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by Influenza Hemagglutinin-Specific CD8 Effector T Cells Influences the Development of Pulmonary Immunopathology
From the Trudeau Institute,*
Saranac Lake, New York; the
Immunobiology Department,
DNAX Research
Institute, Palo Alto, California; and the College of Veterinary
Medicine,
Michigan State University, East
Lansing, Michigan
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Abstract |
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-deficient Tc1- or Tc2-CD8
effector cells responding to influenza pneumonia. The adoptive transfer
of influenza hemagglutinin-specific Tc1 effectors afforded protection
and elicited only minimal impairment of lung function.
IFN--deficient Tc1 effector cells were equally protective,
but were associated with an eosinophil influx and slightly more lung
function impairment early in the response. Relative to Tc1, Tc2
effector cells were less protective, elicited an eosinophil
influx and a greater impairment of lung functions. IFN--deficient
Tc2 effector cells were not protective and were associated with the
severest impairment of lung function throughout the response,
an accumulation of neutrophils, and extensive pulmonary
vasculitis and alveolar hemorrhaging. Deletion of IFN- was
associated with a delay in effector cell recruitment and the
elicitation of a more intense inflammatory response that resulted in
more severe lung function impairment in the recipients of either
Tc1 or Tc2 IFN--deficient effector cells. Thus, during
influenza infections, IFN- production by the responding CD8
T cells is associated with effector cell recruitment and mitigation of
the associated inflammation and of the resulting impairment in lung
functions but is not necessary for optimal protection.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The inflammatory sequelae associated with the resolution of influenza virus infections is attributed in part to the activity of host immune cells at the site of infection.8 After the adoptive transfer of CD4-Th1 cells, clearance of an influenza infection is associated with a diminished level of inflammatory sequelae, whereas the transfer of CD4-Th2 cells is not protective and elicits an exacerbated pulmonary eosinophilia.7 Infection of CD8 T-cell transgenic RAG-1 knockout mice with a high-dose inoculum of influenza virus (107 pfu) induces an exacerbated pulmonary inflammation, loss of normal lung structure, and eventual death of the animal. The administration of 100 pfu elicits significantly less immunopathology and the animals eventually recover from infection.8 These studies suggest that the viral burden and the responding viral-specific T cells are critical factors in the development of influenza virus-associated immunopathology.
Differentiation of cytotoxic CD8 T cells along the Th1- and Th2-like cytokine secretion pattern results in the production of CD8-Tc1 and CD8-Tc2 subsets.9 We have found in adoptive transfer studies using influenza hemagglutinin (HA)-specific CD8 effector T cells that CD8-Tc1 cells are more efficient at viral clearance than CD8-Tc2 cells despite their equivalent in vitro cytolytic activity before transfer.10 The CD8-Tc1 effectors are rapidly recruited into the lung and migrate to the epithelial sites of infection, whereas CD8-Tc2 effectors are associated with delayed cellular migration and localization in the lung at sites distant from the infected airway epithelium. The differences observed in chemokine receptor mRNA expression of these CD8 effector subsets may account for these differences in cell recruitment. Despite these findings, characterization of the CD8-Tc1- and CD8-Tc2-associated immunopathology, the effect of the cytokines associated with these CD8 effector subsets on the inflammatory sequelae and the extent to which the subsequent CD8-Tc1- or CD8-Tc2-mediated inflammation impairs critical lung function during resolution of influenza viral infection remains unresolved.
In this investigation, we used an adoptive transfer model to further
our understanding of how the recruitment of CD8-Tc1- and CD8-Tc2
HA-specific effectors impact the progression of influenza-related
pneumonitis. Our model was designed to compare the immune-mediated
inflammatory sequelae attributable to responding wild-type (+/+) or
interferon (IFN)--deficient (-/-) CD8 effector T cell subsets with
the subsequent impairment of lung functions observed in the recipient
groups of mice. Influenza virus HA-specific CD8 effector T cells from
+/+ and IFN--/- mice were polarized in vitro before
adoptive transfer into influenza infected mice. The CD8-Tc1+/+ and
CD8-Tc1-IFN--/- effectors provided effective protection against
viral infection and did not induce extensive lung damage or prolonged
impairment of lung functions in recipient mice. Although early
recruitment of the Tc1+/+ effectors was more efficient than seen for
the Tc1-IFN--/- effectors, both effector subpopulations were
present in the lung at effective anti-viral levels later in the
infection. The recipients of the CD8-Tc2+/+ effectors displayed a
delayed partial protection against viral infection that was accompanied
by a significant level of lung damage and by a protracted period of
recovery of lung functions. In contrast, recipients of
CD8-Tc2-IFN--/- effectors did not clear the virus and they
continued to display the most severe indications of lung damage and
compromise lung function whereas recipients of all other CD8 effectors
exhibited signs of recovery. The Tc2+/+ effectors were more efficiently
recruited into the lung than Tc2-IFN--/- effectors throughout the
infection process although neither group cleared the virus during the
course of the experiment. These results indicate that the
HA-specific CD8-Tc1 effectors were of greater benefit to the host in
resistance to influenza infection, regardless of their ability to
produce IFN-. On the other hand, the ability of the effectors to
produce even small amounts of IFN- was crucial for the enhancement
of the protective capabilities of CD8-Tc2 effectors and for mitigating
the severity of the inflammatory sequelae attributable to the presence
of the CD8-Tc2 effector cells in the lung.
