A highly conserved intraspecies homolog of the Saccharomyces cerevisiae elongation factor-3 encoded by the HEF3 gene.

A paralog (intraspecies homolog) of the Saccharomyces cerevisiae YEF3 gene, encoding elongation factor-3, has been sequenced in the course of the yeast genome project, and identified by database searching; this gene has been designated HEF3. Bioinformatic and Northern blot analysis indicate that the HEF3 gene is not expressed during vegetative growth. Deletion of the HEF3 gene reveals no growth defects, nor any defects in mating or sporulation. A high copy 2 mu clone of HEF3 was constructed, and was shown to be unable to complement a null allele of yef3. Finally, an in vitro assay for ribosome-stimulated ATPase activity was performed with isogenic HEF3 and delta hef3 strains; no difference in biochemical activity could be detected in these strains. From these results, we conclude that the HEF3 gene does not encode a functional homolog of YEF3.

BMS-182123, a fungal metabolite that inhibits the production of TNF-alpha by macrophages and monocytes.

A fungal metabolite, BMS-182123, which inhibited bacterial endotoxin-induced production of tumor necrosis factor (TNF-alpha) in murine macrophages and human peripheral blood monocytes (in vitro), was isolated from the culture broth of Penicillium chrysogenum strain V39673. The effective BMS-182123 concentration (IC50) resulting in 50% inhibition of lipopolysaccharide-induced TNF-alpha production in murine macrophages and human monocytes was 600 ng/ml and 4.0 microgram/ml, respectively. BMS-182123 suppressed the lipopolysaccharide-induced TNF-alpha promoter activity and did not affect the stability of posttranscriptional mRNA. Addition of hydrophobic resin, Amberlite XAD-8 (1%), to the fermentation enhanced the production of BMS-182123 by 5.5 fold. A total of 577 mg pure BMS-182123 was recovered from a 250-liter fermentation supplemented with 1% Amberlite XAD-8.

Immune complexes of LDL induce atherogenic responses in human monocytic cells.

The ability of immune complexes of LDL or acetylated LDL (acLDL), together with antibodies to LDL, to induce a proatherogenic phenotype in human monocytic cells has been explored. Treatment of THP-1 monocytic cells or peripheral human monocytes with LDL immune complexes containing intact anti-LDL markedly enhanced the ability of these cells to subsequently bind and take up LDL, whereas aggregated LDL or LDL immune complexes prepared with F(ab')2 fragments of anti-LDL had no significant effect. Activation of THP-1 cells with intact LDL immune complexes also stimulated mRNA expression for the scavenger receptor. Additionally, activation of THP-1 cells with insoluble immune complexes of LDL or LDL stimulated generation of reactive oxygen intermediates that, in turn, could oxidize exogenous LDL. These results indicate that the binding of lipoprotein immune complexes to Fc receptors on monocytic cells activates a series of responses that could accelerate the initiation or progression of atherosclerosis.

Prostacyclin agonists reduce early atherosclerosis in hyperlipidemic hamsters. Octimibate and BMY 42393 suppress monocyte chemotaxis, macrophage cholesteryl ester accumulation, scavenger receptor activity, and tumor necrosis factor production.

We determined the effects of two prostacyclin agonists (octimibate and BMY 42393) on the progression of the fatty streak in vivo and on macrophage function in vitro. Hamsters were fed chow plus 0.05% cholesterol and 10% coconut oil. Control hamsters were compared with animals receiving either octimibate (10 or 30 mg/kg per day) or BMY 42393 (30 mg/kg per day). After 10 weeks of treatment, octimibate decreased plasma total cholesterol and triglycerides by 43% and 32%, respectively. Neither agonist affected blood pressure or heart rate. Lesion-prone aortic arches were stained with hematoxylin and oil red O and examined en face. Compared with controls, octimibate and BMY 42393 on average decreased mononuclear cells attached to the luminal surface by 44% and reduced subendothelial macrophage-foam cell number by 56%, foam cell size by 38%, and fatty streak area by 63%. Since octimibate is a putative inhibitor of acyl coenzyme A cholesterol acyltransferase, we studied the effect of both agents on cholesteryl ester metabolism in murine macrophages. At 10 microM, octimibate and BMY 42393 decreased cholesteryl ester accumulation in macrophages by 90% and 41%, respectively. Octimibate inhibited cholesteryl ester synthesis by 96% and increased the rate of cholesteryl ester degradation by 52%. Both prostacyclin agonists reduced macrophage scavenger receptor-mediated uptake of acetylated low density lipoprotein by 24-66% and increased cyclic adenosine monophosphate levels. Octimibate and BMY 42393 inhibited the secretion of tumor necrosis factor by 80-88% when macrophages were activated with lipopolysaccharide. At 10 microM, both agents decreased human monocyte chemotaxis to N-formyl-methionyl-leucyl-phenylalanine by 64-79%. The in vitro results with octimibate and BMY 42393 are consistent with the low number of small foam cells quantified in vivo. We suggest that octimibate and BMY 42393 suppress monocyte-macrophage atherogenic activity and cytokine production and thus inhibit the development of early atherosclerosis.

