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* Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095; and Departments of
Pathology and Laboratory Medicine and
Internal Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Medical School, Ann Arbor, MI 48109
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(MIG)/CXC chemokine ligand (CXCL)9,
IP-10/CXCL10, and IFN-inducible T cell 
chemoattractant
(ITAC)/CXCL11) act through their shared receptor, CXCR3. Because
they are potent leukocyte chemoattractants and are involved in other
inflammation/fibroproliferative diseases, we hypothesized that the
expression of these chemokines during an allogeneic response promotes
the persistent recruitment of mononuclear cells, leading to chronic
lung rejection. We found that elevated levels of MIG/CXCL9,
IFN-inducible protein 10 (IP-10)/CXCL10, and ITAC/CXCL11 in human
bronchoalveolar lavage fluid were associated with the continuum from
acute to chronic rejection. Translational studies in a murine model
demonstrated increased expression of MIG/CXCL9, IP-10/CXCL10, and
ITAC/CXCL11 paralleling the recruitment of CXCR3-expressing mononuclear
cells. In vivo neutralization of CXCR3 or its ligands MIG/CXCL9 and
IP-10/CXCL10 decreased intragraft recruitment of CXCR3-expressing
mononuclear cells and attenuated BOS. This supports the notion that
ligand/CXCR3 biology plays an important role in the recruitment of
mononuclear cells, a pivotal event in the pathogenesis of
BOS. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BOS is a chronic inflammatory process characterized by persistent peribronchiolar leukocyte infiltration that eventually invades and disrupts the basement membrane, submucosa, and airway epithelium (1, 2). Fibroproliferation follows, with increased numbers of mesenchymal cells, matrix deposition, and granulation tissue formation, ultimately leading to fibro-obliteration of airways (1, 2). Acute rejection is characterized by an acute perivascular/bronchiolar leukocyte infiltration and is the main risk factor for the development of BOS (1, 2). Classically, BOS is described as an ongoing immunological event; however, potent immunosuppressive therapy has not changed the natural history of this disorder (1, 2). Moreover, the specific mediators that orchestrate the persistent recruitment of leukocytes leading to fibro-obliteration have not been fully elucidated. MIG/CXC chemokine ligand (CXCL)9, IP-10/CXCL10, and ITAC/CXCL11 are glutamine-leucine-arginine (ELR)-negative CXC chemokines and are potent chemoattractants for mononuclear cells, specifically activated T cells and NK cells (3, 4, 5, 6, 7). All three chemokines share ability to signal through a G protein-coupled receptor, CXCR3 (7, 8). CXCR3 biology has been shown to be important during acute rejection in a murine model of acute cardiac allograft rejection (9, 10). Because of this finding, we hypothesized that the continuum of lung allograft rejection from acute to chronic (BOS) resulting from a persistent immunologic/inflammatory insult to the allograft airways with subsequent aberrant airway repair is due, in part, to the expression of MIG/CXCL9, IP-10/CXCL10, and ITAC/CXCL11 through their interaction with the chemokine receptor CXCR3. The persistent expression of these CXC chemokines leads to a chronic peribronchiolar leukocyte infiltration that promotes the eventual fibrotic obliteration of the airways. Our studies extend the results of previous murine studies involving acute cardiac rejection and demonstrate an association of elevated levels of MIG/CXCL9, IP-10/CXCL10, and ITAC/CXCL11 in human bronchoalveolar lavage fluid (BALF) from transplantation recipients with acute and chronic rejection. Furthermore, in vivo neutralization studies of CXCR3 and its ligands in a murine model of chronic lung allograft rejection significantly attenuated the pathogenesis of BOS by reducing the intragraft recruitment of CXCR3-expressing mononuclear cells. These findings may ultimately result in novel therapies designed to modulate CXCR3 receptor and ligand biology and impact on the development of BOS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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With Institutional Review Board approval and informed written consent from all patients involved in our study, we prospectively enrolled all patients undergoing both lung and heart-lung transplantation at the University of Michigan hospital from June 1992 through January 2000. Patients were eligible for this study if they survived at least 6 mo posttransplantation. One hundred fifty-three patients were evaluated, of which 108 were included: 53 females and 55 males. Forty-five patients were excluded from the study because of technical problems in coordinating the collection of their BALF samples and/or because the sample was deemed infected/colonized (see criteria below). All transplanted patients were routinely followed according to a standard protocol devised at the University of Michigan. This protocol included clinical visits weekly for the first 3 wk, then at 6 wk, and then at 3, 6, 9, and 12 mo. Subsequently, they were seen every 4 mo for the second year and thereafter annually. The clinic visit involved a history, physical, pulmonary function testing, and a bronchoscopy. Bronchoscopy was also performed at times when either infection or rejection was suspected.