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Materials and Methods |
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BALB/c Thy 1.1+ mice were bred at and
obtained from the Animal Breeding Facility at the Trudeau Institute,
Saranac Lake, NY. Clone 4 TCR transgenic mice expressing the
Vß8.2/V
10 chains in the TCR of CD8 T cells specific for the
influenza virus HA2 transmembrane peptide at residues 518 to 528
(IYSTVASSL)11
were originally provided by Dr. Linda
Sherman (The Scripps Research Institute, La Jolla, CA). The mice were
subsequently bred at and obtained from the Trudeau Institute’s Animal
Breeding Facility. These mice were backcrossed for eight generations
onto a BALB/c+/+ Thy1.2+ background and onto a
BALB/c IFN--/- Thy1.2+ background.
Preparation of Tc1 and Tc2 Effector Cells
The preparation of Tc1 and Tc2 effector cells was performed as
previously described.10
Briefly, an enriched CD8 T cell
population was prepared from splenocyte and lymph node preparations
obtained from the clone 4 HA-specific transgenic BALB/c+/+ or
IFN--/- Thy 1.2+ donor mice. The cell
preparations were passed over nylon wool followed by
complement-mediated antibody depletion using anti-CD4 (RL172.4),
anti-heat-stable antigen (J11D) and anti-major histocompatibility
complex class II (D3.137, M5114, CA4) monoclonal antibodies (mAbs). The
CD8 T cell population harvested from the Percoll gradients (Sigma
Chemical Co., St. Louis, MO) were 90 to 95%
CD8+Vß8+ T cells. Antigen
presenting cell (APC) preparations were prepared from T-cell-depleted
splenocytes using complement, anti-Thy 1.2 (HO13.14, F7D5), anti-CD4
(RL172.4) and anti-CD8 (3.155) mAb treatment followed by
lipopolysaccharide (25 [m]g/ml) and dextran sulfate (25 µg/ml)
stimulation for 48 hours. The HA peptide (11 µmol/L) was loaded onto
the APC population for 30 minutes at 37°C after which the cells were
mitomycin treated for 40 minutes at 37°C. After three washes, the APC
populations were cultured in RPMI 1640 media (Irving Scientific Co.,
Santa Ana, CA) supplemented with penicillin, streptomycin,
glutamine, 2-mercapto ethanol Hepes, and 10% fetal calf serum.
Effector Tc1 cells were generated by stimulation of CD8 clone 4
transgenic T cells (2 x 105
cells/ml) with
HA-peptide-loaded APCs (2 x 105
cells/ml)
in the presence of interleukin (IL)-2 (10 ng/ml), IL-12 (2 ng/ml), and
anti-IL-4 mAb (10 µg/ml). Effector Tc2 cells were likewise generated
by co-culture of CD8 T-cell- and HA-peptide-loaded APC (both at 2
x 105
cells/ml) populations in the presence of
IL-2 (10 ng/ml), IL-4 (5.6 ng/ml), and anti-IFN- mAb (10 µg/ml).
At day 4 of culture, the effector populations were 99%
CD8+Vß8+ T cells.
Analysis of Cytokine Production by Tc Effector Cells
The production of IFN-, IL-4, and IL-5 by Tc1 and Tc2+/+ and
IFN--/- effector populations was assessed 4 days after their
generation. Effector populations (2 x 105
cells/ml) were restimulated with mitomycin-treated P815 APCs with or
without previous loading of the HA peptide. The cytokine-specific
enzyme-linked immunosorbent assays were performed as previously
described.12
RNase protection assays were carried out as
previously described.10
The probe set mCK-3 (PharMingen,
San Diego CA) was used to measure specific cytokine mRNA levels. The
bands were detected using the densitometric feature of the Quantity One
software (Bio-Rad Laboratories, Hercules, CA) and normalized against
the housekeeping gene L32.
