Transcript profiling of CD16-positive monocytes reveals a unique molecular fingerprint.

CD16-positive (CD14(++) CD16(+) and CD14(+) CD16(++) ) monocytes have unique features with respect to phenotype and function. We have used transcriptional profiling for comparison of CD16-positive monocytes and classical monocytes. We show herein that 187 genes are greater than fivefold differentially expressed, including 90 genes relevant to immune response and inflammation. Hierarchical clustering of data for monocyte subsets and CD1c(+) myeloid blood dendritic cells (DCs) demonstrate that CD16-positive cells are more closely related to classical monocytes than to DCs. Reverse transcriptase polymerase chain reaction for ten genes with the strongest differential expression confirmed the pattern including a lower messenger RNA level for CD14, CD163, and versican in CD16-positive monocytes. The pattern was similar for CD16-positive monocytes at rest and after exercise mobilization from the marginal pool. By contrast, alveolar macrophages, small sputum macrophages, breast milk macrophages, and synovial macrophages all showed a different pattern. When monocyte-derived macrophages (MDMs) were generated from CD16-positive monocytes by culture with macrophage colony-stimulating factor in vitro, then the MDMs maintained properties of their progeny with lower expression of CD14, CD163, and versican compared with CD14(++) CD16(-) MDMs. Furthermore, CD16-positive MDMs showed a higher phagocytosis for opsonized Escherichia coli. The data demonstrate that CD16-positive monocytes form a distinct type of cell, which gives rise to a distinct macrophage phenotype.

Chemokine expression by small sputum macrophages in COPD.

Small sputum macrophages represent highly active cells that increase in the airways of patients with inflammatory diseases such as chronic obstructive pulmonary disease (COPD). It has been reported often that levels of cytokines, chemokines and pro-teases are increased in sputum supernatants of these patients. In COPD, the small sputum macrophages may contribute to these supernatant proteins and recruit additional cells via specific chemokine expression patterns. We therefore investigated the expression profile of chemokines in sputum macrophages obtained from COPD patients in comparison to cells from healthy donors and cells isolated after inhalation of lipopolysaccharide (LPS). We used the minimally invasive procedure of sputum induction and have purified macrophages with the RosetteSep technology. Using macrophage purification and flow cytometry we show that in COPD small sputum macrophages account for 85.9% ± 8.3% compared with 12.9% ± 7.1% of total macrophages in control donors. When looking at chemokine expression we found, for the small macrophages in COPD, increased transcript and protein levels for CCL2, CCL7, CCL13 and CCL22 with a more than 100-fold increase for CCL13 mRNA (P < 0.001). Looking at active smokers without COPD, there is a substantial increase of small macrophages to 60% ± 15% and, here, chemokine expression is increased as well. In a model of airway inflammation healthy volunteers inhaled 20 µg of lipopolysaccharide (LPS), which resulted in an increase of small sputum macrophages from 18% ± 19% to 64% ± 25%. The pattern of chemokine expression was, however, different with an upregulation for CCL2 and CCL7, while CCL13 was downregulated three-fold in the LPS-induced small macrophages. These data demonstrate that sputum macrophages in COPD show induction of a specific set of CCL chemokines, which is distinct from what can be induced by LPS.

Darkfield-confocal microscopy detection of nanoscale particle internalization by human lung cells.

