Ilaria Puxeddu

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.

New insights on the involvement of Nerve Growth Factor in allergic inflammation and fibrosis.

Nerve Growth Factor (NGF), that was originally discovered for its properties of stimulating growth and differentiation of neurons, is now also considered responsible for several activities in the immune system and beyond. Mast cells and eosinophils, key cells of allergic inflammation, are a source of NGF and are influenced by it. These observations have prompted studies on NGF in allergy and tissue repair. Recent evidences link NGF and these two processes. While NGF is clearly a new tool in the management of untreatable ulcers, its role in allergic inflammation, although appearing to be pro-inflammatory, is still not clearly defined.

Osteopontin is expressed and functional in human eosinophils.

To cite this article: Puxeddu I, Berkman N, Ribatti D, Bader R, Haitchi HM, Davies DE, Howarth PH, Levi‐Schaffer F. Osteopontin is expressed and functional in human eosinophils. Allergy 2010; 65: 168–174.

The pro‐fibrogenic effect of nerve growth factor on conjunctival fibroblasts is mediated by transforming growth factor‐β

Background Nerve growth factor (NGF) and nerve growth factor receptor (NGFR) expressions have been found to be increased in sub‐conjunctival scarring.

The role of eosinophil major basic protein in angiogenesis

Background:  Eosinophil‐derived major basic protein (MBP) plays an active role in allergic inflammation and tissue remodelling. However, its role in angiogenesis has not been established as yet. Therefore our objective was to investigate whether MBP exhibits any direct pro‐angiogenic effects.

Mast cells induce activation of human lung fibroblasts in vitro.

Mast cells are able to induce proliferation of skin fibroblasts; however, their effect on lung fibroblasts has not been clearly established. Using in vitro cocultures of rat or human mast cells with lung fibroblasts, the authors determined whether mast cells alter proliferation, collagen synthesis, and metalloproteinase production from lung fibroblasts. Mast cells enhanced the proliferation of human fibroblasts (mean±SEM: 90%±4.7% increase, P < .001) while inhibiting fibroblast collagen synthesis (48.1%±4.2% decrease, P < .001). Histamine, but not tryptase, significantly enhanced fibroblast proliferation: 92%±5.8% (P < .001) and 39.2%±4.3% (P > 0.05), respectively. Rat mast cell sonicate added to lung fibroblasts induced the activation of metalloproteinase-9 while inhibiting that of metaloproteinase-2. The addition of lipopolysaccharide (LPS)-stimulated lung macrophage supernatant further enhanced the proliferative effect of mast cells on fibroblasts (by 60%±7.8%, P < .001) and induced synthesis of collagen from these cells (190%±28% increase versus control, P < .05). This study demonstrates that mast cells influence several aspects of lung fibroblast function in vitro.

Eotaxin-2/CCL24 and eotaxin-3/CCL26 exert differential profibrogenic effects on human lung fibroblasts

BACKGROUND Eotaxin-2/CCL24 and eotaxin-3/CCL26 play an important role in eosinophil chemotaxis and activation in asthma. We previously demonstrated that eotaxin/CCL11 is profibrogenic for human lung fibroblasts. The effect of eotaxin-2/ CCL24 and eotaxin-3/CCL26 on lung fibroblasts has not yet been investigated. OBJECTIVE To evaluate whether eotaxin-2/CCL24 and eotaxin-3/CCL26 modulate fibrotic properties of lung fibroblasts. METHODS Fibroblast proliferation was evaluated by means of 3-hydroxythymidine incorporation. Collagen production was assessed by means of 3-hydroxyproline incorporation and biochemical staining. Chemotaxis was determined using Boyden chambers. Expression of alpha-smooth muscle actin was evaluated by means of immunostaining. Transforming growth factor beta1 release was assessed using enzyme-linked immunosorbent assay. Parametric analysis of variance, followed by the Tukey-Kramer multiple comparisons test, was used to calculate statistical significance. RESULTS Eotaxin-2/CCL24 but not eotaxin-3/CCL26 stimulated human lung fibroblast proliferation and collagen synthesis. In contrast, eotaxin-3/CCL26 but not eotaxin-2/CCL24 promoted fibroblast migration. Neither eotaxin-2/CCL24 nor eotaxin-3/ CCL26 induced the expression of alpha-smooth muscle actin or transforming growth factor beta1 from lung fibroblasts. CONCLUSIONS Eotaxin-2/CCL24 and eotaxin-3/CCL26 have differential profibrogenic effects on human lung fibroblasts. These CC chemokines may, therefore, contribute to airway remodeling in asthma.

