Serotype-Dependent Induction of Pulmonary ... - Journal of Virology

JOURNAL OF VIROLOGY, Oct. 1996, p. 7079–7084 0022-538X/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 70, No. 10

Serotype-Dependent Induction of Pulmonary Neutrophilia and Inflammatory Cytokine Gene Expression by Reovirus ANTHONY L. FARONE,1* CHARLES W. FREVERT,1† MARY B. FARONE,1 MERRIBETH J. MORIN,2‡ BERNARD N. FIELDS,2,4,5§ JOSEPH D. PAULAUSKIS,1 AND LESTER KOBZIK1,3 Department of Environmental Health, Harvard School of Public Health,1 Department of Microbiology and Molecular Genetics, Harvard Medical School,2 and Department of Pathology,3 Shipley Institute of Medicine,4 and Department of Medicine,5 Brigham and Women’s Hospital, Boston, Massachusetts 02115 Received 1 April 1996/Accepted 11 July 1996

Reovirus type 3 Dearing (T3D) causes a prominent neutrophil influx, substantially greater than seen with reovirus type 1 Lang (T1L) in a rat model of viral pneumonia. We sought to measure reovirus-mediated increases in chemokine mRNA expression in pulmonary cells. We found that the neutrophilia induced by T1L and T3D infection in vivo correlated directly with increased levels of chemokine mRNA expression in T3Dinfected compared with those of T1L-infected lungs. In vitro, reovirus-infected normal alveolar macrophages (AMs) and the rat AM cell line NR8383 expressed greater levels of macrophage inflammatory protein 2, KC, and tumor necrosis factor alpha mRNA. A synergism between reovirus and lipopolysaccharide was also detected for macrophage inflammatory protein 2 and KC mRNA expression. Tumor necrosis factor protein secretion was also increased to a greater extent by T3D than by T1L in primary rat AMs and the NR8383 cells. We conclude that the virus-mediated inflammatory cytokine induction suggests a role for these cytokines in the neutrophil influx observed in the rat reovirus pneumonia model. have also been expressed as recombinant proteins and are chemotactic for PMNs in rats both in vitro and in vivo (11, 12). In the studies described here, we first compared the induction of proinflammatory cytokine mRNA and protein expression by T1L and T3D in vivo and in vitro. We found that cytokine mRNA induction and tumor necrosis factor (TNF) protein secretion were greater with T3D than with T1L, suggesting a role for these molecules in the rat pneumonia model.

Viral pneumonitis is characterized in part by a prominent neutrophil (PMN) influx. The mechanisms and mediators regulating the recruitment of PMNs in response to viral infections have not been well characterized. The mammalian reoviruses are nonenveloped, icosahedral viruses with a segmented double-stranded RNA genome (30). These viruses provide an excellent model for understanding PMN recruitment because (i) their pathogenic effects in other organ systems have been well characterized (39), (ii) a rat lung reovirus pneumonia model has been recently described (25), (iii) mammalian reoviruses have been associated with clinical pulmonary infections and have been isolated from humans and other species experiencing respiratory illnesses (39), and (iv) reassortant viruses are available for analysis of gene segment and type contributions to pathogenesis. The rat pneumonitis model showed that both reoviruses type 1 Lang (T1L) and type 3 Dearing (T3D) induce a prominent inflammatory response in acutely infected lungs. However, a nearly threefold increase in PMNs was demonstrated in animals inoculated with reovirus T3D compared with T1L (25). Several inflammatory mediators have been implicated in PMN recruitment during acute pulmonary inflammation (21, 22, 28, 41). The C-X-C family of cytokines mediates PMN chemotaxis to inflammatory sites both in vitro and in vivo (2, 11, 12, 23). In rodents, the two major C-X-C chemokines are KC and macrophage inflammatory protein 2 (MIP-2) (6, 16– 18, 36, 42, 43, 46, 47). In rat models of pulmonary inflammation, induction of KC and MIP-2 mRNA levels in vitro and in vivo in macrophages exposed to bacterial endotoxin or industrial pollutants has been described (8, 16, 17). Both cytokines

