Chemotactic activity of guinea pig eosinophils for the ECF-A acidic tetrapeptides, histamine, histamine metabolites, and the effect of H1- and H2-receptor antagonists.

Histamine and one of its major metabolites, imidazoleacetic acid, were selectively chemotactic for guinea pig eosinophils, whereas L-histidine, 1,4-methylimidazoleacetic acid, 1,4-methylhistamine and N-acetylhistamine were inactive. The response to histamine was unaffected by concentrations of eosinophils of between 30 and greater than 90% but it was abrogated by preincubation of the cells with histamine prior to assay (self-deactivation). Eosinophilotaxis was also inhibited by H1-(mepyramine-) and H2-)burimamide)-receptor antagonists at high doses (10(-3)m), although at lower concentrations (10(-5) M) inhibition was principally associated with burimamide. The human tetrapeptide, alanine-glycine-serine-glutamic acid, and the analogue, valine-glycine-aspartic acid-glutamic acid, were inactive whereas alanine-glycine-serine-glutamic acid was chemotactic for the guinea pig eosinophil. These results support the concept that the tissue accumulation of eosinophils following anaphylaxis depends on a complex interaction of factors, which in part may be mediated by H2 receptors on the target cells. There may be species differences in the composition of ECF-A.     

The effects of an ECF-A and formyl methionyl chemotactic peptides on oxidative metabolism of human eosinophils and neutrophils.

An ECF-A tetrapeptide (Val-Gly-Ser-Glu) and the synthetic bacterial analogue, formyl Met-Leu-Phe (agents previously recognized to be chemotactic and to enhance complement receptors on human eosinophils and neutrophils), were tested for their capacity to evoke a spontaneous burst of light emission (chemiluminescence), and to affect oxygen-consuming reactions induced by contact with serum-treated zymosan (STZ). Superoxide anion (O-2) production by neutrophils induced by STZ was significantly enhanced in both a time- and concentration-dependent fashion by F-Met-Leu-Phe, and to a lesser extent, by F-Met-Met-Phe. The dipeptide F-Met-Phe and unformylated Met-Leu-Phe were inactive. In addition, significant enhancement of eosinophil O-2 generation, by the valyl- ECF-A tetrapeptide was demonstrable and, in addition, this peptide appeared to have an inhibitory effect on neutrophil superoxide anion generation. Eosinophils and neutrophils both produced a burst of chemiluminescence when treated with F-Met-Leu-Phe. With both cell types the magnitude of the responses was similar although with eosinophils peak activity occurred within 60 sec as compared to 2-6 min for neutrophils. No chemiluminescent response was achieved with the valyl- ECF-A tetrapeptide using either cell type. These experiments (1) suggest that ECF-A and formyl methionyl peptides amplify reactions associated with the respiratory burst of eosinophils and neutrophils respectively, and (2) support the view that the generally greater metabolic activity of eosinophils may be related to the special role that this cell may play in the killing of helminthic parasites.     

Eosinophil chemotaxis to an ECF-A tetrapeptide and histamine: the response in various disease states

Using the micropore filter technique of Boyden, the eosinophil chemotactic responses to an ECF-A tetrapeptide (Val-Gly-Ser-Glu) and histamine were studied in thirty-two patients with a peripheral blood eosinophilia. The eosinophilia was associated with extrinsic asthma (twelve patients), neoplasia (seven), helminth disease (five) and a miscellaneous group (eight) which included three patients with pulmonary eosinophilia, one of whom also had asthma. Both the tetrapeptide and histamine gave two types of dose–response. With Val-Gly-Ser-Glu this was either a single peak at 10^-8, or two peaks at 10^-4 and 10^-7 mol/1, respectively. Histamine gave either a linear dose–response with maximal chemotaxis at the highest concentration or a peak response at 10^-5 mol/1 with inhibition at higher doses. The two types of response given by the peptide were not related to the disease state and were reproducible when tested on more than one occasion. However, with histamine, linear dose–responses were observed in ten out of twelve patients with asthma, four out of five with helminth disease and two of the three with pulmonary eosinophilia. This was also reproducible when tested on subsequent occasions. Therefore, in these diseases, which are known to be associated with exogenous antigens and raised IgE levels, eosinophils from 80% of the patients studied (sixteen out of twenty) gave a linear rather than a ‘bell-shaped’ response to histamine. In contrast, only four of twelve patients(33%) with eosinophilia in association with other diseases (which included seven with neoplasia) gave this type of response. If this observation can be confirmed with larger numbers of patients it may be useful diagnostically in eosinophilia of unknown origin.     

