Mast cells in allergy and beyond

Mast cells (MC) are highly granulated tissue dwelling cells, widely distributed throughout the body in connective tissues and on mucosal surfaces. They are derived from bone marrow progenitors that migrate into the blood and subsequently into the tissues, where they undergo final maturation. Mast cell proliferation, differentiation, survival and activation are regulated by stem cell factor, the ligand for the c-kit tyrosine kinase receptor, expressed on the mast cell surface. They release a large number of pro-inflammatory and immunoregulatory mediators after activation induced by either immunoglobulin E-dependent or immunoglobulin E-independent mechanisms. Mast cells have been most widely studied in the context of allergic reactions and parasite infections, but there is now compelling evidences that they are important players in innate and acquired immunity, wound healing, fibrosis, tumors and autoimmune diseases. This review will discuss current advances in these fields.Cell facts

• Mast cells are high affinity IgE receptor bearing tissue dwelling cells containing prominent cytoplasmic granules and key cells in allergy.

• Mast cell proliferation, differentiation, survival and activation are regulated by stem cell factor.

• Mast cells and their mediators participate in innate and acquired immunity, wound healing, tissue remodeling, angiogenesis and autoimmune diseases.

     

Azelastine potentiates the prostaglandin-induced increase of cyclic AMP content in human platelets and in guinea-pig alveolar macrophages.

The effect of azelastine on intracellular cyclic AMP concentration and on various indexes of cell activation was evaluated in guinea-pig alveolar macrophages and in human platelets. The effect of azelastine was further investigated on adenylate cyclase activity using membranes and homogenates from guinea-pig alveolar macrophages. Pretreatment of alveolar macrophages with azelastine prevented the activation induced by PAF-acether and by the chemotactic peptide fMLP as estimated by the reduced liberation of arachidonic acid metabolites formed by the cyclooxygenase and the lipoxygenase pathways. The effect of azelastine was concentration-dependent (50 to 500 microM) and reversible. Similarly, a short pretreatment with azelastine (100 microM) prevented arachidonic acid-induced platelet aggregation. This effect was also reversible after washing the platelets. In guinea-pig alveolar macrophages, azelastine induced a concentration-dependent (10 to 500 microM) increase in intracellular cyclic AMP and markedly potentiated the increase induced by PGE2. In human platelets, azelastine alone increased intracellular cyclic AMP concentration marginally only but, as in the case of macrophages, synergized with PGI2. Azelastine did not activate significantly adenylate cyclase unless a cytosolic factor was included within the membrane fraction. This effect of azelastine was not due to Ca2+ movements and was not modified by GTP. Our findings show that azelastine interferes with cell activation through a mechanism related to an increase in intracellular cyclic AMP concentration. The increase in cyclic AMP was induced by azelastine in intact cells and in homogenates but not in a crude membrane fraction. Those results indicate that azelastine modifies a cytosolic factor that may be phosphodiesterase. In addition, similarities between the effects of azelastine and those of reference phosphodiesterase inhibitors (theophylline, isobutyl-methyl-xanthine) are shown in this study, suggesting that azelastine might behave as a phosphodiesterase inhibitor.     

Flow cytometry is a rapid and reliable method for evaluating heat shock protein 70 expression in human monocytes

The increasing interest in stress/heat shock proteins (Hsps) as markers of exposure to environmental stress or disease requires an easily applicable method for Hsp determination in peripheral blood cells. Of these cells, monocytes preferentially express Hsps upon stress. An appropriate fixation/permeabilization procedure was developed, combined with immunofluorescence staining and flow cytometry for the detection of the inducible, cytosolic, 72 kDa Hsp (Hsp70) in human monocytes. Higher relative fluorescence intensity was observed in cells exposed to heat shock (HS), reflecting a higher expression of Hsp70 in these cells as compared with cells kept at 37°C. The heat-inducible increased Hsp70 expression was temperature- and time-dependent. Expression of Hsp70 was not uniform within the monocyte population, indicating the presence of subpopulations expressing variable levels of Hsp70 in response to HS. Simultaneous measurements of intracellular Hsp70 and membrane CD14 expression revealed that the higher Hsp70 inducibility coincided with the higher CD14 expression. Comparisons performed with biometabolic labelling, Western blotting, immunofluorescence and immunoperoxidase microscopic analysis, showed a high concordance between these different methods; however, cytometry was more sensitive for Hsp70 detection than Western blotting. Flow cytometric detection of intracellular Hsp70 is a rapid, easy and quantitative method, particularly suited for the determination of protein levels in individual cells from an heterogeneous population such as peripheral mononuclear blood cells, and applicable to cohort studies.     

