Virus-induced alterations in macrophage production of tumor necrosis factor and prostaglandin E2 JAMES R. PANUSKA, FABIO MIDULLA, NICK M. CIRINO, ALBERT0 VILLANI, ILEEN A. GILBERT, E. R. MCFADDEN, JR., AND YUNG T. HUANG Airway Disease Center and Departments of Medicine and Pathology of University Hospitals and Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

PANUSKA, JAMES R., FABIO MIDULLA, NICK M. CIRINO, ALBERTO VILLANI, ILEEN A. GILBERT, E. R. MCFADDEN, JR., AND YUNG T. HUANG. Virus-induced alteration in macrophuge

production of tumor necrosis factor and prostaglandin E2. Am. J. Physiol. 259 (Lung Cell. Mol. Physiol. 3): L396-L402,1990.The cellular immune response to respiratory syncytial virus (RSV) is felt to contribute to viral clearance and/or the inflammation accompanying pulmonary infections with this virus. Both tumor necrosis factor (TNF) and prostaglandin EP (PGE2) are important regulatory mediators of the cellular immune response. We examined the production of these mediators from purified human alveolar and blood mononuclear phagocytes (MP) after RSV infection in vitro and compared production induced by virus with that induced by lipopolysaccharide (LPS). RSV infection of alveolar MP did not alter PGE, production but increased expression of TNFcv mRNA paralleled by increased secretion of immunoreactive and biologically active TNF. TNF production by alveolar MP was dependent on the infectious dose of virus and occurred early in the viral replication cycle. In contrast, RSV had minimal effects on blood MP production of TNF and PGE,. However, blood MP (and not alveolar MP) infected with RSV and costimulated with LPS demonstrated a 1.7-fold increase in PGEz levels compared with LPS alone (P < 0.001). Therefore, RSV has differential effects on human alveolar and blood MP production of these immunoregulatory molecules. alveolar

macrophage;

cytokines;

lipopolysaccharide

PHAGOCYTES (MP) are thought to have a critical role in defense against microbial invasion. MP from separate anatomic origins may encounter pathogens invading that anatomic domain (6, 7) and may respond with altered biological functions that are distinct from the responses of MP from other sites (20, 21, 31, 32). Respiratory syncytial virus (RSV) is an important human pathogen tropic for the respiratory tract where it can induce bronchoconstriction and pulmonary inflammation associated with elevated leukotrienes and histamine in respiratory secretions (30). The alveolar MP is thought to defend the lower respiratory tract against pathogens and may be exposed to RSV in vivo through inhalation or viral replication (17). In vitro studies have demonstrated that alveolar MP can be infected with RSV (17) and can support viral replication (23). It remains unknown whether the alveolar MP has a role in the immunopathogenesis of RSV-induced pulmonary disease. MONONUCLEAR

L396

Tumor necrosis factor-a (hereafter referred to as TNF) and prostaglandin Ez (PGEz) are produced by both alveolar and blood MP and are important mediators of immune and inflammatory responses (19, 21). It has not previously been shown whether any virus can alter alveolar MP production of TNF, although it has been demonstrated that human immunodeficiency virus and Sendai virus, a murine paramyxovirus related to RSV, are capable of inducing TNF production from human monocytes or MP cell lines (9, 15, 18). The alveolar MP is a more potent source of TNF than the blood MP (13, 24, 29). A local pulmonary role for TNF has been suggested by in vivo studies demonstrating that stimuli administered to the lung result in TNF production and sequestration within the respiratory tract (22). TNF mediates a broad range of biological activities including neutrophil activation and adherence to endothelium, decreased fibroblast proliferation with enhanced production of PGEP, and enhanced MP production of interleukin 1 (IL-l), PGEt2, and platelet-activating factor (5, 26). Additional functions for TNF and PGE, separately, or in concert with interferon, include modulation of inflammation and inhibition of infection or replication of both DNA and RNA viruses (16, 27, 33). Because TNF and PGEz could function to modify virus-induced inflammation and/or restrict viral replication, we examined whether human alveolar and blood MP after in vitro infection with RSV demonstrated altered production of TNF and PGEz and compared these responses with that induced by LPS. Our results indicate that RSV-infected alveolar MP upregulate production of TNF and that PGE2 production by RSV-infected, LPSexposed blood MP is increased. MATERIALS

