Effective Model of Food Allergy in Mice Sensitized with Ovalbumin and Freud's Adjuvant.

To better explore the pathophysiology of FA and its therapy, we aimed to establish a simple and practicable FA model with Freund's adjuvant and introduce an easy and reliable laboratory evaluation method for assessment of inflammation in intestinal segments at different anatomical locations. BALB/c mice were sensitized with ovalbumin combined with Freund's adjuvant. Complete Freund's adjuvant was chosen for the first sensitization and two weeks later incomplete Freund's adjuvant was used for a second sensitization. Two weeks later, the sensitized mice were challenged with 50 mg ovalbumin every other day. After the 6 challenge, all mice were assessed for systemic anaphylaxis, and then sacrificed for sample collection. All sensitized mice showed anaphylactic symptoms and markedly increased levels of serum ovalbumin-specific IgE and IgG1. The activity of mast cell protease-1 (mMCPT-1) was significantly increased in the serum and interstitial fluid of the duodenum, jejunum, ileum, and colon. A successful FA model was established, of which inflammation occurred in the duodenum, jejunum, ileum, and colon. This model provides a reliable and simple tool for analysis of the mechanism of FA and methods of immunotherapy. Moreover, combined detection of ovalbumin-specific antibody and local mMCPT-1 levels could potentially be used as the major indicator for assessment of food allergy.

Shedding Light into the Subcutis: A Mass Spectrometry Based Immunocapture Assay Enabling Full Characterization of Therapeutic Antibodies after Injection in Vivo.

An understanding of what happens to therapeutic antibodies in vivo after subcutaneous injection is of high interest. Therefore, we applied the open flow microperfusion technique to extract interstitial fluid from the subcutaneous tissue. In order to analyze those biological samples, a specific and sensitive workflow was required. In this study, we present a complete workflow that enables full characterization of therapeutic antibodies after subcutaneous injection. Compared to classical pharmacokinetic approaches where only a limited number of peptides are detected, our workflow provides full sequence coverage and even enables the identification of potential quality attributes. The efficiency to purify therapeutic antibodies from biological matrixes of two different antibody capture molecules and two types of magnetic beads was compared. Furthermore, several desalting protocols were tested in the development of a miniaturized peptide map procedure. The best results were achieved using a commercial anti-human capture mAb fragment in combination with streptavidin coated magnetic beads, providing capture efficiencies of 90-100%. The optimized peptide map protocol that requires <1 µg of mAb includes two desalting steps and showed sequence coverages of 95-100%. The final method was successfully used for analysis of interstitial fluid and serum samples after a subcutaneous injection of a therapeutic antibody into a domestic pig.

Characterization of the Micro-Environment of the Testis that Shapes the Phenotype and Function of Testicular Macrophages.

Macrophages are important in the activation of innate immune responses and in a tissue-specific manner in the maintenance of organ homeostasis. Testicular macrophages (TM), which reside in the testicular interstitial space, comprise the largest leukocyte population in the testes and are assumed to play a relevant function in maintaining testicular immune privilege. Numerous studies have indicated that the interstitial fluid (IF) surrounding the TM has immunosuppressive properties, which may influence the phenotype of TM. However, the identity of the immunosuppressive molecules present in the IF is poorly characterized. We show that the rat testicular IF shifted GM-CSF-induced M1 toward the M2 macrophage phenotype. IF-polarized M2 macrophages mimic the properties of TM, such as increased expression of CD163, high secretion of IL-10, and low secretion of TNF-α. In addition, IF-polarized macrophages display immunoregulatory functions by inducing expansion of immunosuppressive regulatory T cells. We further found that corticosterone was the principal immunosuppressive molecule present in the IF and that the glucocorticoid receptor is needed for induction of the testis-specific phenotype of TM. In addition, TM locally produce small amounts of corticosterone, which suppresses the basal expression of inflammatory genes as a means to render TM refractory to inflammatory stimuli. Taken together, these results suggest that the corticosterone present in the testicular environment shapes the immunosuppressive function and phenotype of TM and that this steroid may play an important role in the establishment and sustenance of the immune privilege of the testis.