The transplant recipients were divided into three groups: BOS, acute rejection, and healthy transplantation recipients. The first group was comprised of lung transplantation recipients with BOS. We used the first BALF sample that met criteria for BOS without concomitant infection/colonization or treatment with either pulse Solu-Medrol, monoclonal (OKT3), or polyclonal rabbit anti-thymocyte globulin Ab within 30 days of BALF collection (n = 25). The second group was comprised of lung transplant recipients with acute lung allograft rejection that had not thus far developed BOS. We used the first BALF sample that met criteria for acute rejection without concomitant infection or colonization or treatment with pulse Solu-Medrol within 30 days of BALF collection (n = 45). The third group was comprised of healthy lung transplant recipients who thus far had never had an episode of acute rejection or BOS (n = 38). We used the last available BALF sample that met criteria for being healthy without concomitant infection or colonization. We chose the last available BALF in this healthy group to try to ensure an effective comparison of duration (in months) posttransplantation between the healthy transplant recipients and patients with BOS. BALF from patients with infection/colonization were excluded (see exclusion criteria below). The exclusion of any BALF and the assignment to the proper group was performed prospectively without knowledge of the BALF cytokine levels or cell counts. Because of multiple BALF samples from each patient, we designed our study by choosing only one BALF sample from each patient. This avoids the use of multiple samples from the same patient at different time points with different diagnoses (i.e., BOS, acute rejection, and healthy recipients). The patient’s diagnosis was determined by the last abnormal BALF collected unless the specimens remained healthy throughout all time points. The BALF that met the criteria for the diagnosis of BOS, acute rejection, or healthy was then used for all subsequent analyses. The BALF diagnosed with BOS received preference over acute rejection, which received preference over healthy transplant recipients. For instance, if a patient had a diagnosis of being healthy at 3 mo, acute rejection at 6 mo, and then healthy at 9 mo, we used the 6-mo BALF with diagnosis of acute rejection for that patient and did not use the healthy BALFs. However, if a patient had a diagnosis of being healthy at 3 mo, acute rejection at 6 mo, healthy at 9 mo, and BOS at 12 mo, we used the 12-mo BALF with the diagnosis of BOS for that patient. We would not use the early healthy and acute rejection BALFs from this same patient.
Diagnosis of BOS, acute lung allograft rejection, and healthy lung transplant recipients
Patients were diagnosed with BOS on the basis of an unexplained and sustained decrease in the FEV1 to a level of 80% or less of the peak predicted value after transplantation with or without pathologic evidence of BOS as previously described (11). The diagnosis of acute rejection was based on pathologic findings by transbronchial biopsy without concomitant infection/colonization or evidence of BOS as previously described (11). Healthy transplantation recipients were those patients undergoing surveillance bronchoscopy without ever having evidence of biopsy-proven acute rejection or BOS.
Our experimental protocol was designed to reduce bias related to infectious disease episodes and/or colonization. Our protocol excluded any BALF performed at a time when infection and/or colonization was diagnosed with the following criteria: a positive BALF or transbronchial biopsy; microbiology Gram stain; and culture for bacterial, acid fast bacillus, fungus, CMV culture by shell vial technique, transbronchial or cytological evidence of CMV, other virus, or Pneumocystis carinii pneumonia by methenamine silver stain.
Immunosuppression and prophylactic antimicrobials
Patients were placed on a standard pretransplantation and posttransplantation immunosuppression protocol including cyclosporine, methylprednisolone, azathioprine, prophylactic antibiotics, and prophylactic antivirals, as previously described (12, 13). All episodes of acute lung allograft rejection were treated with a 3-day pulse of methlyprednisolone 1 g/day without adjustment of daily prednisone (12, 13).
BALF
BALF was obtained from lung transplant recipients with BOS, acute lung allograft rejection, and healthy lung allografts by methods previously described (12, 13, 14). The cell-free solution was aliquoted and frozen immediately at 70°C until thawed for MIG/CXCL9, IP-10/CXCL10, and ITAC/CXCL11 ELISAs (15).
Reagents
Abs to human MIG/CXCL9 and IP-10/CXCL10 were purchased from R&D
Systems (Minneapolis, MN) and human ITAC/CXCL11 was from PeproTech
(Rocky Hill, NJ). Polyclonal rabbit anti-murine CXCR3 was produced
by the immunization of a rabbit with a 16-mer peptide
(PYDYGENESDFSDSPP) constituting the NH2 terminus
of murine CXCR3 (mCXCR3). Polyclonal goat anti-murine MIG
and anti-murine IP-10 specific anti-serum were produced by the
immunization of goats with the appropriate recombinant murine
chemokines (MIG/CXCL9 or IP-10/CXCL10; CRG2) (R&D Systems). The rabbit
and goats were immunized in multiple intradermal sites with CFA
followed by at least three boosts in IFA (14, 16). Direct
ELISA was used to evaluate antisera titers, and serum was used for
Western blot, ELISA, and neutralization assays when titers had reached
greater than 1/1,000,000. The specificity of anti-mCXCR3 Abs was
assessed by Western blot analysis against cells expressing mCXCR3 and a
panel of other human and murine recombinant cytokines. The specificity
of anti-mMIG and anti-mIP-10 Abs was assessed by Western blot
analysis against a panel of human and murine recombinant cytokines. The
anti-mMIG and anti-mIP-10 Abs were specific in our sandwich
ELISA without cross-reactivity to a panel of cytokines including human
and murine IL-1Ra, IL-1, IL-2, IL-6, IL-4, TNF-, IFN-, and
members of the CXC and CC chemokine families. Furthermore, murine
splenocyte chemotaxis assay was preformed to determine the specificity
of the Abs. Briefly, splenocytes were stimulated with IL-2 for 10 days
with evidence for CXCR3 expression by FACS analysis. A total of 50
ng/ml of mIP-10, mMIG, mIP-10 and mMIG, stromal cell derived
factor-1
(SDF-1), or mRANTES was preincubated with either control
Abs or Abs to mIP-10, mMIG, or mCXCR3 for 30 min at 37°C. Chemotaxis
was performed as previously described with 5.0-µm polycarbonate
filters coated with fibronectin (ICN Biomedicals, Irvine, CA)
(12). The anti-mCXCR3 Abs demonstrated a specific
neutralizing capacity against mIP-10/CXCL10 (PeproTech), mMIG/CXCL9
(PeproTech), or the combination of mMIG/CXCL9 and mIP-10/CXCL10, as
determined by inhibiting the chemotaxis of IL-2-stimulated,
CXCR3-expressing splenocytes. In contrast, the anti-mCXCR3 Abs did
not inhibit the chemotaxis of IL-2-stimulated splenocytes to
either mSDF-1/CXCL12 (PeproTech) or mRANTES/CC chemokine
ligand (CCL)5 (PeproTech) (Table I
).
