Georg Varga

Staphylococcus aureus Panton-Valentine Leukocidin Is a Very Potent Cytotoxic Factor for Human Neutrophils

The role of the pore-forming Staphylococcus aureus toxin Panton-Valentine leukocidin (PVL) in severe necrotizing diseases is debated due to conflicting data from epidemiological studies of community-associated methicillin-resistant S. aureus (CA-MRSA) infections and various murine disease-models. In this study, we used neutrophils isolated from different species to evaluate the cytotoxic effect of PVL in comparison to other staphylococcal cytolytic components. Furthermore, to study the impact of PVL we expressed it heterologously in a non-virulent staphylococcal species and examined pvl-positive and pvl-negative clinical isolates as well as the strain USA300 and its pvl-negative mutant. We demonstrate that PVL induces rapid activation and cell death in human and rabbit neutrophils, but not in murine or simian cells. By contrast, the phenol-soluble modulins (PSMs), a newly identified group of cytolytic staphylococcal components, lack species-specificity. In general, after phagocytosis of bacteria different pvl-positive and pvl-negative staphylococcal strains, expressing a variety of other virulence factors (such as surface proteins), induced cell death in neutrophils, which is most likely associated with the physiological clearing function of these cells. However, the release of PVL by staphylococcal strains caused rapid and premature cell death, which is different from the physiological (and programmed) cell death of neutrophils following phagocytosis and degradation of virulent bacteria. Taken together, our results question the value of infection-models in mice and non-human primates to elucidate the impact of PVL. Our data clearly demonstrate that PVL acts differentially on neutrophils of various species and suggests that PVL has an important cytotoxic role in human neutrophils, which has major implications for the pathogenesis of CA-MRSA infections.

Apolipoprotein E Induces Antiinflammatory Phenotype in Macrophages

Objective—Apolipoprotein E (apoE) exerts potent antiinflammatory effects. Here, we investigated the effect of apoE on the functional phenotype of macrophages. Methods and Results—Human apoE receptors very-low-density lipoprotein receptor (VLDL-R) and apoE receptor-2 (apoER2) were stably expressed in RAW264.7 mouse macrophages. In these cells, apoE downregulated markers of the proinflammatory M1 phenotype (inducible nitric oxide synthase, interleukin [IL]-12, macrophage inflammatory protein-1&agr;) but upregulated markers of the antiinflammatory M2 phenotype (arginase I, SOCS3, IL-1 receptor antagonist [IL-1RA]). In addition, M1 macrophage responses (migration, generation of reactive oxygen species, antibody-dependent cell cytotoxicity, phagocytosis), as well as poly(I:C)- or interferon-&ggr;-induced production of proinflammatory cytokines; cyclooxygenase-2 expression; and activation of nuclear factor-&kgr;B, I&kgr;B, and STAT1, were suppressed in VLDL-R- or apoER2-expressing cells. Conversely, the suppression of the M2 phenotype and the enhanced response to poly(I:C) were observed in apoE-producing bone marrow macrophages derived from VLDL-R-deficient mice but not wild-type or low-density lipoprotein receptor–deficient mice. The modulatory effects of apoE on macrophage polarization were inhibited in apoE receptor-expressing RAW264.7 cells exposed to SB220025, a p38 mitogen-activated protein kinase inhibitor, and PP1, a tyrosine kinase inhibitor. Accordingly, apoE induced tyrosine kinase-dependent activation of p38 mitogen-activated protein kinase in VLDL-R- or apoER2-expressing macrophages. Under in vivo conditions, apoE−/− mice transplanted with apoE-producing wild-type bone marrow showed increased plasma IL-1RA levels, and peritoneal macrophages of transplanted animals were shifted to the M2 phenotype (increased IL-1RA production and CD206 expression). Conclusion—ApoE signaling via VLDL-R or apoER2 promotes macrophage conversion from the proinflammatory M1 to the antiinflammatory M2 phenotype. This effect may represent a novel antiinflammatory activity of apoE.

