Necrotic cell-derived high mobility group box 1 attracts antigen-presenting cells but inhibits hepatocyte growth factor-mediated tropism of mesenchymal stem cells for apoptotic cell death.

Tissue damage due to apoptotic or necrotic cell death typically initiates distinct cellular responses, leading either directly to tissue repair and regeneration or to immunological processes first, to clear the site, for example, of potentially damage-inducing agents. Mesenchymal stem cells (MSC) as well as immature dendritic cells (iDC) and monocytes migrate to injured tissues. MSC have regenerative capacity, whereas monocytes and iDC have a critical role in inflammation and induction of immune responses, including autoimmunity after tissue damage. Here, we investigated the influence of apoptotic and necrotic cell death on recruitment of MSC, monocytes and iDC, and identified hepatocyte growth factor (HGF) and the alarmin high mobility group box 1 (HMGB1) as key factors differentially regulating these migratory responses. MSC, but not monocytes or iDC, were attracted by apoptotic cardiomyocytic and neuronal cells, whereas necrosis induced migration of monocytes and iDC, but not of MSC. Only apoptotic cell death resulted in HGF production and HGF-mediated migration of MSC towards the apoptotic targets. In contrast, HMGB1 was predominantly released by the necrotic cells and mediated recruitment of monocytes and iDC via the receptor of advanced glycation end products. Moreover, necrotic cardiomyocytic and neuronal cells caused an HMGB1/toll-like receptor-4-dependent inhibition of MSC migration towards apoptosis or HGF, while recruitment of monocytes and iDC by necrosis or HMGB1 was not affected by apoptotic cells or HGF. Thus, the type of cell death differentially regulates recruitment of either MSC or monocytes and iDC through HGF and HMGB1, respectively, with a dominant, HMGB1-mediated role of necrosis in determining tropism after tissue injury.

Antimicrobial effects of murine mesenchymal stromal cells directed against Toxoplasma gondii and Neospora caninum: role of immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs).

Mesenchymal stromal cells (MSCs) have a multilineage differentiation potential and provide immunosuppressive and antimicrobial functions. Murine as well as human MSCs restrict the proliferation of T cells. However, species-specific differences in the underlying molecular mechanisms have been described. Here, we analyzed the antiparasitic effector mechanisms active in murine MSCs. Murine MSCs, in contrast to human MSCs, could not restrict the growth of a highly virulent strain of Toxoplasma gondii (BK) after stimulation with IFN-γ. However, the growth of a type II strain of T. gondii (ME49) was strongly inhibited by IFN-γ-activated murine MSCs. Immunity-related GTPases (IRGs) as well as guanylate-binding proteins (GBPs) contributed to this antiparasitic effect. Further analysis showed that IFN-γ-activated mMSCs also inhibit the growth of Neospora caninum, a parasite belonging to the apicomplexan group as well. Detailed studies with murine IFN-γ-activated MSC indicated an involvement in IRGs like Irga6, Irgb6 and Irgd in the inhibition of N. caninum. Additional data showed that, furthermore, GBPs like mGBP1 and mGBP2 could have played a role in the anti-N. caninum effect of murine MSCs. These data underline that MSCs, in addition to their regenerative and immunosuppressive activity, function as antiparasitic effector cells as well. However, IRGs are not present in the human genome, indicating a species-specific difference in anti-T. gondii and anti-N. caninum effect between human and murine MSCs.

Human but not murine multipotent mesenchymal stromal cells exhibit broad-spectrum antimicrobial effector function mediated by indoleamine 2,3-dioxygenase.