Macrophage-derived factors increase low density lipoprotein uptake and receptor number in cultured human liver cells.

Recent evidence suggests the possibility that macrophages can influence lipoprotein metabolism. Therefore we investigated the ability of cultured macrophages to alter low density lipoprotein (LDL) uptake in a human liver cell line (HepG2). Conditioned media from phlogogenic-induced mouse peritoneal macrophages or from a human macrophage cell line stimulated with endotoxin increased HepG2 LDL uptake by as much as 60-70%. The increase was due, in part, to a significant macrophage-induced 40% increase in the number of LDL receptors per cell. Although macrophage conditioned media inhibited HepG2 cholesterol synthesis, the LDL receptor up-regulation did not appear to be due to the effects on cholesterol synthesis. The LDL receptor stimulatory activity was sensitive to proteolysis and heat. Its molecular mass was approximately 20 kDa based on gel filtration. Several macrophage secretory proteins were tested in HepG2 cultures for LDL uptake stimulation. Of these, oncostatin M (approximately 18 kDa by gel filtration) gave the strongest response. The rank order for LDL uptake stimulation was oncostatin M much greater than interleukin 6 = interleukin 1 = transforming growth factor-beta 1. A neutralizing antibody directed against oncostatin M inhibited the ability of conditioned media to up-regulate LDL receptors by 85%. Thus, our results indicate that macrophages can secrete several proteins that up-regulate LDL receptors in HepG2 cells and that most of the up-regulatory activity in macrophage conditioned media appears to be due to oncostatin M.

Lipopolysaccharide (LPS) alters phosphatidylcholine metabolism in elicited peritoneal macrophages.

We investigated the effects of LPS on mouse peritoneal macrophage phospholipids using radiolabeled precursors. LPS (200 ng/ml) stimulated incorporation of [32P] into all classes of phospholipids within 0.5 hr, and after 2 hr the increase was 60% greater than controls. Separation of the phospholipid classes by thin-layer chromatography revealed a selective increase in incorporation of label into phosphatidylcholine (PC) (90% increase compared to approximately 50% in the other phospholipids). In macrophages labeled with [3H]-choline, LPS stimulated both the incorporation of label into PC and the release of incorporated label into the medium. The time dependencies of stimulated [3H] release and [32P] incorporation were similar. These data are consistent with the hypothesis that LPS activates macrophages via a PC-specific phospholipase-dependent mechanism.

Protection of mice against Listeria monocytogenes infection by recombinant human tumor necrosis factor alpha.

Recombinant human tumor necrosis factor alpha (rHuTNF-alpha) administered intravenously to mice resulted in enhanced resistance to a lethal challenge infection of Listeria monocytogenes given 24 h later. The observed protection was lost following treatment of the rHuTNF-alpha preparations with rabbit polyclonal antibody rHuTNF-alpha but not with normal rabbit immunoglobulin G.

Immunotoxicity of organophosphorus compounds. Modulation of cell-mediated immune responses by inhibition of monocyte accessory functions.

Several organophosphorus compounds (OP) used commercially as flame retardants and plasticizers and related chemicals were evaluated for their effects on human in vitro cell-mediated immune responses. At nontoxic concentrations ranging from 0.1 to 20 microM, two of the tested compounds, triphenylphosphine oxide (TPPO) and tetra-o-cresylpiperazinyl diphosphoamidate (TCPD) caused significant suppression of antigen-specific lymphocyte proliferation (P less than 0.01). Mitogenesis was less sensitive to OP treatment and was affected only by TCPD. When monocytes and lymphocytes were treated separately with OP, washed, and recombined, it appeared that these OP mediated their suppressive effects by interfering with a monocyte function rather than acting directly on lymphocytes. Further, triphenyl phosphate (TPP), triphenyl thiophosphate (TPTP) as well as TPPO and TCPD were tested for direct inhibition of monocyte antigen presentation, and all four compounds were found to cause significant inhibition at concentrations as low as 1 microM (P less than 0.001).

