Gabriele Zwadlo-Klarwasser

Rapid uptake of gold nanorods by primary human blood phagocytes and immunomodulatory effects of surface chemistry.

Nanoparticle-based in vivo applications should consider the omnipresence of the phagocytes in the bloodstream and tissue. We have studied the nanoparticle uptake capacities of the most important human primary leukocyte populations using a nanoparticle library encompassing both rod-shaped and spherical gold nanoparticles with diameters between 15 and 50 nm and a variety of surface chemistries. Cetyltrimethylammoniumbromide (CTAB)-stabilized nanoparticles were internalized rapidly within 15 min and in large amounts by macrophages and to a lower extent also by monocytes. Interestingly, we found that the uptake of nanorods by macrophages was more efficient than that of nanospheres. Blocking experiments and electron microscopic studies revealed macropinocytosis as the major uptake mechanism. Grafting of poly(ethylene oxide) (PEO) onto the nanorods was found to significantly delay their internalization for several hours. The long-term uptake of PEO-coated nanoparticles with positively or negatively charged end groups was almost identical. Particle surface chemistry strongly influenced the expression of inflammation-related genes within 1 day. Furthermore, the macrophage phenotype was significantly affected after 7 days of culture with nanorods depending on the surface chemistry. Thus, in vivo application of nanoparticles with certain surface functionalities may lead to inflammation upon particle accumulation. However, our data also suggest that chemical modifications of nanoparticles may be useful for immunomodulation.

Cytochrome P450 1B1: a major P450 isoenzyme in human blood monocytes and macrophage subsets

In this study, cytochrome P450 (CYP; EC 1.14.14.1)-dependent activities and P450 isoenzyme patterns were determined in human monocytes and macrophages, which play a major role in antigen processing including small molecular weight compounds which cause contact dermatitis or drug-allergic reactions. Using reverse transcriptase-polymerase chain reaction (RT-PCR) we determined the mRNA expression of eight CYPs (1A1, 1A2, 1B1, 2B6/7, 2E1, 3A3/4, 3A7 and 4B1) in human blood monocytes and macrophage subsets 27E10 and RM3/1. To study the influence of known P450 inducers, monocytes were incubated in vitro with ethanol, dexamethasone, cyclosporin A (CSA), benzanthracene (BA), phenobarbital (PB), lipopolysaccharide (LPS) and 12-O-tetradecanoyl-phorbol-13-acetat (TPA) for 24 hr. Percoll density gradient isolated monocytes as well as the pro-inflammatory macrophage subtype 27E10 expressed 1B1, 2E1 and 2B6/7. On the other hand, in the anti-inflammatory macrophage subtype RM3/1, predominantly 1B1 and to some extent 2B6/7 were found. Treatment with cyclosporin A, phenobarbital, benzanthracene or ethanol resulted in induction of the expression of 3A3/4. CYP1B1 was the predominant isoenzyme in all monocytes and macrophages. In monocytes purified by adherence or induced by benzanthracene, lipopolysaccharide or 12-O-tetradecanoyl-phorbol-13-acetat, 1A1 was also expressed. Northern blot analysis confirmed the presence of CYP1B1 in monocytes and macrophages, a presence which was also demonstrated on the protein level by immunoblot and by immunohistochemical staining of the cells. The expression of several CYPs in monocytes/macrophages suggests that these cells may be important in the metabolism of small molecular weight compounds, which play a role in allergic contact dermatitis and drug reactions. Of particular interest is the remarkably strong expression of the recently identified dioxin inducible CYP1B1, known to be present in a wide range of malignant tumors.

Phagocytosis Independent Extracellular Nanoparticle Clearance by Human Immune Cells

It has recently been discovered that human immune cells, especially neutrophil granulocytes, form neutrophil extracellular traps (NETs) that abolish pathogens. Our study provides evidence that extracellular traps formed by neutrophils, monocytes and macrophages act as physical barriers for nanoparticles, thus presenting a new nanomaterial clearance mechanism of the human immune system. While particle shape is of minor importance, positive charges significantly enhance particle trapping.