AND

METHODS

Cell collection and preparation, exposure of cells to RSV and immunofluorescence. Following informed consent, a total of 25 healthy, nonsmoking adult volunteers provided alveolar and blood MP for these studies after bronchoalveolar lavage and venipuncture. These studies were approved by the institutional review board, University Hospitals of Cleveland. Peripheral blood mononuclear cells isolated by Ficoll-Hypaque centrifugation were incubated for 1 h at 37°C on plastic Petri dishes precoated with AB sera (Whittaker MA Bioproducts, Walkersville, MD) as previously described (17). Nonadherent

1040-0605/90 $1.50 Copyright 0 1990 the American Physiological Society

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RSV-INDUCED

MACROPHAGE

cells were removed by washing three times with RPM1 1640 (GIBCO, Grand Island, NY), 10% (vol/vol) fetal calf serum (FSC) (Hyclone, Logan, UT), and adherent cells were harvested by scraping with a rubber policeman. Alveolar macrophages were isolated from bronchoalveolar lavage fluid following centrifugation at 500 g for 10 min and adherence to plastic for 1 h at 37°C (24). Alveolar and blood MP preparations were >95% viable and >90% nonspecific esterase positive. Blood and alveolar MP (lo6 cells/ml) were incubated with or without lipopolysaccharide (LPS) from Escherichia coli (Difco, Detroit, MI) (20 ,ug/ml) in RPM1 containing 10% (vol/vol) FCS, 2 mM glutamine, 1 mM nonessential amino acids, 100 U/ml penicillin, 100 pg/ ml streptomycin, and 0.25 pg/ml amphotericin B (culture media) (all supplements from Sigma, St. Louis, MO) for the times indicated in the text at 37°C in 5% CO, and then used in experiments. MP were infected with RSV (strain A2, originally from R. Chanock) as previously described (17). Briefly, blood and alveolar MP (lo6 cells/ml) were exposed for 2 h at 37°C to medium containing virus at 0 to 10 plaqueforming units (pfu)/cell. Cells were then washed once in RPM1 without serum and incubated in culture media at 37°C in 5% CO2. After infection with RSV, alveolar and blood MP were incubated for the times indicated in the text and aliquoted, and portions fixed with ice-cold acetone for 10 min. The fixed cells were washed twice with phosphatebuffered saline (PBS) and reacted for 30 min with a mixture of mouse monoclonal antibodies to RSV proteins conjugated to fluorescein isothiocyanate (Bartels Immunodiagnostic Supplies, Sacramento, CA). The percent fluorescent cells was determined as previously described (17) Akay for TNF bioactivity. TNF cytotoxic activity was measured by minor modifications of methods previously described (24). Briefly, 2.5 X 10” murine L929 cells in modified essential medium (MEM) (M. A. Bioproducts) supplemented with 2 mM L-glutamine, 100 pg/ml gentamicin, 25 mM N-2-hydroxyethylpiperazine-N’ -2-ethanesulfonic acid (HEPES), 1 mM nonessential amino acids, 2 mM pyruvate (complete MEM), and 15% (vol/ vol) FCS were added to each well of a 96-well flat-bottom microtiter plate, incubated at 37°C in 5% CO* for 24 h, and then 0.2 lug actinomycin D (Sigma Chemical) was added to each well. MP supernatants were twofold serially diluted in culture media and 100 ~1 added to each well in replicates of nine. Plates were incubated for 20 h at 37°C in 5% COZ, after which 0.05 ml of freshly prepared 0.033% (wt/vol) neutral red in PBS was added to each well. Plates were incubated for 1 h, emptied by inversion, and rinsed twice with 0.2 ml of PBS at 37OC. One well on each plate received 100 ~1 of 5 M guanidine HCl as a control. Remaining cells were lysed by addition of 0.1 ml of 50 mM sodium phosphate, 50% (vol/vol) ethanol, and optical density at 570 nm was determined with an automated reader. TNF activity, expressed as half-maximal units per lo6 cells (U/lo6 cells), was determined by comparison to human recombinant TNF and calculated by modified probit analysis (24). In selected experiments, the specificity of the TNF activity was

