1Department of Thoracic Medicine, Royal Adelaide Hospital; 2Department of Medicine, University of Adelaide, Adelaide 5001; and 3Haematology Department, Women's and Children's Hospital, North Adelaide 5006, South Australia, Australia
Submitted 16 December 2002 ; accepted in final form 8 March 2003
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ABSTRACT |
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apoptosis; blood; cytokine; chronic obstructive pulmonary disease; transforming growth factor-
There have been reports of increased apoptosis of lymphocytes obtained from the airways by bronchoalveolar lavage in COPD (24, 34). Lymphocytes are known to traffic from the bloodstream to the bronchoalveolar space and then may later rejoin the peripheral circulation (21). On the basis of this known trafficking of lymphocytes between the airways and the peripheral blood and the increased rates of apoptosis in airway-derived T cells, we hypothesized that we could detect an increase in the propensity of peripherally derived T cells from COPD patients to undergo apoptosis compared with cells from subjects without this disease.
Several pathways have been reported to be involved in inducing apoptosis of
T cells. These include transforming growth factor (TGF)-/TGF receptor
(TGFR) 1, TNF-
/TNFR1, and Fas/Fas ligand (FasL)
(22,
36,
37). Increased production of
TGF-
and TNF-
has been reported in the airways in COPD
(19,
43). We thus hypothesized that
increased activation of these proapoptotic pathways might contribute to
increased T-cell apoptosis in the airways. In the present study, we
investigated production of these apoptotic mediators and expression of their
receptors in the peripheral blood from COPD subjects. These factors may
contribute not only to T-cell apoptosis but also to apoptosis of alveolar wall
cells, thereby contributing to the development of emphysema
(17,
24).
Alterations in lymphocyte subsets in the peripheral blood and airways of patients with COPD have also been reported (32). The CD4/CD8 ratio is significantly decreased with the percentage of CD8 lymphocytes increased. These ratio changes in COPD could be due to a number of factors, including relatively increased rates of apoptosis of CD4 vs. CD8 T cells. Alternatively, the changed ratio could primarily be due to an absolute increase in CD8 T-cell numbers. We therefore specifically investigated apoptosis of the CD4 and CD8 subsets of T cells in the COPD patients and control subjects.
We used multiparameter flow cytometry to determine cytokine, cytokine receptor, and apoptosis levels as previously reported (8, 12, 15). It was important to use whole blood, as we have previously shown that purification of peripheral blood mononuclear cells (PBMCs) results in increased levels of apoptosis (13). By investigating peripherally derived cells in this way, we hope to gain further insight into the role of apoptosis in COPD.
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MATERIALS AND METHODS |
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Reagents. Phycoethyrin-conjugated monoclonal antibodies (MAbs)
against TGF- (IQ Products), Fas (CD95), and CD132 (Pharmingen, San
Diego, CA); TNF-
[Becton Dickinson (BD), San Jose, CA]; TNFR1
(R&D); IL-4 (BD), IL-4R (CD124) (Immunotech); CD45 (BD); and CD8 (BD) were
used for flow cytometry. FITC-conjugated monoclonal antibodies to TNF-
(BD) and TNF-R2 (R&D) were also employed. Peridin chlorophyll protein
(PC)-5-conjugated MoAbs to the monocyte/macrophage marker CD14 and the T-cell
marker CD3 (Immunotech) were also included. FITC-conjugated annexin V
(Pharmingen) and 7-aminoactinomycin D (7-AAD; Sigma, St. Louis, MO) were used
for investigation of apoptosis for stimulated T cells by flow cytometry.
