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Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of California School of Medicine, Los Angeles, CA 90095;
Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine and
Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109; and
§
Department of Inflammation Research, Merck & Co., Inc., Rahway, NJ 07065
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Acute lung allograft rejection is characterized by an infiltration of mononuclear cells that ultimately contributes to the loss of lung architecture and function. However, the mediators that orchestrate the extravasation of leukocytes have not been fully elucidated. RANTES was the first CC chemokine to be characterized to induce significant T lymphocyte chemotactic activity and is a potent chemoattractant for monocytes (7, 8, 9, 10, 11, 12). RANTES has two major receptors for mononuclear cell chemoattraction (CCR1 and CCR5), both of which are seven-transmembrane G protein-coupled receptors (11, 13, 14, 15, 16, 17, 18, 19, 20). We hypothesized that the allogeneic response leading to acute lung allograft rejection and extravasation of mononuclear cells is due in part to the expression of RANTES and its interaction with its receptors, CCR1 and CCR5.
We first determined whether RANTES was present in human lung allografts during acute rejection. Human bronchoalveolar lavage specimens from lung transplant recipients demonstrated that RANTES protein levels were 2.3-fold greater during acute lung allograft rejection compared with those in healthy lung allografts. To determine whether RANTES together with the expression of its two receptors, CCR1 and CCR5, were significant factors involved in the mononuclear cell recruitment, we used a well characterized in vivo model of rat orthotopic lung transplantation (21, 22, 23, 24, 25). We found a marked time-dependent increase in levels of RANTES mRNA and protein compared with those in syngeneic control lungs. The increase in RANTES correlated with the recruitment of mononuclear cells and the expression of CCR1 and CCR5. Furthermore, in vivo neutralization of RANTES attenuated acute lung allograft rejection and reduced allospecific responsiveness by decreasing mononuclear cell recruitment. These experiments support the idea that RANTES and the expression of its receptors, CCR1 and CCR5, have important roles in the pathogenesis of acute lung allograft rejection. These findings may ultimately result in novel therapies designed for intervention and possible prevention of both acute and chronic lung allograft rejection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Twenty-four patients with single lung transplantation at the
University of Michigan were involved in this study. They were all
placed on a standard pre- and post-transplantation immunosuppression
protocol. Cyclosporine was begun preoperatively and continued
postoperatively to maintain levels at 
300 ± 50 ng/ml
throughout the first 6 wk, followed by a reduction to maintain levels
at 200 ± 50 ng/ml. Methylprednisone was begun intraoperatively
and was tapered to 20 mg/day by the third postoperative month. Attempts
were made to further taper the dose between 5–10 mg/day by 12 mo.
Azathioprine was begun at 2 mg/kg/day 8 h pretransplantation, then
adjusted to maintain a white blood cell count of
4000/mm3. All episodes of acute rejection were
treated with methylprednisone (1 g/day) for 3 days. All patients had
routine surveillance bronchoscopy preformed at 6 wk and 3, 6, 9, and 12
mo. Patients also had bronchoscopy preformed at times when infection or
rejection was suspected. Twelve bronchoalveolar
lavages3 (BALs) came
from healthy single lung transplant patients having routine
surveillance bronchoscopy and 12 BALs came from patients with biopsy
proven acute lung allograft rejection. The diagnosis and grading of
acute lung allograft rejection were based on histology from
transbronchial biopsies as previously described (26).
There was no evidence of infection (bacterial, fungal,
Pneumocystis carinii pneumonia, or virus) seen on BAL
culture or smears, BAL cytology, or transbronchial biopsy.
BAL fluid was obtained from lung transplant recipients with acute allograft rejection and from healthy lung allografts by methods previously described (27). Briefly, the patients were premedicated with meperidine and glycopyrrolate and nebulized with 2% xylocaine. Midazolam was used to obtain an appropriate degree of sedation. We performed flexible fiberoptic bronchoscopy via a transnasal approach and wedged the scope into a subsegmental bronchus of the middle lobe or lingula of the transplanted lung. BAL was then performed with 240 ml of normal saline in 60-ml aliquots; each retrieved by low suction. After discarding the initial aliquot, an average return of 100–125 ml was obtained. The BAL solution was split into two aliquots. One aliquot was sent to clinical laboratories for evaluation of cytology, bacterial, fungal, and viral cultures and CMV shell vial. The other aliquot was placed on ice and transported to the research laboratory. The recovered BAL fluid was filtered through sterile gauze, then centrifuged for 10 min at 2000 rpm. The cell-free solution was aliquoted and frozen immediately at 70°C until batched and thawed for cytokine ELISA (27).
Orthotopic single lung transplantation model
To assure immunogenic standardization and histoincompatibility,
these studies employed inbred, unmodified, specific pathogen-free rats,
Brown Norway (BN; RT1n) and Lewis (LEW;
RT11). Orthotopic rat left lung transplantation
was performed using a modification of the procedure reported by Marck
et al. and Prop et al., with >85% postoperative survival (21, 28). The 15% mortality associated with this procedure was
directly attributable to surgical mortality and usually occurred within
8 h post-transplantation. Neutralization of RANTES in vivo did not
impact on postoperative survival. A minimum of six rats at each time
point or for each manipulation was used. Animals weighed between 200
and 300 g. Preoperative anesthesia consisted of 0.5 mg of atropine
(s.c.); anesthesia was induced with 4% halothane in a mixture of 40%
O2/60% NO2. The animal’s
trachea was intubated with an 18-gauge endotracheal tube. Anesthesia
was maintained throughout the operation using 0.5–1% halothane in
40% O2/60% NO2 mixture
through a Harvard rodent ventilator (Natick, MA). The body temperature
of the animal was maintained using a warming mattress. Intravascular
volume was assured by i.v. infusion of normal saline (3 ml/h) via tail
vein. The operation on the donor animal was performed by removing the
anterior chest wall and infusing 1000 U (USP) of heparin into the right
atrial appendage of the heart and allowing time to circulate. The right
ventricle was then flushed with 50 ml of normal saline after
trans-section of the pulmonary vein, followed by extraction of the left
lung. The operation on the recipient animal was performed via a left
posterior-lateral thoracotomy in the fourth or fifth intercostal space.
