Institute of Immunology, Philipps University, D-35037 Marburg, Germany
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In chronic
silicosis, mechanisms leading to lymphocyte activation are still poorly
understood, although it is well known that not only the lung but also
the draining lymph nodes are affected. In the present study, we
investigated T-cell activation by analysis of cytokine expression in
the enlarged thoracic lymph nodes of rats 2 mo after an 8-day silica
aerosol exposure. In the case of helper T cell (Th) type 1 cytokines, we found a significant increase in interferon (IFN)- mRNA
expression, whereas interleukin (IL)-2 expression remained unchanged.
In contrast, gene transcription for the Th2-type cytokines IL-4 and
IL-10 was diminished. In addition, with use of an in vitro
lymphocyte-macrophage coculture system, an enhanced IFN-
and a
reduced IL-10 release were shown with cells from silicotic animals.
With regard to IFN-
-inducing cytokines, we observed enhanced IL-12
mRNA levels in vivo, whereas IL-18 gene expression was slightly
decreased. These data indicate that a persistent shift toward an
IFN-
-dominated type 1 (Th1/cytotoxic T cell type 1) T-cell reaction
pattern occurred within the thoracic lymph nodes of silicotic animals.
Thus a mutual activation of lymphocytes and macrophages may maintain
the chronic inflammatory changes that characterize silicosis.
interferon-; chronic lung inflammation; particles; T
lymphocytes; cytokines; interleukins; helper T cell type 1
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SILICOSIS IS A CHRONIC FIBROTIC DISORDER of the
pulmonary parenchyma caused by prolonged inhalation of crystalline
particles (43). Whereas the contribution of macrophages to the
pathogenesis of silicosis has been extensively studied over the last
years (5), only little is known about the putative role that
lymphocytes may play in the initiation and maintenance of the disease.
However, clear evidence has accumulated indicating an involvement of
lymphocytes in the progression of silicosis. Using an
immunohistological examination of the lung, Kumar (23) observed a
significant increase in total lymphocyte number after intratracheal
instillation of silica particles. Kinetic analyses revealed that the
influx of T cells preceded the occurrence of B lymphocytes.
Furthermore, an increased number of lymphocytes expressing the
-chain of the interleukin (IL)-2 receptor (CD25) was reported (24).
These results indicate the presence of an augmented proportion of
activated lymphocytes in the total lymphocyte population of the lung.
It is known that inhaled silica particles not only affect the lung but also other organs, most importantly, the mediastinal lymph nodes and, to a lesser extent liver, spleen, kidneys, and thymus (43). It has been proposed that particle-laden macrophages leave the lung and enter the draining lymph nodes via lymphatic capillaries. Indeed, in a rat inhalation model of silicosis, Absher et al. (1) demonstrated the occurrence of silica particles within mediastinal lymph nodes, and Friedetzky et al. (13) showed that those particles were located within macrophages. Thus the appearance of silica-stimulated macrophages may represent the initial signal for the dramatic changes that subsequently occur within the draining lymph nodes. Friedetzky et al. (13) observed recently that, concomitant with the alterations in the lung, the affected thoracic lymph nodes increased tremendously in size and dramatic histological changes took place. One year after exposure, thoracic lymph nodes of silicotic animals displayed a 35-fold weight increase compared with the corresponding lymph nodes of control animals. Histologically, the normal lymph node structure had disappeared, and T- and B-cell areas including germinal centers were no longer detectable. Instead, granuloma-like structures consisting almost exclusively of macrophages, a portion of which contained silica particles, became the most prominent feature within these lymph nodes.
The lymph node enlargement was due to an increase in cell number rather
than an augmentation of extracellular connective tissue matrix (14). In
accordance with the situation in the lung, the increase in T cells,
especially of the CD4+ phenotype, marginally preceded
B-cell infiltration (13). Finally, all major lymph node cell
populations contributed evenly to the enhancement of lymph node cell
number, and no significant alterations between the relative portions of
the different cell types were observed. Moreover, by cytofluorometric
analysis with antibodies to the cell surface activation markers CD25
(IL-2 receptor -chain) and CD54 (intercellular adhesion molecule-1)
of lymph node cells obtained from animals 12 mo after silica exposure,
we demonstrated an elevation in the relative number of activated T
lymphocytes, especially the CD8+ phenotype, and, to a
lesser extent, CD4+ T cells. In addition, increased
interferon (IFN)-
mRNA expression was found at this late time point,
whereas IL-2 and IL-4 mRNA levels remained unaltered (14).
Because these previous data represent the final stage of silicosis, the present study was designed to examine possible T-cell activation at an early time point (2 mo after silica exposure) by analyzing the cytokine gene expression and protein release of cells from affected lymph nodes compared with those from unaffected lymph nodes from the same animal or from healthy control animals. The aim was to examine whether a shift to either a helper T cell (Th) type 1-like or a Th2-like profile occurred that could further delineate the contribution of T cells to the development of the chronic state of silicosis.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals. Silica exposure was carried out at the Votey Animal Inhalation Facility of the University of Vermont (Burlington, VT) with male Fischer 344 rats obtained from the National Cancer Institute (Bethesda, MD). The rats weighed ~165 g at this time. Subsequently, the animals were shipped to Germany by aircraft and housed at the animal facilities of the Institute of Immunology (University of Marburg, Marburg, Germany) where they were maintained in wire cages at a temperature of 18-22°C in a 12:12-h light-dark cycle. Food and water were given ad libitum. The animals appeared healthy during the time of the experiments and gained weight normally. Housing conditions and mineral exposure met National Institutes of Health and German guidelines.
