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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 IntroductionMaterial and MethodsResultsDiscussionReferences |
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, IL-4, and IL-5 in PBLN cell
cultures were monitored. Transfer of T cells from RSV-infected mice
resulted in increased eosinophil influx into the lungs, increased IL-5
production, and development of AHR following airway sensitization to
allergen. Transfer of CD8 but not CD4 T cells from the PBLN of
RSV-infected mice also resulted in AHR following 10 days of OVA
exposure. Further, depletion of CD8 T cells prevented these
consequences of RSV infection while CD4 T cell depletion reduced them.
We conclude that T cells, in particular CD8 T cells, are critical in
mediating RSV-induced development of lung eosinophilia and AHR
following allergic airway sensitization. |
Introduction |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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To delineate the role of T cells in this model we used two approaches. In one approach, we adoptively transferred T cells from the peribronchial lymph nodes (PBLN), the draining lymph nodes of the lung, from RSV-infected mice into naive animals which were then sensitized to OVA via the airways. In a second approach, we depleted mice of CD4 or CD8 T cells following the acute phase of the RSV infection but prior to airway sensitization. Following airway sensitization to allergen in either experimental approach, airway responsiveness to inhaled methacholine (MCh) was assessed using barometric whole body plethysmography and influx of inflammatory cells into the lungs and cytokine production in PBLN cell cultures was monitored.
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Material and Methods |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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Female BALB/c mice, 8–12 wk of age, free of specific pathogens, and obtained from The Jackson Laboratory (Bar Harbor, ME), were maintained on OVA-free diets. 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 respiratory syncytial virus 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 the American Type Culture Collection (ATCC; Manassas, VA) in medium containing FCS from Life Technologies (Grand Island, NY). The virus was purified as described (16). Briefly, cells and supernatant were harvested, the cells were disrupted by ultrasonic manipulation, and the suspension was clarified by centrifugation (8000 x g for 20 min). The supernatant was layered over 30% sucrose in STEU buffer (0.1 M sodium chloride, 0.01 M Tris, 0.001 M EDTA, and 1 M urea, all obtained from Sigma (St. Louis, MO)) and centrifuged 100,000 x g for 1 h at 10°C. The pellet was resuspended in PBS, aliquoted, and frozen at -70°C. The suspension was adjusted to contain 4 x 106 PFU of RSV/ml as assessed by quantitative plaque-forming assay.
Infection of mice
Mice were infected under light anesthesia (2.5% Avertin (Aldrich, Milwaukee, WI), 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 (17): briefly, on day 4 and day 8 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 on day 4 but not in mice sham-infected with PBS. On day 8 live virus could not be isolated anymore from the lungs of RSV-infected mice. No difference was detected between RSV-infected mice treated with anti-CD4, anti-CD8 or control Ab.
Experimental protocols
Mice were infected with RSV or sham-infected on day 0. Seven or 14 days postinfection, mice were sacrificed, PBLN was harvested, mononuclear cells were (MNC) isolated, and T cells were purified. MNC (107/mouse) or T cells (5 x 106/mouse) were adoptively transferred into naive mice by i.v. injection. Control T cells were purified (>90% CD3+ T cells) from spleens of naive mice, because only small numbers of T cells, insufficient for transfer, could be isolated from the PBLN of noninfected mice. In additional experiments, CD4+ and CD8+ T cells were isolated from the PBLN of infected mice using magnetic beads 14 days after RSV exposure. These cells were >96% CD4 or CD8, respectively, with no cross contamination. In these experiments, 1.5 x 106 cells were transferred. Twenty-four hours after cell transfer, mice were exposed to OVA (Sigma) via the airways for 10 consecutive days using an AeroSonic Nebulizer 5000 D from DeVilbiss (Sommerset, PA), as described (1%, in 7 ml PBS, 20 min/day) (18). A control group of mice was not exposed to allergen following adoptive cell transfer.
