Biomedical subjects

D J Loegering

Publications and source records attributed to D J Loegering.

At least 19 recordsLinked to original sources

PubMed2001

Peptide-based analysis of amino acid sequences important to the biological activity of eosinophil granule major basic protein.

Synthetic peptides corresponding to amino acid sequences in eosinophil granule major basic protein (MBP) were evaluated for cytotoxic activity toward K562 cells and for ability to stimulate basophil mediator release. Results obtained using 14 peptides spanning the 117-amino acid sequence of MBP in overlapping fashion indicated that the activities mapped to peptide sequences near the amino and carboxy termini of MBP. The activity of these regions was confirmed using two peptides corresponding to MBP residues 18-45 and 89-117. A 20-h incubation with 5 microM peptide 18-45 or peptide 89-117 caused approximately the same levels (>60%) of cytotoxicity in K562 cells as 5 microM MBP. Similarly, a 30-min incubation with peptides 18-44 and 89-117 stimulated basophil histamine release in a concentration-dependent manner over the range of 5-20 microM. The level of release stimulated by 20 microM peptide 89-117 approached that stimulated by 2 microM MBP. A 20 microM concentration of peptide 89-117 also stimulated leukotriene C4 (LTC4) production by the basophils. Neither peptide 18-45 nor peptide 89-117 was cytotoxic for basophils under the experimental conditions for histamine and LTC4 release, as determined by 51Cr release. These results indicate that two MBP peptide sequences, including one (89-117) that contains a unique carbohydrate-binding region, share the biologic activities of MBP.

Amino Acid Sequence↗

PubMed2001

Fcgamma-receptor signaling augments the LPS-stimulated increase in serum tumor necrosis factor-alpha levels.

The phagocytosis of IgG-coated erythrocytes (EIgG) has been shown to augment the bacterial lipopolysaccharide (LPS)-stimulated increase in serum tumor necrosis factor-alpha (TNF-alpha) levels. The present study evaluated the role of Fcgamma-receptor (FcgammaR) signaling and complement activation in the effect of EIgG on the TNF-alpha response to LPS. The role of FcgammaR was determined using FcR gamma-chain knockout mice that lack functional FcgammaRI and FcgammaRIII. In wild-type animals, EIgG caused a 16-fold augmentation of the serum TNF-alpha response to LPS, whereas there was no augmentation in the FcgammaR-deficient animals. Heat-damaged erythrocytes also augmented the TNF-alpha response to LPS. This effect was absent in FcgammaR-deficient animals. An IgG antibody against heated erythrocytes was detected in mouse serum. The complement activation caused by EIgG had little effect on the LPS-stimulated increase in serum TNF-alpha levels as indicated by activation of complement with cobra venom factor or IgM-coated erythrocytes as well as studies with C5-deficient mice. These results indicate that FcgammaR signaling primarily mediates the augmented serum TNF-alpha response to LPS caused by EIgG.

Animals↗

PubMed2000

Differential requirement for classic and novel PKC isoforms in respiratory burst and phagocytosis in RAW 264.7 cells.

The binding of Ab (IgG)-opsonized particles by FcgammaRs on macrophages results in phagocytosis of the particles and generation of a respiratory burst. Both IgG-stimulated phagocytosis and respiratory burst involve activation of protein kinase C (PKC). However, the specific PKC isoforms required for these responses have yet to be identified. We have studied the involvement of PKC isoforms in IgG-mediated phagocytosis and respiratory burst in the mouse macrophage-like cell line, RAW 264.7. Like primary monocyte/macrophages, their IgG-mediated phagocytosis was calcium independent and diacylglycerol sensitive, consistent with novel PKC activation. Respiratory burst in these cells was Ca2+ dependent and inhibited by staurosporine and calphostin C as well as by the classic PKC-selective inhibitors Gö 6976 and CGP 41251, suggesting that classic PKC is required. In contrast, phagocytosis was blocked by general PKC inhibitors but not by the classic PKC-specific drugs. RAW 264.7 cells expressed PKCs alpha, betaI, delta, epsilon, and zeta. Subcellular fractionation demonstrated that PKCs alpha, delta, and epsilon translocate to membranes during phagocytosis. In Ca2+-depleted cells, only novel PKCs delta and epsilon increased in membranes, and the time course of their translocation was consistent with phagosome formation. Confocal microscopy of cells transfected with green fluorescent protein-conjugated PKC alpha or epsilon confirmed that these isoforms translocated to the forming phagosome in Ca-replete cells, but only PKC epsilon colocalized with phagosomes in Ca2+-depleted cells. Taken together, these results suggest that the classic PKC alpha mediates IgG-stimulated respiratory burst in macrophages, whereas the novel PKCs delta and/or epsilon are necessary for phagocytosis.

