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Zelluläre Regulation und klinische Aspekte des monocyten-/macrophagenspezifischen Proteins CD163
(2005)
In der vorliegenden Arbeit wurde die zelluläre Regulation des monocyten-/ macrophagenspezifischen Oberflächenproteins CD163 untersucht und klinische Aspekte der löslichen Form des CD163 (sCD163) diskutiert. sCD163 wird in vivo durch einen inflammatorischen Reiz von der Zelloberfläche abgespalten. Bislang waren jedoch noch keine Mediatoren charakterisiert worden, die immer unter Entzündungsbedingungen vorhanden sind. In den eigenen Untersuchungen des Shedding von CD163 konnten für die Generierung des sCD163 neue endogene Aktivatoren identifiziert werden. Sowohl reaktive Sauerstoffspezies, wie Wasserstoffperoxid oder Stickstoffmonoxid, als auch das Produkt von endogenen Oxidationsreaktionen mit reaktiven Sauerstoffspezies 8-iso Prostaglandin F2a erwiesen sich als potente Aktivatoren des Shedding von CD163. Neben den bekannten physiologischen Funktionen des 8-iso Prostaglandin F2a konnte erstmals eine neue Funktion bei Entzündungen definiert werden. Dieser Effekt wurde spezifisch durch 8-iso Prostaglandin F2a hervorgerufen, da das isomere Prostaglandin F2a unter gleichen Bedingungen keinen Einfluss auf das Shedding von CD163 ausübte. Das Immunsuppressivum Cyclosporin A konnte ebenfalls als Induktor des Shedding von CD163 ermittelt werden. Damit konnte zusätzlich zur bekannten immunmodulatorischen Wirkung des Cyclosporin A eine weitere antiinflammatorische Wirkung über Monocyten/Macrophagen aufgezeigt werden. Durch Untersuchungen der Inhibierung des Shedding von CD163 konnten Gemeinsamkeiten bezüglich der in die sCD163-Generierung involvierten Mediatoren dargestellt werden. Obwohl die untersuchten Verbindungen wahrscheinlich über unterschiedliche Signalwege das Shedding von CD163 induzieren, waren die Anwesenheit von reaktiven Sauerstoffspezies und intrazellulärem Calcium und die Beteiligung einer TIMP-3-sensitiven Metalloproteinase an diesem Prozess essentiell. Bei einem Vergleich zwischen dem Shedding von CD163 und Tumor necrosis factor-a (TNF-a) ergaben sich Gemeinsamkeiten durch die Induktion des Shedding beider Verbindungen durch 8-iso Prostaglandin F2a und in sehr viel geringerem Maße durch Wasserstoffperoxid. Im Gegensatz dazu waren deutliche Unterschiede in dem Ausmaß der induzierten Stimulation des Shedding durch Stickstoffmonoxid, Prostaglandin F2a und Cyclosporin A zu erkennen. Im Hinblick auf die entgegengesetzten Wirkungen von sCD163 als antiinflammatorisch wirkende Verbindung und TNF-a als proinflammatorisches Cytokin, konnte dargestellt werden, dass die Freisetzung durch unterschiedliche Aktivatoren erfolgt. Nach Bestimmung der Konzentrationen von sCD163 und 8-iso PGF2a in bronchoalveolärer Lavage-Flüssigkeit von Patienten mit Cystischer Fibrose konnten keine abschließende Aussagen über die Eignung beider Parameter als biologische Marker für chronische Entzündungen der Lunge bei diesen Patienten getroffen werden. Weiterführend zu der Kenntnis, dass sCD163 die antiinflammatorische Wirkung über eine Interaktion des Proteins mit humanen T-Lymphocyten und nachfolgender Hemmung der Proliferation dieser Zellen ausübt, wurde diese Wechselwirkung genauer untersucht. In quantitativen Bestimmungen des sCD163 in isolierten T-Lymphocyten verschiedener Spender konnte erstmals gezeigt werden, dass sCD163 zu einem Teil konstitutiv in die T-Lymphocyten aufgenommen wird und dass diese Aufnahme durch proinflammatorische Aktivierung der T-Lymphocyten stark gesteigert werden kann. Durch fluoreszenzmikroskopische Aufnahmen unstimulierter T-Lymphocyten konnte die intrazelluläre Lokalisation des sCD163 visualisiert werden. Nach Aktivierung der Zellen mit einem proinflammatorischen Reiz fand innerhalb der Zellen eine Translokalisation des sCD163 aus dem cytoplasmatischen Bereich zur Zellmembran statt. Damit konnte erstmals gezeigt werden, dass abhängig vom Aktivierungsstatus der T-Lymphocyten eine Umverteilung des sCD163 innerhalb der Zellen erfolgt. Eine quantitative Bestimmung des sCD163 und seines Bindungspartners in T-Lymphocyten nichtmuskuläres Myosin Typ IIA gelang mittels eines neu entwickeltem ELISA, der spezifisch sCD163 und Myosin ausschließlich im Komplex erfasst. Damit konnte durch die in dieser Arbeit beschriebenen Untersuchungen ein grundlegender Beitrag zur Charakterisierung der Regulation der Proteinexpression und des Shedding von CD163 in humanen Monocyten sowie der Interaktion des sCD163 mit T-Lymphocyten geleistet werden.
