Frank A. Redegeld

Immunoglobulin-free light chains elicit immediate hypersensitivity-like responses

Immunoglobulin (Ig)-free light chains IgLC are present in serum and their production is augmented under pathological conditions such as multiple sclerosis, rheumatoid arthritis and neurological disorders. Until now, no (patho)physiological function has been ascribed to circulating Ig light chains. Here we show that IgLCs can confer mast cell–dependent hypersensitivity in mice. Antigenic stimulation results in plasma extravasation, cutaneous swelling and mast-cell degranulation. We show that IgLCs have a crucial role in development of contact sensitivity, which could be completely prevented by a novel IgLC antagonist. Although IgE and IgG1 are central to the induction of immediate hypersensitivity reactions, our results show that IgLCs have similar activity. IgLCs may therefore be a novel factor in the humoral immune response to antigen exposure. Our findings open new avenues in investigating the pathogenesis of autoimmune diseases and their treatments.

Recovery of extracellular vesicles from human breast milk is influenced by sample collection and vesicle isolation procedures

Extracellular vesicles (EV) in breast milk carry immune relevant proteins and could play an important role in the instruction of the neonatal immune system. To further analyze these EV and to elucidate their function it is important that native populations of EV can be recovered from (stored) breast milk samples in a reproducible fashion. However, the impact of isolation and storage procedures on recovery of breast milk EV has remained underexposed. Here, we aimed to define parameters important for EV recovery from fresh and stored breast milk. To compare various protocols across different donors, breast milk was spiked with a well-defined murine EV population. We found that centrifugation of EV down into density gradients largely improved density-based separation and isolation of EV, compared to floatation up into gradients after high-force pelleting of EV. Using cryo-electron microscopy, we identified different subpopulations of human breast milk EV and a not previously described population of lipid tubules. Additionally, the impact of cold storage on breast milk EV was investigated. We determined that storing unprocessed breast milk at −80°C or 4°C caused death of cells present in breast milk, leading to contamination of the breast milk EV population with storage-induced EV. Here, an alternative method is proposed to store breast milk samples for EV analysis at later time points. The proposed adaptations to the breast milk storage and EV isolation procedures can be applied for EV-based biomarker profiling of breast milk and functional analysis of the role of breast milk EV in the development of the neonatal immune system.

Functional Expression of Neurokinin 1 Receptors on Mast Cells Induced by IL-4 and Stem Cell Factor

It is widely accepted that neurokinin 1 (NK1) receptors are not generally expressed on mast cells but little is known about their expression in inflammation. The present study shows expression of NK1 receptors on bone marrow-derived mast cells (BMMC) under the influence of IL-4 or stem cell factor (SCF). Highest expression was found when both cytokines are present. Six days of coculture with the cytokines IL-4 and SCF showed significant expression of NK1 receptors (NK1 receptor+/c-kit+ BMMC; control: 7%, IL-4/SCF: 16%), while 12 days of cytokine coculture increased this expression to 37% positive cells. A longer coculture with IL-4 and SCF did not give an additional effect. Increased expression in IL-4/SCF-treated BMMC was further confirmed using Western blot analysis. Next, we demonstrated the functional relevance of NK1 receptor expression for mast cell activation, resulting in an enhanced degranulation upon stimulation by substance P. BMMC activation was significantly diminished by the NK1 receptor antagonist RP67580 (10 μM) when stimulated with low concentrations of substance P. The inactive enantiomer RP65681 had no effect. In addition, BMMC cultured from bone marrow of NK1 receptor knockout mice showed significantly decreased exocytosis to low concentrations of substance P. The present study clearly shows that NK1 receptor-induced activation contributes significantly at low physiological substance P concentrations (<100 μM). In conclusion, BMMC were shown to express NK1 receptors upon IL-4/SCF coculture. This expression of NK1 receptors has been demonstrated to be of functional relevance and leads to an increase in the sensitivity of BMMC to substance P.

