Department of Clinical and Experimental Medicine, Padua Hospital, Padua University School of Medicine, 35128 Padua, Italy
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ABSTRACT |
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The accessory function of antigen-presenting
cells depends on the presence of a number of costimulatory molecules,
including members of the B7 family (CD80 and CD86) and the CD5 coligand CD72. The aim of this study was to evaluate the regulation of T
cell-antigen-presenting cell costimulatory pathways in the lung of
patients with a typical Th1-type reaction, i.e., sarcoidosis. Although
normal alveolar macrophages (AMs) did not bear or bore low levels of
costimulatory molecules, AMs from sarcoid patients with CD4 T-cell
alveolitis upmodulated CD80, CD86, and CD72 and expressed high levels
of interleukin (IL)-15; lymphocytes accounting for T-cell alveolitis
expressed Th1-type cytokines [interferon (IFN)-
and/or IL-2] and bore high levels of CD5 and CD28 but not of
CD152 molecules. In vitro stimulation of AMs with Th1-related cytokines
(IL-15 and IFN-
) upregulated the expression of CD80 and CD86
molecules. However, stimulation with IL-15 induced the expression of
Th1-type cytokines (IFN-
) and CD28 on sarcoid T cells, suggesting a
role for this macrophage-derived cytokine in the activation of the
sarcoid T-cell pool. The hypothesis that CD80 and CD86 molecules
regulate the sarcoid T-cell response was confirmed by the evidence that
AMs induced a strong proliferation of T cells that was inhibited by
pretreatment with CD80 and CD86 monoclonal antibodies. To account for
these data, it is proposed that locally released cytokines provide AMs
with accessory properties that contribute to the development of sarcoid
T-cell alveolitis.
interleukin-15; Th1 reaction; sarcoidosis; costimulatory molecules
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INTRODUCTION |
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ALVEOLAR MACROPHAGES (AMs) are the
representatives of the mononuclear phagocyte system in the lung and are
peculiar among the mononuclear phagocytes in different body
compartments because they are strategically located at the air-tissue
interface and are regularly exposed to inhaled antigens (19, 20).
Although pulmonary macrophages are thought to play a critical role in
the initiation of immune responses within the lung, AMs from healthy individuals function poorly as accessory cells (ACs) for the
presentation of common recall antigens. They utilize multiple
mechanisms to suppress intra-alveolar T-cell immune responses,
including transforming growth factor- secretion and lack of
expression of counterreceptors that provide costimulatory signaling to
lung T cells, including CD80 (9, 30).
Although normal AMs are poor ACs, in patients presenting with hypersensitivity reactions related to sarcoidosis, they act as professional antigen-presenting cells (APCs) for T cell-dependent immune responses (16, 32). The sarcoid process is characterized by a persistent accumulation of CD4+ Th1-type T cells in the lung (22, 23), and it has been speculated that the interaction between AMs and T cells is a critical factor in initiating the in loco T-cell activation and proliferation leading to the development of CD4+ T-cell alveolitis (3, 32). T cell-AM interaction depends on the presence of a number of costimulatory molecules on pulmonary cells with APC capacity, including members of the B7 family (CD80 and CD86), some molecules of the tumor necrosis factor (TNF)-receptor superfamily (CD40 and CD27), and the CD5 coligand CD72 (11, 26, 28). Although there are phenotypic data on the expression of the members of the TNF-receptor and B7 families by AMs (5, 24), little is known about the molecules that regulate B7 and CD72 costimulatory molecules during sarcoid inflammation.
In this study, we evaluated the role of the CD72/CD5 and
CD80-CD86/CD28-CD152 pathways in the development and maintenance of
T-cell alveolitis in sarcoidosis. Our results demonstrate that sarcoid
AMs increase their expression of these molecules that endow macrophages
with AC function for T-cell activation and proliferation. Interleukin
(IL)-15 and interferon (IFN)- are likely to be involved in the local
cytokine networks leading to the increased expression of costimulatory molecules.