Topographical control of human macrophages by a regularly microstructured polyvinylidene fluoride surface

In this study we investigated the influence of surface topography on the inflammatory response of human macrophages. We generated different polyvinylidene fluoride (PVDF) surfaces including (i) a smooth surface of PVDF spherulites as a control, (ii) a randomly nanotextured surface with alumina particles, and (iii) a microstructure using laser ablation. The identical chemistry of all PVDF surfaces was demonstrated by X-ray photoelectron spectroscopy. The topography was evaluated by white light interferometry and X-profile analysis. Macrophages were cultured on the different surfaces including lipopolysaccharide (LPS) treatment as an inflammatory activator. Our results demonstrate that the microstructured surface but not the nanotexured significantly affects the activation of primary human macrophages by inducing a specific cytokine and gene expression pattern. This activation resulted in a subtype of macrophages with pro- but also anti-inflammatory properties. Interestingly, the response on the topography differed from that triggered by LPS, pointing to a different activation state of the cells. Our data clearly show that a particular topography induces an inflammatory response. This suggests that the modification of topography could influence the inflammatory potency of a biomaterial and hence could affect the biocompatibility of implants.

Ambroxol inhibits the release of histamine, leukotrienes and cytokines from human leukocytes and mast cells.

Abstract.Objectives and Design: The effects of the mucolytic agents ambroxol and N-acetylcystein (NAC) were studied on the release of histamine, leukotrienes, cytokines and superoxide anions from a variety of cells involved in the pathogenesis of allergic inflammation.¶Subjects: Mast cells were isolated from human adenoids and skin (nu2009=u20095–6). Basophils, monocytes and granulocytes were obtained from Buffy-coat blood obtained from healthy blood donors (nu2009=u20094–7) and enriched by density centrifugation.¶Treatment and Methods: Ambroxol or NAC were added to the cells for different periods before stimulation with various immunological and non-immunological secretagogues. Histamine release from mast cells, basophils and monocytes was assayed either by radioimmunoassay or spectrofluorometrically. LTC4 (basophils), LTB4 (neutrophil/eosinophil granulocytes or monocytes), IL-4 and IL-13 (basophils) were measured by ELISA.¶Results: Ambroxol inhibited histamine release by more than 50% from human adenoidal mast cells (1000u2009μM ambroxol) and skin mast cells (100u2009μM ambroxol) stimulated by Con A and compound 48/80, respectively. Ambroxol (100u2009μM) strikingly inhibited anti-IgE induced release of both histamine, LTC4, IL-4 and IL-13 from basophils and reduced both histamine and LTB4 release induced by C5a or Zymosan in monocytes. The drug also reduced LTB4 and superoxide anion production in granulocytes stimulated by zymosan or fMLP. In all cell types studied, ambroxol was more efficacious following a short preincubation (5–15u2009min) of the drug with the cells before stimulation. In contrast, NAC produced no clear effects on any of the different cell types studied, regardless of the preincubation period, the concentration or the stimulus employed.¶Conclusions: Unlike NAC, ambroxol is able to not only inhibit acute mediator release from mast cells and leukocytes but also reduce immunomodulatory cytokine generation from basophils and may have beneficial effects in the treatment of allergic respiratory diseases.

Inducing healing-like human primary macrophage phenotypes by 3D hydrogel coated nanofibres

Immune cells are present in the blood and in resident tissues, and the nature of their reaction towards biomaterials is decisive for materials success or failure. Macrophages may for example be classically activated to trigger inflammation (M1), or alternatively activated which supports healing and vascularisation (M2). Here, we have generated 3D nanofibrous meshes in different porosities and precisely controlled surface chemistries comprising PLGA, hydrogel-coated protein repellant and protein repellant endowed with the bioactive peptide sequences GRGDS or GLF. We also prepared 2D substrates with corresponding surface chemistry for a systematic evaluation of primary human macrophage adhesion, migration, transcriptome expression, cytokine release and surface marker expression. Our data show that material morphology is a powerful means in biomaterial design to influence immune cell response. Flat substrates lead to an increased number of M2 classified CD163(+) macrophages. However, these M2 cells released large amounts of pro-inflammatory cytokines. In contrast, 3D nanofibres with corresponding surface chemistry yielded M1 classified 27E10(+) macrophages with a significantly increased release of pro-angiogenic chemokines and angiogenesis related molecules and a strong decrease of pro-inflammatory cytokines. We thus suggest that, for macrophages in contact with biomaterials, cytokine release is taken as main criterion instead of surface-markers for macrophage classifications.

The chorioallantoic membrane of the chick embryo as a simple model for the study of the angiogenic and inflammatory response to biomaterials.

