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Regular Articles |
From the Departments of Clinical and Experimental
Medicine,*
Clinical Immunology Branch,
Pathology,
and Thoracic
Surgery,
Padua University School of Medicine,
Padua, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and
stimulates the directional migration of activated T cells,
plays a role in regulating the trafficking of effector T cells during
lung allograft rejection episodes. Immunohistochemical examination
showed that areas characterized by acute cellular rejection (grades 1
to 4) and active obliterative bronchiolitis (chronic rejection,
Ca) were infiltrated by T cells expressing CXCR3, ie,
the specific receptor for CXCL10. In parallel, T cells
accumulating in the bronchoalveolar lavage of lung transplant
recipients with rejection episodes were CXCR3+ and exhibited a strong
in vitro migratory capability in response to CXCL10. In
lung biopsies, CXCL10 was abundantly expressed by
graft-infiltrating macrophages and occasionally by epithelial cells.
Alveolar macrophages expressed and secreted definite levels of CXCL10
capable of inducing chemotaxis of the CXCR3+ T-cell line 300-19; the
secretory capability of alveolar macrophages was up-regulated by
preincubation with interferon-. Interestingly, striking
levels of CXCR3 ligands could be demonstrated in the fluid
component of the bronchoalveolar lavage in individuals with rejection
episodes. These data indicate the role of the CXCR3/CXCL10
interactions in the recruitment of lymphocytes at sites of lung
rejection and provide a rationale for the use of agents that block the
CXCR3/CXCL10 axis in the treatment of lung allograft
rejection.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The central event in the pathogenesis of pulmonary T cell responses is
the organ-specific traffic of T lymphocytes.6
Recently, a
number of chemoattractants for leukocytes have been described. These
molecules belong to the superfamily of chemokines that can be divided
into four groups (C, CC, CXC, and CX3C), according to the positioning
of the cysteine residues in the mature protein.7-10
Expression of chemokine receptors on the cell surface of pulmonary
immunocompetent cells and interactions with their specific ligands are
involved in the mechanisms accounting for the accumulation of
inflammatory cells within the lung microenvironment and the
establishment of local hypersensitivity reactions.6
Although the interactions of chemokine receptors are often
characterized by considerable promiscuity, a human chemokine receptor
that is selectively involved in the recruitment of T cells is
CXCR3.11,12
CXCR3 is selectively expressed by activated T
lymphocytes, and, in turn, its ligands interferon (IFN)--inducible
protein-10 (IP-10, CXCL10 according to the new nomenclature of
chemokine and chemokine receptors,13
), monokine induced by
IFN- (Mig/CXCL9), interferon-inducible T-cell 
-chemoattractant
(I-TAC/CXCL11) are specifically chemotactic for activated T cells.
In this study, using cells recovered from the bronchoalveolar lavage (BAL) and related biopsy specimens, we have evaluated the role of local CXCR3/CXCL10 receptor interactions in the immunological events ultimately leading to lung allograft rejection. We have shown that T cells accumulating in the lower respiratory tract of patients with acute lung allograft rejection, as well as in individuals suffering from BOS, express a functional CXCR3 because they are able to migrate in response to CXCL10. Furthermore, the specific CXCR ligand CXCL10 was abundantly expressed by graft-infiltrating macrophages of patients showing T cell infiltrate.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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According to our protocol, 56 consecutive transbronchial biopsies and BALs were performed during the follow-up of 24 lung allograft recipients (14 men and 10 women) who underwent surveillance bronchoscopy for clinical suspicion of infection or rejection. All patients underwent immunosuppression with rabbit anti-thymocyte globulin postoperatively, followed by a triple immunosuppression regimen of cyclosporin A or tacrolimus, azathioprine or RAD, and prednisone.
Transbronchial biopsies (average, 6.1; range, 5.0 to 11.0) were taken from each lobe and serial sections were used for histological diagnosis and immunohistochemical analysis. Biopsy samples were evaluated according to the revised International Society for Heart and Lung Transplantation working formulation.14 Overall, 47 of 58 biopsy samples showed no histological signs of rejection and eight showed acute rejection ISHLT grade A1 (n = 3), A2 (n = 3), and A3 (n = 2). Three patients were classified as suffering from BOS.