Uric acid induces NADPH oxidase-independent neutrophil extracellular trap formation.

Neutrophil extracellular traps (NETs) are composed of extracellular DNA fibers with antimicrobial peptides that capture and kill microbes. NETs play a critical role in innate host defense and in autoimmune and inflammatory diseases. While the mechanism of NET formation remains unclear, reactive oxygen species (ROS) produced via activation of NADPH oxidase (Nox) are known to be an important requirement. In this study, we investigated the effect of uric acid (UA) on NET formation. UA, a well-known ROS scavenger, was found to suppress Nox-dependent ROS release in a dose-dependent manner. Low concentrations of UA significantly inhibited Nox-dependent NET formation. However, high concentrations of UA unexpectedly induced, rather than inhibited, NET formation. NETs were directly induced by UA alone in a Nox-independent manner, as revealed by experiments using control neutrophils treated with ROS inhibitors or neutrophils of patients with chronic granulomatous disease who have a congenital defect in ROS production. Furthermore, we found that UA-induced NET formation was partially mediated by NF-κB activation. Our study is the first to demonstrate the novel function of UA in NET formation and may provide insight into the management of patients with hyperuricemia.

A serpinB1 regulatory mechanism is essential for restricting neutrophil extracellular trap generation.

NETosis (neutrophil extracellular trap [NET] generation), a programmed death pathway initiated in mature neutrophils by pathogens and inflammatory mediators, can be a protective process that sequesters microbes and prevents spread of infection, but it can also be a pathological process that causes inflammation and serious tissue injury. Little is known about the regulatory mechanism. Previously, we demonstrated that serpinb1-deficient mice are highly susceptible to pulmonary bacterial and viral infections due to inflammation and tissue injury associated with increased neutrophilic death. In this study, we used in vitro and in vivo approaches to investigate whether SerpinB1 regulates NETosis. We found that serpinb1-deficient bone marrow and lung neutrophils are hypersusceptible to NETosis induced by multiple mediators in both an NADPH-dependent and -independent manner, indicating a deeply rooted regulatory role in NETosis. This role is further supported by increased nuclear expansion (representing chromatin decondensation) of PMA-treated serpinb1-deficient neutrophils compared with wild-type, by migration of SerpinB1 from the cytoplasm to the nucleus of human neutrophils that is coincident with or preceding early conversion of lobulated (segmented) nuclei to delobulated (spherical) morphology, as well as by the finding that exogenous human recombinant SerpinB1 abrogates NET production. NETosis of serpinb1-deficient neutrophils is also increased in vivo during Pseudomonas aeruginosa lung infection. The findings identify a previously unrecognized regulatory mechanism involving SerpinB1 that restricts the production of NETs.

Extracellularly delivered single-stranded viral RNA causes neurodegeneration dependent on TLR7.

Innate immune receptors represent an evolutionarily ancient system that allows organisms to detect and rapidly respond to pathogen- and host-derived factors. TLRs are predominantly expressed in immune cells and mediate such a response. Although this class of pattern recognition receptors is involved in CNS disorders, the knowledge of ligands leading to activation of TLRs and to subsequent CNS damage is limited. We report in this study that ssRNA causes neurodegeneration and neuroinflammation dependent on TLR7 in the CNS. TLR7 is not only expressed in microglia, the major immune cells of the brain, but also in neurons of the CNS. Extracellularly delivered ssRNA40, an oligoribonucleotide derived from HIV and an established ligand of TLR7, induces neuronal cell death dependent on TLR7 and the central adapter molecule MyD88 in vitro. Activation of caspase-3 is involved in neuronal damage mediated by TLR7. This cell-autonomous neuronal cell death induced by ssRNA40 is amplified in the presence of microglia that mount an inflammatory response to ssRNA40 through TLR7. Intrathecal administration of ssRNA40 causes widespread neurodegeneration in wild-type but not in TLR7(-/-) mice, confirming that neuronal cell death induced by ssRNA40 through TLR7 occurs in vivo. Our results point to a possible mechanism through which extracellularly delivered ssRNA contributes to CNS damage and determine an obligatory role for TLR7 in this pathway.