The left lung hilum was identified, dissected, and cross-clamped. The
recipient’s left native lung was removed. The reimplantation of the
donor’s lung entailed the successive anastomosis of the following
structures: 1) pulmonary vein and artery were anastomosed with
continuous 10-0 Ethilon, followed by removal of the clamp and
restoration of perfusion; and 2) the bronchus was anastomosed with
interrupted 9–0 Ethilon, followed by reinflation at a maximal pressure
of 30 cm H2O. The allograft/isograft ischemic time was
maintained at <120 min and recorded for each recipient animal. The
thorax was closed over an 18-gauge chest tube, which was removed before
the animal recovered from anesthesia (21, 22, 23, 24, 25).
Postoperatively the animals were kept in an oxygenated cage for the
first 24 h. The animals were allowed to eat and drink ad libitum
through the course of the experiment. At the time of sacrifice, animals
were euthanized with 50 mg of ketamine (i.p.). For a separate
experiment, goat polyclonal Ab to the carboxyl terminus of rat RANTES
was generated using a 14-mer peptide constituting the carboxyl terminus
(KKWVQEYINYLEMS). Polyclonal antiserum against rat RANTES was generated
following s.c. injections of 250 µg of the peptide in CFA, followed
by at least three boosts of 100 µg of the peptide in IFA. Direct
ELISA was used to evaluate antisera titers, and sera were drawn when
titers had reached >1/1,000,000. The polyclonal antiserum against rat
RANTES did not cross-react with any other human and murine chemokines
tested, including MIP-2, MIP-1
, MIP-1ß, MCP-1, inflammatory
protein-10 (IP-10), KC, IL-8, epithelial neutrophil activating
protein-78 (ENA-78), growth-related oncogene 
(GRO), or GRO.
In addition, the antiserum did not cross-react with rodent cytokines
TNF- or IL-1ß. The results of this neutralizing anti-rat
RANTES Ab were similar to those of the anti-human RANTES Ab used in
inhibiting LEW rat adjuvant-induced arthritis (29). The
specificity of the polyclonal Ab was tested using Western blot
analysis. One hundred nanograms of recombinant rat RANTES (PeproTech,
Rocky Hill, NJ) showed a strong band at the appropriate m.w. using a
1/1000 dilution of goat polyclonal anti-rat RANTES. Twenty
milligrams of this anti-rat RANTES has a specific neutralizing
capacity of 10 µg of rat RANTES (PeproTech) by blocking mononuclear
cell infiltration in the peritoneum in response to (i.p.)
administration of rat RANTES. In contrast, 20 mg of this anti-rat
RANTES did not block mononuclear cell infiltration in the peritoneum in
response to 10 µg (i.p.) administration of rat MCP-1 (PeproTech). In
the rat transplant model in vivo neutralization of RANTES was achieved
by inhibiting the biological effect of RANTES by passive immunization
(i.p.). Animals received 20 mg of goat polyclonal IgG anti-rat
RANTES or an equivalent quantity of control goat polyclonal IgG Abs on
days 0, 2, and 4 and were sacrificed on day 6, 8, or 10
post-transplantation.
Histopathology
Ten random 5-µm paraffin-embedded tissue sections for eight different lung allografts were stained with hematoxylin and eosin, and blindly reviewed and graded as previously described (26). Briefly, the histopathology grading of allograft rejection was: A0 = normal, no significant abnormality; A1 = minimal, infrequent perivascular infiltrates; A2 = mild, frequent perivascular infiltrates around arterioles and venules; A3 = moderate, dense perivascular infiltrates with extension into alveolar septae; and A4 = severe, diffuse perivascular, interstitial, and alveolar infiltrates with alveolar pneumocyte damage, potential parenchyma necrosis, infarction, or necrotizing vasculitis.
RANTES, MIP-1, and MIP-1ß ELISAs
RANTES, MIP-1, and MIP-1ß protein were quantitated using a
modification of a double-ligand method as previously described
(30). Briefly, flat-bottom 96-well microtiter plates
(Immuno-Plate I 96-F, Nunc, Naperville, IL) were coated with 50
µl/well of the goat polyclonal anti-murine RANTES, rabbit
anti-murine MIP-1, or rabbit anti-murine MIP-1ß (R&D
Systems, Minneapolis, MN; 1 ng/µl in 0.6 M NaCl, 0.26 M
H3B04, and 0.08 N NaOH, pH
9.6) for 24 h at 4°C and then washed with PBS (pH 7.5) and
0.05% Tween 20 (wash buffer). The specificity of the polyclonal
anti-murine RANTES Ab (R&D Systems) to rat RANTES was tested using
Western blot analysis. One hundred nanograms of recombinant rat RANTES
(PeproTech) showed a strong band at the appropriate m.w. for RANTES
using a 1/200 dilution of goat polyclonal IgG anti-murine RANTES
(R&D Systems). Also, the polyclonal anti-murine RANTES Ab was shown
to have an ELISA standard curve equivalent to those of both rat RANTES
(PeproTech) and murine RANTES (R&D Systems). Microtiter plate
nonspecific binding sites were blocked with 2% BSA in PBS and
incubated for 60 min at 37°C. Plates were rinsed three times with
wash buffer, and undiluted and diluted (1/10) samples (50 µl/well)
were added, followed by incubation for 1 h at 37°C. Plates were
washed three times; 50 µl/well of biotinylated polyclonal goat
anti-murine RANTES Ab, anti-murine MIP-1, or anti-murine
MIP-1ß (R&D Systems; 3.5 ng/µl in PBS (pH 7.5), 0.05% Tween 20,
and 2% FCS) was added; and plates were incubated for 45 min at 37°C.
