Gender differences in circulating levels of neutrophil extracellular traps in serum of multiple sclerosis patients.

Neutrophil extracellular traps (NETs) trap and kill pathogens very efficiently but also activate dendritic cells and prime T cells. Previously, we demonstrated that neutrophils are primed and circulating NETs are elevated in relapsing remitting multiple sclerosis (RRMS), a T cell-mediated autoimmune disease. Here, we demonstrate gender specific differences in circulating NETs but not in neutrophil priming in RRMS patients. Although the results from our systematic and in depth characterization of these patients argue against a major role of circulating NETs in this disease, they suggest that NETs may underlie gender-specific differences in MS pathogenesis.

Displacement chromatography as first separating step in online two-dimensional liquid chromatography coupled to mass spectrometry analysis of a complex protein sample--the proteome of neutrophils.

Displacement chromatography provides some advantages over elution chromatography such as the opportunity to enrich trace amounts of molecules and to elute molecules in highest concentrations achievable with liquid chromatography. In a previous study we demonstrated that displacement chromatography is a well-suited alternative to gradient elution in an offline two-dimensional (2D-)LC-MS approach for the analysis of proteomes. In this study we present a method for applying displacement chromatography in an online 2D-LC-MS system including a cation exchange (CEX) column and a reversed phase column. We circumvented the problem of determining the sample capacity of the CEX column by repeated injection (pulses) of sample aliquots monitored by an LC-MS analysis of each flow-through fraction of the CEX column. Elution of tryptic peptides from the CEX column was achieved by repeated injection (pulses) of the displacer spermine. Pulsed displacer injections offer the advantage through physical separation of preventing post-column mixing of already separated compounds. As a proof of principle we analyzed the cytosolic proteome of human neutrophils.

T lymphocyte priming by neutrophil extracellular traps links innate and adaptive immune responses.

Polymorphonuclear neutrophils constitute the first line of defense against infections. Among their strategies to eliminate pathogens they release neutrophil extracellular traps (NETs), being chromatin fibers decorated with antimicrobial proteins. NETs trap and kill pathogens very efficiently, thereby minimizing tissue damage. Furthermore, NETs modulate inflammatory responses by activating plasmacytoid dendritic cells. In this study, we show that NETs released by human neutrophils can directly prime T cells by reducing their activation threshold. NETs-mediated priming increases T cell responses to specific Ags and even to suboptimal stimuli, which would not induce a response in resting T cells. T cell priming mediated by NETs requires NETs/cell contact and TCR signaling, but unexpectedly we could not demonstrate a role of TLR9 in this mechanism. NETs-mediated T cell activation adds to the list of neutrophil functions and demonstrates a novel link between innate and adaptive immune responses.

Neutrophils in multiple sclerosis are characterized by a primed phenotype.

Neutrophils are armed with proteases with indiscriminate histotoxic potential, and to minimize tissue injury, their activation involves priming with inflammatory mediators before cells are fully activated in a second step. Here, we show that neutrophils in multiple sclerosis patients are more numerous and exhibit a primed state based on reduced apoptosis, higher expression of TLR-2, fMLP receptor, IL-8 receptor and CD43, enhanced degranulation and oxidative burst as well as higher levels of neutrophil extracellular traps in serum. The chronic inflammatory environment in multiple sclerosis probably underlies this inappropriate neutrophil priming, which may result in enhanced neutrophil activation during infection.

Induction of long-lived allergen-specific plasma cells by mucosal allergen challenge.

BACKGROUND: Allergen-specific IgE antibodies are responsible for the pathogenesis of type I hypersensitivity. In patients with allergy, IgE titers can persist in the apparent absence of allergen for years. Seasonal allergen exposure triggers clinical symptoms and enhances allergen-specific IgE. Whether allergen-specific plasma cells originating from seasonal allergen exposures can survive and become long-lived is so far unclear. OBJECTIVE: We analyzed the localization and lifetimes of allergen-specific IgE-secreting, IgA-secreting, and IgG(1)-secreting plasma cells after allergen inhalation in an ovalbumin-induced murine model of allergic asthma. METHODS: Ovalbumin-specific IgG(1)-secreting, IgA-secreting, and IgE-secreting cells in lungs, spleen, and bone marrow were isolated and tested for antibody secretion by the ELISpot technique. Longevity of ovalbumin-specific plasma cells was determined by cyclophosphamide treatment, which depletes proliferating plasmablasts but leaves plasma cells untouched. Ovalbumin aerosol-induced infiltrates in lungs were localized by confocal microscopy. RESULTS: Long-lived ovalbumin-specific plasma cells were generated by systemic sensitization and survived in bone marrow and spleen, maintaining systemic ovalbumin-specific titers of IgG, IgA, and IgE. On inhalation of ovalbumin-containing aerosol, sensitized mice developed airway inflammation and more ovalbumin-specific IgG(1)-secreting, IgA-secreting, and IgE-secreting cells in the lungs and in secondary lymphoid organs. These plasma cells joined the pool of ovalbumin-specific plasma cells in the bone marrow and became long-lived-that is, they are resistant to cyclophosphamide. Termination of ovalbumin inhalation depleted ovalbumin-specific plasma cells from the lungs, but they persisted in spleen and bone marrow. CONCLUSION: Our results show that inhalation of aerosolized allergen generates long-lived, allergen-specific IgG(1)-secreting, IgA-secreting, and IgE-secreting plasma cells that survive cytostatic treatment.