Cytotoxicity Assay
The cytolytic activity of each of the effector populations was
assessed 4 days after their generation. P815 APCs were incubated or not
with HA peptide (11 µmol/L) at 37°C for 30 minutes. The target APCs
were then incubated with 3.7 mBq of 51Cr for 1
hour at 37°C. After three washes, the P815 target APCs
(104
cells/well) were incubated with CD8 effector
cells at the indicated effector:target (E:T) ratios. After a 4-hour
incubation at 37°C the supernatants were collected and the level of
radioactivity was detected by counting. The percentage of
cytotoxicity was calculated as follows: 100 x (cpm
experimental - cpm spontaneous)/cpm total - cpm
spontaneous).
Viral Infection of Mice and Adoptive Cell Transfer
Influenza virus (A/PR/8/34) was grown in the allantoic fluid of
10-day-old embryonated chicken eggs. BALB/c
Thy1.1+ mice were inoculated intranasally with 10
LD50 in 100 µl (10-3
dilution of the viral stock) while under light halothane anesthesia.
Within 1 hour after viral infection, 4-day-old +/+ or IFN--/- Tc1
or Tc2 HA transgenic Thy1.2+ cells (107
cells by
intravenous tail injection in 0.5 ml of phosphate-buffered saline) were
adoptively transferred. The cells were washed twice before adoptive
transfer.
Lung Function Measurements
Respiration rates were measured using a whole body plethysmograph. Mice were confined within the chamber so that respirations per minute could be calculated from steady strip-chart recordings. Blood gases were measured from arterial blood samples taken from the ventral tail artery.13 Animals were warmed for 10 minutes in an enclosed warming chamber at 37 to 40°C to produce vasodilatation of the ventral tail artery. The artery was carefully lacerated and arterial blood was collected into heparinized capillary tubes. Blood gas analysis was done on an IL 1620 series blood-gas analyzer (Instrumentation Laboratories, Lexington, MA).
Recovery of Cells from Infected Lungs
Five mice from each experimental group were killed on day 3 and day 5 after infection. The difference between the starting and final body weight of each mouse was recorded as the change in body weight during the course of the experiment. At the designated time points, the mice were placed under deep halothane anesthetization and exsanguinated by perforation of the abdominal aorta. The trachea was exposed and cannulated and the lungs were lavaged with two 1-ml volumes of cold 0.85% NaCl. The total cell recovery was recorded and an aliquot for differential cell counts was taken and stained using Diff-Quik (Baxter, Miami, FL). In a second series of experiments, the whole lung was removed and passed through a mesh screen to obtain a single cell suspension. The cells were then washed in Hanks’ balanced salt solution (HBSS), resuspended in 2 ml of 80% Percoll, and overlaid with 2 ml of 40% Percoll. The gradient was then centrifuged for 20 minutes at 1,700 rpm on a Sorval H1000B rotor (Du Pont, Wilmington, DE). The lymphocyte population recovered at the 80 to 40% interface of the gradient was washed in HBSS and use for fluorescence-activated cell sorting analysis.
Flow Cytometry
Analysis by flow cytometry of the lymphocyte population recovered in the lung homogenate was performed using a biotin-conjugated mAb specific for mouse CD90.2 (anti-Thy 1.2, clone 53-2.1) (PharMingen, San Diego, CA), and allophycocyanin-streptavidin (Caltag Laboratories, Burlingame, CA). All plot analyses were gated on live lymphocyte populations. Analysis of the stained cells was done on a FACScan using Cell Quest software (Becton Dickinson, San Jose, CA).
Lung Histology
Lung tissue sections were cut from the left lobe of whole lungs fixed in phosphate-buffered 10% formalin and embedded in paraffin. Two 4-µm thick sections from different regions of the left lobe were taken from each animal in each group. Sections were stained with hematoxylin and eosin for histopathological evaluation by an independent observer in a blinded and randomized manner. Immunohistochemical staining for influenza viral antigen was done using frozen tissue sections cut from cryopreserved lungs. Tissue sections were fixed in 4% formaldehyde for 10 minutes. The viral antigen was detected using a biotin-conjugated goat polysera specific for H1N1 influenza viruses (Accurate Chemical & Scientific Corp., Westbury, NY) and an avidin-conjugated alkaline phosphatase-staining reagent (Vectastain ABC Kit; Vector Laboratories, Burlingame, CA).