Comparison of in vitro and in vivo histamine methylation by tissues

A method for determining the histamine-methylating enzyme (HME) using crude enzyme, and minute quantities of the substrate, was applied to tissues of mice, guinea-pigs and rats. Since high levels of endogenous histamine can affect the results, tissue homogenates were dialyzed prior to incubation. Findings were compared with in vivo data on methylating ability of individual tissues; most of this in vivo data is published but a new test of guinea-pig tissues was made using amodiaquine as an inhibitor. The correlation was good, better than that obtained by other procedures. It was observed that dialysis caused an increase in HME for some guinea-pig tissues, but a loss for some mouse tissues. Possible explanations are considered. Quinacrine N-mustard, a derivative of a known HME inhibitor, was tested in mice; it altered the distribution of injected¹⁴C-histamine but showed no evidence of HME inhibition.     

Selective attraction of eosinophils and synergism between eosinophil chemotactic factor of anaphylaxis (ECF-A) and a fragment cleaved from the fifth component of complement (C5a)

ECF-A and C5a were chemotactic both for eosinophils and neutrophils. However, when eosinophils comprised approximately 10 per cent or more of a mixed leucocyte population, they were preferentially attracted by both of these agents.

Marked synergism was observed between ECF-A and C5a in their ability to attract eosinophil leucocytes.

     

A morphometric study of normodense and hypodense human eosinophils that are derived in vivo and in vitro.

Hypodense eosinophils were obtained from two patients with the idiopathic hyperosinophilic syndrome (IHES), and hypodense eosinophils were derived by culturing normodense human eosinophils from control donors in the presence of endothelial cells alone, granulocyte/macrophage-colony-stimulating factor (GM-CSF) alone, or GM-CSF and fibroblasts. These eosinophils were examined ultrastructurally and stereologically for alterations in the volume density (Vv) of their electron-dense granules, the Vv of their lucent granules, the Vv of their lipid droplets, the numerical density of their granules with respect to cytoplasm (Nv), and the plasma membrane surface area-to-cell volume ratio (Sv) that might account for their decreased sedimentation density. The hypodense eosinophils that were obtained from the two patients with IHES exhibited a one-third reduction in granule Vv relative to normodense eosinophils from control donors, primarily because of a decrease in granule size. The culture-derived hypodense eosinophils exhibited 10% to 16% decreases in their granule Vv, significant increases in their lucent granules, and a approximately 7.5% decrease in their Sv. Calculation of the cell volume from cross-sectional area measurements showed that the eosinophils that had been cocultured with fibroblasts in the presence of GM-CSF increased their volume by approximately 15%. The eosinophils that had been cocultured with endothelial cells exocytosed some of their granules. In conclusion, a composite of factors including cell swelling, a decrease in the volume of the cytoplasm occupied by granules, and an increase in granule lucency contributes to the hypodense phenotype in vitro, but only cell swelling and hypogranulation are seen in cells from patients with IHES. The latter could reflect the response of 'primed' hypodense eosinophils in vivo to pertinent tissue ligands.     

Cutaneous eosinophil accumulation in atopic and non-atopic individuals: the effect of an ECF-A tetrapeptide and histamine

The numbers of eosinophils recruited locally to abraded human skin were measured in eight atopic and eight non-atopic volunteers, at time intervals over 24 hr, following the application of an ECF-A tetrapeptide (Val-Gly-Ser-Glu) or histamine. In all subjects the higher doses of the peptide (10^-4 and 10^-6 mol/1) or histamine (10^-3 and 10^-5 mol/1) produced significantly greater counts than the Tyrode's diluent alone. The counts produced with the lowest dose of peptide (10^-8 mol/1) or histamine (10^-7 mol/1) were not significantly different from the control. The peptide or histamine evoked a greater local eosinophilia in the atopics than the non-atopics. This effect was probably independent of the peripheral blood eosinophil counts since at the time of study the numbers of circulating eosinophils between the two groups were not significantly different. In the atopics, histamine in doses of 10^-3 and 10^-5 mol/1 were required to give the same eosinophil response as that obtained with 10^-4 and 10^-6 mol/1 of the peptide, respectively. It is suggested that the relative paucity of eosinophils recruited by locally applied ECF-A peptide or histamine, when compared to antigen-induced eosinophilia, is due either to an inability to mimic the events associated with the release of these mediators from mast-cells or the involvement of other pharmacological agents.     

CD69 is expressed by human eosinophils activated in vivo in asthma and in vitro by cytokines.

