Productive infection of isolated human alveolar macrophages by respiratory syncytial virus.

Respiratory syncytial virus (RSV) is a significant cause of lower respiratory tract disease in children and individuals with cell-mediated immunodeficiencies. Airway epithelial cells may be infected with RSV, but it is unknown whether other cells within the lung permit viral replication. We studied whether human alveolar macrophages supported RSV replication in vitro. Alveolar macrophages exposed to RSV demonstrated expression of RSV fusion gene, which increased in a time-dependent manner and correlated with RSV protein expression. RSV-exposed alveolar macrophages produced and released infectious virus into supernatants for at least 25 d after infection. Viral production per alveolar macrophage declined from 0.053 plaque-forming units (pfu)/cell at 24 h after infection to 0.003 pfu/cell by 10 d after infection and then gradually increased. The capability of alveolar macrophages to support prolonged RSV replication may have a role in the pulmonary response to RSV infection.

Respiratory syncytial virus induces interleukin-10 by human alveolar macrophages. Suppression of early cytokine production and implications for incomplete immunity.

Respiratory syncytial virus (RSV) causes repeated infections thought to be due to an ineffective immune response. We examined the hypothesis that incomplete immunity may result, in part, from RSV-infected alveolar macrophage production of IL-10 which can interfere with the production of immunoregulatory cytokines. We also assessed whether RSV induced the expression of the 2',5' oligoadenylate (2-5A)-dependent RNase L, an endoribonuclease involved in the antiviral activities of interferons. Human alveolar macrophages were exposed to medium (uninfected control), RSV, LPS, and RSV + LPS then were assessed for expression of the cytokines TNF-alpha, IL-1 beta, IL-8, IL-10, as well as 2-5A-dependent RNase L. LPS up-regulated the expression of protein and mRNA for all cytokines. RSV stimulated the protein levels of TNF-alpha, did not alter IL-1 beta, and decreased IL-8. RSV markedly stimulated protein expression of IL-10 and 2-5A-dependent RNase L. RSV had minor effects on the steady state mRNA levels of TNF-alpha, IL-1 beta, and IL-8, yet potently induced IL-10. Cells costimulated with RSV + LPS demonstrated reduced protein and mRNA levels of TNF-alpha, IL-1 beta, IL-8 but synergistically increased IL-10 levels compared to RSV- or LPS-activated cells. Kinetic analysis indicated that RSV induced a delayed and sustained increase in IL-10 transcripts. Furthermore, RSV-infected alveolar macrophage supernatants suppressed IL-1 beta and IL-8 production by LPS-stimulated alveolar macrophages as did recombinant IL-10. Anti-IL-10 neutralized these effects. These studies indicate that RSV is capable of suppressing production of early immunoregulatory cytokines through induction of IL-10 perhaps mediated by 2-5A-dependent RNase L (or other endoribonucleases) accounting for the ineffective immune response to this virus.

Cytokines in the nasal washes of children with respiratory syncytial virus bronchiolitis.

Although respiratory syncytial (RS) virus is the major cause of bronchiolitis and pneumonia in young children, the factors that regulate the associated lung inflammation have not been defined. The levels of interleukin (IL)10, IL-12, and interferon (IFN) were determined in the nasal wash samples from 20 infants with a clinical diagnosis of bronchiolitis, seven with confirmed RS virus infections and 9 control children without respiratory illnesses. IL-10 levels were significantly higher in acute nasal wash samples (1-4 d post hospitalization) from RS virus-infected infants than in convalescent samples from these children (14-21 d post-hospitalization), from children with other forms of bronchiolitis and from control children. In contrast, only one RS virus-infected infant had detectable IL-12 in an acute nasal wash sample. IFN activity was not detected in any samples from RS virus-infected children. RS virus infection stimulates IL-10 expression but not IL-12 and IFN, possibly contributing to an ineffective cell-mediated immune response.