Glucocorticoids induce an activated, anti-inflammatory monocyte subset in mice that resembles myeloid-derived suppressor cells

Glucocorticoids (GC) are still the most widely used immunosuppressive agents in clinical medicine. Surprisingly, little is known about the mechanisms of GC action on monocytes, although these cells exert pro‐ and anti‐inflammatory effects. We have shown recently that GC induce a specific monocyte phenotype with anti‐inflammatory properties in humans. We now investigated whether this also applies for the murine system and how this subset would relate to recently defined murine subtypes. After treatment with dexamethasone for 48 h, monocytes up‐regulated scavenger receptor CD163 and Gr‐1, down‐regulated CX3CR1, and shared with human GC‐treated monocytes functional features such as low adhesiveness but high migratory capacity. They specifically up‐regulated anti‐inflammatory IL‐10, but not TGF‐β, and in contrast to their human counterparts, they down‐regulated IL‐6. Although GC‐induced monocytes down‐regulated CX3CR1, a distinctive marker for classical/proinflammatory human and murine monocytes (CX3CR1loCCR2+Ly6Chi), they differed from this physiologically occurring subset, as they remained Ly6Cmed and unactivated (CD62 ligand++). In addition to their immunosuppressive effects, they were CD11b+Gr‐1+ and expressed the IL‐4Rα chain (CD124), a recently described, signature molecule of tumor‐induced myeloid‐derived suppressor cells (MDSC). We therefore generated murine MDSC in B16 melanoma‐bearing mice and indeed found parallel up‐regulation of CD11b+Gr‐1+ and CD124 on GC‐induced monocytes and MDSC. These data allow us to speculate that the GC‐induced subtype shares with inflammatory monocytes the ability to migrate quickly into inflamed tissue, where they, however, exert anti‐inflammatory effects and that similarities between GC‐induced monocytes and MDSC may be involved in progression of some tumors observed in patients chronically treated with GC.

A hemodynamic response to intravenous adenovirus vector particles is caused by systemic Kupffer cell-mediated activation of endothelial cells.

Intravascular injection of adenoviral vectors may result in a toxic and potentially lethal reaction, the mechanism of which is poorly understood. We noted that mice demonstrated a transient change in behavior that was characterized by inactivity and lethargy within minutes after intravenous injection of relatively low doses of adenoviral vectors (including high-capacity gutless vectors). Moreover, immediately after vector injection a significant drop in blood pressure was measured that most probably was caused by the systemic activation of endothelial cells as monitored by detection of phosphorylated Akt/PKB kinase, activated endothelial nitric oxide synthase (eNOS), and nitrotyrosine. The activation of the endothelium was the result of the interaction of viral particles with Kupffer cells, which are resident macrophages of the liver representing the first line of defense of the innate immune system. Surprisingly, the uptake of vector particles by Kupffer cells not only resulted in their strong activation, but also in their nearly complete disappearance from the liver. Our results suggest that the toxicity of intravenously injected adenoviral vectors may be directly linked to the activation and destruction of Kupffer cells.

β2 integrins are required for skin homing of primed T cells but not for priming naive T cells

Beta2 integrins are of critical importance for leukocyte extravasation through vascular endothelia and for T cell activation. To elucidate the role of beta2 integrins in T cell-mediated immune responses, allergic contact dermatitis (ACD), irritant dermatitis, and delayed-type hypersensitivity (DTH) were assessed in mice lacking the beta2 integrin subunit, CD18. ACD and DTH responses, but not edema formation, were severely suppressed in CD18(-/-) mice. Extravasation of CD18(-/-) T cells into eczematous skin lesions was greatly impaired, whereas migration of Langerhans cell precursors and dendritic cells was normal in CD18(-/-) mice. CD18(-/-)lymph nodes (LNs) contained an abnormal population of CD3(-)CD44(high) lymphocytes and showed evidence of widespread T cell activation. T cells from regional LNs of sensitized CD18(-/-) mice proliferated in response to hapten challenge, and subcutaneous injection of sensitized syngeneic LN cells directly into ears of hapten-challenged naive recipients restored the defective ACD in CD18(-/-) mice, suggesting that CD18 is not required for priming of naive T cells but is indispensable for T cell extravasation. Thus, a dysfunction of T cells, in addition to granulocytes, may contribute to the pathophysiology of leukocyte adhesion deficiency type I, which arises from mutations in the human CD18 gene.

