CCR7-guided neutrophil redirection to skin-draining lymph nodes regulates cutaneous inflammation and infection.

Neutrophils are the first nonresident effector immune cells that migrate to a site of infection or inflammation; however, improper control of neutrophil responses can cause considerable tissue damage. Here, we found that neutrophil responses in inflamed or infected skin were regulated by CCR7-dependent migration and phagocytosis of neutrophils in draining lymph nodes (dLNs). In mouse models of Toll-like receptor-induced skin inflammation and cutaneous Staphylococcus aureus infection, neutrophils migrated from the skin to the dLNs via lymphatic vessels in a CCR7-mediated manner. In the dLNs, these neutrophils were phagocytosed by lymph node-resident type 1 and type 2 conventional dendritic cells. CCR7 up-regulation on neutrophils was a conserved mechanism across different tissues and was induced by a broad range of microbial stimuli. In the context of cutaneous immune responses, disruption of CCR7 interactions by selective CCR7 deficiency of neutrophils resulted in increased antistaphylococcal immunity and aggravated skin inflammation. Thus, neutrophil homing to and clearance in skin-dLNs affects cutaneous immunity versus pathology.

A systematic electron microscopic study on the uptake of barium sulphate nano-, submicro-, microparticles by bone marrow-derived phagocytosing cells.

Nanoparticles can act as transporters for synthetic molecules and biomolecules into cells, also in immunology. Antigen-presenting cells like dendritic cells are important targets for immunotherapy in nanomedicine. Therefore, we have used primary murine bone marrow-derived phagocytosing cells (bmPCs), i.e. dendritic cells and macrophages, to study their interaction with spherical barium sulphate particles of different size (40 nm, 420 nm, and 1 µm) and to follow their uptake pathway. Barium sulphate is chemically and biologically inert (no dissolution, no catalytic effects), i.e. we can separate the particle uptake effect from potential biological reactions. The colloidal stabilization of the nanoparticles was achieved by a layer of carboxymethylcellulose (CMC) which is biologically inert and gives the particles a negative zeta potential (i.e. charge). The particles were made fluorescent by conjugating 6-aminofluoresceine to CMC. Their uptake was visualized by flow cytometry, confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and correlative light and electron microscopy (CLEM). Barium sulphate particles of all sizes were readily taken up by dendritic cells and even more by macrophages, with the uptake increasing with time and particle concentration. They were mainly localized inside phagosomes, heterophagosomes, and in the case of nanoparticles also in the nearby cytosol. No particles were found in the nucleus. In nanomedicine, inorganic nanoparticles from the nanometer to the micrometer size are therefore well suited as transporters of biomolecules, including antigens, into dendritic cells and macrophages. The presented model system may also serve to describe the aseptic loosening of endoprostheses caused by abrasive wear of inert particles and the subsequent cell reaction, a question which relates to the field of nanotoxicology. STATEMENT OF SIGNIFICANCE: The interaction of particles and cells is at the heart of nanomedicine and nanotoxicology, including abrasive wear from endoprostheses. It also comprises the immunological reaction to different kinds of nanomaterials, triggered by an immune response, e.g. by antigen-presenting cells. However, it is often difficult to separate the particle effect from a chemical or biochemical reaction to particles or their cargo. We show how chemically inert barium sulphate particles with three different sizes (nano, sub-micro, and micro) interact with relevant immune cells (primary dendritic cells and macrophages). Particles of all three sizes are readily taken up into both cell types by phagocytosis, but the uptake by macrophages is significantly more prominent than that by dendritic cells. The cells take up particles until they are virtually stuffed, but without direct adverse effect. The uptake increases with time and particle concentration. Thus, we have an ideal model system to follow particles into and inside cells without the side effect of a chemical particle effect, e.g. by degradation or ion release.

Selective detection of NADPH oxidase in polymorphonuclear cells by means of NAD(P)H-based fluorescence lifetime imaging.