Strategies to radiolabel interleukin 1 and 2.

The lymphocytotrophic hormones IL-1 and IL-2 have central functions in initiating the immune response. At present, very little is known about their in vivo effects in immunologically mediated diseases. Serum levels of these hormones have not been measured by radioimmunoassay methods, and cell proliferation assays of their levels in serum are difficult to interpret. Production of radioligands of high specific activity and preserved biological function may soon be possible.

Radioiodination of proteins by reductive alkylation.

The use of the aliphatic aldehyde, para-hydroxyphenylacetaldehyde as the reactive moiety in the radioiodination of proteins by reductive alkylation is described. The para-hydroxyphenyl group is radiolabeled with 125I, reacted through its aliphatic aldehyde group with primary amino groups on proteins to form a reversible Schiff base linkage which can then be stabilized with the mild reducing agent NaCNBH3. The introduction of the methylene group between the benzene ring and the aldehyde group increases its reactivity with protein amino groups permitting efficient labeling at low aldehyde concentrations. Using this method, radioiodinated proteins with high specific activity can be produced. The reductive alkylation procedure is advantageous in that the labeling conditions are mild, the reaction is specific for lysyl residues, and the modification of the epsilon-ammonium group of lysine results in ionizable secondary amino groups avoiding major changes in protein charge.

A DNA-dependent ATPase from T4-infected Escherichia coli. Purification and properties of a 63,000-dalton enzyme and its conversion to a 22,000-dalton form.

A DNA-dependent ATPase with a molecular weight of 63,000 has been purified to near homogeneity from T4-infected Escherichia coli. This enzyme is not present in uninfected E. coli. It is present in E. coli infected with amber mutants of gene 44 or gene 62 and with the data mutant. The enzyme can be eluted from single-stranded DNA-cellulose at NaCl molarities greater than 0.6 M and from ATP agarose with 80 mM MgATP. The enzyme hydrolyzes MgATP or MgdATP to the diphosphates with equal maximum velocities and the Km values are 0.30 and 0.75 mM, respectively. The enzyme hydrolyzes MgCTP with 13% and MgUTP with 7% of the velocity observed with MgATP. Apparent Km values are reported for single-stranded T4 DNA and for a series of synthetic polynucleotides. The 63,000-dalton enzyme can be cleaved proteolytically to an active 22,000-dalton form. This can be demonstrated by storage of infected cells, by mixing of cell extracts, and by inhibition with phenylmethylsulfonyl fluoride. The Mr = 22,000 enzyme cross-reacts with antibody made to the Mr = 63,000 enzyme. This antibody inhibits the 22,000-dalton species. The 22,000-dalton enzyme forms a complex with gene 32 protein in the absence of DNA.

Secreted proteins of human monocytes. Analysis by two-dimensional gel electrophoresis and effect of lipopolysaccharide.

A monocyte-rich preparation from the adherent cell fraction of human peripheral blood leukocytes was incubated for 1-8 h with [35S]methionine or [3H]leucine in the presence and absence of bacterial lipopolysaccharide (LPS). The macromolecules released into the supernatant were analysed by two-dimensional gel electrophoresis and radioautography. A complex labelling pattern involving at least 20 easily demonstrable and apparently distinct products with a broad range of molecular masses and isoelectric points was observed. LPS or LPS plus actinomycin in combination markedly stimulated the labelling and release of at least twelve different macromolecules ranging in apparent Mr from 12,000 to 46,000. Studies with monocytes that had been additionally purified by centrifugal elutriation and with the monocyte-like human cell line U-937 indicated that monocytes rather than contaminating cells were the source of these products. The majority of the secreted products were unique and did not cross-react with antibodies to interleukin 1 or tumour necrosis factor. The high resolving capacity of two-dimensional gel electrophoresis may be useful to define further the diverse biological activities and potential monokines released from monocytes at various stages of their differentiation and activation.