BAL was performed according to the technical recommendations and guidelines for the standardization of BAL procedures.15 Briefly, a total of 200 ml of saline solution was injected in 25-ml aliquots via fiberoptic bronchoscopy, with immediate vacuum aspiration after each aliquot. Immediately after the BAL, the fluid was filtered through gauze and the volume measured. A volume of 100 to 200 ml of BAL recovery and a sample of 50% of the instilled volume with a minimum of 50 ml was considered acceptable. The percentage of BAL recovery was 53.7 ± 4.2% and 55.1 ± 3.7% of the injected fluid in allograft recipients and control patients, respectively. Cells recovered from the BAL were washed three times with phosphate-buffered saline (PBS), resuspended in endotoxin tested RPMI 1640 (Sigma Chemical Co., St. Louis, MO) supplemented with 20 mmol/L HEPES and L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum (ICN Flow, Costa Mesa, CA), and then counted.
A standard morphological and immunological analysis of BAL cellular components was performed and included cell recovery; differential count of macrophages, lymphocytes, neutrophils, and eosinophils; and flow cytometry analysis of CD4/CD8 BAL T cell ratio.
Purification of Alveolar Macrophages (AMs) and T Cells
AMs and T cells were enriched from the BAL cell suspensions by rosetting with neuraminidase-treated sheep red blood cells followed by F/H gradient separations and removing residual CD3+ lymphocytes using high-gradient magnetic separation columns (Mini MACS; Miltenyi Biotec, Germany).16 After this multistep selection procedure >95% of the above cells were viable, as judged by the trypan blue exclusion test. Staining with mAb showed that >99% of purified lymphocytes were CD3+ T cells.
Monoclonal Antibodies and Cytokines
The commercially available conjugated or unconjugated mAbs used
belonged to the Becton Dickinson and PharMingen series and included:
CD3, CD4, CD8, CD45R0, CD45RA, and isotype-matched controls.
Anti-IL-4 and anti-IFN- mAbs were purchased from PharMingen
(San Diego, CA). Purified rabbit anti-human CXCL10 polyclonal antibody
(R&D Systems Inc., Minneapolis, MN) and anti-hCXCR3 mAb (R&D Systems
Inc.) were also used.
Immunohistochemical Analysis of CXCR3+ Cells and CXCL10-Producing Cells
Expression of CXCR3 and CXCL10 was measured by permanent section immunohistochemistry with anti-CXCR3 and anti-IP10 antibodies. Four-µm thick paraffin-embedded sections were used for immunostaining using the standard avidin-biotin complex method (Vectastain ABC Kit; Vector Laboratories, Burlingame, CA). The reliability of both antibodies in paraffin sections was compared with cryostatic (frozen) lung sections of two patients who died from persistent acute airway rejection (A3) and active obliterative bronchiolitis (Ca), respectively. Sections were deparaffinized in xylene (5 minutes, three times) and rehydrated through graded ethanol (twice for 5 minutes in 100% ethanol, 3 minutes in 95% ethanol, 3 minutes in 70% ethanol, and 5 minutes in distilled H20). For the microwave antigen retrieval procedure, slides were placed in a 2-L glass beaker containing 0.01 mol/L citrate buffer, pH 5.9, and microwaved at full power (800 W for 5 minutes, three times) before cooling and equilibration in PBS.
To neutralize endogenous peroxidase activity, slides were pretreated with 3% hydrogen peroxide for 5 minutes. Primary antibodies were applied at the following concentrations: anti-hCXCR3 mAb 1:100 and anti-hIP-10/CXCL10 polyclonal 1:100 for 1 hour in a humidified chamber at 37°C. Immunoreactivity was detected using biotinylated secondary antibodies (1:50 rabbit anti-goat and 1:1,000 dilution goat anti-rabbit in PBS-bovine serum albumin buffer) incubated for 45 minutes followed by a 30-min incubation with avidin-peroxidase (1:200) and visualized by a 7-minute incubation with the use of 0.1% 3,3'-diaminobenzidene tetrahydrochloride as the chromogen. The intensity of antibody staining was classified in three groups: strong, weak, and negative. Parallel control slides were prepared either lacking primary antibody or lacking primary and secondary antibodies, or stained with normal sera to control for background reactivity.