PRODUCTION

OF

TNF

AND

PGE,

L397

assessed by incubating aliquots of supernatants with anti-TNF (a kind gift of Dr. Leo Lin, Cetus) or preimmune serum (both at a 1:400 final dilution) for 16 h at 4°C and determining cytotoxic activity as described above. A solid phase radioimmunoassay (RIA) for TNFcv (Centocor, Malvern, PA) that does not cross-react with lymphotoxin, IL-la or IL-lp was used exactly as described by the manufacturer, and results are expressed as nanograms per 10” cells. mRNA levels determined by whole cell dot blot analysis. MP were twofold serially diluted in PBS and applied by vacuum to Gene Screen membranes prewet in 0.3 M sodium chloride, 0.03 M citric acid, pH 7.0 (2 x standard saline citrate, SSC), 10% (vol/vol) formaldehyde (EM Science, Gibbstown, NJ) on a dot-blot Minifold (Shleicher and Schuell, Keene, NH) as previously described (23). The following solutions were sequentially passed through the filter: 40 ml of 2 x SSC, 0.2% sodium dodecyl sulfate (SDS); 40 ml of 2 x SSC, 15% (vol/vol) formamide (BRL, Gaithersburg, MD), 60°C; and 40 ml of 2 x SSC. The members were then incubated in 10% (vol/vol) formaldehyde and 20 x SSC at 60°C for 15 min followed by two washes (100 ml each) in 2 X SSC and then baked at SOOC for 2 h. Prehybridization and hybridizations were performed as previously described (23) employing 32P-labeled cDNA probes for human TNFcu, the 750-bp Hind111 cDNA fragment from pAW739 (ATCC), a-actin, the 2,OOO-bp cDNA fragment from pA1 (obtained form J. Sedor, CWRU), or a fragment of RSV fusion (F) gene cloned in pUC9 (3). Labeling of the probes was performed by random primer extension (Boehringer Mannheim, Indianapolis, IN), and specific activities exceeded 10’ cpm/ pg. After hybridization, the membranes were washed sequentially for 20 min in 2 x SSC, 0.5% SDS; 1 X SSC, 0.5% SDS; and finally 0.1 X SSC, 0.5% SDS at 60°C and exposed to Kodak XAR-5 film with two intensifying screens at -40°C. After autoradiography, the membranes were incubated in 0.002% (wt/vol) Ficoll 400 (Pharmacia), 0.002 % (wt/vol) polyvinylpyrrolidone (Sigma), 0.002% (wt/vol) bovine serum albumin (Sigma), 5 mM tris(hydroxymethyl)aminomethane (Tris) (pH 8.0), 0.2 mM EDTA, and 0.05% (wt/vol) sodium pyrophosphate with agitation for 2 h at 75°C to remove previously hybridized probe. Membranes were then prehybridized for 2 h and hybridized with the next 32P-cDNA probe as described above. PGE2 determination. PGE2 was measured in 24-h supernatants by RIA as previously described (17). Statistical analysis. Results are presented as means t standard error of the mean (SE). The effects of dose were determined by analysis of variance (ANOVA), and comparisons between means were performed by paired and unpaired Student’s t test and P < 0.05 considered significant. RESULTS

Alveolar macrophage expression of TNF after RSV and LPS exposure. It was initially determined whether RSV infection of alveolar MP altered TNF expression. RSV infection was assessedby determining the kinetics of

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L398

RSV-INDUCED

MACROPHAGE

PRODUCTION

expression of RSV F RNA. This gene codes for the fusion protein of RSV and has previously been shown to correlate with RSV infection using whole cell dot blots of alveolar MP (23). In these experiments, virus was added to equal numbers of unstimulated alveolar MP (3 pfu/ cell) and either immediately removed (designated -2 h in Fig. 1) or incubated for 2 h and then removed and replaced with fresh media (designated the 0 h in Fig. 1). The cultures were then incubated for the hours indicated after addition of virus (Fig. 1). At each indicated time, alveolar MP were analyzed by whole cell dot blots and hybridized with an RSV F cDNA probe. Four hours after viral addition, F RNA began to increase and was maximal by 24 h, whereas cells unexposed to virus (designated U in Fig. 1) demonstrated nearly undetectable signals (Fig. 1A). Rehybridization of this same dot blot with a TNF cDNA probe demonstrated maximal expression of TNF mRNA at 2 h after addition of virus, which rapidly