In a separate set of experiments, mice were infected with RSV and depleted of CD4 or CD8 T cells by i.p. injection of anti-CD4 from clone GK 1.5 (200 µg/dose) obtained from ATCC, or from ascites containing anti-CD8 from clone YTS 169 (200 µl/dose), kindly provided by Dr. Terry Potter (National Jewish Medical and Research Center, Denver, CO). Rat IgG was used as a control. The Abs were injected on days 15, 17, and 19 postinfection. From day 21 to day 30 postinfection, mice were exposed to OVA by inhalation as described above. In an attempt to sustain depletion during sensitization, mice were further treated with the respective Abs on days 23, 26, and 29 postinfection using the same doses as prior to sensitization. The extent of CD4 or CD8 T cell depletion was determined in PBLN cells harvested on day 21 postinfection before sensitization and on day 32 following sensitization. MNC were incubated in staining buffer (PBS, 2% FCS, and 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). In both experimental approaches, airway responsiveness to inhaled MCh was assessed 48 h after completion of airway sensitization and the following day animals were sacrificed to harvest lungs and PBLN.
Cell Preparation
PBLN or spleens were harvested and MNC were purified by passing the tissue through a stainless steel mesh, followed by density gradient centrifugation (Organon Teknika, Durham, NC). Cells were washed three times with PBS and resuspended in RPMI 1640 medium (Life Technologies). For T cell purification, spleens and PBLN were carefully teased apart, RBC were lysed with Grey’s solution, and lysis-resistant cells were passed through nylon wool columns (19). The cell suspension from spleens contained >90%, and from PBLN >95%, CD3+ T cells as determined by FACS analysis following staining with anti-CD3 (FITC-145-2C11, PharMingen).
Determination of airway responsiveness
Airway responsiveness was assessed using a single chamber whole body plethysmograph obtained from Buxco (Troy, NY) as described (20). Enhanced pause (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 5000 D ultrasonic nebulizer (DeVilbiss). 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 (PenhPBS). The PenhPBS values varied very little between groups (0.78 ± 0.22).
Lung cell isolation
Lung cells were isolated by collagenase digestion as previously described (21) 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.
In vitro cytokine production
MNC were cultured for 48 h in 96-well round-bottom plates
at a concentration of 400,000 cells/well in the presence or absence of
the combination of ionomycin (0.5 µM) from Calbiochem (La Jolla, CA)
and phorbol 12,13-dibutyrate (10 ng/ml, Sigma). Supernatants were
harvested and frozen at -20°C. The concentrations of IFN-, IL-4,
and IL-5 in these culture supernatants were assessed by ELISA as
described (22). 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 and 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-HRP (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
Pairs of groups were compared by Student’s t test, comparison of more than two groups was performed by Tukey-Kramer HSD test. The p values for significance were set at 0.05. Values for all measurements are expressed as the mean ± SD except for values of airway responsiveness (Penh) and of cytokine concentrations, which are expressed as the mean ± SEM.
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Results |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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Mice were sensitized to OVA via the airways following RSV
infection, following sham-infection, or following adoptive transfer of
MNC or T lymphocytes from the PBLN of RSV-infected mice or from
noninfected controls. Airway responsiveness to MCh was assessed by
barometric whole body plethysmography following airway sensitization,
11 days after cell transfer. As demonstrated previously, airway
sensitization following RSV infection resulted in increased airway
responsiveness, whereas airway sensitization in the absence of prior
RSV infection did not result in an increased response to MCh. Penh in
response to 50 mg/ml MCh increased 12.8 ± 2.3-fold in
RSV-infected and sensitized mice compared to a 4.8 ± 0.5-fold
increase in mice sensitized without prior infection. Adoptive transfer
of 107 MNC from RSV-infected mice, 14 days postinfection,
into naive mice prior to airway sensitization resulted in a 9.2 ±
0.9-fold increase in Penh in response to 50 mg/ml MCh (Fig. 1
A). Virtually identical
results were obtained following transfer of 5 x 106 T
cells. In contrast, transfer of MNC or T cells from noninfected mice
did not result in increased airway responsiveness to MCh, nor did
adoptive transfer of MNC from RSV-infected mice without subsequent
allergen exposure (Fig. 1A). Surprisingly, transfer of MNC
or T cells from RSV-infected mice obtained only 7 days after infection
failed to significantly increase responsiveness to MCh (Fig. 1B), in contrast to the cells obtained 14 days after
infection (Fig. 1A).