Silica exposure.
The animals were exposed in horizontal flow chambers for 6 h/day for 8 days to aerosols of -cristobalite (C & E Mineral, King of Prussia,
PA) as previously described in detail (17, 18, 28). Matched groups of
rats were simultaneously exposed to the silica aerosol or to the
carrier air (sham control). The aerosol was generated with a Wright
dust feed apparatus and contained particles of respirable size; ~77%
of the particles were <3.3 µm and 55% were <2.0 µm. The mean
air concentration of silica particles was 9.28 mg/m3. Two
months after exposure, the animals were killed by CO2
intoxication. At this time point, the lungs of silica-exposed animals
showed moderate changes typical of chronic silicosis in rodents (43). Lung and lymph node histology and characterization of lymph node cell
populations have been previously described in detail (13). Normal
histology was observed in the lungs and lymph nodes of control animals.
RNA preparation. Thoracic and cervical lymph nodes of six control and six silica-exposed animals were shock-frozen in liquid nitrogen immediately after removal. Frozen tissues were homogenized with nitrogen-chilled pestles and mortars, and total RNA was prepared by use of the RNA clean preparation kit (Angewandte Gemtechnologie Systeme, Heidelberg, Germany) according to the manufacturer's guidelines. RNA yield was determined spectrophotometrically at 260 nm (40).
RT-PCR. First, mRNA from 2 µg of total RNA from each sample was reverse transcribed into cDNA by use of an oligo(dT)20 primer (MWG Biotech, Ebersberg, Germany) and SuperScript II RT (Life Technologies, Gaithersburg, MD) as previously described (14).
For each of the first-strand cDNAs, the following PCRs were set up with specific primers for the sequences of rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH), IFN-
|
Northern blotting. Due to the presumably low copy numbers of the genes of interest within the total lymph node RNA, it was necessary to pool equal amounts of RNA of identical lymph nodes from three comparable animals to obtain sufficient signals. For each sample, 10 µg of total RNA (2 µg for GAPDH Northern blot) were denatured with a formamide-containing sample buffer and subsequently separated electrophoretically on 1% agarose-formaldehyde gels with a running buffer consisting of 20 mM 4-(N-morpholino)propanesulfonic acid, 5 mM sodium acetate, and 1 mM EDTA, pH 7.0 (40). RNAs were transferred to positively charged Biodyne Plus membranes (Pall Biosupport Division, Port Washington, NY) by capillary blotting with 10× saline-saline citrate as a blotting buffer. After drying and ultraviolet cross-linking, the membranes were hybridized with digoxygenin (Dig)-labeled antisense riboprobes in a solution containing 50% formamide at 68°C under continuous rotation in a hybridization oven (Biometra, Göttingen, Germany) as previously described (44). Thereafter, the membranes were carefully washed in 0.1× saline-saline citrate-0.1% sodium dodecyl sulfate at the same temperature, and the bound probes were visualized with the Dig nucleic acid detection kit (Boehringer Mannheim) and CDP-Star chemiluminescence substrate (Tropix, Bedford, MA). Semiquantitative analysis was performed as described in RT-PCR.
Generation of Dig-labeled riboprobes.
Antisense riboprobes corresponding to rat GAPDH, IFN-, IL-2, IL-4,
and IL-10 were generated by RT-PCR, cloning of the respective PCR
products, and subsequent in vitro transcription. Briefly, 1 µg of
total RNA from concanavalin A (Con A)-stimulated rat spleen cells was
reverse transcribed into cDNA followed by PCR with the PCR conditions
mentioned in RT-PCR to amplify the respective
sequences. PCR products were cloned into the TA cloning site of the pCR
II vector with the use of a TA cloning kit (Invitrogen, Leek, The Netherlands). Specificity and cloning direction were confirmed by
sequencing. Dig-labeled RNA probes were generated with SP6 or T7 RNA
polymerase with a Dig-RNA labeling kit (Boehringer Mannheim) with 1 µg of linearized vector as a template.
Cell culture. For preparation of lymphocyte suspensions, thoracic, cervical, and mesenteric lymph nodes were removed aseptically and immediately transferred into cold (4°C) phosphate-buffered saline (PBS) without Ca2+ and Mg2+, pH 7.2. The lymph nodes were dissected and passed through a 70-µm nylon cell strainer (Falcon, Becton Dickinson Labware) with a syringe plunger. The enlarged thoracic lymph nodes of silicotic rats delivered ample cells for study purposes. Thoracic lymph nodes of sham-exposed rats were too small to yield sufficient control cells. Therefore, the lymphocytes from the cervical and mesenteric lymph nodes of individual animals were pooled and served as control cells. The resulting cell suspensions were centrifuged at 300 g for 10 min at 4°C. The cell pellets were resuspended in culture medium (RPMI 1640 medium supplemented with 2 mM L-glutamate, 10 mM HEPES, 1 mM sodium pyruvate, 1× nonessential amino acids, 100 U/ml of penicillin, and 100 mg/ml of streptomycin; all purchased from Biochrom, Berlin, Germany) without fetal calf serum (FCS), filtered through a 70-µm nylon mesh to remove aggregates, and subsequently washed twice with the above-mentioned centrifugation conditions. Thereafter, the lymph node cells were resuspended in culture medium with 20% FCS and adjusted to a cell concentration of 2.5 × 106 cells/ml.