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MATERIALS AND METHODS |
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Study populations. Twenty-one patients with sarcoidosis were analyzed (7 men and 14 women; average age 35.4 ± 7.1 yr). In all cases, the diagnosis was made from a biopsy obtained either from the lungs or from lymph nodes showing noncaseating epithelioid granulomas with no evidence of inorganic material known to cause granulomatous diseases. According to the staging system for sarcoidosis by Agostini et al. (4), each patient underwent bronchoalveolar lavage (BAL) fluid analysis.
Fifteen sequentially enrolled sarcoid patients presenting with an episode of pulmonary involvement were evaluated at the onset of the disease. They were defined as having an active disease on the basis of the following characteristics: 1) lymphocytic alveolitis (>30 × 103 lymphocytes/ml), 2) positivity to 67Ga scan, and 3) lung CD4-to-CD8 ratio > 5.0. Apart from the BAL fluid analysis, the assessment of disease activity included clinical features, chest radiograph, lung function tests, high-resolution computed tomography, and routine blood studies.
BAL samples were also obtained from six patients with previously
diagnosed pulmonary sarcoidosis who repeated the BAL fluid analysis
during their follow-up period. All these patients were in the inactive
phase of the disease because they had normal lung function, normal BAL
fluid cell numbers, negative 67Ga
scan, and no clinical signs of acute disease. Four of these five
patients were previously given steroid therapy (prednisone, 1 mg · kg1 · day
1),
but no patient received immunosuppressive therapy for 6 mo before the
BAL fluid analysis. The average period of follow-up for this group of
patients was 35 ± 17.1 mo (range 18-46 mo).
Twelve healthy adult individuals were selected (6 men and 6 women; average age 30.2 ± 6.1 yr; 4 nonsmoking healthy persons and 8 subjects evaluated for complaints of cough without lung disease). They had normal physical examinations, chest X rays, lung function tests, and BAL cell numbers.
Preparation of cell suspensions. After
administration of local anesthesia, BAL was performed as previously
described (1). Briefly, a total of 150-200 ml of saline solution
was injected via fiber-optic bronchoscopy, the fluid was filtered
through gauze, and its volume was measured. The amount of injected
fluid recovered was 55.9 ± 6.4%. Cells recovered from
the BAL fluid were washed three times with PBS, resuspended in
endotoxin-tested RPMI 1640 medium (Sigma, St. Louis, MO) supplemented
with 20 mM HEPES and 0.532 g/l of
L-glutamine,
100 U/ml of penicillin, 100 µg/ml of streptomycin, and 10% FCS (ICN
Flow, Costa Mesa, CA), and then counted. Macrophages, lymphocytes,
neutrophils, and eosinophils on cytocentrifuged smears stained with
Wright-Giemsa were differentially counted, for a total count of 300 cells, according to morphological criteria. Table
1 reports differential BAL cell counts in
our patients and control subjects.
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Enrichment of AMs and T cells from the BAL cell suspensions. AMs and T cells were purified by rosetting with neuraminidase-treated sheep red blood cells followed by Ficoll-Hypaque gradient separations (1). AMs were further enriched by removing residual CD3+, CD16+, and CD56+ lymphocytes with magnetic separation columns (MiniMACS, Miltenyi Biotec) as previously described (1). Staining with monoclonal antibodies (MAbs) showed that after this multistep selection procedure, >98% of AMs expressed the AM-associated CD68 antigen, whereas >98% of the rosetting population was CD3+.
Peripheral blood mononuclear cells were obtained from six control subjects by centrifugation on Ficoll-Hypaque gradient as previously reported (1). T cells were enriched from the cell suspensions by rosetting with neuraminidase-treated sheep red blood cells followed by Ficoll-Hypaque gradient separations as previously described (1).
MAbs and flow cytometry analysis. The
commercially available conjugated or unconjugated MAbs used belonged to
the Becton Dickinson, Immunotech, and PharMingen (San Diego, CA) series
and included CD3, CD4, CD8, CD28, CD69 (FN50), CD80 (BB-1/B7-1), CD86
(B70/B7-2), CD152 (CTLA-4), and HLA-DR and isotype-matched control.
Anti-IL-15 M110 MAb (IgG1) was kindly provided by Dr. A. Troutt
(Immunex, Seattle, WA); anti-TNF- (MAb11), anti-IL-2 (MQ1-17H12),
anti-IL-4 (8D4-8), and anti-IFN-
(4S.B3) MAbs were purchased from PharMingen.