Similarly, anti-mMIG had a specific neutralizing capacity of
mMIG/CXCL9 by inhibiting splenocyte chemotaxis response to mMIG/CXCL9.
In contrast, anti-mMIG did not inhibit chemotaxis in response to
mIP-10/CXCL10, mSDF-1/CXCL12, or mRANTES/CCL5 (Table I). The
anti-mIP-10 had a specific neutralizing capacity of mIP-10/CXCL10
by inhibiting chemotaxis in response to mIP-10/CXCL10. In contrast,
anti-mIP-10 did not inhibit chemotaxis in response to mMIG/CXCL9,
mSDF-1/CXCL12, or mRANTES/CCL5 (Table I).
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We used a well-established and reproducible murine model of BOS involving heterotopic s.c. trachea transplantation studies as previously described (12). The MHC class I- and class II-disparate combination wasBALB/c (H-2d) to C57BL/6 (H-2b) (allografts) and C57BL/6 (H-2b) to C57BL/6 (H-2b) (syngeneic control).
In separate experiments, animals received 1 ml of anti-mMIG/CXCL9, anti-mIP-10/CXCL10, anti-mCXCR3, or appropriate control Abs on days 0, 2, 4, 6, 8, 10, and 12 by i.p. injection.
Histopathologic grading of BOS
Three random 3-µm paraffin-embedded tissue sections for five
different trachea allografts were stained with Masson’s Trichrome and
H&E at three time points: days 7, 14, and 21. The histopathology was
blindly reviewed with a modified histologic scoring system as
previously described (12). All qualitative histological
changes were noted. In addition, four easily identifiable pathologic
processes were scored on a scale of 0–4 (0, normal; 1, mild; 2,
moderate; 3, severe; and 4, very severe) as previously described
(12): 1) airway lining epithelial loss, 2) deposition of
extracellular matrix (ECM), 3) leukocyte infiltration, and 4) luminal
obliteration due to granulation tissue formation and/or fibrosis (Table II). An overall score of BOS was obtained
based on the summation of all the scores, and then a mean ± SEM
was generated from the cohort of tracheal allografts (three sections of
each trachea, five tracheas per group) at each time point to generate a
cumulative histological BOS score.
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MIG/CXCL9, IP-10/CXCL10, and ITAC/CXCL11 protein was quantitated using a modification of a double-ligand method as previously described (14, 16).
FACS analysis of infiltrating leukocyte populations
Tracheal single-cell suspension preparations were made using a method previously described (12, 14). Mouse CD45 MicroBeads (Miltenyi Biotec, Auburn, CA) were used for positive selection of CD45-expressing cells. Single-cell suspensions were stained with PE-conjugated mouse anti-murine CD3e, CD4, CD8a, NK1.1 (BD PharMingen, San Diego, CA), FITC-conjugated anti-murine MOMA-2 (Seratec, Raleigh, NC), and anti-murine CXCR3 (Santa Cruz Biotechnology, Santa Cruz, CA), which was FITC labeled with N-hydroxy succinimide-Flurescein (Pierce, Rockford, IL) (17). Single-cell suspensions were analyzed on a FACScan flow cytometer (BD Biosciences, San Jose, CA) using CellQuest software (BD Biosciences), as previously described (14).
Total RNA isolation and real-time quantitative PCR
Total cellular RNA was isolated as previous described (14, 16). Total RNA was determined, reverse transcribed into cDNA, and amplified using TaqMan reverse transcription reagents (PE Applied Biosystems, Foster City, CA). Real-time quantitative PCR was performed using specific TaqMan primers and probes, the ABI Prism 7700 sequence detector, and SDS analysis software (PE Applied Biosystems). Negative controls with no template cDNA were performed. The primers and probe sequences (forward primer, reverse primer, TaqMan probe) used were as follows: mMIG/CXCL9 primers, forward, ATG CAC GAT GCT CCT GCA, reverse, AGG TCT TTG AGG GAT TTG TAG TGG, probe 6FAMCAG CAC CAG CCG AGG CAC GATAMRA; mIP-10/CXCL10 primers, forward, GGA GTG AAG CCA CGC ACA C, reverse, ATG GAG AGA GGC TCT CTG CTG T, probe 6FAMCCC CGG TGC TGC GAT GGA TGTAMRA; mITAC/CXCL11 primers, forward, GGG CGC TGT CTT TGC ATC, reverse, AAG CTT TCT CGA TCT CTG CCA T, probe 6FAM CCC CGG GAT GAA AGC CGT CAA MGBNFQ; mCXCR3 TaqMan per developed assay reagent (PE Applied Biosystems); and housekeeping gene 18s TaqMan per developed assay reagent (PE Applied Biosystems).
