of the Mac-1 activation epitope, indicating PLD was activated. This up-regulation was dose-dependently prevented by incubation of neutrophils in 1-butanol, an inhibitor of PLD activity. Despite this effect on Mac-1, 1-butanol did not prevent neutrophil migration across acellular filters. Incubation in 1-butanol did inhibit fMLP but not C5a-mediated migration across intestinal epithelial cell monolayers, showing that transepithelial migration to fMLP but not C5a is dependent on PLD. The addition of phosphatidic acid, a reaction product of PLD, partially restored fMLP-mediated transepithelial migration in the presence of 1-butanol but not the migration of Mac-1-deficient neutrophil-differentiated HL-60 cells. Thus PLD control over expression of the Mac-1 activation epitope is critical for neutrophil migration to fMLP but not C5a. Moreover, as PLD controls other neutrophil functions, such as the oxidative response, degranulation, and protease release, we could exclude these functions as being important in neutrophil transepithelial migration to C5a.

Membrane-activated complex 1 (Mac-1; CD11b/CD18) is a β2 integrin implicated in the pathophysiology of neutrophil-mediated tissue injury whose functional capacity is determined by stimulus-induced conformational activation rather than up-regulation. Mac-1 up-regulation and conformational activation, together with shedding of L-selectin, are reported after in vitro neutrophil activation. However, their regulation on circulating human neutrophils during acute inflammation is unclear. Using flow cytometry, we investigated neutrophil expression of Mac-1, its activation-reporter neo-epitope CBRM1/5, and L-selectin during the inflammatory stimulus of cardiac surgery. A subpopulation of circulating neutrophils expressed CBRM1/5 (CBRM1/5+) under basal conditions (6.28±2.59%) and was persistently expanded (9.95±4.0%–15.2±4.2%; P

Lifestyle changes which include daily exercise training have been shown to slow the progression of coronary artery disease. We designed a study to examine the effects of a multifactorial intervention on atherogenic adhesion molecules on the surface of monocytes in patients with coronary artery disease.We randomized 39 patients with coronary artery disease to (i) an intervention program which consisted of 4 weeks of daily 6x15 min ergometer training at submaximal intensity in addition to a 1 h/week group exercise session, followed by 5 months of home-based ergometer training of 30 min/day again in addition to a 1 h/week group exercise session or (ii) conventional therapy. All patients received a statin. Monocyte-bound cellular adhesion molecules LFA-1 (CD11a), MAC-1 (CD11b), VLA-4 (CD49d) and L-selectin (CD62L) were assessed by fluorescence activated cell sorting analysis.After 4 weeks the multifactorial intervention led to a significant improvement of maximal work capacity, lipid profile, body mass index, blood pressure, fasting glucose and hemoglobin A1c. This was associated with a reduced expression of MAC-1 and VLA-4. After 5 months of a home-based intervention the beneficial effects of the cardiovascular risk profile were still apparent, whereas the effects on the expression of adhesion molecules were blunted.In patients treated with statins, 4 weeks of high frequency and long duration exercise training led to a diminished expression of atherogenic adhesion molecules MAC-1 und VLA-4. After 5 months of home-based exercise training of moderate frequency and duration, these effects were blunted. Our data suggest that our patients in cardiac rehabilitation programs might further benefit from the antiatherogenic effects of an even higher amount of exercise training.

