University of Groningen
Airway epithelium in obliterative airway disease
Qu, Ning
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Chapter 2
Integrity of Airway Epithelium is Essential
against Obliterative Airway Disease in
Transplanted Rat Tracheas
Ning Qu; Paul de Vos; Maaike Schelfhorst; Aalzen de Haan;
Wim Timens; Jochum Prop
Journal of Heart and Lung Transplantation. 2005, Aug.
CHAPTER 2
22
Integrity of airway epithelium is essential
Abstract
Background
The pathogenesis of obliterative bronchiolitis following lung transplantation
requires further elucidation. In this study we used the rat trachea
transplantation to examine the role of epithelium in the progression of
obliterative airway disease.
Methods
Normal and denuded (i.e., epithelium removed) trachea grafts from Lewis
(LEW) and Brown Norway (BN) rats were transplanted subcutaneously into
LEW rats. Viable trachea epithelial cells (to recover epithelium) were seeded
into the lumen of some of the denuded tracheas. Grafts were removed at
different time points between 2 days to 8 weeks after transplantation.
Histological analysis was performed to evaluate the cellular infiltration of
inflammatory cells, loss of epithelium, and obliteration of trachea lumen.
Results
Obliteration was found to occur in trachea transplants after loss of epithelium,
caused by rejection in allografts or by enzymatic denudation in isografts. In
these situations, fibroblasts started to proliferate and to migrate into the lumen
in the second week after transplantation. Obliteration could be prevented
when the epithelial integrity was restored by seeding epithelial cells; no
obliteration occurred when denuded trachea isografts were seeded with
epithelial cells, whereas non-seeded denuded tracheas were obliterated at
day 6 after transplantation.
Conclusions
We conclude that integrity of airway epithelium is essential for rat trachea
transplants to be safeguarded from obliterative airway disease. For clinical
lung transplantation the results of our study suggest that protection of the
integrity of airway epithelium may be important in prevention of the
development of obliterative bronchiolitis.
23
CHAPTER 2
Introduction
Clinical lung transplantation is associated with obliterative bronchiolitis (OB) in
50% of the transplanted patients, resulting in graft failure and high mortality of
the recipients (1). Up to now, observations in human lung allograft recipients
indicate that OB is an immune-mediated process characterized by infiltration
of inflammatory cells, fibroproliferation, and in severe cases occlusion of
airways, most notably of the bronchioles (2). The only therapy available for
OB is augmentation of immunosuppression to arrest the functional decline of
the transplanted lung (3-5). Unfortunately, when obliterative bronchiolitis is
established augmented immunosuppression is ineffective in most cases (3,5).
More insight into the pathogenesis of OB is required to design adequate
treatment modalities.
The pathogenesis of human OB after lung transplantation has been
investigated in experimental studies using the model of obliterative airway
disease (OAD) in trachea grafts (6-11). These studies suggest that the
integrity of airway epithelium is essential for the success of lung allografts. It
was found that loss of epithelium as a result of rejection in allografted rat
tracheal transplants preceded obliteration of the tracheal lumen (12,13). Also,
it was found that exogenous injury of epithelial cells in tracheal isografts
induced obliteration of the lumen that resembled OAD in rejected allografts
(14,15). These findings suggest that the airway epithelium has a regulatory
role in preventing obliteration of airways in lung and trachea transplants. This
may well be accomplished by reducing fibroblasts growth and extracellular
matrix production, since fibroblasts are mainly responsible for the obliteration
of the airway lumen in OB and OAD (13,16).
In the present study a series of three experiments was undertaken in
rats to test the hypothesis that the integrity of airway epithelium is essential to
prevent OAD in trachea transplants. Firstly, we compared epithelial integrity
and luminal obliteration in rejecting trachea allografts (without immune
suppression) and in normal trachea isografts during a prolonged posttransplant period (from 4 days up to 8 weeks after transplantation). Then, we
investigated if epithelial injury (induced by enzymatic denudation) accelerated
the progression of OAD in allo- and isografted tracheas. Finally, we studied
whether healthy epithelial cells seeded into denuded trachea isografts could
prevent the development of OAD.