Stimulation of peripheral blood. Aliquots of 500 µl of blood were added to 500 µl of RPMI 1640 culture medium (Gibco-BRL) containing 1% wt/vol penicillin-streptomycin (Gibco) in 10-ml conical polypropylene tubes (Johns Professional Products, Sydney, Australia) and incubated for 24 h at 37°Cin5% CO2 in air. The samples were stimulated with 25 ng/ml PMA (Sigma) plus 1 µg/ml ionomycin (Calbiochem, La Jolla, CA) for T-cell cytokine analysis, or 10 µg/ml phytohemagglutinin (PHA) for T-cell surface marker and apoptosis analysis or 100 ng/ml Escherichia coli LPS, serotype 0111:B4 (Sigma) for monocyte stimulation. For intracellular cytokine investigation, cells were cultured in the presence of brefeldin A (1 µg/ml) (Sigma Chemicals, Castle Hill, Australia) as a "Golgi block" to inhibit intracellular transport and thus retain cytokines produced during activation inside the cell.
Staining with MAbs to surface markers. Markers to identify cell types (T-cell markers CD3, CD4, and CD8 and monocyte marker CD14), cytokine receptors [TNFR1, TNFR2, and IL-4R (CD124, CD132)], and Fas (CD95) were stained as previously reported (9). Briefly, 200-µl aliquots of blood were stained with 3 µl of directly conjugated MAbs to surface markers of interest, lysed with FACSlyse (BD), washed, then acquired immediately by flow cytometry.
Staining for apoptosis with annexin V. Staining with annexin V [a natural ligand for phosphatidylserine (PTS)] was used for evaluation of apoptosis. In early apoptosis, membrane alteration exposes phospholipids, such as PTS, on the outer cell membrane. Staining for cell type identification was carried out as described above. Cells were further washed and stained with annexin V as we have previously reported (11, 14).
Staining for apoptosis with 7-AAD. Staining with MAbs for cell type identification and 7-AAD for apoptosis was carried out as we have previously reported (14).
Staining for intracellular cytokine production. After surface
staining as described above, cells were stained for cytokine production as we
have previously reported (10).
In brief, red blood cells were lysed, then cells were permeabilized with 500
µl of FACSperm (BD), washed, then stained for 20 min with directly
conjugated MAbs to TGF-, IL-4, TNF-
, and isotype-matched
controls. Cells were washed, and events were acquired immediately. As
TNF-
and Fas have been reported to induce apoptosis of CD8+
T cells (23), production of
these mediators was investigated for CD3+, CD4+, and
CD8+ T cells.
Flow cytometric analysis. We carried out flow cytometric analysis using a FACSCalibur flow cytometer (BD) equipped with an air-cooled 488-nm argon ion laser. We collected 10,000 events in list mode using CellQuest software (BD) for analysis. Cells were initially gated on the basis of forward scatter and side scatter characteristics, with gates set to remove debris and platelets (Fig. 1A). Results were expressed as a percentage of cells exhibiting positive fluorescence. Analysis of intracellular cytokine production was carried out on T cells and monocytes, identified by staining characteristics with MAbs (Fig. 1B). T lymphocytes were gated based on known staining characteristics with CD3 PC-5 versus side scatter. CD8+ events were identified, then CD4+ events gated by CD3+CD8-1 staining characteristics. Monocytes were gated based on known staining characteristics with CD14 PC-5.
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ELISA measurement of soluble TGF-. Plasma from 24-h
LPS-stimulated blood was removed after centrifugation at 1,800 g for
5 min. Plasma was stored at -20°C before batch testing.
"Active" TGF-
(i.e., nonpolymerized) was quantified with a
Quantikine immunoassay kit (R&D), following instructions supplied by the
manufacturer.
Determination of TGFR expression by flow cytometry. We obtained PBMCs by diluting peripheral blood with an equal volume of RPMI 1640, layering 7 ml over 3 ml of Lymphoprep (Nycomed Pharma, Oslo, Norway), and centrifuging for 15 min at 500 g. The monolayer was removed and washed twice with 10 ml of RPMI 1640 containing 10% FCS (Gibco). The cells were resuspended in RPMI and incubated for 24 h, at 37°C in 5% CO2 in air. We quantified TGFR using a sensitive indirect kit (Fluorokine, R&D) following instructions supplied from the manufacturer.
