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Prepublished online as a Blood First Edition Paper on January 2, 2003; DOI 10.1182/blood-2002-08-2465.
TRANSPLANTATION
From the Department of Pediatrics, Division of
Hematology-Oncology, Blood and Marrow Transplant Program, University of
Minnesota, Minneapolis; the Department of Physiology, University of
Minnesota, Minneapolis; and the Lineberger Comprehensive Cancer Center,
University of North Carolina, Chapel Hill.
Idiopathic pneumonia syndrome (IPS) is a significant
cause of morbidity and mortality after bone marrow transplantation
(BMT) in humans. We developed a murine IPS model in which lethal
pre-BMT conditioning and allogeneic T cells results in the recruitment of host monocytes and then donor T cells into the lung by day 7 after
BMT, concomitant with development of severe lung dysfunction. We
reported the T cell-dependent production of the T
cell-attracting chemokine macrophage inflammatory protein-1 Idiopathic pneumonia syndrome (IPS) remains a
major complication after bone marrow transplantation (BMT) and is a
significant cause of morbidity and mortality.1 Risk
factors for developing IPS are related to the intensity of the
conditioning regimen used and the degree of alloreactivity of the donor
graft.2 We have characterized a murine model of IPS caused
by the influx of host monocytes and donor T cells into the lungs of
lethally irradiated mice early after allogeneic BMT.3
Intensifying the pre-BMT conditioning with cyclophosphamide potentiates
the development of IPS. Lung dysfunction in our model presents as
reduced specific compliance, decreased total lung capacity, and
increased wet and dry lung weights. Histologically, IPS is associated
with injured alveolar type II (ATII) cells and increased frequencies of
cells expressing B7 ligands that are costimulatory for T
cells and cells expressing granzyme B, indicative of cytotoxic
T-lymphocyte function.3,4 Bronchoalveolar lavage (BAL)
fluid of mice with IPS contains elevated levels of inflammatory
cytokines as well as increased levels of nitrite, lactate
dehydrogenase, and protein, indicative of lung injury.5
We reported that monocyte- and T cell-attracting chemokines are
produced in the lung during the generation of IPS in our
model.6 Macrophage inflammatory protein-1 Since both CD4+ and CD8+ donor T cells
infiltrate the lung during the generation of IPS,3,4 and
the increase in MIP-1 Mice
Pre-BMT treatment and conditioning
BMT Our BMT protocol has been described previously.12 Briefly, donor C57BL/6 BM was T cell-depleted (TCD) with anti-Thy 1.2 monoclonal antibody (mAb; clone 30-H-12, rat IgG2b, kindly provided by Dr David Sachs, Charlestown, MA) plus complement (Nieffenegger, Woodland, CA). Wild-type BM was given to all mice to avoid problems in interpretation of outcome due to the qualitative difference of MIP-1 in supporting hematopoiesis.13,14
Recipient mice received transplants via the caudal vein with
20 × 106 TCD C57BL/6 (H2b) marrow
with or without 15 × 106 natural killer (NK)
cell-depleted (PK136, anti-NK1.1 + complement) spleen cells (BMS)
from either MIP-1 / or wild-type
(MIP-1+/+) C57BL/6 mice (GFP Tg or non-Tg) as a source
of IPS-causing T cells. The cellular composition of the
MIP-1/ and MIP-1+/+ spleen cell
inocula did not differ as assessed by flow cytometry (CD4, CD8, CD19, CD11b).
Lung weights Mice were humanely killed with sodium pentobarbitol and the thoracic cavity partially dissected. Lungs were exsanguinated by perfusion with 1.0 mL saline via the right ventricle of the heart. To maximize use of mice, the right lung (bilobed) was used for weight determinations while the left lobe was processed for histopathology (see "Frozen tissue preparation"). For each mouse, the wet weight was taken immediately after the right lung was removed from the thorax. Lungs were dried overnight to a constant weight at 80°C followed by determination of dry weight. The wet-dry weight ratio was calculated and taken as a measure of the severity of lung injury.15 No correction for extravascular blood content was used in the calculations.Pressure-volume curves Following full heart-lung excision, the lungs were suspended via the trachea and kept moist with saline. Pressure-volume (P-V) curves of air- and liquid-filled lungs were determined as previously described.3 Air was delivered into the lungs via a tracheal cannula in 0.05-mL increments with a syringe while intratracheal pressure was measured with a transducer until 25 to 30 cm H2O pressure was reached (total lung capacity). Air was then withdrawn in 0.05-mL increments until pressure was atmospheric. This was repeated 3 times and data procured from the third series. Specific lung compliance was calculated from the slope of the third deflation P-V curve from points flanking 5 cm H2O pressure (which is considered normal breathing range) as follows: ( Volume / Pressure)/Av volume, where volume is in milliliters,
pressure is in centimeters of H20, and Av volume is average
volume over the pressure range used to generate the slope of the P-V
curve (ie, Volume / Pressure).