Viral Recovery
Virus recovered from the lungs of influenza infected mice was detected by plaque assay on Madin-Darby canine kidney cells. Lungs were removed from infected mice on days 3, 5, and 6 after infection and snap-frozen in serum-free minimal essential medium supplemented with 0.2% bovine serum albumin, 20 mmol/L Hepes, 100 U/ml penicillin, and 100 µg/ml streptomycin. Snap-frozen lungs were stored at -70°C until analyzed. Madin-Darby canine kidney cells were grown in 12-well plates (Becton Dickinson) to 90% confluency in minimal essential medium supplemented with 10% fetal calf serum, 20 mmol/L Hepes, 100 U/ml penicillin, and 100 µg/ml streptomycin. The snap-frozen lungs were homogenized and 100 µl samples from 10-fold dilutions of the homogenates were used to inoculate the Madin-Darby canine kidney monolayers. After incubation of the monolayers for 1 hour at 35°C, a 1-ml overlay of minimal essential medium containing 0.2% bovine serum albumin, 20 mmol/L Hepes, 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml DEAE Dextran (Sigma Chemical Co.), 1 µg/ml TPCK trypsin (Life Technologies, Inc., Grand Island, NY), and 0.6% agar (Becton Dickinson) was added. The assay was then incubated for 4 to 5 days at 35°C after which 1 ml of 20% acetic acid was added to each well. The assay was incubated overnight and then stained with 0.2% crystal violet on the next day. The limit of detection in the assay was 20 viral particles per lung.
Statistics
The data are expressed as the means ± SD. At each time point, six groups of mice containing five mice per group were taken. Statistically significant differences between the means ± SD of the treatment groups were determined using either a Student’s t-test or a Kruskal-Wallis one analysis of variance on ranks followed by the Dunnett’s method of all pairwise multiple comparisons. Differences were considered significant if P < 0.05.
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Results |
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Analysis of cytokine production by 4-day-old effector cells
revealed that the Tc1+/+ effectors produced significant levels of
IFN- when restimulated by HA peptide-loaded target cells whereas the
Tc2+/+ effectors produced only low levels of IFN- under similar
conditions (Figure 1A)
. The significant
production levels of IL-4 and IL-5 were equivalent in the Tc2+/+ and
Tc2-IFN--/- effectors, whereas production of these cytokines by
Tc1+/+ and Tc1-IFN--/- effectors was not detected (Figure 1, B and C)
. The in vitro production levels of IFN-, IL-4, and
IL-5 by the various effector subsets was HA-peptide-specific and was
proportional to the number of peptide-loaded target cells.
Quantification of mRNA levels for Th1-associated
cytokines10
by RNase protection assay indicated that
Tc1+/+ and Tc1-IFN--/- effectors produced equivalent levels of
tumor necrosis factor- and lymphotoxin- (LT-) mRNA. The
cytokine mRNA levels in the Tc1+/+ and Tc1-IFN--/- effectors,
normalized to the expression level of the L32 housekeeping gene, were
45.10% versus 45.63% for LT- and 38.42%
versus 39.21% for tumor necrosis factor- mRNA,
respectively.
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Four days after their generation, both the Tc1 and Tc2+/+ effector
populations had equivalent in vitro cytolytic activity
levels and both were slightly greater than that of the Tc1-IFN--/-
effectors (Figure 2)
. All three of these
populations showed increased target cell lysis in proportion to the
number of effector cells present. The Tc2-IFN--/- effectors
maintained a low level of cytolytic activity against all titrations of
HA peptide-loaded target cells.
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At day 3 after infection, the loss in body weight in the
recipients of Tc1+/+ (-3.6 ± 0.3 g), Tc2+/+ (-3.3 ±
0.4 g), and Tc1-IFN--/- (-3.6 ± 0.4 g) effectors
was significantly greater (P < 0.05) than that
recorded for the infected control group, (-2.8 ± 0.3 g). By
day 5 after infection, only the loss in body weight observed in the
recipients of the Tc1+/+ (-3.2 ± 0.3 g) and Tc2+/+
(-3.4 ± 1.2 g) effectors was significantly less than
(P < 0.05) the weight loss recorded for the
infected control mice (-5.0 ± 0.8 g).
