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Department of Clinical and Experimental Medicine, Padova University School of Medicine, Padova, Italy;
Institutes of General Pathology and
Department of Pathology, Verona University School of Medicine, Verona, Italy;
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Research and Discovery LeukoSite Inc., Cambridge, MA 02142; and
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Infectious Disease Unit, Massachusetts General Hospital, Department of Medicine, Harvard Medical School, MA 02129
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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secreted
definite levels of IP-10 capable of inducing T cell chemiotaxis.
Interestingly, alveolar lymphocytes recovered from patients with active
sarcoidosis were CD4+ T cells expressing Th1 cytokines
(IL-2 and IFN-) and high levels of CXCR3. Taken together, these data
suggest the potential role of IP-10 in regulating the migration and
activation of T cells toward sites of sarcoid inflammatory process and
the consequent granuloma formation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the etiology of sarcoidosis is still unknown, the
characteristic immunologic and immunohistologic patterns of the
granulomatous lesions suggest that they are the result of an Ag-driven
response. Studies of sarcoid macrophages have shown that these cells
behave as versatile secretory cells that, acting as APCs, release a
great variety of cytokines, including TNF-
, IL-12, IL-15, and growth
factors (3, 4, 5, 6). There is also evidence that sarcoid T cells, which are
biased in expression of genes for the ß-chain V region of the TCR
(7, 8, 9), spontaneously release IFN- and IL-2 (10, 11). Taken
together, these data have suggested the concepts that 1) sarcoid
granulomas are formed in response to a persistent and poorly degradable
antigenic stimulus that locally induces an oligoclonal Th1-type T
cell-mediated immune response; 2) as a consequence of their chronic
stimulation, macrophages locally release mediators of inflammation,
leading to accumulation of Th1 cells at sites of ongoing inflammation
and contributing to the development of the granuloma structure.
The superfamily of chemokines consists of an array of chemoattractant
proteins that has been divided into four branches (C, CC, CXC, and
CXXXC) on the basis of the relative position of the cysteine residues
in the mature protein (12, 13). Structural variations of chemokines
have been demonstrated to be associated with differences in their
ability to regulate the trafficking of immune cells during inflammatory
responses (13). For instance, IFN-inducible protein 10
(IP-10)3, a CXC chemokine
that is induced in monocytes/macrophages by IFN- (14), has been
shown to be chemotactic for IL-2-activated human T cells, but not for
neutrophils (15). The fact that IP-10 favors the migration of T cells
to sites of inflammation makes it an attractive candidate to
investigate the mechanisms promoting the development of sarcoid
granuloma. Interestingly, IP-10 expression has been seen in the
epidermis of patients with another delayed-type hypersensitivity
granulomatous disorder, i.e., tuberculoid leprosy (16).
In this study, we investigated whether IP-10 is expressed by inflammatory cells at sites of delayed-type hypersensitivity cellular immune response characterizing sarcoidosis. Specifically, we evaluated the in situ expression of IP-10 in tissues presenting granulomatous lesions related to sarcoidosis, the relationship between the levels of IP-10 in the bronchoalveolar lavage (BAL) fluid and the degree of sarcoid CD4+ T cell alveolitis, and the in vitro production of IP-10 by sarcoid macrophages. Furthermore, we evaluated whether sarcoid T cells express CXCR3, i.e., the receptor for IP-10 and other related lymphotactic chemokines (Mig, I-TAC) (13, 17, 18). Data reported herein suggest the direct involvement of macrophage-derived IP-10 in the local accumulation of CD4+ T cells and formation of sarcoid granuloma.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Twenty-four patients with sarcoidosis were analyzed (6 male and 18 female; mean age 37.3 ± 7.4 yr; 4 smokers and 20 nonsmokers). In all cases, the diagnosis was made by biopsy obtained either from the lungs or from lymph nodes and showing noncaseating epithelioid granulomas, with no evidence of inorganic material known to cause granulomatous diseases. According to our staging system for sarcoidosis (19), each patient underwent BAL analysis.