NADPH oxidase (NOX2) is a multisubunit membrane-bound enzyme complex that, upon assembly in activated cells, catalyses the reduction of free oxygen to its superoxide anion, which further leads to reactive oxygen species (ROS) that are toxic to invading pathogens, for example, the fungus Aspergillus fumigatus. Polymorphonuclear cells (PMNs) employ both nonoxidative and oxidative mechanisms to clear this fungus from the lung. The oxidative mechanisms mainly depend on the proper assembly and function of NOX2. We identified for the first time the NAD(P)H-dependent enzymes involved in such oxidative mechanisms by means of biexponential NAD(P)H-fluorescence lifetime imaging (FLIM). A specific fluorescence lifetime of 3670 +/- 140 picoseconds as compared to 1870 picoseconds for NAD(P)H bound to mitochondrial enzymes could be associated with NADPH bound to oxidative enzymes in activated PMNs. Due to its predominance in PMNs and due to the use of selective activators and inhibitors, we strongly believe that this specific lifetime mainly originates from NOX2. Our experiments also revealed the high site specificity of the NOX2 assembly and, thus, of the ROS production as well as the dynamic nature of these phenomena. On the example of NADPH oxidase, we demonstrate the potential of NAD(P)H-based FLIM in selectively investigating enzymes during their cellular function.

Migration, cell-cell interaction and adhesion in the immune system.

Migration is an essential function of immune cells. It is necessary to lead immune cell precursors from their site of generation to the places of maturation or function. Cells of the adaptive immune system also need to interact physically with each other or with specialized antigen presenting cells in lymphatic tissues in order to become activated. Thereby a complex series of controlled migration events, adhesive interactions and signalling responses is induced. Finally cells must be able to leave the activating tissues and re-enter the bloodstream from which they extravasate into inflamed tissue sites. Cells of the innate immune system can function directly without the need for previous activation. However, these cells have to adapt their function to a panoply of pathogens and environmental niches which can be invaded. The current review highlights the central aspects of cellular dynamics underlying adaptive and innate cellular immunity. Thereby a focus will be put on recent results obtained by microscopic observation of live cells in vitro or by intravital 2-photon microscopy in live animals.

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)-1beta, 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.

Dendritic cells in cancer immunotherapy.

Antigen presentation is a critical regulatory element for the induction of cellular immune responses. Thus, one of the principal current goals of tumor immunotherapy is to control and enhance tumor antigen presentation. In this respect, dendritic cells (DC) are now being widely investigated as immunotherapeutic agents for the treatment of disseminated malignancies. At present, numerous ways to employ DCs for tumor immunotherapy are being tested, ranging from direct in situ expansion and activation of DCs to adoptive transfer of ex vivo generated DCs, and numerous techniques have been designed to optimize DC activation, tumor antigen delivery to DCs, and induction of tumor-specific, as well as helper immune responses, in vivo. However, the results of recent preclinical studies and the diversity of the clinical phase I trials that are currently underway indicate that little is still known about the exact mechanisms by which DCs modulate tumor immunity and pose the concern that premature clinical trials might not yield the desired results and might be harmful to, rather than promote, the concept of DC-based tumor immunotherapy. This review summarizes some of the current approaches to induce tumor immunity by DC-based vaccination and discusses their advantages and concerns.

Two-step negative enrichment of CD4+ and CD8+ T cells from murine spleen via nylon wool adherence and an optimized antibody cocktail.