Background:
Ureaplasma species have been associated with chorioamnionitis and preterm birth and have been implicated in the pathogenesis of neonatal short and long-term morbidity. However, being mostly commensal bacteria, controversy remains on the pro-inflammatory capacity of Ureaplasma. Discussions are ongoing on the incidence and impact of prenatal, perinatal, and postnatal infection. The present study addressed the impact of Ureaplasma isolates on monocyte-driven inflammation.
Methods:
Cord blood monocytes of term neonates and adult monocytes, either native or LPS-primed, were cultured with Ureaplasma urealyticum (U. urealyticum) serovar 8 (Uu8) and Ureaplasma parvum serovar 3 (Up3). Using qRT-PCR, cytokine flow cytometry, and multi-analyte immunoassay, we assessed mRNA and protein expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-8, IL-12p40, IL-10, and IL-1 receptor antagonist (IL-1ra) as well as Toll-like receptor (TLR) 2 and TLR4.
Results:
Uu8 and Up3 induced mRNA expression and protein release of TNF-α, IL-1β and IL-8 in term neonatal and adult monocytes (p < 0.01 and p < 0.05). Intracellular protein expression of TNF-α, IL-1β and IL-8 in Ureaplasma-stimulated cells paralleled those results. Ureaplasma-induced cytokine levels did not significantly differ from LPS-mediated levels except for lower intracellular IL-1β in adult monocytes (Uu8: p < 0.05). Remarkably, ureaplasmas did not induce IL-12p40 response and promoted lower amounts of anti-inflammatory IL-10 and IL-1ra than LPS, provoking a cytokine imbalance more in favor of pro-inflammation (IL-1β/IL-10, IL-8/IL-10 and IL-8/IL-1ra: p < 0.01, vs. LPS). In contrast to LPS, both isolates induced TLR2 mRNA in neonatal and adult cells (p < 0.001 and p < 0.05) and suppressed TLR4 mRNA in adult monocytes (p < 0.05). Upon co-stimulation, Uu8 and Up3 inhibited LPS-induced intracellular IL-1β (p < 0.001 and p < 0.05) and IL-8 in adult monocytes (p < 0.01), while LPS-induced neonatal cytokines were maintained or aggravated (p < 0.05).
Conclusion:
Our data demonstrate a considerable pro-inflammatory capacity of Ureaplasma isolates in human monocytes. Stimulating pro-inflammatory cytokine responses while hardly inducing immunomodulatory and anti-inflammatory cytokines, ureaplasmas might push monocyte immune responses toward pro-inflammation. Inhibition of LPS-induced cytokines in adult monocytes in contrast to sustained inflammation in term neonatal monocytes indicates a differential modulation of host immune responses to a second stimulus. Modification of TLR2 and TLR4 expression may shape host susceptibility to inflammation.