Seventeen 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 x 103 lymphocytes/ml); 2) positivity to 67Ga scan; and 3) lung CD4/CD8 ratio more than 5. Apart from the BAL analysis, the assessment of disease activity included clinical features, chest radiograph, lung function tests, high resolution computed tomography, and routine blood studies.
Other seven BAL samples were obtained by as many patients with previously diagnosed pulmonary sarcoidosis who repeated BAL analysis during their follow-up period. All of these patients were in the inactive phase of the disease because sequential BAL data performed during their follow-up period showed a regression of the CD4 alveolitis either spontaneously or with therapy. In particular, at the time of our analysis, all patients had normal lung function, normal BAL cell numbers, negative 67Ga scan, and no clinical signs of acute disease. Four of these seven patients were previously given steroid therapy (prednisone 1 mg/kg/day), but no patients received immunosuppressive therapy for 6 mo before the BAL analysis. The average period of follow-up for this group of patients was 35 ± 17.1 mo (range, 18–46 mo).
Six normal subjects were accepted as controls for the BAL studies (3 male and 3 female; mean age 29.8 ± 5.8 yr; 2 nonsmoking healthy persons and 4 subjects evaluated for cough complaints without lung disease). All had normal physical examination, chest x-ray, lung function tests, and BAL cell numbers.
Preparation of cell suspensions
Following administration of local anesthesia, the BAL was performed as previously described (20). Briefly, a total of 150–200 ml of saline solution was injected via fiberoptic bronchoscopy in 25-ml aliquots, with immediate vacuum aspiration after each aliquot. The fluid was filtered through gauze, and its volume was measured: 55.1 ± 4.9% of the injected fluid was recovered. Cells recovered from the BAL were washed three times with PBS, resuspended in endotoxin-tested RPMI 1640 (Sigma Chemical, St. Louis, MO) supplemented with 20 mM HEPES and L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FCS (ICN Flow, Costa Mesa, CA), and then counted. Alveolar macrophages (AMs), lymphocytes, neutrophils, and eosinophils were differentially counted in a total count of 300 cells, according to morphologic criteria, in cytocentrifuged smears stained with Wright-Giemsa.
AMs and BAL T cells were enriched from the entire mononuclear cell suspensions by rosetting with neuraminidase-treated SRBC, followed by Ficoll-Hypaque gradient separations, as previously described (20). Following this multistep selection procedure, more than 95% of the above cells were viable, as judged by trypan blue exclusion. Staining with CD3 (Immunotech, Marseille, France) mAb showed a percentage of CD3 T cells ranging from 98–100% in purified BAL T lymphocytes, while more than 98% of purified AMs expressed the macrophage-associated PAM-1 Ag (kindly provided by Dr. A. Mantovani, Milan, Italy).
IP-10 protein levels in BAL fluid
IP-10 protein levels were measured in the fluid component of BAL using a specific double-determinant RIA, as previously reported (21). Briefly, flat-bottom 96-well plates (MaxiSorp, Nunc, Naperville, IL) were coated with 100 µl/well of goat anti-IP-10 Ab (R&D Systems, Minneapolis, MN) (5 µg/ml in 0.1 M carbonate buffer, pH 9.5) for 24 h at 4°C and then extensively washed with PBS, pH 7.5, 0.05% Tween 20 (washing buffer). A total of 100 µl/well of either IP-10 standards (Peprotech, Rocky Hill, NJ) or BAL fluid was then added, followed by an overnight incubation at 4°C. Plates were rinsed with washing buffer before addition of 100 µl 125I-labeled affinity-purified rabbit anti-human IP-10 polyclonal Ab (0.6 µg/ml in PBS-Tween with 50% FCS) and incubated overnight at 4°C (22, 23). After washings of the plates, 100 µl of 1 N NaOH was added into each well; samples were harvested after 30 min, and read in a gamma counter. This RIA had a detection limit of 10 pg/ml.
Immunohistochemical analysis of IP-10-producing cells
Fresh frozen tissue samples were obtained from lung and lymph nodes of five patients. Fragments of the tissue samples were covered with OCT medium (Ames, Elkhart, IN), snap frozen in liquid nitrogen, and stored at -80°C until analyzed. Five-micron cryostat sections were dried onto glass covered with polylysine adhesive (Sigma) and fixed in cold chloroform/acetone mixed in 1:1 ratio.