Viral Recovery from Influenza Infected Mice
Three days after viral infection and adoptive cell transfer, the
infected control mice had a viral burden of 3.1 ± 0.5 x
107
pfu of virus in their lungs (Figure 3)
. At this time, the viral burden in the
lungs of the recipients of the Tc1+/+ effectors (1.36 ± 0.8
x 106
pfu) and the Tc1-IFN--/- effectors
(2.7 ± 1.0 x 106
pfu) was <10% of
that observed in the infected control mice. The viral burden in the
lungs of the recipients of the Tc2+/+ effectors (8.1 ± 6.7
x 106
pfu) and that observed in the recipients
of the Tc2-IFN--/- effectors (1.9 ± 0.9 x
107
pfu) were equivalent to 25 and 60%,
respectively, of the viral burden in the lungs of the infected control
mice at day 3. By day 5, the viral burden in the lungs of the
recipients of Tc1+/+ effectors (1.6 ± 3.3 x
103
pfu) and Tc1-IFN--/- effectors
(2.24 ± 3.30 x 102
pfu) was reduced
to <0.0001% of the viral burden in the lungs of the infected control
mice (2.12 ± 2.70 x 107
pfu). At this
time, recipients of Tc2+/+ effectors had 
3% (5.9 ± 6.8
x 105
pfu) of the viral burden of the infected
control mice whereas the recipients of the Tc2-IFN--/- effectors
still had at least 60% (1.32 ± 1.74 x
107
pfu) of the viral burden observed in the
infected control mice. At 6 days after infection, none of the
recipients of the Tc1-IFN- effectors had any detectable virus in
their lungs and only one recipient of the Tc1+/+ effectors had a
detectable viral burden in its lung. The recipients of the Tc2+/+
effectors had 2% (7.04 ± 9.37 x 103
pfu) of the viral burden observed in the infected control mice
(3.9 ± 3.6 x 105
pfu) whereas the
viral burden in the lungs of the recipients of the Tc2-IFN--/-
effectors was slightly greater than in the infected control mice at
this time.
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The recovery of neutrophils in the BALF of the Tc1-IFN--/-
recipients was greater than the recovery from the Tc1+/+ recipients on
both days 3 and 5 (Table 1)
. In contrast,
equivalent numbers of neutrophils were recovered from the BALF of
Tc2+/+ and Tc2-IFN--/- recipients only on day 3. The intensity of
the neutrophil accumulation in all infected groups at day 5, with the
exception of the Tc2-IFN--/- recipients was drastically reduced.
The neutrophil accumulation level in the recipients of Tc2-IFN--/-
effectors was significantly greater than in all other groups at this
time.
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-/-,
and Tc2-IFN--/- recipients was significantly greater than from the
infected control mice on both day 3 and day 5 (Table 1)
. The number of
eosinophils recovered from the Tc1+/+ recipients on these days was
minimal and not significantly different from the infected control mice.
The recovery of eosinophils from the BALF of the Tc2+/+ and
Tc1-IFN--/- recipients increased substantially from day 3 to day
5, whereas in the remaining groups, the level of eosinophil recovery
remained close to day 3 levels. Recruitment of Thy1.2+ Cells into the Infected Lungs
Three days after infection and effector cell-transfer, the
recruitment of effector cells was greatest in the recipients of Tc1+/+
effectors (Table 2)
. The recruitment of
Tc2+/+ effector cells was half that seen for the Tc1+/+ recipients. The
recipients of both IFN--/- subsets had equivalent effector
recruitment levels that were significantly lower relative to their
respective +/+ recipients. By day 5 after infection and effector cell
transfer, the recruitment of Tc1+/+ effectors was significantly greater
than that observed in any of the other recipient groups. The
recruitment of Tc2-IFN--/- effectors was significantly less than
that observed for any other recipient group. The recruitment of
effector cells by the recipients of Tc1-IFN--/- and Tc2+/+
effectors to the lung was not significantly different.
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Three days after infection, the epithelium of the airways, up to
and including the terminal bronchial epithelium of all groups of mice,
stained positive for influenza antigen. Only occasionally was influenza
antigen staining seen in the alveoli and then, only in alveolar
macrophages. At day 5, the infected control mice stained for influenza
antigen in all bronchial epithelium (arrows), the epithelium of the
alveoli closest to the terminal bronchioles (arrowheads) and the
alveolar macrophages of the distal alveoli (Figure 4E)
. The recipients of either the Tc2+/+
(Figure 4C)
or Tc2-IFN--/- (Figure 4D)
effectors had a similar
pattern of staining. In mice given either Tc1+/+ (Figure 4A)
or
Tc1-IFN--/- (Figure 4B)
effectors, influenza antigen staining was
limited primarily to the bronchial epithelium down to the terminal
bronchial epithelium. Only occasional influenza antigen staining was
seen in the alveoli of either of these two groups of mice. Thus, the
virus seemed to spread from the bronchial epithelium to the alveolar
epithelium in the infected control mice and in the mice given
Tc2+/+ or IFN--/- effectors whereas the spread of the virus was
limited to the bronchial epithelium in the mice given the Tc1+/+ or
IFN--/- effectors.