24
Integrity of airway epithelium is essential
Material and methods
Design of the study
For the first set of experiments in the study, with the aim to correlate integrity
of airway epithelium and luminal obliteration with inflammatory cell infiltration
in OAD, trachea isografts and allografts were transplanted subcutaneously
from Lewis (LEW) (n=19) and Brown Norway (BN) (n=20) donor rats,
respectively into LEW recipient rats (n=39). Grafts were harvested at 4 days
and 1,2,3,4, and 8 weeks after transplantation to assess the integrity of the
epithelium, the luminal obliteration and the inflammatory infiltration.
Next, the influence of loss of epithelial integrity on luminal obliteration
was investigated in 32 LEW isografts and 31 BN allografts by removal of
airway epithelial cells before transplantation of the tracheas into LEW recipient
rats (i.e., denuded tracheas). Grafts (3 to 8 at each time point) were harvested
at 2,4,6,10,14, and 21 days after transplantation for histological analysis.
Finally, to study whether epithelial cells can prevent the obliteration in
denuded trachea transplants, we seeded epithelial cells into denuded rat
tracheas which were subsequently transplanted isogenically into LEW rats.
Grafts were harvested at 2,4, and 6 days (n=5 at each time point) after
transplantation and histologically analyzed for the integrity of the epithelium
and for luminal obliteration. Denuded trachea isografts (n=2 to 5 at the same
time points) filled with medium instead of epithelial cells served as controls.
Animals
Inbred LEW and BN rats (male, specific-pathogen free) were obtained from
Harlan (Harlan Netherlands, Horst, The Netherlands) and were used in the
experiments at 12 weeks old. The rats were housed in the Central Animal
Laboratory of Groningen University. All animals received care in compliance
with the Dutch regulations and laws. Experimental protocols were approved
by the institutional animal ethical review committee.
Chemicals and antibodies
Protease (Protease Streptomyces griseus, p8811), 4 units/mg solid powder
and collagenase type IV were purchased from Sigma-Aldrich Chemie B.V.
Zwijndrecht, Netherlands. The enzymes were diluted in Hank’s Balanced Salt
25
CHAPTER 2
Solution (HBSS) (GIBCO, Cat. No.14025-092) to obtain appropriate
concentration.
For detection of specific inflammatory cells in the cellular infiltrates the
following primary mouse anti rat antibodies were applied: anti-CD3 for T cells,
anti-His 48 for granulocytes, anti-ED1 for macrophages and the antibodies for
epithelial cells staining, keratin-18 specific monoclonal antibody-RGE53
(Cat.No.69861N) were purchased from BD Pharmingen Alphen aan den Rijn,
The Netherlands. The anti-alpha-actin for myofibroblasts was purchased from
Roche Molecular Biochemicals, Almere, The Netherlands.
Trachea excision, denudation, epithelial cell seeding and transplantation
Donor and recipient rats were anesthetized with halothane and N2O/O2 gas.
Tracheas of donor rats were exposed by anterior midline incision and carefully
dissected from the esophagus and other surrounding tissues. The tracheas
were excised by transsection at the thyroid cartilage and the carina bifurcation
and were immediately brought into cold saline, stored at 4 oC until further
processing or transplantation.
Denudation of tracheas and the isolation of epithelial cells were
performed by enzymatic digestion of the extracellular matrix between the
epithelial cell layer and the submucosa tissue in the trachea. The lumen was
washed 5 times with 1 ml cold HBSS to remove blood and other contaminants,
filled with enzyme using a 25 G needle, and ligated at both ends with a 3-0
polypropylene ligature. The tracheas were brought into HBSS for incubation.