CD69 is an early activation marker for T cells and cross-linking of CD69 on platelets triggers aggregation and mediator release. Expression of a number of membrane receptors is induced on eosinophils after culture with certain cytokines. Therefore, we investigated whether cytokine-activated eosinophils expressed CD69. Unstimulated, peripheral blood eosinophils did not express CD69, as determined by immunofluorescence and flow cytometry (n = 15). CD69 expression was induced on eosinophils by granulocyte-macrophage colony-stimulating factor (GM-CSF) in a time- and dose-dependent manner. After 1 day in culture, expression was significant at concentrations of 10(-11) M and above. CD69 expression could be detected after stimulation with GM-CSF for only 1 hr, was significant after 2 hr and was sustained over 1-2 days in culture. CD69 expression was also induced by interleukin-3 (IL-3), IL-5 and interferon-gamma (IFN-gamma), but stimulation of eosinophils with platelet-activating factor (PAF) (10(-6) M) for up to 2 hr did not induce CD69 expression. Cycloheximide (10(-6) M) significantly inhibited GM-CSF-induced CD69 expression, suggesting a requirement for protein synthesis. However, unlike up-regulation of CR3 expression, GM-CSF-induced CD69 expression was not inhibited by dexamethasone. CD69 was present on eosinophils from the bronchoalveolar lavage (BAL) fluid of patients with mild asthma (5/5), suggesting that the in vitro findings may have biological relevance in vivo. Therefore, CD69 can be used as a marker of eosinophil activation by cytokines and is a candidate receptor for triggering eosinophil mediator release in the airways in asthma.     

Human skin mast cells: in vitro and in vivo studies.

OBJECTIVE: This short review surveys our current knowledge on the development and heterogeneity of human mast cells, the distribution of mast cells within human skin and the properties of human skin mast cells both in vitro and in vivo. It also examines the effects of antihistamines in the wheal-and-flare response in the skin provoked by bradykinin. RESULTS: Mast cells derive from mononuclear precursor cells which undergo their final phase of their differentiation in the tissues. In normal skin, mast cells, which are primarily of the MC(TC) subtype, occur in the greatest density in the superficial dermal zone. Like all other mast cells, human skin mast cells bind IgE with high affinity to specific FcepsilonRI receptors, but unlike those from lung, tonsils, adenoids or intestine, they also express the C5a receptor (CD88) and activation sites for substance P, VIP, somatostatin, and compound 48/80. Both IgE-dependent stimulation by activating tyrosine kinases, and non-immunologic stimulation by activating G-proteins induce a characteristic compound exocytosis resulting in the liberation of the preformed mediators. Production of prostaglandin D2 and leukotriene C4, however, occurs only with IgE-dependent stimulation. In vivo, dermal microdialysis and scanning laser Doppler imaging have been used to assess the role of histamine in the wheal-and-flare response. These techniques were also used to show that low concentrations of intradermal bradykinin release negligible quantities of histamine. The results showed that although the resultant flare was inhibitable by antihistamines, low concentrations of bradykinin released negligible quantities of histamine. This suggests a potentially novel mechanism of action of antihistamines that requires further detailed investigation.     

Human recombinant interleukin-2 induces maturation and activation signals for feline eosinophils in vivo

Immunotherapy, with interleukin-2 (IL-2) or IL-2 plus lymphokine-activated killer (LAK) cells, has been used to treat cancer and acquired immunodeficiency syndrome (AIDS) in man. Similarities between feline leukemia virus (FeLV) infection in the cat and human immunodeficiency virus (HIV) infection in man have prompted immunotherapeutic studies in the cat. To develop baseline data on hematological responses to infused IL-2, cats were given daily (1-14 days) i.v. injections of 5 x 10(4) U/kg of recombinant human IL-2 (rHulL-2). Complete blood cell (CBC) counts were done weekly. Red blood cell (RBC), neutrophil, and lymphocyte numbers did not change appreciably over the course of the study. In contrast, rHulL-2 caused an eosinophilia in all but the 1 day treatment group. Treatment for 3 days generated a transient eosinophilia on day 7 that returned to baseline by 3 weeks. Five day and 7 day treatments generated an eosinophilia by day 7 that peaked on day 14 and returned to normal values by day 28. Treatment of cats for 14 days did not increase the magnitude or duration of the eosinophilia beyond the 5 or 7 day treatments. Bone marrow (BM) biopsies from rHulL-2-treated cats revealed a marked selective hyperplasia of eosinophil precursors. In the 5 day treatment group, all maturation stages of eosinophils were elevated by week 1 of treatment. By week 2, the early stages had returned to normal, whereas the late stage cells remained elevated, suggesting an ordered maturation response. Numbers of all eosinophil precursors approximated pretreatment numbers by weeks 3-4. Thus the BM hyperplasia preceded the blood eosinophilia by 1 week, suggesting that an enhanced maturation response of BM eosinophil precursors is a major contributor to the rHulL-2-induced blood eosinophilia. In addition to a maturation signal, rHulL-2 induces a potent activation signal for eosinophils as measured by a decrease in density and an increase in longevity in culture. The significance of the activated eosinophil in the therapeutic or toxicologic response to rHulL-2 infusion is discussed.     