Animals↗

PubMed2000

IgG-coated erythrocytes augment LPS-stimulated TNF-alpha secretion, TNF-alpha mRNA levels, and TNF-alpha mRNA stability in macrophages.

Previous studies have shown that IgG-coated erythrocytes (EIgG) augment the LPS-stimulated increase in serum TNF-alpha levels in animals and the LPS-stimulated secretion of TNF-alpha by isolated macrophages. The present study evaluated the mechanism for the effect of EIgG on LPS-stimulated TNF-alpha secretion in the murine macrophage cell line, RAW 264.7. Incubation of the macrophages with EIgG or IgG-coated glass beads caused a dose-dependent augmentation of LPS-stimulated TNF-alpha secretion. The addition of EIgG increased the rate of LPS-stimulated TNF-alpha protein secretion between 2 and 4 hr after LPS. Accordingly, EIgG increased the levels of TNF-alpha mRNA at 2 and 3 hr after LPS. The increase in the LPS-stimulated TNF-alpha mRNA levels caused by EIgG was associated with an increase in TNF-alpha mRNA stability. Thus, the augmentation of LPS-stimulated TNF-alpha secretion by EIgG was associated with an increase in TNF-alpha mRNA levels which at least partly resulted from an increase in the stability of TNF-alpha mRNA.

Animals↗

PubMed1999

A phagocytic challenge with IgG-coated erythrocytes depresses macrophage respiratory burst and phagocytic function by different mechanisms.

A phagocytic challenge with IgG-coated erythrocytes (EIgG) previously has been shown to cause impaired macrophage respiratory burst capacity and FcgammaR-mediated phagocytic function. Because both the respiratory burst and FcgammaR-mediated phagocytosis are dependent on the release of arachidonate (AA), we evaluated the effects of impaired AA release on the depression of macrophage function caused by a phagocytic challenge. Challenge with EIgG caused a depression of A23187-stimulated AA release that was associated with impaired phorbol myristate acetate (PMA)-stimulated H2O2 production and FcgammaR-mediated phagocytic function. In contrast, challenge with IgG-coated glass beads (BIgG) had no effect on either AA release or H2O2 production but did depress phagocytic function. Exogenous AA prevented the depression of H2O2 production but had no effect on phagocytic function. Phospholipase A2 (PLA2) activity was depressed under conditions where AA release was impaired. The depression of phagocytic function was correlated with a depression of both EIgG binding and FcgammaR expression. Thus, a phagocytic challenge with EIgG results in macrophage dysfunction by depressing PLA2 activity and depleting FcgammaR.

Animals↗

PubMed1999

Role of an oxidative stress in the macrophage dysfunction caused by erythrophagocytosis.

A phagocytic challenge with immunoglobulin G (IgG)-coated erythrocytes (EIgGs) has been shown to cause a subsequent depression of macrophage respiratory burst capacity and phagocytic function. The present study evaluated the hypothesis that this macrophage dysfunction is caused by an oxidative stress. An oxidative stress induced by ferric ammonium citrate (FAC) plus cumene hydroperoxide (CHP) caused a depression of macrophage function that was attenuated by antioxidants and iron chelators. In contrast, the same antioxidants and iron chelators did not alter changes caused by a challenge with EIgGs. EIgG challenge caused an increase in lipid peroxidation but failed to deplete glutathione (GSH) or decrease the activity of glyceraldehyde-3-phosphate dehydrogenase (GA-3-PD), suggesting that there was only a slight oxidative stress. Inhibition of the Fc gamma receptor (Fc gammaR) stimulated respiratory burst by removing calcium during the challenge did not attenuate the changes caused by an EIgG challenge. A phagocytic challenge with nonerythrocyte particles, IgG-coated beads (BIgGs), did not depress the respiratory burst capacity but did depress phagocytic function. Fc gammaR expression was depressed following a phagocytic challenge but not an oxidative stress. Thus, an oxidative stress can depress macrophage function, but the dysfunction caused by a phagocytic challenge with EIgGs involves Fc gammaR depletion and the erythrocyte contents rather than an oxidative stress.