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No lesions were observed in the lungs from the uninfected control
mice that did not receive any effector cells (Figure 5A)
. At 5 days after infection and
effector cell transfer, the principal morphological lesions in the
examined lung sections of infected control mice were marked
degeneration, necrosis, and exfoliation of the luminal surface
epithelium lining the large and small conducting airways (ie, axial
airway, and preterminal and terminal bronchioles) throughout the lung
(Figure 5B)
. Regenerative epithelium, characterized by basophilic,
squamoid to low cuboidal basal cells with occasional mitotic figures,
partially lined some of these altered airways. Associated with the
airway epithelial lesions was a moderate mononuclear inflammatory cell
infiltrate in the interstitial space surrounding the airways and blood
vessels. The infiltrating inflammatory cells were predominantly
lymphocytes and monocytes, with considerably lesser numbers of
neutrophils and eosinophils. Although the inflammatory response was
principally perivascular and peribronchiolar, there was mild extension
of this cellular inflammation into the tissue and airspaces of the
surrounding alveolar parenchyma. There was also a mild increase of
alveolar macrophages in the affected parenchymal regions.
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-/- effectors (Figure 5, C and D
, respectively). The extent and severity of the virus-induced airway
epithelial lesions were similar among the Tc1+/+ and Tc1-IFN--/-
recipients. In addition, there was a slightly greater inflammatory
response in the recipients of the Tc1-IFN--/- effectors.
Although the necrotizing injury to the surface epithelium lining the
conducting airways of the lung was extensive and similar in the Tc1+/+
and Tc2+/+ recipients, the associated inflammatory cell response was
conspicuously different between these two groups. In contrast to the
mononuclear (lymphocytes and monocytes) inflammatory cell response in
the Tc1+/+ recipients, Tc2+/+ recipients had a marked perivascular and
peribronchiolar cell infiltrate composed principally of numerous large
macrophages, epithelioid cells, eosinophils, and widely scattered
multinucleated giant cells (Figure 6, A and B)
. Lymphocytes, monocytes, and neutrophils were also present, but
in lesser numbers. This inflammatory response often extended into the
adjacent alveolar parenchyma (ie, alveolitis with associated type two
cell hyperplasia) and sometimes to the outer pleural surface (ie,
pleuritis with mesothelial cell hyperplasia). Fibrinoid necrosis of the
walls of the pulmonary vasculature, most notably in the pulmonary
arteries (Figure 6, A–D
, arrows) and widely scattered perivascular and
alveolar hemorrhage (Figure 6, A and D
, arrowhead) were additional
features of the Tc2+/+ and Tc2-IFN--/- recipients.
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-/- recipients (Figure 6, C and D)
were generally more
severely affected than similarly treated Tc2+/+ recipients, both in the
extent and severity of these induced lung lesions. In some areas of the
lungs of the Tc2-IFN--/- recipients, the alveolar airspaces were
completely effaced by the infiltration and accumulation of large
macrophages, epithelioid cells, eosinophils, and
hypertrophic/hyperplastic type two cells. Multinucleated macrophages
and giant cells were often widely scattered in these affected regions.
Accumulation of fibrinous material was also observed in some of the
severely affected alveolar airspaces. Respiratory Rates
All of the mice that received +/+ or IFN--/- effectors
exhibited an approximate 20% increase in their respiratory rates
immediately after infection and effector cell transfer (Figure 7, A and B)
. Between day 1 and 2, all of
these mice displayed a decrease in their respiration rates. The
respiration rate of the infected control mice increased to its maximum
level during this same time (Figure 7C)
. After day 2, the respiration
rates of the recipients of Tc1+/+ and Tc1-IFN--/- (Figure 7A)
, and
Tc2+/+ (Figure 7B)
effectors increased to and were then maintained
within a maximal range until day 5. Recipients of Tc1+/+ and
Tc1-IFN--/- effectors displayed more rapid increases to slightly
higher respiration rates than did the Tc2+/+ recipients. Only the
Tc2-IFN--/- recipients displayed a diminution in their respiration
rate relative to levels that were equivalent to those observed with the
infected control mice after (Figure 7, B and C)
.
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Arterial blood pO2
levels in the recipients of Tc1+/+ effectors were equivalent to that
found for the uninfected control mice throughout the course of the
experiment (Figure 8A)
. A significantly
depressed pO2 level was
observed at day 3 in the Tc1-IFN--/- recipients. The
pO2 levels in these animals
increased thereafter to levels equivalent to that recorded in the
uninfected controls and Tc1+/+ recipients by day 6. At day 3 the
recipients of Tc2+/+ effectors had significantly lower
pO2 levels than any of the
other groups (Figure 8B)
. The arterial
pO2 levels in these animals
significantly increased by day 5 and were maintained to day 6. Although
the Tc2-IFN--/- recipients had only a slightly reduced
pO2 level at day 3, their
pO2 levels by day 5 were
the lowest of the recorded levels for this group during the experiment.