BACKGROUND: Concerns over the health effects of nanomaterials in the environment have created a need for microscopy methods capable of examining the biological interactions of nanoparticles (NP). Unfortunately, NP are beyond the diffraction limit of resolution for conventional light microscopy (~200 nm). Fluorescence and electron microscopy techniques commonly used to examine NP interactions with biological substrates have drawbacks that limit their usefulness in toxicological investigation of NP. EM is labor intensive and slow, while fluorescence carries the risk of photobleaching the sample and has size resolution limits. In addition, many relevant particles lack intrinsic fluorescence and therefore can not be detected in this manner. To surmount these limitations, we evaluated the potential of a novel combination of darkfield and confocal laser scanning microscopy (DF-CLSM) for the efficient 3D detection of NP in human lung cells. The DF-CLSM approach utilizes the contrast enhancements of darkfield microscopy to detect objects below the diffraction limit of 200 nm based on their light scattering properties and interfaces it with the power of confocal microscopy to resolve objects in the z-plane. RESULTS: Validation of the DF-CLSM method using fluorescent polystyrene beads demonstrated spatial colocalization of particle fluorescence (Confocal) and scattered transmitted light (Darkfield) along the X, Y, and Z axes. DF-CLSM imaging was able to detect and provide reasonable spatial locations of 27 nm TiO2 particles in relation to the stained nuclei of exposed BEAS 2B cells. Statistical analysis of particle proximity to cellular nuclei determined a significant difference between 5 min and 2 hr particle exposures suggesting a time-dependent internalization process. CONCLUSIONS: DF-CLSM microscopy is an alternative to current conventional light and electron microscopy methods that does not rely on particle fluorescence or contrast in electron density. DF-CLSM is especially well suited to the task of establishing the spatial localization of nanoparticles within cells, a critical topic in nanotoxicology. This technique has advantages to 2D darkfield microscopy as it visualizes nanoparticles in 3D using confocal microscopy. Use of this technique should aid toxicological studies related to observation of NP interactions with biological endpoints at cellular and subcellular levels.

Standardized single-platform assay for human monocyte subpopulations: Lower CD14+CD16++ monocytes in females.

We present a novel single-platform assay for determination of the absolute number of human blood monocyte subpopulations, i.e., the CD14(++)CD16(-) and the CD14(+)CD16(++) monocytes. A four-color combination of antibodies to CD14, CD16, CD45, and HLA-DR reduces the spill-over of natural killer cells and of granulocytes into the CD14(+)CD16(++) monocyte gate. For these CD14(+)CD16(++) monocytes, the intra-assay coefficient of variation (CV) was 4.1% and the inter-assay CV was 8.5%. Looking at a cohort of 40 donors aged 18-60 years, we found no age dependence. There was however an effect of gender in that females had lower CD14(+)CD16(++) monocytes (45.4 +/- 13.5 cells/microl) compared with males (59.1 +/- 20.3 cells/microl) (P < 0.02). Using this novel approach, we can confirm that exercise will lead to more than three-fold increase of the CD14(+)CD16(++) monocytes. Also, we show that therapy with low doses of glucocorticoids will deplete these cells. This robust single-platform assay may be a useful tool for monitoring the absolute number of monocyte subpopulations in health and disease.

Tissue-specific induction of ADAMTS2 in monocytes and macrophages by glucocorticoids.

The regulated expression of ADAMTS2 (a disintegrin and metalloproteinase with thrombospondin motifs), a secreted metalloproteinase involved in the processing of procollagen to collagen, was studied in peripheral blood mononuclear cells (PBMC). Stimulation with glucocorticoids (GC) resulted in a pronounced dose- and time-dependent increase of ADAMTS2 mRNA levels in PBMC. The increase of ADAMTS2 expression was specific for CD14++ monocytes (440-fold) and alveolar macrophages (200-fold), whereas CD3+ (T lymphocytes), phytohemagglutinin-activated CD3+ (T lymphocytes), and CD19+ (B lymphocytes) showed no significant changes in ADAMTS2 mRNA after GC treatment. Treatment of monocyte-derived macrophages (MDM) with GC also resulted in an increase of ADAMTS2 protein in the culture tissue media. Using the GC analog RU486, GC-mediated induction of ADAMTS2 mRNA was blocked, implicating that GC acts specifically via the GC-receptor. In agreement with findings in blood monocytes, cell lines of the monocytic lineage (MM6, THP-1) showed significant GC-induced significant increases in ADAMTS2 mRNA, while in epithelial cells (A549, Calu-3, Colo320, BT-20) and fibroblast (MRC-5, WI-38, and two NHDF-c cell types from adult cheek and upper arm), they showed no or little responsiveness to GC. As macrophages have important functions in immune defense and tissue homeostasis, these findings suggest that GC-mediated specific induction of ADAMTS2 in these cells may play a crucial role in the resolution of inflammation and wound repair.

