Biomedical subjects

J C Fantone

Publications and source records attributed to J C Fantone.

At least 19 recordsLinked to original sources

PubMed1995

Activation of human neutrophils through L-selectin and Mac-1 molecules.

The effect of selective cell activation through L-selectin and Mac-1 molecules cross-linking on neutrophil superoxide anion (O2-) generation and changes in intracellular calcium [Ca2+]i was investigated. Cross-linking of L-selectin using different mAbs induced a rapid and transient increase in [Ca2+]i and O2- generation by neutrophils that were dependent on the extent of L-selectin cross-linking and mAb epitope binding. In addition, cross-linking of L-selectin induced an up-regulation of surface Mac-1 expression on neutrophils. Cross-linking of Mac-1 molecules with mAb also induced significant changes in [Ca2+]i levels and O2- generation by neutrophils, which was also dependent on the epitope binding by mAb. Pretreatment of neutrophils with LPS caused a marked decrease in the expression of L-selectin molecules and significant inhibition (i.e., 59 +/- 4%) of anti-L-selectin mAb-dependent O2- generation and [Ca2+]i signal. In contrast, LPS pretreatment caused a significant up-regulation of Mac-1 molecules on neutrophil and enhanced O2- generation by neutrophils. The data indicate that cross-linking of L-selectin and Mac-1 initiates changes in [Ca2+]i and O2- production in neutrophils and suggest that these distinct adhesion molecules independently may play important regulatory roles in modulating neutrophil-endothelial cell interactions, transmigration, and neutrophil function at sites of tissue injury.

Animals↗

PubMed1995

The effect of pass/fail grading and weekly quizzes on first-year students' performances and satisfaction.

BACKGROUND: In 1992-93 the University of Michigan Medical School revised its first-year curriculum. An evaluation system using honors, high-pass, pass, and fail grading and only two examinations (a midterm and a final) was replaced with a system using pass/fail grading and weekly quizzes in addition to the two examinations. The objective was to increase students' satisfaction while maintaining a high level of achievement. METHOD: Students' performance scores and survey data from the final year of the former system (1991-92, 222 students) and the first year of the new system (1992-93, 195 students) were used to investigate whether overall performance decreased and whether the students liked the new approach to grading. Statistical methods used were one-sample t-tests, Student's t-test, and Fisher's Z-test. RESULTS: Under the new system, the average scores for courses remained well above passing, and no evidence was found that the students achieved at lower levels than had their predecessors with the former, more traditional grading system. Also, higher cumulative pre-final scores (i.e., scores on the weekly quizzes as well as the midterm) did not predict lower, "just passing" achievement on final examinations. The students' responses to the surveys included comments that pass/fail grading eased anxiety and reduced competition while encouraging the students' co-operation. CONCLUSION: Despite concerns that implementing pass/fail grading for all first-year courses would result in lower overall performance and decreased motivation among students, during the first year of implementation these fears proved to be unfounded as the students continued to perform well and reported greater satisfaction with the new system.

Attitude↗

PubMed1995

In vivo regulation of replicative Legionella pneumophila lung infection by endogenous tumor necrosis factor alpha and nitric oxide.

The in vivo role of endogenous tumor necrosis factor alpha (TNF-alpha) and reactive nitrogen intermediates (RNIs) in modulation of growth of Legionella pneumophila in the lung was assessed using a murine model of replicative L. pneumophila lung infection. Intratracheal inoculation of mice with L. pneumophila resulted in induction of endogenous TNF-alpha, which preceded clearance of L. pneumophila from the lung. Inhibition of endogenous TNF-alpha activity, via in vivo administration of TNF-alpha neutralizing antibody, or inhibition of endogenous RNIs, via administration of the nitric oxide (NO) synthetase inhibitor N-monomethyl-L-arginine (NMMA), resulted in enhanced growth of L. pneumophila in the lung at > or = 3 days postinfection (when compared with untreated L. pneumophila-infected mice). Because of the similar kinetics of enhanced pulmonary growth of L. pneumophila in mice treated in vivo with either anti-TNF-alpha antibody or NMMA, the immunomodulatory effect of NO on endogenous TNF-alpha activity in the lung was assessed. Administration of NMMA to L. pneumophila-infected mice resulted in a significant decrease in endogenous TNF-alpha activity in the lung during replicative L. pneumophila infections in vivo. However, administration of exogenous TNF-alpha to NMMA-treated mice failed to significantly enhance clearance of L. pneumophila from the lung. Results of these studies indicate that both endogenous NO and TNF-alpha facilitate resolution of replicative L. pneumophila lung infections and that regulation of L. pneumophila replication by TNF-alpha is mediated, at least in part, by NO.