Animals↗

PubMed1999

IgG-coated erythrocytes augment the lipopolysaccharidestimulated increase in serum tumor necrosis factor-alpha.

Previous studies have shown that the injection of IgG-coated erythrocytes (EIgG) caused an increase in the mortality rate due to bacterial lipopolysaccharide (LPS). This observation led to the present evaluation of the effect of EIgG on the LPS-stimulated increase in serum tumor necrosis factor-alpha (TNF-alpha) levels and TNF-alpha secretion by macrophages. The prior injection of EIgG augmented the increase in LPS-stimulated serum TNF-alpha levels ninefold at 1 h after LPS. Serum TNF-alpha levels were augmented when LPS was injected 2 or 6 h after EIgG but not at 0.5 or 12 h after EIgG. Complement activation caused by EIgG may contribute to the priming for TNF-alpha, because activation of complement with cobra venom factor caused a threefold augmentation of the LPS-stimulated serum TNF-alpha levels. Isolated macrophages that had ingested EIgG or were adherent to immobilized IgG showed augmented TNF-alpha secretion in response to LPS. Thus clearance of immune complexes from the blood can augment the LPS-stimulated increase in serum TNF-alpha levels that is due, in part, to complement activation and signaling via FcgammaR.

Animals↗

PubMed1998

Blood flow and glucose uptake in denervated, insulin-resistant muscles.

To investigate whether changes in blood flow contribute to the insulin resistance in denervated muscles, basal and insulin-stimulated 2-deoxy-D-glucose (2-DG) uptake in vivo and blood flow were measured in soleus (slow twitch), plantaris (fast twitch), and gastrocnemius (fast twitch) muscles at 1 and 3 days after a right hindlimb denervation in the rat. Muscles of the contralateral sham hindlimb served as an internal control. Sham plantaris and gastrocnemius muscles showed 32 and 60% lower basal 2-DG uptake, 46 and 66% lower insulin-stimulated 2-DG uptake, and 79 and 81% lower blood flow, respectively, compared with sham soleus muscle. At 1 day after denervation, soleus, plantaris, and gastrocnemius muscles exhibited an 80, 64, and 42% decrease in insulin-stimulated 2-DG uptake, respectively, in the presence of 63, 323, and 304% higher blood flow, respectively. At 3 days after denervation, soleus muscle showed a 60% decrease in basal 2-DG uptake, complete unresponsiveness to insulin, and an 86% decrease in blood flow. In contrast, the denervated plantaris and gastrocnemius muscles exhibited a 262 and 105% increase in basal 2-DG uptake, respectively, no change in insulin-stimulated 2-DG uptake, and no change in blood flow compared with corresponding contralateral sham muscles. The results demonstrate that muscle blood flow is influenced by muscle fiber population and time after denervation and that changes in blood flow do not contribute to the insulin resistance in the denervated muscles.

Animals↗

PubMed1997

Lysosomotropic agents ameliorate macrophage dysfunction following the phagocytosis of IgG-coated erythrocytes: a role for lipid peroxidation.

Phagocytosis of IgG-coated erythrocytes (EIgG) can depress several macrophage functions. Our previous studies have suggested that this macrophage dysfunction may be due to an oxidative stress caused by the interaction of hemoglobin-derived iron with superoxide and/or hydrogen peroxide. Since lysosomotropic agents are capable of altering iron handling by macrophages, the present study evaluated the ability of these agents to prevent the macrophage dysfunction and lipid peroxidation caused by a phagocytic challenge with EIgG. Elicited rat peritoneal macrophages showed a depression of PMA-stimulated hydrogen peroxide production, calcium ionophore-stimulated arachidonate release and Fc receptor-mediated phagocytosis. The lysosomotropic agents; chloroquine, quinacrine, ammonium chloride and methylamine all prevented the depression of hydrogen peroxide production and arachidonate release but did not alter the depression of phagocytic function. These agents also prevented the increase in lipid peroxidation products caused by a phagocytic challenge with EIgG. These results suggest that the ability of lysosomotropic agents to prevent some aspects of macrophage dysfunction after a phagocytic challenge may be due to their ability to block the oxidative stress caused by the challenge.