The frequency of BAL cells positive for the above reagents was determined by flow cytometry as previously described (1, 3, 5). Briefly, 10 µl of MAb (10 µg/ml) were added to 106 cells, and the mixture was incubated for 60 min at 4°C. The cells were then washed twice and resuspended in 0.2 ml of PBS for fluorescence-activated cell sorting (FACS) analysis as indicated below. For direct fluorescence analysis, FITC- or phycoerythrin-conjugated control isotype-matched mouse MAbs were used to set the negative control (IgG1, IgG2a, and IgG2b; Becton Dickinson). In the indirect fluorescence analysis, cells were incubated with control purified isotype-matched MAbs to determine the background fluorescence (Becton Dickinson).
For FACS analysis, 10 × 103 cells were acquired, and the analysis was determined by overlaying the histograms of the samples stained with the different reagents. Both BAL fluid lymphocytes and pulmonary macrophages were gated in flow cytometry analysis with two different approaches: 1) physical characteristics of cells and 2) expression of the T cell-associated CD3 and macrophage-associated PAM-1 antigens in the area of lymphocytes and pulmonary macrophages, respectively. The purity of the gates was always >98% cells.
Cells were scored with a FACScan analyzer (Becton Dickinson), and data
were processed with the Macintosh CELLQuest software program
(Becton Dickinson). Expression of the cytoplasmic cytokines was
evaluated after permeabilization of the cell membranes with 1:2 diluted
PermeaFix (Ortho, Raritan, NJ) for 40 min. After permeabilization procedures, anti-IL-15, anti-TNF-, anti-IFN-
, anti-IL-4, and anti-IL-2 MAbs were added.
Because activation markers, molecules involved in CD72/CD5 and CD80-CD86/CD28-CD152 pathways, and cytoplasmic cytokines are expressed by BAL fluid macrophages and lymphocytes in a unimodal expression pattern, indicating that both cell populations exhibit relatively homogeneous fluorescence, the percentage of positive cells does not represent the most accurate way of enumerating cells bearing these antigens. For this reason, the mean fluorescence intensity (MFI) was used to compare the positivity of these specific antigens on different cell populations. The analysis was determined by overlaying the histograms of the samples stained with the different reagents. Mean MFI values were obtained by subtracting the MFI of the isotype control sample from the MFI of the positively stained sample. Furthermore, to evaluate whether the differences between the peaks of positively stained cells and the peaks of control purified isotype-matched MAbs were significant, the Kolmogorov-Smirnov statistical test for analysis of histograms was used according to the CELLQuest software user's guide (Becton Dickinson).
Effect of cytokines on the expression of costimulatory
molecules by sarcoid AMs. To assess the effects of
cytokines on the expression of CD72, CD80, and CD86, a time-course
experiment of the effects of cytokines on the expression of
costimulatory molecules by AMs and BAL fluid T cells was performed. AMs
and BAL fluid T cells at a concentration of 1 × 106 cells/ml were cultured in
medium in 24-well plates (Corning, Corning, NY) for 24 h at 37°C in
a 5% CO2 atmosphere. After 24 h
of incubation, the plates were centrifuged, and after removal of the
supernatants, the BAL fluid cells were further cultured for 12 h with
medium alone, IL-2 (100 IU/ml), IL-15 (100 ng/ml), IFN- (100 IU/ml),
TNF-
(100 IU/ml), anti-IL-15 MAb, or anti-IFN-
MAb. The frequency
of BAL fluid cells positive for the above reagents was determined by
flow cytometry as described in MAbs and flow cytometry
analysis at time
0, after 24 h of incubation, and after restimulation
with cytokines. Log MFI was obtained by subtracting the MFI of the
isotype control sample from the MFI of the positively stained sample.
In this way, the values of MFI represent the relative increase in
fluorescence over the background value, reported as zero. Furthermore,
to evaluate whether the differences between the peaks of the cells were
significant with respect to the control values, the Kolmogorov-Smirnov
test for analysis of histograms was used according to the CELLQuest
software user's guide (Becton Dickinson).