Animals↗

PubMed1995

Regulation of monocyte chemoattractant protein-1 gene expression and secretion in rat pulmonary alveolar macrophages by lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-1 beta.

Chemotactic cytokines coordinate the recruitment of leukocytes into the lung during pulmonary inflammation. In a previous study, we determined that rat pulmonary alveolar macrophages (PAMs) facilitate monocyte recruitment and activation in the lung during acute inflammatory lung injury, in part, through the inducible expression of monocyte chemoattractant protein-1 (MCP-1). MCP-1 is an 11 to 15 kD basic peptide that specifically mediates monocyte chemotaxis and activation. Inflammatory mediators that regulate the expression and secretion of MCP-1 by rat PAMs have not been identified. We determined that stimulation of resident rat PAMs with bacterial lipopolysaccharide (LPS), murine tumor necrosis factor-alpha, or human interleukin-1 beta resulted in the inducible expression of MCP-1 mRNA and the secretion of biologically active MCP-1. In contrast, phorbol myristate acetate, a nonphysiologic leukocyte activator, was significantly less effective in stimulating either enhanced MCP-1 mRNA expression or secretion of MCP-1. These results indicate that the expression of MCP-1 mRNA and the secretion of MCP-1 by rat PAMs are regulated by bacterial products (LPS) and inflammatory cytokines. Further, these results suggest PAMs are regulated by bacterial products (LPS) and inflammatory cytokines. Further, these results suggest that resident PAMs, through elaboration of MCP-1, may play a pivotal role in regulating recruitment and activation of monocytes in the lung during acute inflammatory lung injury.

Alkaloids↗

PubMed1993

Expression of monocyte chemoattractant protein-1 (MCP-1) by rat alveolar macrophages during chronic lung injury.

Using a well-characterized model of bleomycin-induced pulmonary fibrosis in the rat, we determined that there was a time-dependent elaboration of monocyte chemotactic activity in bronchoalveolar lavage fluid. Northern hybridization analysis revealed markedly increased expression of rat monocyte chemoattractant protein-1 (MCP-1) mRNA in alveolar macrophages (AMs) from rats following induction of pulmonary fibrosis. Monocyte chemotactic activity was also significantly increased in conditioned media from AMs retrieved from injured rat lungs. These data suggest that one important role of AMs in the pathogenesis of chronic inflammatory lung injury and pulmonary fibrosis is the regulation of monocyte recruitment and activation within the lung secondary to secretion of monocyte chemoattractants including MCP-1.

Animals↗

PubMed1992

Membrane association of active 5-lipoxygenase in resting cells. Evidence for novel regulation of the enzyme in the rat alveolar macrophage.

The enzyme 5-lipoxygenase (5-LO) catalyzes the first two steps in the metabolism of arachidonic acid to leukotrienes, substances which play pivotal roles both in normal host defense and in pathologic states of inflammation. Recent studies in granulocytic cells have shown that activation of 5-LO involves its Ca(2+)-dependent translocation from cytosol to membrane compartments. However, little information exists about the molecular regulation of 5-LO in macrophages, even though these cells comprise the resident effector cell population of most organs. We therefore examined the levels of 5-LO activity and immunoreactive protein in cytosol and membrane fractions of resident rat alveolar (AM) and peritoneal macrophages (PM) and compared them with the well studied human neutrophil (polymorphonuclear leukocyte). In the resting state, PM resembled polymorphonuclear leukocyte in that most of their cell-free 5-LO activity, as well as protein content, were localized to the cytosol fraction. By contrast, resting AM contained most of their activity and almost half of their immunoreactive protein in the crude membrane fraction. The inability of the drug MK-886 to reverse this membrane association suggested that the 5-LO-activating protein was not the site of binding in the resting cell; however, this drug completely inhibited leukotriene B4 synthesis in ionophore A23187-stimulated AM, indicating that an interaction between 5-LO and 5-LO-activating protein was nonetheless required for product synthesis upon stimulation. Translocation of cytosolic 5-LO protein could not be convincingly demonstrated in A23187-stimulated AM, suggesting that the pool of 5-LO enzyme responsible for product formation originated in the membrane rather than the cytosol fraction of the resting cell. The AM therefore represents the first mammalian cell in which 5-LO has been recovered from the membrane fraction (a) of a resting cell and (b) in active form. These novel findings extend our understanding of the molecular regulation of 5-LO and may be of importance in designing strategies to limit inflammation in the lung and other sites.

