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Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, 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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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secreting cells (18, 19, 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 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 x 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 x g, 1 h, 10°C). The pellet was resuspended in PBS, aliquoted, and frozen at -70°C. The suspension was adjusted to contain 4 x 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 µl 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 µl 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).
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 -20°C. The concentrations of
IFN-, 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--(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-- (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 peroxidase (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 ± SD except for values of airway responsiveness (Penh), which are expressed as the mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 ± 1.4-fold over PBS in infected mice compared
with a 3.4 ± 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 RSV-induced AHR. In fact,
the dose-response curve to MCh was shifted to the left compared with
nondepleted, RSV-infected mice (Fig. 1).
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To investigate changes in pulmonary inflammatory cells during
acute RSV infection, lung cells were isolated and differential cell
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.
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We measured the levels of IFN-, IL-4, and IL-5 in BAL fluid
collected on day 7 postinfection. In RSV-infected controls, levels of
IL-5 and IFN- 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-
levels were also decreased. IL-5 treatment of these mice did not
increase IL-5 or IFN- levels in BAL fluid (data not shown). IL-4 was
not detectable in any of the groups.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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- and IL-5 were detected in BAL fluid following RSV
infection. The increases in IFN- 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
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- 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- 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- producers that balance Th2 type immune responses to
RSV (34, 35, 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, 41, 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- 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 demonstrated to induce lung eosinophilia
in models of murine lymphocytic choriomeningitis virus infection (21)
and in allergen-induced 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 RSV-induced 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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Footnotes |
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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:
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.
Received for publication September 1, 1998. Accepted for publication December 30, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) cytokines in bronchoalveolar lavage and bronchial biopsies from atopic asthmatics and normal control subjects. Am. J. Respir. Cell Mol. Biol. 12:477.[Abstract]
/ß and /
T cell subsets in viral immunity. Annu. Rev. Immunol. 10:123.[Medline]
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, aCD8/RSV+IL5 vs aCD8/RSV;
¶, aCD8/RSV+IL5 vs RSV.
