Vincenzo Patella

Stem Cell Factor in Mast Cells and Increased Mast Cell Density in Idiopathic and Ischemic Cardiomyopathy

BACKGROUND We compared cardiac mast cell (HHMC) density and the immunological and nonimmunological release of mediators from mast cells isolated from heart tissue of patients with idiopathic dilated (DCM) (n=24) and ischemic cardiomyopathy (ICM) (n = 10) undergoing heart transplantation and from control subjects (n = 10) without cardiovascular disease. METHODS AND RESULTS HHMC density in DCM (18.4+/-1.6 cells/mm2) and ICM (18.4+/-1.5 cells/mm2) was higher than that in control hearts (5.3+/-0.7 cells/mm2; P<.01). The histamine and tryptase contents of DCM and ICM hearts were higher than those of control hearts. The histamine content of the hearts was correlated with mast cell density (r(s)=.91; P<.001). Protein A/gold staining of heart tissue revealed stem cell factor (SCF), the principal growth, differentiating, and activating factor of human mast cells, in HHMC secretory granules. Histamine release from cardiac mast cells caused by immunological (anti-IgE and rhSCF) and nonimmunological stimuli (Ca2+ ionophore A23187) was higher in patients with DCM and ICM compared with control subjects. Immunological activation of HHMC induced a significantly greater release of tryptase and LTC4 in patients with DCM and ICM compared with control subjects. CONCLUSIONS Histamine and tryptase content and mast cell density are higher in failing hearts than in control hearts. SCF, present in secretory granules of HHMC, might represent an autocrine factor sustaining mast cell hyperplasia in heart tissue in these patients. The increased local release of fibrogenic factors (eg, histamine, tryptase, and leukotriene C4) might contribute to collagen accumulation in the hearts of patients with cardiomyopathy.

HIV-1 gp120 induces IL-4 and IL-13 release from human Fc epsilon RI+ cells through interaction with the VH3 region of IgE.

HIV-1 glycoprotein (gp) 120 from different clades is a potent stimulus for IL-4 and IL-13 release from basophils purified from healthy individuals seronegative for Abs to HIV-1 and HIV-2. IL-4 mRNA, constitutively present in basophils, was increased after stimulation by gp120 and was inhibited cyclosporin A and tacrolimus. IL-4 and IL-13 secretion from basophils activated by gp120 was not correlated. There was a correlation between the maximum gp120- and anti-IgE-induced IL-4 release from basophils. The average t1/2 gp120-induced IL-4 release was lower than for IL-13 release. Basophils from which IgE had been dissociated by brief exposure to lactic acid no longer released IL-4 in response to gp120 or to anti-IgE. The response to a mAb cross-linking the α-chain of high-affinity receptor for IgE (FcεRI) was unaffected by this treatment. Three human VH3+ monoclonal IgM inhibited gp120-induced secretion of IL-4 from basophils. In contrast, VH6+ monoclonal IgM did not inhibit the release of IL-4 induced by gp120. Synthetic peptides distant from the NH2 and COOH termini of gp120MN inhibited the activating property of gp120MN. These results indicate that gp120, which acts as a viral superantigen, interacts with the VH3 region of IgE to induce the release of IL-4 and IL-13 from human FcεRI+ cells.