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, only the transfer of CD8 T cells from
the infected mice resulted in the development of AHR following exposure
to OVA. Transfer of CD4 T cells was not effective.
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To assess whether changes in airway responsiveness were associated
with changes in pulmonary inflammatory cells following RSV infection or
adoptive transfer, lung cells were isolated from these animals and
differential cell counts were performed. RSV infection prior to airway
sensitization resulted in an influx of eosinophils into the lung (from
0.66 ± 0.28 x 106 eosinophils/lung following
sensitization alone to 1.57 ± 0.33 x 106
eosinophils/lung after RSV infection and sensitization). Adoptive
transfer of MNC or T cells derived from the PBLN of RSV-infected mice
14 days postinfection into naive mice prior to airway sensitization
resulted in significant increases in numbers of lung eosinophils as
well (Fig. 3). Transfer of either MNC or
T cells from noninfected mice or from RSV-infected mice harvested 7
days postinfection did not result in lung eosinophilia following
sensitization (day 7 T cell transfer; 0.67 ± 0.28 x
106 eosinophils/lung; day 7 MNC transfer, 0.55 ±
0.1 x 106 eosinophils/lung). Adoptive transfer of MNC
from RSV-infected mice without subsequent allergen exposure also did
not result in increased lung eosinophils (0.59 ± 0.34 x
106 eosinophils/lung). RSV infection prior to airway
sensitization also resulted in the influx of neutrophils into the lungs
from 1.64 ± 0.56 to 3.22 ± 0.68 x 106
neutrophils/lung. Transfer of MNC or T cells from RSV-infected mice
failed to significantly alter lung neutrophil numbers (Fig. 3).
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To monitor local cytokine production, MNC isolated from PBLN
following adoptive transfer and airway sensitization were cultured and
stimulated with phorbol dibutyrate/ionomycin. Concentrations of
IFN-, IL-4, and IL-5 in culture supernates were measured by ELISA.
Transfer of MNC from RSV-infected mice 14 days postinfection and
subsequent airway sensitization resulted in increased production of
IL-5 in culture (Fig. 4). In contrast, no
such effect was observed following transfer of control cells from
noninfected animals or from RSV-infected mice 7 days postinfection.
Differences in IL-4 and IFN- production did not reach statistical
significance between groups, but interestingly there was also a
noticeable increase in IFN- levels following MNC transfer 14 days
after RSV infection and subsequent airway sensitization (from
1035.2 ± 826.2 pg/ml to 3429.3 ± 2844.9 pg/ml).
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To investigate the involvement of individual T cell subsets in
mediating the effects of RSV infection on subsequent allergic
sensitization via the airways, mice were depleted of CD4 or CD8 T cells
beginning day 15 postinfection, i.e., following the acute phase of
RSV-induced disease. From day 21 postinfection, mice were sensitized to
OVA via the airways over 10 days. Anti-CD4 treatment resulted in a 98%
depletion of CD4 T cells in the PBLN assessed prior to OVA exposure and
in a 72% depletion when assessed following the completion of airway
sensitization, without affecting numbers of CD8 T cells. Following
anti-CD8 treatment, CD8 T cells were depleted by 96% throughout
the experimental protocol and numbers of CD4 T cells were not altered.
