Anna Ciccarelli

Human heart mast cells: a definitive case of mast cell heterogeneity.

Mast cells and their chemical mediators play a role in cardiac and systemic anaphilaxis. Perivascular and cardiac mast cells have been implicated in the pathogenesis of coronary artery spasm, atherosclerosis, myocardial ischemia, and cardiomyopathy. Despite this, nothing is known about the immunological and biochemical characteristics of the human heart mast cell (HHMC). We have isolated and partially purified HHMC and compared them with mast cells isolated from lung (HLMC) and skin (HSMC) tissues. Cross-linking of the high-affinity receptor for IgE (Fc epsilon RI) by a polyclonal anti-Fc epsilon antibody caused the release of preformed (histamine and tryptase) and de novo synthesized mediators [peptide leukotriene C4 (LTC4) and prostaglandin D2 (PGD2)]. The tryptase content of HHMC (19.4 +/- 1.5 micrograms/10(6) cells) was lower than HSMC (33.4 +/- 2.5 micrograms/10(6) cells) and higher than HLMC (10.6 +/- 1.9 micrograms/10(6) cells). Maximal stimulation of HHMC with anti-IgE led to the release of LTC4 (17.5 +/- 5.1 ng/10(6) mast cells) and PGD2 (17.8 +/- 5.0 ng/10(6) mast cells, whereas HSMC synthesized more PGD2 (65.0 +/- 6.8 ng/10(6) mast cells) and much less LTC4 (< 5 ng/10(6) cells). Recombinant human C5a anaphylatoxin and protamine induced histamine release from HHMC and HSMC, but not from HLMC. Substance P and morphine selectively induced the release of histamine from HSMC, but not from HHMC and HLMC. Compound 48/80 caused histamine release from HSMC and HHMC, but not from HLMC. The pattern of mediators synthesized and the responsiveness of HHMC to different secretagogues appear unique providing strong evidence of human mast cell heterogeneity.

Cyclosporin H is a potent and selective competitive antagonist of human basophil activation by N-formyl-methionyl-leucyl-phenylalanine

BACKGROUND Cyclosporin A (CsA) binds with high affinity to cyclophilin, a critical step in the molecular mechanism of action of cyclosporins, where cyclosporin H (CsH) has extremely low affinity for cyclophilin. CsH differs from CsA by the substitution of the L-methyl valine at position 11 with it D-isomer. METHODS We compared the effects of CsA and CsH on the release of performed (histamine) and de novo synthesized inflammatory mediators (peptide leukotriene C4) from peripheral blood basophils activated by N-formyl-methionyl-leucyl-phenylalanine (FMLP). RESULTS CsH (8 to 800 nmol/L) concentration-dependently inhibited histamine and leukotriene C4 release from purified and unpurified basophils activated by FMLP, whereas CsA (8 to 800 nmol/L) had little inhibitory effect on histamine release from basophils challenged with FMLP. Inhibition of histamine release from basophils challenged with FMLP was extremely rapid and was abolished by washing the cells (three times) before challenge. CsH (8 to 800 nmol/L) had no effect on the release of histamine caused by C5a, platelet activating factor, monocyte chemotactic activating factor, RANTES, IL-8, bryostatin 1, and phorbol myristate. Preincubation of basophils with granulocyte-macrophage colony-stimulating factor (30 and 100 pmol/L), but not IL-1 beta (30 and 100 ng/ml), concentration-dependently reversed the inhibitory effect of CsH on FMLP-induced histamine release. CsH competitively inhibited the effect of FMLP on histamine release from basophils. The dissociation constant (Kd) for the CsH-FMLP receptor complex was approximately 9 x 10(-8) mol/L, more than 10-fold lower than that (approximately equal to 1.3 x 10(-6) mol/L) of N-t-BOC-methionyl-L-leucyl-phenylalanine (BocMLP), a known formyl peptide receptor antagonist. CsH inhibited tritiated FMLP binding to human polymorphonuclear leukocytes with a concentration required to inhibit binding by 50% of approximately 5.4 x 10(-7) mol/L, whereas BocMLP was less potent with a concentration required to inhibit binding by 50% of approximately 9.1 x 10(-5) mol/L. Scatchard analysis revealed that the decreased tritiated FMLP binding caused by CsH was due to a decrease in the Bmax (0.22 +/- 0.04 nmol/L/5 x 10(6) cells vs 0.09 +/- 0.01 nmol/L/5 x 10(6) cells; p < 0.05), without a significant difference in the Kd (5.16 +/- 1.22 nmol/L vs 6.32 +/- 2.42 nmol/L; p = NS). CONCLUSION CsH is a potent and selective inhibitor of mediator release from basophils induced by activation of the formyl peptide receptor; it acts by interfering with agonist binding to FMLP receptors.

