CD8 T Cells Are Essential in the Development of Respiratory Syncytial Virus-Induced Lung Eosinophilia and Airway Hyperresponsiveness1 Ju¨rgen Schwarze, Grzegorz Cieslewicz, Anthony Joetham, Toshihide Ikemura, Eckard Hamelmann, and Erwin W. Gelfand2 Viral respiratory infections can cause bronchial hyperresponsiveness and exacerbate asthma. In mice, respiratory syncytial virus (RSV) infection results in airway hyperresponsiveness (AHR) and eosinophil influx into the airways. The immune cell requirements for these responses to RSV infection are not well defined. To delineate the role of CD8 T cells in the development of RSV-induced AHR and lung eosinophilia, we tested the ability of mice depleted of CD8 T cells to develop these symptoms of RSV infection. BALB/c mice were depleted of CD8 T cells using anti-CD8 Ab treatment before intranasal administration of infectious RSV. Six days postinfection, airway responsiveness to inhaled methacholine was assessed by barometric body plethysmography, and numbers of lung eosinophils and levels of IFN-g, IL-4, and IL-5 in bronchoalveolar lavage fluid were monitored. RSV infection resulted in airway eosinophilia and AHR in control mice, but not in CD8-depleted animals. Further, whereas RSV-infected mice secreted increased amounts of IL-5 into the airways as compared with noninfected controls, no IL-5 was detectable in both bronchoalveolar lavage fluid and culture supernatants from CD8-depleted animals. Treatment of CD8-depleted mice with IL-5 fully restored both lung eosinophilia and AHR. We conclude that CD8 T cells are essential for the influx of eosinophils into the lung and the development of AHR in response to RSV infection. The Journal of Immunology, 1999, 162: 4207– 4211.
A
sthma exacerbations and bronchial hyperreactivity in nonasthmatics can be triggered by viral respiratory tract infections (1, 2). Respiratory syncytial virus (RSV)3 is the most common respiratory virus in infants. It has been implicated in the development of childhood asthma (3, 4). The immune cell requirements for the development of airway inflammation and bronchial hyperresponsiveness triggered by viral respiratory infections are not well defined. There are several lines of evidence implicating T cells in the development of asthma. In asthmatics, increased numbers of activated T cells in the peripheral blood (5) and recruitment of both CD41 and CD81 T cells to the airways has been demonstrated (6, 7). In atopic asthma, increases in Th2 type cells in the airways may be of particular importance in inducing eosinophilic inflammation and bronchial hyperresponsiveness (8, 9). In murine models, a role for T cells (10) and for both CD41 (11) and CD81 T cell subsets (12) in the development of allergic airway inflammation and airway hyperresponsiveness (AHR) has been demonstrated. Both cell types have been shown to be capable of producing Th2 type cytokines, especially IL-5 (13,
Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206 Received for publication September 1, 1998. Accepted for publication December 30, 1998. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Grants HL-36577 and HL-61005 from the National Institutes of Health. J.S. was supported by the Deutsche Forschungs-gemeinschaft (Schw 597/1-1). 2 Address correspondence and reprint requests to Dr. Erwin W. Gelfand, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail address:
[email protected] 3 Abbreviations used in this paper: RSV, respiratory syncytial virus; AHR, airway hyperresponsiveness; BAL, bronchoalveolar lavage; MCh, methacholine; PBLN, peribronchial lymph nodes; PFU, plaque-forming units; aCD8, anti-CD8.
Copyright © 1999 by The American Association of Immunologists
14), which is thought to play a central role in eosinophil recruitment to the lung and development of AHR in allergic airway inflammation (Refs. 10, 15, and 16; reviewed in Ref. 17). Viral infections elicit strong CD8 T cell responses and these CD8 T cells are predominantly cytotoxic, IFN-g secreting cells (18 –20). Interestingly, recent studies demonstrated that a Th2 cytokine environment can transform virus-specific CD8 T cells from cytotoxic cells into noncytotoxic, IL-5-producing cells in vitro (14) and in vivo (21). These latter CD8 T cells were able to induce airway eosinophilia. This suggests that CD8 T cells may also play a role in the development of virus-induced AHR. To investigate the cellular mechanisms linking viral respiratory infections to the development of AHR, we employed a murine model of acute RSV infection that allows the investigation of airway responsiveness, the assessment of pulmonary inflammation, and the study of local cytokine production in the bronchoalveolar lavage (BAL) (22). We reported recently in this model that acute RSV infection results in eosinophil and neutrophil influx into the lung and in AHR to inhaled methacholine (MCh). Here, we use this model to define the role of CD8 T cells in the development of RSV-induced AHR.