Hydroxytyrosol is a phenol found in olive oil. To verify the effect of hydroxytyrosol on the development of atherosclerosis, two groups of apo E deficient male mice on a standard chow diet were used: the control group receiving only water, and the second group an aqueous solution of hydroxytyrosol in order to provide a dose of 10 mg/kg/day to each mouse. This treatment was maintained for 10 weeks. At the moment of sacrifice, blood was drawn and heart removed. Plasma lipids, apolipoproteins and monocyte Mac-1 expression were assayed as well as aortic atherosclerotic areas in both groups. Data showed no significant changes in HDL cholesterol, paraoxonase, apolipoprotein B or triglyceride levels. However, hydroxytyrosol administration decreased apolipoprotein A-I and increased total cholesterol, atherosclerotic lesion areas and circulating monocytes expressing Mac-1. The latter was highly correlated with lesion areas (r = 0.65, P < 0.01). These results indicate that administration of hydroxytyrosol in low cholesterol diets increases atherosclerotic lesion associated with the degree of monocyte activation and remodelling of plasma lipoproteins. Our data supports the concept that phenolic-enriched products, out of the original matrix, could be not only non useful but also harmful. Our results suggest that the formulation of possible functional foods should approximate as much as possible the natural environment in which active molecules are found., This research was supported by grants FEGA-FEOGA (CAO99-0014), CICYT (SAF2004-08173-C03-02 and AGL2002-00495), Junta de Andalucía (CAO01-002), FISS 01/0202, Redes DGA (A-26) and FISS de investigación cooperativa C03-01 and G03-140 and by Fundación Española del Corazón., This research was supported by grants FEGA-FEOGA (CAO99-0014), CICYT (SAF2004-08173-C03-02 and AGL2002-00495), Junta de Andalucía (CAO01-002), FISS 01/0202, Redes DGA (A-26) and FISS de investigación cooperativa C03-01 and G03-140 and by Fundación Española del Corazón. R.C., S.A., M.A.N. and N.G. were recipient of DGA, FEGA-FEOGA and Fundación Cuenca Villoro fellowships.

Francisella tularensis is one of the most infectious human pathogens known. Although much has been learned about the immune response of mice using an attenuated live vaccine strain (LVS) derived from F. tularensis subspecies holarctica (Type B), little is known about the responses of human monocyte-derived immature dendritic cells (DC). Here, we show that optimal phagocytosis of LVS by DC is dependent on serum opsonization. We demonstrate that complement factor C3-derived opsonins and the major complement receptors expressed by DC, the integrins CR3 (CD11b/CD18) and CR4 (CD11c/CD18), play a critical role in this adhesion-mediated phagocytosis. LVS induced proinflammatory cytokine production and up-regulation of costimulatory surface proteins (CD40, CD86, and MHC Class II) on DC but resisted killing. Once taken up, LVS grew intracellularly, resulting in DC death. DC maturation and cytokine production were induced by direct contact/phagocytosis of LVS or interaction with soluble products of the bacteria, and enhanced activation was seen when LVS was pretreated with serum. Sonicated LVS and supernatants from LVS cultures were potent activators of DC, but LVS LPS failed to activate DC maturation or cytokine production. Serum-treated LVS rapidly induced (within 6 h) a number of cytokines including IL-10, a potent suppressor of macrophage functions and down-regulator of Th1-like responses and the Th1 response inducer IL-12. These results suggest that the simultaneous production of an activating (IL-12, IL-1β, and TNF-α) and a suppressing (IL-10) cytokine profile could contribute to the immunopathogenesis of tularemia.

Background: Theproteins ofthe complementcascade playan importantrolein inflammationand the immune response. Theyhavebeen shown to be activated during cardiopulmonary bypass (CPB), and may be responsible for the inflammatory response to CPB. We looked at the effect of APT070, an anti-complement agent, on human blood during in vitro CPB. Materials and methods: Four hundred millilitres of blood was venesected from healthy human volunteers and heparinised. To the blood was added either APT070 to a concentration of 50 mg/ml (n =5 ) or vehicle control (n = 4). The blood was entered into an in vitro CPB circuit and circulated for 90 min. Results: Our results showed that after 90 min of in vitro bypass APT070 significantly inhibited the activation of compliment as demonstrated by C3a (p = 0.03) and sC5b-9 (p = 0.01) levels, and reduced neutrophil stimulation as measured by CD11b expression (p = 0.04 at 90 min).Conclusion: APT070 significantly inhibits complement and neutrophilactivation.Thisresultmayhave considerableimplications, especially ifit canbe shownto decreasetheinflammatory sequelaeofCPB. # 2006 Published by Elsevier B.V.