To obtain alveolar macrophages, the lungs of animals were lavaged with 100 ml of prewarmed (37°C) PBS without Ca2+ and Mg2+. The resulting cell suspensions were washed twice with PBS. Subsequently, mononuclear cells were prepared by density gradient centrifugation with Lympholyte-Rat (Cedarlane, Hornby, ON) followed by two washing steps, the first in PBS and the second in culture medium without FCS. After resuspension in FCS-free culture medium, the cells were adjusted to 2.5 × 105 cells/ml. Purity of the alveolar macrophages was >98%, and as shown in a previous study (28), ~30% of alveolar macrophages from silicotic animals contained detectable silica particles. One hundred microliters of the alveolar macrophage suspensions were added to each well of 96-well cell culture plates (Corning Costar, Cambridge, MA) and incubated at 37°C in a humid atmosphere containing 5% CO2. Two hours later, 100 µl of the different lymph node cell suspensions were added, resulting in a macrophage-to-lymphocyte ratio of 1:10. For the control cells, similar numbers of lymphocytes and macrophages were cultured separately. Finally, 1 µg/ml of indomethacin (Sigma-Aldrich, Deisenhofen, Germany) to block production of suppressive prostaglandin E2 (15) and 2.5 µg/ml of Con A (Sigma-Aldrich) dissolved in culture medium with 10% FCS were added, yielding a total volume of 250 µl/well. The final FCS concentration for all cultures was 10%. Cocultures were incubated under the above-mentioned conditions, and 24 or 48 h later, the culture supernatants were removed and stored atCytokine quantitation by ELISA.
Culture supernatants were tested for cytokine content with ELISAs
specific for rat IFN-, IL-2, IL-4, and IL-10. Matched antibody pairs
with a biotinylated monoclonal detection antibody and recombinant standard proteins were purchased from either Biosource (IFN-
; Camarillo, CA) or PharMingen (IL-2, IL-4, and IL-10; San Diego, CA).
Sandwich ELISAs were set up according to standard procedures. Briefly,
Maxisorp immunoplates (Nunc, Roskilde, Denmark) were coated with 50 µl/well of the relevant capture antibody diluted to a predetermined
concentration and incubated overnight at 4°C. All subsequent steps
were performed at room temperature. After three washes with PBS-5%
Tween 20, the nonspecific binding sites were blocked by the addition of
250 µl of blocking buffer (1% bovine serum albumin in PBS) to each
well and 30 min of incubation. The plates were washed three times, and
25 µl of sample buffer (0.5% Tween 20 in blocking buffer) were added
followed by 25 µl of sample or standard (diluted in culture medium
with 10% FCS). Four hours later, the plates were washed three times,
and 50 µl of the appropriately diluted biotinylated detection
antibody were added per well. After incubation for 1 h and 4 washing
steps, 100 µl of horseradish peroxidase-conjugated streptavidin/well (1:10,000 diluted in sample buffer; Boehringer Mannheim) were applied
and incubated for 30 min. After four washes, 100 µl of substrate
solution, 1 mg/ml of o-phenylenediamine (Sigma-Aldrich) in
substrate buffer (0.05 M phosphate-citrate buffer, pH 5.0 with 0.5 µl/ml of 30% H2O2), were applied. The
reaction was stopped 15-30 min later by the addition of 25 µl of
2.5% sulfuric acid. Adsorption was read with microplate reader MR7000
(Dynatech Laboratories, Denkendorf, Germany) at 570 nm with a
630-nm reference filter.
Statistics. All data were calculated and are expressed as means ± SD. After analysis of the sample values for Gaussian distribution, determination of significance was carried out with Student's t-test or the paired t-test.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Th1/Th2 cytokine gene expression in lymph nodes of silicotic animals. As previously shown in rats 12 mo after silica exposure, the enormous enlargement of two lung-draining lymph nodes coincided with an apparent T-lymphocyte stimulation (14). The present investigation was designed to examine these lymph nodes earlier, 2 mo after silica exposure, and to study in detail Th1 and Th2 cytokine gene expression pattern by RT-PCR and Northern blot analysis.
In the first set of experiments, we examined the specific cytokine mRNA
content by semiquantitative RT-PCR, which was the only available method
that allowed the individual detection of mRNA expression in the
particularly small thoracic lymph nodes of sham-exposed control rats.
We compared cytokine gene transcription in the thoracic and cervical
lymph nodes of six silicotic and six control animals. As shown in Fig.
1, a significantly higher mRNA expression
for the Th1 cytokine IFN- was found in the thoracic lymph nodes of
silicotic animals compared with that in the cervical lymph nodes of
silicotic animals or both types of lymph nodes of control rats (Fig.