Hypersensitivity reaction to desipramine.

Adverse reactions to the tricyclic antidepressant drugs imipramine and desipramine have been described and include eosinophilia, pulmonary infiltrates with eosinophilia, and elevated total serum IgE levels. The immunologic mechanism accounting for these adverse reactions has not been elucidated. This article describes a patient manifesting bronchospasm, profound eosinophilia, and elevated serum IgE levels after therapy with desipramine that resolved rapidly after withdrawal of the drug. Immunologic investigations failed to demonstrate specific IgE directed against a protein conjugate of desipramine but demonstrated the ability of desipramine to induce mast cell degranulation with direct intradermal skin challenges.

Respiratory syncytial virus replication in human lung epithelial cells: inhibition by tumor necrosis factor alpha and interferon beta.

Respiratory syncytial virus (RSV) is the major pathogen causing severe lung disease in children. RSV initially replicates efficiently in the respiratory tract but becomes undetectable by 7 to 21 d after infection in normal children, suggesting that intrinsic cellular mechanisms, as yet undefined, may restrict virus replication. To provide an in vitro model to examine mechanisms that restrict RSV replication, three human lung epithelial cell lines were exposed to RSV in vitro and virus replication proceeded in a dose- and time-dependent manner, although less efficiently than the highly permissive CV-1 cell line (monkey kidney epithelial cell). Tumor necrosis factor alpha (TNF alpha) and/or interferon beta (IFN beta) markedly inhibited RSV replication in a dose- and time-dependent manner. TNF alpha combined with IFN beta essentially aborted RSV replication in A549 epithelial cells. TNF alpha and/or IFN beta did not induce cell membrane damage, cause cell lysis, or inhibit cellular protein synthesis. RSV-infected human alveolar macrophages, which produce TNF alpha, failed to productively infect lung epithelial cells in co-culture. Together these studies suggest that endogenous TNF alpha coupled with exogenous IFN beta could restrict RSV replication in lung epithelium.

Respiratory syncytial virus infection of alveolar macrophages in adult transplant patients.

Pulmonary epithelial cells are thought to be the primary cellular targets for infection by respiratory syncytial virus (RSV) in vivo. To determine whether other pulmonary cells are infected by RSV, bronchoalveolar lavage cells from six adult transplant patients, four of whom had acute RSV infection, were examined by in situ immunohistochemistry to identify infected lung cells. Both alveolar macrophages and epithelial cells were infected with RSV in vivo. At the single-cell level, three-color immunofluorescent studies revealed that both RSV-infected epithelial cells and alveolar macrophages expressed Class II molecules of the major histocompatibility complex, but only the alveolar macrophage coexpressed interleukin-1 beta. Paraformaldehyde-fixed bronchoalveolar lavage cells from RSV-infected but not uninfected patients induced a marked proliferative response by cloned T cells indicating that in vivo infected cells expressed bioactive interleukin-1. Together, these studies indicate that the alveolar macrophage may have a critical role in the lung immune response to RSV.

Sphingolipid metabolites differentially regulate extracellular signal-regulated kinase and stress-activated protein kinase cascades.

The mitogen-activated protein kinase (MAPK) signalling pathway serves to translocate information from activated plasma-membrane receptors to initiate nuclear transcriptional events. This cascade has recently been subdivided into two analogous pathways: the extracellular signal-regulated kinase (ERK) cascade, which preferentially signals mitogenesis, and the stress-activated protein kinase (SAPK) cascade, which is linked to growth arrest and/or cellular inflammation. In concurrent experiments utilizing rat glomerular mesangial cells (MCs), we demonstrate that growth factors or sphingosine activate ERK but not SAPK. In contrast, inflammatory cytokines or cell-permeable ceramide analogues activate SAPK but not ERK. Ceramide, but not sphingosine, induces interleukin-6 secretion, a marker of an inflamed phenotype. Moreover, ceramide can suppress growth factor- or sphingosine-induced ERK activation as well as proliferation. These studies implicate sphingolipid metabolites as opposing regulators of cell proliferation and inflammation through activation of separate kinase cascades.