Animals↗

PubMed1992

Transferrin: a potential source of iron for oxygen free radical-mediated endothelial cell injury.

The ability of transferrin to potentiate oxygen free radical-mediated endothelial cell injury was assessed. 51Cr-labeled endothelial cells derived from rat pulmonary arteries (RPAECs) were incubated with hydrogen peroxide (H2O2) in the presence and absence of holosaturated human transferrin, and the effect of transferrin on H2O2-mediated endothelial cell toxicity was determined. Addition of holosaturated transferrin potentiated H2O2-mediated RPAEC cytotoxicity at concentrations of H2O2 greater than 10 microM, suggesting that transferrin may provide a source of iron for free radical-mediated endothelial cell injury. Free radical-mediated injury is dependent on non-protein-bound iron. The ability of RPAECs to facilitate the release of iron from transferrin was assessed. We determined that RPAECs facilitate the release of transferrin-derived iron by reduction of transferrin-bound ferric iron (Fe3+) to ferrous iron (Fe2+). The reduction and release of transferrin-derived Fe2+ were inhibited by apotransferrin and chloroquine, indicating a dependence on receptor-specific binding of transferrin to the RPAEC cell surface, with subsequent endocytosis, acidification, and reduction of transferrin-bound Fe3+ to Fe2+. The release of transferrin-derived Fe2+ was potentiated by diethyldithiocarbamate, an inhibitor of intracellular superoxide dismutase (SOD). In contrast, exogenous SOD did not alter iron release, suggesting that intracellular superoxide anion (O2-) may play an important role in mediating the reduction and release of transferrin-derived iron. Results of this study suggest that transferrin may provide a source of iron for oxygen free radical-mediated endothelial cell injury and identify a novel mechanism by which endothelial cells may mediate the reduction and release of transferrin-derived iron.

Animals↗

PubMed1992

Myocardial glutathione depletion impairs recovery of isolated blood-perfused hearts after global ischaemia.

This study was performed to determine whether depletion of myocardial glutathione would impair recovery of left ventricular function of blood-perfused, isolated hearts after reversible ischaemic injury. Cats were treated with either vehicle or buthionine sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in the synthesis of glutathione. The feline isolated hearts were perfused with the blood of normal donor cats before and after 40 min of global myocardial ischaemia. The myocardial concentration of glutathione of the BSO group, 178 +/- 38 ng/mg tissue, was significantly less than that of the control group, 292 +/- 38 ng/mg tissue (P < 0.05). The peak left ventricular developed pressure (LVDP) 1 h after reperfusion, expressed as a fraction of the peak LVDP before ischaemia, was 0.87 +/- 0.10 for the control group and 0.64 +/- 0.08 for the BSO group (P = 0.05 vs. control). The peak left ventricular dP/dt after reperfusion, expressed as a fraction of the peak dP/dt before ischaemia, was 1.08 +/- 0.14 for the control group and 0.78 +/- 0.09 for the BSO group (P = 0.05 vs. control). The myocardial creatine kinase activity of the BSO group, 1046 +/- 46 U/g tissue, was not significantly different from that of the control group, 1038 +/- 17 U/g tissue (P = 0.87). Thus, depletion of myocardial glutathione resulted in impaired post-ischaemic contractile function that cannot be attributed to a greater extent of irreversible cell injury.

Animals↗

PubMed1992

Effect of acute inflammatory lung injury on the expression of monocyte chemoattractant protein-1 (MCP-1) in rat pulmonary alveolar macrophages.