1A). Interestingly, the level of gene transcription of IL-2,
the second important Th1/cytotoxic T cell (Tc) type 1-derived cytokine,
remained unaltered (Fig. 1B). In contrast, the mRNAs for IL-4
and IL-10, which are dominantly expressed by Th2/Tc2 cells,
were found to be significantly less expressed in the thoracic lymph
nodes of silicotic animals (Fig. 1, C and D).
|
In the second series of experiments, Northern blot analyses were
performed to confirm the semiquantitative RT-PCR data on cytokine gene
transcription. Due to the limited availability of total RNA from the
lymph nodes of control animals and the weak expression of some genes of
interest, we had to pool RNA from the lymph nodes of three individual
animals to obtain detectable signals. When comparing cytokine gene
expression in the enlarged thoracic lymph nodes of silicotic animals
with that in pooled cervical and mesenteric lymph nodes from either
silicotic or healthy rats, we could entirely confirm the results of the
RT-PCR experiments. We found increased mRNA levels for IFN-, no
changes for IL-2, and decreased levels of IL-10 (Fig.
2, A-C). IL-4 mRNA was
not detectable, even with 10 µg total RNA/lane (data not shown). This is in accordance with the RT-PCR results, where 35 PCR cycles were
necessary to obtain just-visible bands of IL-4-specific PCR products.
Thus cytokine mRNA results obtained by RT-PCR corresponded well with
Northern blot data.
|
Cytokine release from in vitro stimulated cells. To establish that enhanced cytokine gene expression resulted in increased production and release of cytokines, we set up in vitro cultures with cells from enlarged thoracic lymph nodes from silicotic animals and compared them with pooled cells from cervical and mesenteric lymph nodes. The lymphocytes were cultured alone or in the presence of alveolar macrophages at a 10:1 ratio with and without the addition of Con A as a mitogenic stimulus. Lymphocytes and alveolar macrophages were obtained from silicotic and control rats, and various combinations were tested with Con A at an optimal (2.5 µg/ml) concentration.
When the lymphocytes and macrophages were cultured alone or in combination for 24 or 48 h in the absence of Con A, no measurable amount of cytokines was released into the culture medium (data not shown). The addition of Con A to the lymph node cells induced the release of moderate amounts of IFN-
|
|
|
|
IL-12 and IL-18 gene expression in lymph nodes of silicotic animals.
To investigate possible mechanisms for the specific increase in IFN-
gene expression and subsequent protein release within the enlarged
thoracic lymph nodes of silicotic rats, we examined the level of gene
transcription for the two major IFN-
-inducing cytokines IL-12 and
IL-18 by semiquantitative RT-PCR (Fig. 7). Interestingly, the gene expres-sions of these two cytokines were regulated in quite opposite directions. Whereas for IL-12, a
significant increase in mRNA amounts could be detected (Fig.
7A), IL-18 expression seemed to be less pronounced within the
silicotic thoracic lymph nodes compared with that in control lymph
nodes (Fig. 7B). Unfortunately, no antibodies against these rat
cytokines are as yet available to detect actual IL-12 and IL-18
release.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although it is known from patients as well as from experimental animal data that in silicosis the lung-draining thoracic lymph nodes are also affected, the underlying mechanisms and the possible involvement of lymphocytes in the pathogenesis of the disease are still poorly understood. At present, the available data remain controversial, which indicates that an in-depth investigation of the role of lymphocytes in silicosis and related lung diseases is urgently required. Kumar et al. (24) observed an activation of lymphocytes in the pulmonary response to silica, and Li et al. (26) supposed that lymphocytes contribute to the regulation of macrophage activities. This concept was further supported by data from Suzuki et al. (46), who showed that athymic mice developed less severe silica-induced interstitial pneumonitis and inflammatory responses than euthymic mice. In contrast, in the rather artificial model of quartz injection into the footpads and subsequent analysis of the popliteal lymph node reaction, Weirich et al. (50) ignored a possible participation of T lymphocytes in the development of silicotic nodules within the lymph nodes. Also, Corsini et al. (3) described a protective role for T lymphocytes in asbestos-induced pulmonary inflammation and collagen deposition in mice.
Garn et al. (14) recently demonstrated that the enlargement of thoracic lymph nodes after silica exposure was mainly caused by an influx of mononuclear cells. Initial lymph node changes could be observed as early as 2 wk after particle exposure (13). Even 1 yr after silica exposure, an increased number of activated T lymphocytes could be detected with flow cytometric analysis by the expression of surface activation markers (14). The present study was designed to examine in detail the cytokine profile within the enlarged thoracic lymph nodes 8 wk after silica exposure. Our main interest was focused on the expression of cytokines that may drive an immune response in either the Th1 or a Th2 direction.
The most important finding of the present study was the differential
regulation of IFN- and IL-10 within the affected thoracic lymph
nodes of silicotic animals. In the case of IFN-
, an increase in gene
expression could be demonstrated by Northern blot analysis and
semiquantitative RT-PCR (Figs. 1 and 2). In addition, further evidence
for an important role for IFN-
was obtained by in vitro stimulation
experiments. Lymph node cells from the enlarged silicotic lymph nodes
released significantly higher amounts of IFN-
into the culture
medium compared with similar cells obtained from unaffected lymph nodes
from either control or silica-exposed animals (Fig. 3). Opposite
results were observed for IL-10 and IL-4, which showed reduced mRNA
amounts (Figs. 1 and 2), and in the case of IL-10, a decrease in
protein release after in vitro stimulation could be demonstrated (Fig.