We developed a method to highly purify CD4+ and CD8+ T cells from murine spleen by negative enrichment strategy. Single-cell suspensions of spleen cells were depleted from erythrocytes by ammonium chloride-mediated lysis. The obtained cell suspension contained approximately 28% CD4+ cells and 14% CD8+ cells. Passing of these cells over a nylon wool column removed up to 75% of all cells, leading to a suspension containing approximately 50% CD4+ and 23% CD8+ cells. These cells were further purified by a single immunomagnetic depletion step using a panel of eight antibodies in combination with MACS magnetic beads and an autoMACS machine. After purification, cells were viable and mostly non-activated based on the expression of activation markers and did not or only minimally respond to polyclonal stimuli such as soluble anti-CD3 antibodies or Concanavalin A. With this method, 19-38% of all CD4 cells and 10-29% of all CD8 cells in a spleen cell suspension were recovered at the mentioned purity. The whole procedure is fast (<4 h of preparation), simple and cost effective, as all antibodies and the magnetic beads have been titrated to the minimal concentration needed for purification. The method is highly reproducible, routinely leading to CD4+ cells with >97% purity (range 97.4-99%) and CD8+ cells with >96% purity (range 95.6-96.7%). The described protocol should facilitate studies aiming at the physiology of "untouched" murine T cells.

Dendritic cells and tumor immunity.

Researchers and clinicians have tried for decades to use the mechanisms of immunity for the fight against cancer. Early attempts aimed at the instrumentation of soluble immune mediators such as antibodies or cytotoxic proteins for the therapy of malignancies. Major improvements in understanding the induction and regulation of cellular immunity have now made it possible to generate effector cells in cancer patients which are specific for the neoplastic disease. At the beginning of every cellular immune reaction against cancers tumor antigens have to be presented to T cells in order to activate them and drive them into clonal expansion. This is done by antigen presenting cells, the most powerful of which is the dendritic cell (DC). While DC were hard to isolate initially, they can be generated in large numbers in vitro today and manipulated in multiple ways before given back to a patient to induce tumor immunity. Thus, a great amount of hope lies in the use of DC as inducers of tumor immunity. However, the first clinical studies, which have now been completed with only limited success make clear, that still a lot of open questions remain to be answered. This review tries to give an overview of this rapidly developing field, mentioning the major conceptual approaches and techniques, but also discussing important caveats. The next years will show whether we can improve our understanding of DC biology and the mechanisms of immune induction strongly enough to effectively employ DC for immunotherapy of cancer.

Interaction of T cells with APCs: the serial encounter model.

Primary immune responses are initiated by specific physical interaction of antigen-specific T cells and professional antigen-presenting cells (APCs). Productive interactions can be a dynamic process that combines physical T-cell binding to APCs with vigorous crawling across and scanning of the APC surface, resulting in signal induction. After T-cell detachment, subsequent migratory contacts to the same or neighboring dendritic cells (DCs) allow the accumulation of sequential signals and interaction time. Here, we develop a serial encounter model of T-cell activation and discuss how the summation of multiple signals provides an efficient strategy to control an ongoing immune response.

Antigen presentation in extracellular matrix: interactions of T cells with dendritic cells are dynamic, short lived, and sequential.

Cognate interactions of naive T cells with antigen-presenting dendritic cells require physical cell-cell contacts leading to signal induction and T cell activation. Using a three-dimensional collagen matrix videomicroscopy model for ovalbumin peptide-specific activation of murine and oxidative mitogenesis of human T cells, we show that T cells maintain vigorous migration upon cognate interactions to DC (dendritic cell), continuously crawl across the DC surface, and rapidly detach (median within 6-12 min). These dynamic and short-lived encounters favor sequential contacts with the same or other DC and trigger calcium influx, upregulation of activation markers, T blast formation, and proliferation. We conclude that a tissue environment supports the accumulation of sequential signals, implicating a numeric or "digital" control mechanism for an ongoing primary immune response.

Evidence for functional relevance of CTLA-4 in ultraviolet-radiation-induced tolerance.