Tissue-resident ecto-5' nucleotidase (CD73) regulates leukocyte trafficking in the ischemic brain.

Ectoenzymes expressed on the surface of vascular cells and leukocytes modulate the ambient nucleotide milieu. CD73 is an ecto-5' nucleotidase that catalyzes the terminal phosphohydrolysis of AMP and resides in the brain on glial cells, cells of the choroid plexus, and leukocytes. Though CD73 tightens epithelial barriers, its role in the ischemic brain remains undefined. When subjected to photothrombotic arterial occlusion, CD73(-/-) mice exhibited significantly larger (49%) cerebral infarct volumes than wild-type mice, with concordant increases in local accumulation of leukocyte subsets (neutrophils, T lymphocytes, macrophages, and microglia). CD73(-/-) mice were rescued from ischemic neurologic injury by soluble 5'-nucleotidase. In situ, CD73(-/-) macrophages upregulated expression of costimulatory molecules far more than wild-type macrophages, with a sharp increase of the CD80/CD86 ratio. To define the CD73-bearing cells responsible for ischemic cerebroprotection, mice were subjected to irradiative myeloablation, marrow reconstitution, and then stroke following engraftment. Chimeric mice lacking CD73 in tissue had larger cerebral infarct volumes and more tissue leukosequestration than did mice lacking CD73 on circulating cells. These data show a cardinal role for CD73 in suppressing ischemic tissue leukosequestration. This underscores a critical role for CD73 as a modulator of brain inflammation and immune function.

Extracellular ATP exerts opposite effects on activated and regulatory CD4+ T cells via purinergic P2 receptor activation.

It has been reported that ATP inhibits or stimulates lymphoid cell proliferation depending on the cellular subset analyzed. In this study, we show that ATP exerts strikingly opposite effects on anti-CD3/CD28-activated and regulatory CD4(+) T cells (T(regs)), based on nucleotide concentration. We demonstrate that physiological concentrations of extracellular ATP (1-50 nM) do not affect activated CD4(+) T cells and T(regs). Conversely, higher ATP concentrations have a bimodal effect on activated CD4(+) T cells. Whereas 250 nM ATP stimulates proliferation, cytokine release, expression of adhesion molecules, and adhesion, 1 mM ATP induces apoptosis and inhibits activated CD4(+) T cell functions. The expression analysis and pharmacological profile of purinergic P2 receptors for extracellular nucleotides suggest that activated CD4(+) T cells are induced to apoptosis via the upregulation and engagement of P2X7R and P2X4R. On the contrary, 1 mM ATP enhances proliferation, adhesion, migration, via P2Y2R activation, and immunosuppressive ability of T(regs). Similar results were obtained when activated CD4(+) T cells and T(regs) were exposed to ATP released by necrotized leukemic cells. Taken together, our results show that different concentrations of extracellular ATP modulate CD4(+) T cells according to their activated/regulatory status. Because extracellular ATP concentration highly increases in fast-growing tumors or hyperinflamed tissues, the manipulation of purinergic signaling might represent a new therapeutic target to shift the balance between activated CD4(+) T cells and T(regs).

Involvement of cross-linked ribosomal protein S19 oligomers and C5a receptor in definitive erythropoiesis.

We performed a series of experiments under a working hypothesis that cross-linked oligomers of ribosomal protein S19 (RP S19) play an essential role in definitive erythropoiesis as a ligand of the C5a receptor of erythroblasts and macrophages. We found molecules functionally and immunologically indistinguishable from RP S19 oligomers in the extracellular fluid of porcine and guinea pig bone marrow. When an increased hematopoietic state was induced in guinea pigs by bloodletting, the bone marrow RP S19 oligomer concentration was concomitantly increased. However, when the RP S19 oligomers were immunologically neutralized or the C5a receptor was pharmacologically antagonized, hyper-erythropoiesis induced by bloodletting was prevented and the anemic state was retarded in guinea pigs. When the RP S19 oligomers were neutralized in mice after bloodletting, the reactive hyper proliferation of erythroblasts in the spleen was prevented. Proerythroblasts and erythroblasts prepared by bone marrow aspiration from healthy individuals were found to express significant levels of the C5a receptor and type 2 transglutaminase genes. Majority of erythroblasts in cord blood of healthy newborns bore the C5a receptor. Taken together, these results support our hypothesis.