Plates were washed three times, streptavidin-peroxidase conjugate
(Jackson ImmunoResearch Laboratories, West Grove, PA) was added, and
the plates were incubated for 30 min at 37°C. Plates were washed
three times, and 3,3',5,5'-tetramethylbenzidine chromogen substrate
(Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added. The
plates were incubated at room temperature to the desired extinction,
and the reaction was terminated with 50 µl/well of 3 M
H2SO4 solution. Plates were
read at 450 nm in an automated microplate reader (Bio-Tek Instruments,
Winooski, VT). Standards were 0.5-log dilutions of rat RANTES
(PeproTech), murine RANTES, murine MIP-1, murine MIP-1ß (R&D
Systems) from 100 ng to 1 pg/ml (50 µl/well). This ELISA method
consistently detected specific cytokine concentrations in a linear
fashion >10 pg/ml. All Abs were tested against cross-reactivity with
all other human or murine chemokines. All tissue homogenate specimens
were run in parallel to determine total protein content
(30).
FACS analysis for CCR1, CCR5, and leukocyte surface markers
Before removal, transplanted lungs were perfused with normal saline and dissected free of the thoracic cavity. The transplanted lung was minced and incubated for 1 h in Dispase (Collaborative Biomedical Products, Two-Oak Park, MA). The cell suspension and undigested fragments were further dispersed by drawing them up and down through the bore of a 10-ml syringe. Cells were then pelleted at 600 x g for 10 min, resuspended in sterile water for 30 s to lyse remaining RBCs, washed in 1x PBS, and resuspended in RPMI 1640 (BioWhittaker, Walkersville, MD) with 5% FCS. Cells were counted with a hemocytometer, transferred at a concentration of 5 x 106 cells/ml to fluorescent Ab buffer (Difco, Detroit, MI; 1% FCS, and 0.1% azide), and maintained at 4°C for the remainder of the staining procedure. Single-cell suspensions of 100 µl were stained with Tri-color-conjugated mouse anti-rat CD45 (leukocytes; Caltag, South San Francisco, CA) followed by FITC-conjugated mouse anti-rat CD3 (T cells), CD4 (T cells), CD8 (T cells), CD45RA (naive T cells/B cells), NKR-P1A (NK cells; PharMingen, San Diego, CA), ED1 (mononuclear phagocytes; Seratec, Kidlington, U.K.), rabbit anti-rat PMN (Accurate, Westbury, NY), or goat anti- CCR1 or CCR5 (Santa Cruz Biotechnology, Santa Cruz, CA). Double staining allowed for live gating on CD45-positive cells (leukocytes), thus permitting further analysis of subset populations. FITC-conjugated mouse, rabbit, and goat IgG were used as control Abs. Unbound Ab was washed with 1% FCS, and 0.1% azide, and the cell suspension was analyzed with a flow cytometer (Becton Dickinson, Bedford, MA). The data were expressed as the percentage of cells staining positively, using a modification previously described (31).
Total RNA isolation and RT-PCR amplification
Total cellular RNA from lung tissue was isolated by lysis of cells in a Trizol solution according to the manufacturer’s instructions (Roche, Indianapolis, IN). The RNA was alcohol precipitated, and the pellet was dissolved in diethylpyrocarbonate-water. Total RNA was determined by spectrometric analysis at 260 nm. Five micrograms of total RNA was reversed transcribed into cDNA, and amplification was preformed using the Access RT-PCR kit (Promega, Madison, WI) and specific primers for rodent CCR1, CCR5, or RANTES. The specific primers for the housekeeping gene, ß-actin, was used as an internal control. The primers used were 5'-TTG TAA CCA ACT GGG ACG ATA TGG-3' (sense) and 5'-GAT CTT GAT CTT CAT GGT GCT AGG-3' (antisense) for ß-actin, 5'-GAC CAG CAT CTA CCT GTT CA-3' (sense) and 5'-GCA GAA ACA AAT ACA CTC AG-3' (antisense) for CCR-1, 5'-GCT GAA GAG CGT GAC TGA TA-3' (sense) and 5'-CCC TGT CAA GAG TTG ACA CA-3' (antisense) for CCR-5, and 5'-CCA TAT GGC TCG GAC ACC A-3' (sense) and 5'-GCT CAT CTC CAA ATA GTT G-3' (antisense) for rat RANTES, giving amplified products of 764, 587, 364, and 221 bp, respectively (32). The cDNA was amplified after determining the optimal number of cycles. The mixture was first incubated for 45 min at 48°C and for 2 min at 95°C, followed by cycling 30 times at 95°C for 30 s and 60°C for 1 min, and then elongating at 68°C for 2 min. This format allowed optimal amplification with little or no nonspecific amplification of contaminating DNA, as determined by the absence of bands when RT was excluded. After amplification, the sample (20 µl) was separated on a 2% agarose gel containing 0.3 µg/ml (0.003%) of ethidium bromide, and bands were visualized and photographed using a UV transilluminator (33, 34, 35).