MATERIALS AND METHODS Animals. Female, juvenile (25- to 28-day-old), viral antigen-free SpragueDawley rats weighing 75 to 100 g (Taconic Laboratories, Germantown, N.Y.) were used in the pneumonia model. To avoid prior exposure to reovirus, animals were used within 24 h of arrival and housed in filtertop cages in a laboratory separate from other animals. Adult female Sprague-Dawley rats (Charles River Breeding Laboratories, Inc., Wilmington, Mass.) weighing 200 to 250 g were used for collection of primary rat alveolar macrophages (AMs) by bronchoalveolar lavage (BAL). Animals were housed in an air-conditioned room (22 6 28C; relative humidity, 50 6 10%) with a 12-h light-dark cycle and fed conventional laboratory chow and tap water ad libitum. All animal experiments and protocols were approved by the appropriate institutional organizations. Virus. The prototype strains of reovirus T1L and T3D used in these studies have been previously described (27). Reovirus stocks of T1L and T3D were purified and titers were determined as previously described (14, 33). Virus particle number was determined by measurement at an optical density of 260 nm by the method of Smith et al. (35). Virus particle-to-PFU ratios were approximately 1,000:1 for all preparations. All virus stocks were assayed for lipopolysaccharide (LPS) contamination by a modified Limulus amoebocyte assay (24) prior to use in vivo or in vitro (all stocks used had LPS levels of #20 pg/ml). Reovirus pneumonia model. Pneumonia in rats was produced by intratracheal (i.t.) administration of reovirus according to the protocol of Morin et al. (25). Briefly, animals were exposed to vaporized halothane (Halocarbon Laboratories, River Edge, N.J.) until unconscious and i.t. instilled with 108 PFU of reovirus in 0.1 ml of LPS-free Dulbecco’s phosphate-buffered saline (DPBS; Sigma Chemical Co., St. Louis, Mo.), LPS (10 mg/ml; Sigma) in DPBS, or DPBS alone with a blunt-ended, 5-cm catheter. Animals were euthanized 6 or 24 h after infection by intraperitoneal injection of sodium pentobarbital (50 mg/kg; Anpro Pharmaceuticals, Arcadia, Calif.), and BAL or tissue samples were collected. BAL and analysis. BAL was performed with six 2-ml lavages of sterile DPBS. Lavage fluid from each animal was pooled, immediately placed on ice, and subsequently centrifuged (400 3 g) at 48C for 10 min. The supernatants were removed, and the cell pellets were resuspended in 10 ml of DPBS for the determination of cell number and differential staining. Cell samples were diluted to 106/ml in DPBS, and 100 ml was placed in a cytocentrifuge (Shandon, Pittsburgh, Pa.) for preparation of microscope slides. Slides were air dried, fixed in

* Corresponding author. Present address: Biology Department, Middle Tennessee State University, Murfreesboro, TN 37132. Phone: (615) 898-5343. Fax: (615) 898-5093. † Present address: Pulmonary and Critical Care Medicine, VA Medical Center, Seattle, WA 98108. ‡ Present address: Virus Research Institute, Cambridge, MA 02138. § Deceased, January 31, 1995. 7079