Paraffin-embedded blocks from the thalamus of 9 control patients, 9 moderately disabled, 12 severely disabled, and 10 vegetative head-injured patients assessed using the Glasgow Outcome Scale and identified from the Department of Neuropathology archive. Neurons, astrocytes, macrophages, and activated microglia were differentiated by Luxol fast blue/cresyl violet, GFAP, CD68, and CR3/43 staining and stereological techniques used to estimate cell number in a 28-microm-thick coronal section. Counts were made in subnuclei of the mediodorsal, lateral posterior, and ventral posterior nuclei, the intralaminar nuclei, and the related internal lamina. Neuronal loss occurred from mediodorsal parvocellularis, rostral center medial, central lateral and paracentral nuclei in moderately disabled patients; and from mediodorsal magnocellularis, caudal center medial, rhomboid, and parafascicular nuclei in severely disabled patients; and all of the above and the centre median nucleus in vegetative patients. Neuronal loss occurred primarily from cognitive and executive function nuclei, a lesser loss from somatosensory nuclei and the least loss from limbic motor nuclei. There was an increase in the number of reactive astrocytes, activated microglia, and macrophages with increasing severity of injury. The study provides novel quantitative evidence for differential neuronal loss, with survival after human head injury, from thalamic nuclei associated with different aspects of cortical activation.

Hyaluronan (HA), a high-molecular-weight glycosaminoglycan ubiquitously present in the extracellular matrices (ECMs) of animals, plays important roles in ECM organization and cell behavior through binding to hyaluronan-binding proteins (HABPs). We previously reported that HA has anti-inflammatory effects on guinea pig phagocytes, although the nature of guinea pig HABPs was unknown. In this study, we characterized guinea pig HABPs on peritoneal polymorphonuclear leukocytes (PMNs) and blood neutrophils by flow cytometry and affinity chromatography. It was found that PMNs express diverse HABPs with different molecular weights. These HABPs maximally bound with HA over a wide pH range (6-8), and recognized HAs as small as the pentadisaccharide units of d-glucuronic acid and N-acetyl-d-glucosamine. Furthermore, they could be divided into Mg(2+)-dependent and Ca(2+)/Mg(2+)-independent groups. Interestingly, two proteins in the Mg(2+)-dependent group were found to be the two subunits of complement receptor type 3 (CR3, CD11b/CD18). Unlike PMNs, blood neutrophils expressed several functionally inactive HABPs. Among these inactive HABPs, Mg(2+)-dependent proteins including CR3 but not Ca(2+)/Mg(2+)-independent proteins were activated on phorbol ester-stimulation. These results show the existence of diverse HABPs on guinea pig neutrophils and the cell activation-dependent activation of HABPs. It is also suggested that the CR3-HA interaction is possibly involved in the regulation of neutrophil function.

Tuberculous pleuritis usually shows lymphocytic preponderance, but neutrophils are also present. Therefore, pleuritis is a good model for the study of neutrophil fate at sites of active Mycobacterium tuberculosis infection. We have previously demonstrated in vitro that M. tuberculosis-induced neutrophil apoptosis involves p38 mitogen protein kinase activation through Toll-like receptor 2. Herein, we demonstrate that, in tuberculous pleuritis, neutrophil apoptosis increases together with the expression of Toll-like receptor 2 and phosphorylated p38 (p-p38) kinase. In addition, receptors associated with activation/apoptotis (CD11b, CD64, tumor necrosis factor receptor, and Fas ligand) are up-regulated, together with a loss of CD16 expression. However, neutrophils express CD86, CD83, and major histocompatibility complex class II antigens, acquiring dendritic cell (DC) characteristics. Therefore, the cytokine milieu in the pleural space may influence signaling pathways on activated neutrophils, thereby inducing apoptosis and inhibiting their proinflammatory capacity, as well as allowing them acquire DC characteristics that influence the immune response.