BAL Mice were humanely killed with sodium pentobarbitol and the thoracic cavity partially dissected. The trachea was cannulated with a 19-gauge needle and infused with 0.5 mL PBS and withdrawn. This was repeated 2 more times and a total of 1.5 mL BAL fluid was collected per mouse. BAL fluid was centrifuged at 4°C for 10 minutes at 1000g to pellet the cells and stored at 80°C.
Lung protein extracts After exsanguination and BAL, the left lung was homogenized in 1 mL PBS containing protease inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN) and centrifuged at 3000 rpm for 10 minutes. The supernatant was filtered through a 1.2-µm syringe filter and stored at80°C.
Serum collection Blood was collected by cardiac puncture, placed immediately at 4°C, the serum separated at 4°C, and stored at80°C.
Chemokine/cytokine level determination Serum, BAL, and lung protein extract levels of monocyte attractants MCP-1 (CCL2) and MCP-3 (CCL7); T-cell attractants MIP-1 (CCL3), MIP-1 (CCL4), RANTES (CCL5), eotaxin (CCL11),
lymphotactin (XCL1), C10 (CCL6), exodus-2 (CCL21), and IP-10 (CXCL10);
neutrophil attractants KC (CXCL1) and MIP-2 (CXCL2); proinflammatory T
helper cell type 1 (Th1)-type cytokines interferon- (IFN-),
tumor necrosis factor-alpha (TNF-), interleukin-1 (IL-1), and
IL-6; and anti-inflammatory Th2-type cytokines IL-13 and IL-10 were
determined by sandwich enzyme-linked immunosorbent assay
(ELISA) using mouse-specific commercial kits (R&D Systems, Minneapolis,
MN; sensitivity 1.5 to 3 pg/mL) or by sandwich ELISA (sensitivity 1 pg/mL) empirically developed using specific monoclonal antibodies and
results interpolated from standard curves of the relevant recombinant
proteins (R&D Systems).
In vivo imaging Mice receiving GFP Tg MIP-1+/+ or
MIP-/ spleen cells were humanely killed with sodium
pentobarbitol, a midline incision was made, and the ribcage was opened.
Whole images of internal organs (lungs, spleen, and bronchus-associated
lymphoid tissue [BALT] are shown) were taken with a Magnafire color
camera (Optronics, Goleta, CA) mounted onto a Leica MZFLIII
stereomicroscope (1.9-second exposures) using a GFP-bandpass filter.
Imaging was done on days 1, 3, 5, and 7 after BMT. Mice receiving BM
only (MIP-1+/+, non-GFP) served as controls for
background autofluorescence in the green channel.
Frozen tissue preparation The thoracic cavity was partially dissected and a mixture of 0.5 mL Optimal Cutting Temperature (OCT) compound (Miles, Elkhart, IN) and PBS (3:1) was infused via the trachea into the lungs. Lung tissue, as well as liver, colon, and skin tissue, was taken on days 3 and 7 after BMT, embedded in OCT, snap-frozen in liquid nitrogen, and stored at80°C.
Histologic assessment Cryosections (6 µm) were acetone-fixed (5 minutes at room temperature), stained by hematoxylin and eosin, and tissues were assessed for GVHD on a scale of 0 to 4+ by a scoring system previously described.16Immunohistochemistry Following fixation in acetone, cryosections (6 µm) were immunoperoxidase-stained using biotinylated mAbs essentially as described17 using avidin-biotin blocking reagents, ABC-peroxidase conjugate, and DAB chromogenic substrate purchased from Vector Laboratories (Burlingame, CA). The biotinylated mAbs used were as follows: anti-CD4 (clone GK1.5), anti-CD8 (clone 2.43), anti-Mac-1 (CD11b, clone M1/70), and anti-Gr-1 (clone RB6-8C5), all purchased from Pharmingen (San Diego, CA). The number of positive cells in the lung was quantitated as the percentage of nucleated cells under × 200 magnification (× 20 objective lens). Four fields per lung were evaluated.In situ hybridization (ISH) Cryosections (6 µm) were hybridized with digoxigenin-labeled antisense RNA probes.18 The corresponding ribonucleotide sequences used were 80-910 for granzyme A and 239-775 for granzyme B. Immunological detection of digoxigenin-labeled RNA duplexes was accomplished with antidigoxigenin antibody (alkaline-phosphatase conjugated; Boehringer Mannheim) and NBT/BCIP (nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate) substrate.Flow cytometry Phenotyping of splenocytes and BAL cells was evaluated on days 3, 5, and 7 by quantitation of donor cells using biotin-labeled anti-H2k mAb (clone 11-4.1) and host cells using phycoerythrin (PE)-labeled anti-H2b (clone EH144). The T-cell composition was determined using fluorochrome-labeled mAb (fluorescein isothiocyanate [FITC] or PE) directed to CD3 (clone 1452C11), CD4 (clone GK1.5), and CD8 (clone 53-6.72). Flow cytometry was done on a FACSCalibur (BD Biosciences, Mountain View, CA) with 10 000 events analyzed (determined by forward and side scatter).Statistical analysis Survival data were analyzed by life-table methods using the Mantel-Peto-Cox summary of 2. Other data were analyzed
by analysis of variance (ANOVA) or Student t test.