Western blot analysis of RANTES, CCR1, and CCR5
Total protein extracts were made by homogenizing lungs in TNE lysis buffer (20 mM Tris-HCl (pH 8), 150 mM NaCl, 1% Nonidet P-40, and 2.5 mM EDTA) supplemented with 2 ng/ml aprotinin and 35 ng/ml PMSF. Cell extracts were incubated on ice for 30 min, followed by centrifugation at 4°C for 30 min. Supernatants were then removed and assayed for total protein content using bicinchoninic acid protein assay reagents (Pierce, Rockford, IL) and comparison to known amounts of BSA. One hundred twenty micrograms of total protein was loaded in each well of a 12% polyacrylamide gel, and extracts were subjected to SDS-PAGE. The separated proteins were transferred to polyvinylidene fluoride membrane (Pierce) by electrophoretic transfer overnight in Tris-glycine buffer (20 mM Tris, 150 mM glycine (pH 8.0), and methanol added to a final concentration of 20% (v/v)). Blots were blocked in 5% skim milk in TBST buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.05% Tween 20) for 2 h at room temperature, followed by incubation in either primary goat anti-CCR1 (Santa Cruz Biotechnology; 1/100), or goat anti-CCR5 (Santa Cruz Biotechnology; 1/50) in blocking solution for 2 h at room temperature. Blots were washed for three 10-min washes in TBST and were incubated for 1 h at room temperature in rabbit anti-goat HRP-conjugated secondary Ab (Dako, Carpinteria, CA; 1/2000). Blots were again washed for four 10-min washes in TBST, and proteins were visualized after incubation of the blots in SuperSignal chemiluminescent substrate solution according to the manufacturer’s protocol (Pierce) and exposure to XAR-5 film (Eastman Kodak, Rochester, NY). In a separate experiment 100 ng of recombinant rat RANTES (PeproTech) and 100 ng of recombinant murine RANTES (R&D Systems) were loaded in each well of a 12% polyacrylamide gel and subjected to SDS-PAGE. The proteins were transferred to polyvinylidene fluoride membrane (Pierce) by electrophoretic transfer overnight in Tris-glycine buffer (20 mM Tris, 150 mM glycine (pH 8.0), and methanol added to a final concentration of 20% (v/v)). Blots were blocked in 5% skim milk in TBST buffer for 2 h at room temperature, followed by incubation in either primary goat anti-rat RANTES (generated by our laboratory; 1/1000) or primary goat anti-murine RANTES (R&D Systems; 1/200) in blocking solution for 2 h at room temperature. Blots were washed for three 10-min washes in TBST and were incubated for 1 h at room temperature in rabbit anti-goat HRP-conjugated secondary Ab (Dako; 1/2000). Blots were again washed for four 10-min washes in TBST, and proteins were visualized following incubation of the blots in SuperSignal chemiluminescent substrate solution according to the manufacturer’s protocol (Pierce) and exposure to XAR-5 film (Kodak).
Mixed lymphocyte reaction (MLR)
Rat spleens were harvested and placed on ice in RPMI 1640. Spleens were passed through wire mesh, resulting in a single-cell suspension. Red cells were lysed using ammonium chloride lysis buffer. The recovered cells were washed three times with RPMI 1640, counted, and assessed for purity and viability. MLR was set up in 96-well flat-bottom tissue culture plates for proliferative studies and in six-well plates for cytokine analysis. Responder cells were mixed 1/1 with either irradiated (2000 rad) stimulator cells (one-way MLR) or nonirradiated stimulator cells (two-way MLR) in a total volume of 200 µl for 96-well plates and 1 ml for six-well plates. RPMI 1640 supplemented with 1 mM L-glutamine, 10 mM HEPES, antibiotics, and 10% FCS was used in the assay. Dilution studies of both the responder and stimulator cells determined that a 1/1 ratio provided optimal proliferative response. Cells were cultured at 105 cells/well for proliferative responses and 3 x 106 cells/well for cytokine elicitation. For determination of proliferation, cultured cells were pulsed with 0.5 µCi of [3H]thymidine 18 h before harvest on day 6. Cells were harvested, and [3H]thymidine incorporation was determined. For cytokine determination, culture supernatants were harvested from 35-mm plates, centrifuged, and stored at -20°C until cytokine concentrations were assessed. As a control, donor cells stimulated with their own irradiated cells failed to mount a proliferative response or a significant peak in cytokine production (36). In vitro neutralization of rat RANTES during the two-way allogeneic MLRs were performed by administering either anti-rat RANTES Abs at a dilution of 1/1000 or an equivalent quantity of control goat polyclonal Abs on days 0 through 6. In separate ex vivo MLR experiments we determined whether in vivo neutralization of RANTES could reduce allospecific responsiveness to a rechallenge with either second-party irradiated BN splenocytes or third-party irradiated Fischer splenocytes. Responder cells were splenocytes harvested from LEW rats transplanted with a BN lung and then treated in vivo with either anti-rat RANTES or control Abs. Both anti-rat RANTES and control Ab were give on day 0 and every 48 h until animals were sacrificed on day 6, and spleens were harvested. Responder cells were mixed 1/1 with either irradiated first-party self splenocytes, irradiated second-party BN splenocytes, or irradiated third-party Fischer rat splenocytes. Ex vivo MLR proliferation was determined as described above.