NT agonist regulates expression of nuclear high-affinity neurotensin receptors.

Neurotensin (NT) exerts multiple functions in the central nervous system and peripheral tissues. Its actions are mainly mediated by a high-affinity G-protein-coupled receptor, the NT-1 receptor. In this study we demonstrated a nuclear NT binding site in different cellular models. We first noted that a large percentage of NT-1 receptor cell body immunoreactivity was located in the nuclear soma and nuclear envelope of rat substantia nigra, a brain area rich in NT-containing axon terminals. The NT-1 receptor was also visualized in purified nuclei from CHO cells stably transfected with NT-1 receptor coupled to the enhanced green fluorescence protein by immunocytochemistry. We observed that both the nuclear envelope and the nuclear soma were labeled, and the labeling intensity significantly increased after NT agonist treatment. These results suggested that NT-1 receptors, present in both the nuclear soma and the nuclear envelope, can be modulated by the ligand. Lastly, [(125)I]-NT binding experiments performed on isolated nuclei from a human lung cancer cell line endogenously expressing NT-1 receptor and NT, LNM35, revealed the existence of nuclear Gpp(NHp)-sensitive binding sites. These binding sites markedly decreased when cells were chronically treated with an NT-1 receptor antagonist, SR 48692. Taken together, these data suggest that the agonist regulates the expression of nuclear NT-1 receptors.     

2B4 (CD244) is expressed and functional on human eosinophils

Eosinophils are present in parasitic, allergic, various immunological, and malignant disorders as well as in a variety of idiopathic hypereosinophilic syndromes. However, their exact role in some of these conditions remains elusive. They can be activated both in vivo and in vitro by various agonists, such as Igs, lipid mediators, and cytokines. By phenotyping the surface of the eosinophils, it may be possible to better define their function(s) in different pathophysiological settings. In the present work we screened eosinophils with a panel of Abs recognizing CD2 subfamily receptors usually present on a number of hemopoietic cells. We have demonstrated that human peripheral blood eosinophils, but not basophils or neutrophils, express NTB-A. In addition eosinophils express 2B4, CD84, CD58, and CD48, but not signaling lymphocytic activation molecule or CD2, on their surface (FACS). Cross-linking of 2B4 on eosinophils elicited a significant release of eosinophil peroxidase (30 min), IFN-gamma, and IL-4 (18 h). Moreover, activation of eosinophils via 2B4 induced eosinophil-mediated cytotoxicity toward two malignant cell lines, i.e., mouse mastocytoma P815 and EBV-infected 721.221 B cell lines. Cross-linking of 2B4 on the surface of eosinophils or pervenadate treatment elicited ERK and tyrosine phosphorylation, respectively. Furthermore, we showed that eosinophils express slam-associated protein. The demonstration that human eosinophils express a functional 2B4 receptor indicates a broader role for these cells in health and disease.     

Mitochondrial alterations precede Bordetella pertussis-induced apoptosis

Bordetella pertussis, the causative agent of whooping cough in humans, secretes a number of toxins, including adenylate cyclase-hemolysin (AC-Hly), and induces macrophage apoptosis. We investigated the effects of B. pertussis on mitochondrial membrane potential (deltapsim) and ATP levels, as possible determinants of cell death. Using the fluorescent probe JC-1, we found that infection of human monocytes by B. pertussis lead to a disruption in host-cell deltapsim. deltapsim alterations were preceded by a massive increase in cyclic AMP, a moderate decrease in ATP, and was independent from oxidative stress. These changes were observed when human monocytes were infected by the parental B. pertussis 18323 but not when infected by the mutants deficient in the expression of AC-Hly. Exposure of human monocytes to purified AC-Hly induced changes comparable to those observed with the B. pertussis parental strain. Our results provide a mechanistic relationship between AC-Hly, ATP, and deltapsim disruption in the cascade of events leading to B. pertussis-induced apoptosis.     