Evaluation of the involvement of IL-15 and costimulatory molecules in the accessory function of pulmonary macrophages. Highly purified T cells at a concentration of 1 × 106 cells/ml were cultured in 96-round-bottom-well plates for 72 h at 37°C in a 5% CO2 atmosphere with 12.5 × 103 AMs in the presence of mitogens (concanavalin A, 10 µg/ml; Sigma) as previously reported (1). In inhibition experiments, anti-CD80 (10 µg/ml), anti-CD86 (10 µg/ml), anti-CD72 (20 µg/ml), or anti-IL-15 (10 µg/ml) antibody or control isotype-matched IgG1 was added at the beginning of the culture. Each experiment was carried out in quadruplicate. For the last 18 h of culture, the plates were pulsed with 1 µCi/well of [3H]thymidine as previously reported (3).
Statistical analysis. Data are expressed as means ± SD. Values were compared with ANOVA. A P value < 0.05 was considered significant.
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RESULTS |
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Table 1 summarizes the results of the differential BAL fluid cell counts and BAL fluid T-cell subpopulations retrieved from the lung of healthy patients and patients with sarcoidosis. Due to the presence of CD4+ high-intensity T-cell alveolitis, cell recovery was significantly higher in patients with active sarcoidosis compared with that in control subjects and patients with inactive disease. As a consequence of the increase in the absolute number of CD4+ T cells, the BAL fluid CD4-to-CD8 ratio was significantly increased in patients with active disease compared with that in patients with inactive disease and healthy control subjects. As previously reported (2), >98% of T lymphocytes recovered from the BAL fluid of sarcoid patients were CD4+/CD45R0+ memory T cells (data not shown).
Expression of costimulatory molecules by normal BAL fluid cells. As a first step in the investigation of the role of coreceptor systems in local immune competence, we evaluated the expression of these surface receptors on AMs and BAL fluid lymphocytes isolated from healthy subjects. In all normal individuals, the total cell recovery ranged from 8.5 to 12 × 106 BAL fluid cells; <10% of BAL fluid cells were lymphocytes (Table 1). Both CD4 helper-related and CD8 cytotoxic- or suppressor-related cells were present in approximately the same proportions as in the peripheral blood (pulmonary CD4-to-CD8 ratio 1.95 ± 0.44).
Figure 1 shows flow cytometry analysis of
BAL fluid cells in a representative control subject; a consistent
pattern of expression was seen in all control subjects. Normal AMs did
not bear CD80 (Fig. 1B), and <5%
of AMs were CD72+ or
CD86+ (Fig. 1,
A and
C, respectively). In addition,
pulmonary macrophages from healthy subjects did not show cytoplasmic
expression of IL-15, TNF-, and IFN-
(Fig. 1,
D-F,
respectively). Normal BAL fluid T cells showed a strong expression of
CD5 antigen (Fig. 1G). A percentage
ranging between 25.3 and 41.5% of lung lymphocytes bore the CD28
molecule (Fig. 1H). Nonetheless,
Kolmogorov-Smirnov analysis demonstrated that the CD28 histograms were
significantly shifted compared with the control histograms. The
findings reported above indicate that, albeit at low levels, CD28 is
constitutively expressed by the normal lung T-cell population. Normal
BAL fluid T cells did not express the CD5-ligand CD72 or the CD152
antigen (Fig. 1I); furthermore, they
did not express cytoplasmic cytokines (Fig. 1,
J-L,
respectively).
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Sarcoid macrophages coexpress costimulatory molecules
involved in their APC capacity and cytokines implicated in the
triggering of T-cell immune response. As reported in
Table 1, an alveolitis characterized by the accumulation of CD4 cells
represents the hallmark of sarcoidosis. Profiles shown in Figs.