Materials and Methods Animals Female BALB/c mice, 8 –12 wk of age, free of specific pathogens, were obtained from The Jackson Laboratories (Bar Harbor, ME). All experimental animals used in this study were under a protocol approved by the Institutional Animal Care and Use Committee of the National Jewish Medical and Research Center.
Virus Human RSV A (Long strain), free of chlamydia or mycoplasma contamination, was obtained from the Viral Diagnostics Laboratory, Health Sciences Center, University of Colorado (Denver, CO). The virus was cultured on Hep 2 cells from American Type Culture Collection (Manassas, VA) in medium containing FCS from Life Technologies (Grand Island, NY). It 0022-1767/99/$02.00
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ESSENTIAL ROLE FOR CD8 T CELLS IN RSV-INDUCED AHR
was purified as described (23). Briefly, cells and supernatant were harvested, the cells were disrupted by ultrasonic manipulation, and the suspension was clarified by centrifugation (8000 3 g, 20 min). The supernatant was layered over 30% sucrose in STEU buffer (sodium chloride 0.1 M, Tris 0.01 M, EDTA 0.001 M, and urea 1 M, all obtained from Sigma (St. Louis, MO)) and centrifuged (100,000 3 g, 1 h, 10°C). The pellet was resuspended in PBS, aliquoted, and frozen at 270°C. The suspension was adjusted to contain 4 3 106 plaque-forming units (PFU) of RSV/ml as assessed by quantitative plaque-forming assay.
Infection of mice Mice were infected under light anesthesia (avertin 2.5%, 0.015 ml/g body weight) by intranasal inoculation of RSV (105 PFU in 50 ml PBS). Controls were sham infected with PBS in the same way. Efficacy of this infection procedure was regularly tested by qualitative plaque-forming assays (24): briefly, on day 4 postinfection mice were sacrificed, the lungs were removed, homogenized, centrifuged, and the supernatant was added to Hep 2 cell cultures. Infection could be demonstrated by cell pathogenic effects in all infected animals tested but not in mice sham infected with PBS.
Experimental protocols Mice were infected on day 0. Airway responsiveness was assessed on day 6 postinfection, and animals were sacrificed the following day for BAL and the removal of peribronchial lymph nodes (PBLN) and lungs. To deplete CD8 T cells, mice were treated i.p. with 200 ml of ascites fluid containing anti-CD8 (aCD8) from clone YTS 169 (kindly provided by Dr. Terry Potter, National Jewish Medical and Research Center, Denver, CO) 6, 4, and 2 days before and 3 days after infection. Control animals were injected with 0.2 mg rat IgG (Sigma) at the same time points. A group of RSV-infected mice, depleted of CD8 T cells, were treated i.v. with murine IL-5 (40 ng/dose) on the day of infection and 3 days after infection. Murine IL-5 was kindly provided by Dr. J. Lee (Mayo Clinic, Scottsdale, AZ).
FIGURE 1. Depletion of CD8 T cells during acute RSV infection prevents development of AHR, which is restored by IL-5 treatment. BALB/c mice depleted of CD8 T cells (aCD8/RSV, n 5 12) or normal controls (RSV, n 5 12) were infected with RSV. Sham infection with PBS served as a control (C, n 5 12). Some CD8-depleted, RSV-infected mice were treated with IL-5 during the infection (aCD8/RSV1IL5, n 5 8). On day 6 postinfection, airway responsiveness to increasing concentrations of inhaled MCh (3–50 mg/ml) was assessed by barometric body plethysmography and Penh values were calculated. Baseline Penh values in the mice did not differ between the groups (C, 0.9 6 0.16; RSV, 0.86 6 0.12; aCD8/RSV, 0.87 6 0.08; aCD8/RSV/IL-5, 0.83 6 0.07). Means 6 SEM of Penh values from three independent experiments are expressed as the percentage of baseline Penh values observed after PBS exposure. Significant differences (p , 0.05) were noted for: p, RSV vs C and aCD8/RSV; ‡, aCD8/RSV1IL5 vs aCD8/RSV; ¶, aCD8/RSV1IL5 vs RSV.