Statistical analysis
Data were analyzed on a Macintosh Power PC computer using the
StatView 4.5 statistical package (Abacus Concepts, Berkeley, CA).
Comparisons were evaluated by both the unpaired t test and
nonparametric (Mann-Whitney) analyses for statistical significance.
Data were considered statistically significant at p
0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We determined whether RANTES was present in the lungs of patients
undergoing acute lung allograft rejection. We examined the BALs from
both healthy single lung transplant patients (n = 12)
and single lung transplant patients with clinical, chest
roentgenographic and histologic evidence of acute lung allograft
rejection (n = 12). All patients were on standard
protocol post-transplantation immunosuppressive regimens. Histologic
grading from transbronchial biopsies ranged from A0 to A1 in the
healthy group and from A2 to A3 in the group with clinical acute lung
allograft rejection. There was no evidence of P. carinii,
viral, bacterial, or fungal infections in these patients. RANTES
protein levels in the unconcentrated BALs were 2.3-fold greater in the
human acute lung allograft rejection group than in healthy lung
allograft recipients (Fig. 1
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To determine whether RANTES involvement in human lung
allograft rejection was translational to an animal model we evaluated a
unique model system of orthotopic rat lung transplantation. This rat
model system has been previously studied and determined to be a
reproducible, immunogenetically standardized model of acute lung
allograft rejection with many characteristics of human acute lung
allograft rejection. We determined the full kinetics of RANTES in our
rat model of acute allograft rejection. LEW rats were subjected to lung
transplantation with allografts from BN rats or from syngeneic donors
as previously described (21, 22, 23, 24, 25). Animals were sacrificed
on days 1, 4, and 6 post-transplantation, and lungs were harvested for
isolation of RANTES mRNA by RT-PCR and protein measurement by specific
ELISAs. In lung allografts, RANTES mRNA was barely detectable on day 1;
however, RANTES mRNA expression was detectable by day 4, with maximal
expression occurring on day 6 (Fig. 2I). In contrast, RANTES mRNA
expression was essentially undetectable from syngeneic lungs throughout
the full 6-day time course (Fig. 2I). Similarly, there was a
temporal increase in RANTES protein in lung allografts on days 1, 4,
and 6, whereas RANTES protein levels in syngeneic controls were
significantly lower throughout the same time course (Fig. 2IIA).
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and
MIP-1ß by ELISA. Although protein levels of MIP-1 were
significantly elevated in allografts compared with syngeneic controls
on day 6, the levels of MIP-1 were 170-fold lower than those of
RANTES (Fig. 2
II, A and B). In
contrast, MIP-1ß protein levels were lower in the allografts compared
with those in syngeneic controls (Fig. 2IIC). Expression of RANTES parallels mononuclear cell infiltration during acute lung allograft rejection
The temporal expression of RANTES mRNA and protein in the lung
allografts paralleled mononuclear cell infiltration into the lung
allograft. This was seen qualitatively by histology assessment of the
lung allografts on days 1, 4, and 6 post-transplantation. Day 1
demonstrated only minimal interstitial and alveolar edema, hemorrhage,
and leukocyte infiltration compatible with ischemic-reperfusion injury
(Fig. 3A) (37).
Day 4 demonstrated a significant perivascular and peribronchial
mononuclear cellular infiltration, compatible with grades A1–A2
allograft rejection (Fig. 3B) (2, 26). Day 6
demonstrated enormous perivascular and peribronchial mononuclear cell
infiltration with architectural destruction, compatible with grade A4
acute allograft rejection (Fig. 3C) (2, 26).
The syngeneic controls on day 1 showed the same histology as the
allografts: minimal interstitial and alveolar edema, hemorrhage, and
leukocyte infiltration compatible with ischemic-reperfusion injury
(37). However, day 4 and 6 samples showed essentially
normal lung histology (2, 26). The amount of mononuclear
cell recruitment in both lung allografts and syngeneic controls seen on
histology was quantitated by FACS analysis using double staining to
permit leukocyte subset analysis. The specific cell markers CD4 (T
cells), CD3 (T cells), CD8 (T cells), CD45RA (naive T cell/B cells),
ED1 (mononuclear phagocytes), and NKR-P1A (NK cells) showed a temporal
increase in the number of cells on days 1, 4, and 6 (Fig. 4). In contrast the syngeneic controls
started out with comparable numbers on day 1, then were markedly
reduced throughout the rest of the time course (Fig. 4).
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To determine whether mononuclear cell infiltration was associated
with cells expressing the appropriate receptors for RANTES, we assessed
the expression of CCR1 and CCR5 during acute lung allograft rejection.
LEW rats were subjected to lung transplantation with allografts from BN
rats or from LEW syngeneic donors as previously described
(21, 22, 23, 24, 25). Animals were sacrificed on days 1, 4, and 6
post-transplantation, and lungs were harvested for analysis of CCR1 and
CCR5 expression by RT-PCR, FACS, and Western blot analysis. In lung
allografts, CCR1 and CCR5 mRNA were barely detectable on day 1;
however, both CCR1 and CCR5 mRNA expression were detectable by day 4,
with maximal expression occurring on day 6, the time coincidental with
maximal mononuclear cell infiltration and acute lung allograft
rejection (Fig. 5I). In
contrast, CCR1 mRNA and CCR5 mRNA expression were essentially
undetectable from syngeneic lungs throughout the full 6-day time course
(Fig. 5I). FACS analysis of CCR1 in the lung allografts
showed no significant changes on days 1 and 4, but had a significant
increase of 100% on day 6 (Fig. 5IIIA). FACS analysis of
CCR5 in the lung allografts showed 170, 350, and 160% increases
compared with syngeneic controls on days 1, 4, and 6, respectively
(Fig. 5IIIB). Western blot analysis of CCR1 and CCR5 protein
in the lung allograft homogenates showed a temporal increase on days 1,
4, and 6 (Fig. 5II, A and B). In
contrast, these receptors showed minimal to moderate amounts of protein
on day 1 and then were virtually undetectable on days 4 and 6 in
syngeneic controls (Fig. 5II, A and
B).