2 and 3 are
representative of nine patients with active sarcoidosis. AMs isolated
from patients with active sarcoidosis and T-cell alveolitis showed an
upmodulation of CD72, CD80, and CD86 molecules (Fig. 2,
A-C,
respectively) and cytoplasmic cytokines (IL-15 and TNF-; Fig. 2,
G and
H, respectively) involved in the
triggering of T-cell immune responses. Interestingly, in all patients,
the CD86 molecule was found to be expressed by AMs at a higher
intensity than CD80 as compared by Kolmogorov-Smirnov analysis
(P < 0.001). Figure 2 also points
out that sarcoid AMs coexpress high levels of markers related to their
activation state (CD14, CD54, and CD58;
D-F,
respectively). Pulmonary macrophages from the patients with inactive
sarcoidosis did not express or expressed very low levels of CD72, CD80,
and CD86 molecules, and <5% of AMs of patients with inactive disease
showed cytoplasmic cytokines (TNF-
, IFN-
, and IL-15).
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Sarcoid T cells show an upmodulation of CD28 and CD5
molecules and a Th1-like profile. Flow cytometry
analysis of BAL fluid T lymphocytes showed that sarcoid
CD4+ T cells expressed CD5
molecules (Fig. 3A);
Kolmogorov-Smirnov analysis demonstrated that the CD5 histograms were
significantly shifted compared with the CD5 histograms of normal
subjects and patients with inactive disease. A percentage ranging from
96 to 100% of BAL fluid CD4+
cells bore CD28 at high density (Fig.
3B; P < 0.001 with respect to normal BAL T cells), whereas they lacked
CD152 (Fig. 3C). The enhanced
expression of CD28 was associated with an increased cell surface
density of activation antigens, including CD69, CD103, and class II
major histocompatibility complex molecule HLA-DR (Fig. 3,
D-F,
respectively), and with a Th1-type cytokine profile (Fig. 3,
G-I).
In fact, T cells from patients with active disease exhibited a striking
polarization of Th1-type immune response because they expressed
cytoplasmic IL-2 and IFN- but not IL-4. Less than 5% of T cells
from patients with inactive disease showed cellular IL-2, IFN-
, or
IL-4.
Macrophage-derived cytokines upregulate the expression
of B7 family molecules on sarcoid macrophages. Because
cytokines are capable of regulating B7 expression leading to APC
activation, we investigated whether cytokines expressed by pulmonary
cells enhanced the expression of molecules involved in APC-T cell
contact. Specifically, in a time-course experiment, we evaluated
whether macrophage-derived (i.e., IL-15 and TNF-) and T cell-derived cytokines (IL-2 and IFN-
) modulate the expression of CD72, CD80, and
CD86 ligands on highly purified AMs from five patients with active sarcoidosis.
Preliminary time-course experiments showed that after 24-36 h of
culture, medium-cultured sarcoid AMs partially lose the expression of
CD80, CD86, and CD72. In fact, Fig.
4A
shows that the MFI of CD80+ and
CD86+ AMs decreased if they were
maintained in medium without cytokine stimulation (CD80 MFI 72.4 ± 10.1 and 23.2 ± 6.6 and CD86 MFI 161.5 ± 30.3 and
35.9 ± 12.2 at time 0 and after 24 h of culture in medium, respectively); furthermore, as previously
reported (1), when maintained in the absence of stimulation, the
cytoplasmic expression of cytokines by AMs progressively drops (data
not shown).
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The progressive loss of the activation state of sarcoid AMs was
reversed by cytokine incubation because exposure to IFN- and, in
particular, IL-15 reinduced the expression of B7 molecules. As
demonstrated in Fig. 4, after restimulation with IL-15, sarcoid AMs
reexpressed enhanced levels of CD80 (MFI 81.5 ± 12.9 after restimulation with IL-15; P < 0.001 compared with the histogram of unstimulated AMs by Kolmogorov-Smirnov
analysis) and CD86 (MFI 151.7 ± 99.7 after restimulation with
IL-15; P < 0.001). IFN-
also
induced an upregulation of CD80 (67.2 ± 11.0;
P < 0.001) and CD86 (140.7 ± 40.3; P < 0.001). By contrast,
restimulation with TNF-
and IL-2 did not modify the expression of
CD80 [26.3 ± 10.6 and 31.3 ± 7.4, respectively;
P = not significant (NS) compared with
the histograms of unstimulated AMs] and CD86 (22.1 ± 9.1 and
41.2 ± 11.3, respectively; P = NS). Furthermore, cytokine stimulation did not or only slightly
influenced CD72 expression on AMs in all cases except two patients in
whom IL-15 induced a slight upregulation of CD72 expression (data not
shown). Although IFN-
and, in particular, IL-15 reinduced the
expression of B7 molecules on sarcoid macrophages, cytokine incubation
did not induce CD80, CD86, and CD72 expression on normal macrophages
isolated from five healthy subjects. In particular, stimulation with
IL-15 and IFN-
did not modify the expression of CD80 (31.2 ± 11.1 and 33.4 ± 6.8, respectively;
P = NS compared with the histograms of
unstimulated AMs), CD86 (29.7 ± 8.9 and 33.3 ± 11.3, respectively; P = NS), and CD72
(42.3 ± 7.7 and 41.4 ± 8.4, respectively).