FACS analysis To determine the extent of CD8 T cell depletion, PBLN cells were harvested on day 7 postinfection, and mononuclear cells were purified by passing the tissue through a stainless steel mesh, followed by density gradient centrifugation (Organon Teknika, Durham, NC). For FACS analysis, cells were pooled from four mice per group and incubated in staining buffer (PBS, 2% FCS, 0.2% sodium azide) with anti-CD4 (FITC-RM4-4, PharMingen, San Diego, CA) or anti-CD8 (FITC-53-6.7, PharMingen) at 4°C. Stained cells were analyzed using an Epics cytofluorograph (Coulter Electronics, Hialeah, FL).
Determination of airway responsiveness Airway responsiveness was assessed using a single-chamber whole-body plethysmograph obtained from Buxco (Troy, NY) as described (25). Penh was used as the measure of airway responsiveness in this study. In the plethysmograph, mice were exposed for 3 min to nebulized PBS and subsequently to increasing concentrations of nebulized MCh (Sigma) in PBS using an AeroSonic ultrasonic nebulizer (DeVilbiss, Sommerset, PA). After each nebulization, recordings were taken for 3 min. The Penh values measured during each 3-min sequence were averaged and are expressed for each MCh concentration as the percentage of baseline Penh values following PBS exposure.
Lung cell isolation Lung cells were isolated by collagenase digestion as previously described (26) and counted with a hemocytometer. Cytospin slides were stained with Leukostat from Fisher Diagnostics (Pittsburgh, PA), and differential cell counts were performed in a blinded fashion by counting at least 300 cells under light microscopy.
Cytokine assessment in BAL fluid Tracheas were dissected, and a lung lavage with 1 ml of HBSS (Life Technologies) was performed. Cells in BAL fluid were pelleted, and supernatants were frozen at 220°C. The concentrations of IFN-g, IL-4, and IL-5 in BAL fluid and culture supernatants were assessed by ELISA as described (27). Briefly, Immulon-2 plates from Dynatech (Chantilly, VA) were coated with anti-IFN-g-(R4-6A2, PharMingen), anti-IL-4- (11B11, PharMingen) or anti-IL-5-Abs (TRFK-5, Dr. R. Coffman, Palo Alto, Ca) and blocked with PBS/10% FCS overnight. Samples were added, biotinylated anti-IFN-g- (XMG 1.2, PharMingen), anti-IL-4- (BVD6-24G2, PharMingen), or anti-IL-5- Abs (TRFK-4, PharMingen) were used as detecting Abs, and the reactions were amplified with avidin-horseradish per-
oxidase (Sigma). Cytokine levels were calculated by comparison with known cytokine standards (PharMingen). The limit of detection in the assay was 4 pg/ml for each cytokine.
Statistical analysis Groups were compared by Tukey-Kramer HSD test. Values of p were considered significant at 0.05. Values for all measurements are expressed as the mean 6 SD except for values of airway responsiveness (Penh), which are expressed as the mean 6 SEM.
Results Depletion of CD8 T cells prevents the development of AHR following RSV infection Anti-CD8 treatment of BALB/c mice resulted in 97% depletion of CD8 T cells in PBLN, while treatment with rat IgG as a control did not affect numbers of CD8 T cells. Numbers of CD4 T cells were not affected by either treatment. Following depletion, mice were infected by intranasal instillation of RSV (105 PFU). The airway response to MCh in mice infected with RSV and in sham-infected controls was assessed by barometric whole-body plethysmography on day 6 of the acute infection. The airways of nondepleted mice infected with RSV were significantly more reactive than the airways of sham-infected controls (Fig. 1). Penh in response to 50 mg/ml MCh increased 7.6 6 1.4-fold over PBS in infected mice compared with a 3.4 6 0.6-fold increase in mice sham infected with PBS. In contrast, in mice depleted of CD8 T cells, acute RSV infection did not result in increased airway responsiveness to MCh. Treatment of CD8-depleted mice with IL-5 fully restored RSVinduced AHR. In fact, the dose-response curve to MCh was shifted to the left compared with nondepleted, RSV-infected mice (Fig. 1). Acute RSV infection does not result in influx of eosinophils to the lung in mice depleted of CD8 T cells To investigate changes in pulmonary inflammatory cells during acute RSV infection, lung cells were isolated and differential cell
The Journal of Immunology
4209 Depletion of CD8 T cells results in decreased IL-5 levels in BAL fluid We measured the levels of IFN-g, IL-4, and IL-5 in BAL fluid collected on day 7 postinfection. In RSV-infected controls, levels of IL-5 and IFN-g in BAL fluid were significantly increased (Fig. 3). Following depletion of CD8 T cells in RSV-infected mice, no IL-5 could be detected in BAL fluid and IFN-g levels were also decreased. IL-5 treatment of these mice did not increase IL-5 or IFN-g levels in BAL fluid (data not shown). IL-4 was not detectable in any of the groups.