Role of Extracellular ATP and P1 and P2 Classes of Purinergic Receptors in T‐cell Development and Cytotoxic T Lymphocyte Effector Functions

The extracellular ATP (ATPQ) model of cell-mediated cytotoxicity was originally suggested as an aiternalive cytotoxic pathway (Trenn et al. 1987) that does not require extracellular Ca-^ and complements the perforin-mediated pathway (FilIppini et al. 1990a,b, diVirgilio et al. 1990). The straightforward consideration of ATPo as a CTL-produced. lethal hit-delivering lytic intermediate has evolved into the more general model of ATPo involvement (as the source of extracellular adenosine. as a phosphate donor, and as a transmembrane signaling ligand) in both T-cell development and elTector functions. Extracellular ATP (ATP,)) is strongly implicated as a fast excitatory transmitter at synapses between neurons (Edwards et al. 1992) and into the ATP-reccptor mediated cell-to-cell spread of calcium signals between mast cells (Osipchuk & Cahalan 1992). Extracellular ATP and e.xtraccllular phosphorylation were suggested to be involved in the cell-cell contact interactions leading to the lymphocyte activation and effector functions of T lymphocytes {Filippini et al. 1990a.b. Redegeld et al. !991. 1993). The presence of highly active ecto-ATPase (Filippini etal. 1990a) and the Ag-receptor-triggered accumulation of extracellular ATP (Filippi-

Ecto-protein kinases: ecto-domain phosphorylation as a novel target for pharmacological manipulation?

An increasing number of studies document the presence of protein kinases facing outwards at the cell surface of a diverse array of cells. These ecto-protein kinases phosphorylate cell-surface proteins and soluble extracellular substrates, and thus could affect many physiological processes involving cell-cell contacts, cellular differentiation and proliferation, ion fluxes and cellular activation. To date, only limited attention has been paid to exploring ecto-protein kinases as possible pharmacological targets. Here, the identification and physiological role of ecto-protein kinases in different biological systems is described; it is suggested that ecto-protein kinases are attractive and novel candidates for pharmacological manipulation under various (patho)physiological conditions.

Antigen-specific, antibody-coated, exosome-like nanovesicles deliver suppressor T-cell microRNA-150 to effector T cells to inhibit contact sensitivity

BACKGROUND T-cell tolerance of allergic cutaneous contact sensitivity (CS) induced in mice by high doses of reactive hapten is mediated by suppressor cells that release antigen-specific suppressive nanovesicles. OBJECTIVE We sought to determine the mechanism or mechanisms of immune suppression mediated by the nanovesicles. METHODS T-cell tolerance was induced by means of intravenous injection of hapten conjugated to self-antigens of syngeneic erythrocytes and subsequent contact immunization with the same hapten. Lymph node and spleen cells from tolerized or control donors were harvested and cultured to produce a supernatant containing suppressive nanovesicles that were isolated from the tolerized mice for testing in active and adoptive cell-transfer models of CS. RESULTS Tolerance was shown due to exosome-like nanovesicles in the supernatants of CD8(+) suppressor T cells that were not regulatory T cells. Antigen specificity of the suppressive nanovesicles was conferred by a surface coat of antibody light chains or possibly whole antibody, allowing targeted delivery of selected inhibitory microRNA (miRNA)-150 to CS effector T cells. Nanovesicles also inhibited CS in actively sensitized mice after systemic injection at the peak of the responses. The role of antibody and miRNA-150 was established by tolerizing either panimmunoglobulin-deficient JH(-/-) or miRNA-150(-/-) mice that produced nonsuppressive nanovesicles. These nanovesicles could be made suppressive by adding antigen-specific antibody light chains or miRNA-150, respectively. CONCLUSIONS This is the first example of T-cell regulation through systemic transit of exosome-like nanovesicles delivering a chosen inhibitory miRNA to target effector T cells in an antigen-specific manner by a surface coating of antibody light chains.

Immunoglobulin free light chains and mast cells: pivotal role in T-cell-mediated immune reactions?

Immunoglobulin (Ig) free light (L)-chains have long been considered as the meaningless remnants of a spillover in the regular Ig production by B cells. The recently discovered role for Ig free L-chains in mediating hypersensitivity-like responses sheds new light on their potential role in immune responses. Ig free L-chains can sensitize mast cells, such that a second encounter with the appropriate antigen results in mast-cell activation. The possible importance of this reaction for the induction of T-cell-mediated immune reactions, leading to contact sensitivity, multiple sclerosis and rheumatoid arthritis is discussed.