Tolerance induced via TLR2 and TLR4 in human dendritic cells: role of IRAK-1.

BACKGROUND: While dendritic cells (DCs) can induce tolerance in T cells, little is known about tolerance induction in DCs themselves. We have analysed tolerance induced in human in-vitro generated DCs by repeated stimulation with ligands for TLR4 and TLR2. RESULTS: DCs stimulated with the TLR4 ligand LPS did show a rapid and pronounced expression of TNF mRNA and protein. When DCs were pre-cultured for 2 days with 5 ng LPS/ml then the subsequent response to stimulation with a high dose of LPS (500 ng/ml) was strongly reduced for both TNF mRNA and protein. At the promoter level there was a reduced transactivation by the -1173 bp TNF promoter and by a construct with a tetrameric NF-kappaB motif. Within the signalling cascade leading to NF-kappaB activation we found an ablation of the IRAK-1 adaptor protein in LPS-tolerant DCs. Pre-culture of DCs with the TLR2 ligand Pam3Cys also led to tolerance with respect to TNF gene expression and IRAK-1 protein was ablated in such tolerant cells as well, while IRAK-4 protein levels were unchanged. CONCLUSION: These data show that TLR-ligands can render DCs tolerant with respect to TNF gene expression by a mechanism that likely involves blockade of signal transduction at the level of IRAK-1.

Monitoring of glucocorticoid therapy by assessment of CD14(+)CD16(+) monocytes: a case report.

Bronchiolitis obliterans with organizing pneumonia (BOOP) is a disease affecting small airways and alveoli. It is characterized by interstitial inflammation rich in foamy macrophages and by fibroblastic connective tissue expanding into the airway and alveolar lumen. We report herein on a 54-year-old male BOOP patient who was treated with glucocorticoids (GCs) and who over a 5-year period had three relapses. At diagnosis the patient showed elevated CD14(+)CD16(+) monocyte numbers (85 cells/microl) and increased serum C-reactive protein (CRP) levels (29.4 mg/l). With GC therapy both parameters decreased within a few days. Diagnosis of relapse was preceded by a rise in CD14(+)CD16(+) monocyte numbers and in CRP levels which again responded to GC treatment. We conclude that determination of CD14(+)CD16(+) monocytes is a useful marker for monitoring of BOOP diagnosis and GC therapy.

Expression of p57-Kip2 in monocytes and macrophages.

The p57-Kip2 gene encodes a cyclin-dependent kinase inhibitor and hence this gene has received much attention in the study of malignancy. We have analysed expression of this gene in human monocytes and macrophages. In comparison to CD14++ monocytes, p57-Kip2 expression was higher in both CD14+16+ monocytes and alveolar macrophages. p57-Kip2 expression decreased in CD14++ monocytes after stimulation with lipopolysaccharide but increased after incubation with methylprednisolone. The results indicate that p57-Kip2 may be involved in regulating the inflammatory response of monocytic cells.

Diesel exhaust particles increase LPS-stimulated COX-2 expression and PGE2 production in human monocytes.

Little is known about health effects of ultrafine particles (UFP) found in ambient air, but much of their action may be on cells of the lung, including cells of the monocyte/macrophage lineage. We have analyzed the effects of diesel exhaust particles (DEP; SRM1650a) on human monocytes in vitro. DEP, on their own, had little effect on cyclooxygenase (COX)-2 gene expression in the Mono Mac 6 cell line. However, when cells were preincubated with DEP for 1 h, then stimulation with the Toll-like receptor 4 (TLR4) ligand lipopolysaccharide (LPS) induced an up-to fourfold-higher production of COX-2 mRNA with an average twofold increase. This costimulatory effect of DEP led to enhanced production of COX-2 protein and to increased release of prostaglandin E(2) (PGE(2)). The effect was specific in that tumor necrosis factor gene expression was not enhanced by DEP costimulation. Furthermore, costimulation with the TLR2 ligand Pam3Cys also led to enhanced COX-2 mRNA. DEP and LPS showed similar effects on COX-2 mRNA in primary blood mononuclear cells, in highly purified CD14-positive monocytes, and in monocyte-derived macrophages. Our data suggest that UFP such as DEP may exert anti-inflammatory effects mediated by enhanced PGE(2) production.