Erythromycin-induced suppression of pulmonary antibacterial defenses. A potential mechanism of superinfection in the lung.

Erythromycin is a broad-spectrum antibiotic commonly used in patients with respiratory infections. Certain of these patients become colonized with new microorganisms and develop superinfections. Antibiotics have a number of effects other than simply killing or inhibiting the growth of bacteria and may have direct effects upon host cells, including phagocytes. In vitro and in vivo studies have demonstrated that erythromycin decreases polymorphonuclear leukocyte (PMN) directed migration. To test the hypothesis that erythromycin inhibits normal PMN migration into the alveoli in response to a bacterial challenge, mice were challenged by aerosol inhalation with Proteus mirabilis or Staphylococcus aureus and injected intravenously with erythromycin (50 or 100 mg/kg). Pulmonary bactericidal activity and total lavaged lung cell and differential counts were determined 4 h after bacterial challenge. In control mice, only 24 +/- 2% of the initial inoculum of P. mirabilis was viable at 4 h. At a dose of 100 mg/kg, lung defenses after erythromycin were ablated, allowing the proliferation of P. mirabilis to 113 +/- 5% of the initial inoculum. The number of PMN obtained by lavage after P. mirabilis challenge was also inhibited by erythromycin in a dose-dependent manner. In untreated animals, 5.0 +/- 0.2 x 10(6) PMN were recovered as compared with 3.1 +/- 0.4 x 10(6) and 1.1 +/- 0.3 x 10(6) with increasing doses of erythromycin. Intrapulmonary bactericidal activity against S. aureus was not impaired by erythromycin.(ABSTRACT TRUNCATED AT 250 WORDS)

Alveolitis induced by influenza virus.

Previous studies of influenza virus infections have focused on the acute pathologic manifestations associated with the virus pneumonia; however, there is evidence suggestive of persistent pathologic processes with possible long-term consequences. Herein we have examined the long-term outcome of virus pneumonia in mice infected by aerosol inhalation of a sublethal dose of influenza A/PR8/34 virus. At 3, 5, 7, 9, 15, 30, 60, 90, 120 days, and a year thereafter, the lavageable lung cell populations and differential counts were quantitated. Consistent with previous studies we demonstrated an inflammatory cellular response during the acute phase of the infection. However, this inflammatory response did not completely resolve, the pulmonary leukocytosis remaining stable from Day 30 through a year after virus infection. For example, on Day 30, virus-infected lungs yielded 12.4 +/- 0.9 X 10(5) cells per lavage of which 15 +/- 3% were polymorphonuclear leukocytes, 18 +/- 4% were lymphocytes, and 67 +/- 5% were alveolar macrophages. In contrast, 7.2 +/- 0.5 X 10(5) cells per lavage were obtained from uninfected lungs of which more than 98% were alveolar macrophages. Histopathologic examination of virus-infected lungs showed an ongoing inflammatory response resulting in patchy mononuclear interstitial pneumonia, deposition of collagen in the affected areas, and marked hyperplasia of bronchial-associated lymphoid tissue. Infectious virus could not be recovered after Day 9. However, in contrast to loss of infectivity, viral antigen persisted at high concentrations in the lung. We conclude that influenza virus infection induced a long-term alveolitis that is associated with persistence of viral antigen. These data open the possibility that influenza virus infections may play a role in interstitial lung disease.

Pulmonary inflammatory responses during viral pneumonia and secondary bacterial infection.

Pulmonary bactericidal mechanisms are reduced during viral pneumonia. It has been proposed that secondary bacterial pneumonia occurs because the host's ability to mount an inflammatory response is suppressed. These studies examine the pathobiology of parainfluenza 1 virus and viral-associated staphylococcal pneumonia in a murine model. The sequence of leukocyte and fluid protein changes were studied in the lung and blood. An influx of polymorphonuclear leukocytes into the lungs occurred early in the viral infection, and coincided with lung macrophages aggregation. Maximal increases in pulmonary leukocytes occurred during the period associated with maximum suppression of lung bactericidal mechanisms (days 7-9). During this period, the host was capable of mounting an additional inflammatory response to staphyloccal challenges. Finally, viral pneumonia resulted in a prolonged elevation in the numbers of pulmonary macrophages, lymphocytes, and granulocytes. Thus, changes in lung biology persisted well after resolution of the initiating infections.