Respiratory syncytial virus lung infection in infants: immunoregulatory role of infected alveolar macrophages.

Thirteen previously healthy children with acute onset of severe lower respiratory tract signs and symptoms underwent bronchoscopy and bronchoalveolar lavage (BAL) for diagnostic purposes. BAL samples were assessed for viral, bacterial, mycobacterial, and fungal cultures. Cytospin preparations of BAL cells were assessed for expression of respiratory syncytial virus (RSV), HLA-DR, interleukin-1 beta, and tumor necrosis factor proteins. Purified alveolar macrophages from 2 RSV-infected children were assessed for viral replication. Three children had bacterial pneumonia and 6 were infected with RSV. BAL cells from RSV-infected children demonstrated viral protein expression. Alveolar macrophages were the predominant cell type recovered by BAL and demonstrated coexpression of RSV, HLA-DR, interleukin-1 beta, and tumor necrosis factor proteins. Purified alveolar macrophages from 2 RSV-infected children replicated RSV by infectious center assays. Thus, alveolar macrophages are infected by RSV in vivo and coexpress potent immunomodulatory molecules that potentially regulate the local immune response or lung injury due to this virus.

Respiratory syncytial virus infection of human mononuclear phagocytes stimulates synthesis of platelet-activating factor.

Production of platelet-activating factor 1-O-alkyl-2-acetyl-sn-glycero-3- phosphocholine (PAF), a potent mediator of inflammation, by mononuclear phagocytes varies with their stage of cellular differentiation and the nature of the eliciting stimulus. The human monocytic cell line U937 can be induced to differentiate to a macrophage-like cell following phorbol myristate acetate exposure, and after differentiation, these cells efficiently support replication of respiratory syncytial virus (RSV). U937 cells induced to differentiate with phorbol myristate acetate demonstrated a time-dependent decrease in PAF synthesis. RSV infection of these differentiated U937 cells caused a sustained stimulation of PAF synthesis that paralleled viral replication and was dependent on infectious virus. Virus increased the activity of lyso-PAF:acetyl-CoA acetyl-transferase (PAF acetyltransferase) in cell lysates, thus enhancing the anabolic pathway of PAF synthesis without altering the activity of PAF acetylhydrolase, which regulates PAF catabolism. RSV infection of human monocytes also caused a marked increase in [3H] monocytes also caused to uninfected monocytes. Thus, virus infection serves as a novel stimulus to induce PAF synthesis in human mononuclear phagocytes and suggests that increased PAF production may have a critical role in the inflammatory response to RSV.

Normal bronchial epithelial cells constitutively produce the anti-inflammatory cytokine interleukin-10, which is downregulated in cystic fibrosis.

Interleukin-10 (IL-10) is a potent regulatory cytokine that decreases inflammatory responses and T-cell stimulation. We have found that respiratory epithelial lining fluid (ELF) from patients with cystic fibrosis (CF) contains significantly less soluble IL-10 than ELF of healthy control subjects. Although macrophages from the chronically infected lungs of CF patients appear to be one source of IL-10, little or no intracellular IL-10 was found in bronchoalveolar lavage macrophages from healthy control subjects, suggesting that there must be another source of this cytokine in healthy lungs. We found that bronchial epithelial cells from healthy control subjects constitutively produce IL-10, which appears to be downregulated in CF patients. It is thus likely that the bronchial epithelium plays an important role in regulating the local immune response, producing IL-10 to decrease inflammation in the healthy lung. Conversely, downregulation of epithelial IL-10 production in CF airways may contribute to enhancing local inflammation and tissue damage.

Respiratory syncytial virus infection of human cord and adult blood monocytes and alveolar macrophages.