Zymosan is a β-glucan, mannan-rich yeast particle widely used to activate the inflammatory response of immune cells. We studied the zymosan-binding potential of human dendritic cells (hDCs) by using specific carbohydrate inhibitors and blocking monoclonal antibodies. We show that DC-specific intercellular adhesion molecule-grabbing nonintegrin (DC-SIGN) is a major nonopsonic recognition receptor for zymosan on hDCs. Indeed, blocking of DC-SIGN inhibited the inflammatory response of DCs to zymosan. We compared the zymosan-binding capacity of hDC-SIGN to that of Dectin-1 and complement receptor 3 (CR3), which are receptors involved in the nonopsonic recognition of these yeast-derived particles. Dectin-1- and DC-SIGN-K562 cells bound to zymosan particles, whereas CR3-K562 cells did not. DC-SIGN and Dectin-1 were also expressed in COS cells to compare their ability to trigger particle internalization in a nonphagocytic cell line. DC-SIGN transfectants were unable to internalize bound particles, indicating that DC-SIGN is primarily involved in recognition but not in particle internalization. Zymosan induced a rapid DC aggregation that was accompanied by a dramatic change of DC-SIGN distribution in the plasma membrane. Under resting conditions, DC-SIGN was diffusely distributed through the cell surface, displaying clusters at the free leading edge. Upon zymosan treatment, DC-SIGN was markedly redistributed to cell–cell contacts, supporting an adhesion role in DC–DC interactions. The mechanism(s) supporting DC-SIGN-mediated intercellular adhesion were further investigated by using DC-SIGN-K562 aggregation. DC-SIGN was highly concentrated at points of cell–cell contact, suggesting a role for enhanced avidity during DC-SIGN-mediated intercellular adhesion.

Recruitment of leukocytes from bloodstream to extrahematic sites is tightly regulated by a variety of adhesion molecules that are expressed on the leukocytes and the vessel walls. In this manuscript, wedescribe the interactions between natural killer (NK) cells and activated, autologous platelets under physiologic flow. We found that surface-adherent human platelets are capable of recruiting human NK cells from flow and that this recruitment is characterized by an initial tethering followed by a rolling phase. Both phases were dependent on the adhesion molecule P-selectin and its counter-ligand on the NK cells (P-selectin glycoprotein ligand 1). Activation of rolling NK cells with inflammatory mediators commonly found in atherosclerotic plaques (interleukin-12 and leukotriene B4) causes immediate cessation of the rolling process and conversion to stationary adhesion. Blocking antibodies to the adhesion molecules membrane-activated complex-1 and leukocyte function antigen-1 inhibited this conversion. Our data suggest that platelets deposited at sites of vascular injury may provide an alternative substrate to endothelial cells for initial recruitment of NK cells to the vessel wall. This may result in extravasation of the NK cells if the appropriate chemotactic signal is applied. These data implicate the P-selectin and integrin family of adhesion molecules in the recruitment of NK cells to atherosclerotic sites.

The peripheral benzodiazepine receptor (PBR) has been used as a sensitive marker to visualize and measure glial cell activation associated with various forms of brain injury and inflammation. Previous studies have shown that increased PBR levels following brain injury are specific to areas expressing activated glial cells. However, the contribution of glial cell types responsible for the increases in PBR levels following brain injury is not well defined. In the present study, we used a murine model of cuprizone-induced demyelination to broaden the application of PBR as a marker of brain injury and to validate the relationship between PBR levels and glial cell types. C57BL/6J mice were maintained on a cuprizone-containing or control diet and sacrificed at specific time points after initiation of treatment. Quantitative autoradiography of the PBR-selective ligand [(3)H]-(R)-PK11195 and [(125)I]-(R)-PK11195 showed that increased PBR levels were associated with the degree of demyelination assessed by Black-Gold histochemistry and activation of glial cells assessed by glial fibrillary acidic protein (GFAP) immunohistochemistry for astrocytes and CD11b (Mac-1) for microglia. Our findings indicate that brain PBR levels increased as a function of dose and duration of cuprizone treatment and it was detectable prior to observable demyelination. Increased PBR levels were associated with the degree of demyelination and temporal activation of glial cell types in different anatomical regions. In the corpus striatum, we found a close anatomical correlation between microglial activation and increased PBR levels in demyelinating fibre tracts. In the deep cerebellar nuclei, the temporal increases in PBR paralleled demyelination and microglia and astrocyte activation. On the other hand, in the corpus callosum there was an apparent temporal shift in the increase in PBR levels by different glial cell types from an early and predominantly microglial contribution to a late microglial and astrocytic response. High-resolution emulsion autoradiography of [(3)H]-(R)-PK11195 binding to PBR coupled with GFAP or Mac-1 immunohistochemistry showed that demyelination-induced increases in PBR levels were co-localized to both microglia and astrocytes. These findings support the notion that PBR is a sensitive and specific marker for the in vitro and in vivo visualization and quantification of neuropathological changes in the brain.