Modulation of human lung mast cell function by the c-kit receptor ligand

The gene product of the steel locus is a growth factor for mast cells and a ligand for the c- kit proto-oncogene receptor, a member of the tyrosine kinase receptor class of oncogenes

Ciclosporin A Inhibits Mediator Release from Human FcεRI+ Cells by Interacting with Cyclophilin

Ciclosporin A (CsA), a natural cyclic undecapep-tide originally used to prevent human organ transplant rejection [1], has more recently proved to be effective in the treatment of such autoimmune diseases as type I diabetes [2], uveoretinitis [3], rheumatoid arthritis [4] and Crohn’s disease [5]. The mechanism of action of CsA is largely unknown. Part of the immunosuppressive effect of CsA is generally attributed to the inhibition of lymphokine mRNA transcription [6]; however, CsA appears to have multiple sites of action and could exert its effects by binding to specific cytoplasmic receptor(s). Following the purification by Handschumacher et al. [7] of cyclophilin, a cytoplasmic protein which possesses peptidyl-prolylris’-írαns-isomerase (PPIase) activity [8, 9], it was suggested that CsA binding to PPIase/cyclophilin is a critical step in the molecular mechanism of action [10]. We examined the effect of CsA and of a series of analogs (CsG, CsC, CsD and CsH), which bind with decreasing affinity to cyclophilin [11], to evaluate the involvement of this protein in the release of chemical mediators from human inflammatory cells possessing high affinity receptors for IgE (FcεRI+ cells) (i.e., basophils and mast cells). Pharmacological concentrations (8–250 nM) of CsA concentration-dependently inhibited (5–60%) the release of histamine and the de novo synthesis of peptide leukotriene C4 (LTC4) from human basophils (fig. 1) and mast cells isolated from lung parenchyma challenged with anti-IgE. When basophils were activated by the ionophore A23187, which bypasses the channel-operated influx of Ca2+ [12], CsA was more potent (93 vs. 60%) and more effective at low concentrations (IC4o = 24 vs. 100 nM). The inhibition of CsA on mediator release was extremely rapid, similar to the rate of inhibition of PPIase activity [8, 9], and was not abolished by washing the cells before anti-IgE or A23187 challenge. The direct activation of different protein kinase C isoforms induced by phorbol myristate (TPA) and bryostatin 1 [13] was unaffected by preincubation with ciclospo-rins, which suggests that CsA affects biochemical step(s) that are bypassed by direct PKC activation.

In vitro and in vivo characterization of the anti-inflammatory effects of cyclosporin A