Although a slight increase in their
pO2 levels was noted for
these animals between day 5 and day 6, they still remained
significantly depressed relative to the uninfected control mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production associated with responding CD8 effector cells mitigated the
inflammatory sequelae affiliated with the presence of these effectors
in the lung. This in turn was reflected in a diminution of the
subsequent level of impairment in lung function. Critical differences
in the inflammatory sequelae and impaired lung functions were more
evident between the recipients of Tc2+/+ and Tc2-IFN--/-
effectors. This mitigating role for IFN- was most apparent with
responding CD8-Tc2+/+ effector cells than with the responding Tc1+/+
effector cells.
The comparable level of mRNA expression for the Tc1-associated
cytokines, tumor necrosis factor-, and LT-10
suggested that polarization of the Tc1+/+ and Tc1-IFN--/-
effectors was equivalent. Likewise, the similar IL-4 and IL-5
production levels in the Tc2+/+ and Tc2-IFN--/- effectors
suggested equivalent polarization of these subsets. The low level of
IFN- production in the Tc2+/+ effector preparations has been
detected previously.10,14
It has been suggested that small
numbers of contaminating Tc1 effector cells may account for
this.12
Adoptively transferred Thy1.2+ CD8 Tc1 and Tc2
cells recovered from the spleen 3 days after influenza infection still
retained the same IFN- and IL-4 secretion pattern as the polarized
effector cells before their transfer (unpublished observations). In
earlier studies, the recovery of Tc1 and Tc2 CD8 memory cells from the
lymphoid tissues of uninfected adoptively transferred hosts
demonstrated that the cytokine secretion pattern of the polarized
effector cells persisted for up to 13 weeks after adoptive
transfer.12
Although the Tc1+/+ and Tc2+/+ effectors had equivalent in
vitro cytolytic activity levels, the mice that received Tc2+/+
effectors exhibited a delayed pattern of viral clearance. In
vivo factors not accounted for in the in vitro assays
may have differentially regulated the ability of the CD8-Tc1 and
CD8-Tc2 effector cells to lyse infected target cells in the lung. This
pattern of viral clearance is similar to that found
previously.10
The deletion of the IFN- gene was
coincident with a slight reduction in the in vitro cytolytic
activity of the Tc1-IFN--/- effectors relative to the Tc1+/+
effectors. This circumstance was also true of the Tc2+/+ and
Tc2-IFN--/- effectors except that the coincident loss of cytolytic
activity in the Tc2-IFN--/- effectors was always substantially
greater than that seen in the Tc1-IFN--/- effectors. The low
in vitro cytolytic activity of the Tc2-IFN--/-
effectors was reflected in the significant amount of virus recovered
from the recipients of these cells. The means by which the loss of the
IFN- gene results in the diminution of the cytolytic activity in the
CD8 effector cells remains unresolved.
The recovery of eosinophils in this study was confined to the
recipients of effector CD8 T cells whereas the recovery of neutrophils
was noted in the infected recipient mice as well as in the infected
controls. This suggested that the eosinophilia was dependent on the
recruitment into the lung of the transferred CD8 effector T cells
whereas host-derived factors15-18
in the lung were
sufficient for the recruitment of neutrophils. At day 5, the recipients
of Tc2+/+ and Tc1-IFN--/- effectors had equivalent eosinophil
recovery levels despite their opposing polarity. In addition, although
both Tc2+/+ and Tc2-IFN--/- effectors produce similar amounts of
the potent eosinophil chemoattractant, IL-5, their disparate levels of
eosinophil recovery remain unexplained. The evidence from this study
suggests that networks of complex regulatory pathways involving
resident lung cells and recruited CD8 effector T cells are involved in
the recruitment of eosinophils into the inflamed lungs.
The trend in physical deterioration of some mice and the recovery of
others can be extrapolated to lung function and viral burden.
Respiration rates increased in all infected groups shortly after
infection as a means of compensating for the loss of functional
alveolar surface area normally available for gas exchange. Although the
recipients of Tc2+/+ effectors displayed a significantly low arterial
pO2 level at day 3, these
mice were able to sustain a continual increase in their compensatory
respiration rate after day 2. This resulted in the gradual increase of
their arterial pO2 levels
to slightly less than the maximum seen for the Tc1+/+ and IFN--/-
recipients by day 5. During this same time period all three of these
groups exhibited diminishing viral burdens in their lungs. The
recipients of Tc2-IFN--/- effectors and the infected control mice
both failed to maintain their compensatory respiration rates during the
course of the experiment. The respiration rates of both of these groups
fell to levels below the normal resting respiration rate of the
uninfected controls. In addition, both of these infected groups of mice
displayed equivalent high viral burdens and decreasing arterial
pO2 levels. The
viral-induced damage in the infected control mice and the combination
of the viral-induced and immune-associated lung damage in the
recipients of the Tc2 -IFN--/- effectors compromised the lung
function of these mice. The increasing loss of functional alveolar
surface area in these infected mice would result in an inadequate gas
exchange and lead to the inability of these mice to maintain their
compensatory respiration rate and the eventual death of the animal.
