Loems Ziegler-Heitbrock

Nomenclature of monocytes and dendritic cells in blood

Monocytes and cells of the dendritic cell lineage circulate in blood and eventually migrate into tissue where they further mature and serve various functions, most notably in immune defense. Over recent years these cells have been characterized in detail with the use of cell surface markers and flow cytometry, and subpopulations have been described. The present document proposes a nomenclature for these cells and defines 3 types of monocytes (classical, intermediate, and nonclassical monocytes) and 3 types of dendritic cells (plasmacytoid and 2 types of myeloid dendritic cells) in human and in mouse blood. This classification has been approved by the Nomenclature Committee of the International Union of Immunological Societies, and we are convinced that it will facilitate communication among experts and in the wider scientific community.

The CD14+ CD16+ blood monocytes: their role in infection and inflammation

Blood monocyte subpopulations have been defined in man initially, and the two major types of monocytes are the CD14++ CD16− and the CD14+ CD16+ monocytes. These cells have been shown to exhibit distinct phenotype and function, and the CD14+ CD16+ were labeled proinflammatory based on higher expression of proinflammatory cytokines and higher potency in antigen presentation. The current review describes these properties, including the relationship to dendritic cells, and summarizes the host of publications about CD14+ CD16+ monocytes in inflammation and infectious disease in man, all of which suggest a crucial role of these cells in the disease processes. The review also covers the more recent description of homologues of these cells in other model species, which is expected to better define the role of monocyte subsets in disease.

Hypoxia-Induced Gene Expression in Human Macrophages: Implications for Ischemic Tissues and Hypoxia-Regulated Gene Therapy

Macrophages accumulate in ischemic areas of such pathological tissues as solid tumors, atherosclerotic plaques and arthritic joints. Studies have suggested that hypoxia alters the phenotype of macrophages in a way that promotes these lesions. However, the genes up-regulated by macrophages in such hypoxic tissues are poorly characterized. Here, we have used cDNA array hybridization to investigate the effects of hypoxia on the mRNAs of 1185 genes in primary human monocyte-derived macrophages. As shown previously in other cell types, mRNA levels for vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT-1) were up-regulated by hypoxia. However, the mRNAs of other genes were also up-regulated including matrix metalloproteinase-7 (MMP-7), neuromedin B receptor, and the DNA-binding protein inhibitor, Id2. The promoters of GLUT-1 and MMP-7 confer hypoxic inducibility on a reporter gene in RAW 264.7 macrophages, indicating that the hypoxic up-regulation of these mRNAs may occur, at least in part, at the transcriptional level. GLUT-1 and MMP-7 mRNA were also shown to be up-regulated in hypoxic macrophages in vitro by real-time RT-PCR, and these proteins were elevated in hypoxic macrophages in vitro and in hypoxic areas of human breast tumors. The hypoxia up-regulated genes identified could be important for the survival and functioning of macrophages in hypoxic diseased tissues, and their promoters could prove useful in macrophage-delivered gene therapy.

Toward a refined definition of monocyte subsets

In a nomenclature proposal published in 2010 monocytes were subdivided into classical and non-classical cells and in addition an intermediate monocyte subset was proposed. Over the last couple of years many studies have analyzed these intermediate cells, their characteristics have been described, and their expansion has been documented in many clinical settings. While these cells appear to be in transition from classical to non-classical monocytes and hence may not form a distinct cell population in a strict sense, their separate analysis and enumeration is warranted in health and disease.

IL-10 induces IL-10 in primary human monocyte-derived macrophages via the transcription factor Stat3

IL-10 is an important immunosuppressive cytokine that can down-regulate expression of other cytokines and has been shown to down-regulate itself. We show, in this study, that treatment of human monocyte-derived macrophages with IL-10 induces IL-10 mRNA in a dose- and time-dependent manner with an optimum induction at 100 ng/ml and at 6 h, whereas IL-10-induced IL-10 protein can be detected at 18 h. In the same cells, IL-10 can partially suppress IL-10 mRNA induced by LPS, but only down to the level of IL-10-induced IL-10. An adenoviral luciferase reporter construct driven by the −195 IL-10 promoter, which contains a Stat motif, was readily induced by both IL-10 and LPS. Mutation of this Stat motif ablated IL-10 activation of this promoter, but not the LPS activation. Finally, we show that overexpression of a dominant-negative Stat3 protein will prevent IL-10 induction, but not LPS induction, of IL-10 mRNA. These data show that IL-10 induces IL-10 in monocyte-derived macrophages in an autocrine manner via activation of the transcription factor Stat3.

A deletion defining a common Asian lineage of Mycobacterium tuberculosis associates with immune subversion.

Six major lineages of Mycobacterium tuberculosis appear preferentially transmitted amongst distinct ethnic groups. We identified a deletion affecting Rv1519 in CH, a strain isolated from a large outbreak in Leicester U.K., that coincidentally defines the East African-Indian lineage matching a major ethnic group in this city. In broth media, CH grew less rapidly and was less acidic and H2O2-tolerant than reference sequenced strains (CDC1551 and H37Rv). Nevertheless, CH was not impaired in its ability to grow in human monocyte-derived macrophages. When compared with CDC1551 and H37Rv, CH induced less protective IL-12p40 and more antiinflammatory IL-10 and IL-6 gene transcription and secretion from monocyte-derived macrophages. It thus appears that CH compensates microbiological attenuation by skewing the innate response toward phagocyte deactivation. Complementation of Rv1519, but none of nine additional genes absent from CH compared with the type strain, H37Rv, reversed the capacity of CH to elicit antiinflammatory IL-10 production by macrophages. The Rv1519 polymorphism in M. tuberculosis confers an immune subverting phenotype that contributes to the persistence and outbreak potential of this lineage.