Objective: We have previously shown that macrophage colony stimulating factor (M-CSF), a potent survival and mitogenic factor for monocytes/macrophages (MM), enables MM to induce vascular smooth muscle cell (VSMC) apoptosis. The killing requires the binding of MM to VSMC via Mac-1 (CD11b/CD18) on MM and intracellular adhesion molecule-1 (ICAM-1) on VSMC. We hypothesized that, in addition to Mac-1 binding, the killing process requires the activation of the Fas–death receptor pathway, which can be blocked at the level of Fas–Fas ligand interaction. Methods and Results: Human peripheral blood monocytes and VSMC were isolated and cultured as previously described. Soluble Fas (sFas) was overexpressed in VSMC by transduction using adenovirus specifying soluble Fas (Ad3hsFas). M-CSF markedly increased the expression of ICAM-1 in VSMC, resulting in enhanced clustering of MM on the surface of VSMC (≥3 MM per VSMC). MM, but not VSMC, expressed Fas-ligand (FasL), and VSMC apoptosis was inhibited by secretion of sFas by VSMC upon Ad3sFas transduction. Conclusions: MM and M-CSF-induced VSMC killing requires MM binding to VSMC mediated by Mac-1 and ICAM-1, and Fas–FasL interaction.

Methimazole (methyl-mercapto-imidazole, MMI), a compound used clinically in therapy of Graves’ thyroiditis, was found to inhibit development of several autoimmune diseases in animal models. It was suggested on the basis of in vitro data that inhibition is through down-regulation of interferon-γ (IFN-γ)-induced expression of major histocompatibility complex class I and class II molecules. Here, we investigate the effect of MMI on experimental autoimmune uveoretinitis (EAU) and study its mechanism(s). Treatment of EAU with MMI administered in drinking water inhibited induction of the disease and associated antigen (Ag)-specific proliferation and cytokine production by draining lymph node cells (LNCs). The treatment was protective only if administered during the first but not during the second week after immunization, suggesting an effect on the induction phase of EAU. It is interesting that MMI inhibited disease in IFN-γ knockout mice, indicating that the in vivo protective effect is IFN-γ-independent. Flow cytometric analysis of draining LNCs extracted 5 days after immunization showed that MMI partly to completely reversed the increase in Mac-1+/class I+/class II+ cells induced by immunization and reduced the proportion of B7-1 and CD40-positive cells, suggesting a deficit in the Ag-presenting cell (APC) population. APC from untreated mice largely restored antigen-specific proliferation of MMI-treated LNCs. We suggest that MMI inhibits EAU at least in part by preventing the recruitment and/or maturation of APC, resulting in reduced generation of Ag-specific T cells.

Glucosamine, an amino monosaccharide naturally occurring in the connective and cartilage tissues, contributes to maintaining the strength, flexibility, and elasticity of these tissues. In recent years, glucosamine has been used widely to treat osteoarthritis in humans and animal models. Neutrophils, which usually function as the primary defenders in bacterial infections, are also implicated in the destructive, inflammatory responses in arthritis. In this study, we have evaluated the effects of glucosamine on neutrophil functions using human peripheral blood neutrophils. Glucosamine (0.01–1 mM) dose-dependently suppressed the superoxide anion generation induced by formyl-Met-Leu-Phe (fMLP) or complement-opsonized zymosan and inhibited the phagocytosis of complement-opsonized zymosan or IgG-opsonized latex particles. Furthermore, glucosamine inhibited the release of granule enzyme lysozyme from phagocytosing neutrophils and suppressed neutrophil chemotaxis toward zymosan-activated serum. In addition, glucosamine inhibited fMLP-induced up-regulation of CD11b significantly, polymerization of actin, and phosphorylation of p38 mitogen-activated protein kinase (MAPK). In contrast, N-acetyl-glucosamine, an analogue of glucosamine, did not affect these neutrophil functions (superoxide generation, phagocytosis, granule enzyme release, chemotaxis, CD11b expression, actin polymerization, and p38 MAPK phosphorylation) at the concentrations examined (1–10 mM). Together these observations likely suggest that glucosamine suppresses the neutrophil functions, thereby possibly exhibiting anti-inflammatory actions in arthritis.