After incubation, the tracheas were cut open at both ends and the lumen was
washed with HBSS to flush out the dissociated cells. For epithelial cells
isolation, 1 ml cold Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco
BRL, Grand Island, NY, USA) was used to flush. The collected cells were
washed by centrifugation at 300g for 10 minutes and subsequently
resuspended in 25 cm2 culture flask coated with Collagen I gel. Epithelial cells
growth factor enriched culture medium DMEM/F12 (17) was added to allow
only the epithelial cells to recover and grow. Before seeding, the epithelial
cells were cultured for 24 hours. Cells viability was tested using trypan blue
staining.
In a pilot study the procedures for denudation and for the isolation of
epithelial cells were optimized. Therefore, different concentrations of enzymes
(collagenease IV and protease), temperatures, and incubation times were
tested (see Table 1). The efficacy of the denudation procedure was assessed
26
Integrity of airway epithelium is essential
histologically by assessing the remaining epithelial cells lining the trachea
lumen and scoring the integrity of the submucosal tissue. The applicability of
the isolation procedure of epithelial cells was assessed based on the viability
of the isolated epithelial cells after the enzymatic digestion. We found that the
best procedure for denudation of the tracheas was incubation with 4 units of
protease at 37 0C for 1 hour, since this procedure resulted in a complete
removal of all epithelial cells from the trachea without significant damage of
the submucosal tissue of the grafts. For the isolation of epithelial cells, the
best procedure was incubation with 4 units of protease at 4 0C of 18 hours,
yielding epithelial cells with a viability of 93%. (Table 1). Therefore, these
procedures were applied in later experiments for isolation and subsequent
seeding of epithelial cells.
Table 1. Effect of denudation procedures of rat tracheas and isolation of trachea epithelial
cells by enzymatic treatment.*
Procedures
Results
n
enzyme
(unit/ml)
incubation
time
incubation
temperature
epithelium
presence
submucosa
presence
epithelial
cells viability
8
Collagenase IV
100u
1h
37◦C
++
++
<10%
7
Collagenase IV
500u
1h
37◦C
+
4
Collagenase IV
1000u
1h
37◦C
-
-
<20%
6
Protease 4u
1h
37◦C
-
++++
<40%
9
Protease 8u
1h
37◦C
-
+
<35%
1
4
Protease 4u
18h
4◦C
+
+
>93%
<10%
* Histology was scored on a scale from – to ++++ representing: no epithelium/submucosa (-)
to intact epithelium/submucosa (++++) presence. Shown are the median values from the
number of experiments given in the table. Cells viability is given as a percentage of total cells
determined by trypan blue exclusion.
Seeding of epithelial cell into denuded tracheas was performed as
follows. Tracheal epithelial cells, harvested and cultured as described above,
were prepared at 2x106 cells/ml. Subsequently, the epithelial cells were
27
CHAPTER 2
injected into the lumen of denuded Lewis tracheas. Control denuded tracheas
were filled with DMEM/F12 medium only. The tracheas were closed at both
ends with surgical clips (Ligation clip 316l, Ethicon, Cincinnati, OH, USA) for
transplantation.
For transplantation of the trachea, a small incision was made at the
lateral side of the back of the recipient rat. By blunt dissection, a
subcutaneous pouch was made and the clipped trachea was put into the
pouche. The incision was closed by 2 single stitches (5-0 polypropylene
suture). One trachea graft was implanted per recipient.
Histology and immunohistochemistry
Tracheas from transplantation experiments were explanted by meticulous
excision, and were cut into two segments. One segment was fixed in 10%
formalin for paraffin embedding and Haematoxylin & Eosin (H&E) and
Verhoeff's elastin staining while the second segment was snap frozen in liquid
nitrogen for immunohistochemical staining. Tracheas for testing denudation
procedures were cross cut into three segments (i.e., an upper, a middle, and a
lower part) and fixed with 10% formalin for 24 hours. These tracheas were
embedded in paraffin and were only H&E stained.