An essential role of STIM1, Orai1, and S100A8-A9 proteins for Ca2+ signaling and FcγR-mediated phagosomal oxidative activity.

Phagocytosis is a process of innate immunity that allows for the enclosure of pathogens within the phagosome and their subsequent destruction through the production of reactive oxygen species (ROS). Although these processes have been associated with increases of intracellular Ca(2+) concentrations, the mechanisms by which Ca(2+) could regulate the different phases of phagocytosis remain unknown. The aim of this study was to investigate the Ca(2+) signaling pathways involved in the regulation of FcγRs-induced phagocytosis. Our work focuses on IgG-opsonized zymosan internalization and phagosomal ROS production in DMSO-differentiated HL-60 cells and neutrophils. We found that chelation of intracellular Ca(2+) by BAPTA or emptying of the intracellular Ca(2+) store by thapsigargin reduced the efficiency of zymosan internalization. Using an small interfering RNA strategy, our data establish that the observed Ca(2+) release occurs through two isoforms of inositol 1,4,5-triphosphate receptors, ITPR1 and ITPR3. In addition, we provide evidence that phagosomal ROS production is dependent on extracellular Ca(2+) entry. We demonstrate that the observed Ca(2+) influx is supported by ORAI calcium release-activated calcium modulator 1 (Orai1) and stromal interaction molecule 1 (STIM1). This result suggests that extracellular Ca(2+) entry, which is required for ROS production, is mediated by a store-operated Ca(2+) mechanism. Finally, our data identify the complex formed by S100A8 and S100A9 (S100 calcium-binding protein A8 and A9 complex), two Ca(2+)-binding proteins, as the site of interplay between extracellular Ca(2+) entry and intraphagosomal ROS production. Thus, we demonstrate that FcγR-mediated phagocytosis requires intracellular Ca(2+) store depletion for the internalization phase. Then phagosomal ROS production requires extracellular Ca(2+) entry mediated by Orai1/STIM1 and relayed by S100A8-A9 as Ca(2+) sensor.

Extracellular ATP may contribute to tissue repair by rapidly stimulating purinergic receptor X7-dependent vascular endothelial growth factor release from primary human monocytes.

Extracellular ATP has been proposed to act as a danger signal to alert the immune system of cell damage. Release of high local concentrations of ATP activates the nucleotide receptor, purinergic receptor X7 (P2RX7), on monocytic cells, which promotes the processing/release of proinflammatory mediators. Although the proinflammatory actions of P2RX7 are well recognized, little is known regarding the potential function of P2RX7 in repair responses. Because the resolution of inflammation is characterized by monocytic cell-dependent production of proangiogenic factors, we evaluated the contribution of P2RX7 to this process. We observed that both short-term and long-term P2RX7 activation promotes the robust release of vascular endothelial growth factor from primary human monocytes. This vascular endothelial growth factor release is calcium dependent and associated with reactive oxygen species production. This previously unrecognized action of P2RX7 suggests that it may not only participate in inflammation and cell death, but that it is also likely to be important in the control of angiogenesis and wound repair.

A New Method for Obtaining Tumor Interstitial Fluid Applied to Cytokine Analysis of Breast Carcinoma Samples.