In a third set of experiments, we evaluated whether anti-cytokine
antibodies can reduce the expression of CD80 and CD86 on sarcoid AMs.
As shown in Fig. 5, macrophages showed a
progressive decrease in the expression of CD80 and CD86 coligands after
medium incubation; however, neutralizing antibodies against IL-15 and IFN- did not further decrease the intensity of expression of these
costimulatory molecules (P = NS
compared with medium alone).
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IL-15 and IFN- modulate CD28 expression on sarcoid
T cells. After 24 h of culture, the expression of CD28
partially decreased in medium-cultured sarcoid T cells (CD28 MFI
51.6 ± 8.8 and 27.1 ± 7.3 at time
0 and after 24 h of culture in medium, respectively; Fig. 4B).
When T cells were restimulated for a further 12 h with IL-15 and
IFN-, the cytokine milieu was able to reinduce the expression of
CD28 molecules on sarcoid T cells (MFI 41.2 ± 6.1 and 37.2 ± 6.3, respectively; P < 0.001). In
three of the five patients, IL-2 increased CD28 expression, whereas
TNF-
did not modify the shift of the B7 ligand histograms
(P = NS compared with unstimulated BAL
fluid T cells). Furthermore, IL-2, IFN-
, IL-15, and TNF-
stimulation did not modulate CD154 expression on sarcoid T cells. Figure 5 shows that after the addition of anti-IFN-
and anti-IL-15 neutralizing antibodies, the expression of CD28 by T cells was downregulated, but the difference was not significant compared with the
corresponding values obtained after incubation of AMs with medium alone
(P = NS).
B7 and CD72 molecules expressed by sarcoid macrophages
regulate the proliferative activity of T cells. Taken
together, the above findings suggest that macrophage-derived cytokines
endow a self-perpetuating cycle of inflammation in the sarcoid lung where IFN- and IL-15 favor the expression of costimulatory molecules on APCs involved in T-cell activation and IL-15, in turn, induces proliferation of sarcoid T cells (1). The possibility that the
cytokine-induced expression of B7 molecules may favor the APC capacity
of AMs was investigated by an in vitro coculture assay in which the
mitotic activity of highly purified allogeneic T cells was investigated
in the presence of sarcoid macrophages (Fig.
6). The purity of the T-lymphocyte
populations used in the proliferation assays exceeded 99%, with
virtually no detectable residual monocytes/macrophages. Highly purified
T cells did not proliferate when stimulated with concanavalin A unless
accessory cells were added. However, sarcoid AMs induced the
proliferation of highly purified T cells in the presence of mitogen;
these data were not surprising given the known accessory function of
pulmonary macrophages from patients with interstitial lung
disease in mitogen assays (13, 30).
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Figure 6 shows the blocking effect of anti-CD72, anti-CD80, anti-CD86, and anti-IL-15 MAbs on the accessory function of AMs isolated from the BAL fluid of six patients with sarcoidosis and T-cell alveolitis. It is readily apparent that, when added to the mitogen assay at the beginning of culture, anti-CD72, anti-CD80, anti-CD86, and anti-IL-15 MAbs inhibited the proliferation of T cells; the inhibitory ability of a control isotype-matched IgG added at the beginning of the culture was always <2% (data not shown). Besides confirming the functional importance of the expression of IL-15 by AMs (3), these data indicate the role of CD80-CD86/CD28 pathways in maintaining the T-cell immune response within the alveolar spaces of patients with active sarcoidosis.