Using a well-characterized rat model of immune complex-mediated acute inflammatory lung injury, we determined that there is a time-dependent elaboration of monocyte chemotactic activity in bronchoalveolar lavage fluid. Monocyte chemotactic activity is also significantly enhanced in culture supernatants from pulmonary alveolar macrophages (PAMs) from injured rat lungs. Northern hybridization analysis revealed markedly increased expression of rat monocyte chemoattractant protein 1 (MCP-1) mRNA in PAMs obtained from rats with immune complex-induced lung injury. The increased expression of MCP-1 mRNA and associated increase in monocyte chemotactic activity present in culture supernatants of PAMs from injured rat lungs suggest that PAMs may participate in the pathogenesis of acute inflammatory lung injury by the secretion of monocyte chemoattractants including MCP-1.

Animals↗

PubMed1992

Oxygen radical scavengers selectively inhibit interleukin 8 production in human whole blood.

The hydroxyl radical (OH.) scavenger dimethyl sulfoxide (DMSO) was found to dose-dependently inhibit interleukin 8 (IL-8) production in LPS-stimulated human whole blood. At a concentration of 1% (vol/vol), DMSO blocked IL-8 release by approximately 90% in the presence of 1 microgram/ml LPS at a 24-h time point, but did not affect cell viability or reduce the production of tumor necrosis factor (TNF), interleukin 6, or interleukin-1 beta (IL-1 beta). DMSO was found to directly inhibit IL-8 expression at the level of transcription. Furthermore, this effect was not LPS-specific, in that IL-8 production was reduced by DMSO to a similar extent upon stimulation of blood with phytohemagglutinin, aggregated immune complexes, TNF, or IL-1 beta. Other oxygen radical scavengers that have been shown to inhibit OH.-dependent reactions (dimethyl thiourea, thiourea, mannitol, and ethanol) also inhibited IL-8 production. Conversely, addition of H2O2 caused a dose-dependent stimulation of IL-8 release. These results provide evidence that reactive oxygen metabolites play an important role in the regulation of IL-8 production and suggest that reduction of IL-8 release may contribute to the beneficial effects of antioxidants in experimental models of inflammation and ischemia/reperfusion injury.

Blood↗

PubMed1992

Insoluble immune complex-stimulated neutrophil leukotriene B4 production is dependent on Fc gamma RII and Fc gamma RIII and independent of pertussis toxin-sensitive signal transduction pathways.

Leukotriene B4 (LTB4) release initiated by interaction of immune complexes (ICs) with Fc gamma RII and Fc gamma RIII receptors on human neutrophils was studied using well-defined complexes. Immune complexes consisting of polyclonal rabbit antibody to human albumin were prepared at equivalence (insoluble complex) and at five times antigen excess (soluble complex). Incubation of human neutrophils with soluble and insoluble ICs led to the synthesis of LTB4 from endogenous arachidonic acid (AA). LTB4 release induced by ICs was markedly inhibited by monoclonal antibodies against either Fc gamma RII or Fc gamma RIII receptor. Treatment of neutrophils with pertussis toxin significantly inhibited the release of LTB4 induced by soluble ICs. However pertussis toxin treatment minimally inhibited the LTB4 release induced by insoluble ICs. Crosslinking of either Fc gamma RII and Fc gamma RIII receptors on neutrophil surfaces induced LTB4 release. This is the first experimental observation showing that both Fc gamma RII and Fc gamma RIII directly induce neutrophil LTB4 metabolism in the absence of exogenous AA. These studies also suggest the involvement of novel pertussis toxin insensitive signal transduction pathways in insoluble ICs stimulation of neutrophils.

Antigen-Antibody Complex↗

PubMed1991

Monocyte chemotaxis and activating factor production by keratinocytes in response to IFN-gamma.