A

RSV F

OF TNF AND PGEz

declined and had returned to baseline by 48 h. When this blot was stripped and reprobed a third time with [32P]~actin, there were increased levels of actin between 2 and 24 h after viral addition, reflecting either slight differences in the numbers of cells recovered or possible differences in steady-state levels (Fig. 1B). Nevertheless, changes in actin expression were minor (by densitometry) compared with the changes in RSV F or TNF expression at these same time points. Similar results were seen in three additional donors where TNF mRNA was maximally expressed between 2 and 4 h after addition of virus before significant expression of RSV F gene. TNF mRNA accumulation induced by LPS and RSV was directly compared. Whole cell dot blot analysis of alveolar MP exposed for 4 h to RSV + LPS (3 pfu/cell, 20 cl.g/ml, respectively) demonstrated high levels of TNF (Fig. 2, lane A) that were greater than the levels seen in cells exposed only to LPS (lane B). Alveolar MP exposed to RSV alone (Fig. 2, lane C) demonstrated increased TNF expression compared with cells unexposed to either stimuli (lane D) but less than that observed with RSV + LPS or LPS alone. The results are representative of those seen in four separate donors where densitometry indicated that TNF mRNA was increased 3.1 + l.O-fold by RSV, 6.9 + 2.4-fold by LPS, and 7.2 +- 1.8-fold by RSV + LPS (means + SE), all significantly greater than uninfected controls (P < 0.05). There were no significant differences between RSV + LPS and LPS alone. Immunoreactive

‘NF

Hours after

Z

Hours

after

Add ition of

Addition

levels of TNF after RSV exposure.

TNF secretion into alveolar MP supernatants was examined to determine whether TNF mRNA expression resulted in release of bioactive TNF. Alveolar MP (lo6 cells/condition from 6 donors) were unexposed or exposed to RSV (3 pfu/cell) and/or LPS (20 pg/ml) for 2 h, placed into fresh media, and 24 h later cell-free supernatants were analyzed for immunoreactive TNF. RSV,

Virus

of Virus

FIG. 1. Kinetics of expression of respiratory syncytial virus F RNA, TNF, and actin at varying times after infection with virus. Alveolar macrouhaees (lO”/ml) were unexuosed (U) or exposed to RSV (3 vfu/ cell) for -> h‘(viral inoculum was applied and immediately removed) or for 2 h (0 h as indicated) and viral inoculum removed, replaced with culture media, and incubated for indicated times. Alveolar macrophages were then washed, serially 2-fold diluted, and applied to Gene Screen nylon membranes at cell concentrations indicated. Dot blots were evaluated sequentially by hybridization, autoradiography, stripped of previous probe, and finally rehyhridized. Order of hybridization was first RSV F, then TNFol (A), and finally ol-actin (B). Results are from a single donor representation of results from 4 separate donors. See text for definitions.

FIG. 2. Dot blot analysis of alveolar macrophages evaluated with a ‘*P-cDNA probe for TNFor. Alveolar macrophages were exposed for 4 h to RSV (3 pfu/cell) and LPS (20 pg/ml) (lane A), LPS (lane B), RSV (lane C), or unexposed (lone D). Equal numbers of cells were serially diluted from 106/ml to 0.07 X 106/ml and applied to Gene Screen membranes, hybridized with a 32P-cDNA probe for human TNFol, and then washed and autoradiographed for 12 h. Results are from a single donor representative of results seen in 4 separate donors.