Probability (P) values less than or equal to .05 were
considered statistically significant.
Acceleration of GVHD-mediated mortality in the absence of donor
MIP-1 / splenocytes had the
capacity to induce GVHD that is associated with IPS in a fully
allogeneic setting, cyclophosphamide (Cy)/TBI-conditioned
B10.BR mice received transplants of C57BL/6 BM plus either
MIP-1+/+ or MIP-1/ splenocytes.
Wild-type BM was given to all mice to avoid problems in interpretation
of outcome due to the qualitative difference of MIP-1 in supporting
hematopoiesis.13,14 Recipients of
MIP-1/ splenocytes had accelerated mortality
compared with recipients of MIP-1+/+ cells (Figure
1; P < .0007).
Post-BMT lung dysfunction occurs early in recipients of
MIP-1 ,
Cy/TBI-conditioned B10.BR mice received transplants of C57BL/6 BM plus
either MIP-1+/+ or MIP-1/
splenocytes. We measured wet and dry lung weights (reflecting lung
injury due to edema and inflammation), total lung capacity, and
specific compliance. On day 10 after BMT, the wet and dry lung weights
and wet-dry weight ratios of recipients of MIP-1/
cells did not differ from those of recipients of wild-type cells (Table
1). This indicates that giving mice
MIP-1/ cells did not diminish the lung injury
capacity of the allogeneic cells, leading to barrier disruption and
edema. Because recipients of MIP-1/ cells exhibited
accelerated mortality, we questioned whether specific compliance and
total lung capacity were also compromised earlier in such recipients.
Recipients of MIP-1/ cells had significantly lower
specific compliance as early as day 3 after BMT (Figure
2A), but total lung capacity had not yet been affected (Figure 2B), indicating that the inflammatory process compromised the elasticity of the lung before affecting the total lung
volume. By day 7 after BMT, specific compliance and total lung capacity
were compromised to the same degree in recipients of
MIP-1/ cells as in recipients of
MIP-1+/+ cells.
Acceleration of donor T-cell expansion in recipients of
MIP-1 / spleen cells
had accelerated mortality and decreased specific compliance in the
lungs (both are allogeneic T cell-dependent outcomes in this mouse
model), we wanted to know whether absence of MIP-1 affects donor
cell expansion after BMT. We used GFP-Tg spleen cells from
MIP-1+/+ and MIP-1/ donor mice and
imaged the migration/expansion of these GFP-Tg cells in allogeneic and
syngeneic recipients on days 1, 2, 3, 5, and 7 after BMT, using in vivo
whole body imaging under a stereomicroscope equipped with a color
camera and a GFP-bandpass filter. By day 1 after transplantation,
GFP+ cells were easily seen in lung, bronchial-associated
lymphoid tissue (BALT), and spleen in all allogeneic and syngeneic
recipients of GFP-Tg cells (Figure 3A).
By day 3 after BMT, the presence of GFP+ cells was
increased in the lung and spleen of all allogeneic recipients of GFP-Tg
splenocytes (Figure 3B). In contrast, such increases were limited in
syngeneic recipients at this time point and had dissipated considerably
compared with day 1 (compare Figure 3B with 3A). Furthermore,
recipients of allogeneic MIP-1/ cells, compared with
recipients of MIP-1+/+ cells, exhibited accelerated
migration/expansion of GFP-Tg cells in the lymphoid tissues (BALT and
spleen) and the lungs, and these differences were evident by day 3 after BMT (Figure 3B). By day 7 after BMT, GFP-Tg cells were present in
great abundance in these tissues in allogeneic recipients. However,
differences between recipients of MIP-1/ splenocytes
and recipients of MIP-1+/+ splenocytes were more
difficult to discern (not shown).
