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Notes: To assess human mast-cell (MC) behavior after repetitive activation, we cocultured human foreskin MC (SMC) with human foreskin fibroblasts (F). Under these conditions, we have previously demonstrated that SMC keep their viability and functional activity for up to 8 days. SMC were presensitized with atopic serum and repeatedly activated by consecutively increasing concentrations of anti-IgE antibodies (α-IgE, 0.0002–0.1). This treatment, which mimics the “rush desensitization” procedure, led to complete SMC unresponsiveness to activation by a-IgE at optimal concentrations, as evaluated by histamine release. However, presensitization of SMC with IgE antibodies before exposure to α-IgE restored their sensitivity to this stimulus. These data indicate that desensitization was probably due to lack of membrane-bound IgE rather than to downregulation of intracellular mechanisms. In fact, SMC challenged by an optimal concentration of α-lgE could release histamine upon a second activation by 2 h after the first activation, if the cells had been presensitized before the second challenge. SMC incubation with increasing concentrations of compound 48/80 (0.2–10μg/ml) led to MC unresponsiveness to an optimal concentration of this stimulus. Furthermore, SMC activated by an optimal concentration of compound 48/80 and rechallenged with the same agent were insensitive to the second activation for at least 24 h. In summary, we have shown that it is possible to induce “desensitization” in SMC to both IgEdependent and IgE-independent stimuli by incubating the cultures with consecutively increasing concentrations of the activator. SMC can release histamine when reactivated with a-IgE antibodies after presensitization by 2 h after the first challenge, while they reacquire their susceptibility to reactivation with compound 48/80 in only 2–3 days.

Notes: Prominent blood and tissue eosinophilia is manifested in a number of inflammatory states, particularly in allergic diseases. Eosinophils are a source of numerous cytokines and growth factors, thus in principle they can display both pro-inflammatory and anti-inflammatory activities as well as immunoregulatory ones. In this review, we will discuss the cross-talk between eosinophils and other cell types that they come in contact with in the inflammatory milieu, such as mast cells, fibroblasts and endothelial cells. ‘New’ roles for eosinophils in cancer and novel activatory signals will also be described.

Notes: Background In vivo rush desensitization is a procedure widely employed to quickly desensitizo allergic patients by administering increasing doses of the offending antigen at short intervals. The mechanism(s) underlying this process and the possible role of mast cells in it have not been well delineated.Objective To define an ex vivo model for rush desensitization utilizing the mast cell-fibroblast coculture system.Methods Rat peritoneal mast cells were cultured on 3T3 fibroblasls and were pre-incubated with saturating or suboptimal concentrations of IgE anti-DNP antibodies. Mast cells were then repeatedly challenged at 30 min intervals, with increasing amounts (0.001 100ng/mL) of the antigen DNP-HSA, in the presence of calcium ions. Parallel control cultures were stimulated only once by each antigen concentration. In another set of experiments, mast cells were repeatedly activated with increasing concentrations (0.1-1000 ng/mL) of compound 48/80. Supernatants and cell sonicates were assessed for histamine content and the percentage of histamine released was calculated.Results When saturating concentrations of IgE anti-DNP antibodies were used, mast cells challenged repeatedly with DNP-HSA did not release significant amounts of histamine up to an antigen concentration of I ng/mL. At this stage they were partially desensitized, releasing only 108.3 . 17.1 ng histamine/plate (7.9±0.8%). A marked desensitization was observed at optimal antigen concentration (100 ng/mL). where experimental mast cells released only 45.6±10.9ng/plate, compared with 661.9±48.2ng/plate in firstly challenged cultures. Desensitization was probably not due to mast cells histamine depletion, since the cells still contained large amounts of histamine (579.5±108.6ng/plate) at the end of the procedure. A similar pattern of desensitization was observed when mast cell were preincubated with a subotimal concentration of IgE antibodies. Activation of mast cells with increasing amounts of the IgE-independent secretagogue, compound 48/80. did not cause desensitization since at each concentration both repetitively challenged and control cultures released similar amounts of histamine. Furthermore, challenge of antigen-desensitized mast cells with compound 48/80 caused the release of 75.9±4.9% histamine comparable to the percentage of histamine released from controls (79.5±6.7).Conclusion Repeated exposure of mast cells to gradually increasing amounts of antigen induces their refractoriness. This observation would suggest a role for mast cells in rush desensitization procedure in vivo. Our coculture system may serve as a useful model for studying this process.

Notes: Background Nerve growth factor (NGF) and nerve growth factor receptor (NGFR) expressions have been found to be increased in sub-conjunctival scarring.Objective The aim of this study was to investigate the in vitro effects of NGF on some pro-fibrogenic properties of human conjunctival fibroblasts.Methods Expression of NGF, trkANGFR and p75NTR on human fibroblasts grown from conjunctival biopsies and incubated for 2 or 6 days with NGF were evaluated by immunofluorescence, RT-PCR, flow cytometry and ELISA. The fibrogenic effect of NGF on conjunctival fibroblasts was investigated by evaluating their migration (wound model), proliferation ([3H]-thymidine incorporation), collagen production (3H]-proline incorporation), expression of alpha-smooth muscle actin (α-SMA) (cell surface ELISA) and contraction of 3D collagen gels.Results NGF induced the expression of p75NTR in the fibroblasts that constitutively expressed only trkANGF and increased the migration of wounded fibroblasts, but not their proliferation and collagen production. NGF induced the conversion of fibroblasts into myofibroblasts expressing α-SMA, and enhanced their contraction of a collagen matrix. Interestingly, chronic NGF treatment induced transforming growth factor-β1 (TGF-β1) production by fibroblasts, and following specific TGF-β neutralization, all the NGF-induced effects were completely abrogated.Conclusion Our findings indicate that NGF, via TGF-β induction, is likely to be involved in the healing or fibrotic processes occurring in conjunctiva during some pathological conditions.

Notes: Background The amniotic membrane (AM), which is the innermost layer of the placenta, was shown to possess anti-inflammatory and anti-fibrotic properties in various in vitro and clinical studies.Purpose To evaluate the anti-fibrotic and anti-inflammatory effects of the AM matrix (AMM) on human conjunctival and lung fibroblasts in an in vitro system that tests fibrotic and inflammatory responses at the effector stages of allergic inflammation.Methods Human conjunctival or lung fibroblasts were seeded on plastic or on the stromal aspect of the AM, which was mounted on plastic inserts. Sonicates of human peripheral blood eosinophils activated with lipopolysacharide (LPS), or human mast cell (HMC-1) leukaemia cell sonicates, were added to sub-confluent fibroblast monolayers. Proliferation of the sub-confluent fibroblasts was assessed using the [3H]-thymidine incorporation assay. The production of transforming growth factor (TGF)-β1, granulocyte–macrophage colony-stimulating factor (GM-CSF) and IL-8 in conjunctival or lung fibroblasts was measured in conditioned media from these cultures by ELISA.Results After 4 days in culture, the [3H]-thymidine incorporation assay indicated a reduced proliferation of activated conjunctival and lung fibroblasts when cultured directly on the AMM. The production of both TGF-β1 and IL-8 was significantly suppressed in activated conjunctival fibroblasts cultured on the AMM compared with those cultured on plastic, while the production of both TGF-β1 and GM-CSF was decreased in human lung fibroblast cultured on the AMM.Conclusions The AMM is capable of suppressing fibrotic responses in an in vitro system of effector stages of ocular allergic inflammation. These data may provide a basis for exploring matrix components in the AM for the treatment of allergic eye disease.