Quantitative analysis of gene expression was done using the comparative
CT (
CT)
methods, in which CT is the threshold cycle
number (the minimum number of cycles needed before the product can be
detected) (18). The arithmetic formula for the
CT method is described as the difference
in threshold cycles for a target (i.e., mMIG) and an endogenous
reference (i.e., our housekeeping gene 18s). The amount of target
normalized to an endogenous reference (i.e., mMIG in allografts at day
3) and relative to a calibration normalized to an endogenous reference
(i.e., mMIG in syngeneic controls at day 3) is given by
2-CT
(18). The following is an example for comparing mMIG/CXCL9
expression from allografts and syngeneic controls at day 3. Both mMIG
from the allografts and syngeneic controls at day 3 are normalized to
18s: CT =
CT (mMIG from allograft at day 3
normalized to endogenous 18s) - CT
(mMIG from syngeneic control at day 3 normalized to endogenous 18s).
The calculation of
2-CT
then gives a relative value when comparing the target to the
calibrator, which we designate in this context as fold increase of
allograft to syngeneic controls of the target mRNA relative
quantification.
Hydroxyproline assay
Before removal, heterotopically transplanted tracheas were dissected free of surrounding tissue, and total tracheal collagen was determined by analysis of hydroxyproline as previously described (12, 16).
Statistical analysis
Data were analyzed on a Dell PC computer using the Statview 4.5 statistical package (Abacus Concepts, Berkeley, CA). All human group comparisons were evaluated by the nonparametric Kruskal-Wallis test with the post hoc analysis Dunn. Data were expressed as mean ± SEM and displayed using a box plot summary. The plot’s horizontal line represents the median, the box encompasses the 25th to 75th percentile, and the error bars encompass the 10th to the 90th percentile. Continuous data were expressed as mean ± SEM. Data were considered statistically significant if p values were 0.05 or less. All animal group comparisons were evaluated by the ANOVA test with the post hoc analysis Bonferroni/Dunn. Data were expressed as mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We determined whether the ligands for CXCR3 were elevated in the
BALF from patients with BOS and acute lung allograft rejection, as
compared with healthy lung transplantation recipients. There were
significant differences in these ELR-negative CXC chemokines in BALF
using a three-group comparison: recipients with BOS, recipients with
acute rejection, and healthy transplantation recipients. The levels of
MIG/CXCL9 were significantly different when comparing all three groups:
955.00 ± 215.70 vs 586.00 ± 85.09 vs 218.50 ± 51.41
pg/ml, respectively (p = 0.0002) (Fig. 1A). Specifically, MIG/CXCL9
levels from the BALF of patients with BOS and acute rejection were
significantly higher than those of healthy lung transplant recipients
(p < 0.001) and (p <
0.01), respectively (Fig. 1A). Similarly, the levels of
IP-10/CXCL10 were significantly different when comparing all three
groups: 1228.00 ± 170.80 vs 806.70 ± 81.26 vs 301.60
± 66.05 pg/ml, respectively (p < 0.0001)
(Fig. 1B). IP-10/CXCL10 levels from the BALF of patients
with BOS and acute rejection were significantly higher than those of
healthy lung transplant recipients (p < 0.001)
and (p < 0.001), respectively (Fig. 1B). The levels of ITAC/CXCL11 were significantly different
when comparing all three groups: 78.32 ± 30.14 vs 45.44 ±
7.03 vs 34.07 ± 10.54 pg/ml, respectively
(p = 0.0016) (Fig. 1C). ITAC/CXCL11
levels from the BALF of patients with BOS and acute rejection were
significantly higher than those of healthy lung transplant recipients
(p < 0.01) and (p <
0.05), respectively (Fig. 1C). In regard to MIG/CXCL9, using
a cutoff of 100 pg/ml for a positive result of rejection (BOS/acute
rejection), we found only 4 of 25 patients with MIG/CXCL9 levels <100
pg/ml with BOS and only 8 of 45 patients with MIG/CXCL9 levels <100
with acute rejection. Similarly, for IP-10/CXCL10, using a cutoff of
100 pg/ml for a positive result of rejection, we found only 2 of 25
patients with IP-10/CXCL10 levels <100 pg/ml with BOS and only 7 of 45
patients with IP-10/CXCL10 levels <100 pg/ml with acute rejection. In
addition, for ITAC/CXCL11, using a cutoff of 50 pg/ml for a
positive result of rejection, we found 8 of 25 patients with
ITAC/CXCL11 levels <50 pg/ml with BOS and 21 of 45 patients with
ITAC/CXCL11 levels <50 pg/ml with acute rejection. These results
demonstrate the importance of both MIG/CXCL9 and IP-10/CXCL10, as
compared with ITAC/CXCL11 during rejection. Moreover, in regard to
MIG/CXCL9 levels, using a cutoff of 100 pg/ml for a positive result of
rejection (BOS/acute rejection), there were 58 true positives and only
17 false positives, yielding a predictive value of 77%. With
IP-10/CXCL10 levels, using a cutoff of 100 pg/ml for a positive result
of rejection, there were 84 true positives and only 18 false positives,
yielding a predictive value of 82%. Lastly, in regard to ITAC/CXCL11
levels, using a cutoff of 50 pg/ml for a positive result of rejection,
there were 28 true positives and 29 false positives, yielding a
predictive value of 50%.