To examine the integrity of the epithelium, trachea cross sections
stained by H&E were analyzed and epithelium was scored in five degrees
categorized from 0 to 4 for low to high integrity. The degree of epithelial cells
differentiation (the presence of well ciliated and pseudostratified epithelium
indicates high differentiation representing normal epithelium, otherwise, the
presence of single layer of flattened non-ciliated epithelium indicates poor
differentiation representing abnormal epithelium), and the epithelium lining of
the trachea lumen (in percentage) were evaluated.
To examine the degree of cellular infiltration in the H&E stained grafts
we assessed the infiltration level on a semi-quantitative scale of 0 to 4 (18).
The scale corresponds to the following infiltration grades: 0-no infiltration, 1minimal infiltration: scattered or diffuse cells infiltrates, 2-mild infiltration:
diffuse cell infiltrates with one area of dense infiltrates, 3-moderate infiltration:
more than one area infiltrates, 4-severe infiltration: a thick layer of dense cell
infiltrates.
The degree of luminal obliteration of the trachea was assessed by
examining the thickness of the submucosa (i.e., between epithelium and
cartilage) on a scale of 0 to 4 (18). The scores represent the following grades:
28
Integrity of airway epithelium is essential
0-no thickening of submucosa, 1-minimal thickening: focal submucosa
thickening, 2-mild thickening: submucosa thickening equal to the thickness of
the cartilage ring, 3-moderate thickening, submucosa thickening resulting in a
small lumen, 4-severe thickening: total lumen obliteration.
Immunohistochemistry was performed on frozen sections of tracheas.
The tracheas were sectioned and processed as previously described (18).
Sections were fixed in acetone and washed 3 times in 0.01M phosphatebuffered saline (PBS). Subsequently, we applied the appropriate primary
antibodies in 1:50 dilutions. After 60 minutes incubation with the primary
antibody the slides were washed with PBS. Next, we applied the peroxidaseconjugated secondary rabbit anti mouse antibody at 1:50 dilution (Dako,
Glostrup, Denmark). After 60 minutes incubation, the slides were washed with
PBS, and processed for staining, using amino-aethyl carbazole (AEC) (Sigma,
0.5mg in 3.75 ml DMF plus 70ml Acetate buffer, PH =4.9) together with H2O2,
as a reagent giving a reddish-brown precipitate. Slides were counterstained
with haematoxylin (Mayers’ haematoxylin 1:10). The slides were examined for
number of positive cells from low to high numbers as 0 to grade 5 (six levels).
Epithelial cells were stained by keratin-18 specific monoclonal antibody
(RGE53) and myofibroblasts were stained by anti-alpha-actin specific
antibody. The staining procedure was identical to the protocol described
above. All histology counting and scoring was done by two to three
independent observers.
Statistical analysis
Results are expressed either as mean ± SEM or as median plus range (table).
Statistical comparisons for difference between groups were evaluated by
Mann-Whitney U test. A p-value < 0.05 was considered statistically significant.
Results
Transplantation induced OAD in allografts but not in isografts
In trachea iso- and allografts we investigated how the integrity of the tracheal
epithelium and the degree of luminal obliteration correlated with the
inflammatory cell infiltration at 4 days and at 1,2,3,4,8 weeks after
transplantation. Isografting was not associated with significant cellular
infiltration or loss of integrity of the epithelium (Table 2A), despite the ischemic
and surgical injury at the time of transplantation. We did observe a few
29
CHAPTER 2
granulocytes, macrophages in the isografts. In a minority of the tracheas, we
observed some abnormal architecture of the epithelium as demonstrated by
loss of cilia and the absence of pseudostratified epithelium. This was
completely restored in the first week after transplantation.
grading of infiltration,
epithelium and myofibroblast
found in the grafts
Figure 1.
5
4
3
2
1
0
0.5 1
2
3
4
8
weeks after transplantation
Figure 1. Composition of cellular infiltrates and epithelium presence in rat trachea allografts.