Chronic Nerve Growth Factor Exposure Increases Apoptosis in a Model of In Vitro Induced Conjunctival Myofibroblasts

In the conjunctiva, repeated or prolonged exposure to injury leads to tissue remodeling and fibrosis associated with dryness, lost of corneal transparency and defect of ocular function. At the site of injury, fibroblasts (FB) migrate and differentiate into myofibroblasts (myoFB), contributing to the healing process together with other cell types, cytokines and growth factors. While the physiological deletion of MyoFB is necessary to successfully end the healing process, myoFB prolonged survival characterizes the pathological process of fibrosis. The reason for myoFB persistence is poorly understood. Nerve Growth Factor (NGF), often increased in inflamed stromal conjunctiva, may represent an important molecule both in many inflammatory processes characterized by tissue remodeling and in promoting wound-healing and well-balanced repair in humans. NGF effects are mediated by the specific expression of the NGF neurotrophic tyrosine kinase receptor type 1 (trkANGFR) and/or the pan-neurotrophin glycoprotein receptor (p75NTR). Therefore, a conjunctival myoFB model (TGFβ1-induced myoFB) was developed and characterized for cell viability/proliferation as well as αSMA, p75NTR and trkANGFR expression. MyoFB were exposed to acute and chronic NGF treatment and examined for their p75NTR/trkANGFR, αSMA/TGFβ1 expression, and apoptosis. Both NGF treatments significantly increased the expression of p75NTR, associated with a deregulation of both αSMA/TGFβ1 genes. Acute and chronic NGF exposures induced apoptosis in p75NTR expressing myoFB, an effect counteracted by the specific trkANGFR and/or p75NTR inhibitors. Focused single p75NTR and double trkANGFR/p75NTR knocking-down experiments highlighted the role of p75NTR in NGF-induced apoptosis. Our current data indicate that NGF is able to trigger in vitro myoFB apoptosis, mainly via p75NTR. The trkANGFR/p75NTR ratio in favor of p75NTR characterizes this process. Due to the lack of effective pharmacological agents for balanced tissue repairs, these new findings suggest that NGF might be a suitable therapeutic tool in conditions with impaired tissue healing.

Airway eosinophil accumulation and eotaxin-2/CCL24 expression following allergen challenge in BALB/c mice.

Eotaxin-1/CCL11 is important for early eosinophil recruitment to the airways of asthmatics. In order to clarify whether eotaxin-2/CCL24 accounts for prolonged airway eosinophilia, the authors determined the expression of CCL11 and CCL24 in lung tissue and bronchoalveolar lavage (BAL) as well as eosinophil infiltration over 14 days in BALB/c mice sensitised (intraperitonealy) and challenged (inhalations) with ovalbumin (OVA). Allergen exposure induced perivascular, peribronchial, and BAL eosinophilia for up to 7 days. CCL11 and CCL24 were highly expressed in lung tissue from 6 and up to 72 hours. Peak expression of CCL11 protein was 1557 ± 109 pg/mL for OVA (mean ± SEM) versus 404 ± 73 pg/mL in controls (SAL) (P <.001) and 1690 ± 54 versus 455 ± 165 pg/mL for CCL24 (P <.01). In BAL, only eotaxin-2/CCL24 was significantly increased (1623 ± 85 pg/mL for OVA versus 157 ± 22 pg/mL for SAL, P <.01). Peak eosinophilia and CCL24 expression occurred later in BAL than in lung tissue. These data suggest that both CCL11 and CCL24 are important for recruitment of eosinophils to perivascular and peribronchial tissue seen up to 72 hours. This finding implies redundancy between these chemokines rather than differentially regulated expression over time. In contrast, only CCL24 seems important for recruitment of eosinophils into BAL. Specific inhibition of CCL11 alone is therefore unlikely to inhibit eosinophil recruitment to the airways.

Effects of dexamethasone on TNF-alpha-induced release of cytokines from purified human blood eosinophils

SummaryBackgroundTNF-alpha is an important mediator in allergy also for its effects on eosinophils.MethodsThe effect of dexamethasone on TNF-alpha induced eosinophils survival, degranulation (ECP), cytokines release (IL-8, GM-CSF) and adhesion to VCAM-1, ICAM-1 and IgG coated wells (EPO release) were evaluated.ResultsThe drug inhibited IL-8 and GM-CSF production, but not viability, degranulation or adhesion in human peripheral blood eosinophils.ConclusionThese results indicate that part of the activity of glucocorticosteroids on eosinophils may be mediated by their ability to inhibit cytokine secretion that in turn is important for the perpetuation of the allergic inflammation.