We studied the permissiveness of human leukocytes, blood monocytes, alveolar macrophages, and cord blood monocytes to infection with respiratory syncytial virus (RSV). Specific immunofluorescence was used to determine the percentage of infected leukocytes. The results indicated that monocytes were the most susceptible human leukocyte to in vitro infection with RSV. Polymorphonuclear leukocytes demonstrated no specific fluorescent staining after 24 h of exposure to RSV, whereas peripheral blood nonadherent mononuclear cells demonstrated a low percentage of positive cells, with a mean of 6 +/- 1% SE. In contrast, 37 +/- 5% of monocytes expressed RSV antigen after viral exposure. Exposure of monocytes to lipopolysaccharide (LPS) for 1 h prior to RSV increased the percentage of infected cells to 48 +/- 6% and stimulated their secretion of prostaglandin E2 (PGE2) and alpha tumor necrosis factor (TNF). Intrinsic mononuclear phagocytic factors influencing the permissiveness to RSV were studied by determining infection of adult and cord blood and alveolar mononuclear phagocytes (MP). Alveolar and blood MP simultaneously isolated from adult donors were similarly infected by RSV, which varied with the viral dose. Cord blood MP were more susceptible to RSV infection than were adult MP, 58 +/- 9% infected versus 37 +/- 5%, respectively (p less than 0.05). Treatment with LPS for 1 h prior to RSV exposure did not increase infection of cord blood MP as seen with adult blood MP. However, LPS can induce human monocytes to secrete cytokines with antiviral activity, and our results indicate that both gamma interferon and TNF, independently or in combination, prevented infection of monocytes in a dose-dependent manner.

The accessory function of B lymphocytes is resistant to the adverse effects of UV radiation.

The effect of UV radiation on the accessory activities of B lymphoblastoid cell lines (B-LCL) was investigated in three types of in vitro T lymphocyte proliferation assay, each of which differed in its accessory requirements. In contrast to monocytes whose accessory function was universally sensitive to UV radiation, B-LCL were resistant to UV in oxidative mitogenesis and staphylococcal enterotoxin B assays, in which stimulus processing was not a requirement. Expression of membrane interleukin (IL) 1 and HLA-DR antigens by B-LCL and monocytes was not affected by UV, nor was surface membrane expression of intercellular adhesion molecule-1 (ICAM-1) on B-LCL. These results were in marked contrast to monocytes in which there was a greater than 65% reduction in ICAM-1 expression. When UV-irradiated B-LCL were employed as antigen-presenting cells for tetanus toxoid-dependent T cell stimulation, a reduction in antigen-presenting function was observed. However, pulsing of B-LCL with tetanus toxoid prior to UV irradiation preserved their antigen-presenting capacity in this system also. These findings indicate that there is differential UV sensitivity among accessory cells which may be explained by different effects of UV radiation on antigen processing and adhesion molecule expression.

Dyscoordinate expression of tumor necrosis factor-alpha by human blood monocytes and alveolar macrophages.

Previous studies have shown that human alveolar macrophages produce less interleukin-1 (IL-1) in response to lipopolysaccharide (LPS) than do their precursors, blood monocytes. The purpose of this study was to compare the capacities of alveolar macrophages and blood monocytes to synthesize tumor necrosis factor (TNF) in response to LPS. Alveolar macrophages were obtained by bronchoalveolar lavage of healthy nonsmoking subjects, and blood monocytes were obtained by adherence of mononuclear cells to plastic. TNF activity was measured in supernatants and cell lysates as cytotoxicity to L929 fibroblasts (uptake of neutral red at 570 nm). TNF activity of alveolar macrophages stimulated at 10(6) cells/ml with LPS (10 micrograms/ml) for 16 h was 596 +/- 367, and of blood monocytes it was 60 +/- 84 U/ml (mean +/- SD, p less than 0.005). At no concentration of LPS and at no period of stimulation did alveolar macrophages express less TNF activity than did blood monocytes. In concurrent experiments, supernatants of LPS-stimulated alveolar macrophages contained less IL-1 activity than did blood monocytes. Lysates of both cell types contained less than 20% of total TNF activity. The TNF activity of LPS-stimulated alveolar macrophages was neutralized greater than 99% by monoclonal antibody to TNF-alpha; control monoclonal antibody OKT3 had no effect. Next, alveolar macrophages and blood monocytes were biosynthetically labeled with [3H]leucine during incubation with LPS; supernatants were immunoprecipitated with anti-TNF, and precipitates were electrophoresed on polyacrylamide gels. Autoradiographs indicated that immunoreactive TNF was produced by both blood monocytes and alveolar macrophages and that the relative molecular weights were identical (17,000).(ABSTRACT TRUNCATED AT 250 WORDS)