4). These data indicate that in silicosis those lymphocytes that
produce the Th1 cytokine IFN-
were preferentially stimulated,
whereas the number or activity of Th2 cytokine-producing cells seemed
to be diminished. IFN-
itself is known to efficiently suppress
a Th2/Tc2 response by inhibiting the transcription and release of IL-4 and IL-10 (2, 29, 32, 35), the first of which is
considered to be necessary for the onset of a Th2 response (25, 49).
Thus IFN-
may be responsible for the downregulation of IL-4
and IL-10.
Because of its inhibitory effects on inflammatory responses, the decreased expression of IL-10 is of special interest. IL-10 is known to inhibit several functions of macrophages such as synthesis of proinflammatory cytokines, release of oxygen radicals and nitric oxide, and antigen-presenting capacity (4, 9). In addition, IL-10 may reduce the production of Th1/Tc1-derived cytokines (12, 29) but, on the other hand, may stimulate B-cell proliferation (8, 38). Thus IL-10 is clearly involved in the shift of an immune reaction toward a Th2/Tc2 response. Using a quartz instillation model in mice, Huaux et al. (20) showed increased levels of IL-10 mRNA and protein within the lung tissue and in the cells of bronchoalveolar lavage fluid 24 h after instillation of particles. These data indicate a regulatory role for IL-10 in the early inflammatory response that was further confirmed by Driscoll et al. (10), who demonstrated an attenuating effect of exogenously applied IL-10, whereas the application of an anti-IL-10 antiserum increased the inflammatory reaction. Beneficial effects of IL-10 have also been described in other inflammatory lung diseases such as adult respiratory distress syndrome (27) and acute lung injury caused by acute necrotizing pancreatitis (34). Compared with the results by Huaux et al. (20) and Driscoll et al. (10), our data clearly indicate a differential expression of IL-10 in the immediate inflammatory response in the lung and the chronic state of the disease when studied in the lung-draining lymph nodes. However, it cannot be excluded that a principally different IL-10 expression pattern may occur in different tissue compartments and at different time periods during the development of silicosis. On the basis of our results, we postulate that the decreased expression of IL-10 at later time points after silica exposure is a crucial point for the development of the chronic state of silicosis. Whether differences in IL-10 expression in the lung and lymph nodes are caused by a differential regulation in a distinctive cell population or are rather due to an involvement of different cells (Th2/Tc2 lymphocytes, macrophages, epithelial cells) has to be investigated in the future. The extremely weak mRNA expression for IL-4 and, subsequently, the lack of IL-4 in culture supernatants of in vitro stimulated cells may be due to the animal model employed here because our data are in agreement with results by Odinot et al. (31), who observed in an infection model almost no IL-4 expression in spleen and lymph nodes when using Fischer 344 rats compared with Lewis or Norway rats.
The fact that no changes in IL-2 gene transcription or protein release
after in vitro stimulation of the lymph node cells from silicotic lymph
nodes were detected raises the question of which cell populations were
responsible for the increased IFN- release. Generally, IFN-
may
be produced by CD4+ Th1-lymphocytes, CD8+ Tc1
lymphocytes, and natural killer cells (11). Because these cell types
may also produce IL-2 (30), special signaling mechanisms inducing a
selective IFN-
gene expression have to be proposed. To further
address this question, we examined mRNA expression for the cytokines
IL-12 and IL-18, both of which are strong inducers of IFN-
(33).
Interestingly, we found increased levels of IL-12 mRNA, whereas IL-18
gene transcription was even slightly diminished (Fig. 7). IL-12 is
known to preferentially regulate the proliferation of Th1 T-cell clones
(22, 47), which subsequently produce IFN-
. In contrast, IL-18 acts
mainly as an amplifier of IFN-
production, for example, in
IL-12-activated Th1 lymphocytes (33). Thus an increase in IL-12 release
by activated macrophages within the affected thoracic lymph nodes could
be responsible for activation of IFN-
-producing Th1 cells.
In addition, the stimulatory role of alveolar macrophages for the
activation of T lymphocytes is of particular interest. In contrast to
previous findings (37) describing an inhibitory influence of alveolar
macrophages on T-cell functions (19), our results show significant
stimulating effects of alveolar macrophages, independent of whether
they are derived from silicotic or normal animals, at least in the
release of the cytokines IFN-, IL-2, and IL-10 by mitogen-stimulated
T lymphocytes (Figs. 3-5). One reason for this discrepancy might
be that we introduced indomethacin into the cell cultures to abolish
the inhibitory effects caused by the release of prostaglandin
E2 by macrophages (15, 41). Because alveolar macrophages
from silicotic animals are more potent in aiding cytokine production by
lymphocytes, it seems likely that activated macrophages exert their
stimulatory effects by the release of cytokines. However, we could not
detect any IL-1 in the culture supernatants (data not shown), which
indicates a minor role for proinflammatory cytokines at this stage of
the disease. Instead, regulatory cytokines such as IL-12 may be
responsible candidates for the apparent lymphocyte stimulation. In
addition to cytokine-mediated mechanisms, specific physical cell-cell
interactions, i.e., by costimulatory cell surface molecules and/or
adhesion molecules, have to be taken into account. In vivo, macrophage properties may change when activated lung macrophages leave the lung
and migrate to the lymph nodes. Because alveolar macrophages have been
described to have poor accessory activity (36) and suppressive
properties with respect to T-cell activation (45), it is feasible that
they will become more activated when they leave the lung and enter the
lymph nodes. Additional experiments are in progress to further analyze
the properties of lymph node macrophages and to investigate the
mechanisms of in situ and in vitro macrophage-lymphocyte interactions.