The alkaline phosphatase/anti-alkaline phosphatase or avidin-biotin peroxidase techniques were used for revealing IP-10-producing cells together with differentiation Ag (CD4 and CD68) on cell membranes on several sections, as previously reported (24). Specifically, to evaluate IP-10 immunohistochemistry, we covered cryostat sections with diluted swine serum to avoid nonspecific binding of Abs and then incubated them for 60 min in a moist chamber with a 1/750 dilution of unconjugated affinity-purified rabbit anti-human IP-10 polyclonal Ab.
Flow cytometry analysis of BAL cells
The commercially available conjugated or unconjugated mAbs used
belonged to the Becton Dickinson (Sunnyvale, CA) and Immunotech
series and included: CD3, CD4, CD8, CD29, and CD45R0. The unconjugated
affinity-purified rabbit anti-human IP-10 polyclonal Ab,
anti-IL-2 mAb (purchased from PharMingen, San Diego, CA),
anti-IFN- mAb (PharMingen), anti-IL-4 mAb (PharMingen),
anti-IL-15 mAb (M112; Genzyme, Boston, MA), and anti-hCXCR3 mAb
(1C6; Leukosite, Cambridge, MA) were also used.
The frequency of BAL cells positive for the above reagents was
determined by flow cytometry, as previously described (5). Briefly,
10 x 103 cells were acquired, and the analysis was
determined by overlaying the histograms of the samples stained with the
different reagents. Both BAL lymphocytes and AMs were gated in flow
cytometry analysis using these two different approaches: 1) physical
characteristics of cells, and 2) expression of the T-associated CD3 and
AM-associated PAM-1 Ags on the area of lymphocytes and AMs,
respectively. For direct fluorescence analysis, FITC- or
phycoerythrin-conjugated control, isotype-matched mouse mAbs were used
to set the negative control (IgG1, IgG2a, IgG2b; Becton Dickinson). In
the indirect fluorescence analysis, cells were incubated with control
purified isotype-matched mAb (Becton Dickinson). The proper control for
affinity-purified rabbit anti-human IP-10 polyclonal Ab was
represented by 100 µg/ml of rabbit IgG myeloma protein (Serotec,
Oxford, U.K.). The second-step reagents were represented by
F(ab')2 anti-rabbit IgG (Caltag, South San Francisco,
CA) for IP-10 Ab staining. To eliminate nonspecific binding of Abs to
cytokines (IP-10, IFN-, IL-2, IL-4, IL-15) bound to the surface of
cells, the cells were washed in 40 mM citrate containing 140 mM NaCl,
pH 4, to remove possible bound cytokines.
The purity of the gates was always higher than 98% cells. Cells were
scored using a FACScan analyzer (Becton Dickinson), and data were
processed using the Macintosh CELLQuest software program (Becton
Dickinson). The expression of cytoplasmic cytokines was evaluated
following permeabilization of cell membranes using 1/2 diluted Permea
Fix (Ortho, Raritan, NJ) for 40 min. After the permeabilization
procedure, affinity-purified rabbit anti-human IP-10 polyclonal Ab,
anti-IL-2, anti-IL-4, anti-IL-15, and anti-IFN- mAbs
were added, as previously reported (5).
In vitro production of IP-10 by pulmonary cells
To verify the ability of AMs to release the chemokine,
unstimulated AMs (1 x 106/ml) were isolated both from
sarcoid patients and healthy subjects, resuspended in RPMI medium, and
cultured for 24 h in 24-well plates at 37° in 5%
CO2. In separate experiments, AMs were stimulated with
IFN- (100 U/ml) and LPS (10 µg/ml; Difco, Detroit, MI). Following
the incubation period, supernatants were harvested, filtered through a
0.45-µm Millipore filter (Molsheim, France), and
immediately stored at -80°C. At the end of the culture time, AM
viability was always greater than 95%. IP-10 protein levels in
supernatants were measured using the above reported double-determinant
RIA.