Discussion
FIGURE 2. Eosinophil influx into the lung during acute RSV infection is prevented by CD8 depletion and restored by IL-5 treatment. Lung cells were isolated from the same mice 7 days after sham infection (C, n 5 12) or RSV infection (RSV, n 5 12) without or with CD8 depletion (aCD8/ RSV, n 5 12) and IL-5 treatment (aCD8/RSV1IL5, n 5 8). Numbers of eosinophils and neutrophils per lung were determined. Illustrated are means 6 SD of the numbers of these cells from three independent experiments. Significant differences (p , 0.05) were noted for: p, RSV vs C and aCD8/RSV; ‡, aCD8/RSV1IL5 vs all other groups.
counts performed. In BALB/c mice acutely infected with RSV, the numbers of eosinophils and neutrophils were significantly increased in lung cell isolates compared with sham-infected controls (Fig. 2). The increases in numbers of eosinophils and neutrophils were 2.6- and 1.8-fold, respectively. The total numbers of isolated lung cells did not differ significantly between the groups. In contrast, in mice depleted of CD8 T cells, no increase in numbers of pulmonary eosinophils was observed, whereas RSV infection still resulted in a small increase in numbers of neutrophils in the lung. Treatment of RSV-infected, CD8-depleted mice with IL-5 resulted in eosinophil influx into the lung even exceeding the numbers observed in nondepleted, RSV-infected controls. Administration of IL-5 did not result in increased numbers of lung neutrophils.
In the present study, we monitored airway responsiveness, pulmonary inflammation, and local cytokine production in a murine model of RSV infection. We recently reported that RSV infection results in AHR and eosinophil influx into the lung in this model (22). Here, we used this approach to address the question whether virus-induced AHR is dependent on the presence of CD8 T cells. To address this issue, we compared the responses to RSV infection in CD8 depleted and nondepleted mice. Airway responsiveness to aerosolized MCh was assessed using barometric whole-body plethysmography in unrestrained animals, and pulmonary inflammation and cytokine levels in BAL fluid were monitored. Acute RSV infection in normal BALB/c mice resulted in significant increases in airway responsiveness to MCh, and this was associated with the infiltration of both eosinophils and neutrophils in the lung. These findings parallel those reported previously in BALB/c mice (22, 28) and in other models of respiratory tract viral infection (29). Increased levels of IFN-g and IL-5 were detected in BAL fluid following RSV infection. The increases in IFN- g levels in BAL fluid during acute RSV infection are in keeping with observations made earlier that showed increased production of total IFN in BAL fluid from RSV-infected mice (30) and our own previous findings in cultures of PBLN cells (22). The increase in IL-5 in BAL fluid in RSV-infected mice may be more physiologically relevant to the development of airway eosinophilia than decreases in IL-5 production in PBLN cell cultures in acute RSV infection that we reported previously (22). Depletion of CD8 T cells following anti-CD8 was very effective, resulting in almost complete
FIGURE 3. Depletion of CD8 T cells during RSV infection results in decreased levels of IL-5 and IFN-g in BAL fluid. BAL was performed in the same CD8-depleted (aCD8/RSV, n 5 12) or normal mice (RSV, n 5 12) 7 days after RSV infection or following sham infection (C, n 5 12). Concentrations of IFN-g and IL-5 were determined in BAL fluid by ELISA. Illustrated are means 6 SD of the cytokine concentrations from three independent experiments. p, Significant differences (p , 0.05) were noted for RSV vs C and aCD8/RSV.