Phenotypic Evaluation of BAL Cells
The frequency of BAL cells positive for the above reagents was
determined by overlaying the flow cytometry histograms of the samples
stained with the different reagents as previously
reported.16
Cells were scored using a FACScan analyzer
(Becton Dickinson, Mountain View, CA), and data were processed using
the Macintosh CELLQuest software program (Becton Dickinson). The
expression of cytoplasmic cytokine was evaluated after permeabilization
of cell membranes using 1:2 diluted PermeaFix (Ortho, Raritan, NJ) for
40 minutes. After permeabilization procedures, anti-IL-4, anti-IFN-,
and anti-CXCL10 antibodies were added. Because pulmonary cells bore
cytoplasmic cytokines in a unimodal expression pattern, indicating that
the entire cell population exhibits relatively homogeneous
fluorescence, the percentage of positive cells does not represent the
most accurate way of enumerating positive cells. The mean fluorescence
intensity was used to compare the positivity of these specific antigens
on different cell populations. To evaluate whether the shift of the
positive cell peak was statistically significant, the
Kolmogorov-Smirnov test for analysis of histograms was used according
to the Macintosh CELLQuest software user’s guide (Becton Dickinson).
For immunofluorescence analysis, control IgG1 and IgG2a and IgG2b were obtained from Becton-Dickinson; control rat antiserum consisted of ascites containing an irrelevant rat IgG2b; control rabbit antiserum consisted of rabbit IgG (purified protein) purchased from Serotec (Serotec, UK); goat-anti-rabbit IgG and goat F(ab')2 anti-rat IgG were obtained from Immunotech (Marseille, France).
Generation of Macrophage Supernatants
To verify the ability of AMs to release CXCL10, AMs (1 x
106/ml) were isolated from the BALs of allograft
recipients, resuspended in RPMI medium, and cultured for 24 hours in
24-well plates at 37° in 5% CO2. In separate
experiments AMs were stimulated with IFN- (100 U/ml), phorbol
myrisate acetate (10 ng/ml), and lipopolysaccharide (10 µg/ml; Difco
Labs., Detroit, MI). After the incubation period, supernatants were
harvested, filtered through a 0.45-µm Millipore filter, and
immediately stored at -80°C. At the end of the culture time AM
viability was always >95%. Chemotactic activity of supernatants was
determined as reported below.
Migration Activity of Pulmonary T Cells in Response to CXCR Chemokines
T-cell migration was measured in a 48-well modified Boyden chamber (AC48 Neuro Probe Inc.). The chamber is made of two sections: different chemotactic stimuli were loaded in the bottom section while cells were added in the top compartment. Polyvinylpyrrolidone-free polycarbonate membranes with 3- to 5-µm pores (for lung T cells obtained from allograft patients and the CXCR3+ and CXCR- T cell lines, respectively; Osmonics, Livermore, CA) and coated with fibronectin were placed between the two chamber parts. Only the bottom face of filters was pretreated with fibronectin; the fibronectin pretreatment maximizes attachment of migrating cells to filters, avoiding the possibility that they may not adhere. Using this procedure in preliminary experiments we demonstrated that only a trivial number of cells may be recovered in the bottoms of the wells. To avoid the shedding of fibronectin, fibronectin-treated filters were extensively washed. In preliminary experiments, fibronectin-treated filters did not induce spontaneous chemotaxis in absence of chemokines.
To evaluate the migratory properties of pulmonary T lymphocytes rhIP-10/CXCL10 (200 ng/ml) were used. The CXCR- and CXCR3+ cell lines (300-19, kindly provided by Dr. B. Moser, Theodor-Kocher Institute, University of Bern, Switzerland) were used as negative and positive controls. Thirty µl of chemokines or control medium were added to the bottom wells, and 50 µl of 5.0 x 106 cells/ml T cells or CXCR-/+ cells resuspended in RPMI 1640 were added to the top wells. The chamber was incubated at 37°C with 5% CO2 for 2 hours. The membranes were then removed, washed with PBS on the upper side, fixed, and stained with DiffQuik (Dade AG, Düdingen, Switzerland). Cells were counted in three fields per well at x800 magnification. All assays were performed in triplicate. In blocking experiments, cell suspensions were preincubated before chemotaxis assay for 30 minutes at 4°C with anti-human CXCR3 mAb at the concentration of 20 µg/ml.
Chemotactic Activity of the Fluid Component of BAL and Macrophage Supernatants
The CXCR- and CXCR3+ cell lines were also used to evaluate both the chemotactic activities of macrophage supernatants and the fluid component of BAL samples. Supernatants from cell cultures and the fluid components of BALs were obtained as reported above and used undiluted; different concentrations of CXCL10 were used as a positive control. Chemotactic assays were performed as reported above. In blocking experiments, anti-CXCL10 was added to the cell supernatants before chemotaxis assay at the concentration of 20 µg/ml.
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 analysis of variance test. A P value <0.05 was considered as significant.