Key Role for Mast Cells in Nonatopic Asthma

The mechanisms involved in nonatopic asthma are poorly defined. In particular, the importance of mast cells in the development of nonatopic asthma is not clear. In the mouse, pulmonary hypersensitivity reactions induced by skin sensitization with the low-m.w. compound dinitrofluorobenzene (DNFB) followed by an intra-airway application of the hapten have been featured as a model for nonatopic asthma. In present study, we used this model to examine the role of mast cells in the pathogenesis of nonatopic asthma. First, the effect of DNFB sensitization and intra-airway challenge with dinitrobenzene sulfonic acid (DNS) on mast cell activation was monitored during the early phase of the response in BALB/c mice. Second, mast cell-deficient W/Wv and Sl/Sld mice and their respective normal (+/+) littermate mice and mast cell-reconstituted W/Wv mice (bone marrow-derived mast cells→W/Wv) were used. Early phase mast cell activation was found, which was maximal 30 min after DNS challenge in DNFB-sensitized BALB/c, +/+ mice but not in mast cell-deficient mice. An acute bronchoconstriction and increase in vascular permeability accompanied the early phase mast cell activation. BALB/c, +/+ and bone marrow-derived mast cell→W/Wv mice sensitized with DNFB and DNS-challenged exhibited tracheal hyperreactivity 24 and 48 h after the challenge when compared with vehicle-treated mice. Mucosal exudation and infiltration of neutrophils in bronchoalveolar lavage fluid associated the late phase response. Both mast cell-deficient strains failed to show any features of this hypersensitivity response. Our findings show that mast cells play a key role in the regulation of pulmonary hypersensitivity responses in this murine model for nonatopic asthma.

Evidence for the involvement of free light chain immunoglobulins in allergic and nonallergic rhinitis

BACKGROUND Allergic rhinitis is characterized by mast cell degranulation induced by antigen cross-linking of IgE. It has been proposed that some patients with rhinitis show nasal allergy in the absence of systemic markers of atopy, termed entopy. Recent murine studies suggest the existence of an IgE-independent hypersensitivity response involving antigen-induced mast cell activation, mediated by immunoglobulin free light chains (FLCs). OBJECTIVES To determine whether FLC is associated with mast cell-mediated nasal hypersensitivity and its relationship with eosinophilic activity in allergic and nonatopic rhinitis. METHODS Patients with allergy and nonallergic rhinitis with eosinophilia syndrome (NARES) had levels of soluble FLC measured in nasal secretions and serum. In addition, levels of the nasal inflammatory mediators mast cell tryptase and eosinophil cationic protein were quantified. Cellular expression of kappa and lambda FLC was characterized in the nasal mucosa of allergic and nonatopic idiopathic rhinitis and control subjects by using immunohistochemistry. Immunopositive cells were phenotyped by using laser microdissection and PCR. RESULTS Free light chain was significantly increased in nasal secretions of subjects with allergy and NARES, and in serum of patients with NARES. Nonatopic patients with allergy showed significantly increased nasal mast cell tryptase and eosinophil cationic protein. FLC-positive cells were significantly increased in allergic and nonatopic mucosa, and were shown to be mast cells and plasma cells. CONCLUSION Nasal FLC is significantly increased in allergic and nonatopic rhinitis nasal mucosa, suggesting a role in nasal hypersensitivity. Further studies are needed to identify which allergens trigger FLC-mediated responses in nonatopic rhinitis.

Stem cell factor and Interleukin-4 induce murine bone marrow cells to develop into mast cells with connective tissue type characteristics in vitro

In this study, we have developed a method to obtain mast cells with connective tissue type mast cell (CTMC) characteristics directly from mouse bone marrow (BM) cells. BM cells were grown for 3 weeks in presence of interleukin-4 (IL-4) plus stem cell factor (SCF). SCF alone poorly supported growth and development of mast cells. IL-4 dose-dependently enhanced the expression of c-kit and high-affinity receptor for IgE (Fc(epsilon)RI) on the cell surface of SCF-cultured BM cells. Furthermore, cytoplasmic granulation and histamine synthesis of BM-derived mast cells were increased in presence of IL-4 and SCF. Histochemical staining demonstrated that granules were safranin positive. BM-derived mast cells could be activated for granule exocytosis (beta-hexosaminidase release) and lipid mediator generation (LTC4 production) via Fc(epsilon)RI after sensitization with IgE and subsequent crosslinking with multivalent antigen. In addition, mast cells derived from BM cells cultured with SCF plus IL-4 could be activated by substance P, a nonimmunologic stimulus, to release beta-hexosaminidase. The results presented indicate that IL-4 and SCF both have a prominent role in the development of mast cells from murine BM cells in vitro. Mast cells can directly be derived from BM cells in presence of SCF and IL-4 and the cultured cells show typical hallmarks of CTMC, indicating that precursor cells for CTMC may be present in BM. The described culture procedure may be useful to investigate the molecular aspects of the development of committed mast cell lineages.