4210 elimination of CD8 T cells. As a consequence, RSV infection of CD8-depleted mice did not result in the influx of eosinophils into the lung nor in the development of AHR. This was not due to a lack of infection because RSV infection in the lungs could be demonstrated by plaque-forming assay (data not shown) and still resulted in increased production of IFN-g in these animals. The slight reduction in neutrophil influx into the lung in these mice may be secondary to the lack of eosinophil influx, because activated eosinophils are a source of IL-8 (31), a strong neutrophil chemoattractant (32, 33). CD8 depletion also altered the local cytokine profile following RSV infection by preventing IL-5 secretion into the BAL fluid and the production of IL-5 by cultured and stimulated PBLN cells. These data indicate that CD8 T cells are either a major source of IL-5, as has been shown in other models (12, 14, 21), or that they stimulate other cells to secrete IL-5. In addition, IFN-g was not detectable in the BAL fluid of CD8-depleted, RSV-infected mice. This observation parallels other reports suggesting that during RSV infection, CD8 T cells are IFN-g producers that balance Th2 type immune responses to RSV (34 –36). The cytokine data imply that the lack of CD8 T cells results in an inhibition of local IL-5 production in the airways. IL-5 appears critical for RSV-induced (22) and allergen-triggered (10, 15, 16) eosinophil recruitment to the lung and for the development of AHR as recent data indicate. If true, then predictably administration of IL-5 should render CD8-depleted mice susceptible to the effects of RSV infection. Reconstitution of CD8-depleted mice with IL-5 during RSV infection restored both lung eosinophilia and AHR. The eosinophil influx into the lung of these mice exceeded the levels of eosinophilia in nondepleted, RSV-infected mice possibly due to higher IL-5 levels following treatment than may have been generated following RSV infection. The results of the present study contrast with two recently published reports demonstrating the ability of CD8 T cells to inhibit Th2-type cytokine production and to prevent pulmonary eosinophilia in a model of RSV infection (35, 36). The model employed in these studies provides insight into the secondary response to isolated RSV Ags, because mice were first immunized with recombinant vaccinia virus expressing single RSV proteins before challenge with live RSV infection. In our model, a primary infection with infectious RSV was used in which several RSV Ags are capable of inducing immune responses. Used separately for immunization, different RSV Ags may induce divergent immune responses upon subsequent live RSV challenge. Immunization with the two major glycoproteins of RSV, RSV-F, a fusion protein, and RSV-G, an attachment protein, has been studied extensively. RSV-F induces cytotoxic CD8 T cells (34) and Th1 CD4 T cells (37, 38), resulting in a mononuclear cell infiltrate in the lungs with little pathology. In contrast, RSV-G induces no CD8 T cells (39) but Th2 CD4 T cells (40 – 42), resulting in lung eosinophilia and severe pathology. The RSV-G-induced immune response seems to be dominant over the response to RSV-F as indicated by simultaneous adoptive transfer of T cell lines specific to either Ag (40). Primary infection with live RSV containing these two but likely other Ags also may trigger both a cytotoxic CD8 T cell response and increased IFN-g production as well as induction of Th2 CD4 T cells (for example, in response to RSV-G). The latter cell population (through IL-4) could result in the expansion of a population of IL-5-producing CD8 T cells. Indeed, the changes in the cytokine profile in BAL fluid following CD8 depletion and RSV infection observed in this study imply that two distinct populations of CD8 T cells may be present during RSV infection: one inducing the production of Th1-type cytokines and the other release of Th2-type cytokines. CD8 T cells producing Th2-type cytokines have been
ESSENTIAL ROLE FOR CD8 T CELLS IN RSV-INDUCED AHR demonstrated to induce lung eosinophilia in models of murine lymphocytic choriomeningitis virus infection (21) and in allergeninduced AHR (12) and could play a similar role in RSV infection. Interestingly, CD8 T cells primed to produce IL-4 and IL-5 can evolve into stable memory cells that will exhibit a “Th2” phenotype upon restimulation (43). Such memory cells might be an important trigger of eosinophilic airway inflammation and AHR in repeated viral infections of the respiratory tract. In summary, we present a murine model of airway inflammation and AHR following acute RSV infection. Using mice depleted of CD8 T cells, we show that these animals fail to develop RSVinduced lung eosinophilia and AHR, establishing the role of CD8 T cells in the development of altered airway responsiveness to acute RSV infection. Further, diminished IL-5 production in the airways following CD8 depletion and the ability of exogenous IL-5 to render CD8-depleted mice susceptible to the effects of RSV infection indicates that CD8 T cells exert their effect on lung eosinophilia and AHR via IL-5 in response to RSV.
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