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D). As expected, there were differences in
duration posttransplantation between the acute rejection and healthy
groups and between the acute rejection and BOS groups
(p < 0.001) and (p <
0.001), respectively (Fig. 1D). However, there was no
difference in duration posttransplantation between the BOS and healthy
group (p > 0.05) (Fig. 1D). MIG/CXCL9, IP-10/CXCL10, and ITAC/CXCL11 are elevated during murine BOS
Our human data demonstrated that elevated levels of MIG/CXCL9,
IP-10/CXCL10, and ITAC/CXCL11 are important and are associated with the
continuum of acute to chronic (BOS) rejection. On this basis, we
hypothesized that these chemokines promote BOS. To test this
contention, translational studies were performed in a murine model of
BOS to determine whether these chemokines, by interacting with their
shared receptor CXCR3, play a functional role in promoting the
persistent recruitment of peribronchial mononuclear cells during the
pathogenesis of BOS. We first performed FACS analysis on single-cell
suspensions of tracheal digests to quantitate the magnitude and
characteristics of leukocyte infiltration into allografts. We found a
significant increase in numbers of lymphocytes including CD3, CD4, CD8,
and NK cells infiltrating the allografts at days 7 and 14, as compared
with syngeneic controls (Fig. 2, A–D). In addition,
there was a significant increase in numbers of mononuclear phagocytes
infiltrating the allografts that peaked at days 7 and 14; however,
their presence remained significantly elevated throughout our 21-day
time course, as compared with syngeneic controls (Fig. 2E).
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I). All three chemokines had
similar patterns of expression. They all peaked at day 7 and remained
significantly elevated throughout the rest of the time course (Fig. 3I). Importantly, the increase of IP-10/CXCL10 was more
dramatic than either MIG/CXCL9 or ITAC/CXCL11, a trend similar to what
was seen for our human protein data (Figs. 1, A–C, and 3I).
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II). Furthermore,
similar to the human BOS data, IP-10/CXCL10 protein levels were greater
than MIG/CXCL9 (Figs. 1, A and B, and
3II). CXCR3 expression on infiltrating mononuclear cells within the allograft parallels the expression of its ligands during murine BOS
CXCR3 is the shared receptor for the ligands (MIG/CXCL9,
IP-10/CXCL10, and ITAC/CXCL11). Real-time quantitative PCR of tracheal
homogenates demonstrated a significant increase of CXCR3 mRNA
expression from allografts, as compared with syngeneic controls (Fig. 4I). CXCR3 expression peaked
at day 7 and remained persistently elevated throughout the rest of the
time course, paralleling the expression of its three ligands and
intragraft infiltration of mononuclear cells (Figs. 2, 3, and 4I). These finding were confirmed by performing FACS
analysis on single-cell suspensions of whole tracheal digests for the
cell surface expression of CXCR3 protein. There were significant
increases in cell surface expression of CXCR3 from allografts at day 7
that remained markedly elevated throughout the rest of the time course
(Fig. 4II). Knowing that MIG/CXCL9, IP-10/CXCL10, and
ITAC/CXCL11 exert a specific chemotactic effect on activated
lymphocytes expressing CXCR3, dual-colored FACS analysis was used to
determined whether the infiltrating intragraft lymphocytes were a major
source of CXCR3 expression (7, 19, 20). There were
significant increases of CXCR3 cell surface expression on CD3, CD4,
CD8, and NK cells infiltrating the allografts, as compared with the
syngeneic controls (Fig. 4III).
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With MIG/CXCL9, IP-10/CXCL10, and ITAC/CXCL11 levels correlating
with the recruitment of mononuclear cells and the expression of CXCR3
on infiltrating lymphocytes, we determined whether inhibiting
ligands/CXCR3 biology could attenuate leukocyte recruitment during the
pathogenesis of BOS. In vivo neutralization studies of endogenous
mCXCR3, as compared with control Abs, were performed. In all
experiments, C57BL/6 mice were the recipients of BALB/c tracheas. The
recipients received specific anti-mCXCR3 or control Abs at days 0,
2, 4, 6, 8, 10, and 12. Kinetic investigation of FACS analysis data on
single-cell suspensions of whole tracheal digests at days 7 and 14
demonstrated marked attenuation of infiltrating CD3, CD4, CD8, NK
cells, and mononuclear phagocytes in allografts from animals treated
with neutralizing anti-mCXCR3 Abs, as compared with allografts from
animals treated with control Abs (Fig. 5). However, by day 21 there was no
significant difference in numbers of infiltrating intragraft
mononuclear cells between the groups (Fig. 5). Importantly, the
increase in mononuclear cells in allografts from animals treated with
anti-mCXCR3 never reached the peak levels seen in the
allografts from animals treated with control Abs (Fig. 5). These
results were further confirmed by quantitative analysis of
histopathologic sections demonstrating significant reductions in
infiltrating leukocytes in allografts from animals treated with
anti-mCXCR3 Abs, as compared with allografts from animals treated
with control Abs (Figs. 6 and 7IA). To rule out the theoretical possibility that
the Abs to mCXCR3 could be, in part,
acting by mediating leukocyte clearance through the reticular
endothelial system, FACS analysis on PBMCs for CXCR3-expressing
lymphocytes was performed on mice with allograft rejection treated with
either anti-mCXCR3 or control Abs at day 7. The number of
circulating lymphocytes expressing CXCR3 were not significantly
different between these two groups: 8.3 x
104 cells vs 9.0 x
104 cells expressing CXCR3, respectively
(p = 0.62).