Ammonium Chloride↗

PubMed1996

Macrophage dysfunction following the phagocytosis of IgG-coated erythrocytes: production of lipid peroxidation products.

The phagocytosis of erythrocytes may contribute to the increased susceptibility to life-threatening infections in patients with burn injury, sickle cell anemia, and malaria. The phagocytosis of immunoglobulin G-coated erythrocytes (EIgG) is followed by a transient depression of several macrophage functions including phagocytosis, respiratory burst capacity, and killing of bacteria. The present study suggests the possibility that after erythrophagocytosis hemoglobin-derived iron conspires with reactive oxygen products of the macrophage respiratory burst to cause oxidant damage to the phagocyte. Challenge of elicited peritoneal macrophages with EIgG phagocytosis was followed by an increase in lipid peroxidation as assessed by thiobarbituric acid-reactive substances (TBARS). Doses of EIgG associated with increased TBARS also caused a depression of Fc receptor-mediated phagocytosis and phorbol myristate acetate (PMA)-stimulated hydrogen peroxide production. Time course experiments demonstrated that the increase in TBARS coincided with the depression of macrophage function. There was no increase in TBARS following the phagocytosis of IgG-coated erythrocyte ghosts, suggesting that hemoglobin iron is involved in the generation of TBARS. The phagocytosis of erythrocyte ghosts did not depress macrophage function. Since complement receptor-mediated phagocytosis does not stimulate the respiratory burst, the role of the respiratory burst in causing lipid peroxidation was assessed using the phagocytosis of complement-coated erythrocytes. Phagocytic challenge with complement-coated erythrocytes caused neither an increase in TBARS nor a depression of macrophage function. However, there was an increase in TBARS when the respiratory burst was stimulated with PMA following complement receptor-mediated phagocytosis of erythrocytes. These results suggest that hemoglobin iron and phagocyte-generated oxidants collaborate to cause the depression of macrophage function following EIgG phagocytosis.

Animals↗

PubMed1995

Diethyldithiocarbamate ameliorates the effect of lipopolysaccharide on both increased nitrite production by vascular smooth muscle cells and decreased contractile response of aortic rings.

The depression of vasoconstrictor responsiveness caused by bacterial lipopolysaccharide (LPS) is mediated, in part, by the induction of nitric oxide synthase (NOS) and the resultant increase in nitric oxide production by vascular smooth muscle. The present study evaluated the ability of the antioxidant, diethyldithiocarbamate (DDTC), to attenuate the LPS-stimulated induction of NOS in cultured vascular smooth muscle cells (VSMC) and the depression of in vitro vascular reactivity caused by LPS administration to rats. The LPS-stimulated increase in nitrite production by cultured VSMC was inhibited 85% by DDTC (100 microM). When VSMC were stimulated with a combination of LPS, interferon-gamma (INF) and tumor necrosis factor (TNF) nitrite production was 5-fold greater than with LPS alone. DDTC inhibited 49% of the increase caused by LPS plus INF and TNF. Aortic rings taken from animals injected with LPS showed a depression of maximum force in response to phenylephrine which was reversed by inhibition of NOS activity. Pretreatment of animals with DDTC attenuated this depression of vascular reactivity. The DDTC treatment did not reduce the increase in serum TNF levels caused by LPS. These results suggest that DDTC can attenuate the LPS-stimulated induction of NOS in vascular smooth muscle and may thereby ameliorate the impairment of vascular reactivity.

Amino Acid Oxidoreductases↗

PubMed1995

The antioxidant, U74389, ameliorates the depression of vascular reactivity caused by lipopolysaccharide.