Statistical analysis. The Wilcoxon nonparametric test for paired
data was used to analyze the data, using SPSS software. P values
0.05 were considered significant.
Investigation of the influence of age on apoptosis and cytokine production. There was a difference in the mean age of control subjects (mean age 41 yr) tested in parallel with the COPD subjects (mean age 66 yr) in this study. To assess the influence of age, we investigated levels of apoptosis and production of cytokines in peripheral blood from five normal, nonsmoking volunteers aged 3545 yr, seven aged 4555 yr, and seven aged 5565 yr as described above.
Investigation of the influence of sex of the subjects on apoptosis and cytokine production. There was a difference in the ratio of male-female control subjects (male-female, 8:8) tested in parallel with the COPD subjects (male-female, 15:3) in this study. To investigate the influence of sex, we investigated levels of apoptosis and production of cytokines in peripheral blood from eight normal, nonsmoking volunteers and eleven males as described above.
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RESULTS |
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Quantitation of apoptosis by flow cytometry. Apoptosis, identified by staining with annexin V, was higher for PHA-stimulated T cells from patients with COPD than those from the control group (P = 0.006, Table 2). Apoptosis of stimulated T cells was significantly increased in both CD4 and CD8 T cells from COPD subjects (P = 0.043 for both subsets). There was no significant difference in levels of apoptosis between CD4 and CD8 T-cell subsets in COPD patients. To confirm these findings, we carried out 7-AAD staining in parallel. There was good correlation between the two methods (Fig. 2). Apoptosis, identified by staining with 7-AAD, was higher for PHA-stimulated T cells from COPD subjects than from control subjects (COPD 70.0% ± SD 23.1% vs. control 58.2% ± SD 15.9%, P = 0.043).
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Expression of Fas. Having found an increase in the propensity of T cells from COPD subjects to undergo apoptosis, we next investigated a number of potential pathways that may explain this. To investigate the role of the Fas pathway in apoptosis of T cells in peripheral blood, we measured Fas (CD95) expression for CD3+, CD4+, and CD8+ T cells, and CD14+ monocytes. T cells from COPD patients expressed Fas (CD95) at a higher frequency than control subjects (Table 2). This difference was significant for both CD4 and CD8 T cells (Table 2). However, Fas expression by monocytes was not significantly different for COPD and control groups.
Quantification of intracellular cytokines and cytokine receptors by
flow cytometry. In addition to Fas, TNF- has also been reported to
induce apoptosis of T cells
(44), thus we hypothesized
that changes in the levels of this mediator in COPD might be involved in the
alteration of T-cell apoptosis rates and/or the CD4/CD8 ratio. Using flow
cytometry, we found significantly increased production of TNF-
by
peripheral blood T cells in COPD (P = 0.008,
Fig. 3). There was no
significant difference noted for CD4+ or CD9+ T cells
(CD4, COPD 17.8 ± 20.1% vs. control 3.6 ± 2.9%; CD8, COPD 15.2
± 24.0% vs. control 2.0 ± 15.2%, not significant). The effect of
TNF-
is dependent on which TNFR is activated, with TNFR1 and TNFR2
having pro- and antiapoptotic roles, respectively. Increased expression of
TNFR1 was observed for CD3+ T cells from COPD subjects compared
with controls (P = 0.003, Table
3). There was no significant difference in expression of TNFR2
between COPD and control groups.
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We also investigated the role of TGF- and its receptor in induction
of apoptosis of peripheral blood T cells in COPD. Flow cytometric analysis
showed low production of TGF-
by activated T cells and no significant
difference between COPD and control groups
(Fig. 3). However, monocyte
production of TGF-
was significantly increased in COPD
(Fig. 3). In contrast to
TGF-
, TGFR expression was significantly increased for T cells from COPD
subjects compared with controls (P = 0.018,
Table 3).
Evaluation of secreted, active TGF- by ELISA.