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Leukostat (Fisher Scientific, Pittsburgh, Pa.), and stained with Diff-Quik (Baxter, Miami, Fla.). The slides were examined by light microscopy to determine the proportion of PMNs and mononuclear cells. Virus treatment of rat AMs. Rat AMs were collected from adult animals by sterile BAL. Yields and viability were determined by hemacytometer counts and trypan blue exclusion. The cells were cultured at 106/ml in 100-mm-diameter tissue culture dishes in 8 ml of RPMI 1640 supplemented with 0.1% bovine serum albumin and antibiotics (100 U of penicillin per ml and 100 mg of streptomycin per ml; Sigma) overnight at 378C and 5% CO2 to permit cells to adhere. Medium was then aspirated and replaced with complete medium (RPMI 1640 containing 5% fetal equine serum [HyClone Laboratories, Logan, Utah] and antibiotics), and cells were incubated for an additional 24 h prior to treatment. The rat AM cell line NR8383 (15) (generously provided by R. Helmke) was maintained in complete medium at 378C in 5% CO2. For cytokine mRNA expression studies, NR8383 cells or primary macrophages from BAL were incubated as described above with either T1L or T3D at a particle-to-cell ratio of 1,000:1 in DPBS or with DPBS alone. For the time course studies of cytokine mRNA induction, cells were incubated with virus for 12, 18, or 24 h. In a separate experiment to determine the effect of combining virus and LPS, cells were treated with virus for 24 h with LPS (10 mg/ml) in DPBS or with DPBS alone for the last 4 h of incubation. Following incubation, cells were prepared for total RNA extraction by a modified guanidium method (4). Briefly, cells were lysed in 3 ml of guanidine thiocyanate (Fluka, Ronkonkoma, N.Y.)–25 mM sodium citrate (pH 7)–0.5% N-lauryl sarcosine–0.1 M 2-mercaptoethanol (Sigma) and stored at 2708C for RNA isolation. RNA was isolated by layering the mixture on 5.7 M CsCl–0.1 M EDTA and centrifuging it at 47,000 rpm (268,000 3 g) for 4 h. Pelleted RNA was resuspended in diethylpyrocarbonate-treated TE buffer (10 mM Tris, 1 mM EDTA [pH 7.4]) and stored at 2708C. Northern (RNA) blotting and hybridization. Total RNA from rat lungs, primary AMs, or NR8383 cells was denatured, resolved in 0.8% agarose-formaldehyde gels (10 mg per lane), transferred to Nytran filters (Schleicher & Schuell, Keene, N.H.), and UV cross-linked to the membrane. The quantity of RNA loaded in each lane was evaluated by ethidium bromide staining of rRNA or by hybridization with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) DNA as an internal standard (37). We have previously described the MIP-2 and KC DNAs used in this study and the predicted mRNA transcript sizes (16). Human TNF-a and murine interleukin-1a (IL-1) DNAs (19, 26) were also used. Cytokine and GAPDH DNA was labeled by random priming (Life Technologies, Bethesda, Md.) and [32P]dATP (6,000 Ci/mmol). Prehybridization and hybridization were carried out in 0.5 M NaPO4 with 10 mM EDTA, 7% sodium dodecyl sulfate (SDS), and 150 mg of tRNA per ml (5) at 658C. Blots were washed in 0.13 saline sodium citrate (SSC; 13 SSC is 0.15 M NaCl plus 0.015 sodium citrate) with 1% SDS at 378C and twice at 528C for 15 min before autoradiography. Quantitation of cytokine mRNA was achieved by using Desk Scan II (HewlettPackard, Greeley, Colo.), Adobe Photoshop 2.5.1 (Adobe Systems, Inc., Mountain View, Calif.), and NIH Image software (National Institutes of Health, Bethesda, Md.). Individual mRNA values were normalized to that for the GAPDH internal standard to determine differences in cytokine mRNA expression. TNF assay. TNF activity in the treated culture supernatants was measured by a microplate cytotoxicity assay using the WEHI clone 164 cell line (7). Briefly, cells were plated at 4 3 104 per well in 0.2 ml of RPMI 1640 containing 10% fetal bovine serum (HyClone) in 96-well plates (Costar, Cambridge, Mass.) and cultured for 24 h at 378C and 5% CO2. The medium was removed and replaced with serial dilutions of filtered (0.2 mm-pore-size filter) culture supernatants or recombinant human TNF (Biological Response Modifiers Program standard) in RPMI 1640 containing 10% fetal bovine serum and 1 mg of actinomycin D (Sigma) per ml. To determine the effect of introduction of virus in this assay, infectious T1L and T3D virus samples were incubated with the WEHI cells as a control, and no detectable cytotoxicity was exhibited. Following overnight culture, cytotoxicity was determined by addition of the tetrazolium salt XTT (Sigma) and measurement of optical density at 570 nm. TNF activity (nanograms per milliliter) was determined by comparison with the standard curve. Statistical analysis. Data were analyzed by using the statistical software package StatView (Abacus Concepts, Inc., Berkeley, Calif.). Mean separation was accomplished with analysis of variance and the Scheffe F test (3). Statistical significance was considered at P , 0.05.