BACKGROUND/AIM: Tumor interstitial fluid (TIF), a component of the tumor microenvironment, is a valuable source of molecules and substances that help in diagnosis and prognosis of solid tumors. There is still no consensus on the optimal method for collecting TIF. Therefore, this study aimed to evaluate the effectiveness of a new method of collecting TIF in invasive ductal carcinoma (IDC) samples for cytokine interleukin 1ß (IL1ß) quantification. MATERIALS AND METHODS: Forty women allowed the collection of TIF using absorbent paper strips during the performance of the core biopsy. The samples were stored at a temperature of -80°C and then analyzed using an enzyme-linked immunoassay. RESULTS: The mean values for IL1ß and total protein were 11.39 mg/ml and 2.15 mg/ml, respectively. CONCLUSION: it was possible to quantify the cytokine IL1ß and the total protein concentration present in the tumor tissue through TIF collection with the use of absorbent paper filters, demonstrating the effectiveness of this new method in oncology.

Pathophysiology of tissue fluid accumulation in inflammation.

The potential role of extravascular factors for the local as well as systemic response to an inflammatory stimulus is addressed here in light of recent data from the trachea, serving as a surrogate for lower airways, and spleen, because of its role in the immune response and fluid volume regulation. From analysis of interstitial fluid from trachea it is apparent that the colloid osmotic pressure is high relative to plasma, suggesting a significant buffering capacity against oedema formation, and also that there is a significant local production of proinflammatory mediators to a systemic inflammatory stimulus. Inflammatory stimuli may furthermore result in a rapid reduction in interstitial fluid pressure, thus leading to increased filtration and oedema formation. Knowledge regarding the fluid phase within the spleen microenvironment can be gathered via analysis of drained lymph. During a septic response induced by lipopolysaccharide injection, the spleen contributes significantly to the production of pro- and anti-inflammatory cytokines, and may induce protracted inflammation because of a dominant role in IL-6 production. Significant amounts of immune cells exit via lymph, and acquire specific activation signatures having been exposed to the spleen microenvironment. Although often overlooked, extravascular or interstitial factors may therefore contribute significantly to the inflammatory process and thus the ensuing oedema associated with inflammation.

Binding of factor H to tubular epithelial cells limits interstitial complement activation in ischemic injury.

Factor H is a regulator of the alternative pathway of complement, and genetic studies have shown that patients with mutations in factor H are at increased risk for several types of renal disease. Pathogenic activation of the alternative pathway in acquired diseases, such as ischemic acute kidney injury, suggests that native factor H has a limited capacity to control the alternative pathway in the kidney. Here we found that an absolute deficiency of factor H produced by gene deletion prevented complement activation on tubulointerstitial cells after ischemia/reperfusion (I/R) injury, likely because alternative pathway proteins were consumed in the fluid phase. In contrast, when fluid-phase regulation by factor H was maintained while the interaction of factor H with cell surfaces was blocked by a recombinant inhibitor protein, complement activation after renal I/R increased. Finally, a recombinant form of factor H, specifically targeted to sites of C3 deposition, reduced complement activation in the tubulointerstitium after ischemic injury. Thus, although factor H does not fully prevent activation of the alternative pathway of complement on ischemic tubules, its interaction with the tubule epithelial cell surface is critical for limiting complement activation and attenuating renal injury after ischemia.

NADPH oxidase NOX2 mediates rapid cellular oxidation following ATP stimulation of endotoxin-primed macrophages.

The phagocytic NADPH oxidase (NOX2) plays a fundamental role in host defense and innate immunity. Here we demonstrate that external ATP triggers rapid cellular oxidation inhibited by diphenyleneiodonium in endotoxin-primed J774 macrophages and primary murine bone marrow-derived macrophages. To identify the source of reactive oxygen species (ROS), we compared responses between wild-type and NOX2-deficient macrophages. ATP-mediated ROS production was strongly attenuated in NOX2-deficient macrophages where responses were comparable to inhibition with diphenyleneiodonium. Notably, spatial differences in superoxide anion formation were observed where ROS formation was partially antagonized by extracellular superoxide dismutase in primary bone marrow-derived macrophages but unaffected in J774 macrophages. Loss of NOX2 was not observed to affect ATP-induced cell death. However, ATP-evoked cell death was found to be partially dependent on caspase-1 and cathepsin B activation. In conclusion, NOX2 plays a fundamental role in conferring macrophages with the ability to respond to extracellular ATP stimulation with robust changes in cellular oxidation.