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Reductions in the recruitment of mononuclear cells in the
allografts from animals treated with anti-mCXCR3 Abs led us to
assess whether these reductions affected ECM deposition, airway
obliteration, and epithelial cell injury. Histopathologic analysis of
tracheal allografts from the anti-mCXCR3-treated animals
demonstrated marked reductions in ECM deposition, airway obliteration,
and loss of epithelial cell integrity, as compared with tracheal
allografts treated with control Abs with overall cumulative scores
(i.e., leukocyte infiltration + ECM deposition + airway obliteration +
epithelial injury): day 7 (3.1 ± 0.6 vs 8.2 ± 0.4;
p = 0.001), day 14 (4.6 ± 0.7 vs 11.3 ±
0.8; p = 0.003), and day 21 (8.2 ± 0.4 vs
12.3 ± 0.8; p = 0.01), respectively (Figs. 6 and
Fig. 7II). We further quantitated these findings of airway
fibroplasia/fibro-obliteration by measuring hydroxyproline levels from
whole tracheal allograft homogenates. There was a significant reduction
in hydroxyproline levels from tracheal allografts at day 21 from the
animal treated with anti-mCXCR3 Abs, as compared with the animal
treated with control Abs (Fig. 7III).
In vivo neutralization of either mMIG or mIP-10 inhibits the recruitment of mononuclear cells during murine BOS
Our data demonstrate the importance of CXCR3 and the expression of
its corresponding CXC chemokine ligands during the pathogenesis of BOS.
Specifically, by inhibiting CXCR3, we attenuated intragraft mononuclear
cell recruitment, thus attenuating fibro-obliteration. To confirm our
results of blocking CXCR3, passive immunization and neutralization
experiments of either endogenous mMIG/CXCL9 or IP-10/CXCL10, as
compared with control Abs, were performed. The identical protocols as
described for in vivo neutralization of mCXCR3 were used. Similar to
our anti-mCXCR3 studies, FACS analysis on single-cell suspensions
of whole tracheal digests at days 7 and 14 demonstrated a marked
attenuation of infiltrating CD3, CD4, CD8, NK cells, and mononuclear
phagocytes in allografts from animals treated with anti-mMIG or
anti-mIP-10 Abs, as compared with allografts from animals treated
with control Abs (Fig. 8). However, by
day 21 there was no significant difference in numbers of infiltrating
intragraft mononuclear cells between the groups (Fig. 8). Moreover, our
anti-mCXCR3 data suggest that infiltrating lymphocytes expressing
CXCR3 are important during the pathogenesis of BOS. To further assess
this, we determined whether in vivo depletion of either endogenous mMIG
or mIP-10 would inhibit the number of CXCR3-expressing mononuclear
cells in the allograft. There were marked reductions of CXCR3
expressing CD3, CD4, CD8, and NK cells in the allografts from animals
treated with either anti-mMIG or anti-mIP-10 Abs, as compared
with allografts from animals treated with control Abs (Fig. 9). These results were further confirmed
by quantitative analysis of histopathologic sections of tracheal
allograft demonstrating significant reductions in infiltrating
leukocytes from animals treated with either anti-mMIG or
anti-mIP-10 Abs, as compared with animals treated with control Abs
(Figs. 10 and Fig. 11IA).
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and 11I). Furthermore, there were significant differences in the
overall cumulative histopathologic scores in a three-group comparison:
anti-MIG, anti-IP-10, and control Abs at days 7, 14, and 21.
Day 7 (3.7 ± 0.4 vs 3.6 ± 0.3 vs 7.8 ± 0.7,
respectively; p < 0.001; day 14 (5.5 ± 0.4 vs
5.0 ± 0.4 vs 11.0 ± 0.7, respectively; p <
0.001); and day 21 (9.3 ± 0.7 vs 9.0 ± 0.8 vs 11.8 ±
0.4, respectively; p = 0.04) (Figs. 10 and 11II). Specifically, there were significant reductions in
overall cumulative scores for anti-MIG vs control Ab and
anti-IP-10 vs control Ab at day 7 (p <
0.0001 and p < 0.0001, respectively), day 14
(p < 0.0001 and p < 0.0001,
respectively); and day 21 (p < 0.0001 and
p = 0.0005, respectively) (Figs. 10 and Fig. 11II). We further quantitated these findings of
fibro-obliteration and, similar to the anti-mCXCR3-treated animals,
there was a significant reduction in hydroxyproline levels from
allografts at day 21 from the animals treated with either anti-mMIG
or anti-mIP-10, as compared with allografts from animals treated
with control Abs (Fig. 11III). These findings further
support the importance of the CXCR3/CXCR3 ligand biology in
mediating BOS. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We determined that ligands/CXCR3 interactions contribute to the pathogenesis of human chronic lung rejection (BOS) by performing proof of concept studies using a murine model of BOS. We have previously found that tracheal allografts undergoing BOS have increased procollagen expression and ECM deposition (12). We further characterized this model system and found increased numbers of infiltrating mononuclear cells (CD3, CD4, CD8, NK cells, and mononuclear phagocytes) peaking at days 7 and 14. With this finding, together with our results from the human studies suggesting that MIG/CXCL9, IP-10/CXCL10, and ITAC/CXCL11 are important for the continuum of acute to chronic rejection, we determined the kinetics of expression of the three CXCR3 ligands in a murine model of BOS. Consistent with the human data, we found markedly elevated levels of all three chemokines in the tracheal allografts undergoing BOS. In addition, their expression paralleled the recruitment of mononuclear cells into the tracheal allografts. Furthermore, the magnitude of expression was similar to what we have seen in the human data with IP-10/CXCL9>MIG/CXCL10>>ITAC/CXCL11. The association between increased expression of CXCR3 ligands and allograft mononuclear cell infiltration is supported by murine studies of acute cardiac and skin allograft rejection (9, 10, 24, 25). We have now extended these studies to chronic lung rejection and demonstrated an important relationship between persistent expression of MIG/CXCL9, IP-10/CXCL10, and ITAC/CXCL11, mononuclear cell infiltration, and the continuum of acute to chronic allograft rejection.