The interplay of specific leukocyte subpopulations, resident cells and proalgesic mediators results in pain in inflammation. Proalgesic mediators like reactive oxygen species (ROS) and downstream products elicit pain by stimulation of transient receptor potential (TRP) channels. The contribution of leukocyte subpopulations however is less clear. Local injection of neutrophilic chemokines elicits neutrophil recruitment but no hyperalgesia in rats. In meta-analyses the monocytic chemoattractant, CCL2 (monocyte chemoattractant protein-1; MCP-1), was identified as an important factor in the pathophysiology of human and animal pain. In this study, intraplantar injection of CCL2 elicited thermal and mechanical pain in Wistar but not in Dark Agouti (DA) rats, which lack p47phox, a part of the NADPH oxidase complex. Inflammatory hyperalgesia after complete Freund's adjuvant (CFA) as well as capsaicin-induced hyperalgesia and capsaicin-induced current flow in dorsal root ganglion neurons in DA were comparable to Wistar rats. Macrophages from DA expressed lower levels of CCR2 and thereby migrated less towards CCL2 and formed limited amounts of ROS in vitro and 4-hydroxynonenal (4-HNE) in the tissue in response to CCL2 compared to Wistar rats. Local adoptive transfer of peritoneal macrophages from Wistar but not from DA rats reconstituted CCL2-triggered hyperalgesia in leukocyte-depleted DA and Wistar rats. A pharmacological stimulator of ROS production (phytol) restored CCL2-induced hyperalgesia in vivo in DA rats. In Wistar rats, CCL2-induced hyperalgesia was completely blocked by superoxide dismutase (SOD), catalase or tempol. Likewise, inhibition of NADPH oxidase by apocynin reduced CCL2-elicited hyperalgesia but not CFA-induced inflammatory hyperalgesia. In summary, we provide a link between CCL2, CCR2 expression on macrophages, NADPH oxidase, ROS and the development CCL2-triggered hyperalgesia, which is different from CFA-induced hyperalgesia. The study further supports the impact of CCL2 and ROS as potential targets in pain therapy.
Macrophages predominate the inflammatory landscape within multiple sclerosis (MS) lesions, not only regarding cellularity but also with respect to the diverse functions this cell fraction provides during disease progression and remission. Researchers have been well aware of the fact that the macrophage pool during central nervous system (CNS) autoimmunity consists of a mixture of myeloid cells. Yet, separating these populations to define their unique contribution to disease pathology has long been challenging due to their similar marker expression. Sophisticated lineage tracing approaches as well as comprehensive transcriptome analysis have elevated our insight into macrophage biology to a new level enabling scientists to dissect the roles of resident (microglia and non-parenchymal macrophages) and infiltrating macrophages with unprecedented precision. To do so in an accurate way, researchers have to know their toolbox, which has been filled with diverse, discriminating approaches from decades of studying neuroinflammation in animal models. Every method has its own strengths and weaknesses, which will be addressed in this review. The focus will be on tools to manipulate and/or identify different macrophage subgroups within the injured murine CNS.
Background
Invasive fungal infections with Candida albicans (C. albicans) occur frequently in extremely low birthweight (ELBW) infants and are associated with poor outcome. Phagocytosis of C.albicans initializes apoptosis in monocytes (phagocytosis induced cell death, PICD). PICD is reduced in neonatal cord blood monocytes (CBMO).
Hypothesis
Phagocytosis of C. albicans causes PICD which differs between neonatal monocytes (CBMO) and adult peripheral blood monocytes (PBMO) due to lower stimulation of TLR-mediated immune responses.
Methods
The ability to phagocytose C. albicans, expression of TLRs, the induction of apoptosis (assessment of sub-G1 and nick-strand breaks) were analyzed by FACS. TLR signalling was induced by agonists such as lipopolysaccharide (LPS), Pam3Cys, FSL-1 and Zymosan and blocked (neutralizing TLR2 antibodies and MYD88 inhibitor).
Results
Phagocytic indices of PBMO and CBMO were similar. Following stimulation with agonists and C. albicans induced up-regulation of TLR2 and consecutive phosphorylation of MAP kinase P38 and expression of TNF-α, which were stronger on PBMO compared to CBMO (p < 0.005). Downstream, TLR2 signalling initiated caspase-3-dependent PICD which was found reduced in CBMO (p < 0.05 vs PBMO).
Conclusion
Our data suggest direct involvement of TLR2-signalling in C. albicans-induced PICD in monocytes and an alteration of this pathway in CBMO.