Although we found increased cellular TGF-
levels, TGF-
is produced
in a latent form and is biologically inactive and unable to bind to its
receptors until it has been activated
(2). The flow cytometric method
utilized an MAb to both active and latent TGF-
and hence measured total
TGF-
. To measure secreted, active TGF-
in the peripheral blood, we
applied ELISA techniques to measure plasma levels. Release of active
TGF-
in the peripheral blood in COPD was significantly increased
compared with the control group (P = 0.015)
(Fig. 4).
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Investigation of the influence of age on apoptosis and cytokine
production. There was no significant difference in the levels of
apoptosis and production of IL-4 or TGF- for the various age groups
(Table 4). TNF-
production for peripheral blood-derived T cells increased with age, but the
increase did not reach statistical significance (P = 0.087, 55- to
65-yr-old age group compared with the 35- to 45-yr-old group)
(Table 4). Monocyte TNF-
production was significantly lower for the 55- to 65-yr-old age group compared
with the 35- to 45-yr-old age group (Table
4).
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Investigation of the influence of sex of the subjects on apoptosis and cytokine production. There was no significant difference in levels of apoptosis or cytokine production between males and females (Table 4).
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DISCUSSION |
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We have previously found that the detectable level of apoptosis of T cells (identified by annexin V staining) in peripheral blood samples is <1% (11). Direct comparisons of unstimulated blood samples between groups of subjects have not previously been reported due to the low levels of apoptosis detectable, which is likely a reflection of the rapid removal of apoptotic cells from the circulation. Such comparisons are thus likely to miss important differences between groups. The present study utilized PHA stimulation of peripheral blood T cells from COPD and control subjects, using flow cytometry and annexin V and 7-AAD staining methods. PHA has been widely reported for studies of the potential of T cells to undergo apoptosis when stimulated (1, 5, 7, 16, 18, 25, 26).
Common contaminants of cigarette smoke, such as LPS, induce an increase in activated T cells in the airways in COPD (33). This increase may result from local proliferation of T cells in the lung or enhanced trafficking from the bloodstream (40), although the rapidity of the increase following LPS instillation (within a few hours) suggests that enhanced entry from the bloodstream is more likely (29). This trafficking of T cells also occurs from the airways to the bloodstream. Lymphocytes from the bronchoalveolar space have been reported to reenter the lung tissue, migrate to regional lymph nodes, and rejoin the systemic immune system (21). Therefore, it is possible that increased apoptosis of T cells in the peripheral blood in COPD may result either from local apoptotic stimuli in the airways or alternatively from apoptotic stimuli in the peripheral blood. The link between T cells in the peripheral blood and the airways is still a controversial area, and further studies are thus warranted. We are currently investigating the changes in intracellular apoptotic pathways in lymphocyte subsets in both airways and peripheral blood in COPD to further clarify apoptotic mechanisms associated with this disease.
Activation of the Fas/FasL pathway induces apoptosis of mature CD4+ T cells after repeated antigenic stimulation (38), thus the raised levels of Fas we report here may be significant for apoptosis induction in the peripheral blood in COPD (42).
Apoptosis of CD8+ T cells can be mediated by either TNF-
or Fas pathways (44). The
effect of TNF-
is dependent on which TNFR is activated, with TNFR1 and
TNFR2 having pro- and antiapoptotic roles, respectively. In the present study,
production of TNF-
and expression of TNFR1 by peripheral blood T cells
were increased in COPD. These results are consistent with previous reports of
increased circulating TNF-
in the peripheral blood in COPD
(39). However, the
significance of these findings is not clear, as increased age was found to be
associated with increased T-cell production of TNF-
(but not TGF-
or IL-4), although the difference did not reach statistical significance.
Therefore, the observed increase in T-cell production of TNF-
in COPD
subjects (mean age 66 yr) compared with control subjects (mean age 41 yr) may
have been influenced by age differences between the groups. Further studies
using age-matched controls are warranted.