BN trachea allografts (n= 3 to 9) were analyzed by immunohistochemistry at each time point
after subcutaneous transplantation into LEW rats. Open squares (□) represent the epithelium
integrity; black triangles (▲) represent the macrophages; open triangles (∆) represent T cells;
asterisks (*) represent myofibroblasts; black circles (●) represent granulocytes. A value of 0
represents absence of infiltrate while value 5 represents a severe infiltration of the graft.
Values are medians of at least 3 experiments.
The pathology of rejecting allografts (tracheas transplanted from BN to
LEW rats) was very different from that of isografts: severe inflammatory cell
infiltration was associated with loss of airway epithelium within the first weeks
after transplantation, resulting in severe luminal obliteration at 3 weeks (Table
2B). Cellular infiltration during the first week after transplantation was most
obvious in the tissue outside of the tracheal cartilage. The infiltration inside of
the trachea started later, to reach its highest level at 2 weeks.
Immunohistochemical analysis of the infiltrating cells showed that these were
mainly macrophages, granulocytes and T cells (Figure 1), a pattern consistent
with acute rejection. The intensity of the infiltration was associated with the
30
Integrity of airway epithelium is essential
higher loss of integrity of the airway epithelium. In the first week after
transplantation, the appearance of the epithelium was flattened and part of the
epithelial cells was lost. The loss of epithelium was complete at 2 weeks after
transplantation. Simultaneously, the lumen of the trachea became obliterated
(Table 2B). At 1 week, the obliteration could be explained by thickening of the
tracheal submucosa by infiltrating cells (Figure 1). As of the moment of
disappearance of epithelial cells from the lumen (2 weeks posttransplantation), the obliteration was caused by large numbers of
myofibroblasts and increase of connective tissue in the tracheas (Figure 1).
Denudation of tracheal epithelial cells and OAD
In order to investigate the relation between the integrity of epithelium and
luminal obliteration in more detail, we next studied the obliteration after
grafting of tracheas in the absence of epithelial cells (i.e., denuded tracheas).
Obliteration was assessed in normal and in denuded tracheas at short
intervals after transplantation (at 2,4,6,10,14, and 21 days, n=3 to 8). The
amount of epithelial cells profoundly decreased in control trachea isografts in
the first days after transplantation (Figure 2 A). This loss was compensated by
fast regeneration of the epithelial cells during the first week after
transplantation, resulting in complete recovery of the epithelial layer by day
10. In control allografts, the loss of epithelial cells showed a similar pattern of
recovery after transplantation (Figure 2 B). We found an initial loss of the
epithelium at day 2 with gradual recovery up to day 6. Then, the epithelial
presence dropped sharply to disappear completely by day 10 after
transplantation. These data are in agreement with the less detailed
observations in the first experiment of this study.
As expected, the denuded iso- and allografts showed virtually no epithelium in
the period immediately after transplantation (Figure 2 C, D, n=4 to 5) although
in spite of the enzymatic removal of epithelium, denuded isografts showed
some epithelial cells in the lumen at day 4 and day 6 (Figure 2 C). These
epithelial cells may be responsible for the complete recovery of the epithelial
layer seen at day 21 after implantation. There was no epithelium regeneration
observed in denuded allografts after transplantation (Figure 2 D).
31
CHAPTER 2
Figure 2.
□ epithelium coverage
● obliteration
X: days after transplantation
32
Integrity of airway epithelium is essential
Figure 2. Integrity of the epithelium (open squares □) and degree of luminal obliteration
(black circles ●) after transplantation of normal and denuded LEW iso- and BN allografts into
LEW recipients. The integrity of the epithelium is expressed as the percentage of luminal area
that is covered with epithelial cells. The obliteration level is expressed from low to high as 0-4,
i.e. a value of 0 represents absence of obliteration while 4 represents complete luminal
obliteration. Values represent medians and ranges of 3 to 9 experiments.