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DISCUSSION |
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In this study, we demonstrate that macrophages and T cells isolated from the lungs of patients with sarcoidosis are preactivated cells showing an upmodulation of the CD72/CD5 and CD80-CD86/CD28 costimulatory systems, which, in turn, regulate the proliferation of sarcoid T cells. In addition, pulmonary cells expressed high levels of cytokines, which favored the expression of accessory molecules, suggesting a role for the cytokine-driven modulation of CD72/CD5 and CD80-CD86/CD28 counterreceptors in the compartmentalization of the T-cell response within sarcoid lungs.
T-cell activation requires at least two membrane signals. The first signal is generated by the T-cell receptor after engagement with antigen presented by APCs. In addition, T cells require additional costimulatory signals provided by a set of costimulatory molecules expressed by APCs, including the B7 family and the CD72/CD5 coreceptor system (17). The members of the B7 family belong to the immunoglobulin gene superfamily; under physiological conditions, their expression is restricted to cells that can function as APCs, and no B7 expression was found in normal human lungs. The pattern of CD80 and CD86 expression shown by pulmonary macrophages of patients with interstitial lung disease (CD86 molecule was detected on AMs at a higher intensity than CD80) is consistent with that of conventional APCs because it has been previously demonstrated that CD80 is commonly expressed at lower levels than CD86 on cells with accessory function (13, 17). This suggests the potential role of the B7 family in the pulmonary microenvironment. Likely, the expression of CD80 and CD86 may allow AMs to act as APCs in the stimulation of the local Th1-type T-cell response to antigens as recently demonstrated in other organs (34). In fact, a similar pattern of B7 expression takes place in the intestinal tract during Helicobacter pylori infection of human gastric mucosal epithelial cells, which is characterized by an increase in gastric epithelial T cells (34). Furthermore, an increased expression of B7 molecules on dendritic cells from patients with T cell-mediated skin diseases (21) and in APCs isolated from the synovial fluid of patients with rheumatoid arthritis has been reported (33).
Our data also point out the relevance of the CD5/CD72 costimulatory pathway in the generation of the pulmonary T-cell response taking place in the sarcoid lung. CD5 is a 67-kDa type I transmembrane glycoprotein physically and functionally coupled with the T-cell receptor-CD3 signal transducer complex that is involved in T-cell proliferation. In fact, its triggering, besides inducing IL-2 secretion and upregulation of the IL-2 receptor, signals T cells to proliferate (27). Although it is known that the CD5 coligand CD72 is expressed by B-cell subsets with antigen-presenting capacity (10), the role of this costimulatory molecule in mucosal immunity is less clear. In our study, AMs from patients with sarcoidosis overexpressed the CD5 ligand CD72 and preincubation with anti-CD72 MAb resulted in a partial loss of the macrophagic accessory capacity. To our knowledge, this is the first demonstration of an enhanced expression of the CD72 molecule by cells involved in local mucosal T-cell immune responses. Based on the increased CD72 on AMs, it may be postulated that this molecule allows macrophages to interact with local CD5+ T cells. Just the opposite, the absence of both B7 and CD72 on normal AMs may partially explain their inability to respond to recall antigens. In particular, because the respiratory mucosa represents the largest interface between the external environment and the internal milieu and is exposed daily to over 10,000 liters of inhaled air, it is likely that the lack of these molecules may be important in the induction of local tolerance mechanisms.
The regulation of the expression of costimulatory molecules by both
APCs and T cells is controlled by several cytokines. Both molecules of
the B7 family are expressed on monocytes after IFN- activation (12,
13), whereas IL-10 blocks both CD80 and CD86 antigens on peritoneal
macrophages and downregulates CD86 but not CD80 expression on dendritic
cells (8). However, CD28 and CD152 expression increases on T cells
after activation (18, 31). In turn, CD28 engagement has been shown to
enhance the production of several cytokines including IL-2, IL-4,
TNF-
, and IFN-
(29). Differentiation of human Th1 and Th2 T-cell
subsets is also thought to be dependent on CD28 binding. In particular, there are data suggesting that in the absence of CD28 signaling, naive
T cells are biased toward a Th1 phenotype (28).