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Immunohistochemical and Flow Cytometry Analysis of the Expression of CXCR3 by Pulmonary Immunoinflammatory Cells during Lung Allograft Rejection Episodes
Immunohistochemical analysis was used to investigate the pattern
of expression of this chemokine receptor. Grafts with histological
evidence of rejection (presence of perivascular lymphocytic
infiltrates, which in more severe cases, spilled over into the
interstitium and alveolar air spaces) showed an infiltrate
characterized by T cells expressing CXCR3 (Figure 1, a and b)
. Positively marked
lymphocytes showed more intense staining in patients with a higher
grade of acute rejection than in patients with lower grade rejection
(A3>A2>A1). Strongly stained infiltrates of CXCR3+ lymphocytes were
also seen in active obliterative bronchiolitis (Figure 1, c and d)
.
Frozen lung samples showed staining intensity and distribution similar
to the formalin-fixed lung biopsies used in the study.
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and bore IFN- but not interleukin
(IL)-4, a pattern that is characteristic of Tc1/Th1 cells. BAL T cells
of patients with BOS expressed CXCR3 at high density as well as
cytoplasmic IFN- (Figure 2)
. CXCR3 was found to be expressed at
higher intensity in BAL T cells of patients with higher grade acute
rejection than in patients with lower grade rejection, the differences
in the mean fluorescence intensity of CXCR3+ peaks being significant in
the Kolmogorov-Smirnov analysis (P < 0.01).
Although CXCR3 was highly expressed by patients with T cell alveolitis,
normal BAL T cells and T cells from patients with normal cellular
recovery expressed low or insignificant levels of CXCR3 at the
Kolmogorov-Smirnov analysis.
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To characterize the biological properties of CXCR3, highly purified T cells isolated from the BALs of patients with allograft rejection and T cell alveolitis were assessed for their migratory capabilities in response to different concentrations of CXCL10. The evaluation of the migratory capabilities of pulmonary T cells in normal patients or patients with no signs of rejection was prevented by the low recovery of pulmonary T cells from the BAL. For this reason, the 300-19 T cell lines expressing high levels of CXCR3 or not expressing CXCR3 were used as positive and negative controls for the in vitro chemotaxis assay.
As shown in Figure 3
, pulmonary T cells
purified from the BAL of patients with grade A1 to A3 and BOS exhibited
a definite migratory capability in response to CXCL10. The migratory
capability was influenced by CXCR expression. In fact, the blocking of
the CXCR3 receptor with specific antibodies determined a marked
inhibition of CXCL10-induced chemotaxis. These data suggest that
pulmonary T cells infiltrating lung allografts express a functional
CXCR3 receptor that induces migration in response to CXCL10.
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In a second set of experiments we evaluated which inflammatory
cells infiltrating lung allograft express CXCL10 during rejection
episodes. The expression of CXCL10 was observed in
macrophage-infiltrating lung biopsies of patients with histological
evidence of A1-A3 rejection (Figure 4)
or
with BOS; interestingly, also a few epithelial cells expressed CXCL10.
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, macrophages
isolated from the BAL of recipients with rejection episodes or BOS
expressed CXCL10.
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Cell-free supernatants were obtained from 24-hour cultured AMs and
tested for their capabilities of inducing T cell migration.
Supernatants obtained from AMs of patients with acute rejection
episodes or BOS exerted significant chemotactic activity on the CXCR3+
cell line (Figure 6)
; the CXCR3-negative
cell line did not migrate in the presence of supernatants (data not
shown). The addition of an anti-CXCL10 neutralizing antibody (Figure 6)
, but not of a control antibody, inhibited chemotactic activities of
supernatants (data not shown). The inhibitory activity shown by the
neutralizing antibody was not complete (range, 46 to 79%). This
suggests that other CXCR3 ligands, including CXCL9 and CXL11, are
likely to be present in conditioned media and interact with the CXCR3+
T cell clone, mediating its migration. When macrophages were cultured
in the presence of IFN- the chemotactic activity of cell-free
supernatants was higher than the levels obtained from unstimulated AMs
(46.7 ± 12.3 versus 58.0 ± 9.1 number of
migrating CXCR3+ cells/high-powered field; mean ± SD of pooled
results obtained in different lung allograft recipients;
P < 0.01). Again, the chemotactic activity of
conditioned medium obtained by IFN- stimulated AMs was only
partially inhibited by the pretreatment with the anti-CXCL10 antibody,
suggesting the presence of other CXCR3 ligands in the supernatants. As
a confirmation, the addition of an anti-CXCL9 antibody also
inhibited chemotactic activities of supernatants (data not shown).