Human multipotent mesenchymal stromal cells (MSCs) exhibit multilineage differentiation potential, support hematopoiesis, and inhibit proliferation and effector function of various immune cells. On the basis of these properties, MSC are currently under clinical investigation in a range of therapeutic applications including tissue repair and immune-mediated disorders such as graft-versus-host-disease refractory to pharmacological immunosuppression. Although initial clinical results appear promising, there are significant concerns that application of MSC might inadvertently suppress antimicrobial immunity with an increased risk of infection. We demonstrate here that on stimulation with inflammatory cytokines human MSC exhibit broad-spectrum antimicrobial effector function directed against a range of clinically relevant bacteria, protozoal parasites and viruses. Moreover, we identify the tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) as the underlying molecular mechanism. We furthermore delineate significant differences between human and murine MSC in that murine MSC fail to express IDO and inhibit bacterial growth. Conversely, only murine but not human MSC express inducible nitric oxide synthase on cytokine stimulation thus challenging the validity of murine in vivo models for the preclinical evaluation of human MSC. Collectively, our data identify human MSC as a cellular immunosuppressant that concurrently exhibits potent antimicrobial effector function thus encouraging their further evaluation in clinical trials.

Monitoring bacterial contamination of blood components in Germany: effect of contamination reduction measures.

BACKGROUND AND OBJECTIVES: National guidelines for monitoring bacterial contamination of blood components were introduced in Germany in 1997. Between 1998 and 2002, numerous measures were implemented to prevent bacterial contamination. This study investigates their impact on contamination rates. MATERIALS AND METHODS: Culture-based testing for bacterial detection on a random sample of blood components is part of routine quality control in German blood establishments. Using standardized questionnaires, data from the production periods 1998, 2001 and 2005/2006 were collected and analysed. RESULTS: The bacterial contamination rate of RBCs was reduced from 0·157% in 1998 to 0·029% in 2005/2006 (P<0·001). While the contamination rate of apheresis PCs remained nearly unchanged over the years, it dramatically decreased for pooled PCs by 70% to a contamination rate of 0·158% (P=0·001) within the last observation period, similar to that of apheresis PCs. The contamination rate of plasma decreased from 0·100% in 1998 to 0·019% in 2005/2006 (P=0·002). CONCLUSIONS: Precautionary measures significantly reduced bacterial contamination rates of blood components. Long-term monitoring with standardized methods is appropriate to evaluate the cumulative effect of contamination-preventing measures.

Bacterial contamination of platelet concentrates prepared by different methods: results of standardized sterility testing in Germany.

BACKGROUND AND OBJECTIVES: National guidelines for monitoring the bacterial contamination rate of blood components were introduced in Germany in 1997. The objective of this study was to present and evaluate the results of sterility testing of platelet concentrates (PCs) prepared by different methods. MATERIALS AND METHODS: The analysis of results of sterility testing of blood component production from transfusion medicine centres in Germany in 1998 and 2001 was based on information collected using standardized questionnaires. RESULTS: The bacterial contamination rates for single-donor PCs derived from whole blood and apheresis (0.210% vs. 0.156%) were comparable and showed no significant difference. However, pooled PCs produced from four buffy coats using the sterile docking procedure showed a significantly higher bacterial contamination rate compared with single-donor PCs derived from whole blood and apheresis (0.184% vs. 0.604%). CONCLUSIONS: Use of standardized methods for sterility monitoring is sufficient to assess collection and production processes in terms of hygiene and yields reliable data on bacterial contamination rates of blood components. The methods described are suitable for using to analyse the efficiency of newly introduced methods to reduce bacterial contamination rates of blood components (e.g. diversion, bacteria screening and pathogen inactivation).

Inhibition of human herpes simplex virus type 2 by interferon gamma and tumor necrosis factor alpha is mediated by indoleamine 2,3-dioxygenase.

Genital herpes simplex virus type 2 (HSV-2) is a significant clinical problem. Infection in pregnancy may result in disseminated infection of the newborn with encephalitis. We analyzed the antiviral effects induced by interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in cervix carcinoma cells (HeLa) and astrocytoma cells (86HG39). We found that replication of HSV-2 in HeLa cells and in 86HG39 cells is inhibited after stimulation of the cells by IFN-gamma and TNF-alpha. The antiviral effect of IFN-gamma is enhanced in the presence of TNF-alpha, while stimulation by TNF-alpha alone did not induce antiviral activity. We found that IFN-gamma induces a strong activation of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) and in addition, that the IFN-gamma-induced IDO activity was enhanced in the presence of TNF-alpha. Furthermore, we found that the induction of IDO activity is responsible for the inhibition of herpes simplex virus replication, since the presence of excess amounts of l-tryptophan abrogates the antiviral effect induced by IFN-gamma and the combination of IFN-gamma and TNF-alpha. We therefore conclude that the antiviral effect against HSV-2 mediated by type II interferon and TNF-alpha are dependent on IDO activation.