In conclusion, activated alveolar macrophages appear to play an
important role in the initiation of pathological changes within the
lung as well as in the lung-draining lymph nodes during silicosis. Activated macrophages enter these thoracic lymph nodes (16) where they
most likely contribute to the observed lymphocyte stimulation. The
initiating mechanisms of lymphocyte activation still remain unresolved.
An antigenic structure has not been described on silica particles,
which suggests that other mechanisms such as oligoclonal or polyclonal
T-cell activation by macrophage-derived cytokines, e.g., IL-12, may
have taken place (48). Once activated, T lymphocytes produce IFN-,
which leads to an amplifying mechanism driven by the mutual stimulation
of macrophages and lymphocytes within the affected thoracic lymph nodes
that has been initiated and, in an unknown way, maintained by silica
particles. This continuous process may be responsible for the chronic
changes within the lymph nodes of silicotic animals and patients.
Furthermore, activated lymphocytes are able to leave the lymph nodes
via the efferent lymph and may enter the lung. Indeed, increased
lymphocyte numbers (23) with an augmented portion of activated (24) and
IFN-
-producing (6) lymphocytes have been described in the lungs of
silicotic rodents. Within the lung, those lymphocytes may sustain the
macrophage infiltrate (21) and contribute to macrophage activation via release of IFN-
. Thus IFN-
may be the postulated
lymphocyte-derived cytokine stimulating the alveolar
macrophage-secreted fibroblast mitogenic activity (26). This view is
supported by most recent observations of Davis et al. (7), who showed
significantly reduced lung fibrosis in IFN-
knockout mice. The
presence of IFN-
-activated macrophages and macrophage-derived
mediators appear to form the basis for continuous fibrotic and
inflammatory processes and subsequent pathological changes in the lung.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. G. S. Davis and Dr. D. R. Hemenway (University of Vermont, Burlington, VT) for exposure of the rats at the Votey Animal Inhalation Facility and the delivery of the animals to Germany.
![]() |
FOOTNOTES |
---|
This study was supported by Deutsche Forschungsgemeinschaft Grant Ge 354/9-1 and German Ministry for Education and Research Grant 07ITU06/8.
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: D. Gemsa, Institute of Immunology, Philipps Univ. of Marburg, Robert-Koch-Str. 17, D-35037 Marburg, Germany (E-mail: gemsa{at}mailer.uni-marburg.de).
Received 2 August 1999; accepted in final form 21 January 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Absher, MP,
Hemenway DR,
Leslie KO,
Trombley L,
and
Vacek P.
Intrathoracic distribution and transport of aerosolized silica in the rat.
Exp Lung Res
18:
743-757,
1992[ISI][Medline].
2.
Boehm, U,
Klamp T,
Groot M,
and
Howard JC.
Cellular responses to interferon-.
Annu Rev Immunol
15:
749-795,
1997[ISI][Medline].
3.
Corsini, E,
Luster MI,
Mahler J,
Craig WA,
Blazka ME,
and
Rosenthal GJ.
A protective role for T lymphocytes in asbestos-induced pulmonary inflammation and collagen deposition.
Am J Respir Cell Mol Biol
11:
531-539,
1994[Abstract].
4.
Cunha, FQ,
Moncada S,
and
Liew FY.
Interleukin-10 (IL-10) inhibits the induction of nitric oxide synthase by interferon-gamma in murine macrophages.
Biochem Biophys Res Commun
182:
1155-1159,
1992[ISI][Medline].
5.
Davis, GS,
and
Gemsa D.
Immunopathogenesis of silicosis.
In: Immunopathology of Lung Disease, edited by Kradin RL,
and Robinson BWS. Boston, MA: Butterworth-Heinemann, 1996, p. 445-467.
6.
Davis, GS,
Pfeiffer LM,
and
Hemenway DR.
Expansion of interferon--producing lung lymphocytes in mouse silicosis.
Am J Respir Cell Mol Biol
20:
813-824,
1999
7.
Davis, GS,
Pfeiffer LM,
and
Hemenway DR.
Reduced silicosis in interferon- (IFN-
) knock-out mice (Abstract).
Am J Respir Crit Care Med
159:
A24,
1999[ISI].
8.
Defrance, T,
Vanbervliet B,
Briere F,
Durand I,
Rousset F,
and
Banchereau J.
Interleukin 10 and transforming growth factor beta cooperate to induce anti-CD40-activated naive human B cells to secrete immunoglobulin A.
J Exp Med
175:
671-682,
1992[Abstract].
9.