The essential role of the presence of epithelial cells in preventing
obliteration is also demonstrated in Figure 2. With the grafting of denuded
isografts and allografts, we found a pronounced and progressive obliteration
of the lumen (Figure 2 C, D), whereas in normal isografts we found only
minimal obliteration, disappearing after the complete recovery of the
epithelium (Figure 2 A). In the control allografts, obliteration developed more
slowly than in the denuded allografts (Figures 2 B, D). The best illustration of
the role of luminal epithelial cells in preventing obliteration is given by the
denuded isografts. Here obliteration developed quickly after transplantation,
but this stopped by day 10 which coincided with the recovery of the epithelium
(Figure 2 C).
We also evaluated those grafts in which the epithelium was not
completely removed before transplantation (i.e., incompletely denuded group).
The epithelium in these grafts started to recover immediately after
transplantation at day 2, and was mostly restored within 6 days (Figure 2E,
n=2 to 3). The obliteration of the lumen remained at minimal to mild level
during the whole study period, similar to the control isografts.
Transplantation of epithelial cells into denuded trachea grafts to prevent
OAD
The experiments described above suggest a regulatory role of epithelial cells
in preventing luminal obliteration in OAD. In order to confirm this role of airway
epithelium in preventing obliteration, we studied whether seeding of isolated,
viable epithelial cells could prevent the early obliteration in trachea isografts.
In the epithelial cell seeded, denuded isografts investigated at 2 days
after transplantation, the lumen of the trachea was filled with proteinacous
material containing loose cells, amongst which were many granulocytes and
erythrocytes. At that moment no epithelium layer could be recognized, but
already on day 4 after transplantation a thin layer of epithelial cells lined the
partially open lumen of the treated trachea (Figure 3, seed d4). The epithelial
layer was still irregular, at some places with flattened epithelial cells, at other
33
CHAPTER 2
places with cells in a multilayer up to 5 cells thick without nuclear polarity. The
remainder of the trachea lumen was filled with similar material as seen on day
2, with decreasing numbers of granulocytes. At day 6, the epithelium was
restored to normal as demonstrated by the presence of pseudostratified
epithelium with ciliated epithelial cells; the lumen of the tracheas was open
without significant fibrotic obliteration (Figure 3, seed d6). In the control
denuded isografts without cell seeding, luminal obliteration at day 4 after
transplantation (Figure 3, denu d4) progressed at day 6, with many cells in the
lumen showing the morphology of fibroblasts (Figure 3, denu d6). In these
tracheas no epithelium regeneration was observed at these early time points
after transplantation.
Table 2. Inflammation, integrity of epithelium, and obliteration in trachea iso- and allografts at
4 days, 1, 2, 3, 4, and 8 weeks after transplantation*.
A. Isografts
Inflammation
Epithelium
integrity
Luminal
obliteration
Time
n
Median(Range)
Median(Range)
Median(Range)
D4
3
0
(0-0)
3
(3-4)
0
(0-0)
1w
3
0
(0-0)
4
(4-4)
0
(0-0)
2w
3
0
(0-0)
4
(4-4)
0
(0-0)
3w
3
0
(0-0)
4
(4-4)
0
(0-0)
4w
3
0
(0-0)
3
(0-4)
0
(0-0)
8w
4
0
(0-0)
3.5
(3-4)
0
(0-0)
B. Allografts
Inflammation
inside trachea
Inflammation
outside trachea
Epithelium
integrity
Luminal
obliteration
Time
n
Median(Range)
Median(Range)
Median(Range)
Median(Range)
D4
3
0
(0-0)
3.5
(3-4)
2
(0-2)
0
(0-0)
1w
3
0
(0-4)
4
(4-4)
2
(2-2)
0
(0-4)
2w
3
3
(0-3)
4
(4-4)
0
(0-0)
0
(0-4)
3w
3
2
(2-2)
4
(4-4)
0
(0-0)
4
(2-4)
4w
3
3
(2-4)
4
(3-4)
0
(0-0)
4
(2-4)
8w
5
0
(0-2)
2
(0-3)
0
(0-0)
4
(2-4)
* Scores represent grading from low to high as 0-4.