RESULTS Serotype-dependent induction of pulmonary neutrophil influx. To characterize the response to T1L and T3D viruses in vivo, female Sprague-Dawley rats were intratracheally instilled with 108 PFU of T1L, T3D, or DPBS. The lungs of the animals were lavaged after 24 h, and the numbers of AMs and PMNs were determined (Fig. 1A). As previously reported (25), we found a statistically significantly increased neutrophilia in animals (n 5 3 per group, P , 0.05) receiving T3D compared with T1L. There were no significant increase in the numbers of

J. VIROL.

FIG. 1. Neutrophilia and MIP-2 gene expression in the lungs of animals after treatment with reovirus. (A) Animals (three per group) were instilled i.t. with 108 PFU of reovirus T1L or T3D in 0.1 ml of DPBS or DPBS alone. Cell infiltrate was collected by BAL after 24 h. Neutrophil cell numbers were determined from cytospin counts and averaged. Statistically significant differences in PMN numbers between T3D and T1L are indicated (p). Results are representative of at least two experiments. (B) For chemokine gene expression, rats (one animal per lane) were instilled i.t. with DPBS (lanes 1 to 3), 10 mg of LPS per ml (lane 4), reovirus T1L (lanes 5 to 7), or reovirus T3D (lanes 8 to 10). Virus treatments were with 108 PFU/0.1 ml. After 6 h, total RNA was collected and resolved (10 mg per lane) on 0.8% agarose under denaturing conditions and probed with MIP-2 DNA. Loading of lanes was compared by ethidium bromide (EtBr) staining. Computer-generated images were prepared by using Desk Scan II (Hewlett-Packard) and Adobe Photoshop 2.5.1 (Adobe Systems) software and printed on a Codonics Printer (Toyo Spectrum, Santa Clara, Calif.).

macrophages in any viral treatment group compared with the DPBS control. Reovirus-induced chemokine gene expression in rat lungs. To characterize the chemokine mRNA expression in response to reovirus in vivo, mRNA expression of MIP-2 was determined by Northern analysis (Fig. 1B). Total RNA from lung homogenates of the animals (three per group) was collected after rats were instilled i.t. with 108 PFU of the parental reovirus T1L or T3D. LPS was instilled as a positive control for chemokine mRNA induction, and DPBS was instilled as a negative control. Although PMNs are detected 24 h after virus instillation, no detectable chemokine mRNA was expressed in the lung homogenates at 24 h (data not shown). To determine whether chemokine genes were being expressed at an earlier time point, total RNA was collected at 6 h following virus inoculation. Figure 1B shows that both serotypes of reovirus induced chemokine mRNA expression. Notably, reovirus T3D caused a greater expression of MIP-2 compared with T1L. Reovirus-induced cytokine gene expression in primary rat AMs. Normal rat AMs from adult animals were treated in vitro with T1L, T3D, or DPBS for 24 h. Northern analysis was performed to characterize cytokine mRNA expression in these

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FIG. 3. Inflammatory cytokine mRNA expression and TNF protein expression in NR8383 cells. (A) NR8383 cells were treated with DPBS (lane 1) for 24 h, DPBS for 24 h with the addition of LPS (10 mg/ml) for the final 4 h of incubation (lane 2), reovirus T1L for 24 h (lane 3), reovirus T1L for 24 h with the addition of LPS (10 mg/ml) for the final 4 h of incubation (lane 4), reovirus T3D for 24 h (lane 5), or reovirus T3D for 24 h with the addition of LPS (10 mg/ml) for the final 4 h of incubation (lane 6). All reovirus treatments were at a ratio of 1,000 particles per cell. Total RNA (10 mg per lane) was resolved on 0.8% agarose under denaturing conditions and probed with MIP-2, KC, TNF-a, IL-1, or GAPDH DNA as an internal standard. Results are representative of at least two experiments. Computer-generated images were prepared by using Desk Scan II (Hewlett-Packard) and Adobe Photoshop 2.5.1 (Adobe Systems) software and printed on a Codonics Printer (Toyo Spectrum). (B) Culture supernatants from NR8383 cells treated with DPBS, T1L, or T3D for 24 h were analyzed for TNF bioactivity in a WEHI cytotoxicity assay. TNF activity was calculated from a standard curve, and statistical significance is presented as the mean 6 standard deviation (n 5 4). Statistically significant differences (P , 0.05) between virus treatment and the control are indicated (p).