Biologic activity of AM supernatants and BAL fluids on the CXCR3+2F12 T cell clone
The 2F12 (CD4+) T cell clone (kindly provided by Dr. James Kurnick, Massachusetts General Hospital, Charlestown, MA) was used to evaluate the chemotactic activities of AM supernatants and BAL fluids. The cells were grown in RPMI 1640 supplemented with 1% glutamine, 5% human serum, 1% kanamycin, and 100 U/ml human rIL-2. Cells were periodically expanded by restimulation with PHA (1 µg/ml) in the presence of irradiated blood mononuclear cells (10:1 ratio of feeder cells:2F12) and used for experiments after a culture period of 10 to 14 days.
Cell migration was measured in a 48-well modified Boyden chamber.
Chemokines were diluted in HEPES-buffered RPMI 1640 supplemented with
0.1% BSA. Supernatants from cell cultures and BAL samples were used
undiluted; different concentrations of IP-10 were utilized as positive
control. Polyvinylpyrrolidone-free polycarbonate membranes (pore size,
3 µm) coated with type IV collagen were used. A total of 30 µl of
samples was added to the bottom wells, and 50 µl of 2F12 cells
resuspended in the HEPES-buffered RPMI 1640 were added to the top wells
(
100,000 cells/ml). Chambers were incubated at 37°C with 5%
CO2 for 1 h. The membrane was then removed, washed
with PBS on the upper side, fixed, and stained with DiffQuik (Baxter
Dasle, München, Germany). Cells were counted
microscopically at x40 magnification in four fields per well. All
assays were performed in duplicate.
Statistical analysis
Data were analyzed with the assistance of the Statistical Analysis System. Data are expressed as mean ± SD. Mean values were compared using the ANOVA test. To investigate the correlation coefficients (r) between IP-10 levels and BAL cell findings, the nonparametric Spearman rank correlation test was used. A p value <0.05 was considered as significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Table I
summarizes the results of
the BAL findings. Due to the presence of CD4+ high
intensity T cell alveolitis, cell recovery was significantly higher in
patients with active sarcoidosis with respect to control subjects and
patients with inactive disease. As a consequence of the increase in the
absolute number of CD4+ T cells, the BAL CD4/CD8 ratio was
significantly increased in patients with active disease (6.9 ±
2.1) with respect to patients with inactive disease (1.71 ± 0.41;
p < 0.001) and healthy controls (1.65 ± 0.35;
p < 0.001). As previously reported (2), more than 98%
of T lymphocytes recovered from the BAL of sarcoid patients were
CD4+/CD45R0+/CD29+ memory T cells
(data not shown).
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). They were significantly higher than in the BAL of patients
with inactive disease (range, 0–128 pg/ml of BAL fluid). Only three of
the six control subjects showed detectable levels of IP-10 in the BAL
fluid (18, 14, and 16 pg/ml of BAL fluid, respectively). Spearman rank
correlation coefficients between BAL cell findings and levels of IP-10
were also calculated. A positive significant correlation was
demonstrated between the concentrations of IP-10 and the percentages
and absolute numbers of BAL lymphocytes (r, 0.845 and
p < 0.001; and r, 0.816 and
p < 0.001, respectively) and the percentages and
absolute numbers of CD4+ T cells (r, 0.815 and
p < 0.001; and r, 0.825 and
p < 0.001, respectively). As a consequence of the high
intensity alveolitis, AMs and CD8 T cells significantly increased in
their absolute number (Table I); however, correlation coefficients
between IP-10 levels and alveolar macrophages and CD8+ T
cell numbers were -0.802 and -0.693, respectively, indicating a
negative correlation. Most likely, other chemokines are involved in the
recruitment of these cell subsets as well as in the mechanisms leading
to the increase of neutrophils (Table I) that have been involved in the
mechanisms leading to fibrogenic events occurring in the lungs of
patients with sarcoidosis (1, 2). IP-10 expression by immunoinflammatory cells in sarcoid granuloma
Immunohistochemical analysis was used with an Ab recognizing IP-10
to investigate the membrane interactions occurring among macrophages
within granulomas (Fig. 1). The central
core of sarcoid granuloma was made up of a number of
monocytes/macrophages at various states of activation and
differentiation, as well as epithelioid cells and multinucleated giant
cells, while a number of CD4 lymphocytes and plasma cells surrounded
the central core of the granuloma. IP-10 was preferentially expressed
by macrophages and epithelioid cells located inside the granuloma,
whereas giant cells were mainly nonreactive.