Vitamin D receptor signaling contributes to susceptibility to infection with Leishmania major

We have previously reported that 1α,25‐dihydroxyvitamin D3 (1α,25(OH)2D3) can selectively suppress key functions of interferon‐gamma (IFN‐γ) activated macrophages. To further explore this mechanism for its relevance in vivo, we investigated an infection model that crucially depends on the function of IFN‐γ activated macrophages, the infection with the intracellular protozoan Leishmania mαjor. 1α,25(OH)2D3 treatment of L. major infected macrophages demonstrated a vitamin D receptor (Vdr) dependent inhibition of macrophage killing activity. Further analysis showed that this was a result of decreased production of nitric oxide by 1α,25(OH)2D3‐treated macrophages due to Vdr‐dependent up‐regulation of arginase 1 ex‐pression, which overrides NO production by Nos2. When analyzing the course of infection in vivo,we found that Vdr‐knockout (Vdr‐KO) mice were more resistant to L. major infection than their wild‐type littermates. This result is in agreement with an inhibitory influence of 1α,25(OH)2D3 on the macrophage mediated host defense. Further investigation showed that Vdr‐KO mice developed an unaltered T helper cell type 1 (Th1) response on infection as indicated by normal production of IFN‐γ by CD4+ and CD8+ T cells. Therefore, we propose that the absence of 1α,25(OH)2D3‐mediated inhibition of macrophage microbicidal activity in Vdr‐KO mice results in increased resistance to Leishmania infection.

Proinflammatory S100A12 Can Activate Human Monocytes via Toll-like Receptor 4

RATIONALE S100A12 is overexpressed during inflammation and is a marker of inflammatory disease. Furthermore, it has been ascribed to the group of damage-associated molecular pattern molecules that promote inflammation. However, the exact role of human S100A12 during early steps of immune activation and sepsis is only partially described thus far. OBJECTIVES We analyzed the activation of human monocytes by granulocyte-derived S100A12 as a key function of early inflammatory processes and the development of sepsis. METHODS Circulating S100A12 was determined in patients with sepsis and in healthy subjects with experimental endotoxemia. The release of human S100A12 from granulocytes as well as the promotion of inflammation by activation of human monocytes after specific receptor interaction was investigated by a series of in vitro experiments. MEASUREMENTS AND MAIN RESULTS S100A12 rises during sepsis, and its expression and release from granulocytes is rapidly induced in vitro and in vivo by inflammatory challenge. A global gene expression analysis of S100A12-activated monocytes revealed that human S100A12 induces inflammatory gene expression. These effects are triggered by an interaction of S100A12 with Toll-like receptor 4 (TLR4). Blocking S100A12 binding to TLR4 on monocytes or TLR4 expressing cell lines (HEK-TCM) abrogates the respective inflammatory signal. On the contrary, blocking S100A12 binding to its second proposed receptor (receptor for advanced glycation end products [RAGE]) has no significant effect on inflammatory signaling in monocytes and RAGE-expressing HEK293 cells. CONCLUSIONS Human S100A12 is an endogenous TLR4 ligand that induces monocyte activation, thereby acting as an amplifier of innate immunity during early inflammation and the development of sepsis.

Agonists of proteinase-activated receptor-2 modulate human neutrophil cytokine secretion, expression of cell adhesion molecules, and migration within 3-D collagen lattices

Proteinase‐activated receptor‐2 (PAR2) belongs to a novel subfamily of G‐protein‐coupled receptors with seven‐transmembrane domains. PAR2 can be activated by serine proteases such as trypsin, mast cell tryptase, and allergic or bacterial proteases. This receptor is expressed by various cells and seems to be crucially involved during inflammation and the immune response. As previously reported, human neutrophils express functional PAR2. However, the precise physiological role of PAR2 on human neutrophils and its implication in human diseases remain unclear. We demonstrate that PAR2 agonist‐stimulated human neutrophils show significantly enhanced migration in 3‐D collagen lattices. PAR2 agonist stimulation also induced down‐regulation of L‐selectin display and up‐regulation of membrane‐activated complex‐1 very late antigen‐4 integrin expression on the neutrophil cell surface. Moreover, PAR2 stimulation results in an increased secretion of the cytokines interleukin (IL)‐1β, IL‐8, and IL‐6 by human neutrophils. These data indicate that PAR2 plays an important role in human neutrophil activation and may affect key neutrophil functions by regulating cell motility in the extracellular matrix, selectin shedding, and up‐regulation of integrin expression and by stimulating the secretion of inflammatory mediators. Thus, PAR2 may represent a potential therapeutic target for the treatment of diseases involving activated neutrophils.