Here, we assessed whether 36 single nucleotide polymorphisms (SNPs) within the TNFSF4 and MAPKAPK2 loci influence the risk of developing invasive aspergillosis (IA). We conducted a two-stage case control study including 911 high-risk patients diagnosed with hematological malignancies that were ascertained through the aspBIOmics consortium. The meta-analysis of the discovery and replication populations revealed that carriers of the TNFSF4\(_{rs7526628T/T}\) genotype had a significantly increased risk of developing IA (p = 0.00022). We also found that carriers of the TNFSF4\(_{rs7526628T}\) allele showed decreased serum levels of TNFSF14 protein (p = 0.0027), and that their macrophages had a decreased fungicidal activity (p = 0.048). In addition, we observed that each copy of the MAPKAPK2\(_{rs12137965G}\) allele increased the risk of IA by 60% (p = 0.0017), whereas each copy of the MAPKAPK2\(_{rs17013271T}\) allele was estimated to decrease the risk of developing the disease (p = 0.0029). Mechanistically, we found that carriers of the risk MAPKAPK2\(_{rs12137965G}\) allele showed increased numbers of CD38+IgM-IgD- plasmablasts in blood (p = 0.00086), whereas those harboring two copies of the allele had decreased serum concentrations of thymic stromal lymphopoietin (p = 0.00097). Finally, we also found that carriers of the protective MAPKAPK2\(_{rs17013271T}\) allele had decreased numbers of CD27-IgM-IgD- B cells (p = 0.00087) and significantly lower numbers of CD14+ and CD14+CD16- cells (p = 0.00018 and 0.00023). Altogether, these results suggest a role of the TNFSF4 and MAPKAPK2 genes in determining IA risk.
Schmerz ist eine klassische Komponente von Entzündungsreaktionen. Im Rahmen des Entzündungsgeschehens werden Zytokine und Chemokine freigesetzt, die Leukozyten zum Entzündungsort rekrutieren. Über die Freisetzung weiterer proalgetischer Mediatoren tragen diese zur Aktivierung und Sensitivierung von Nozizeptoren und damit zur Schmerzentstehung bei. Das Monozyten-rekrutierende Chemokin CCL2 verursachte in Verhaltensexperimenten eine Hyperalgesie bei Ratten. Die Hyperalgesie war durch den Cox-2 Inhibitor Parecoxib vollständig reversibel. Daher wurde in dieser Arbeit die Rolle von Monozyten und Prostaglandinen in der Entstehung dieser Hyperalgesie untersucht. Dazu wurde in vitro die Cox-2 Expression und die Prostaglandin-Bildung in humanen Monozyten und Peritonealmakrophagen der Ratte nach CCL2 Stimulation bestimmt. Zudem wurde in vivo die Cox-2 Expression im Rückenmark und in der Rattenpfote nach CCL2 Injektion in die Pfote untersucht.
Background
Natural surfactant preparations, commonly isolated from porcine or bovine lungs, are used to treat respiratory distress syndrome in preterm infants. Besides biophysical effectiveness, several studies have documented additional immunomodulatory properties. Within the near future, synthetic surfactant preparations may be a promising alternative. CHF5633 is a new generation reconstituted synthetic surfactant preparation with defined composition, containing dipalmitoyl-phosphatidylcholine, palmitoyl-oleoyl-phosphatidylglycerol and synthetic analogs of surfactant protein (SP-) B and SP-C. While its biophysical effectiveness has been demonstrated in vitro and in vivo, possible immunomodulatory abilities are currently unknown.
Aim
The aim of the current study was to define a potential impact of CHF5633 and its single components on pro- and anti-inflammatory cytokine responses in human CD4\(^+\) lymphocytes.
Methods
Purified human CD4\(^+\) T cells were activated using anti CD3/CD28 antibodies and exposed to CHF5633, its components, or to the well-known animal-derived surfactant Poractant alfa (Curosurf®). Proliferative response and cell viability were assessed using flow cytometry and a methylthiazolyldiphenyltetrazolium bromide colorimetric assay. The mRNA expression of IFNγ, IL-2, IL-17A, IL-22, IL-4, and IL-10 was measured by quantitative PCR, while intracellular protein expression was assessed by means of flow cytometry.