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Immunochemical analysis of IP-10-producing cells in the BAL
demonstrated that the majority of AMs retrieved from the lungs of
patients with sarcoidosis showed a strong expression of this chemokine
(Fig. 2A).
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, B–E). In particular, as demonstrated by
the Kolmogorov-Smirnov analysis, lung macrophages expressed IP-10 since
the peak of positive cells was significantly shifted with respect to
the negative controls (percentage ranging from 51–78% of AMs bore
IP-10). Profiles shown in Fig. 2 are representative of membrane IP-10
staining in 17 patients with active sarcoidosis (nonpermeabilized AMs).
Histogram profiles of cytoplasmic IP-10 following cell permeabilization
were superimposable (data not shown). Pulmonary macrophages from two of
the seven patients with inactive sarcoidosis (41 and 49%,
respectively) and two of the six controls expressed low levels of
cytoplasmic IP-10 (35 and 27%, respectively), but not membrane IP-10.
Fig. 2 shows pulmonary macrophages from two representative control
subjects who express low levels of IP-10 (G) or do not
(F). BAL T cells from both sarcoid patients and controls did
not show IP-10 expression.
To determine whether increased IP-10 expression was associated with an
enhanced expression of cytokines that are actively released in sarcoid
lung (4, 5), BAL cells were analyzed for the expression of IL-2, IL-4,
IFN-, and IL-15. Fig. 3 shows
cytoplasmic expression of these cytokines by sarcoid AMs and T cells. A
percentage ranging from 47–58% and from 55–84% of AMs from five
patients with active sarcoidosis expressed cytoplasmic IFN- and
IL-15, respectively (mean 45.1% ± 6.3 and 66.5% ± 8.1,
respectively). By contrast, less than 5% of AMs isolated from patients
with inactive sarcoidosis and normal AMs showed cellular IFN- or
IL-15. Concerning pulmonary T cells, BAL T cells from patients with
active sarcoidosis bore IL-2 (percentage ranging from 17–35%; mean
29% ± 13.8) and IFN- (percentage ranging from 21–39%; mean
32.3% ± 11.7), but did not express IL-4 (data not shown). Less than
1% of normal BAL T cells express this cytokine. Thus, sarcoid T cells
show a striking polarization of Th1-type immune response that parallels
IP-10 expression.
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Alveolar macrophages have never been reported to release IP-10 in vitro. To confirm our immunochemical and flow cytometry data, we evaluated whether sarcoid AMs can produce IP-10 both at resting conditions and following stimulation.
Fig. 4 shows the in vitro production of
IP-10 by AMs. Cell-free supernatants were obtained from AMs cultured in
different experimental conditions and tested for the presence of IP-10.
After 24 h of culture, unstimulated AMs from patients with active
sarcoidosis were able to produce higher amounts of IP-10 (mean
354.7 ± 146.9 pg/ml) than AMs isolated from patients with
inactive disease (108.3 ± 75.6 pg/ml; p < 0.05)
and healthy subjects (27.6 ± 12.1 pg/ml; p <
0.001). Both AMs from patients with active and inactive disease
increased IP-10 release following LPS stimulation, and there were no
significant differences in the production of IP-10 between patients
with active and inactive disease (1714 ± 1330 pg/ml vs 710.5
± 624.6 pg/ml, respectively; p = NS). In contrast,
normal LPS-stimulated AMs showed a slight increase in the release of
IP-10 (54.4 ± 9). When AMs were cultured in the presence of
IFN-, the amount of IP-10 in cell-free supernatants from
patients with active disease was 10-fold higher (69,571 ± 25,255
pg/ml) than the levels obtained from IFN--stimulated AMs from
controls (6,722 ± 2,323 pg/ml, p < 0.001).