There is growing evidence that an oxidant stress contributes to the deleterious effects of bacterial lipopolysaccharide (LPS). The present study evaluated the ability of the antioxidant, U74389, to prevent the depression of vascular reactivity caused by LPS. Aortic rings taken from rats given LPS showed a depression of maximum force in response to phenylephrine that was reversed by an inhibitor of nitric oxide synthase. Pretreatment of animals with U74389 attenuated this depression of vascular reactivity. U74389 did not limit the increase in serum tumor necrosis factor levels caused by LPS. These results show that U74389 can ameliorate the depression of vascular reactivity caused by LPS possibly by interfering with the induction of nitric oxide synthase.

Animals↗

PubMed1994

Effect of IgG-coated erythrocytes and lipopolysaccharide (LPS) tolerance on the depression of vascular reactivity caused by LPS.

Previous studies have shown that the injection of IgG-coated erythrocytes (EIgG) increased the mortality rate caused by bacterial lipopolysaccharide (LPS) and that animals made tolerant to LPS show a decrease in the mortality rate caused by LPS. The present study determined the effect of the injection of EIgG and of LPS tolerance on the depression of vascular reactivity caused by LPS in vivo or in vitro. For in vivo studies, LPS was injected intravenously into rats 2 h after the injection of E or EIgG. Aortae were removed 90 min after the injection of LPS, and cumulative concentration-response curves to phenylephrine were performed in helically cut aortic strips. LPS (.1 mg/kg) caused a 21% decrease in the maximum tension developed in response to phenylephrine in aortae from animals given erythrocytes. In contrast, animals given EIgG showed a 63% decrease in maximum tension following the injection of this dose of LPS. For in vitro studies, the depression of vascular reactivity caused by incubation with LPS of aortae from rats injected with EIgG was determined. As with the in vivo studies, there was a greater depression of vascular reactivity caused by incubation with LPS of aortae taken from animals injected with EIgG. Similar studies were carried out with rats made tolerant to LPS. Tolerance was induced by giving a regimen of five daily injections of LPS. Following the injection of LPS (1 mg/kg), the maximum tension of aortae from sham tolerant animals was depressed 63%, while aortae from LPS tolerant animals were depressed only 16%.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗

PubMed1993

Depression of macrophage respiratory burst capacity and arachidonic acid release after Fc receptor-mediated phagocytosis.

The phagocytosis of IgG-coated erythrocytes (ElgG) by macrophages results in a subsequent depression of macrophage phagocytic function, respiratory burst capacity, and bactericidal activity. Our study was carried out to determine the importance of impaired arachidonic acid release in the depression of the respiratory burst after ElgG phagocytosis. The depression of triggered H2O2 production after ElgG phagocytosis was not due to cyclooxygenase products because indomethacin or aspirin did not modify the depression. Further studies revealed that the depression of triggered H2O2 production after ElgG phagocytosis was associated with a depression in the ability of macrophages to release arachidonic acid in response to PMA, zymosan, or calcium ionophore. The addition of exogenous arachidonic acid partially prevented the depression of triggered H2O2 production after ElgG phagocytosis. Unlike phagocytosis mediated by FcR, complement receptor-mediated phagocytosis did not alter H2O2 production or arachidonic acid release. Ligation of FcR was not sufficient to depress triggered H2O2 production and arachidonic acid release because these functions were not depressed when phagocytosis was inhibited with cytochalasin D. Thus, it was found that the depression of triggered H2O2 production by macrophages after FcR-mediated phagocytosis was associated with impaired release of arachidonic acid and that H2O2 production could be partially restored by the addition of arachidonic acid. These results suggest that the impairment of arachidonic acid release after FcR-mediated phagocytosis contributes to the depression of macrophage respiratory burst capacity after FcR-mediated phagocytosis.

Animals↗

PubMed1992

Respiratory burst capacity of activated macrophages is resistant to depression by erythrocyte phagocytosis.

The present study evaluated whether macrophage activation would reduce the depression in the capacity of macrophages to produce H2O2 following EIgG phagocytosis. Macrophage activation was accomplished by exposing inflammatory rat peritoneal macrophages to 10 units of IFN gamma for 72 h. IFN gamma treatment caused a four to fivefold increase in phorbol myristate acetate (PMA)-triggered H2O2 production, but Fc receptor phagocytic function was unaltered. IFN gamma-activated macrophages were able to phagocytize a greater number of EIgG before a decrease in PMA-triggered H2O2 production was observed and the level of H2O2 production did not fall below that of untreated-inflammatory macrophages that had not received an EIgG phagocytic challenge. The depression in Fc receptor phagocytic function was unaltered with macrophage activation. These results indicate that activated macrophages are resistant to the depression of respiratory burst capacity caused by erythrocyte phagocytosis and suggests that IFN gamma treatment may be effective in preventing the impairment of host defense against bacterial infection that is associated with erythrocyte phagocytosis.