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.

A defect of CD16-positive monocytes can occur without disease.

The CD16-positive monocytes have been first described in 1988 but to date no selective defect in the number of these cells in blood has been reported. We now describe a family in which three of four siblings lack both CD16-positive monocyte subsets, i.e. the nonclassical and the intermediate monocytes. All three had CD16-positive monocytes of 2 cells/µl or less as compared to 52±18 cells/µl in healthy controls. The index case was affected by recurrent pleural effusion and infections and had evidence of an auto-inflammatory condition but no mutation of any of the relevant candidate genes. The other two siblings without CD16-positive monocytes were apparently healthy. There was no defect in serum M-CSF levels and no mutation in the M-CSF and M-CSFR genes. The data indicate that the absence of CD16-positive monocytes in blood does not lead to disease.

All-trans retinoic acid up-regulates Prostaglandin-E Synthase expression in human macrophages.

All-trans retinoic acid (ATRA) is a potent retinoid, which has been used successfully in different clinical settings as a potential drug to treat COPD and emphysema. In the present study, we analyzed genes modulated by ATRA by performing mRNA expression array analysis on alveolar macrophages after treatment with ATRA. Here we observed a 375-fold up-regulation of Prostaglandin-E Synthase (microsomal PGES-1, NM_004878 PTGES) which mediates the conversion of prostaglandin H(2) (PGH(2)) to Prostaglandin E(2) (PGE(2)). We furthermore studied the expression of PTGES after treatment with ATRA in human monocyte-derived macrophages (MDMs) and bronchoalveolar lavage (BAL) cells. ATRA up-regulated PTGES mRNA expression in MDMs generated with M-CSF by 2500-fold whereas in M-CSF+IL-13 macrophages the up-regulation was only 20-fold. Similarly, ATRA up-regulated PTGES mRNA expression by factor 1524 in BAL cells. The up-regulation of PTGES mRNA expression by ATRA is both time and dose dependent. IL-13 suppressed the ATRA induced PTGES expression at both mRNA and protein level in MDM and BAL cells. We also observed that LPS acts synergistically with ATRA in MDMs and strongly induces PTGES expression. ATRA had little impact on cyclooxygenase-1 and -2 (COX-1 and -2) expression as compared to PTGES expression under the same experimental conditions. Furthermore, we observed an induction of PGE(2) levels by ATRA in BAL cells. These data indicate that ATRA is a potent inducer of PTGES expression in human macrophages but not in alternatively activated macrophages and suggest that the eicosanoid pathway is important for ATRA action in macrophages.

Differential inflammatory response to inhaled lipopolysaccharide targeted either to the airways or the alveoli in man.