The interrelationship between the cytokine network and the
costimulatory pathways is confirmed by our data because the addition of
exogenous IL-15 and IFN- increased CD80 and, in particular, CD86
expression on BAL fluid macrophages and sarcoid
CD4+ T cells showed a Th1-type
profile (6, 22; present study). Interestingly, although IFN-
and, in
particular, IL-15 reinduced the expression of B7 molecules on sarcoid
macrophages, cytokine incubation did not modify the expression on
normal AMs. To explain this negative data, a number of hypotheses can
be proposed: 1) sarcoid macrophages are inherently different from their normal counterparts,
2) the presence of an unknown
antigen(s) that triggers the sarcoid inflammatory process is needed to
induce the IL-15-dependent expression of CD80 and CD86, and
3) newly recruited monocytes
[i.e., the main mononuclear cells that account for the sarcoid
macrophagic alveolitis (2)] but not well-differentiated
macrophages (as AMs resident in the lung of healthy subjects) respond
to IL-15 stimulation with an increased expression of accessory
molecules. Further studies are in progress in our laboratories to
verify these possibilities and to evaluate the importance of other
cytokines in the regulation of costimulatory pathways. In particular,
because IL-12 synergizes with B7-CD28 interaction in inducing efficient
proliferation and cytokine production by T cells (14) and IL-12 is
actively released in the sarcoid lung (21), we are planning to evaluate
whether codependence mechanisms between IL-12 and IL-15 may be involved in the maintenance of sarcoid inflammatory response. Furthermore, further investigations are needed to compare the pattern of cytokines produced by AMs of patients with sarcoidosis with that of patients showing a typical Th2 response in the lung, for instance, patients with
asthma. This comparative analysis will be crucial in determining whether changes in the expression of cytokines and/or costimulatory molecules by AMs selectively drive the development of Th1 and Th2
immune responses and, consequently, lung granuloma development (15).
It is interesting to note that CD152 was never expressed by lung T cells. Recent data (7, 25) support the role for CD152 as a downregulatory molecule in T-cell activation either by competing with CD28 for its ligand or by inducing apoptosis. Agostini et al. (5) and others (24) recently demonstrated that a disregulation of the expression of the TNF-receptor superfamily members takes place in the lungs of patients with sarcoidosis, and it has been hypothesized that the abnormal expression of these molecules may influence the persistence of the local immune response within the lung (5). Studies on the expression of CD152 by T cells during the different phases of the sarcoid inflammatory process at different stages of disease might help in defining the role of this molecule in the pulmonary immune system. A better understanding of the relationship between the TNF-receptor superfamily and CD80-CD86/CD28-CD152 pathways in the lungs of patients with different interstitial lung diseases is expected to offer new insights into elements controlling the immune response within the respiratory tract.
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ACKNOWLEDGEMENTS |
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We thank colleagues from the Department of Pulmonary Medicine
(Padua Hospital, Padua, Italy) who contributed to this project by
allowing study of their patients and performing the bronchoscopies. We
also thank Biogen (Cambridge, MA) for providing recombinant interleukin
(IL)-2; Knoll (Ludwigshafen, Germany) for providing tumor necrosis
factor-; Dr. A. Troutt (Immunex, Seattle, WA) for providing
recombinant IL-15 and anti-IL-15 M110 monoclonal antibody; and Martin
Donach for help in the preparation of the manuscript.
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FOOTNOTES |
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This study was supported by a 1997 Grant from the Regione Veneto (Venice, Italy).
A. Perin, F. Piazza, M. Siviero, and U. Basso are recipients of a fellowship from Ministero dell'Universitá e della Ricerca Scientifica e Tecnologica (Rome, Italy).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: G. Semenzato, Università di Padova, Dipartimento di Medicina Clinica e Sperimentale, Immunologia Clinica, Via Giustiniani 2, 35128 Padua, Italy (E-mail: giansem{at}ux1.unipd.it).
Received 29 May 1998; accepted in final form 23 March 1999.
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