Hapten sensitization through UV-exposed skin induces hapten-specific tolerance that can be adoptively transferred by injecting T lymphocytes into naive recipients. The exact phenotype of T cells responsible for inhibiting the immune response and their mode of action remain unclear. Evidence exists that CTLA-4 negatively regulates T cell activation. We addressed whether CTLA-4 is involved in the transfer of UV-induced tolerance. Injection of lymph node cells from mice that were sensitized with dinitrofluorobenzene (DNFB) through UV-irradiated skin inhibited induction of contact hypersensitivity against DNFB in the recipient animals. When CTLA-4+ cells were depleted, transfer of suppression was lost. Likewise, significantly fewer lymphocytes enriched for CTLA-4+ cells were necessary to transfer suppression than unfractionated cells. Expression of CTLA-4 appears to be functionally relevant, since in vivo injection of a blocking anti-CTLA-4 Ab was able to break UV-induced tolerance and inhibited transfer of suppression. Upon stimulation with dendritic cells in the presence of the water-soluble DNFB analogue, DNBS, CTLA-4+ T cells from DNFB-tolerized mice secreted high levels of IL-10, TGF-beta, and IFN-gamma; low levels of IL-2; and no IL-4, resembling the cytokine pattern of T regulatory 1 cells. Ab blocking of CTLA-4 resulted in inhibition of IL-10 release. Accordingly, transfer of tolerance was not observed when recipients were treated with an anti-IL-10 Ab. Hence we propose that T cells, possibly of the T regulatory 1 type, transfer UV-mediated suppression through the release of IL-10. Activation of CTLA-4 appears to be important in this process.

Migration of dendritic cells within 3-D collagen lattices is dependent on tissue origin, state of maturation, and matrix structure and is maintained by proinflammatory cytokines.

The function of dendritic cells (DC) depends on active migration through three-dimensional (3-D) extracellular matrices. We have analyzed the migration of murine DC from different tissue origins within 3-D collagen lattices through the use of time-lapse videomicroscopy and single-cell tracking. Directly after incorporation, 50-90% of DC from the spleen (spDC) and Langerhans cells freshly isolated from the epidermis (fLC) displayed active motility in these matrices. Whereas mature spDC showed multilateral pseudopod dynamics as well as fast and heterogeneous migration, immature fLC displayed a spherical shape with faint membrane processes and very homogenous, slow migration characteristics. In the absence of external stimuli, migration of both, spDC and fLC, vanished after >36 h due to cell death. Maintaining fLC viability by external granulocyte-macrophage colony-stimulating factor or tumor necrosis factor alpha prolonged migration up to 5 days. During this period fLC transformed into mature cells with large dendrites, thereby developing a heterogeneous migration pattern more similar to spDC. In randomly polymerized collagen matrices cell paths were without preferential orientation. In contrast, in artificially aligned lattices directional paths in accordance with the forced fiber orientation were observed. Thus, migration is an inherent property of DC, largely influenced by tissue origin, degree of maturity, and the 3-D structure of the environment.

T lymphocytes and neutrophil granulocytes differ in regulatory signaling and migratory dynamics with regard to spontaneous locomotion and chemotaxis.

Chemotactic migration of T lymphocytes and neutrophil granulocytes within a three-dimensional collagen matrix is distinct from spontaneous, matrix-induced migration concerning dynamic parameters and regulatory intracellular signaling. Both spontaneous T lymphocyte locomotion and stromal-cell-derived factor-1 (SDF-1)-induced chemotaxis-involved protein tyrosine kinase (PTK) activity, whereas only SDF-1-induced migration was protein kinase C (PKC) dependent. Spontaneous locomotion of neutrophil granulocytes was independent of PKC and PTK activity, but formyl-methionyl-leucyl-phenylalanine-induced migration involved PKC activity. In addition, the microtubule cytoskeleton was not changed after induction of chemotaxis in both cell types. T lymphocytes had a well-developed microtubule cytoskeleton with the microtubule organizing center located in the uropod, whereas neutrophil granulocytes revealed a clustered tubulin distribution at the leading edge of the migrating cell. Therefore, differences of the microtubule cytoskeleton might contribute to differences in locomotion between T lymphocytes and neutrophil granulocytes but not to differences between spontaneous locomotion and chemotaxis.