Fecundity ofLitomosoides carinii (Nematoda,Filarioidea) in vivo and in vitro

Several parameters concerning the reproduction ofLitomosoides carinii were assessed using quantitatively infected cotton rats (Sigmodon hispidus). The course of embryogenesis from the fertilization of eggs to the delivery of the first microfilariae was observed by daily autopsies during prepatency. The duration of embryogenesis in vivo could thus be determined as 18±2 days. The contents of embryos in the uteri of female worms had been examined at various intervals. At the onset of patency 7–8 weeks p.i. the females were 71±6 mm long and on average contained 308×10³ embryos/female, of which 19% were pathologically altered. In the middle of patency 16–20 weeks p.i. the females had grown up to 100±11 mm in length and now contained 509×10³ embryos/female, 25% of them were pathologically altered, the others were normally developed. A positive correlation between the body length of a female worm and its number of embryos in utero was evident. Additionally the percentage of pathologically altered embryos was increased with respect to the age of the worms. The calculated fecundity of a femaleL. carinii in vivo of around 20×10³ microfilariae/female per day had been confirmed with worms maintained in vitro. Three combinations of media and serum supplements were used and their influence on embryogenesis evaluated.Dedicated to Dr. B.O.L. Duke on the occasion of his 60th birthday     

Interactions between temperature and human leptin physiology in vivo and in vitro

To investigate the possibility that environmental temperature may exert physiologically significant direct, local effects on subcutaneous adipose tissue temperatures, and its secretion of leptin, we exposed healthy males (n=12) to repeated cold-water immersion (study 1), and also incubated surgically removed human subcutaneous adipose tissue samples (n=7) at 27°, 32° and 37°C (study 2). In vivo immersions were conducted over 15 days (60–90 min at 18°C). Regional body temperatures and plasma leptin concentrations were measured before and during immersion. Acute cold exposure suppressed plasma leptin concentration (25 min: –14%, 60 min: –22%, P=0.0001), whilst repeated cold-water immersion was associated with an increase of plasma leptin concentration relative to test day 1 (+19% day 8, +13% day 15, overall P=0.03). Leptin secretion in vitro decreased 3.7-fold as the incubation temperature decreased from 37° to 27°C (P=0.001). In a compartmental model of leptin turnover in vivo, the measured (local) temperature effect on leptin secretion in vitro was more than able to account for the observed cold-induced decrease in leptin concentration in vivo. We therefore conclude that acute and repeated cold-water immersions have separate and opposing effects on circulating leptin concentrations in humans. Under our experimental conditions, the local effects of reduced subcutaneous adipose tissue temperature may be a more important contributor to the acute effects observed in vivo, than the sympathetically mediated suppression of leptin secretion.     

Effects of various S-adenosylmethionine preparations on histamine methylation in vitro and in vivo

To test possible enhancement of in vivo methylation of histamine, mice were injected with S-adenosylmethionine (SAM) of approximately 96% purity. Instead of the expected enhancement, very strong inhibition of methylation was observed. Tests indicated that S-adenosylhomocysteine (SAH) probably was not the inhibitor. Pure SAM, from another source, showed no inhibition of methylation in rats and guinea pigs, and only slight inhibition in mice. Pure SAM was much more effective than the 96% SAM for histamine methylation in vitro. It seems either that injected SAM cannot enter cells containing the histamine methylating enzyme (HME), or that the endogenous supply of SAM is optimal. The inhibitor may be structurally related to SAM, possibly a by-product of SAM synthesis. As it is very effective even in the presence of large amounts of SAM, in pure form it would probably be the most potent inhibitor of histamine methylation available.     

Cardiovascular protection of nonmitogenic human acidic fibroblast growth factor from oxidative damage in vitro and in vivo.