Monocytes accumulate in the epidermis and along the dermo-epidermal junction in several different inflammatory skin diseases. To determine whether human epidermal keratinocytes elaborate a specific chemotaxin responsible for the accumulation of monocytes at these anatomic sites, monocyte chemotactic activity in conditioned 16-h cultured keratinocyte supernatants were assayed using human peripheral blood monocytes as the target cell. Dilutional analysis revealed directed monocyte migration in IFN-gamma-treated (100 U/ml) keratinocyte supernatants (80% maximal FMLP response) which was 10-fold more than IFN-gamma itself or untreated keratinocyte activity alone. Gel filtration chromatography revealed that this activity eluted just ahead of a 12.5-kDa molecular mass marker. Blocking studies demonstrated that a rabbit polyclonal antibody to monocyte chemotaxis and activating factor (MCAF) inhibited all monocyte chemotaxis by greater than 80%. Keratinocytes were metabolically labeled with 35S-cysteine/methionine, and after 16 h incubation the supernatants immunoprecipitated with the same anti-MCAF antibody. MCAF was detected as a protein doublet of 12 and 9 kDa only in IFN-gamma-treated (100 U/ml) keratinocyte supernatants. Incubation with IFN-gamma and TNF-alpha (250 U/ml) in combination resulted in increased production of MCAF protein. By Northern blot analysis, MCAF mRNA was constitutively expressed in keratinocytes and upregulated only in the presence of IFN-gamma. TNF-alpha, IL-1 beta, transforming growth factor-beta and phorbol esters had no positive or negative influence on MCAF mRNA. These studies demonstrate that biologically active MCAF is elaborated by human epidermal keratinocytes upon activation by IFN-gamma, a cytokine also required for the induction of adherence between monocytes and keratinocytes. Keratinocyte-derived MCAF is likely to be important in the regulation of cutaneous monocyte trafficking and may also be responsible for the recruitment of Langerhans cells and dermal dendrocytes, which share many phenotypic features with monocytes/macrophages, to their anatomic locations in skin.

Cells, Cultured↗

PubMed1991

The anaphylatoxins C3a and C5a are vasodilators in the canine coronary vasculature in vitro and in vivo.

The effect of complement fragments on coronary blood flow in vivo and the contraction of coronary arteries in vitro was determined. In pentobarbital anesthetized dogs, intraarterial bolus injection of C3a and C5a, zymosan-activated serum and methylcholine in the coronary vascular bed caused transient and dose-dependent increases in coronary blood flow. Similar increases were obtained with 25 micrograms of C3a (104 +/- 13%, n = 5) and 0.1 microgram of methylcholine (102 +/- 4%, n = 3). Smaller increases in blood flow were elicited by 25 micrograms of C5a (41 +/- 18%, n = 4) and 0.2 ml of zymosan-activated serum (48 +/- 5%, n = 4). None of these responses were associated with significant changes in left ventricular contractile force measured with a strain gauge, arterial blood pressure, and heart rate. C3a dilated the coronary vascular bed in conscious dogs with an activity equal to or greater than that observed in anesthetized dogs. Isolated canine coronary arteries that were precontracted with serotonin relaxed in response to C3a, whether or not the endothelium was intact. Overall these data suggest that physiologically high doses of anaphylactic complement fragments vasodilate the canine coronary circulation.

Animals↗

PubMed1991

Ferrous iron release from transferrin by human neutrophil-derived superoxide anion: effect of pH and iron saturation.

The ability of superoxide anion (O2-) from stimulated human neutrophils (PMNs) to release ferrous iron (Fe2+) from transferrin was assessed. At pH 7.4, unstimulated PMNs released minimal amounts of O2- and failed to facilitate the release of Fe2+ from holosaturated transferrin. In contrast, incubation of phorbol myristate acetate (PMA)-stimulated PMNs with holosaturated transferrin at pH 7.4 enhanced the release of Fe2+ from transferrin eightfold in association with marked generation of O2-. The release of Fe2+ was inhibited by addition of superoxide dismutase (SOD), indicating that the release of Fe2+ was dependent on PMN-derived extracellular O2-. In contrast, at physiologic pH (7.4), incubation of transferrin at physiological levels of iron saturation (e.g. 32%) with unstimulated or PMA stimulated PMNs failed to facilitate the release of Fe2+. The effect of decreasing the pH on the release of Fe2+ from transferrin by PMN-derived O2- was determined. Decreasing the pH greatly facilitated the release of Fe2+ from both holosaturated transferrin and from transferrin at physiological levels of iron saturation by PMN-derived O2-. Release of Fe2+ occurred despite a decrease in the amount of extracellular O2- generated by PMNs in an acidic environment. These results suggest that transferrin at physiologic levels of iron saturation may serve as a source of Fe2+ for biological reactions in disease states where activated phagocytes are present and there is a decrease in tissue pH. The unbound iron could participate in biological reactions including promoting propagation of lipid peroxidation reactions or hydroxyl radical formation following reaction with phagocytic cell-derived hydrogen peroxide.