Role of indoleamine-2,3-dioxygenase in alpha/beta and gamma interferon-mediated antiviral effects against herpes simplex virus infections.

Gamma interferon (IFN-gamma)-mediated indoleamine-2,3-dioxygenase (IDO) activity in human astrocytoma cells and in native astrocytes was found to be responsible for the inhibition of herpes simplex virus replication. The effect is abolished in the presence of excess amounts of L-tryptophan. Both IFN-alpha and IFN-beta restricted herpes simplex virus replication in both cell types, but (in contrast to the results seen with IFN-gamma) the addition of an excess amount of L-tryptophan did not inhibit the induced antiviral effect.

Lack of portosystemic bacterial translocation in patients with liver cirrhosis after placement of transjugular shunt.

The purpose of this study was to clarify whether bacteria are transferred from the portal venous system into central venous blood during the placement of a transjugular portosystemic stent shunt (TIPSS). TIPSS was created in 30 consecutive cirrhotic patients for recurrent variceal bleeding ( n=12), refractory ascites ( n=16), or hepatorenal syndrome ( n=2). Microbiological analysis was performed prospectively on central venous blood before and on portal venous blood immediately after puncture of the portal vein. Twenty minutes after the placement of TIPSS, another sample of central venous blood was obtained. None of the first two sets of blood cultures showed bacterial growth, so that no bacterial transfer was seen at the time of TIPSS placement. Four of the third sets of blood samples showed skin and mouth flora, interpreted as iatrogenic contamination.

[Predictive parameters of infectiologic complications in patients after TIPSS]. / Prädiktive Parameter infektiologischer Komplikationen bei Patienten nach TIPSS-Anlage.

AIM: To define predictive parameters of a complicated clinical course after the TIPSS procedure. METHODS: Blood cultures were drawn prospectively in 41 patients from a central line and from the portal venous blood before stent placement as well as from the central line 20 min after intervention. C-reactive protein (CRP) (mg/dl) and white blood cell count (WBC,/microl) on the day of TIPSS-procedure (d0), the first (d1) and seven (d7) days after TIPSS were compared in patients with a complicated clinical course (spontaneous bacterial peritonitis,pneumonia,sepsis; group I) to patients without clinical complications (group II) RESULTS: Group I showed a significant increase in CRP (d0: 1.8+/-1.0; d1: 3.2+/-1.5; d7: 4.3+/-3.2), and white blood cell count (d0: 7700+/-2600; d1: 10800+/-2800; d7: 7500+/-1800) on the first day after TIPSS-procedure in comparison to group II (CRP: d0: 1.6+/-0.6; d1: 1.8+/-1.0; d7: 1.9+/-0.6. WBC: d0: 6900+/-1500; d1: 8000+/-1600; d7: 7600+/-1400). Microbiological analysis showed in 12% skin or oral flora in the last sample. CONCLUSIONS: The course of CRP and WBC-count during the first week after TIPSS procedure may indicate patients with a potential risk of a complicated clinical course.

Potential role of human brain microvascular endothelial cells in the pathogenesis of brain abscess: inhibition of Staphylococcus aureus by activation of indoleamine 2,3-dioxygenase.

