IL-3 synergises with basophil-derived IL-4 and IL-13 to promote the alternative activation of human monocytes.

Basophil-derived IL-4 is involved in the alternative activation of mouse monocytes, as recently shown in vivo. Whether this applies to human basophils and monocytes has not been established yet. Here, we sought to characterise the interaction between basophils and monocytes and identify the molecular determinants. A basophil-monocyte co-culture model revealed that IL-3 and basophil-derived IL-4 and IL-13 induced monocyte production of CCL17, a marker of alternative activation. Critically, IL-3 and IL-4 acted directly on monocytes to induce CCL17 production through histone H3 acetylation, but did not increase the recruitment of STAT5 or STAT6. Although freshly isolated monocytes did not express the IL-3 receptor α chain (CD123), and did not respond to IL-3 (as assessed by STAT5 phosphorylation), the overnight incubation with IL-4 (especially if associated with IL-3) upregulated CD123 expression. IL-3-activated JAK2-STAT5 pathway inhibitors reduced the CCL17 production in response to IL-3 and IL-4, but not to IL-4 alone. Interestingly, monocytes isolated from allergen-sensitised asthmatic patients exhibited a higher expression of CD123. Taken together, our data show that the JAK2-STAT5 pathway modulates both basophil and monocyte effector responses. The coordinated activation of STAT5 and STAT6 may have a major impact on monocyte alternative activation in vitro and in vivo.

Inhibition of secretory phospholipase A2-induced cytokine production in human lung macrophages by budesonide.

BACKGROUND: Secretory phospholipases A(2) (sPLA(2)) are an emerging class of mediators of inflammation. These enzymes are released in vivo in patients with systemic inflammatory diseases and allergic disorders. sPLA(2)s may activate inflammatory cells by both enzymatic and nonenzymatic mechanisms. The aim of this study was to evaluate the effect of the inhaled glucocorticoid budesonide on sPLA(2)-induced activation of primary human macrophages. METHODS: Macrophages isolated from human lung tissue were preincubated (3-18 h) with budesonide (1-1,000 nM) before stimulation with 2 distinct sPLA(2)s (group IA and group X). At the end of incubation the release of TNF-alpha, IL-6 and IL-8 was assessed by ELISA. Specific mRNA for these products was determined by quantitative RT-PCR. Activation of mitogen-activated kinases ERK 1/2 and p38 was assessed by Western blot. RESULTS: Budesonide inhibited the release of TNF-alpha, IL-6 and IL-8 from sPLA(2)-stimulated macrophages in a concentration-dependent manner. The inhibitory effect of budesonide was due to a reduction of gene expression and was complete after 18 h of preincubation. Budesonide had no effect on sPLA(2)-induced arachidonic acid mobilization and exocytosis, assessed as beta-glucuronidase release. Suppression of cytokine/chemokine production by budesonide was associated with inhibition of sPLA(2)-induced ERK 1/2 and p38 activation. CONCLUSIONS: Budesonide inhibits the production of proinflammatory cytokines/chemokines from human lung macrophages activated by sPLA(2). Budesonide represents the first example of a drug able to block the nonenzymatic effects of sPLA(2) on human inflammatory cells and, therefore, may provide a useful therapeutic options for diseases associated with enhanced release of sPLA(2)s in vivo.

BHK cells transfected with NCX3 are more resistant to hypoxia followed by reoxygenation than those transfected with NCX1 and NCX2: Possible relationship with mitochondrial membrane potential.

The specific role played by NCX1, NCX2, and NCX3, the three isoforms of the Na+/Ca2+ exchanger (NCX), has been explored during hypoxic conditions in BHK cells stably transfected with each of these isoforms. Six major findings emerged from the present study: (1) all the three isoforms were highly expressed on the plasma membranes of BHK cells; (2) under physiological conditions, the three NCX isoforms showed similar functional activity; (3) hypoxia plus reoxygenation induced a lower increase of [Ca2+]i in BHK-NCX3-transfected cells than in BHK-NCX1- and BHK-NCX2-transfected cells; (4) NCX3-transfected cells were more resistant to chemical hypoxia plus reoxygenation than NCX1- and NCX2-transfected cells. Interestingly, such augmented resistance was eliminated by CBDMD (10 microM), an inhibitor of NCX and by the specific silencing of the NCX3 isoform; (5) chemical hypoxia plus reoxygenation produced a loss of mitochondrial membrane potential in NCX1- and NCX2-transfected cells, but not in NCX3-transfected cells; (6) the forward mode of operation in NCX3-transfected cells was not affected by ATP depletion, as it occurred in NCX1- and NCX2-transfected cells. Altogether, these results indicate that the brain specifically expressed NCX3 isoform more significantly contributes to the maintenance of [Ca2+]i homeostasis during experimental conditions mimicking ischemia, thereby preventing mitochondrial delta psi collapses and cell death.