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Because the above studies demonstrated that the production of
RANTES correlated with mononuclear cell infiltration and cells
expressing CCR1 and CCR5, we next assessed whether neutralization of
RANTES would inhibit extravasation of mononuclear cells expressing
these receptors during acute lung allograft rejection. The specificity
of the polyclonal anti-rat RANTES Abs generated in our laboratories
were first assessed and compared with those of commercially available
polyclonal anti-murine RANTES Abs using a Western blot analysis.
Both polyclonal Abs specifically detected 100 ng of recombinant rat
RANTES and murine RANTES (Fig. 6) and did
not cross-react with other murine or rat recombinant chemokines
MIP-1, MIP-1ß, MCP-1, C10, IP-10, MIG, KC, and MIP-2. In addition,
i.p. injection of 20 mg of purified anti-rat RANTES was found to
specifically neutralize rat RANTES (10 µg i.p., concomitant with
neutralizing Abs)-mediated mononuclear cell infiltration in the
peritoneum (Fig. 7). This effect was
specific, as these Abs did not inhibit 10 µg of recombinant rat
MCP-1-induced mononuclear infiltration in the peritoneum (Fig. 7).
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I) as well as FACS
analysis of specific leukocyte surface markers (Fig. 8II)
and of CCR1 and CCR5 (Fig. 8III). Treatment with
anti-rat RANTES Abs demonstrated a significant reduction in acute
lung allograft rejection compared with control Ab-treated animals (Fig. 8I, A, C, and E). The lung
allografts on days 6, 8, and 10 treated with control Ab all
demonstrated a histological rejection grade of A4 (Fig. 8I,
B, D, and F). There was an obvious
temporal increase in perivascular, interstitial, and alveolar
infiltrates throughout this time course in both treated groups.
However, the lung allografts on day 6 treated with neutralizing
anti-rat RANTES Abs showed a dramatic decrease in rejection, with a
histological grade of A1–A3, mild perivascular infiltration, and
occasional extension into the alveolar interstitium (Fig. 8IA). Day 8 and 10 lung allografts treated with neutralizing
anti-rat RANTES Abs also showed markedly reduced histological
evidence of rejection compared with their appropriate controls (Fig. 8, C and E vs D and F,
respectively). We determined the presence of leukocyte subset
infiltration during acute lung allograft rejection by FACS analysis.
Recipient animals treated with neutralizing anti-rat RANTES Abs
demonstrated a significant reduction of infiltrating mononuclear cells
expressing CD3, CD4, CD8, ED1, CD45RA, and NK cell markers at all three
time points (Fig. 8II). Interestingly, FACS analysis of the
lung allografts from anti-rat RANTES-treated recipients showed no
significant change in neutrophil accumulation compared with control
Ab-treated animals at all three time points (Fig. 8II). This
reduction of infiltrating mononuclear cells correlated with the
significant reductions of cells expressing CCR1 and CCR5 on days 6, 8,
and 10 post-transplantation (Fig. 8III).
|
). In contrast,
RANTES levels in the syngeneic two-way MLRs did not increase on day 4
and only increased minimally on day 6 (Fig. 9). We then determined with
in vitro studies whether RANTES had any effect on mononuclear cell
proliferation. We performed in vitro neutralization studies of RANTES
with either neutralizing anti-rat RANTES Abs or an equivalent
quantity of control Abs on days 0–6 in the two-way allogeneic MLRs. We
found no significant difference in the proliferative response to
alloantigens on day 6 between the anti-rat RANTES Ab- and control
Ab-treated MLRs (Table I).
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). In contrast, splenocytes isolated
from transplanted Lewis rats treated in vivo with control Abs
demonstrated a significant proliferative response when challenged with
irradiated second-party splenocytes (Fig. 10). The proliferative
response was normalized to the proliferative response of the syngeneic
control (LEW:LEW splenocytes). Furthermore, splenocytes isolated from
transplanted LEW rats treated with either anti-rat RANTES or
control Abs showed no significant difference in proliferative responses
when stimulated with irradiated third-party splenocytes (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study we first assessed whether RANTES was associated with
human acute lung allograft rejection. Similar to previous
investigators, we found a 2.3-fold increase in RANTES protein levels in
BALs from lung transplant recipients with acute rejection compared with
healthy lung allograft recipients (39). To determine proof
of the principle that RANTES was contributing to acute allograft
rejection, we employed a well-characterized model of orthotopic rat
lung transplantation (21, 22, 23, 24, 25). In our rat lung transplant
model we measured levels of three CC chemokines; RANTES, MIP-1, and
MIP-1ß. RANTES and MIP-1 were both significantly elevated in lung
allografts compared with syngeneic controls, with RANTES protein levels
at least 170-fold greater than MIP-1 levels. This correlates with
the LEW rat adjuvant-induced arthritis model in which RANTES levels
were greater than MIP-1 levels in both blood and synovial joints,
and neutralization of MIP-1 did not ameliorate symptoms
(29). Interestingly, MIP-1ß protein levels, in contrast
to either RANTES or MIP-1 levels, were lower in the lung allografts
compared with the syngeneic controls. In light of the markedly elevated
levels of RANTES during lung allograft rejection, and previous studies
showing that RANTES is 10 times more potent than MIP-1 for inducing
T cell migration (8), we studied the full kinetics of
RANTES during allograft rejection.