34
Integrity of airway epithelium is essential
Figure 3.
Figure 3. The histology of denuded isografts: non-seeding and seeded with epithelial cells
after transplantation. In the denuded non-seeding graft at day 4, no epithelium was present
and the lumen was partially obliterated by cellular debris. At day 6, epithelium remained
absent, and the lumen was largely obliterated by fibrotic tissue. In the epithelial cell seeded
group at day 4, a thin epithelium layer was lining the lumen (arrow). In the lumen was some
cellular debris. On day 6, a totally restored epithelium was lining the lumen, while epithelial
cells regained cilia and polarity. Histology slides were Verhoeff's elastin stained; pictures were
taken at 40x magnification, the insets at 100x magnification.
Discussion
From the three experiments in this study it can be concluded that
development of OAD in trachea transplants correlates with injury and repair of
the airway epithelium. Injury of the epithelium with subsequent luminal
obliteration coincided with inflammatory responses in the trachea grafts. In
this inflammation-induced injury to airway epithelium we distinguish two injury
phases after transplantation of rat tracheas: the “surgical” injury phase (most
obvious in isografts immediately after transplantation) and the rejection injury
phase (most obvious in allografts from 10 days after transplantation).
35
CHAPTER 2
Furthermore, the progression of OAD is facilitated if repair of epithelium is
prevented by denudation of trachea iso- and allografts. In contrast,
development of OAD is prevented when the integrity of epithelium is restored
quickly, in our experiments by seeding of epithelial cells into denuded trachea
isografts. Taken together, these observations demonstrate that integrity of
airway epithelium is essential for trachea transplants to be safeguarded from
OAD.
How does epithelial injury in the two phases lead to OAD? In the
surgical injury phase a non-specific inflammatory reaction with influx of
granulocytes and macrophages develops in the tracheas immediately after
transplantation. This inflammation is the response to ischemia in the period
between removal of the graft from the donor and complete engraftment in the
recipient and to reperfusion in the subsequent period. In this surgical injury
phase epithelial cells are partially lost from the trachea transplants,
irrespective of alloreactivity as its severity is similar in trachea isografts and
allografts. In isografted tracheas, epithelium can recover from the surgical
injury phase; it regrows quickly and covers the trachea lumen completely. The
mild and transient obliteration of the lumen observed in some isografts in our
experiments probably reflects transient swelling of the submucosa by the
inflammatory reaction, without fibroblast proliferation. Even in allografts, the
epithelium recovers from the surgical injury phase during the first
postoperative week. These observations show that inflammation from the
surgical injury phase has no lasting effect on trachea epithelium, provided no
allograft-rejection related inflammation is involved.
In the rejection injury phase the airway epithelium is injured more
severely by the inflammatory reaction as a result of acute rejection.
Alloreactive, antigen-dependent T cells have been shown to play a role in the
development of OAD (19,20). In agreement, a significant proportion of the
inflammatory cells in our trachea allografts have the T cell phenotype.
Remarkably, the inflammation starts in the periphery of the trachea allograft,
to reach the epithelium between 1 and 2 week post-transplantation. It is
exactly in this period that the epithelial cells are injured beyond repair and
disappear from the trachea allografts. Simultaneously, myofibroblasts start to
grow and obliterate the trachea lumen in a process typical of OAD as
described by Boehler et al (21) and King et al (13). During the rejection injury
phase, recovery of the trachea allograft is still possible to a certain degree.
This was shown in a study of Brazelton (22) where OAD in allografts was
prevented when the progression of rejection was stopped. They removed
36
Integrity of airway epithelium is essential
trachea allografts from the recipients before getting irreversible epithelium
damage (day 10) and transplanted the tracheas back to syngeneic recipients
of the donor strain. In these retransplanted grafts the airway epithelium
regenerated and the fibroproliferative obliteration of the lumen did not occur.