Matrix metalloproteinase (MMP)-1 and MMP-3 induce macrophage MMP-9: evidence for the role of TNF-alpha and cyclooxygenase-2.

Matrix metalloproteinase (MMP)-9 (gelatinase B) participates in a variety of diverse physiologic and pathologic processes. We recently characterized a cyclooxygenase-2 (COX-2)-->PGE(2)-->EP4 receptor axis that regulates macrophage MMP-9 expression. In the present studies, we determined whether MMPs, commonly found in inflamed and neoplastic tissues, regulate this prostanoid-EP receptor axis leading to enhanced MMP-9 expression. Results demonstrate that exposure of murine peritoneal macrophages and RAW264.7 macrophages to MMP-1 (collagenase-1) or MMP-3 (stromelysin-1) lead to a marked increase in COX-2 expression, PGE(2) secretion, and subsequent induction of MMP-9 expression. Proteinase-induced MMP-9 expression was blocked in macrophages preincubated with the selective COX-2 inhibitor celecoxib or transfected with COX-2 small interfering RNA (siRNA). Likewise, proteinase-induced MMP-9 was blocked in macrophages preincubated with the EP4 antagonist ONO-AE3-208 or transfected with EP4 siRNA. Exposure of macrophages to MMP-1 and MMP-3 triggered the rapid release of TNF-alpha, which was blocked by MMP inhibitors. Furthermore, both COX-2 and MMP-9 expression were inhibited in macrophages preincubated with anti-TNF-alpha IgG or transfected with TNF-alpha siRNA. Thus, proteinase-induced MMP-9 expression by macrophages is dependent on the release of TNF-alpha, induction of COX-2 expression, and PGE(2) engagement of EP4. The ability of MMP-1 and MMP-3 to regulate macrophage secretion of PGE(2) and expression of MMP-9 defines a nexus between MMPs and prostanoids that is likely to play a role in the pathogenesis of chronic inflammatory diseases and cancer. These data also suggest that this nexus is targetable utilizing anti-TNF-alpha therapies and/or selective EP4 antagonists.

Extracellular Lactate Acts as a Metabolic Checkpoint and Shapes Monocyte Function Time Dependently.

Elevated blood lactate levels are frequently found in critically ill patients and thought to result from tissue hypoperfusion and cellular oxygen shortage. Considering the close relationship between immune cell function and intracellular metabolism, lactate is more than a glycolytic waste molecule but able to regulate the immune response. Our aim was to elucidate the temporal and mechanistic effect of extracellular lactate on monocytes. To this end, primary human monocytes and the human monocytic cell line MonoMac6 were stimulated with various toll-like-receptor agonists after priming with Na-L-lactate under constant pH conditions. As readout, cytokine production was measured, real-time assessment of intracellular energy pathways was performed, and intracellular metabolite concentrations were determined. Irrespective of the immunogenic stimulus, short-term Na-lactate-priming strongly reduced cytokine production capacity. Lactate and hexoses accumulated intracellularly and, together with a decreased glycolytic flux, indicate a lactate-triggered impairment of glycolysis. To counteract intracellular hyperglycemia, glucose is shunted into the branching polyol pathway, leading to sorbitol accumulation. In contrast, long-term priming with Na-L-lactate induced cellular adaption and abolished the suppressive effect. This lactate tolerance is characterized by a decreased cellular respiration due to a reduced complex-I activity. Our results indicate that exogenous lactate shapes monocyte function by altering the intracellular energy metabolism and acts as a metabolic checkpoint of monocyte activation.

Redox remodeling as an immunoregulatory strategy.