The effect of beta-1,3-D-glucans SSG, from Sclerotinia sclerotiorum, or soluble (sMG) or particulate (pMG) MacroGard from baker's yeast on growth of Mycobacterium tuberculosis H37Rv in cultures of peritoneal macrophages from BALB/c mice was examined. After 24 h intracellular bacteria from lysed macrophages were cultured and the number of cfu counted. SSG given with challenge, but not 24 h after, reduced the number of M. tuberculosis cfu significantly. pMG, but not sMG, given with challenge had an even stronger inhibitory effect, which was enhanced after serum opsonization of the particles. The effect of serum-treated pMG was abrogated by addition of a monoclonal antibody to CD11b. The results indicate that beta-glucans inhibit growth of M. tuberculosis in host cells in vitro, probably due to cellular stimulation and/or competitive inhibition of uptake of bacteria via CR3 (CD11b/18).

Previously, we described a novel glycosylphosphatidyl inositol (GPI)-anchored glycoprotein (designated GPI-80) on human neutrophils and monocytes that may regulate β2 integrin-dependent neutrophil adherence and migration. However, the mechanism regulating β2 integrin remains to be clarified. To study this, we examined changes in β2 integrin expression and function caused by cross-linking GPI-80. GPI-80 cross-linking induced up-regulation of CD11b/CD18 (Mac-1) expression on neutrophil surfaces and shedding of L-selectin, which depends on tyrosine phosphorylation and cytoskeleton remodeling. Furthermore, the cross-linking enhanced fMLP-induced human neutrophil adherence. These results suggest that GPI-80 may be a regulator of β2 integrin in neutrophils.

We have investigated the expression of TLR2 and Dectin-1 in retinal microglia and their involvement in Candida albicans phagocytosis using a cytometric approach. The expression of both receptors has been demonstrated in CD11b+ retinal cells. Phagocytosis of pHrodo-labelled C. albicans yeasts by microglial CD11b+ cells of C57BL/6 mice was inhibited both by the Dectin-1 antagonist laminarin and anti-Dectin-1 antibodies, whereas phagocytosis of yeasts by retinal microglia of TLR2 KO mice was unaffected. These data indicate that phagocytosis of C. albicans yeasts by retinal microglia is mediated by Dectin-1, whereas TLR2 does not play a significant role in this process.

SummaryLeucocyte adhesion deficiency (LAD) is a hereditary disorder caused by mutations in the CD18 (β2 integrin) gene. Four missense mutations have been identified in three patients. CD18(A270V) supports, at a diminished level, CD11b/CD18 (Mac-1, αMβ2 integrin) and CD11c/CD18 (p150,95, αXβ2 integrin) expression and function but not CD11a/CD18 (LFA-1, αLβ2 integrin) expression. Conversely, CD18(A341P) supports a limited level of expression and function of CD11a/CD18, but not of the other two CD11/CD18 antigens. CD18(C590R) and CD18(R593C) show a decreasing capacity to associate with the CD11a, CD11c and CD11b subunits. Transfectants expressing the CD11a/CD18 with the C590R and R593C mutations are more adhesive than transfectants expressing wild-type LFA-1, and express the reporter epitope of the monoclonal antibody 24 constitutively. Thus, the four mutations affect CD18 differently in its capacities to support CD11/CD18 expression and adhesion. These results not only provide a biochemical account for the clinical diversity of patients with leucocyte adhesion deficiency, but also offer novel insights into the structural basis of interaction between the α and β subunits, which is an integral component in our understanding of integrin-mediated adhesion and its regulation.