Our receptor data demonstrated the expression of CXCR3 on recruited intragraft lymphocytes that paralleled the expression of the appropriate chemokine ligands. These results are supported by studies that demonstrated that BALF cells from human acute and chronic (BOS) lung allograft rejection have increased cell surface expression of CXCR3 (23). Together these studies demonstrate that augmented levels of MIG/CXCL9, IP-10/CXCL10, and ITAC/CXCL11 are important in the persistent recruitment of mononuclear cells expressing CXCR3 during the pathogenesis of acute rejection to the fibro-obliterative disease, BOS.
To determine a direct role of CXCR3 and its chemokine ligands in leukocyte recruitment during the pathogenesis of murine BOS, we performed in vivo neutralization experiments of endogenous mCXCR3. Allografts from animals treated with anti-mCXCR3 had significant reductions of intragraft mononuclear cells (i.e., CD3, CD4, CD8, NK, and mononuclear phagocytes) throughout the first 14 days of the experiment, which then increased at day 21. Moreover, the infiltrating mononuclear cells in allografts from animal treated with anti-mCXCR3 never reached the peak levels seen in controls. Furthermore, assessment of allografts from animals treated with anti-mCXCR3 demonstrated lower scores for BOS (i.e., leukocyte infiltration, ECM deposition, airway obliteration, and epithelial injury), as compared with control Ab-treated animals. This reduction in fibroplasia/fibro-obliteration was confirmed by demonstrating lower levels of hydroxyproline from tracheal allografts treated with anti-mCXCR3 Abs. These studies demonstrate the importance of ligands/CXCR3 biology during the pathogenesis of chronic lung rejection, BOS. Although the Abs to mCXCR3 could conceivably work by promoting clearance of CXCR3-producing lymphocytes from the bloodstream through the reticular endothelial system, this was ruled out by demonstrating no significant difference in circulating lymphocytes expressing CXCR3 between transplant recipients treated with anti-mCXCR3, as compared with control Abs.
MIG/CXCL9 and IP-10/CXCL10 levels were both found to be an order of magnitude higher than those of ITAC/CXCL11 in the continuum of acute to chronic lung rejection. On this basis, we next determined whether inhibiting CXCR3 ligands through the use of anti-mMIG or anti-mIP-10 Abs would have effects similar to those found with the anti-mCXCR3 Ab. Similar to the findings for neutralization of CXCR3, there were significant reductions of mononuclear cell intragraft infiltration, scores for BOS, and levels of hydroxyproline in allografts from animals treated with either anti-MIG or anti-IP-10 Abs, as compared with control Abs. This is supported by similar results seen using IP-10-/- hearts or anti-MIG Abs in murine models of acute cardiac rejection (24, 25). To confirm that this approach had a direct effect in attenuating the infiltration of mononuclear cells expressing CXCR3, we determined if there were changes in infiltrating lymphocytes expressing CXCR3 into allografts from animals treated with either anti-mMIG or anti-mIP-10 Abs. The neutralizing Abs to mMIG or mIP-10 inhibited the recruitment of CXCR3-expressing lymphocytes in the allografts.
It is interesting that neutralization of ligands MIG/CXCL9 and IP-10/CXCL10 both give similar levels of inhibition of the biological response during BOS. This raises the question of the relative roles of MIG/CXCL9 and IP-10/CXCL10 in lymphocyte recruitment during the pathogenesis of BOS. This is similar to previous findings in cardiac allograft rejection in which inhibition of either IP-10/CXCR3 or MIG/CXCR3 interactions attenuated acute cardiac rejection (9, 25). As MIG/CXCL9 and IP-10/CXCL10 share the same receptor (CXCR3), one possible explanation is homologous desensitization of the receptor by the two ligands, whereby neutralization of MIG/CXCL9 may overexpose the receptor to IP-10/CXCL10 and vice versa, thereby resulting in desensitization of the receptor as is seen in calcium flux and chemotaxis assays at high concentrations of ligand (20, 26). Our results do not show that either MIG/CXCL9 or IP-10/CXCL10 is more important, but merely that they both play an important role when interacting with their shared receptor, CXCR3, during the pathogenesis of BOS.