In Vivo Optical Imaging of Cellular Inflammatory Response in Granuloma Formation Using Fluorescence-Labeled Macrophages

Near-infrared imaging such as fluorescence reflectance imaging (FRI) and fluorescence-mediated tomography (FMT) yields high signal-to-noise ratios (SNRs) and should thus be well suited for cell-tracking studies. Extravasation of monocytes or macrophages (Mϕs) is one of the earliest events in inflammation. The purpose of this study was to assess whether FRI and FMT allow for the visualization and quantification of early inflammatory processes by tracing the migration of fluorescence-labeled murine Mϕs in a cutaneous granuloma model. Methods: Mϕs were labeled with a membrane-selective carbocyanine dye (1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide [DiR]). Cellular viability and function (nitric oxide production, phagocytosis, adherence) were assessed in vitro. Local inflammation was induced in mice by the subcutaneous injection of polyacrylamide gel pellets including or excluding a strong inflammatory stimulus (lipopolysaccharide). Labeled Mϕs were injected intravenously, and FRI and FMT were performed up to 7 d. SNRs were calculated for the pellets, and the 3-dimensional distribution of Mϕs was assessed using FMT. Cells were harvested from gel pellets and analyzed by flow cytometry. Results: DiR labeling did not affect cell viability or cell function. FRI revealed the migration of labeled Mϕs into gel pellets and the homing of Mϕs to different body compartments. The lipopolysaccharide-containing pellets exhibited significantly higher SNRs than did pellets without lipopolysaccharide. FMT showed that Mϕs distributed mainly in the periphery of the pellets. The cellular infiltrates extracted from the harvested pellets revealed the presence of approximately 10%−23% DiR-positive Mϕs-expressing typical markers, confirming the transendothelial migration of injected Mϕs. Conclusion: The tagging of Mϕs with DiR allows the noninvasive tracking of inflammatory cells for several days in vivo. FRI and FMT are versatile techniques to monitor and quantify cellular inflammatory responses in vivo.

CD4+ T Cell-Associated Pathophysiology Critically Depends on CD18 Gene Dose Effects in a Murine Model of Psoriasis

In a CD18 hypomorphic polygenic PL/J mouse model, the severe reduction of CD18 (β2 integrin) to 2–16% of wild-type levels leads to the development of a psoriasiform skin disease. In this study, we analyzed the influence of reduced CD18 gene expression on T cell function, and its contribution to the pathogenesis of this disease. Both CD4+ and CD8+ T cells were significantly increased in the skin of affected CD18 hypomorphic mice. But only depletion of CD4+ T cells, and not the removal of CD8+ T cells, resulted in a complete clearance of the psoriasiform dermatitis. This indicates a central role of CD4+ T cells in the pathogenesis of this disorder, further supported by the detection of several Th1-like cytokines released predominantly by CD4+ T cells. In contrast to the CD18 hypomorphic mice, CD18 null mutants of the same strain did not develop the psoriasiform dermatitis. This is in part due to a lack of T cell emigration from dermal blood vessels, as experimental allergic contact dermatitis could be induced in CD18 hypomorphic and wild-type mice, but not in CD18 null mutants. Hence, 2–16% of CD18 gene expression is obviously sufficient for T cell emigration driving the inflammatory phenotype in CD18 hypomorphic mice. Our data suggest that the pathogenic involvement of CD4+ T cells depends on a gene dose effect with a reduced expression of the CD18 protein in PL/J mice. This murine inflammatory skin model may also have relevance for human polygenic inflammatory diseases.