Activation and proliferation of T cells require a reducing extracellular microenvironment in the immune synapse that is provided by antigen presenting cells, especially dendritic cells. Stimulation of dendritic cells by T cells activates the NF-kappaB pathway in dendritic cells and induces an antioxidant response. It also enhances system x(c)(-)-dependent cystine uptake, leading to enhanced glutathione synthesis, export, and, finally, degradation to cysteine outside the cell. Accumulation of extracellular cysteine supports glutathione synthesis in T cells while also leading to a more reducing redox potential that is needed for T cell proliferation. Naturally occurring regulatory T cells, a suppressor subpopulation of T cells, prevent autoimmune diseases and maintain peripheral tolerance by suppressing self-reactive effector T cells. They also suppress beneficial immune responses to parasites, viruses, and tumors. However, their mechanism of suppression is still not fully understood. Recently, we have found that inhibition by regulatory T cells of dendritic cell-induced extracellular redox remodeling is a component of the regulatory T cell suppression mechanism. In this review, we describe recent advances in our understanding of redox regulation and signaling in the adaptive immune system with a focus on T cell activation by dendritic cells. The role of regulatory T cells in perturbing redox remodeling by dendritic cells and its implications as a general regulatory T cell suppression mechanism are discussed.

Differential cell reaction upon Toll-like receptor 4 and 9 activation in human alveolar and lung interstitial macrophages.

BACKGROUND: Investigations on pulmonary macrophages (MΦ) mostly focus on alveolar MΦ (AM) as a well-defined cell population. Characteristics of MΦ in the interstitium, referred to as lung interstitial MΦ (IM), are rather ill-defined. In this study we therefore aimed to elucidate differences between AM and IM obtained from human lung tissue. METHODS: Human AM and IM were isolated from human non-tumor lung tissue from patients undergoing lung resection. Cell morphology was visualized using either light, electron or confocal microscopy. Phagocytic activity was analyzed by flow cytometry as well as confocal microscopy. Surface marker expression was measured by flow cytometry. Toll-like receptor (TLR) expression patterns as well as cytokine expression upon TLR4 or TLR9 stimulation were assessed by real time RT-PCR and cytokine protein production was measured using a fluorescent bead-based immunoassay. RESULTS: IM were found to be smaller and morphologically more heterogeneous than AM, whereas phagocytic activity was similar in both cell types. HLA-DR expression was markedly higher in IM compared to AM. Although analysis of TLR expression profiles revealed no differences between the two cell populations, AM and IM clearly varied in cell reaction upon activation. Both MΦ populations were markedly activated by LPS as well as DNA isolated from attenuated mycobacterial strains (M. bovis H37Ra and BCG). Whereas AM expressed higher amounts of inflammatory cytokines upon activation, IM were more efficient in producing immunoregulatory cytokines, such as IL10, IL1ra, and IL6. CONCLUSION: AM appear to be more effective as a non-specific first line of defence against inhaled pathogens, whereas IM show a more pronounced regulatory function. These dissimilarities should be taken into consideration in future studies on the role of human lung MΦ in the inflammatory response.

Immune evasion of Enterococcus faecalis by an extracellular gelatinase that cleaves C3 and iC3b.

Enterococcus faecalis (Ef) accounts for most cases of enterococcal bacteremia, which is one of the principal causes of nosocomial bloodstream infections (BSI). Among several virulence factors associated with the pathogenesis of Ef, an extracellular gelatinase (GelE) has been known to be the most common factor, although its virulence mechanisms, especially in association with human BSI, have yet to be demonstrated. In this study, we describe the complement resistance mechanism of Ef mediated by GelE. Using purified GelE, we determined that it cleaved the C3 occurring in human serum into a C3b-like molecule, which was inactivated rapidly via reaction with water. This C3 convertase-like activity of GelE was shown to result in a consumption of C3 and thus inhibited the activation of the complement system. Also, GelE was confirmed to degrade an iC3b that was deposited on the Ag surfaces without affecting the bound C3b. This proteolytic effect of GelE against the major complement opsonin resulted in a substantial reduction in Ef phagocytosis by human polymorphonuclear leukocytes. In addition, we verified that the action of GelE against C3, which is a central component of the complement cascade, was human specific. Taken together, it was suggested that GelE may represent a promising molecule for targeting human BSI associated with Ef.