In contrast to T-cell production, a significantly greater percentage of
monocytes from COPD subjects produced TNF- compared with control
subjects. As production of this cytokine was shown to significantly reduce
with age, the results suggest that monocyte production of TNF-
may play
a role in increased apoptosis in the peripheral blood in COPD.
Although in absolute terms the level of TNFRs seen was low, the difference
between the COPD and control groups was clear. The low levels may be a
reflection of the sensitivity of the flow cytometry technique and does not
discount the potential significance of the findings
(45,
46). Cytokine binding has been
shown to occur through receptors that need only to be expressed at low
concentrations (100 molecules per cell) to transmit activation signals
(45), which is well below the
level of sensitivity of flow cytometry.
TGF-, unlike the other cytokines investigated in this study, is
produced in a latent form and is biologically inactive and unable to bind to
its receptors until it has been activated
(2). The flow cytometric method
utilized a MAb to both active and latent TGF-
and hence measured total
TGF-
. To measure active TGF-
, we also applied ELISA techniques to
measure plasma levels. The TGF-
/TGFR pathway enhances apoptosis of
peripheral blood T cells by inhibiting proliferation at G1S phase
transition (3) and inhibiting
IL-2-induced expression of
- and
-chains of IL-2R- and
IL-2-induced activation of signal transduction molecules janus kinase-1 and
signal transducer and activator of transcription 5
(4). Our findings of increased
production of TGF-
by monocytes, increased release of active TGF-
,
and upregulated expression of TGFR by T cells suggest that this cytokine may
contribute to excess apoptosis of T cells in the peripheral blood in COPD.
Interestingly, we have previously reported that plasma-derived factor VIII
concentrate has apoptosis-promoting effects on T cells
(11). The presence of
TGF-
was shown to be a major component responsible for the apoptotic
effects seen in PHA-stimulated T cells from hemophilia patients receiving
factor VIII prophylaxis therapy
(11).
Recent reports have shown that phagocytosis of apoptotic cells by
macrophages leads to release of TGF-
(6). Monocytes may therefore
increase their secretion of TGF-
in the peripheral blood, as a result of
ingestion of increased numbers of apoptotic cells, potentially explaining our
findings of increased intracellular production of TGF-
by monocytes and
secreted TGF-
in COPD.
The ratio of CD4/CD8 cells has important implications for the host response
to infective and inflammatory stimuli. COPD is associated with a relative
increase in CD8+ T cells
(32), a finding that was
confirmed in the present study. Reasons for this have not been determined.
TNF- and Fas have been shown to induce apoptosis of CD8+ T
cells (44). These mediators
were therefore investigated for CD4 and CD8 lymphocyte subsets. The increase
in CD8+ T cells could not be explained by a relative increase in
apoptosis of CD4+ T cells nor by relative changes in TNF-
and Fas. TGF-
, although well recognized as a growth inhibitor of T
cells, has been demonstrated to be costimulatory for naïve
CD8+ T cells (20).
Thus increased TGF-
in the peripheral blood in COPD, as well as
increased expression of TGFR by CD8+ T cells, may partially explain
the increase of CD8+ T cells in COPD.
COPD is associated with an increased susceptibility to infection
(35). TGF- has broad
inhibitory effects on immune function and may increase susceptibility to
opportunistic infections and malignancies
(23). We can speculate that
elevated TGF-
and TGFR expression in COPD may result in an increase in
T-cell apoptosis following an infection. This would lead to a diminished
immune response to the infective organism and contribute to the increased
frequencies of infection, which are associated with the disease
(35).
In conclusion, we have demonstrated the novel finding of increased propensity of peripheral blood T cells in COPD to undergo apoptosis. Whether this finding represents a systemic effect of COPD on peripheral cells or whether these cells have reentered the circulation after passing through the airway epithelium requires further study.
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ACKNOWLEDGMENTS |
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DISCLOSURES
This study was supported by a Dawes Scholarship from the Royal Adelaide Hospital.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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