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* From the University of Virginia Health Sciences Center, Charlottesville, VA.
Correspondence to: Richard I. Enelow, MD, Department of Medicine, University of Virginia Health System, Box 546, Charlottesville, VA 22908; email: enelow{at}virginia.edu
CD
8+ T cells infiltrate the lung in a variety of inflammatory and
interstitial lung diseases, though little is known about the functional
activities expressed by these cells in the lung
parenchyma.1
2
3
The function of CD8+ T cells in viral
defense is likely mediated by several effector activities, including
cytotoxicity and cytokine secretion, but it is unclear to what extent
these activities participate in injury associated with disease. CD8+
T-cell clearance of experimental respiratory virus infection results in
considerable lung injury,4
though it is difficult to
distinguish the deleterious effects of T-cell–effector activity from
those of the virus infection itself. We have developed a murine model
for the purpose of studying the direct effects of CD8+ T-cell
recognition of antigens presented by alveolar epithelial cells, in the
absence of virus infection. The model antigen is the A/Japan/57
influenza hemagglutinin, expressed under the transcriptional control of
the surfactant protein C (SP-C) promoter, resulting in alveolar
epithelial expression. Adoptive transfer of CD8+ T cells activated
against hemagglutinin into these mice results in severe interstitial
pneumonitis and significant lung injury, characterized by restrictive
respiratory mechanics and significant diminution in carbon monoxide
diffusing capacity.5
The functional deficits correlate
more closely with the host inflammatory influx than with the initial
T-cell infiltration. We have previously shown that neither perforin nor
Fas is necessary for lung injury triggered by CD8+ T-cell transfer, but
that tumor necrosis factor (TNF)-
is essential.6
In this study, we tested the hypothesis that the host inflammatory
infiltrates are recruited, at least in part, by inflammatory mediators
expressed by epithelial target cells in direct response to CD8+ T-cell
recognition of alveolar antigen. We present evidence that the alveolar
cells that are targets of CD8+ T-cell recognition actively express
several chemokines, particularly monocyte chemoattractant protein
(MCP)-1, in lieu of (or in the process of) undergoing apoptosis.
Chemokine production was critically dependent on TNF- (which is
expressed by the T cell specifically on recognition of the antigen on
the epithelial cell). We also demonstrate that expression of MCP-1 by
alveolar cells contributes to the inflammatory influx that occurs after
T-cell engagement, suggesting that the epithelium participates actively
in the amplification and perpetuation of lung inflammation triggered by
CD8+ T-cell recognition of alveolar antigen.
Materials and Methods
T-Lymphocyte Clones
CD8+ T-cell clones, specific for the 210–219 epitope of
A/Japan/57 hemagglutinin, were used in these experiments, and were
generated by limiting dilution and restimulated weekly in
vitro with irradiated syngeneic splenocytes that were infected
with A/Japan/57 influenza. For in vitro assays, the MLE-K
target cell, a mouse type II pneumocyte-derived cell line transfected
with the class I major histocompatibility antigen, Kd,6
was plated in 24-well plates and allowed to adhere
overnight. T cells and synthetic peptide were added to the culture,
incubated, and then trypsin/ethylenediaminetetra-acetic acid was added
in order to collect the cells for RNA extraction. Ribonuclease
protection assays were performed using probes for a panel of chemokines
(Pharmingen; San Diego, CA). For effector/target cell
separation, anti-CD8–coupled magnetic beads were used (Dynal; Lake
Success, NY).
Adoptive Transfer
Hemagglutinin transgenic mice (H-2d),
expressing the A/Japan/57 hemagglutinin under the transcriptional
control of the SP-C promoter, and hemagglutinin-positive p55-/-
(TNF- receptor 1–deficient) mice (H-2d) were used for
in vivo studies.7
CD8+ T-cell clones were
separated from stimulator cells by density gradient centrifugation and
injected via the tail vein into recipient animals. Some animals
received antibody to MCP-1 (Pharmingen) or isotype control by tail vein
injection at the time of T-cell transfer. Lungs were harvested for
histology or homogenized for RNA extraction. At appropriate times after
adoptive transfer, the animals were killed and the airways perfused.
Sections were stained with hematoxylin-eosin or with peroxidase-labeled
antibody to a macrophage marker (F4/80; Caltag; Burlingame, CA).
Tritiated riboprobes were prepared using an MCP-1 complementary DNA,
and in situ hybridization was performed with a 2- to 4-week
exposure.
Results and Discussions
Using a murine model developed for the purpose of studying the
direct effects of CD8+ T-cell recognition of alveolar antigens, in the
absence of virus infection, we have shown that CD8+ T-cell–effector
activities initially result in mild interstitial pneumonia, which
eventually progresses to severe lung injury.5
The model
antigen is the A/Japan/57 influenza hemagglutinin, expressed under the
transcriptional control of the SP-C promoter, resulting in alveolar
epithelial expression. The neoantigen is processed and presented as a
"self" antigen by class I major histocompatibility complex
molecules on epithelial cells, and as a result there is complete CD8+
T-cell tolerance to all class I-restricted epitopes.7
We
have generated hemagglutinin-specific CD8+ T-cell clones from
nontransgenicinfluenza-infected mice, which are then activated in
vitro against the virus, followed by adoptive transfer into
recipients that express hemagglutinin on the alveolar epithelium.