Cyclosporin A (CsA) a cyclic undecapeptide extracted from Tolypodadium inflatum Gams, is effective in the prevention/treatment of graft rejection and in an increasing number of autoimmune and inflammatory disorders. The immunosuppressive effect of CsA was initially attributed to inhibition of lymphokine mRNA transcription. We have demonstrated that pharmacological concentrations of CsA (10–100 ng/ml) also inhibit the in vitro immunologic release of preformed (histamine) and de novo synthesized (leukotriene C4 and prostaglandin D2) mediators of inflammatory reactions from human basophils and mast cells. Furthermore the administration of a single dose of CsA (7 mg/ kg) to 8 normal donors caused a rapid and significant (20–40%) inhibition of histamine release from basophils obtained ex vivo from these subjects and challenged in vitro with anti-IgE, f-Met-Leu-Phe and A23187. The inhibitory effect of CsA peaked at 1–5 h, slowly decreased up to 13 h, and was associated with a sharp increase in blood CsA levels. Thus, CsA exerts remarkable anti-inflammatory effects on the immunologic release of mediators from human basophils and mast cells – properties that may contribute to the drug’s therapeutic effects.

Selective activation of human mast cells by general anesthetics

We investigated thein vitro effects of increasing concentrations of three general anesthetics commonly used in clinical practice, i.e., ketamine (10−6–10−3M), thiopentone (10−5–8×10−4M) and propofol (5–70 μg/ml), on histamine release (HR) from human peripheral blood basophils and mast cells isolated from lung parenchyma (HLMC) and skin tissues (HSMC). None of the drugs induced HR from basophils. Ketamine caused HR from HLMC (p<0.001 compared to spontaneous release [SR]) and HSMC (p<0.05 compared to SR). Thiopentone induced limited HR from HLMC (p<0.05 compared to SR). Propofol induced HR from HLMC (p<0.005 compared to SR) and HSMC (p<0.05 compared to SR). Thus, general anesthetics induce HR selectively from human mast cells.

Modulation of Mediator Release From Human FcεRI+ Cells

Peripheral blood basophils and tissue mast cells are the only human cells that have high-affinity receptors for IgE (FceRI) and synthesize histamine [Marone et al.,1989a]. Human basophils and mast cells appear to differ morphologically, ultrastructurally, immunologically, biochemically and pharmacologically [Dvorak et al., 1983; Marone et al., 1989a] and play distinct roles in different types and phases of allergic reactions involving the respiratory tract [Bascom et al., 1988; Casolaro et al., 1989] and skin tissues [Bedard et al., 1986]. Mast cells presumably play a larger role in immediate reactions, whereas basophils play a major role in late phases of bronchial asthma and skin disorders [Bascom et al., 1988; Charlesworth et al., 1989; Guo et al., 1990]. Basophils are recruited to the site of late-phase reactions and synthesize most of the mediators recovered in nasal lavage and skin blister models [Naclerio et al.,1985; Bascom et al., 1988; Charlesworth et al., 1989].

Allergic sensitization to common pets (cats/dogs) according to different possible modalities of exposure: an Italian Multicenter Study

BackgroundThe query “are there animals at home?” is usually administered for collecting information on anamnesis. This modality to consider exposure to pet allergens constitutes a potential bias in epidemiological studies and in clinical practice. The aim of our study was to evaluate/quantify different modalities of exposure to cat/dog in inducing allergic sensitization.MethodsThirty Italian Allergy units participated in this study. Each centre was required to collect the data of at least 20 consecutive outpatients sensitized to cat/dog allergens. A standardized form reported all demographic data and a particular attention was paid in relieving possible modalities of exposure to cat/dog.ResultsA total 723 patients sensitized to cat/dog were recorded, 359 (49.65%) reported direct pet contact, 213 patients (29.46%) were pet owners, and 146 subjects (20.19%) were exposed to pets in other settings. Other patients were sensitized by previous pet ownership (150–20.75%) or indirect contact (103–14.25%), in 111 subjects (15.35%) any contact was reported.ConclusionsOnly 213 patients (29.46%) would be classified as “exposed to animals” and 510 (70.54%) as “not exposed” according to usual query. Our classification has shown that many “not-exposed” subjects (399–55.19%) were “really exposed”. The magnitude of exposure to pet allergens at home is not related exclusively to pet ownership. These considerations should be taken into account during the planning of epidemiological studies and in clinical practice for the management of pet allergic individuals.