Airway responsiveness to MCh and numbers of lung inflammatory cells
were monitored following completion of airway sensitization. RSV
infection prior to airway sensitization resulted in increases in
numbers of lung eosinophils and neutrophils and in AHR as reported
above. Depletion of CD8 T cells prevented the development of AHR (Fig. 5A) and lung eosinophilia and
neutrophilia (Fig. 5B). Depletion of CD4 T cells, on the
other hand, reduced the increases in airway responsiveness by 51.2
± 16.9% (Fig. 5A). Numbers of lung eosinophils were
reduced to a lesser extent than following CD8 T cell depletion and
increases in neutrophil numbers were also prevented (Fig. 5B).
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Discussion |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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As previously reported, RSV infection prior to airway sensitization
resulted in significant increases in airway responsiveness to MCh, and
this was associated with the infiltration of both eosinophils and
neutrophils in the lung while allergic sensitization via the airways
induced little of these responses (5). Following OVA
exposure alone, OVA-specific IgE and IgG1 Abs were detectable in the
serum at similar levels in both groups, indicating successful
sensitization (data not shown). Transfer of 107 MNC into
naive mice, isolated from PBLN and harvested from mice 14 days after
RSV infection, enhanced the effects of subsequent airway sensitization
resulting in AHR and lung eosinophilia. No such effect was observed if
MNC from noninfected animals were transferred as controls or if the
adoptive transfer from RSV-infected mice was not followed by allergen
exposure via the airways. Further, transfer of 5 x
106 T cells (half the number of MNC) resulted in virtually
identical findings. Because T cells, which account for 
60% of the
cells obtained from PBLN, can transfer these effects, these data
demonstrated that the effect of RSV infection on subsequent airway
sensitization can be transferred to noninfected animals by T cells
alone, emphasizing that T cells are the critical cell type in mediating
RSV-induced effects on allergic airway sensitization. Transfer of cells
14 days postinfection was also associated with increased levels of IL-5
production when PBLN of recipient mice were cultured. IL-5 has been
shown to be essential for eosinophil recruitment and development of AHR
induced by allergen (23, 24, 25) and following RSV infection
(26). Our cytokine data suggest that T cells capable of
producing IL-5 or capable of inducing other cells to do so evolve
following RSV infection and were transferred in these experiments. The
concomitant increase in IFN- production following transfer of cells
14 days postinfection may indicate that different T cell subsets, able
to induce the production of a variety of cytokines, were adoptively
transferred. Interestingly, transfer of MNC or T cells obtained only 7
days postinfection, that is during the height of RSV-induced disease,
did not result in AHR, lung eosinophilia, or increased cytokine
production following airway sensitization. Mononuclear PBLN cells
harvested at this time point produce very high levels of IFN- but
little IL-5 and IL-4 upon stimulation, as we reported previously
(5). Such IFN--producing (Th1) cells when adoptively
transferred may fail to enhance or may inhibit Th2 cytokine responses
induced by airway sensitization, rendering transfer of cells 7 days
postinfection ineffective in inducing AHR.