Severe interstitial pneumonitis occurs (Fig 1
, top right,
B), in a dose-dependent fashion, resulting in significant lung
injury characterized by restrictive respiratory mechanics and
significant diminution in carbon monoxide diffusing
capacity.5
Lung injury was not observed in nontransgenic
control mice receiving the same cell transfer. The genetic background
of the T cells has an impact on the phenotype of the injury. For
example, T cells derived from interferon-
–deficient animals produce
milder injury initially than wild-type T cells, but the injury
progresses in a more protracted fashion resulting in chronic
interstitial and intraluminal fibrosis (data not shown). We have also
demonstrated in vitro that CD8+ T-cell recognition and
cytolysis of alveolar epithelial-derived cells occurs in an exquisitely
antigen-specific fashion, using either influenza-infected or
peptide-loaded targets, and that the majority of this activity is
mediated by TNF- expressed by the T cell.6
In
vivo transfer data using genetically deficient T cells or
recipients indicate that neither perforin nor Fas expression is
necessary for lung injury to evolve after T-cell recognition of
alveolar antigen. Rather, it is critically dependent upon TNF-
expressed by the CD8+ T cell. Interestingly, the injury in this model
is characterized by the significant accumulation of other inflammatory
cells, particularly macrophages in the lung parenchyma (Fig 1
,
middle left, C, and middle right, D), and this
occurs about 3 to 4 days after T-cell transfer.8
Studies
with labeled T cells indicate that the transferred cells are
undetectable in the lung parenchyma after 2 days, and yet the injury is
quite mild at this time point, histologically and physiologically.
Instead, the severe physiologic derangements correlate temporally with
the host inflammatory influx.
|
expressed by
CD8+ T cells is not mediated exclusively by cytotoxicity, but also
through the induction of alveolar target-cell activation and
target-cell expression of inflammatory mediators. Ribonuclease
protection assays were performed on RNA extracted from whole-lung
homogenates after adoptive transfer demonstrated expression of a number
of chemokines. In order to distinguish those expressed specifically by
the alveolar cells, we initially used in vitro assays of
T-cell/alveolar cell interactions. Ribonuclease protection assays were
performed on RNA extracted from in vitro co-cultures of CD8+
T cells and MLE-Kd cells. Varying doses of antigenic
peptide (10-6, 10-8,
10-10, and 10-12 mol/L)
were added to the cultures, and several chemokine messages were
detected by 6 h, including macrophage inflammatory protein
(MIP)-1, MIP-1ß, RANTES (regulated upon activation, normal T-cell
expressed and secreted), MCP-1, and MIP-2. Chemokine expression
became undetectable at 10-12 mol/L peptide.
Ribonuclease protection assays were also performed on RNA extracted
from CD8+ T cells, stimulated by anti-CD3 in the absence of
antigen-presenting cells. T cells expressed several chemokines on
activation, but no MIP-2 or MCP-1 expression was evident. We then used
RNA extracted from in vitro co-cultures of CD8+ T cells and
MLE-Kd cells incubated in the presence of
10-8 mol/L peptide, or after influenza
virus infection of the MLE-Kd cells. After 4 h of
incubation all cells were removed and incubated with anti-CD8–coupled
magnetic beads twice in order to fully separate the two cell
populations. MCP-1 and MIP-2 expression was observed exclusively in the
target cell fraction, and was inhibited by the addition of anti-TNF-
to the media during incubation. Protein expression was confirmed by
enzyme-linked immunosorbent assay. Alveolar chemokine expression
in vivo was confirmed using in situ
hybridization, performed 24 h after T-cell transfer. These
demonstrated MCP-1 expression in hemagglutinin-positive transgenic
recipients, localized to the alveolar septae, most prominently at the
junctions of the alveolar walls (Fig 1
, bottom right, f). No expression was evident in
the nontransgenic recipients (Fig 1
, bottom left,
e). This expression was not evident in
hemagglutinin-positive p55-/- recipients, either by in situ
hybridization at 24 h, or using ribonuclease protection on RNA
extracted from whole-lung homogenates. Though the source of the TNF-
is likely the CD8+ T cell, since MCP-1 expression was observed as early
as 3 h after T-cell transfer, we cannot formally exclude a
contribution of macrophage-derived TNF-, though macrophage influx
was not observed until 2 to 3 days after transfer. Further studies are
underway to distinguish these possibilities. Finally, in order to
establish the physiologic significance of alveolar chemokine expression
after T-cell recognition, we performed in vivo
neutralization of MCP-1 during T-cell transfer. This resulted in
significant abrogation of host parenchymal infiltration, and the
residual infiltrates were essentially devoid of macrophages by
immunohistochemistry. Two interesting implications arise from these data. The first is that "cytotoxic" T cells may not necessarily utilize their cytolytic-effector mechanisms on recognition of their target antigen, or if they do, T-cell–mediated cytotoxicity may not contribute significantly to alveolar injury. The second is that the epithelial target cell may play an important role in amplifying and perpetuating otherwise mild lung injury triggered by CD8+ T-cell recognition of alveolar antigens.
Footnotes
Abbreviations: MCP = monocyte chemoattractant protein; MIP = macrophage inflammatory protein; MLE = mouse lung epithelial; TNF = tumor necrosis factor; SP-C = surfactant protein C
References
: relative insensitivity to Fas ligand. Am J Respir Cell Mol Biol 20,849-858
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