De Waal Malefyt, R,
Abrams J,
Bennett B,
Figdor CG,
and
de Vries JE.
Interleukin 10 (IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes.
J Exp Med
174:
1209-1220,
1991[Abstract].
10.
Driscoll, KE,
Carter JM,
Howard BW,
Hassenbein D,
Burdick M,
Kunkel SL,
and
Strieter RM.
Interleukin-10 regulates quartz-induced pulmonary inflammation in rats.
Am J Physiol Lung Cell Mol Physiol
275:
L887-L894,
1998
11.
Farrar, MA,
and
Schreiber RD.
The molecular cell biology of interferon- and its receptor.
Annu Rev Immunol
11:
571-611,
1993[ISI][Medline].
12.
Fiorentino, DF,
Bond MW,
and
Mosmann TR.
Two types of mouse T helper cells. IV: Th2 clones secrete a factor that inhibits cytokine production by Th1 clones.
J Exp Med
170:
2081-2095,
1989[Abstract].
13.
Friedetzky, A,
Garn H,
Kirchner A,
and
Gemsa D.
Histopathological changes in the enlarged thoracic lymph nodes during the development of silicosis in rats.
Immunobiology
199:
119-132,
1998[ISI][Medline].
14.
Garn, H,
Friedetzky A,
Davis GS,
Hemenway DR,
and
Gemsa D.
T-lymphocyte activation in the enlarged thoracic lymph nodes of rats with silicosis.
Am J Respir Cell Mol Biol
16:
309-316,
1997[Abstract].
15.
Gemsa, D.
Stimulation of prostaglandin E release from macrophages and possible role in the immune response.
Lymphokines
4:
335-375,
1981.
16.
Harmsen, AG,
Muggenburg BA,
Snipes MB,
and
Bice DE.
The role of macrophages in particle translocation from lungs to lymph nodes.
Science
230:
1277-1280,
1985[ISI][Medline].
17.
Hemenway, DR,
and
MacAskill SM.
Design, development and test results of a horizontal flow inhalation toxicology facility.
Am Ind Hyg Assoc J
43:
874-879,
1982[ISI][Medline].
18.
Hemenway, DR,
Sylvester D,
Gale PN,
Vacek P,
and
Evans JN.
Effectiveness of animal rotation in achieving uniform dust exposure and lung deposition in horizontal flow chambers.
Am Ind Hyg Assoc J
44:
655-658,
1993.
19.
Holt, PG.
Alveolar macrophages. II. Inhibition of lymphocyte proliferation by purified macrophages from rat lung.
Immunology
37:
429-436,
1979[ISI][Medline].
20.
Huaux, F,
Louahed J,
Hudspith B,
Meredith C,
Delos M,
Renauld J-C,
and
Lison D.
Role of interleukin-10 in the lung response to silica in mice.
Am J Respir Cell Mol Biol
18:
51-58,
1998
21.
Hubbard, AK.
Role for T lymphocytes in silica-induced pulmonary inflammation.
Lab Invest
61:
46-52,
1989[ISI][Medline].
22.
Kennedy, MK,
Picha KS,
Shanebeck KD,
Anderson DM,
and
Grabstein KH.
Interleukin-12 regulates the proliferation of Th1, but not Th2 or Th0, clones.
Eur J Immunol
24:
2271-2278,
1994[ISI][Medline].
23.
Kumar, RK.
Quantitative immunohistologic assessment of lymphocyte populations in the pulmonary inflammatory response to intratracheal silica.
Am J Pathol
135:
605-614,
1989[Abstract].
24.
Kumar, RK,
Li W,
and
O'Grady R.
Activation of lymphocytes in the pulmonary inflammatory response to silica.
Immunol Invest
19:
363-372,
1990[ISI][Medline].
25.
Launois, P,
Swihart KG,
Milon G,
and
Louis JA.
Early production of IL-4 in susceptible mice infected with Leishmania major rapidly induces IL-12 unresponsiveness.
J Immunol
158:
3317-3324,
1997[Abstract].
26.
Li, W,
Kumar RK,
O'Grady R,
and
Velan GM.
Role of lymphocytes in silicosis: regulation of secretion of macrophage-derived mitogenic activity for fibroblasts.
Int J Exp Pathol
73:
793-800,
1992[ISI][Medline].
27.
Lo, CJ,
Fu M,
and
Cryer HG.
Interleukin 10 inhibits alveolar macrophage production of inflammatory mediators involved in adult respiratory distress syndrome.
J Surg Res
79:
179-184,
1998[ISI][Medline].
28.
Mohr, C,
Gemsa D,
Graebner C,
Hemenway DR,
Leslie KO,
Absher MP,
and
Davis GS.
Systemic macrophage stimulation in rats with silicosis: enhanced release of tumor necrosis factor- from alveolar and peritoneal macrophages.
Am J Respir Cell Mol Biol
5:
395-402,
1991[ISI][Medline].
29.
Morel, PA,
and
Oriss TB.
Crossregulation between Th1 and Th2 cells.
Crit Rev Immunol
18:
275-303,
1998[ISI][Medline].
30.
Mosmann, TR,
and
Coffman RL.
TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties.
Annu Rev Immunol
7:
145-173,
1989[ISI][Medline].