BACKGROUND: In our previous study, a mutant human acidic fibroblast growth factor without mitogenic action (nonmitogenic human acidic fibroblast growth factor) was created, and its protection from the cytotoxic effect of hydrogen peroxide treatment was confirmed in cultured cardiomyocytes. METHODS: The present study was performed to further investigate whether genetically overexpressing nonmitogenic human acidic fibroblast growth factor in cardiomyocytes provides similar protection from the cytotoxic effect of hydrogen peroxide and whether in vivo administration of nonmitogenic human acidic fibroblast growth factor attenuates ischemia/reperfusion-induced cardiac dysfunction and tissue damage and protects the carotid sinus baroreceptor from alcohol-induced damage, as shown by a reduced response of blood pressure to short carotid artery occlusion. RESULTS AND CONCLUSIONS: Cardiomyocytes transfected by nonmitogenic human acidic fibroblast growth factor, with significant increases in the cellular expression and secretion of nonmitogenic human acidic fibroblast growth factor into a culture medium, were resistant to hydrogen-peroxide-induced cytotoxicity, as measured by cell viability. Hearts isolated from rats pretreated with saline, human acidic fibroblast growth factor, or nonmitogenic human acidic fibroblast growth factor for 24 h were subjected to ischemia/reperfusion in the Langendorff system. Ischemia/reperfusion induced cardiac dysfunction in the saline group, but not in the group pretreated with human acidic fibroblast growth factor or nonmitogenic human acidic fibroblast growth factor. Ischemia/reperfusion also caused a release of the cardiac enzyme lactic dehydrogenase into-and an increase in lipid peroxide content in the efflux of-the hearts of saline-treated rats, but not in rats pretreated with human acidic fibroblast growth factor or nonmitogenic human acidic fibroblast growth factor. There was no difference in cardioprotective effects between human acidic fibroblast growth factor and nonmitogenic human acidic fibroblast growth factor. Furthermore, the protective effect of in-vivo-administered nonmitogenic acidic fibroblast growth factor on alcohol-induced damage to the carotid sinus baroreceptor, as shown by the reduced response of blood pressure to short carotid artery occlusion, was also observed. These results suggest that nonmitogenic human acidic fibroblast growth factor, similar to the native human acidic fibroblast growth factor, provides significant cardiovascular protection from oxidative damage in vitro and in vivo.     

Gastric mucosal histamine,histamine formation capacity (HFC) and plasma gastrin after cysteamine administration.

Cysteamine administration is followed by stomach and duodenal ulcers in rats. We aimed to establish a time relationship between changes in gastric mucosal histamine, histamine formation capacity (HFC) and plasma gastrin after cysteamine administration. Up to 4 h after cysteamine s.c., no ulcers were found. Plasma gastrin rose after cysteamine and was higher than in controls 2h after injection. Mucosal histamine fell after 4h; no other significant changes were found in mucosal histamine and HFC. A direct correlation was found between plasma gastrin and HFC in both controls and after cysteamine. It is suggested that the changes indicate that cysteamine releases gastrin, which increases HFC and thus histamine. The fall in histamine seems to indicate utilization of histamine in acid production.     

A tumoral acidic pH-responsive drug delivery system based on a novel photosensitizer (fullerene) for in vitro and in vivo chemo-photodynamic therapy

Fullerene has shown great potential both in drug delivery and photodynamic therapy. Herein, we developed a doxorubicin (DOX)-loaded poly(ethyleneimine) (PEI) derivatized fullerene (C60–PEI–DOX) to facilitate combined chemotherapy and photodynamic therapy in one system, and DOX was covalently conjugated onto C60–PEI by the pH-sensitive hydrazone linkage. The release profiles of DOX from C60–PEI–DOX showed a strong dependence on the environmental pH value. The biodistributions of C60–PEI–DOX were investigated by injecting CdSe/ZnS (Qds) labeled conjugates (C60–PEI–DOX/Qds) into tumor-bearing mice. C60–PEI–DOX/Qds showed a higher tumor targeting efficiency compared with Qds alone. Compared with free DOX in an in vivo murine tumor model, C60–PEI–DOX afforded higher antitumor efficacy without obvious toxic effects to normal organs owing to its good tumor targeting efficacy and the 2.4-fold greater amount of DOX released in the tumor than in the normal tissues. C60–PEI–DOX also showed high antitumor efficacy during photodynamic therapy. The ability of C60–PEI–DOX nanoparticles to combine local specific chemotherapy with external photodynamic therapy significantly improved the therapeutic efficacy of the cancer treatment, the combined treatment demonstrating a synergistic effect. These results suggest that C60–PEI–DOX may be promising for high treatment efficacy with minimal side effects in future therapy.