Cerebral abscess is a rare complication of staphylococcal septicemia in infants associated with high mortality and morbidity. In the pathogenesis of abscess formation, S. aureus, one major causative agent, interacts with endothelial cells of the brain vessels before reaching the central nervous system. This study examined the growth of S. aureus in human brain microvascular endothelial cells (HBMEC) cultures stimulated with cytokines. IFN-gamma inhibited S. aureus replication by the induction of indoleamine 2,3-dioxygenase (IDO) in HBMEC. This activation of IDO in HBMEC could be shown by RT-PCR and by detection of kynurenine in culture supernatants of activated cells. Resupplementation of L-tryptophan abrogated the inhibitory effect of IFN-gamma on the growth of staphylococci, hence confirming the activation of indoleamine 2,3-dioxygenase as being responsible for the induced bacteriostasis. Addition of TNF-alpha enhanced the IFN-gamma mediated antibacterial effects, whereas TNF-alpha alone had no influence on staphylococcal growth. Stimulation of HBMEC with IFN-gamma failed to activate inducible nitric oxide synthase (iNOS) and subsequent production of nitric oxide (NO). Thus, intra- and extracellular depletion of L-tryptophan seems to be an important process in the defense against staphylococcal brain abscesses by means of creating an unfavorable microenvironment.

Restriction of Toxoplasma gondii growth in human brain microvascular endothelial cells by activation of indoleamine 2,3-dioxygenase.

One of the first steps in the development of cerebral toxoplasmosis is the penetration of the blood-brain barrier, which is comprised of microvascular endothelial cells. We examined the capacity of human brain microvascular endothelial cells (HBMEC) to interact with Toxoplasma gondii. We found that stimulation of HBMEC with gamma interferon (IFN-gamma) resulted in the induction of toxoplasmostasis. The capacity of HBMEC to restrict Toxoplasma growth after IFN-gamma stimulation was enhanced in the presence of tumor necrosis factor alpha (TNF-alpha). In addition, we found that IFN-gamma induced a strong induction of indoleamine 2,3-dioxygenase (IDO) activity in HBMEC, and this enzyme activity was enhanced by costimulation with TNF-alpha. The addition of excess amounts of tryptophan to the HBMEC cultures resulted in a complete abrogation of the IFN-gamma-TNF-alpha-mediated toxoplasmostasis. We therefore conclude that IDO induction contributed to the antiparasitic effector mechanism inducible in HBMEC by IFN-gamma and TNF-alpha.

Molecular characterization of TgMIC5, a proteolytically processed antigen secreted from the micronemes of Toxoplasma gondii.

During invasion of host cells, Toxoplasma gondii discharges the contents of small, apically located secretory organelles called micronemes. Micronemal proteins are known to be necessary for both parasite motility and invasion of host cells. To further define the contents of Toxoplasma micronemes, we used cell fractionation and secretion-modulating drugs to identify six novel, putative micronemal proteins. In this paper we describe preliminary characterization of one of these novel proteins, TgMIC5. Molecular cloning and DNA sequence analysis of the TgMIC5 cDNA and gene revealed that it encodes a previously identified immunodominant antigen called H4. TgMIC5 also possesses a consensus sequence unique to members of the parvulin family of peptidyl-prolyl cis-trans isomerases (PPIases). TgMIC5 is expressed as a preproprotein, which is proteolytically processed to a proprotein by signal peptidase before being further processed to a mature protein of 22 kDa. Using a combination of protein secretion experiments, immunofluorescence and immunoelectron microscopy, we demonstrated that TgMIC2 is stored in the micronemes of T. gondii tachyzoites before it is secreted into the surrounding medium. Based on its homology with parvulin-like PPIases, TgMIC5 may assist in the folding of other micronemal proteins that function in invasion of host cells by T. gondii tachyzoites.

Inhibition of indoleamine 2,3-dioxygenase in human macrophages inhibits interferon-gamma-induced bacteriostasis but does not abrogate toxoplasmastasis.