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RSV-INDUCED

MACROPHAGE

PRODUCTION

RSV + LPS, and LPS alone all induced significant increases in alveolar MP secretion of TNF compared with unexposed controls (P c 0.001) (Table 1). TNF secretion by alveolar MP costimulated with RSV + LPS did not significantly differ from LPS alone. Thus RSV induced alveolar MP to release increased amounts of immunoreactive TNF compared with controls. RS V dose-dependent effects on alveolar MP production of TNF activity. TNF cytotoxic activity in alveolar MP supernatants was next assessedto determine whether biologically active TNF was released in parallel with the increased TNF mRNA and immunoreactivity. In addition, we determined whether alveolar MP exhibited altered production of TNF as a function of viral dose. Alveolar MP (lo6 cells/condition, 4 separate donors) were exposed to RSV at the doses indicated in Fig. 3 for 2 h, placed into fresh media, and incubated for 24 h. Cytotoxic activity in supernatants increased in a virus dose-dependent manner (P c 0.05, ANOVA) appearing maximally induced between 3 and 10 pfu/cell (Fig. 3). Cytotoxic activity (U/lo6 cells) produced by alveolar MP was significantly increased by RSV at 1 (52 t 16), 3 (59 1. Supernatant levels of TNF determined by radioimmunoassay from alveolar macrophage uninfected or infected with RSV (3 pfulcell) and unexposed or exposed to LPS (20 pg/ml)

TABLE

TNF (Y, ng. 10” cells-‘. 24 h-l

Condition

Control +RSV +RSV +LPS

0.9kO.2 9.7t2.4* + LPS

20.6&8.4* 16.9k5.4"

Values

are means t SE for 6 experiments. See MATERIALS for definitions and information on radioimmunoassay. compared with controls.

METHODS

0.001

80

4

r

AND

*

P
90% with an antibody to TNF (not shown). Blood MP exposed to RSV demonstrated a minor increase in TNF cytotoxic activity that did not differ significantly from controls. When alveolar and blood MP were exposed to RSV + LPS, TNF production was significantly increased compared with RSV alone (P c 0.01) and controls (P < 0.001). TNF cytotoxic levels from alveolar and blood MP stimulated with RSV + LPS did not differ significantly from LPS alone. In three separate experiments, no significant interactive effects of RSV (3 pfu/cell) on LPSinduced alveolar MP production of TNF at doses of LPS of 0.02, 0.2, and 2.0 pg/ml were seen (data not shown). TNF production by alveolar MP exceeded that produced by blood MP under all conditions (significant for each at P < 0.01, Table 2). Neither alveolar nor blood MP following RSV infection retained detectably increased amounts of intracellular TNF compared with uninfected controls (n = 2). Together these results confirmed prior results observed with TNF mRNA expression and protein production by alveolar MP. To assesswhether the differential effects of RSV on alveolar and blood MP could be accounted for by a PGEz autoregulatory loop (11, 28), separate aliquots of the supernatants from the 10 donors described above were analyzed for PGE2. RSV alone did not alter PGE2 levels from either alveolar or blood MP compared with controls (Table 2) indicating that PGEz production did not ac2. TNF cytotoxic activity and PGE2 levels in supernatants of alveolar and blood mononuclear phagocytes uninfected or infected with RSV at 3 pfulcell and unexposed or exposed to LPS TABLE

TNF Condition

Activity, U. lo6 cells-’ -24 h-l

FIG. 3. TNF cytotoxic activity from alveolar MP exposed to varying doses of RSV. Alveolar MP (10" cells/ml) were exposed for 2 h to indicated doses of RSV, washed, and cultured in fresh media for 24 h. Cell-free supernatants were analyzed for TNF cytotoxic activity using L929 fibroblast assay as described in MATERIALS AND METHODS. Results are means t SE with n = 4 for all conditions except 10 pfu/cell where n = 3. * P < 0.05 by paired and unpaired Student’s t test. See text for definitions.

1lt2 53t11* + LPS

ng. lo6 cells-’ -24 h-’

Alveolar macrophages

Control +RSV +RSV +LPS

PGE2,

1,523&587t

573&208-b

Monocytes

4k2 8k4 85+37t 25&5*

Alveolar macrophages

0.56kO.4 0.41kO.l 22.54&5.1-t 24.39&7.4-l-

Monocytes

3.05t1.2 2.99t1.4 51.07&7.3-i28.78&3.7-f-

Values are means t SE for 10 experiments. Cells were unexposed (control) or exposed to RSV for 2 h with or without LPS (20 yg/ml), washed once and incubated for 24 h, 37°C in 5% CO,. Cell-free supernatants were obtained and TNF activity and PGEz determined as described in MATERIALS AND METHODS. See text for definitions. * P < 0.01 compared with controls. t P < 0.001 compared with controls.