We assessed the kinetics of donor CD4+ and CD8+
T-cell populations in the spleen on days 3, 5, and 7 after BMT by flow
cytometry. As Figure 4 shows, recipients
of MIP-1
Acceleration of T-cell influx into the lungs of recipients of
MIP-1 / splenocytes
exhibited decreased specific compliance in the lung earlier than
recipients of MIP-1+/+ cells, the lungs were examined by
immunohistochemistry to determine whether inflammatory cell influx into
the lungs had been affected. As Figure 5
shows, compared with the MIP-1+/+ recipients, mice
receiving MIP-1/ cells had increased percentages of
CD4+ and CD8+ T cells in their lungs by day 3 after BMT, consistent with the decreased specific compliance present in
this group at this time point (and our in vivo findings, shown in
Figure 3). Mice receiving MIP-1/ cells also had
accelerated influx of cells expressing mRNA for the cytolytic proteins
granzymes A and B, as assessed by ISH (Figure 6; granzyme B shown). In contrast, the
frequencies of Mac-1- or MHC class II-positive cells did not differ,
indicating that the monocyte/antigen-presenting cell (APC)
influx had not been affected. On day 7 after BMT, there were no
differences between the 2 groups of recipients in the percentages of
donor T cells in the lungs, consistent with the lack of differences in
lung function parameters at this time point (Figure 5).
Recipients of MIP-1 / splenocytes after BMT, we sought to
determine whether this was associated with accelerated production of
other chemokines known to attract T cells. In contrast to what we had
anticipated, we found that the lungs of MIP-1/
recipients (BAL fluid [BALF] and parenchyma) had
decreased levels of several T cell-attracting chemokines,
specifically MIP-1, RANTES, and lymphotactin, on day 7 after BMT
(MIP-1 and RANTES levels are shown in Figure
7; lymphotactin levels were 416 ± 148 vs 243 ± 155 pg/mL, mean ± SD for MIP-1+/+ vs
MIP-1/ groups; P < .05). Similar
decreases in these chemokines were seen in the serum (data not shown).
As expected, the level of MIP-1 was undetectable in the lungs of
MIP-1/ recipients, indicating that donor, not host,
cells are the source of MIP-1 in this model. To determine whether
the decrease in MIP-1 may be playing a role in our IPS model, we
injected either recombinant murine MIP-1 (1 ng per mouse injected
intraperitoneally daily for 7 days; R&D Systems) or goat-antimouse
MIP-1-neutralizing antibody (10 ng per mouse injected
intraperitoneally daily for 7 days; R&D Systems) into allogeneic BMT
recipients of MIP-1+/+ or MIP-1/
spleen cells. These injections had no effect on any of the parameters we studied, despite the attainment of systemic levels of MIP-1 in
recipients of MIP-1/ cells equivalent to those of
recipients of MIP-1+/+ cells (data not shown). These
data imply that in our murine IPS model, chemokines may be serving
other functions in addition to those involving attraction of T cells.
Lower levels of the anti-inflammatory cytokine IL-13 in
MIP-1 / cells, we wanted to determine whether
cytokine levels had been affected in ways that may provide clues as to
the mechanism of the accelerated lung injury. We analyzed serum, lung
tissue extracts, and BALF for several Th1 (predominantly
inflammatory) and Th2 (predominantly anti-inflammatory) cytokines. Of
an extensive panel of cytokines examined (see "Materials and
methods"), none were elevated in recipients of
MIP-1/ cells compared with recipients of
MIP-1+/+ cells. But the anti-inflammatory cytokine IL-13
was decreased in the serum of these mice (95.2 ± 12.3 vs
165.9 ± 26.3 pg/mL, mean ± SD for MIP-1/ vs
MIP-1+/+ groups; Figure 8)
as well as in lung tissue extracts (0.8 ± 1.0 vs 3.8 ± 0.9 pg/mL,
mean ± SD for MIP-1/ vs
MIP-1+/+ groups; P = .04). Therefore, this
shift away from an anti-inflammatory milieu may contribute to the
accelerated lung injury.
We have demonstrated that deficiency of donor-derived MIP-1 On the basis of previous data on the potential functions of chemokines
in lung injury, both from our group using BMT models6,9,10 and from others using bleomycin-induced injury,19 we
reasoned that mice receiving MIP-1 The decrease in systemic IL-13 levels in MIP-1 Our current results demonstrating the accelerated expansion of donor T
cells in recipients of MIP-1 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(CCL3) after allogeneic BMT in
mice
Top
Abstract
Introduction
)
plus either MIP-1
) or MIP-1
) splenocytes. Recipients of MIP-1