We found that rat lung allografts expressed RANTES mRNA and protein levels in a time-dependent manner, with maximal expression and levels occurring on day 6 coincidental with maximal lung allograft rejection. We then wanted to investigate whether there was a correlation with mononuclear cell recruitment and expression of the RANTES receptors. RANTES has the ability to bind with multiple receptors (CCR1, CCR3, CCR5, CCR9, and DARC). The amount of expression of CCR3 on mononuclear cells remains controversial, although CCR3 is known to be predominately expressed on eosinophils and basophils. Although RANTES has been shown to bind to CCR9, it does not initiate a Ca2+ flux, and it remains unclear whether RANTES interaction with CCR9 leads to biologically relevant chemotaxis (17, 19, 20, 40). CCR4 was initially thought to bind RANTES, but latter studies have shown that its major ligands are macrophage-derived chemokine (MDC) and thymus and activation-regulated chemokine (TARC) (13, 18, 41). Therefore, we focused our studies on the kinetics of CCR1 and CCR5 expression, because they have previously been shown to be the two major RANTES receptors for mononuclear cell chemotaxis (11, 14, 15, 17, 19, 20). The temporal expression of RANTES mRNA and protein in the rat lung allografts correlated with the mononuclear cell recruitment and expression of RANTES receptors CCR1 and CCR5. In contrast, the mRNA and protein levels of RANTES and its receptors, expressed by the syngeneic controls, were undetectable or decreased throughout the 6-day time course. The early elevation of CCR1 and CCR5 mRNA and protein by Western blot analysis in the syngeneic controls most likely represents ischemic-reperfusion injury, in which there is recruitment, activation, and emigration of neutrophils, which are known to express CCR1 and CCR5 (37, 42). The increase in the amount of CCR1 protein determined by Western blot analysis on days 1 and 4 compared with the amount of CCR1 protein in lung allografts determined by FACS analysis may be due to different strategies used for analyzing these receptors and different pools of receptors (i.e., cell surface vs internalization) (43). We speculate that the discrepancies of the results using different strategies for analysis of CCR expression is related to the fact that Western blot analysis is detecting both cell surface-expressed and internalized receptor protein, whereas FACS analysis is detecting predominating cell surface expression. For example, our results for the kinetics of CCR1 by Western blot and FACS analyses suggest that CCR1 protein expression during allograft rejection may be undergoing significant dynamic changes related to both internalization and cell surface expression. Alternatively, we cannot completely exclude that cells other than mononuclear cells and neutrophils (i.e., stromal or parenchymal cells) during acute lung allograft rejection may express CCR1 that was detected by Western blot analysis and not by FACS analysis.
The association between RANTES and acute lung allograft rejection described here is supported by other studies of solid organ allograft rejection (32, 44, 45, 46, 47). RANTES mRNA was detected in infiltrating mononuclear cells and renal tubular epithelium during acute renal allograft rejection, suggesting that RANTES has a role in human allograft rejection (47). In addition, Kondo examined RANTES mRNA expression using three histoincompatible models of allogeneic skin grafts in mice (44). RANTES mRNA was found to coincide with maximal allograft rejection (infiltration of mononuclear cells) in all three skin allograft models on day 7 (44). These results are similar to ours and demonstrate an important relationship between augmented expression of RANTES and mononuclear cell infiltration. In a rat acute renal allograft rejection model, RANTES mRNA expression displayed a bimodal pattern (46). The first peak occurred at 6 h after engraftment in association with ischemia-reperfusion of the allograft, and the second peak in expression occurred between 3 and 6 days postreimplantation in conjunction with acute renal allograft rejection (46). Infiltrating macrophages and CD4 and CD8 cells were found to peak in the renal allografts by day 5 in parallel with maximal RANTES expression (46). These results paralleled our findings of increased RANTES expression during acute allograft rejection coinciding with increased mononuclear infiltration and allograft rejection.
To prove that RANTES and its receptors are important in the mediation
of acute lung allograft rejection, we determined whether depletion of
endogenous RANTES would attenuate acute lung allograft rejection and
the expression of RANTES receptors on infiltrating mononuclear cells.