Patients with inactive sarcoidosis released intermediate levels of
IP-10 (27,671 ± 5,459 pg/ml).
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Data reported above clearly demonstrated that sarcoid AMs release
IP-10 that interacts with a G protein-coupled cell surface receptor
(CXCR3) expressed on activated T cells and may induce T cell
inflammation in peripheral tissues (13, 18, 25). To evaluate whether T
cells accumulating in the lungs of patients with sarcoidosis express
CXCR3, flow cytometry analysis experiments were performed using a mAb
against CXCR3. Fig. 5 shows that alveolar
CD4+ T cells of patients with active sarcoidosis are CXCR3
positive (Fig. 5, A and B). Interestingly, BAL T
cells of patients with active sarcoidosis (A and
B) showed enhanced levels of CXCR3 expression with respect
to BAL T cells of patients with inactive sarcoidosis (C) and
normal BAL T cells (C and D)
(p < 0.001, as determined by the
Kolmogorov-Smirnon analysis). However, the small number of
CD8+ T cells found in the BAL of patients with active
sarcoidosis also expressed CXCR3, albeit at lower density than
CD4+BAL T cells (data not shown).
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The possibility that the increased levels of IP-10 in AM
supernatants might account for the recruitment of CXCR3+
sarcoid T cells was also investigated by an in vitro chemotaxis assay
that uses a T cell clone (2F12) expressing high levels of CXCR3 (Fig. 6A). As shown in Fig. 6B, supernatants obtained by IFN--stimulated AMs
isolated from six sarcoid patients with T cell alveolitis exerted
significant chemotactic activity on the CXCR3+2F12 clone.
As determined by the RIA test, IP-10 levels ranged from 11,073 to
40,125 pg/ml in these supernatants. However, on the basis of number of
migrating CXCR3+ T cells, the levels of the in vitro
chemotaxis assay seen with purified protein (D) correlated
with IP-10 levels in the supernatants.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Most of the CXC and CC chemokines that have been involved in lymphocyte chemotaxis are not selective for lymphocytes since they are active also on monocytes and granulocytes. The scenario is quite different for IP-10. As recently demonstrated by Loetscher et al. (17), this cytokine favors the selective recruitment of IL-2-activated T cells, which exclusively express the IP-10 receptor. The observations that sarcoid AMs abundantly express IP-10 and the significant correlation demonstrated between the concentration of IP-10 and the number of BAL CD4+ T lymphocytes indicate the role of macrophage-derived IP-10 in regulating the migration of IL-2-activated T cells toward sites of the sarcoid inflammatory process. Most likely, the fact that sarcoid BAL cells express a number of Th1-type cytokines, including IL-2 and IL-15, suggests that IP-10 cooperates with Th1-type cytokines that act as local factors for T cell activation and proliferation (5).
Although our study did not clarify factors regulating the in situ
IP-10 production by sarcoid macrophages, we postulate that the
chemokine release is induced by the chronic IFN--dependent
activation of sarcoid AMs and T cells. AMs from sarcoid patients differ
from normal AMs in a series of parameters related to their functional
properties, such as their phenotypic characteristics, accessory cell
function, and secretory capabilities (26 and our results). The
hypothesis being proposed is that, as a result of the chronic
IFN--dependent stimulation, AMs release IP-10. In this scenario, it
is possible that Th1-type sarcoid T cells that, as herein demonstrated,
express IFN-, IL-2, and CXCR3, could be considered inducers of
production as well as targets of IP-10. IFN-/IP-10 axis might
contribute to the migration of Th1-type sarcoid T cells to the site of
sarcoid reaction, inducing self-perpetuating immune mechanisms that
drive the development of sarcoid granuloma.