Humans↗

PubMed1991

Xanthine oxidase inhibition does not limit canine infarct size.

BACKGROUND: Evidence supporting the role of xanthine oxidase in myocardial reperfusion injury is based on studies with pharmacological interventions used to inhibit enzyme function. Controversy exists, however, regarding the true role of xanthine oxidase in reperfusion injury. This study was performed to determine whether xanthine oxidase inhibition limits myocardial injury due to coronary artery occlusion and reperfusion. METHODS AND RESULTS: Anesthetized dogs underwent coronary artery occlusion (90 minutes) and reperfusion (6 hours). Oxypurinol (28 mg/kg) or amflutizole (30 mg/kg), chemically unrelated inhibitors of xanthine oxidase, or vehicle was infused intravenously 15 minutes before and 3 hours after reperfusion. Regional myocardial blood flow was determined with radiolabeled microspheres. Infarct size was determined with the tetrazolium method. Myocardial infarct size (percent of risk region) was less in oxypurinol-treated dogs, 32 +/- 16%, compared with that of the control group, 46 +/- 15%. Infarct size for the amflutizole-treated dogs, 40 +/- 21%, was not significantly different from that of the control group. There were no differences in rate-pressure product or collateral blood flow to account for differences in infarct size. Uric acid concentration in the coronary venous plasma increased after reperfusion in the dogs treated with vehicle but not in the drug-treated dogs. Xanthine oxidase inhibition was demonstrated in each of the drug treatment groups, but only oxypurinol limited the extent of myocardial injury. CONCLUSIONS: Previously reported cardioprotective effects of allopurinol, noted to occur only when the drug was administered chronically, may be related to a property of oxypurinol, a major metabolite of allopurinol. The beneficial effect of oxypurinol is unrelated to inhibition of superoxide formation during xanthine oxidase-catalyzed oxidation of xanthine and hypoxanthine.

Animals↗

PubMed1991

Modulation of keratinocyte-derived interleukin-8 which is chemotactic for neutrophils and T lymphocytes.

Interactions between T lymphocytes, neutrophils, and epidermal cells are believed to play a central role in the pathophysiology of psoriasis and other inflammatory cutaneous disorders. Although there is strong evidence that lymphocyte-function-associated antigen-1 (LFA-1) positive T cells are retained in the epidermis via intercellular adhesion molecule-1 (ICAM-1) expression induced on keratinocytes, the molecular basis for the directed migration of T cells or neutrophils towards the epidermis is not known. To investigate whether epidermal keratinocyte-derived products may be important in the migration of T cells and neutrophils into the epidermis, human keratinocytes were cultured in the presence of various cytokines and chemotactic activity of the supernatants were assessed. TNF-alpha stimulation produced directed migrational responses for both neutrophils and T-lymphocytes (both CD4 and CD8), but not B lymphocytes; 69% of T-cell movement and 80% of neutrophil migration induced by the TNF-alpha treated keratinocyte cell supernatants could be inhibited by anti-interleukin-8 (IL-8) serum. Using the same antibody, IL-8 was immunoprecipitated from the supernatants of TNF-stimulated 35S-labelled keratinocytes, and a single 7-kd band product detected by SDS-PAGE. In keeping with these biological activities and protein data, Northern blot analysis of total cellular RNA extracted from keratinocyte monolayers hybridized with a 32P-labelled 1-kb cDNA to IL-8 mRNA, revealed induction of the IL-8 gene in the presence of TNF-alpha and IL-1 beta, but not IFN-gamma. The protein kinase C agonist, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a known stimulator of psoriasiform cutaneous inflammation when applied directly to murine epidermis, strongly induced keratinocyte elaboration of IL-8 mRNA. These studies demonstrate that activated human keratinocytes are capable of producing biologically active IL-8, and provide evidence that keratinocytes can play a key role in mediating the influx of T cells and neutrophils into the epidermis.