Endotoxin (Lipopolysaccharide, LPS) is a potent inducer of inflammation and there is various LPS contamination in the environment, being a trigger of lung diseases and exacerbation. The objective of this study was to assess the time course of inflammation and the sensitivities of the airways and alveoli to targeted LPS inhalation in order to understand the role of LPS challenge in airway disease.In healthy volunteers without any bronchial hyperresponsiveness we targeted sequentially 1, 5 and 20 µg LPS to the airways and 5 µg LPS to the alveoli using controlled aerosol bolus inhalation. Inflammatory parameters were assessed during a 72 h time period. LPS deposited in the airways induced dose dependent systemic responses with increases of blood neutrophils (peaking at 6 h), Interleukin-6 (peaking at 6 h), body temperature (peaking at 12 h), and CRP (peaking at 24 h). 5 µg LPS targeted to the alveoli caused significantly stronger effects compared to 5 µg airway LPS deposition. Local responses were studied by measuring lung function (FEV(1)) and reactive oxygen production, assessed by hydrogen peroxide (H(2)O(2)) in fractionated exhaled breath condensate (EBC). FEV(1) showed a dose dependent decline, with lowest values at 12 h post LPS challenge. There was a significant 2-fold H(2)O(2) induction in airway-EBC at 2 h post LPS inhalation. Alveolar LPS targeting resulted in the induction of very low levels of EBC-H(2)O(2).Targeting LPS to the alveoli leads to stronger systemic responses compared to airway LPS targeting. Targeted LPS inhalation may provide a novel model of airway inflammation for studying the role of LPS contamination of air pollution in lung diseases, exacerbation and anti-inflammatory drugs.

An integrated imaging approach to the study of oxidative stress generation by mitochondrial dysfunction in living cells.

BACKGROUND: The mechanisms of action of many environmental agents commonly involve oxidative stress resulting from mitochondrial dysfunction. Zinc is a common environmental metallic contaminant that has been implicated in a variety of oxidant-dependent toxicological responses. Unlike ions of other transition metals such as iron, copper, and vanadium, Zn(2+) does not generate reactive oxygen species (ROS) through redox cycling. OBJECTIVE: To characterize the role of oxidative stress in zinc-induced toxicity. METHODS: We used an integrated imaging approach that employs the hydrogen peroxide (H2O2)-specific fluorophore Peroxy Green 1 (PG1), the mitochondrial potential sensor 5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolylcarbocyanine iodide (JC-1), and the mitochondria-targeted form of the redox-sensitive genetically encoded fluorophore MTroGFP1 in living cells. RESULTS: Zinc treatment in the presence of the Zn(2+) ionophore pyrithione of A431 skin carcinoma cells preloaded with the H(2)O(2)-specific indicator PG1 resulted in a significant increase in H(2)O(2) production that could be significantly inhibited with the mitochondrial inhibitor carbonyl cyanide 3-chlorophenylhydrazone. Mitochondria were further implicated as the source of zinc-induced H(2)O(2) formation by the observation that exposure to zinc caused a loss of mitochondrial membrane potential. Using MTroGFP1, we showed that zinc exposure of A431 cells induces a rapid loss of reducing redox potential in mitochondria. We also demonstrated that zinc exposure results in rapid swelling of mitochondria isolated from mouse hearts. CONCLUSION: Taken together, these findings show a disruption of mitochondrial integrity, H(2)O(2) formation, and a shift toward positive redox potential in cells exposed to zinc. These data demonstrate the utility of real-time, live-cell imaging to study the role of oxidative stress in toxicological responses.

Pivotal Advance: Expansion of small sputum macrophages in CF: failure to express MARCO and mannose receptors.

Macrophages in the airways form an important element of immune defense and inflammation. We analyzed induced sputum from airways of patients with CF for the types of macrophages present, their receptor expression, and phagocytic function. In samples from patients and age-matched controls, macrophages were analyzed by multicolor flow cytometry, scavenger receptor expression was studied at the protein and mRNA level, and receptor function was investigated using fluorescent particles. In adult patients with CF, we discovered a pronounced expansion of the small CD14+ DR+ CD68weak+ macrophages to 73 +/- 18% compared with 16 +/- 8% in healthy controls. Expression of the MARCO and CD206 (mannose receptor) was strongly reduced at the mRNA and protein level in sputum macrophages. Antibody-blocking studies showed that MARCO mediates phagocytosis of unopsonized particles. In line with reduced MARCO expression, sputum macrophages in CF showed a deficient uptake of particles (23+/-9% of cells) compared with healthy controls (71+/-15%). The deficiency of MARCO expression in the predominant small sputum macrophages in CF may lead to impaired clearance of inhaled particles with increased inflammation and damage to the CF lung.