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L400

RSV-INDUCED

MACROPHAGE

count for the differential effects of RSV on alveolar or blood MP expression of TNF. In addition, RSV + LPS significantly increased PGE2 production by both alveolar and blood MP when compared with unexposed controls (P < 0.001). However, RSV + LPS, when compared with LPS alone, only resulted in significantly increased PGE2 by blood MP (1.7fold, P c 0.001) (Table 2). PGE2 production by alveolar MP did not differ significantly from blood MP except in unexposed (control) cells where alveolar MP produced lesser amounts of PGE2 than blood MP (P c 0.04). The effects of RSV on PGE2 production by MP were assessed at varying doses of RSV. As shown in Fig. 4, RSV at the indicated doses had no effect on alveolar or blood MP production of PGEB. RSV combined with LPS did not significantly alter PGE, production by alveolar MP but significantly enhanced blood MP production of PGE2 at doses of 1 and 3 pfu/cell (P c 0.05). Therefore, RSV demonstrated differential effects on both TNF and PGEz production by MP dependent on their anatomic origin. It was unlikely that LPS contamination of viral stocks could account for these results, since PGE, production by RSV-exposed MP was not altered compared with unexposed controls. In addition, when alveolar MP from three additional donors were exposed to ultraviolet (UV) light-inactivated RSV at 3 pfu/cell, TNF activity in supernatants was 8 t 6 (SE) U. lo6 cells-1o 24 h-l, which was comparable to controls (see Table 2). Less than 2% of these cells expressed viral proteins at 24 h after exposure to UV-inactivated RSV similar to results previously published ( 17).

_---*--------

_____ ---_

a

1

2

1

0

3

pfu/celf

4. Supernatant levels of PGE2 from blood and alveolar macrophages exposed to varying doses of respiratory syncytial virus without or with LPS (20 pg/ml). Mononuclear phagocytes (106/ml) were infected with virus at indicated concentrations for 2 h, washed with media, incubated for 24 h, and cell-free supernatants were analyzed for PGE2 by RIA. Blood monocytes (-----), alveolar macrophages (-), solid circles without LPS, squares with LPS. Results are means k SE, n = 4. * P < 0.01. See text for definitions. FIG.

PRODUCTION

OF

TNF

AND

PGE2

DISCUSSION

The results presented here indicated that RSV infection of human alveolar MP induced TNF mRNA expression resulting in elevated levels of TNF protein and bioactivity. Steady-state levels of TNF mRNA from RSV-infected alveolar MP were increased -3.1-fold compared with controls. These findings are in reasonable agreement with the 4.8-fold increase observed with cytotoxic activity determinations (Table 2). However, a RIA revealed a 10.9-fold increase in TNF levels (Table l), demonstrating the lack of strict concordance between these separate assays of TNF activity as has been previously reported (4, 8). Either transcriptional or posttranscriptional regulation of TNF (9) or metabolism of secreted TNF cytotoxic activity resulting in increased immunoreactive forms (8) could account for the observed quantitative differences between these assays of TNF production. Although RSV increased alveolar MP production of TNF, we observed no significant interactive effects of RSV on LPS-induced TNF production. However, our results do not exclude this possibility. We did observe that virus was less potent than LPS in inducing TNF production by MP. The present results differ from studies of Sendai-exposed blood MP that produce greater amounts of TNF than LPS exposed cells (1, 2,9). These contrasting results may be due to differences between these virus and/or the origin of the MP. It is also possible that RSV induces TNF production by a subpopulation of alveolar MP, whereas LPS may induce TNF production by a larger population of cells. This latter hypothesis is supported by the following observations: 1) that alveolar MP costimulated with both RSV and LPS demonstrated significantly increased TNF mRNA levels (Fig. 2) and production of immunoreactive and bioactive TNF compared with cells stimulated with RSV alone (Tables 1 and 2), 2) that because the percentage of infected cells averaged 35% (even when exposed to higher doses of virus), a significant proportion of cells remain uninfected and might be capable of increasing TNF production in response to LPS. Alternatively, LPS may induce TNF production by both infected and uninfected cells, a possibility that would have to be examined at the individual cell level. Our results further demonstrated that alveolar MP expression of TNF mRNA occurred at an early step in the RSV replication cycle and was dependent on infectious virus. UV-inactivated RSV did not increase secretion of TNF nor did infectious RSV induce increased PGE2 production by MP, indicating that LPS contamination of viral stocks was unlikely to account for these results. MP isolated by adherence may have altered cytokine expression and/or production (20,32), but both RSV and LPS more potently stimulated TNF than seen with control cells. Proposed mechanisms by which virus might induce TNF production by MP include virusreceptor interaction (X5), double-stranded RNA (33), or virus-induced inhibition of a protein kinase (9). It is unknown whether RSV enters MP through a receptormediated process. It seems unlikely that UV treatment of RSV (which did not elicit an increase in TNF produc-