Immune cells as a source and target of angiogenic and lymphangiogenic factors.

Angiogenesis and lymphangiogenesis are distinct and complex processes requiring a finely tuned balance between stimulatory and inhibitory signals. Immune and inflammatory cells can contribute to these processes by multiple mechanisms: directly by producing a broad array of angiogenic growth factors, and indirectly by secreting several cytokines, chemokines and other mediators able to coordinate the cell-cell interactions. Immune cells can stimulate or inhibit angiogenesis/lymphangiogenesis, depending on their activation status and subset specificity. We summarize recent findings reporting the expression and activity of angiogenic and lymphangiogenic factors and their receptors and coreceptors in immune cells. It is evident that modulation of angiogenesis and lymphangiogenesis by the innate and adaptive immune cells (mast cells, macrophages, dendritic cells, basophils, eosinophils, and some subsets of T cells) is a highly complex process not yet completely understood.

The role of mast cell-derived secreted phospholipases A2 in respiratory allergy.

Secreted phospholipases A(2) (sPLA(2)s) are molecules released in plasma and biological fluids of patients with systemic inflammatory, autoimmune and allergic diseases. These molecules exert proinflammatory effects by either enzymatic-mechanisms or through binding to surface molecules expressed on inflammatory cells. sPLA(2)s are released at low levels in the normal airways and tend to increase during respiratory allergies (e.g., rhinitis and bronchial asthma) as the result of local secretion. Several sPLA(2) isoforms are expressed in the human lung and some of them (e.g., group IIA and group X) are released in the airways of patients with rhinitis or asthma. Mast cells play a major role in the pathogenesis of respiratory allergies and other chronic inflammatory lung diseases. Recent evidence indicates that mast cells purified from human lung express most of the sPLA(2) isoforms so far described. IgE-mediated activation of these cells induce the release of sPLA(2)s suggesting that mast cells are a main source of extracellular sPLA(2)s during allergic reactions. Once released, sPLA(2)s may contribute to the generation of eicosanoids (e.g., PGD(2) and LTC(4)) and to the release of preformed mediators (e.g., histamine) by an autocrine loop involving the interaction of sPLA(2)s with surface molecules such as heparan sulphate proteoglycans or the M-type receptor. Thus, mast cell-derived sPLA(2)s may play an important role in the initiation and amplification of the inflammatory reactions in patients with allergic rhinitis and bronchial asthma.

Differentiation of monocytes into macrophages induces the upregulation of histamine H1 receptor.

BACKGROUND: Histamine modulates several functions in human monocytes, macrophages, and dendritic cells. However, responses elicited by histamine differ depending on cell type, suggesting variable expression of histamine receptors in the monocyte/macrophage lineage. OBJECTIVE: We sought to examine whether the expression of H(1) receptors was regulated by cell differentiation of human monocytes into macrophages or dendritic cells. METHODS: Expression of H(1) receptor was evaluated by RT-PCR and western blot in monocytes, monocyte-derived macrophages (MDMs), monocyte-derived dendritic cells (DCs) and human lung macrophages (HLMs). RESULTS: Expression of H(1) receptor mRNA and protein was higher in HLMs and DCs than in monocytes. H(1) expression was approximately 15-fold and 4-fold higher in MDMs and HLMs, respectively, as compared with that seen in monocytes. H(1) receptor protein was undetectable in monocytes, whereas it was conspicuous in MDMs. Simultaneous analysis of H(2) and H(1) mRNA expression indicated that the H(2)/H(1) ratio decreased from 202.7 +/- 14.8 in monocytes to 2.2 +/- 0.4 in MDM and 39.5 +/- 5.0 in DCs. Incubation of monocytes with histamine neither affected intracellular Ca(2+) concentrations nor influenced IL-8 production. In contrast, histamine rapidly induced a Ca(2+) signal and stimulated IL-8 production in MDMs. Both effects were inhibited by H(1) blockade with levocetirizine, but not by H(2) blockade with ranitidine. CONCLUSIONS: Differentiation of monocytes into macrophages or dendritic cells is associated with profound changes of histamine receptor expression. Upregulation of H(1) receptors confers on macrophages the capacity of being activated by histamine. CLINICAL IMPLICATIONS: Regulation of H(1) and H(2) receptor expression in the monocyte/macrophage lineage can be relevant to the pathogenesis of allergic inflammation.