Molecular and Cellular Biology of Mast Cells and Basophils

In all mammalian species investigated so far, mast cells and basophils are the only cells that synthesize histamine and express plasma membrane receptors that bind IgE with high affinity (Fc epsilonRI). Human basophils and mast cells derive from distinct precursors that originate in the bone marrow and fetal liver and probably circulate in peripheral blood. There is extensive evidence that mast cells and basophils and their mediators are primary effectors of allergic inflammation. Immunologically activated human basophils release two cytokines: IL-4 and IL-13. Expression of several cytokines has been documented in a number of experimental models of human and rodent mast cells. However, to date few studies have analyzed the mechanisms of gene expression in human Fc epsilonRI+ cells. Some of these studies imply a role for NFAT and GATA family members in the IgE-mediated activation of cytokine gene transcription in basophils and mast cells. Studies of human basophils and mast cells isolated from different anatomic sites have established the different profiles of eicosanoids released by these cells. Recently, the characterization of arachidonic acid pools and the identification of novel enzymes involved in arachidonate remodeling and mobilization clarified in part how eicosanoid productions is regulated in mast cells and basophils. In addition to histamine, human mast cell secretory granules contain the neutral proteases tryptase, chymase and carboxypeptidase that possess several biochemical properties. In particular, tryptase may play a role as a fibrogenic factor and chymase might convert angiotensin I to angiotensin II. Mast cells are present in human heart and in human coronary arteries raising the possibility that local activation of cardiac mast cells might contribute to certain cardiovascular diseases. Recent evidence also suggests that mast cells and basophils can play a role during viral and bacterial infections. It is now evident that in man these two cells not only participate in inflammation associated with allergic disease, but also in chronic and fibrotic disorders affecting several organs and in host defense against bacterial and viral infections.

Human basophil/mast cell releasability. IX. Heterogeneity of the effects of opioids on mediator release.

Opioids differ in their capacity to cause release of histamine. The effects of increasing concentrations of three opioids (morphine, buprenorphine, and fentanyl) were studied on the release of preformed (histamine and tryptase) and de novo synthesized (prostaglandin D2 [PGD2] and peptide-leukotriene C4 [LTC4]) chemical mediators from human peripheral blood basophils and mast cells isolated from skin tissues or lung parenchyma. Basophils released < 5% of their histamine content and did not synthesize significant amounts of LTC4 when incubated with any of the opioids. Mast cells showed markedly different responses to the three opioids. Morphine (10(-5)-3 x 10(-4) M), in a concentration-dependent manner, induced histamine and tryptase release from skin but not from lung mast cells, up to a maximum of 18.2 +/- 1.9% and 13.0 +/- 4.1 micrograms/10(7) cells, respectively. Morphine did not induce de novo synthesis of PGD2 from skin mast cells. Buprenorphine (10(-6)-10(-4) M), in a concentration-dependent manner, caused histamine and tryptase release from lung but not from skin mast cells, to a maximum of 47.6 +/- 7.2% and 35.1 +/- 13.6 micrograms/10(7) cells, respectively. Buprenorphine also induced de novo synthesis of PGD2 and LTC4 from lung mast cells. Fentanyl (10(-5)-10(-3) M) did not induce histamine and tryptase release or the de novo synthesis of PGD2 or LTC4 from any mast cells. Histamine release caused by buprenorphine from lung mast cells was slow (t1/2 = 11.2 +/- 3.6 min) compared with that induced by morphine from skin mast cells (t1/2 < 1 min, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Immunological characterization and functional importance of human heart mast cells

Mast cells are present in normal and even more abundant in diseased human heart tissue and their localization is of particular relevance to their function. Within heart tissue mast cells lie between myocytes and in close contact with blood vessels. They are also found in the coronary adventitia and in the shoulder regions of a coronary atheroma. The density of cardiac mast cells is markedly higher in some patients with myocarditis and dilated cardiomyopathy than in accident victims without cardiovascular diseases. More importantly, in some of these conditions there is in situ evidence of mast cell activation. We have described an original technique to isolate and purify HHMC for in vitro study. This procedure gives viable cells and after stimulation with immunological or non-immunological stimuli they release performed (histamine and tryptase) and newly generated mediators (PGD2 and LTC4). We have demonstrated that HHMC differ from those in other anatomical districts in that they are activated by specific immunological and non-immunological stimuli, and in their relation to the arachidonic acid metabolism, suggesting that the local microenvironment can influence their phenotypic and biochemical characteristics. Our own and other findings suggest that HHMC have complex and significant roles in different pathophysiological conditions involving the cardiovascular system. Direct activation of HHMC by therapeutic and diagnostic substances injected intravenously explains some of the anaphylactoid reactions caused by these agents. HHMC possess Fc epsilon RI and IgE bound to the surface and C5a receptors, which could explain how cardiac mast cells are involved in systemic and cardiac anaphylaxis. Cardiac mast cells and those in human coronary arteries also play a role in the early and late stages of atherogenesis and during ischemic myocardial injury. In conclusion, although studies of HHMC are in their infancy, their in vitro isolation may be useful in identifying additional mediators synthesized and released, stimuli relevant to human pathophysiology, and pharmacological agents selectively modulating the activation of these cells and their mediators. Drugs specifically acting on HHMC or on their mediators may eventually be useful in treating different cardiovascular diseases.