FIG. 2. Inflammatory cytokine mRNA expression in normal rat AMs. (A) Normal rat AMs were treated with 1,000 virus particles of reovirus T1L or reovirus T3D per cell or DPBS (C [control]) for 24 h, and total RNA was characterized by Northern analysis. RNA (10 mg per lane) was resolved on 0.8% agarose under denaturing conditions and probed with MIP-2, TNF-a, and IL-1 DNAs. Loading was compared by ethidium bromide (EtBr) staining. Results are representative of at least two experiments. Computer-generated images were prepared by using Desk Scan II (Hewlett-Packard) and Adobe Photoshop 2.5.1 (Adobe Systems) software and printed on a Codonics Printer (Toyo Spectrum). (B) Culture supernatants from primary rat AM culture supernatants treated with reovirus T1L or T3D at 1,000 virus particles per cell or DPBS (control) were analyzed for bioactivity in a WEHI cytotoxicity assay. TNF activity was calculated from a standard curve, and statistical significance is presented as the mean 6 standard deviation (n 5 4). Statistically significant differences (P , 0.05) between virus treatment and the control are indicated (p).

cells. Virus treatment of AMs resulted in an induction of inflammatory cytokine mRNA expression whose magnitude was type dependent (Fig. 2A). T3D induced greater expression of MIP-2, TNF-a, and IL-1 mRNAs in AMs compared with T1L. We tested culture supernatants from rat AMs treated with T1L or T3D for TNF protein production (Fig. 2B). Virus treatment of cells resulted in the statistically significant increase in production of TNF by reovirus T3D compared with untreated controls (P , 0.05). However, T1L did not cause a significant increase in TNF production compared with the control levels (P 5 0.4784), consistent with the lack of increased TNF-a mRNA after T1L treatment (Fig. 2A).

Reovirus-induced cytokine gene expression in a rat AM cell line. We sought to test the utility of the rat AM cell line NR8383 to study the differential stimulation of cytokine mRNA expression induced by T1L and T3D. The subcloned NR8383 cell line was derived from rat AMs (15) and has been used in our laboratory for similar experiments demonstrating inflammatory cytokine expression (8). For these studies, cells were treated with T1L, T3D, or DPBS for 24 h. Northern analysis of mRNA expression was conducted, and the results were compared with those for primary rat AMs (Fig. 3A, lanes 1, 3, and 5). MIP-2, KC, and TNF-a mRNA levels were induced to greater levels with reovirus T3D than with T1L. To determine if noninfectious virus could induce chemokine mRNA, UV-inactivated T1L and T3D were used in similar experiments but did not induce detectable levels of MIP-2 or KC mRNA (data not shown). Having established that the NR8383 cells respond to reovirus similarly to primary AMs, we were then able to use these cells to characterize the production of TNF protein in response to T1L and T3D, the response to a combined treatment of virus and LPS, and the time course of chemokine gene induction. Serotype-dependent induction of TNF expression in NR8383 cells. TNF bioactivity from T3D-treated NR8383 cells was statistically significantly greater than the bioactivity seen after T1L treatment (Fig. 3B). Although the amount of TNF bioactivity from NR8383 cells was approximately 2-fold higher than in the primary AMs, both cell types treated with T3D exhibited an approximate 10-fold increase in bioactivity compared with T1L-treated cells. These results indicate again that the response of the NR8383 cells to T1L and T3D treatment follows a pattern similar to that for the primary AMs. Inflammatory cytokine mRNA response in NR8383 cells to reovirus and LPS. Because many viral pneumonias are followed by the onset of secondary bacterial infections, resulting