Lung macrophage defense responses during suramin-induced lysosomal dysfunction.

Lysosomes form an integral part of the degradative mechanisms of the phagocytic cells. Mice were injected with suramin, a lysosomotrophic drug, to investigate the effects of lysosomal pathology on the cell biology and in situ bactericidal activity of the pulmonary macrophage. Treatment with suramin resulted in marked alterations in the cell biology of the macrophage: (i) increased vacuolization and protein content, (ii) suppressed intracellular phagosome-lysosome fusion, (iii) decreased activity of the lysosomal enzymes beta-glucuronidase and N-acetyl-glucosaminidase, and (iv) enhanced exocytosis of acid phosphatase during phagocytosis. Addition of suramin, in vitro, to cell lysates resulted in a reduction in the catalytic activities of acid phosphatase, beta-glucuronidase, and N-acetyl-glucosaminidase; thereby suggesting that selective interaction, in vivo, between suramin and lysosomes containing beta-glucuronidase and N-acetyl-glucosaminidase may have occurred. Plasma membrane 5'-nucleotide phosphodiesterase activity was increased in macrophages recovered from suramin-treated animals. Although the "resting-state" reduction of nitroblue tetrazolium (NBT) was lower in these macrophages, cells stimulated by a phagocytic challenge demonstrated normal increases in NBT reduction. Phagocytosis, in vitro, and pulmonary bactericidal activity were not altered. These data demonstrate that suramin altered numerous aspects of the phagocyte's lysosomal system. Despite these changes in the cell biology of the pulmonary macrophage, the cell's defense functions were not reduced.

The participation of antiviral immune mechanisms in alveolar macrophage dysfunction during viral pneumonia.

Virus infections transiently suppress pulmonary antibacterial defenses by causing dysfunctions in the alveolar macrophage phagocytic system. This impairment of pulmonary bactericidal activity is not associated in time with virus proliferation, but rather with the period of time of rapidly declining virus titers and the expression of the antiviral immune response in the lungs. This temporal relationship suggests that the impairment of pulmonary bactericidal activity might be secondary to the antiviral immune response rather than a direct effect of virus replication. Immune depletion of mice during the course of influenza virus pneumonia ameliorated the virus-induced bactericidal defect and prevented bacterial multiplication in the lungs. In contrast, immune reconstitution reestablished the alveolar macrophage phagocytic defect. These data indicate that virus-induced suppression of pulmonary antibacterial defenses may be, in part, immunologically mediated.

Effect of in vivo administration of gold sodium thiomalate on rat macrophage function.

It has been shown that gold accumulates in macrophages. In vitro studies have also shown that long-term anti-inflammatory and immuno-regulatory effects on these cells may be responsible for the effectiveness of gold in the treatment of rheumatoid arthritis. However, the relevance of this information to the in vivo circumstance is largely untested. In this study, the effect of gold sodium thiomalate (AuTM) on rat alveolar macrophage (AM) lysosomal enzymes, bacterial killing, and metabolic activities associated with phagocytosis were assessed after in vivo administration. The activities of beta-glucuronidase, acid phosphatase, and lysozyme were inhibited 1 day following a single AuTM injection (50 mg/kg, subcutaneous). However, lysozyme returned to normal, while the activities of beta-glucuronidase and acid phosphatase were elevated from 4 to 12 days thereafter. When AuTM was administered weekly for 8 weeks, the activities of acid phosphatase and beta-glucuronidase were elevated throughout, while lysozyme was largely unaffected. The increased lysosomal enzyme activities were not due to contamination of polymorphonuclear leukocytes. These long-term effects of AuTm on enzyme activity were in marked contrast to its in vitro effect which inhibited the activities of beta-glucuronidase and acid phosphatase. No effect of AuTM administration on the release of beta-glucuronidase upon phagocytosis of opsonized zymosan was observed. At 1 day following a single AuTM injection or 3 days after a second weekly injection, in vivo bactericidal activity of AM toward S. aureus was diminished. This bacterial killing defect was not due to decrease phagocytosis; the in vivo binding and ingestion of bacteria were normal. The defect correlated with imparied metabolic activities associated with phagocytosis, namely a significant decrease in the reduction of nitroblue tetrazolium and the stimulation of the hexose monophosphate shunt. This may be an attractive anti-inflammatory effect in light of the destructive potential of the reactive oxygen species produced by macrophages in an arthritic circumstance.

Immune-enhanced phagocytic dysfunction in pulmonary macrophages infected with parainfluenza 1 (Sendai) virus.