Loratadine and desethoxylcarbonyl-loratadine inhibit the immunological release of mediators from human FceRI+cells

Background Loratadine, a novel histamine H1‐receptor antagonist, is effective in the treatment of patients with seasonal and perennial rhinitis and some allergic skin disorders. Histamine and other chemical mediators are synthesized and immunologically released by human peripheral blood basophils and tissue mast cells (FcɛRI+ cells).

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.

Human basophil/mast cell releasability. XI. Heterogeneity of the effects of contrast media on mediator release†

BACKGROUND The activation of basophils and mast cells plays a role in the pathogenesis of anaphylactoid reactions occurring during the administration of iodinated radiocontrast media. METHODS We compared the effects of three contrast media (CM), Hexabrix (sodium and meglumine salts of ioxaglic acid), Telebrix (sodium and meglumine salts of ioxitalamic acid), and Optiray (ioversol) on the release of preformed (histamine and tryptase) and de novo synthesized (prostaglandin D2 and leukotriene C4) mediators from human basophils and mast cells isolated from lung, skin, and heart tissue. The commercial preparations were evaluated in parallel with the pure substances. Mannitol was used as a positive control inducing histamine release (HR) by hyperosmolar stimulation. RESULTS Hexabrix (0.1 to 0.3 mol/L), Telebrix (0.1 to 0.5 mol/L), Optiray (0.2 to 0.5 mol/L), and the corresponding pure substances concentration-dependently induced HR from basophils. A positive correlation was found between CM osmolality and HR from basophils. Mast cells isolated from different anatomic sites responded differently to the three CM. Hexabrix and Optiray induced histamine and tryptase release from human lung mast cells, but not from human skin mast cells. No correlation was found between the osmolality of CM and HR from human lung mast cells. There was a significant correlation between the percent of histamine and tryptase release induced by CM from human lung mast cells. Hexabrix, Telebrix, and Optiray also induced histamine and tryptase release from human heart mast cells. None of the CM induced the de novo synthesis of leukotriene C4 or prostaglandin D2 from basophils or mast cells. The kinetics of HR caused by CM differed according to the drug used and the cell (basophils or human lung mast cells) examined. CM-induced HR from basophils and human lung mast cells was temperature-dependent, partially influenced by extracellular Ca2+ concentrations, and not modified by preincubation of basophils with IL-2 or IL-3. CONCLUSIONS These results provide evidence of the heterogeneity of the effects of CM on mediator release from human basophils and mast cells from different anatomic sites. They also suggest that hyperosmolarity may be an important factor in the activation of basophils by CM, but less relevant for mast cells. CM induce only the release of preformed mediators. The measurement of plasma tryptase might be clinically useful for monitoring adverse reactions caused by CM.

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.

The clinical relevance of basophil releasability

area is the possibility that groups of cytokines st imulate specific pat terns of expression of receptors and that this pa t te rn is responsible for behavior of the cell and can be diagnosed by MAb. Third, the possibility of having clinically relevant antagonists is real with the development of powerful blocking M A b and, perhaps more important , of analogues of growth factors that act as antagonists by specifically binding one of the chains of the receptors for these factors (Tables V and VI). Al though each of these reagents may have significant drawbacks in clinical use (the M A b is mur ine and the antagonist must be used in excess over ligand of approximately 100 times), each points the way for the specific design of smaller molecules that may be more po ten t and more easily administered. Finally, the effect of cyclosporine on cells o ther than T lymphocytes also points the way for the inhibition of t ranscript ion of cytokine genes in non-T cells. The particularly attractive possibility is that there exist several pathways of activation only some of the activation pathways are sensitive to cyclosporine. If this were to be the case, it may be possible to target activation pathways specific for allergy.