Angiogenesis is essential in wound healing and a common feature in chronic inflammation which is crucially involved in the biological response to biomaterials. A useful system to evaluate the angiogenic activity and the inflammatory potency of various agents is the chorioallantoic membrane (CAM) of the chick embryo. Here we examined its response to different biomaterials. Smooth materials such as PVC or the polyurethane Tecoflex® either unmodified or modified by an OH- or N(CH3)3+-end group (HEMA or MAPTAC) inhibited angiogenesis and did not induce the formation of granulation tissue. The anti-angiogenic effects of PVC, Tecoflex® and its HEMA modification, however, were only seen at an early stage of development. In contrast, the MAPTAC modified Tecoflex>® inhibited angiogenesis over the whole time. Rough materials, e.g. filter paper or a collagen/elastin membrane, stimulated angiogenesis and induced the formation of inflammatory tissue. Histological analysis revealed that the filter material was homogeneously populated with cells consisiting mainly of macrophages, fibroblasts and endothelial cells. The collagen/elastin membrane was only partially infiltrated with cells. Among those also clusters of granulocytes were present pointing to an acute inflammatory process. These data show that the angiogenic activity and inflammatory response of biomaterials strongly depend on the chemical composition and the physical structure of the material. The CAM assay appears to be a useful tool for studying biocompatibility.

Glucocorticoid-Induced Appearance of the Macrophage Subtype RM 3/1 in Peripheral Blood of Man

The effect of intravenous administration of the glucocorticoid prednylidene on the macrophage subtype RM 3/1 in the peripheral blood of man was studied. Injection of 60 mg steroid resulted in an increase in the proportion of RM 3/1 positive monocytes 12 h after application from the basic level to about 80%. After 24 h the number of RM 3/1 positive cells decreased but remained elevated over the basis rate for a period of at least 15 days. Similar results were obtained after administration of 30 or 6 mg prednylidene even if the peak value occurred with a delay of 12 h after 6 mg. A dose- and time-dependent induction of the RM 3/1 positive subtype could also be demonstrated in vitro by the addition of prednylidene or dexamethasone to cultured purified human monocytes/macrophages. Deoxycortone or indomethacin had no effects. These results suggest that glucocorticoids exert their influence on cells of the mononuclear phagocytic lineage by inducing a distinct monocyte/macrophage subpopulation which seems to be associated with anti-inflammatory functions.

Macrophages and lymphocytes: Alternative sources of histamine

Some recent observations have indicated that cells other than mast cells, notably macrophages, may contain significant amounts of histamine. Using a histamine-specific radioimmunoassay, we found that human blood monocytes and lymphocytes contain about 0.05pg histamine/cell. Various other cells, e.g. fibroblasts, colorectal tumor, kidney and ovarian cells, and murine bone marrow derived macrophages contained markedly less histamine (<0.008 pg/cell). Ionophore A23187 (1 μM) released up to 50% of the total histamine from monocytes and lymphocytes. C5a caused a dose-dependent histamine release of up to 40% in monocytes and up to 20% in lymphocytes. Substance P induced a release only in cells of certain donors. Lipopolysaccharide, concanavalin A, and compound 48/80 had no effect. Culturing of the cells caused a loss of cellular histamine and its releasability. In view of the huge numbers of monocytes and lymphocytes in the blood, the histamine in these cells has to be taken into account under both physiological and pathophysiological conditions.

Induction of specific macrophage subtypes by defined micro-patterned structures.

In this study, we investigated the influence of different perfluoropolyether (PFPE) microstructures on the inflammatory response of human macrophages. We generated four different microstructured PFPE surfaces by replica molding from silicon masters. The function-associated surface markers 27E10 and CD163 were monitored using flow cytometry to measure the pro- and anti-inflammatory reactions. Inflammatory mediator expression was measured at the protein and mRNA level. Lipopolysaccharide treatment served as positive control for pro-inflammatory activation. We observed that each micropattern induced a specific morphology, phenotype and mediator profile. A microstructure of regular grooves induced a pro-inflammatory phenotype (M1) which was not accompanied by release of pro-inflammatory mediators. However, the larger cylindrical posts induced an anti-inflammatory phenotype (M2) with a remarkable down-regulation of CXCL10. Smaller posts with a shorter distance exhibited a stronger pro-inflammatory response than those with a longer distance, on the levels of both phenotype and mediator release. Regression analysis suggests that the geometrical parameters of the microstructures, specifically the period of structures, may play an important role in macrophage response. Optimization of such microstructures may provide a method to invoke a predictable response of macrophages to implants and control the mediator release.