Induction of indoleamine 2,3-dioxygenase (IDO) by IFN-gamma results in growth inhibition of Toxoplasma and Chlamydia spp. as well as tumor cells. This is caused by the degradation, and therefore depletion, of L-tryptophan necessary for cell protein synthesis. Human macrophages stimulated with IFN-gamma express IDO and inhibit the growth of intracellular toxoplasma and chlamydia as well as that of extracellular bacteria such as group B streptococci. Here we describe experiments in which the L-tryptophan analog, 6-chloro-DL-tryptophan (CDLT) caused a dose-dependent inhibition in the IFN-gamma-induced IDO-mediated L-tryptophan degradation in monocyte-derived macrophages and glioblastoma cells. An inhibition of IDO activity of up to 80 % was observed at concentrations of CDLT of 750 microM. Expression of IDO at this concentration, as shown by Northern blot analysis, was unimpaired. This inhibition of IDO was coupled in glioblastoma cells by a complete abrogation of the IFN-gamma-induced toxoplasmastasis in these cells. IDO inhibition by CDLT in human macrophages resulted in a complete abrogation of the IFN-gamma-induced growth inhibition of streptococci and staphylococci. In contrast to this, IFN-gamma-induced toxoplasmastasis was not inhibited in human macrophages by CDLT-mediated IDO inhibition.

Interleukin-1 inhibits gamma interferon-induced bacteriostasis in human uroepithelial cells.

The most prominent gamma interferon (IFN-gamma)-induced antimicrobial effector mechanisms are the induction of nitric oxide (NO) synthase (NOS) and of indoleamine 2,3-dioxygenase (IDO) activity. We have recently found that human glioblastoma cells and human macrophages inhibit the growth of group B streptococci after stimulation with IFN-gamma. In this report, we show that in addition, human RT4 (uroepithelial) cells can inhibit the growth of enterococci. Murine macrophages (RAW cells) are unable to inhibit bacterial growth after IFN-gamma stimulation. Stimulation of human glioblastoma cells, macrophages, and RT4 cells with human IFN-gamma results in a strong expression of IDO activity; however, NO production remains undetectable. In strong contrast, murine RAW cells produce large amounts of NO when stimulated with murine IFN-gamma and IDO activity is not detectable. Interleukin-1 (IL-1) induces NO synthase in human RT4 cells when the cells are costimulated with IFN-gamma. We found that IL-1 inhibits IFN-gamma-stimulated IDO activity and antimicrobial effects in RT4 cells, while in human glioblastoma cells, which lack detectable NO synthase activity, neither of these effects was altered by costimulation with IFN-gamma and IL-1. The IL-1-mediated inhibition of IDO activity and of subsequent antibacterial effect is due to the production of NO. This conclusion was supported by evidence that N(G)-monomethyl-L-arginine, a competitive inhibitor of inducible NOS activity, is able to block the inhibitory action of IL-1 on IFN-gamma-induced bacteriostasis. We therefore conclude that NO production does not inhibit the growth of enterococci but might be involved in the regulation of IDO activity in some human cells.

Inducible anti-parasitic effector mechanisms in human uroepithelial cells: tryptophan degradation vs. NO production.

In murine cells the most important effector mechanism directed against the intracellular pathogen Toxoplasma gondii is the production of toxic nitrogen oxides. In contrast the induction of the tryptophan degrading enzyme indolamine 2,3-dioxygenase (IDO) has been described to be the most effective anti-parasitic mechanism in most human cells. In this report we analysed IDO induction and NO production in the human uroepithelial carcinoma cell line RT4. We found that after stimulation with IFN-gamma these cells were able to restrict toxoplasma growth. This was due to an activation of IDO, and the anti-parasitic effect mediated by RT4 cells was abrogated by the addition of L-tryptophan. In addition we found that the costimulation of RT4 cells with IL-1 and IFN-gamma results in the production of nitric oxide, and that in RT4 cells stimulated with both these cytokines, IDO activity and toxoplasmostasis was lower than in cells stimulated with IFN-gamma alone. This IL-1-mediated inhibition of IFN-gamma-induced IDO activity and toxoplasmostasis could be blocked by monomethyl L-arginine, an inhibitor of NO production. We therefore conclude that the induction of indolamine 2,3-dioxygenase activity in human cells is a very important effector mechanism directed against Toxoplasma gondii, and that in human cells the production of NO might be involved in the regulation of IDO activity.