Having demonstrated that T cells and T cells alone can play a critical
role in mediating RSV-induced AHR following airway sensitization, we
attempted to delineate the involvement of CD4 and CD8 T cells in this
process. We first isolated CD4 and CD8 T cells from the PBLN of
RSV-infected mice and transferred them into naive recipients prior to
OVA inhalation. Only the CD8 but not CD4 T cells conferred the ability
to develop AHR. We also approached this question by depleting mice of
CD4 or CD8 T cells by Ab treatment following the acute phase of RSV
infection but prior to airway sensitization. CD8 T cells were almost
totally depleted throughout the experiment, whereas depletion of CD4 T
cells was almost complete just prior to allergen exposure but could not
be sustained during the sensitization phase. Following CD8 T cell
depletion, no increase in airway responsiveness or eosinophil and
neutrophil recruitment to the lungs was observed in RSV-infected and
allergen-sensitized animals, confirming that the presence of CD8 T
cells was important for the enhancing effects of RSV infection on
subsequent airway sensitization. CD8 T cells have been demonstrated to
be capable of IL-5 production (27) and to be essential for
the development of allergen-induced AHR (13). Further, in
a different viral infection model, CD8 T cells were driven to IL-5
production resulting in eosinophil recruitment (28). We
have recently demonstrated that CD8 T cells are critical for the
development of RSV-induced AHR during acute infection
(14). IL-5-producing CD8 T cells have been shown to
persist for longer periods of time in vivo and to produce IL-5 on
restimulation (29). One explanation of our results is that
such IL-5-producing CD8 T cells persist following the acute infection,
whereas cytotoxic IFN--producing CD8 T cells, which may predominate
during the acute phase of RSV infection (30, 31, 32), actually
decrease in numbers over time. Persistence of IL-5-producing CD8 T
cells may be critical in inducing eosinophilia and AHR in response to
an inflammatory stimulus such as airway sensitization. The balance
between IFN--producing and IL-5-producing CD8 T cells may account
for the differences seen in day 7 and day 14 transfer results. At the
present time, we cannot exclude the possibility that a cell type other
than CD8 T cells was primed by RSV infection to induce AHR and lung
eosinophilia following sensitization mediated by CD8 T cells.
Transfer of CD4 T cells (PBLN) from RSV-infected mice failed to result in AHR on subsequent exposure to OVA. On the other hand, depletion of CD4 T cells in RSV-infected mice reduced AHR to a limited extent. These data indicate that although CD4 T cells may be involved in the development of AHR and lung eosinophilia following the combination of RSV infection and airway sensitization, the transfer of RSV-induced T cell effects appears to be mediated primarily by CD8 T cells. That CD4 T cell depletion only had a partial effect on OVA responsiveness in our study may very well be due to the incomplete depletion during airway sensitization. It is also possible that CD4 T cells play an important role in enhancing Th2 cytokine responses (in CD8 T cells) and, as a result, the recruitment of eosinophils and the development of AHR in this model.
In summary, we present a murine model of RSV-induced enhancement of inflammatory cell recruitment to the lungs and of increased airway responsiveness following allergic airway sensitization. Utilizing adoptive transfer and T cell subset depletion in this model, we demonstrate that the effects of RSV infection on subsequent airway sensitization can be transferred by CD8 T cells from RSV-infected mice or that depletion of CD8 T cells in RSV-infected mice prevents these effects of RSV infection; CD4 T cell transfer was ineffective and CD4 T cell depletion only partially reduced AHR. We conclude that T cells, and in particular the combination of CD8 and CD4 T cells, are essential in mediating the effects of RSV infection on subsequent airway (allergic) sensitization.
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Footnotes |
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2 Current address: Klinik jür Kinder und Jugendmedizin, Rühr-Universität Bochum, Bochum, Germany.
3 Address correspondence and reprint requests to Dr. Erwin W. Gelfand, 1400 Jackson Street, Denver, CO 80206. E-mail address:
4 Abbreviations used in this paper: RSV, respiratory syncytial virus; AHR, airway hyperresponsiveness; PBLN, peribronchial lymph nodes; MCh, methacholine; MNC, mononuclear cells; Penh, enhanced pause.
Received for publication October 26, 1998. Accepted for publication September 1, 1999.
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References |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
,
Tr-CD3-R14/O and Tr-MNC-R14/O vs O, Tr-CD3-C/O, Tr-MNC-C/O, and
Tr-MNC-R14/S. B, Transfer of T cells or MNC from PBLN 7 days
after RSV infection into naive mice does not result in AHR following
airway sensitization. BALB/c mice were treated as described above, but
cells for transfer were harvested 7 days after RSV infection.
Twenty-four hours after completion of airway sensitization airway
responsiveness to increasing concentrations of inhaled MCh (3–50
mg/ml) was assessed and Penh values were calculated. Means ± 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, R/O vs
O.