Cultured alveolar macrophages infected with parainfluenza 1 (Sendai) virus were treated with specific antiviral immune serum and their phagocytic activity for opsonized erythrocytes (EA), Candida krusei, and Staphylococcus epidermidis quantitated. Membrane Fc receptor and candida binding activity were unaffected by the viral infection. In contrast, the virus infection decreased the phagocytic ingestion of EA. The addition of immune serum induced new phagocytic defects in that the treatment of virus-infected macrophages decreased the binding of EA and candida and reduced the ingestion of the yeast and the staphylococci. In addition, treatment with immune serum also enhanced the phagocytic defects induced by the virus infection alone, further reducing the binding and ingestion of EA. Neither virus infection nor treatment with immune serum affected the intracellular killing of S. epidermidis. These data demonstrated that virus infection of alveolar macrophages in vitro induce phagocytic defects that are accentuated by the treatment of the macrophages with antiviral antibody.

Alterations of pulmonary defense mechanisms by protein depletion diet.

Pulmonary defense mechanisms were quantitated in mice that were fed a protein-free diet (PFD) for periods of 2 and 3 weeks. Despite the severe weight loss and emaciation induced by the diet, the bactericidal mechanisms in their lungs were preserved against aerogenic challenges with staphylococcus aureus, Proteus mirabilis, and Listeria monocytogenes. Phagocytic assays of alveolar macrophages that were retrieved by pulmonary lavage from PFD-fed animals showed a decrease in Fc receptor-mediated binding activity but no alteration in the ingestion of sensitized erythrocytes. In contrast, the PFD induced defects in both the attachment phase and the engulfment phase of the phagocytic process when the challenge organism was Candida krusei. The PFD suppressed the pulmonary inflammatory response after mice were infected with influenza virus strain PR8; such mice also failed to eliminate infectious virus from their lungs. Virus infection in control mice suppressed pulmonary antibacterial defenses against challenges with S. aureus and P. mirabilis, and defect that was ameliorated in the lungs of PFD-fed mice with viral pneumonia. The data demonstrated that pulmonary defense mechanisms were modulated by a PFD but that the observed effect was dependent on the agent used to test host defenses.

Lung defenses against viral and bacterial challenges during immunosuppression with cyclophosphamide in mice.

Pulmonary virus infections predispose to secondary bacterial pneumonias by suppressing the antibacterial defenses of the lung. Cyclophosphamide (CY) treatment interferes with antiviral defenses and also impairs pulmonary bactericidal activity. To determine whether CY aggravates secondary bacterial pneumonias, mice were infected by aerosol inhalation with para-influenza 1 (Sendai) virus and injected intraperitoneally with either 0.5, 1.0, 2.5, and 5.0 mg of CY on Days 1 and 5 of the infection. On Day 7, the lungs of control (CY-treated but not infected) and virus-infected mice were lavaged and the total and differential number of free pulmonary cells quantitated. At the same time, other groups of mice were challenged aerogenically with Staphylococcus aureus and the number of initially viable bacteria remaining in their lungs quantitated at 4 and 24 h thereafter. The CY treatment induced a dose-dependent neutropenia, which was paralleled by the number of free pulmonary cells recovered from the lungs. Pulmonary bactericidal activity was also suppressed by CY treatment, with the percentage of staphylococci remaining at 24 h in the lungs of control animals being 0.5 +/- 0.2% and 1.5 +/- 0.5%, 4.0 +/- 1.5%, 8.5 +/- 2%, and 36 +/- 5%, respectively, for the increasing doses of CY. In virus-infected animals, CY treatment suppressed the inflammatory response in a dose-dependent manner, with the total number of free lung cells recovered from the highest dose group being only 5% of that recovered from untreated animals. Virus infection depressed the antibacterial defenses of the lung so that in untreated animals, 80 +/- 9% of the staphylococcal remained at 24 h. Treatment with the 2 higher doses of CY caused the bacteria to proliferate extensively in the lungs to 280 +/- 53% and 792 +/- 112%, respectively, for the 2.5 mg and 5.0 mg CY doses. In contrast, treatment with 0.5 mg and 1.0 mg of CY significantly enhanced the intrapulmonary killing of S. aureus in virus-infected lungs so that the bactericidal values at 24 h were 28 +/- 3% and 21 +/- 2%, respectively. These data demonstrated that immunosuppression modulates virus-induced suppression of pulmonary antibacterial defenses with high doses of CY aggravating and low doses ameliorating the defect.