IFN-gamma activated indoleamine 2,3-dioxygenase activity in human cells is an antiparasitic and an antibacterial effector mechanism.

In nearly all human cells IFN-gamma stimulation leads to an activation of indoleamine 2,3-dioxygenase (IDO) activity, which is responsible for anti-toxoplasma and anti-chlamydia effects. We have recently shown that IDO activation is also a defense mechanism against extracellular beta-hemolytic streptococci groups A, B, C and G in human glioblastoma cells, fibroblasts and macrophages. Similar effects were also seen with enterococci and in approximately 65% of staphylococci tested, including multiresistant strains of both species. In addition, we have found that IDO activity is differentially regulated in different cells. For example we have found that TNF-alpha enhances IFN-gamma induced IDO activity and antimicrobial effect in human glioblastoma cells whereas both IFN-gamma mediated effects were blocked by TNF-alpha as well as by IL-1 in a human uroepithelial cell line. We were able to show that the IL-1 and TNF-alpha mediated inhibition of IFN-gamma-induced IDO activity in uroepithelial cells is due to stimulation of inducible nitric oxide synthase. In human astrocytoma cells, IL-1 and TNF-alpha did not inhibit IDO activity and in concordance with this finding these cells did not show a detectable nitric oxide production.

Cytokine mediated regulation of interferon-gamma-induced IDO activation.

Stimulation of human monocyte-derived-macrophages (MDM) with interferon gamma induces the L-tryptophan degrading enzyme indoleamine 2,3-dioxygenase (IDO). It has been well documented that the growth of some intra-cellular parasites such as Chlamydia and Toxoplasma in human fibroblasts and glioblastoma cells is inhibited by IDO mediated L-tryptophan depletion. We have recently shown that IDO induction in cord blood MDM is also responsible for the growth inhibition of extra-cellular group B streptococci and thus for the first time shown an anti-bacterial effect of IDO activation. In view of this immunological function we sought to investigate the regulation, and in particular the downregulation of IDO by the immune system. We describe here the effect of cytokines on IDO activation and in particular the inhibitory function of IL-10, TGF beta and IL-4.

Inhibition of group B streptococcal growth by IFN gamma-activated human glioblastoma cells.

Group B streptococci are the most important bacteria inducing neonatal septicemia and meningitis. The aim of this study was to assess the role of IFNgamma in the induction of anti-microbial effector mechanisms in human brain tumor cells. Different human glioblastoma/astrocytoma cell lines, stimulated with IFNgamma, restricted the growth of group B streptococci. In addition, we found that TNF alpha is able to enhance the IFNgamma-mediated anti-microbial effect. In contrast to group B streptococci, other bacteria which are also capable of inducing meningitis, like E. coli and all but one of the tested Streptococcus pneumoniae strains, were not influenced by the IFNgamma treated cells. We found that the IFNgamma or the IFNgamma/TNF alpha induced activation of indoleamine 2,3-dioxygenase is responsible for the inhibition of streptococcal growth, since the addition of supplemental L-tryptophan completely blocks the IFNgamma induced bacteriostasis.

Interferon-gamma-induced activation of indoleamine 2,3-dioxygenase in cord blood monocyte-derived macrophages inhibits the growth of group B streptococci.

Neonatal sepsis is most often caused by group B streptococci (GBS) and is a major cause of death in the neonatal period. The response of the immune system in the newborn child has received much attention and is thought to be deficient in a number of ways. The effector response of neonatal monocyte-derived macrophages (MDM) was investigated. Interferon-gamma induced the activation of indoleamine 2,3-dioxygenase in MDM and inhibited the growth of GBS. Both effects were enhanced by the addition of tumor necrosis factor-alpha to the culture conditions. The coincident supplementation of L-tryptophan with the bacteria abrogated the bacterial growth inhibition, thus confirming the causative role of L-tryptophan depletion. Control of the extracellular as well as intracellular L-tryptophan levels may thus be one of the effector mechanisms with which the immune system defends the host against GBS dissemination and disease.