Results
Neither CHF5633 nor any of its phospholipid components with or without SP-B or SP-C analogs had any influence on proliferative ability and viability of CD4\(^+\) lymphocytes under the given conditions. IFNγ, IL-2, IL-17A, IL-22, IL-4, and IL-10 mRNA as well as IFNγ, IL-2, IL-4 and IL-10 protein levels were unaffected in both non-activated and activated CD4+ lymphocytes after exposure to CHF5633 or its constituents compared to non-exposed controls. However, in comparison to Curosurf®, expression levels of anti-inflammatory IL-4 and IL-10 mRNA were significantly increased in CHF5633 exposed CD4\(^+\) lymphocytes.
Conclusion
For the first time, the immunomodulatory capacity of CHF5633 on CD4\(^+\) lymphocytes was evaluated. CHF5633 did not show any cytotoxicity on CD4\(^+\) cells. Moreover, our in vitro data indicate that CHF5633 does not exert unintended pro-inflammatory effects on non-activated and activated CD4+ T cells. As far as anti-inflammatory cytokines are concerned, it might lack an overall reductive ability in comparison to animal-derived surfactants, potentially leaving pro- and anti-inflammatory cytokine response in balance.
Developmentally regulated features of innate immunity are thought to place preterm and term infants at risk of infection and inflammation-related morbidity. Underlying mechanisms are incompletely understood. Differences in monocyte function including toll-like receptor (TLR) expression and signaling have been discussed. Some studies point to generally impaired TLR signaling, others to differences in individual pathways. In the present study, we assessed mRNA and protein expression of pro- and anti-inflammatory cytokines in preterm and term cord blood (CB) monocytes compared with adult controls stimulated ex vivo with Pam3CSK4, zymosan, polyinosinic:polycytidylic acid, lipopolysaccharide, flagellin, and CpG oligonucleotide, which activate the TLR1/2, TLR2/6, TLR3, TLR4, TLR5, and TLR9 pathways, respectively. In parallel, frequencies of monocyte subsets, stimulus-driven TLR expression, and phosphorylation of TLR-associated signaling molecules were analyzed. Independent of stimulus, pro-inflammatory responses of term CB monocytes equaled adult controls. The same held true for preterm CB monocytes—except for lower IL-1β levels. In contrast, CB monocytes released lower amounts of anti-inflammatory IL-10 and IL-1ra, resulting in higher ratios of pro-inflammatory to anti-inflammatory cytokines. Phosphorylation of p65, p38, and ERK1/2 correlated with adult controls. However, stimulated CB samples stood out with higher frequencies of intermediate monocytes (CD14\(^+\)CD16\(^+\)). Both pro-inflammatory net effect and expansion of the intermediate subset were most pronounced upon stimulation with Pam3CSK4 (TLR1/2), zymosan (TR2/6), and lipopolysaccharide (TLR4). Our data demonstrate robust pro-inflammatory and yet attenuated anti-inflammatory responses in preterm and term CB monocytes, along with imbalanced cytokine ratios. Intermediate monocytes, a subset ascribed pro-inflammatory features, might participate in this inflammatory state.
Dendritic cells (DCs) and macrophages (Mph) share many characteristics as components of the innate immune system. The criteria to classify the multitude of subsets within the mononuclear phagocyte system are currently phenotype, ontogeny, transcription patterns, epigenetic adaptations, and function. More recently, ontogenetic, transcriptional, and proteomic research approaches uncovered major developmental differences between Flt3L-dependent conventional DCs as compared with Mphs and monocyte-derived DCs (MoDCs), the latter mainly generated in vitro from murine bone marrow-derived DCs (BM-DCs) or human CD14\(^{+}\) peripheral blood monocytes. Conversely, in vitro GM-CSF-dependent monocyte-derived Mphs largely resemble MoDCs whereas tissue-resident Mphs show a common embryonic origin from yolk sac and fetal liver with Langerhans cells (LCs). The novel ontogenetic findings opened discussions on the terminology of DCs versus Mphs. Here, we bring forward arguments to facilitate definitions of BM-DCs, MoDCs, and LCs. We propose a group model of terminology for all DC subsets that attempts to encompass both ontogeny and function.