Interestingly, all tracheal allografts from animals treated with neutralizing Abs (i.e., anti-mCXCR3, anti-mMIG, or anti-IP-10) demonstrated reductions in mononuclear phagocytes, as compared with tracheal allografts from animals treated with controls. However, mononuclear phagocytes do not express CXCR3 (4, 7, 27). Therefore, the reduction in mononuclear phagocyte recruitment with these Abs may be related to reduced numbers of lymphocytes expressing CXCR3 interacting with other immune and/or stromal cells required to create a proinflammatory cytokine/chemokine milieu that ultimately leads to the recruitment of mononuclear phagocytes. This is analogous to what was demonstrated in murine models of allograft rejection when BALB/c organs were transplanted into CCR1-/- or CCR2-/- or CXCR3-/- recipient mice (10, 12, 28). In these transplant model systems, a significant reduction of intra-allograft cytokine/chemokine expression was seen (10, 12, 28).
In conclusion, we have demonstrated that MIG/CXCL9, IP-10/CXCL10, and
ITAC/CXCL11 through their interaction with their shared receptor,
CXCR3, play a pivotal role in mediating the persistent recruitment of
peribronchiolar mononuclear cells that lead to fibro-obliteration
during BOS. The fact that there are still mononuclear cells in our
allografts from recipients treated with neutralizing Abs, albeit
reduced in number, suggests that other chemokines have a role during
BOS. With regards to alternative chemokines involved in rejection, we
have not found elevated levels of macrophage-inflammatory
protein-1/CCL3 or macrophage-inflammatory protein-1/CCL4 during
human acute or chronic rejection (data not shown). However, we have
previously demonstrated elevated levels of monocyte chemoattractant
protein-1 (MCP-1)/CCL2 during human acute and chronic (BOS) lung
allograft rejection (12). Furthermore, using an animal
model we have previously demonstrated that inhibition of MCP-1/CCR2
interactions attenuates rejection and fibro-obliteration by defective
and specific recruitment of mononuclear phagocytes independent of
lymphocytes (12). Importantly, in this study we have found
elevated levels of lymphocyte-specific chemoattractants, CXCR3 ligands,
in humans with acute and chronic rejection. Translational studies in a
murine model of BOS demonstrate that the inhibition of ligands/CXCR3
interactions also attenuates BOS. Combined, these studies demonstrate
that chemokines (MCP-1/CCR2 specific for mononuclear phagocytes and
IFN-inducible CXC chemokine ligands/CXCR3 specific for lymphocytes) are
acting in parallel during the pathogenesis of acute and chronic (BOS)
rejection. Future studies will determine whether combined inhibitors of
both pathways will ultimately lead to additive or syngeneic reductions
in the pathogenesis of BOS.
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Robert M. Strieter, Department of Medicine, Division of Pulmonary and Critical Care Medicine, Department of Pathology and Laboratory Medicine, University of California, Los Angeles, School of Medicine, Room 14-154 Warren Hall, Box 711922, 900 Veteran Avenue, Los Angeles, CA 90024-1922. E-mail address: rstrieter{at}mednet.ucla.edu
3 Abbreviations used in this paper: BOS, bronchiolitis obliterans syndrome; MIG, monokine induced by IFN-; CXCL, CXC chemokine ligand; IP-10, IFN-inducible protein 10; ITAC, IFN-inducible T cell chemoattractant; ELR, glutamine-leucine-arginine; BALF, bronchoalveolar lavage fluid; m, murine; SDF-1, stromal cell-derived factor-1; CCL, CC chemokine ligand; ECM, extracellular matrix; MCP-1, monocyte chemoattractant protein-1.
Received for publication February 4, 2002. Accepted for publication May 17, 2002.
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J. A. Belperio, M. P. Keane, M. D. Burdick, J. P. Lynch III, D. A. Zisman, Y. Y. Xue, K. Li, A. Ardehali, D. J. Ross, and R. M. Strieter Role of CXCL9/CXCR3 Chemokine Biology during Pathogenesis of Acute Lung Allograft Rejection J. Immunol., November 1, 2003; 171(9): 4844 - 4852. [Abstract] [Full Text] [PDF]
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H. D. Chen, A. E. Fraire, I. Joris, R. M. Welsh, and L. K. Selin Specific History of Heterologous Virus Infections Determines Anti-Viral Immunity and Immunopathology in the Lung Am. J. Pathol., October 1, 2003; 163(4): 1341 - 1355. [Abstract] [Full Text] [PDF]
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G. Valbuena, W. Bradford, and D. H. Walker Expression Analysis of the T-Cell-Targeting Chemokines CXCL9 and CXCL10 in Mice and Humans with Endothelial Infections Caused by Rickettsiae of the Spotted Fever Group Am. J. Pathol., October 1, 2003; 163(4): 1357 - 1369. [Abstract] [Full Text] [PDF]
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 J. Kao, J. Kobashigawa, M. C. Fishbein, W. R. MacLellan, M. D. Burdick, J. A. Belperio, and R. M. Strieter Elevated Serum Levels of the CXCR3 Chemokine ITAC Are Associated With the Development of Transplant Coronary Artery Disease Circulation, April 22, 2003; 107(15): 1958 - 1961. [Abstract] [Full Text] [PDF]
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S. G. Kallapur, A. H. Jobe, M. Ikegami, and C. J. Bachurski Increased IP-10 and MIG Expression after Intra-amniotic Endotoxin in Preterm Lamb Lung Am. J. Respir. Crit. Care Med., March 1, 2003; 167(5): 779 - 786. [Abstract] [Full Text] [PDF]
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