Mechanism of glucocorticoid-induced depletion of human CD14+CD16+ monocytes.

Healthy donors infused with high doses of glucocorticoids [GCs; methyl‐prednisolone (MP); 500 mg/day for 3 days] suffer a selective depletion of the CD14+CD16+ monocytes such that these cells are reduced by 95% on day 5. In vitro studies revealed that at 11 h of culture in the presence of 10−5 M MP, no depletion was observed as yet, but a reduction by 80% was seen after 24 h. In dose‐response analysis, MP still led to a 50% reduction of CD14+CD16+ monocytes at 10−7 M. Depletion could not be overcome by addition of the cytokines interleukin‐1β or macrophage‐colony stimulating factor, and it was independent of CD95. Depletion was, however, inhibited by the caspase 3,8 blocker z‐Val‐Ala‐Asp, suggesting that cell death occurs in a caspase‐dependent manner. Furthermore, blockade of depletion by RU‐486 indicates that the intracellular GC receptor (GCR) is involved. Measurement of GCR by flow cytometry revealed a 50% higher level of expression in the CD14+CD16+ monocytes. Our studies show a selective depletion of CD14+CD16+ monocytes by GC treatment in vivo and in vitro, an effect to which the modestly increased level of GCR may contribute.

Blood Monocytes and Their Subsets: Established Features and Open Questions

In contrast to the past reliance on morphology, the identification and enumeration of blood monocytes are nowadays done with monoclonal antibodies and flow cytometry and this allows for subdivision into classical, intermediate, and non-classical monocytes. Using specific cell surface markers, dendritic cells in blood can be segregated from these monocytes. While in the past, changes in monocyte numbers as determined in standard hematology counters have not had any relevant clinical impact, the subset analysis now has uncovered informative changes that may be used in management of disease.

Tolerance Induced by the Lipopeptide Pam3Cys Is Due to Ablation of IL-1R-Associated Kinase-1

Stimulation of the human monocytic cell line Mono Mac 6 with the synthetic lipopeptide (S)-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser(S)-Lys4-OH, trihydrochloride (Pam3Cys) at 10 μg/ml induces a rapid expression of the TNF gene in a TLR2-dependent fashion. Preculture of the cells with Pam3Cys at 1 μg/ml leads to a reduced response after subsequent stimulation with Pam3Cys at 10 μg/ml, indicating that the cells have become tolerant to Pam3Cys. The CD14 and TLR2 expression is not decreased on the surface of the tolerant cells, but rather up-regulated. Analysis of the NF-κB binding in Pam3Cys-tolerant cells shows a failure to mobilize NF-κB-p50p65 heterodimers, while NF-κB-p50p50 homodimers remain unchanged. Pam3Cys-tolerant cells showed neither IκBα-Ser32 phosphorylation nor IκBα degradation but MyD88 protein was unaltered. However, IRAK-1 protein was absent in Pam3Cys-induced tolerance, while IRAK-1 mRNA was still detectable at 30% compared with untreated cells. In contrast, in LPS-tolerized cells, p50p50 homodimers were induced, IRAK-1 protein level was only partially decreased, and p50p65 mobilization remained intact. It is concluded that in Mono Mac 6 monocytic cells, inhibition of IRAK-1 expression at the mRNA and protein levels is the main TLR-2-dependent mechanism responsible for Pam3Cys-induced tolerance, but not for TLR-4-dependent LPS-induced tolerance.

Mucociliary and long-term particle clearance in airways of patients with immotile cilia.

Spherical monodisperse ferromagnetic iron oxide particles of 1.9 μm geometric and 4.2 μm aerodynamic diameter were inhaled by seven patients with primary ciliary dyskinesia (PCD) using the shallow bolus technique, and compared to 13 healthy non-smokers (NS) from a previous study. The bolus penetration front depth was limiting to the phase1 dead space volume. In PCD patients deposition was 58+/-8 % after 8 s breath holding time. Particle retention was measured by the magnetopneumographic method over a period of nine months. Particle clearance from the airways showed a fast and a slow phase. In PCD patients airway clearance was retarded and prolonged, 42+/-12 % followed the fast phase with a mean half time of 16.8+/-8.6 hours. The remaining fraction was cleared slowly with a half time of 121+/-25 days. In healthy NS 49+/-9 % of particles were cleared in the fast phase with a mean half time of 3.0+/-1.6 hours, characteristic of an intact mucociliary clearance. There was no difference in the slow clearance phase between PCD patients and healthy NS. Despite non-functioning cilia the effectiveness of airway clearance in PCD patients is comparable to healthy NS, with a prolonged kinetics of one week, which may primarily reflect the effectiveness of cough clearance. This prolonged airway clearance allows longer residence times of bacteria and viruses in the airways and may be one reason for increased frequency of infections in PCD patients.