Another interesting question is whether IP-10 acts in concert with other chemokines in regulating the inflammatory infiltrate of sarcoid lesions. It has been demonstrated previously that RANTES, a CC chemokine, is highly expressed in sarcoid tissues (27). Like IP-10, RANTES is known to recruit and activate specific T cell subsets (28). Furthermore, immunocytochemical staining of sarcoid granuloma in lymph node and lung revealed that a chemoattractant factor for CD4+ T cells, i.e., IL-16, is expressed in areas in which there is perivascular accumulation of lymphocytes (29). Finally, our preliminary data have shown the presence of Mig in sarcoid granulomas, a related member of the CXC chemokine family that targets lymphocytes via CXCR3 (30). We have also seen that that small number of CD8+ T cells found in the BAL of sarcoid patients with high intensity alveolitis also express CXCR3, albeit at lower density than CD4+ T cells. This suggests that different T cell-attracting cytokines might play a role in the regulation of the sarcoid granuloma development, perhaps by controlling the trafficking of discrete CD4 and CD8 T cell subsets at sites of sarcoid inflammatory response.
In this regard, data in animal models have shown that Th cells
facilitate, while suppressor T cells down-modulate the growth of the
granulomatous process (2). Furthermore, while CD4+ T cells
predominate in the inner area of sarcoid granulomas, CD8+ T
cells predominate in the outer margin of the lymphocyte rim (1, 2).
From our observations, it may be inferred that IP-10 release is higher
in sarcoid patients characterized by a massive CD4+ T cell
infiltrate than in patients with inactive disease. Sarcoid
CD4+ T cells produce Th1-type cytokines, including IL-2 and
IFN- (our data and 4, 5, 11, 31); the net effect of the Th1-type
response is the development of the hypersensitivity granuloma (32).
However, depending on the host susceptibility, a switch to Th2-type T
cells may occur in sarcoid patients evolving toward lung fibrosis with
concomitant release of cytokines, including IL-4, which stimulates the
production of extracellular matrix proteins and/or are chemoattractants
for fibroblasts (32). Since IL-4 has also been shown to down-regulate
chemokine production (33), studies on the Th1/Th2 pattern during the
different phases of the sarcoid inflammatory process could clarify
whether the Th2 shift has a suppressive effect on the in situ
production of IP-10 and, thus, on the formation of granuloma and the
evolution toward fibrosis.
The present study has shown that sarcoid T cells express CXCR3 at high levels. Five human CXC chemokine receptors (CXCR1 through CXCR5), nine human CC chemokine receptors (CCR1 through CCR9), and one CXXXC chemokine receptor (CX3R1) have been identified to date (13). Since the increase in the secretion of different chemokines and the overexpression and/or down-modulation of some chemokine receptors might have dramatic effects in regulating Th1/Th2 pattern during inflammation (34), further studies are in progress in our labs to verify the importance of other chemokines and chemokine receptors in the pathogenesis of sarcoid inflammatory process.
In conclusion, this study supports the hypothesis that IP-10 participates in the complex network between pulmonary immunocompetent cells and cytokines, which sets the stage for the development of the sarcoid granuloma. A greater understanding of local mechanisms controlling IP-10 release will clarify the sequel of events that lead to sarcoid-associated pulmonary fibrosis. Furthermore, our findings suggest that the in situ release of IP-10 mirrors the development of the Th1 sarcoid inflammatory process. In this context, the use of IP-10 levels in BAL fluid as biologic markers for disease activity in sarcoidosis needs to be validated by a wide and long-term follow-up study.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gianpietro Semenzato, Università di Padova, Dipartimento di Medicina Clinica e Sperimentale, Via Giustiniani 2, 35128 Padova, Italy.
3 Abbreviations used in this paper: AM, alveolar macrophage; BAL, bronchoalveolar lavage; IP-10, interferon-inducible protein 10.
Received for publication October 6, 1997. Accepted for publication July 27, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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interferon-induced protein (IP-10) in delayed immune responses in human skin. J. Exp. Med. 166:1098. chemoattractant (I-TAC): a novel non-ELR chemokine with potent activity on activated T cells through selective high affinity binding to CXCR3. J. Exp. Med. 187:2009. inducible protein-10 (IP-10) chemokine by human neutrophils. Eur. J. Immunol. 27:111.[Medline]
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