Toxicity of cadmium in tobacco smoke: protection by antioxidants and chelating resins

The effects of cadmium, an environmental toxin present in tobacco smoke, were studied in vitro in human monocytes and compared to those of tobacco smoke. Overexpression of the 72 λkDa heat shock/stress protein Hsp70 and cell death occurred with a similar time-course and to a similar extent in human monocytes exposed to either cadmium or tobacco smoke. Cadmium and tobacco smoke-mediated toxicity were associated with a decrease in the cellular content of glutathione and ATP and the glutathione precursor N -acetyl- l -cysteine prevented both cadmium and tobacco smoke-mediated toxicity. Furthermore, tobacco smoke-mediated toxicity was prevented by pretreatment with the cadmium chelator resin Chelex-100, supporting the conclusion that cadmium plays a major role in tobacco smoke-mediated toxicity.     

Adaptive responses of human monocytes infected by bordetella pertussis: the role of adenylate cyclase hemolysin

The activation/adaptive responses of human monocytes exposed to Bordetella pertussis parental or mutant strains were evaluated and correlated to the expression of two bacterial toxins: adenylate cyclase-hemolysin and pertussis toxin. The marked rise in intracellular cyclic adenosine monophosphate (cAMP) observed in monocytes infected by B. pertussis parental strain, inversely correlated with (1) the production of tumor necrosis factor alpha; (2) the release of superoxide anion; and (3) the expression of the 72-kDa heat shock/stress protein, Hsp70. Experiments performed with mutants deficient in adenylate cyclase-hemolysin or with purified bacterial toxins confirmed the key role of adenylate cyclase-hemolysin in the control of monocytes' response to infection by B. pertussis. This bacterial strategy primarily involves evasion from antimicrobial defenses and, eventually, the sacrifice of the host cell.     

Curosurf modulates cAMP accumulation in human monocytes through a membrane-controlled mechanism

The cellular mechanisms by which pulmonary surfactant exerts its effects, including anti-inflammatory or proinflammatory effects, have remained elusive. To address the issue of whether plasma membrane modifications represent a target for these mechanisms, we designed an experimental protocol involving the determination of changes in cAMP levels under membrane-dependent or -independent stimulatory pathways. The effects of a modified natural porcine surfactant, Curosurf, and the major surfactant protein A were evaluated on resting and stimulated cAMP levels of human monocytes. We found that agents that elevate intracellular cAMP exhibit different susceptibilities toward a preexposure to Curosurf. The rise in cAMP induced by membrane-active agents such as cholera toxin or the diterpene forskolin was significantly inhibited by monocyte preexposure to Curosurf. In contrast, the rise in cAMP induced by the membrane-permeant phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine or by the Bordetella pertussis toxin adenylate cyclase-hemolysin was unaffected by Curosurf. Surfactant protein A did not affect either cAMP levels or the inhibitory capacity of Curosurf. We suggest that a plasma membrane-associated event affecting the mechanism underlying the effects of cholera toxin or forskolin is involved in the inhibition of cAMP accumulation caused by Curosurf.     

Inhibition of phospholipase A2 activity of guinea-pig alveolar macrophages by lipocortin-like proteins purified from mice lung

Guinea-pig alveolar macrophages were harvested by bronchoalveolar lavage and purified by differential adhesion. They were labeled with 14C-Arachidonic acid and then exposed to platelet-activating factor or to the calcium ionophore A23187. The activity of cellular phospholipase A2 was considered as the release of free 14C-Arachidonic acid in the cell supernatant. The pretreatment of guinea-pig alveolar macrophages with two lipocortin-like proteins (36 kDa and 40 kDa) purified from mice lung induced a significant inhibition of their phospholipase A2 activity upon platelet-activating factor and calcium ionophore stimulation. These results indicate that lipocortin-like proteins can modulate the phospholipase A2 activity of isolated cells in vitro.