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RSV-INDUCED

MACROPHAGE

tion by alveolar MP) would inhibit virus-receptor interaction. The inability of UV-treated virus to induce TNF could result from UV-damaged RNA interfering with production of double-stranded molecules (14). However, lymphocytic choriomeningitis virus also replicates within MP through double-stranded RNA intermediates but does not induce TNF expression in elicited peritoneal murine MP (12). Virus-induced inhibition of a protein kinase resulting in modifications of transcriptional control factors for TNF (9) is compatible with the kinetics of TNF induction we observed; however, our results do not allow us to address this mechanism directly. Previous studies have shown that alveolar MP produce larger amounts of TNF per cell than blood MP (13, 24, 29), and the present results extend these observations and indicate that MP from alveoli and blood, although of common lineage, differ in TNF production in response to RSV infection. Blood MP show smaller changes in RSV-induced TNF activity than autologous alveolar IMP. The differential effect of RSV on alveolar and blood MP production of TNF did not appear to be regulated by PGE2, since RSV alone did not alter PGE2 production by either cell type. Both alveolar and blood MP stimulated with RSV + LPS, or LPS alone, significantly increased PGEz production compared with unexposed controls yet only blood MP costimulated with RSV + LPS demonstrated significantly enhanced PGEz production compared with LPS alone. Thus RSV appears to have unique stimulatory effects on MP production of TNF and PGE2 based in part on the anatomic origin of the MP. The differences in the secretory capacities and regulation of MP functions dependent on their origin may have direct effects on the local immunological response (6, 7, 13, 19). Paramyxovirus infections, which include RSV, induce both humoral and cell-mediated immunological responses that may have a role in the pathogenesis of disease (14). These immunological responses are felt to be initiated by the interaction of virus or viral antigens with MP resulting in the production of immunomodulatory cytokines (25). RSV induces production of IL-l and IL-l inhibitory molecules by blood MP, suggesting that the initiation of the immunological response to this virus may be complex (25). From the results presented here, RSV alone induces alveolar MP to produce TNF that can both directly lyse virus-infected cells (10) and inhibit viral infection in several cell types (16, 33) as well as restrict RSV infection of human blood MP in a dosedependent manner (17). RSV also replicates in alveolar MP, although a subpopulation of these cells remains resistant to viral replication (23). These combined results suggest that TNF produced by RSV-infected alveolar MP may have a critical role in limiting viral infection or modulating the pulmonary inflammatory response to this virus. The excellent technical assistance of Kimberly Lenner, Jo Ann Nelson, and Jill Patterson is appreciated. This work was supported by National Institutes of Health Grants HL-37117, HL-25830, and MO1 RR-00080 and by a research grant from the American Lung Association (to J. R. Panuska). Present address of F. Midulla: IV Cattedra di Clinica Pediatrica, Universita’ di Roma. La Sanienza. Rome. Italv.

PRODUCTION

OF

Address University Received

TNF

for reprint Hospitals, 13 February

AND

L401

PGE2

requests: J. R. Panuska, Airway Disease Center, 2074 Abington Rd., Cleveland, OH 44107. 1990; accepted

in final

form

5 June

1990.

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Virus-induced alterations in macrophage production of tumor necrosis factor and prostaglandin E2.

The cellular immune response to respiratory syncytial virus (RSV) is felt to contribute to viral clearance and/or the inflammation accompanying pulmon...
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