Treatment with anti-rat RANTES Abs delayed the recruitment of
mononuclear cells involved in acute rejection. This was demonstrated by
histopathology and FACS analysis demonstrating a significant reduction
in mononuclear cells on days 6, 8, and 10. Similar results were
obtained with F(ab')2 neutralizing
anti-RANTES Abs (data not shown). Concomitant with the marked
reduction in mononuclear cell recruitment was a significant reduction
in cells expressing CCR1 and CCR5. The specificity of RANTES for
mononuclear cell recruitment was exemplified by minimal changes in the
presence of neutrophils in rats treated with neutralizing Abs. Although
we did not perform physiological function testing on the lungs during
allograft rejection in the presence or the absence of neutralization of
RANTES, it has been established that physiological measurements (i.e.,
forced expiratory volume at 1 s and forced vital capacity) decline in
direct correlation to the magnitude of acute lung allograft rejection
(i.e., mononuclear cell infiltration) (48). Based on this
information, one can assume that with evidence of attenuated lung
allograft rejection in the context of neutralization of RANTES, lung
physiological function would be less impaired. Interestingly, although
neutralization of RANTES resulted in significant attenuation of acute
allograft rejection, there was still a presence of perivascular,
alveolar, and interstitial leukocytes at later time points. This
suggests that other CC chemokines, CXC chemokines, or other factors may
be either synergistically or independently involved in acute lung
allograft rejection. The attenuation of mononuclear cell recruitment by
in vivo neutralization of RANTES described here is supported by other
studies of disease models of inflammation that have responded to
anti-RANTES Abs (29, 49). In vivo neutralization of
RANTES was shown to significantly decrease the pulmonary influx of
macrophages without an alteration in the number of neutrophils in a
murine model of endotoxemia (49). These findings suggest
the involvement of RANTES in facilitating the accumulation of
macrophages in the lung during allograft rejection. Furthermore, in a
rat model of arthritis, RANTES levels were increased in blood and
synovial joints, and in vivo neutralization of RANTES markedly reduced
arthritis scores, joint radiographic scores, and synovial joint
leukocyte infiltration (29). This emphasizes the pivotal
role of RANTES in mononuclear cell recruitment in two distinct
diseases. Moreover, treatment with a RANTES antagonist reduced
mononuclear cell infiltration as well tubular and vascular damage in a
rat renal transplantation model (50). Also, low dose
RANTES antagonist in conjunction with low dose cyclosporin reduced
inflammatory events compared with the effect of low dose cyclosporin
alone (50). This supports our findings that in vivo
neutralization of RANTES significantly attenuates lung allograft
rejection. Lastly, in vivo neutralization of TNF- in a rat model of
acute lung allograft rejection was shown to attenuate leukocyte
infiltration during allograft rejection (23). This is
consistent with our findings, because TNF- is known to stimulate the
expression of RANTES from a number of cell types (23, 51, 52, 53).
We further confirmed that RANTES was a CC chemokine ligand produced in response to alloantigen by one- and two-way allogeneic MLRs. RANTES protein levels increased in a temporal manner, with maximal levels on day 6. In contrast, there was only a modest elevation of RANTES protein levels on day 6 in the syngeneic two-way MLRs. Interestingly, neutralizing anti-RANTES Abs did not alter the proliferative response of the MLRs. This suggests that the production of RANTES during an alloreactive response is necessary for subsequent recruitment of additional mononuclear cells independent of proliferation. Although previous investigators have shown that RANTES may cause the proliferation of T cells at a concentration of 1 µM (54), we have found that at the concentrations generated during our MLRs or in vivo during lung allograft rejection, there was no alteration of the proliferative response when RANTES was depleted by neutralizing Abs.
Previous work has shown a decreased secretion of RANTES coinciding with Ag-specific anergy and superantigen-induced anergy (38). We therefore performed ex vivo experiments to determine whether the reduction of allograft rejection under conditions of endogenous RANTES depletion was at all related to altered allospecific responsiveness (38). With ex vivo one-way MLRs, we found a significant reduction in the proliferative response to second-party irradiated BN splenocyte stimulator cells when responder cells were splenocytes from transplanted LEW rats treated in vivo with anti-rat RANTES Abs, compared with control Abs. In contrast, ex vivo one-way MLRs showed no significant difference in the proliferative response to third-party irradiated Fisher splenocyte stimulator cells when responder cells were splenocytes from transplanted Lewis rats treated in vivo with either anti-rat RANTES Abs or control Abs. These findings suggest that depletion of endogenous RANTES during lung allograft rejection leads to a reduced allospecific response. However, using an in vitro two-way MLR assay of native LEW and BN splenocytes, we have determined that inhibition of RANTES during the allospecific response does not reduce proliferation. Together, these findings suggest that endogenous depletion of RANTES in vivo during acute lung allograft rejection reduces allospecific responsiveness, perhaps through a mechanism of decreased recruitment of alloresponsive mononuclear cells. This is supported by studies that have found monocytes to represent precursor cells that can differentiate into immature dendritic cells (55, 56, 57). These monocyte-derived immature dendritic cells express CCR1 and CCR5 (57, 58). Decreasing recruitment of mononuclear cells, including immature dendritic cells, will decrease initiation and sustained Ag sampling, thereby decreasing and delaying full activation and priming of effector and memory cells. Furthermore, attenuating the recruitment of these immature dendritic cells may decrease alloantigen presentation and reduce dendritic cell migration to regional lymph nodes, thereby delaying an allospecific response (57, 59).
In conclusion, we have demonstrated that RANTES and its receptors, CCR1 and CCR5, play a major role in mediating mononuclear cell infiltration during acute lung allograft rejection. This study of RANTES and future studies of other CC and CXC chemokines will lead to the development of new paradigms to understand the pathogenesis of acute lung allograft rejection and its relationship to chronic lung allograft rejection. The findings in this study may ultimately result in novel therapies designed for intervention and possible prevention of both acute and chronic lung allograft rejections.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert M. Strieter, Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of California School of Medicine, Room 14-154 WH, Box 711922, 900 Veteran Avenue, Los Angeles, CA 90095-1690.
3 Abbreviations used in this paper: BAL, bronchoalveolar lavage; LEW, Lewis rat; BN, Brown Norway; MIP, macrophage inflammatory protein; MCP, monocyte chemoattractant protein.
Received for publication October 25, 1999. Accepted for publication April 3, 2000.
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