Taken together these data suggest the need to evaluate the role of
other non-ERL chemokines in the pathogenesis of allograft rejection.
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. A definite biological
activity was demonstrated in the samples of patients with acute
rejection or BOS; this activity was partially inhibited by an
anti-CXCL10 neutralizing antibody indicating the presence of other
CXCR3 ligands in the fluid component of BAL.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As recently reviewed,17 BAL associated with transbronchial biopsies may be useful for understanding the natural course of lung transplantation. During the first 3 months after pulmonary transplantation, elevated total cell counts in BAL and neutrophilic alveolitis are common features, representing the cellular response to the graft. Lymphocytic alveolitis with a decreased CD4/CD8 ratio suggests acute rejection, but is also detectable in viral pneumonia and obliterative bronchiolitis. In the case of a combined lymphocytosis and neutrophilia without any evidence of infection, BOS should be taken into account. It is herein shown that graft-infiltrating T cells express CXCR3 whenever a T cell alveolitis occurs. Episodes of grade A2 or greater rejection were associated with a striking expression of this chemokine receptor by pulmonary lymphocytes as well as with cases of patients suffering from BOS and showing BAL lymphocytosis and neutrophilia. We have also shown CXCR3 expression in allograft recipients with clinical signs of viral pneumonia associated with a lymphocytic alveolitis (data not shown).
CXCR3 expression is likely to represent a common mechanism that is involved in the recruitment of activated T cells in the pulmonary microenvironment of lung allograft recipients. The continuous recruitment of Th1-type CXCR3+ T cells might play a role not only in the pathogenesis of acute rejection but also in favoring the persistence of local hypersensitivity reactions that in turn set the stage for the development of the fibroproliferative process. In this context, because acute rejection and viral infection represent major risk factors for BOS,3,18 it may be speculated that the fibroproliferative response represents a relative resolution process of the persistent CXCR3+ T-cell chronic inflammation, which may partially recover obviously at the expense of airway fibrosis.19 This hypothesis may have important therapeutic implications. Because of the role of CXCR3 expression in the migration of T cells into lung allografts, strategies to block CXCR3/CXCL10 interactions are likely to be successful in preventing the development of BOS. It is also important to note that such pathogenetic hypothesis we suggest is not specific for allograft reaction, because CXCR3/CXCL10 interactions have been found to be important in mediating CTL response20 and T cell reactions taking place in the lung of patients with another Th1 disorder that is followed by severe tissue injury and remodeling, ie, sarcoidosis16
A second issue raised by our data deals with mechanisms that regulate
CXCR3/CXCL10 interactions in lung allografts. Recent data, obtained in
the animal model, have shown a distinct pattern of cytokines and
chemokine gene expression in the development of posttransplant airway
obliteration.21
Allografts show a strong and persistent
Th1-type response (expression of IL-2 and IFN- genes), even after
fibrous airway obliteration is complete, suggesting that an ongoing
Th1-mediated alloimmune process persists until late in the
fibroproliferative stage. Other investigators have shown that Th1, Th2,
and cytotoxic lymphocyte subtypes contribute to the development of
obliterative bronchiolitis in the heterotopic mouse trachea
model.22
Our data indicating that graft-infiltrating T
cells show a dominant expression of IFN- suggest a putative role for
IFN- in regulating CXCL10/CXCR3 interactions in the pulmonary
milieu. The recent evidence that detection of IFN- in BAL cells
significantly correlates with early acute rejection,23
coupled to the present finding that in the presence of IFN-
graft-infiltrating macrophages increased their release of chemotactic
factor, further supports the concept that IFN- plays a regulatory
role in the recruitment of CXCR3+ pulmonary T cells. IL-15 is another
ideal candidate that may favor T cell accumulation in lung allografts.