31.
Odinot, PT,
Curfs JHAJ,
Meis JFGM,
Melchers WJG,
and
Hoogkamp-Korstanje JAA
Local expression of cytokine mRNA in spleen and Peyer's patches of rats is involved in resistance against infection with Yersinia enterocolitica.
Cytokine
10:
206-212,
1998[ISI][Medline].
32.
O'Garra, A,
and
Murphy K.
Role of cytokines in determining T-lymphocyte function.
Curr Opin Immunol
6:
458-466,
1994[ISI][Medline].
33.
Okamura, H,
Kashiwamura S,
Tsutsui H,
Yoshimoto T,
and
Nakanashi K.
Regulation of interferon-gamma production by IL-12 and IL-18.
Curr Opin Immunol
10:
259-264,
1998[ISI][Medline].
34.
Osman, MO,
Jacobsen O,
Kristensen JU,
Deleuran B,
Gesser B,
Larsen CG,
and
Jensen SL.
IT 9302, a synthetic interleukin-10 agonist, diminishes acute lung injury in rabbits with acute necrotizing pancreatitis.
Surgery
124:
584-592,
1998[ISI][Medline].
35.
Paludan, SR.
Interleukin-4 and interferon-gamma: the quintessence of a mutual antagonistic relationship.
Scand J Immunol
48:
459-468,
1998[ISI][Medline].
36.
Rich, AR,
Tweardy DJ,
Fujiwara H,
and
Ellner JJ.
Spectrum of immunoregulatory functions and properties of human alveolar macrophages.
Am Rev Respir Dis
136:
258-265,
1987[ISI][Medline].
37.
Roth, MD,
and
Golub SH.
Human pulmonary macrophages utilize prostaglandins and transforming growth factor beta1 to suppress lymphocyte activation.
J Leukoc Biol
53:
366-371,
1993[Abstract].
38.
Rousset, F,
Garcia E,
Defrance T,
Peronne C,
Vezzio N,
Hsu DH,
Kastelein R,
Moore KW,
and
Banchereau J.
Interleukin 10 is a potent growth and differentiation factor for activated human B lymphocytes.
Proc Natl Acad Sci USA
89:
1890-1893,
1992[Abstract].
39.
Ruano, G,
Pagliaro EM,
Schwartz TR,
Lamy K,
Messina D,
Gaensslen RE,
and
Lee HC.
Heat-soaked PCR: an efficient method for DNA amplification with applications to forensic analysis.
Biotechniques
13:
266-274,
1992[ISI][Medline].
40.
Sambrook, J,
Fritsch EF,
and
Maniatis T.
Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989.
41.
Sestini, P,
Tagliabue A,
and
Boraschi D.
Modulation of macrophage suppressive activity and prostaglandin release by lymphokines and interferon: comparison of alveolar, pleural and peritoneal macrophages.
Clin Exp Immunol
58:
573-580,
1984[ISI][Medline].
42.
Siegling, A,
Lehmann M,
Platzer C,
Emmrich F,
and
Volk H-D.
A novel multispecific competitor fragment for quantitative PCR analysis of cytokine gene expression in rats.
J Immunol Methods
177:
23-28,
1994[ISI][Medline].
43.
Silicosis and Silicate Disease Committee.
Diseases associated with exposure to silica and nonfibrous silicate minerals.
Arch Pathol Lab Med
112:
673-720,
1988[ISI][Medline].
44.
Sprenger, H,
Konrad K,
Rischkowsky E,
and
Gemsa D.
Background reduction in Northern analysis by preabsorption of digoxygenin-labeled probes.
Biotechniques
19:
334-340,
1995[ISI][Medline].
45.
Strickland, D,
Kees UR,
and
Holt PG.
Regulation of T-cell activation in the lung: alveolar macrophages induce reversible T-cell anergy in vitro associated with inhibition of interleukin-2 receptor signal transduction.
Immunology
87:
250-258,
1996[ISI][Medline].
46.
Suzuki, N,
Ohta K,
Horiuchi T,
Takizawa H,
Ueda T,
Kuwabara M,
Shiga J,
and
Ito K.
T lymphocytes and silica-induced pulmonary inflammation and fibrosis in mice.
Thorax
51:
1036-1042,
1996[Abstract].
47.
Trinchieri, G.
Interleukin-12 and its role in the generation of TH1 cells.
Immunol Today
14:
335-338,
1993[ISI][Medline].
48.
Trinchieri, G.
Proinflammatory and immunoregulatory functions of interleukin-12.
Int Rev Immunol
16:
365-396,
1998[Medline].
49.
Von der Weid, T,
Beebe AM,
Roopenian DC,
and
Coffman RL.
Early production of IL-4 and induction of Th2 responses in the lymph node originate from an MHC class I-independent CD4+NK1.1-T cell population.
J Immunol
157:
4421-4427,
1996[Abstract].
50.
Weirich, U,
Friemann J,
Rehn B,
Henkelüdecke U,
Lammers T,
Sorg C,
Bruch J,
and
Gleichmann E.
Silicotic lymph node reactions in mice: genetic differences, correlation with macrophage markers, and independence from T lymphocytes.
J Leukoc Biol
59:
178-188,
1996[Abstract].