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FIG. 4. Time course of chemokine mRNA induction by reovirus in the rat AM cell line NR8383. NR8383 cells were treated with DPBS, reovirus T1L, or T3D for 12, 18, and 24 h. Virus treatments were at a ratio of 1,000 particles per cell. Total RNA (10 mg per lane) was resolved on 0.8% agarose under denaturing conditions and probed with MIP-2 or KC DNA. Equivalent loading was confirmed by ethidium bromide (EtBr) staining. Results are representative of at least two experiments. Computer-generated images were prepared by using Desk Scan II (Hewlett-Packard) and Adobe Photoshop 2.5.1 (Adobe Systems) software and printed on a Codonics Printer (Toyo Spectrum).

in a more severe inflammatory response (20), we wanted to assess whether a combination of reovirus and LPS would produce an enhancement of cytokine mRNA expression in rat AMs. NR8383 cells were treated with T1L, T3D, or DPBS for 24 h. LPS at 10 mg/ml in DPBS or DPBS alone was added for the last 4 h of the incubation. Combined treatment with reovirus and LPS produced a similar type-dependent effect in the NR8383 cells when total RNA was analyzed for chemokine and inflammatory cytokine mRNA expression; however, both T1L and T3D acted synergistically with LPS to dramatically enhance levels of MIP-2 and KC (Fig. 3A, lanes 4 and 6). Scanning densitometry of the Northern blots showed that T1L and T3D induced twofold increases in MIP-2 and threefold increases in KC. The combination of virus and LPS resulted in additive effects for TNF-a and IL-1 mRNA expression. Time course of chemokine mRNA expression in NR8383 cells following virus treatment. To determine the time course of chemokine mRNA expression, the NR8383 cell line was treated with T1L, T3D, or DPBS for 12, 18, and 24 h. mRNA expression was detected by Northern analysis. A similar typedependent effect was observed in the NR8383 cells at all time points, with MIP-2 and KC mRNA expression first detectable at 18 h and increasing through 24 h (Fig. 4). Chemokine expression at earlier time points was also measured, with no detectable signal at 4 h (data not shown). Cell viabilities were determined at all time points, and no statistically significant differences were seen between T1L- and T3D-treated cells at any time point. Cytokine mRNA expression after 24 h was not measured because of low cell viability in virus-treated cultures. DISCUSSION We have identified a possible role for inflammatory cytokines in the PMN influx observed during reovirus-induced pneumonia in the rat. In the reovirus pneumonia model, reovirus T3D induces a greater neutrophilia than T1L (25). The C-X-C chemokine MIP-2 has been shown to be an important mediator of PMN influx into the lungs of rats (11). We sought to determine whether T3D induced greater levels of MIP-2 cytokine mRNA in the lungs of reovirus-treated animals (Fig. 1B). Reovirus T3D induced greater levels of MIP-2 mRNA than T1L in the lungs 6 h following instillation of virus. This induction of chemokine mRNA correlated with a subsequent serotype-dependent neutrophilia from BAL fluids of virus-