The pulmonary dysfunction that was evident in the Tc2+/+ and
Tc2-IFN--/- recipients correlated with the histological findings
for these two groups. Both of these recipient groups exhibited
indications of fibrinoid necrosis in their pulmonary vasculature as
well as perivascular and alveolar hemorrhaging. These pathological
features were more severe in the Tc2-IFN--/- recipients.
Abnormalities in gas exchange functions of the lung have been suggested
to result from pulmonary capillary obliteration/thrombosis and
ventilation/perfusion mismatching.19
In view of the
histological and lung function findings, these two factors may well
have contributed to the pulmonary dysfunction seen in these recipient
mice. In addition, the extent of viral replication into the alveolar
spaces of the infected control mice and in the recipients of the Tc2+/+
and IFN--/- effectors will contribute to the pulmonary
dysfunction.
Ascribing a level of responsibility for the inflammatory sequelae and
associated impairment of lung function during influenza virus
infections to CD8-Tc1 or -Tc2 subsets had not been addressed before
this study. In our study, the physical deterioration in the recipients
of the Tc2+/+ and Tc2-IFN--/- effectors was similar to that seen
after adoptive transfer of HA-specific CD8 T cells into transgenic mice
that express influenza HA on their type 2 pneumocytes.6
As
the HA antigen in the transgenic model is never cleared, its presence
continuously stimulates the recruitment of the adoptively transferred
HA-specific cytolytic CD8 T cells. This results in the ongoing
destruction of the HA-expressing type 2 pneumocytes in the alveolar
epithelium and causes the inflammation and impaired lung function in
this model. In our influenza model, the inflammatory sequelae are
initially established by the viral infection but subsequently
exacerbated by the functional characteristics of the recruited
HA-specific CD8 effector cells. In the recipients of Tc1+/+ and
Tc1-IFN--/- effectors, a relatively modest inflammatory response
and a brief impairment of lung function accompanied the rapid clearance
of viral antigen. These effector cells clearly restricted the viral
infection to the level of the bronchial epithelium up to and including
the epithelium of the terminal bronchioles. This effectively preserved
the functional integrity of the alveolar space in these animals. The
inefficient viral clearance observed in the recipients of Tc2+/+ and
Tc2-IFN--/- effectors allowed for the expansion of the viral
infection into the alveolar epithelium and thus for the involvement of
the alveoli in the inflammatory sequelae. The resultant loss of
functional alveolar surface area was manifested by the greater
impairment of lung function observed in these mice.
The recipients of the Tc1+/+ or Tc1-IFN--/- effectors had
equivalent or greater numbers of effector cells in their lungs relative
to that found in the recipients of Tc2+/+ or Tc2-IFN--/-
effectors. Despite this, the later effector cells elicited a greater
inflammatory response. This suggested that it was not the number of
effector cells but rather the characteristics of the effector cells
that were associated with the development of the inflammatory sequelae.
The absence of IFN- production by the Tc1-IFN--/- effectors
resulted in only a small difference in viral clearance and in a
slightly more intense inflammation in that it caused eosinophils to
accumulate.
In contrast, the small amount of IFN- affiliated with the Tc2+/+
effectors played a significant role in the response of these effectors.
The absence of IFN- in the Tc2-IFN--/- effectors was associated
with a decrease in the rate of viral clearance and an exacerbation of
the inflammatory sequelae and lung function impairment relative to that
observed in the Tc2+/+ associated response. The evidence in this study
indicates that IFN- mitigates the inflammatory sequelae in the lung
that are associated with the CD8-Tc1 and -Tc2 cells responding to
influenza virus infections. This mitigating role for IFN- was more
evident with the Tc2+/+ effectors. The evidence from this study
suggests that this mitigating role is associated with the efficiency
that these cells are recruited to the site of infection and with their
cytolytic capabilities. The means by which IFN- had a greater
influence on the Tc2 response is not known and is the subject of future
investigation. Nevertheless, this study indicates that IFN- and
viral-specific cells, particularly CD8-Tc2 effectors, recruited to the
lung during influenza infections play significant roles in development
of lung damage and dysfunction.
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Acknowledgements |
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Footnotes |
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Supported in part by National Institutes of Health grants AI7935 and AI36263 and in part by an American Lung Association grant from the American Lung Association of New York State awarded to J. A. W.
Accepted for publication September 21, 2000.
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References |
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