These data are in line with the hypothesis that rejection-induced inflammation
is the major factor causing epithelial injury that ultimately leads to OAD.
The importance of epithelium for the repair from injury is emphasized
by our observations in two ways. On the one hand, when recovery of
epithelium is hampered by denudation from trachea grafts, OAD develops
both in isografts and in allografts. On the other hand, development of OAD is
effectively prevented if epithelium is allowed to regrow quickly in isografts
seeded with epithelial cells. These experiments show that early recovery of
epithelium can prevent OAD even in situations with severe epithelial injury at
the time of transplantation.
Intact epithelium is likely to play a regulatory role on fibroproliferative
responses in the trachea transplants. We presume that under normal
circumstances, like in the isografted tracheas, fibroblast growth is suppressed
by the luminal epithelium layer, releasing regulating cytokines and growth
factors. This is confirmed by studies showing that myofibroblasts start to
proliferate only after damage or inadequate function of the airway epithelium,
both in vivo (23) and in vitro (24-26). In vitro studies (27,28) have shown that
cytokines produced by epithelial cells, such as prostaglandin E2 (29) and
transforming growth factor beta (28), are successful inhibitors of
fibroproliferation. In the absence of epithelial cells in the rejected or denuded
tracheas, myofibroblasts will have the opportunity to grow into the lumen with
unregulated growth and to obliterate the lumen as a consequence.
Our observation that seeding of viable epithelial cells into the lumen of
tracheas can prevent OAD corroborates the findings of the group of Morris.
They showed successful engraftment without OAD when epithelium was
transplanted into trachea isografts from which the epithelium was partly
digested with protease (15). In that study, however, it could not be excluded
that the prevention of OAD was due to early regeneration of the epithelial cells
in the trachea since the protease-treated tracheas showed epithelial cells at
the time of transplantation. This is in accordance with our incomplete
denudation group in which we found that the epithelium recovered quickly
after grafting without significant obliteration. In an elegant model (30,31) using
trachea allografts flanked with segments of trachea isografts, it was shown
that the epithelium in the rejecting tracheas was regenerated by cells growing
37
CHAPTER 2
from the adjacent isografts and that the lumen remained patent. From these
very different studies we conclude that the replacement of epithelium by wellfunctioning epithelial cells can help to prevent airway obliteration.
This trachea transplantation study may be of relevance for clinical lung
transplantation, although the development of OB in patients is influenced by
many more noxious factors such as infection (32) and HLA specific antibodies
(34),. Yet, we think that also in patients the integrity of the airway epithelium is
an essential component of adequate prevention of OB. The induction of OB is
clearly linked to episodes of acute rejection (2) which was most detrimental for
epithelial integrity in our study. Besides by rejection, OB can be induced by
many more factors. From our experiments with denuded and epithelial-cell
seeded trachea isografts it can be concluded that it is essential to preserve or
repair the epithelial integrity in order to prevent OAD. At this moment it is hard
to envision how seeding of epithelial cells could be performed in recipients of
lung transplants, although the use of recipient stem cells might be a future
perspective for development of a similar treatment. A more plausible approach
at present to prevent OB development would be the protection of airway
epithelial cells by activation of 'protective genes' like HO-1 (35), which might
be capable to reduce local inflammation at the epithelial level in case of
allograft reactivity. This might be achieved by exposure of donor lung to
carbon monoxide, a treatment that was effective in a rat lung transplantation
model (36). The efficacy and possible side effects of such an approach for
prevention of OB after human lung transplantation have yet to be investigated.
38
Integrity of airway epithelium is essential
Acknowledgement
The authors want to thank Gineke Drok and Hans Vos for their help with
histological staining, reading and scoring and Arjen Petersen for the help with
the animal surgery.
39
CHAPTER 2
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