J. VIROL.

treated animals at 24 h (Fig. 1A). Another rat neutrophil chemotactic C-X-C chemokine, KC, also followed a similar type-dependent pattern of mRNA expression (data not shown). These data suggest that the cytokines MIP-2 and KC may be partially responsible for the in vivo observation of a greater PMN influx into reovirus T3D-treated animals. Primary rat AMs treated with T1L or T3D followed a similar serotype-dependent pattern of chemokine mRNA expression (Fig. 2A). However, this induction occurred after 24 h of virus treatment in vitro, indicating that other cells may be involved in the more rapid induction of these cytokines in vivo. The cytokines TNF and IL-1 may indirectly increase PMN recruitment into the air spaces of the lungs by stimulating epithelial cells, fibroblasts, and endothelial cells to produce chemotactic cytokines (1, 13, 29, 34). Both of these cytokine mRNAs were also induced to greater levels by reovirus T3D than by T1L in the primary rat AMs (Fig. 2A), suggesting that TNF and IL-1 may also contribute to the PMN response seen in the rat lung in vivo. When supernatants from these virus treatments were assayed for TNF bioactivity, only supernatants from T3Dtreated rat AMs contained significant increased levels of TNF (Fig. 2B). The induction of TNF-a has also been reported in reovirus T3D-treated murine peritoneal macrophages (9). We then sought to test the utility of the rat AM cell line NR8383 in characterizing the inflammatory cytokine response to reovirus. Our laboratory has previously used the NR8383 cells to characterize the cytokine mRNA expression stimulated by LPS (8). In these experiments, the NR8383 cells were treated with reovirus T1L or T3D. MIP-2, KC, TNF-a, and IL-1 mRNA expression was stimulated in a reovirus T3Ddependent pattern (Fig. 3A). Because the NR8383 cells produced patterns of cytokine expression similar to those of the primary AMs, this cell line provided a convenient in vitro system for further analysis of the cytokine induction by reovirus. Because many viral pneumonias are followed by the onset of secondary bacterial infections, resulting in a more severe inflammatory response, we wanted to assess whether a combination of reovirus and LPS would produce an enhancement of cytokine mRNA expression. Results from these studies suggest that although the type-dependent difference remained, both T1L and T3D showed a dramatic enhancement of chemokine mRNA expression when cells were treated with LPS after virus (Fig. 3A). T1L and T3D exhibited a synergistic effect when combined with LPS in the induction of MIP-2 and KC mRNA in the NR8383 cells. Future studies are needed to investigate a possible synergism of reovirus and LPS in vivo. To determine whether 24 h was the optimal time for detection of chemotactic cytokine gene expression by reovirus, mRNA expression was analyzed at earlier treatment intervals. These studies indicated that the earliest consistent detection of MIP-2 gene expression occurred at 18 h and that mRNA levels increased through 24 h (Fig. 4). The T1L- and T3D-treated cells exhibited similar viabilities from 4 to 24 h following virus treatment, indicating that differential cytopathic effects are not responsible for differences in gene expression. The time course of this induction in response to reovirus is delayed compared with chemotactic cytokine gene expression induced by LPS and industrial pollutants (8, 17), in which case expression is seen after 4 h. A 4-h treatment with either T1L or T3D did not induce any significant chemokine mRNA (data not shown). This finding suggests that reovirus may induce this gene by a different pathway. We also determined the minimum dose of virus necessary to stimulate a detectable level of cytokine mRNA expression (virus particle-to-cell ratio of 1,000:1) (data not shown), which was consistent with previous results (9)

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showing that similar doses of virus were necessary to induce TNF-a protein expression. To determine if infectious virus is necessary to induce chemokine gene expression, UV-inactivated T1L and T3D were used to treat the rat AM cell line. However, when cells were treated with UV-inactivated virus at particle/cell ratios similar to those for the infectious virus, chemokine mRNA was not detected (data not shown). These results correlate well with the results of Morin et al. (25), which suggest that infectious virus is necessary to induce pneumonia. To further characterize the mechanisms occurring during the virus-mediated induction of cytokines, we are currently investigating the contribution of the S1 gene segment because the serotype difference in the PMN response was mapped primarily to the S1 gene of reovirus (25) and because of its importance in pathogenesis (31, 38, 40, 44, 45) and the immune response to the virus (10, 32). Future studies will use a panel of reassortants generated from T1L and T3D, containing individual intertypic gene segments, as well as combinations of the S1 gene and other segments. These reassortants will be used to further define the viral genes responsible for the type differences in cytokine induction and neutrophilia in the rat reovirus pneumonia model. The interactions between viruses and cells of the immune system, particularly the mediators which signal phagocytic cells that virus is present, are not well understood. We have shown here the induction of chemotactic cytokines by reovirus and the effectiveness of a model for exploring interactions in the lungs between reovirus and cells of the immune system.

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ACKNOWLEDGMENTS This research was supported in part by National Institutes of Health grants HL19170, ES-00002, ES-05947, HL-07118, EPA1P42ES05947, and PHS5R37AI13178 and by a Parker B. Francis Fellowship in Pulmonary Research to J. Paulauskis. We thank Hadi Danaee and Roger Morey for technical assistance with this project.

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