Inflammatory cytokines in cystic fibrosis lungs.

Chronic pulmonary infection with Pseudomonas aeruginosa continues to be the major cause of morbidity and mortality in cystic fibrosis (CF). Several characteristics of CF, including the excessive influx of neutrophils into the airways, cachexia, and hyperglobulinemia, could reflect the effects of cytokines, such as interleukin-1 (IL-1), IL-6, IL-8, and tumor necrosis factor (TNF-alpha). We hypothesized that these pro-inflammatory cytokines, produced by alveolar macrophages in response to pseudomonas and/or other microorganisms, promote the destructive inflammatory process in the lung. We evaluated bronchoalveolar lavage (BAL) fluid and BAL macrophages from 22 CF patients and 13 healthy control (HC) subjects, measuring soluble TNF-alpha, IL-1 beta, IL-6, and IL-8 and the regulatory molecules TNF soluble receptor (TNF-sR), IL-1 receptor antagonist (IL-1Ra), and IL-10 (cytokine synthesis inhibitory factor). Levels of the proinflammatory cytokines were higher in CF versus HC BAL (p < or = 0.05 for IL-1, TNF, and IL-8; p = 0.06 for IL-6). In contrast, HC BAL contained significantly more IL-10 than CF BAL (p < 0.05), but TNF-sR and IL-1Ra were similar. Immunocytochemistry demonstrated a higher percentage of CF than control BAL macrophages expressing intracellular cytokines (p < 0.05). Thus, enhanced macrophage production of proinflammatory cytokines and decreased production of the regulatory molecule IL-10 may have important roles in the pathogenesis of CF lung disease.

Restricted replication of respiratory syncytial virus in human alveolar macrophages.

The cellular factors that regulate infection and replication of respiratory syncytial virus (RSV) in human alveolar macrophages were examined. RSV-exposed alveolar macrophages demonstrated a time-dependent expression of viral glycoproteins, maximal by 24 h post-infection resulting in infection of approx. 38% of the cells. Essentially all (33%) of these freshly isolated alveolar macrophages replicated RSV as shown by infectious centre assays. This RSV-permissive subpopulation of alveolar macrophages consisted primarily of major histocompatibility class II-expressing cells as determined by fluorescence-activated cell sorting. Re-infection of alveolar macrophages did not significantly alter the number of cells infected or capable of replicating RSV. However, in vitro differentiation of alveolar macrophages prior to infection resulted in a significant (P < 0.05), time-dependent decrease (approx. sevenfold) in the number of cells that replicated virus. The mechanism by which cellular differentiation restricted RSV replication is unknown. Production of defective interfering particles did not account for this decrease. Alveolar macrophages infected with RSV produce a variety of cytokines potentially contributing to this restricted viral replication. Pretreatment with several of these cytokines did not affect viral infection or replication. However, tumour necrosis factor (TNF alpha) significantly (P < 0.05) decreased viral replication but only by 30 to 60%. Thus RSV replication is reduced by in vitro differentiation of alveolar macrophages and, to a lesser degree, by pretreatment with TNF.