Blotting, Northern↗

PubMed1990

Soluble and insoluble immune complexes activate human neutrophil NADPH oxidase by distinct Fc gamma receptor-specific mechanisms.

Signal transduction initiated by interaction of immune complexes (IC) with Fc gamma RII and Fc gamma RIII receptors on human neutrophils was studied by investigating the capacity of well-defined complexes to stimulate O2- generation in neutrophils. IC consisting of polyclonal rabbit antibody to human albumin were prepared at equivalence (insoluble complexes) and at five times Ag excess (soluble complexes). Stimulation of human neutrophils with soluble and insoluble IC caused a dose-dependent activation of the respiratory burst and O2- generation. Incubation of neutrophils with cytochalasin B significantly enhanced O2- generation in neutrophils stimulated with soluble IC. In contrast, cytochalasin B treatment had a minimal effect on O2- generation in neutrophils stimulated with insoluble IC. Treatment of neutrophils with PGE1 or pertussis toxin (PTx) significantly inhibited O2- generation by soluble IC-stimulated neutrophils. However, neither PGE1 nor PTx treatment significantly altered O2- generation in neutrophils stimulated with insoluble complexes. Although O2- generation induced by soluble IC was significantly inhibited by mAb against both Fc gamma RII and Fc gamma RIII receptor, insoluble IC stimulation of neutrophil O2- generation was significantly diminished only by mAb against Fc gamma RIII receptor. Cross-linking of either Fc gamma RII or Fc gamma RIII receptors on neutrophil surfaces induced O2- generation, and this activation was inhibited by both PGE1 and PTx treatment. These findings indicate that soluble and insoluble ICs induce O2- production in human neutrophils through distinct mechanisms. Soluble IC induce activation of neutrophils through a PTx- and PGE1-sensitive pathway that is dependent upon both Fc gamma RII and Fc gamma RIII receptors. Although insoluble IC induce O2- production through a PTx and PGE1 insensitive pathway mediated primarily through Fc gamma RIII receptor.

Alprostadil↗

PubMed1990

Diminished protein kinase C-activated arachidonate metabolism accompanies rat macrophage differentiation in the lung.

Alveolar macrophages (AM) differ from other macrophage (m phi) populations in their profile of eicosanoids synthesized from arachidonic acid (AA)3. Little information is available regarding possible differences in the regulation of AA metabolism among various m phi populations. In our study, we compared the ability of cultured resident rat AM and peritoneal m phi (PM) to release and metabolize AA in response to exogenous activators of protein kinase C (PKC). When stimulated with PMA, prelabeled PM released free [3H]AA in a dose-dependent manner over the concentration range 1 to 100 nM. As assessed by HPLC, PMA-stimulated PM metabolized AA to a variety of predominantly cyclooxygenase products. The dose-dependent synthesis of PGE2 by unlabeled PM stimulated with PMA was confirmed using RIA. The ability of PMA to trigger AA release and metabolism in PM was a function of its capacity to activate PKC, as indicated by the following: 1) an additional activator of PKC, oleoyl acetylglycerol, also triggered PM AA metabolism, whereas phorbol didecanoate, which lacks the ability to activate PKC, did not; 2) two structurally unrelated inhibitors of PKC activation (staurosporine and sphinganine) both abrogated PMA induced AA release in PM; and 3) pretreatment for 18 h with high dose PMA (used to deplete cellular PKC), but not phorbol didecanoate, rendered PM refractory to subsequent PMA stimulation of AA release. In contrast to PM, AM cultured in identical fashion failed to release or metabolize AA in response to either PMA or oleoyl acetylglycerol. PM and AM were also compared for their ability to release extracellular superoxide anion in response to PMA; once again, PM exhibited significantly greater release than did AM. Inasmuch as this unresponsiveness to activation of PKC distinguishes AM from other m phi populations, we conclude that it is a unique consequence of m phi differentiation in the lung. Moreover, because both AA metabolism and the respiratory burst are affected, this refractoriness appears to reflect a defect at some proximal level in PKC-mediated signaling.

Alkaloids↗