In fact, IL-15, a potent inducer of CXC-chemokines on T
cells,24
may favor the expression of CXCR3 in the lungs of
patients with other Th1-mediated hypersensitivity
reactions.25
Because our preliminary data indicate that
this molecule in actively released during rejection episodes (data not
shown), it is conceivable that IFN-, IL-15, and IFN--inducible
CXC chemokines act in concert to sustain Th1 inflammatory responses in
lung allografts. Accordingly, it has been recently reported that CXCL10
selectively up-regulates human T-cell cytokine synthesis, with
enhancement selectively targeted toward the promotion of IFN-
expression (Th1-like).26
The relationship between the fibroangiogenetic response associated with
the bronchiolitis obliterans and the local release of chemokines is
another important aspect that deserves further investigation. The CXC
chemokine family contains members that on a structural/functional basis
exhibit either angiogenic or angiostatic biological activity. In
particular, it is known that a variety of ELR-containing chemokines are
chemotactic for endothelial cells whereas non-ELR CXC chemokines,
including CXCL9 and CXCL10 are not only nonangiogenic themselves, but
they inhibit the angiogenic effects of other ELR
chemokines.27
As an example, IL-8/CXCL8, which is
expressed in the lung of patients evolving toward BOS,28
has markedly increased angiogenetic activity compared to the
IFN--induced CXCL10 in idiopathic pulmonary fibrosis.29
Furthermore, Th2 cytokines (IL-4 and IL-13) have been involved in the
pathogenesis of idiopathic pulmonary fibrosis whereas Th1 cytokines
(IFN-) seem to have a protective role.30
Our findings
and the data of DiGiovine and colleagues28
suggest that
the production of ordinarily antifibrotic agents (IFN- and CXCL10)
and profibrotic molecules, as CXCL8/IL-8 may be paradoxically
associated during local fibroproliferative repair processes taking
place in chronic lung rejection. These data are consistent with
findings obtained in animal models: although IFN- has antifibrotic
activity in experimental granuloma models,31
a persistent
Th1 immune response (IL-2 and IFN-) characterizes the development of
posttransplant airway obliteration observed in the rat tracheal
transplant model.21
Thus, events mediating fibroangiogenesis are likely to be different in
the posttransplant airway obliteration and in diffuse lung diseases
characterized by an evolution toward fibrosis. Further studies aimed at
evaluating the complex interactions between Th1/Th2 cytokines, CXC
chemokines, and CC chemokines may help to clarify this issue. In
particular, given the ability of other ligands of CXCR3 (CXCL9 and
CXCL11) to favor T cell recruitment, studies are in progress in our
laboratories to evaluate the functional importance of these IFN-
inducible CXC chemokines during the different phases of the rejection
process. The production of CC chemokines should be also investigated.
In a murine model it has been shown that CCL5 and monocyte chemotactic
protein-1 (MCP-1/CCL2) are induced late after transplantation, whereas
up-regulation of macrophage inflammatory protein-2 (MIP-2/CXCL2)
occurs early after transplant.21
Studies in lung allograft
recipients have also demonstrated that RANTES/CCL5 contributes to the
intrapulmonary accumulation of immune cells during acute
rejection32
and CMV pneumonia after lung
transplantation.33
In this context, it is likely that chemokines may be redundant in their action on different lung cells. For instance, CCL2 and CCL5 interacting with CCR1/CCR2 or CCR1/CCR3/CCR5, respectively, may be chemoattractant for different cell targets that have been involved in the pathogenesis of both acute and chronic complications of lung transplantation, including macrophages, T lymphocytes, neutrophils, mast cells, and eosinophils.17,34-37 The issue is complicated by the fact that lung macrophages do not represent the only chemokine source in the lung. As an example, Th1 or Th2 cytokines in the lung tissue may polarize lung fibroblasts to produce either CCL5 or eotaxin (CCL11)38 and the T-cell-specific chemokines CXCL9, CXCL10, and CXCL11 may be produced by pulmonary epithelial cells.39 Given the heterogeneous pattern of pulmonary infiltrate during rejection episodes, a full understanding of the interdependence of the local hyperproduction of chemoattractant molecules may help to clarify not only events leading to acute rejection but also why chronic rejection is associated with a Th1 immune response and evolves toward bronchiolitis obliterans.
Despite the complexity and redundancy of the chemokine system, it is easy to anticipate that further information on chemokine biology will provide pharmacological opportunities to improve long-term survival in transplant recipients. Our findings clearly indicate the effects of CXCR3/CXCL10 interactions on allograft rejection, suggesting that targeting CXCR3 and its ligand with engineered molecules might have therapeutic utility in down-modulating T cell recruitment and thus T-cell-specific inflammatory responses taking place in the pulmonary parenchyma. Considering the importance of T cells in the alloimmune responses, further studies are needed in animal models to explore the therapeutic potential of CXCR3 or CXCL10 antagonists with the ultimate goal of offering new clues for immune intervention in human diseases.
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Acknowledgements |
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Footnotes |
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Supported by grants from the M.U.R.S.T ex40%.
Accepted for publication January 23, 2001.
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