Animals↗

PubMed1992

Corynebacterium parvum can reverse the depression of macrophage hydrogen peroxide production caused by erythrocyte phagocytosis.

Our previous studies have shown that the phagocytosis of IgG-coated erythrocytes (EIgG) in vivo increases the mortality rate with bacterial infection, and EIgG phagocytosis in vitro depresses phorbol myristate acetate (PMA)-triggered H2O2 production. The present study was undertaken to determine if the depression of H2O2 production caused by EIgG phagocytosis could be reversed by exposing macrophages to priming agents. Macrophages exposed to 100 micrograms/ml of C. parvum, it's pyridine-soluble extract (PE), or the pyridine extract residue (PER) for 1 hr showed an enhanced production of H2O2 in response to PMA triggering. The priming effect of C. parvum, PE, and PER lasted for 3-6 hr. 18 hr after exposure to C parvum or PER, PMA-triggered H2O2 production was depressed, however PE did not have this effect. The priming effect of C parvum was not prevented by cycloheximide. EIgG phagocytosis caused a dose dependent depression of PMA-triggered H2O2 production. When macrophages were exposed to C. parvum, PE, or PER following EIgG phagocytosis, the priming of PMA-triggered H2O2 production was reduced but H2O2 production was maintained at levels equal to or greater than that of control macrophages. These results show that phagocytosis did not prevent the action of priming agents on macrophage respiratory burst capacity, and suggests that such agents may preserve macrophage bactericidal function following phagocytosis.

Animals↗

PubMed1991

Macrophage hydrogen peroxide production and phagocytic function are decreased following phagocytosis mediated by Fc receptors but not complement receptors.

Previous in vivo and in vitro studies have shown that the phagocytosis of IgG-coated erythrocytes results in a depression of macrophage function. The present study compared the effect of phagocytosis mediated by Fc receptors with that mediated by complement receptors. The phagocytosis of IgG-coated erythrocytes by elicited peritoneal macrophages depressed their capacity to produce hydrogen peroxide as well as phagocytic function. Phagocytosis of erythrocytes coated with IgM and complement had neither of these effects. These results implicate the intracellular signaling that results from Fc receptor mediated phagocytosis in the depression of macrophage function that is caused by phagocytosis.

Animals↗

PubMed1991

Scavengers of reactive oxygen intermediates do not mediate the depression of macrophage hydrogen peroxide production caused by erythrocyte phagocytosis.

Our previous studies have shown that a phagocytic challenge with IgG-coated erythrocytes (EIgG) depressed macrophage triggered H2O2 production in vitro, and in vivo there was a decrease in the survival rate following bacteremia. The phagocytosis of an equal number of IgG-coated erythrocyte ghosts had none of these effects, indicating that the contents of the erythrocytes are important for these effects. The present study evaluated the role of the scavengers of reactive oxygen intermediates within erythrocytes in the depression of H2O2 production triggered with phorbol myristate acetate following a phagocytic challenge with EIgG. Elicited rat peritoneal macrophages (PM) were challenged with EIgG prepared from normal E or E with inactivated catalase, depleted glutathione, hemoglobin converted to methemoglobin, or fixed with formaldehyde. The depression of triggered H2O2 production was similar when equal numbers of normal EIgG and EIgG with inactivated scavengers were phagocytized. When the phagocytic challenge with normal EIgG was carried out in the presence of cytochalasin B, no depression of triggered H2O2 production was observed. Cytochalasin B partially blocked the phagocytosis of EIgG, so that with larger doses of EIgG there was sufficient ingestion of EIgG to depress H2O2 production in untreated PM. These results indicate that the scavengers of reactive oxygen intermediates present in erythrocytes are neither required nor sufficient to depress H2O2 production by macrophages.

Animals↗